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Spurney, Robert Frank

Overview:

Dr. Spurney’s research has focused on the role of G protein coupled receptors (GPCRs) in regulating cellular physiology both in normal and disease states as well as the regulatory mechanisms that modulate GPCR responsiveness at the molecular level. These studies have centered on two major themes. The first investigates the role of GPCR signaling pathways in regulating podocyte function using cultured podocytes and transgenic animals models. The second area of research involves the role of GPCR kinases (GRKs) and arrestin scaffolding proteins in modulating GPCR signaling in bone forming osteoblasts using cultured osteoblast cell lines as well as genetically modified mice including transgenic models and knockout animals. The long-term goal of these studies is to identify novel therapeutic targets that may useful for treating disease processes such as glomerulonephritis and osteoporosis.

Please see the Duke Nephrology Division website for more detailed information.

Positions:

Professor of Medicine

Medicine, Nephrology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1983

M.D. — Ohio State University

Medical Resident, Medicine

Duke University

Resident In Pathology, Pathology

Duke University

Senior Assistant Resident, Medicine

Duke University

Fellow In Nephrology, Medicine

Duke University

Grants:

Duke Training Grant in Nephrology

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 20, 1995
End Date
June 30, 2022

The Role of Interleukin-15 Receptor-alpha Variants in the Pathogenesis of FSGS

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
June 15, 2017
End Date
June 14, 2022

The role of dendritic cell-mediated T cell activation in hypertension

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
July 01, 2017
End Date
June 30, 2019

Functional and Phenotypic Characterization of a New FSGS Gene

Administered By
Pediatrics, Nephrology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
September 24, 2014
End Date
May 31, 2019

Targeting TRPC6 to treat focal segmental glomerulosclerosis (FSGS)

Administered By
Medicine, Nephrology
AwardedBy
Amgen, Inc.
Role
Principal Investigator
Start Date
December 07, 2016
End Date
December 06, 2018

Gene Discovery in Autosomal Dominant Focal Segmental Glomerulosclerosis

Administered By
Duke Molecular Physiology Institute
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
September 18, 2012
End Date
June 30, 2018

Serial Block Face Scanning Electron Microscope

Administered By
Pathology
AwardedBy
National Institutes of Health
Role
Major User
Start Date
June 01, 2016
End Date
May 31, 2018

Role of Gq Signaling in Promoting Podocyte Injury in Diabetes Mellitus

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 09, 2011
End Date
August 31, 2016

IPA Extensioin - Wang

Administered By
Medicine, Nephrology
AwardedBy
Durham Veterans Affairs Medical Center
Role
Principal Investigator
Start Date
January 01, 2013
End Date
September 01, 2014

IPA - Liming Wang

Administered By
Medicine, Nephrology
AwardedBy
Durham Veterans Affairs Medical Center
Role
Principal Investigator
Start Date
January 01, 2011
End Date
December 31, 2012

A Novel Mouse Model of Podocyte Injury

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2009
End Date
December 31, 2012

Molecular mechanisms of altered calcium sensing in human parathyroid disease

Administered By
Surgery
AwardedBy
National Institutes of Health
Role
Collaborating Investigator
Start Date
June 01, 2010
End Date
January 31, 2012

Regulation of Podocyte Function by Angiotensin II

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 15, 2004
End Date
December 31, 2008

Role of G-Protein coupled receptor kinases in Osteogenesis

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 30, 2001
End Date
April 30, 2006

Pharmacologically Induced Neo-osteogenesis

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
August 01, 1986
End Date
May 15, 2004

Thromboxane Receptor Regulation By Protein Kinases

Administered By
Medicine, Nephrology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 10, 1995
End Date
June 30, 2000
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Publications:

Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease.

Enhanced expression of cyclooxygenase 2 (COX2) in podocytes contributes to glomerular injury in diabetic kidney disease, but some basal level of podocyte COX2 expression might be required to promote podocyte attachment and/or survival. To investigate the role of podocyte COX2 expression in diabetic kidney disease, we deleted COX2 specifically in podocytes in a mouse model of Type 1 diabetes mellitus (Akita mice). Podocyte-specific knockout (KO) of COX2 did not affect renal morphology or albuminuria in nondiabetic mice. Albuminuria was significantly increased in wild-type (WT) and KO Akita mice compared with nondiabetic controls, and the increase in albuminuria was significantly greater in KO Akita mice compared with WT Akita mice at both 16 and 20 wk of age. At the 20-wk time point, mesangial expansion was also increased in WT and KO Akita mice compared with nondiabetic animals, and these histologic abnormalities were not improved by KO of COX2. Tubular injury was seen only in diabetic mice, but there were no significant differences between groups. Thus, KO of COX2 enhanced albuminuria and did not improve the histopathologic features of diabetic kidney disease. These data suggest that 1) KO of COX2 in podocytes does not ameliorate diabetic kidney disease in Akita mice, and 2) some basal level of podocyte COX2 expression in podocytes is necessary to attenuate the adverse effects of diabetes on glomerular filtration barrier function.

Authors
Wang, L; Sha, Y; Bai, J; Eisner, W; Sparks, MA; Buckley, AF; Spurney, RF
MLA Citation
Wang, L, Sha, Y, Bai, J, Eisner, W, Sparks, MA, Buckley, AF, and Spurney, RF. "Podocyte-specific knockout of cyclooxygenase 2 exacerbates diabetic kidney disease." American journal of physiology. Renal physiology 313.2 (August 2017): F430-F439.
PMID
28490532
Source
epmc
Published In
American Journal of Physiology: Renal Physiology
Volume
313
Issue
2
Publish Date
2017
Start Page
F430
End Page
F439
DOI
10.1152/ajprenal.00614.2016

Losing their footing: Rac1 signaling causes podocyte detachment and FSGS.

Selective modulation of Rho GTPase activity in podocytes recapitulates characteristic features of human nephrosis. Using a mouse model, Robins et al. found that high levels of Rac1 activation in podocytes caused podocyte detachment and glomerulosclerosis. Podocyte Rac1 activity was enhanced in biopsy specimens from patients with nephrosis, and serum from this patient population activated Rac1 in cultured podocytes. These data provide a causal link between podocyte Rac1 activation and human nephrotic diseases.

Authors
Hall, G; Spurney, RF
MLA Citation
Hall, G, and Spurney, RF. "Losing their footing: Rac1 signaling causes podocyte detachment and FSGS." Kidney international 92.2 (August 2017): 283-285.
PMID
28709595
Source
epmc
Published In
Kidney international
Volume
92
Issue
2
Publish Date
2017
Start Page
283
End Page
285
DOI
10.1016/j.kint.2017.03.045

Twenty years after ACEIs and ARBs: emerging treatment strategies for diabetic nephropathy.

Diabetic nephropathy (DN) is a serious complication of both type 1 and type 2 diabetes mellitus. The disease is now the most common cause of end-stage kidney disease (ESKD) in developed countries, and both the incidence and prevalence of diabetes mellitus is increasing worldwide. Current treatments are directed at controlling hyperglycemia and hypertension, as well as blockade of the renin angiotensin system with angiotensin-converting enzyme inhibitors (ACEIs), and angiotensin receptor blockers. Despite these therapies, DN progresses to ESKD in many patients. As a result, much interest is focused on developing new therapies. It has been over two decades since ACEIs were shown to have beneficial effects in DN independent of their blood pressure-lowering actions. Since that time, our understanding of disease mechanisms in DN has evolved. In this review, we summarize major cell signaling pathways implicated in the pathogenesis of diabetic kidney disease, as well as emerging treatment strategies. The goal is to identify promising targets that might be translated into therapies for the treatment of patients with diabetic kidney disease.

Authors
Johnson, SA; Spurney, RF
MLA Citation
Johnson, SA, and Spurney, RF. "Twenty years after ACEIs and ARBs: emerging treatment strategies for diabetic nephropathy." American journal of physiology. Renal physiology 309.10 (November 2015): F807-F820. (Review)
PMID
26336162
Source
epmc
Published In
American Journal of Physiology: Renal Physiology
Volume
309
Issue
10
Publish Date
2015
Start Page
F807
End Page
F820
DOI
10.1152/ajprenal.00266.2015

Gq signaling causes glomerular injury by activating TRPC6.

Familial forms of focal segmental glomerulosclerosis (FSGS) have been linked to gain-of-function mutations in the gene encoding the transient receptor potential channel C6 (TRPC6). GPCRs coupled to Gq signaling activate TRPC6, suggesting that Gq-dependent TRPC6 activation underlies glomerular diseases. Here, we developed a murine model in which a constitutively active Gq α subunit (Gq(Q209L), referred to herein as GqQ>L) is specifically expressed in podocytes and examined the effects of this mutation in response to puromycin aminonucleoside (PAN) nephrosis. We found that compared with control animals, animals expressing GqQ>L exhibited robust albuminuria, structural features of FSGS, and reduced numbers of glomerular podocytes. Gq activation stimulated calcineurin (CN) activity, resulting in CN-dependent upregulation of TRPC6 in murine kidneys. Deletion of TRPC6 in GqQ>L-expressing mice prevented FSGS development and inhibited both tubular damage and podocyte loss induced by PAN nephrosis. Similarly, administration of the CN inhibitor FK506 reduced proteinuria and tubular injury but had more modest effects on glomerular pathology and podocyte numbers in animals with constitutive Gq activation. Moreover, these Gq-dependent effects on podocyte injury were generalizable to diabetic kidney disease, as expression of GqQ>L promoted albuminuria, mesangial expansion, and increased glomerular basement membrane width in diabetic mice. Together, these results suggest that targeting Gq/TRPC6 signaling may have therapeutic benefits for the treatment of glomerular diseases.

Authors
Wang, L; Jirka, G; Rosenberg, PB; Buckley, AF; Gomez, JA; Fields, TA; Winn, MP; Spurney, RF
MLA Citation
Wang, L, Jirka, G, Rosenberg, PB, Buckley, AF, Gomez, JA, Fields, TA, Winn, MP, and Spurney, RF. "Gq signaling causes glomerular injury by activating TRPC6." The Journal of clinical investigation 125.5 (May 2015): 1913-1926.
PMID
25844902
Source
epmc
Published In
Journal of Clinical Investigation
Volume
125
Issue
5
Publish Date
2015
Start Page
1913
End Page
1926
DOI
10.1172/jci76767

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

Authors
Hall, G; Gbadegesin, RA; Lavin, P; Wu, G; Liu, Y; Oh, EC; Wang, L; Spurney, RF; Eckel, J; Lindsey, T; Homstad, A; Malone, AF; Phelan, PJ; Shaw, A; Howell, DN; Conlon, PJ; Katsanis, N; Winn, MP
MLA Citation
Hall, G, Gbadegesin, RA, Lavin, P, Wu, G, Liu, Y, Oh, EC, Wang, L, Spurney, RF, Eckel, J, Lindsey, T, Homstad, A, Malone, AF, Phelan, PJ, Shaw, A, Howell, DN, Conlon, PJ, Katsanis, N, and Winn, MP. "A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS." Journal of the American Society of Nephrology : JASN 26.4 (April 2015): 831-843.
PMID
25145932
Source
epmc
Published In
Journal of the American Society of Nephrology : JASN
Volume
26
Issue
4
Publish Date
2015
Start Page
831
End Page
843
DOI
10.1681/asn.2013101053

Mutations in the gene that encodes the F-actin binding protein anillin cause FSGS.

FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1-associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm-associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton.

Authors
Gbadegesin, RA; Hall, G; Adeyemo, A; Hanke, N; Tossidou, I; Burchette, J; Wu, G; Homstad, A; Sparks, MA; Gomez, J; Jiang, R; Alonso, A; Lavin, P; Conlon, P; Korstanje, R; Stander, MC; Shamsan, G; Barua, M; Spurney, R; Singhal, PC; Kopp, JB; Haller, H; Howell, D; Pollak, MR; Shaw, AS; Schiffer, M; Winn, MP
MLA Citation
Gbadegesin, RA, Hall, G, Adeyemo, A, Hanke, N, Tossidou, I, Burchette, J, Wu, G, Homstad, A, Sparks, MA, Gomez, J, Jiang, R, Alonso, A, Lavin, P, Conlon, P, Korstanje, R, Stander, MC, Shamsan, G, Barua, M, Spurney, R, Singhal, PC, Kopp, JB, Haller, H, Howell, D, Pollak, MR, Shaw, AS, Schiffer, M, and Winn, MP. "Mutations in the gene that encodes the F-actin binding protein anillin cause FSGS." Journal of the American Society of Nephrology : JASN 25.9 (September 2014): 1991-2002.
PMID
24676636
Source
epmc
Published In
Journal of the American Society of Nephrology : JASN
Volume
25
Issue
9
Publish Date
2014
Start Page
1991
End Page
2002
DOI
10.1681/asn.2013090976

Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity.

The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr(69)/Thr(70) (Thr(69) in mice and Thr(70) in humans) and Ser(321)/Ser(322) (Ser(321) in mice and Ser(322) in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr(69) in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3',4'-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr(69) dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.

Authors
Hall, G; Rowell, J; Farinelli, F; Gbadegesin, RA; Lavin, P; Wu, G; Homstad, A; Malone, A; Lindsey, T; Jiang, R; Spurney, R; Tomaselli, GF; Kass, DA; Winn, MP
MLA Citation
Hall, G, Rowell, J, Farinelli, F, Gbadegesin, RA, Lavin, P, Wu, G, Homstad, A, Malone, A, Lindsey, T, Jiang, R, Spurney, R, Tomaselli, GF, Kass, DA, and Winn, MP. "Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity." American journal of physiology. Renal physiology 306.12 (June 2014): F1442-F1450.
PMID
24740790
Source
epmc
Published In
American Journal of Physiology: Renal Physiology
Volume
306
Issue
12
Publish Date
2014
Start Page
F1442
End Page
F1450
DOI
10.1152/ajprenal.00212.2013

Special deLIVERy: podocyte injury promotes renal angiotensin II generation from liver-derived angiotensinogen.

The role of the circulating renin-angiotensin system (RAS) in regulating systemic blood pressure and sodium balance is well established. More recently, researchers have turned their focus to the local generation of angiotensin II (Ang II) in specific tissues. Matsusaka et al. revisit the renal RAS and provide evidence that liver-derived angiotensinogen (AGT) is a major determinant of renal Ang II levels in a model of podocyte injury.

Authors
Ortiz-Melo, DI; Spurney, RF
MLA Citation
Ortiz-Melo, DI, and Spurney, RF. "Special deLIVERy: podocyte injury promotes renal angiotensin II generation from liver-derived angiotensinogen." Kidney international 85.5 (May 2014): 1009-1011.
PMID
24786873
Source
epmc
Published In
Kidney international
Volume
85
Issue
5
Publish Date
2014
Start Page
1009
End Page
1011
DOI
10.1038/ki.2013.440

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4-5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease. © 2014 Elsevier Inc. All rights reserved.

Authors
Wang, L; Tang, Y; Eisner, W; Sparks, MA; Buckley, AF; Spurney, RF
MLA Citation
Wang, L, Tang, Y, Eisner, W, Sparks, MA, Buckley, AF, and Spurney, RF. "Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice." Biochemical and Biophysical Research Communications 444.4 (February 21, 2014): 622-627.
Source
scopus
Published In
Biochemical and Biophysical Research Communications
Volume
444
Issue
4
Publish Date
2014
Start Page
622
End Page
627
DOI
10.1016/j.bbrc.2014.01.115

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice.

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4-5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.

Authors
Wang, L; Tang, Y; Eisner, W; Sparks, MA; Buckley, AF; Spurney, RF
MLA Citation
Wang, L, Tang, Y, Eisner, W, Sparks, MA, Buckley, AF, and Spurney, RF. "Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice." Biochemical and biophysical research communications 444.4 (February 2014): 622-627.
PMID
24491571
Source
epmc
Published In
Biochemical and Biophysical Research Communications
Volume
444
Issue
4
Publish Date
2014
Start Page
622
End Page
627
DOI
10.1016/j.bbrc.2014.01.115

Non-immunologic actions of calcineurin inhibitors in proteinuric kidney diseases.

Authors
Spurney, RF
MLA Citation
Spurney, RF. "Non-immunologic actions of calcineurin inhibitors in proteinuric kidney diseases." Frontiers in endocrinology 5 (January 2014): 181-. (Review)
PMID
25429282
Source
epmc
Published In
Frontiers in Endocrinology
Volume
5
Publish Date
2014
Start Page
181
DOI
10.3389/fendo.2014.00181

Erratum: Activation of Gaq-coupled signaling pathways in glomerular podocytes promotes renal injury (Journal of the American Society of Nephrology (2005) 16 (3611-3622))

Authors
Wang, L; Fields, TA; Pazmino, K; Dai, Q; Burchette, JL; Howell, DN; Coffman, TM; Spurney, RF
MLA Citation
Wang, L, Fields, TA, Pazmino, K, Dai, Q, Burchette, JL, Howell, DN, Coffman, TM, and Spurney, RF. "Erratum: Activation of Gaq-coupled signaling pathways in glomerular podocytes promotes renal injury (Journal of the American Society of Nephrology (2005) 16 (3611-3622))." Journal of the American Society of Nephrology 24.12 (December 9, 2013): 2126-.
Source
scopus
Published In
Journal of the American Society of Nephrology : JASN
Volume
24
Issue
12
Publish Date
2013
Start Page
2126
DOI
10.1681/ASN.2013080868

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis.

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.

Authors
Wang, X; Liao, S; Nelson, ER; Schmalzigaug, R; Spurney, RF; Guilak, F; Premont, RT; Gesty-Palmer, D
MLA Citation
Wang, X, Liao, S, Nelson, ER, Schmalzigaug, R, Spurney, RF, Guilak, F, Premont, RT, and Gesty-Palmer, D. "The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis." Biochem Biophys Res Commun 425.2 (August 24, 2012): 407-412.
PMID
22846567
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
425
Issue
2
Publish Date
2012
Start Page
407
End Page
412
DOI
10.1016/j.bbrc.2012.07.111

A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension.

RATIONALE: Human clinical trials using type 1 angiotensin (AT(1)) receptor antagonists indicate that angiotensin II is a critical mediator of cardiovascular and renal disease. However, recent studies have suggested that individual tissue pools of AT(1) receptors may have divergent effects on target organ damage in hypertension. OBJECTIVE: We examined the role of AT(1) receptors on T lymphocytes in the pathogenesis of hypertension and its complications. METHODS AND RESULTS: Deficiency of AT(1) receptors on T cells potentiated kidney injury during hypertension with exaggerated renal expression of chemokines and enhanced accumulation of T cells in the kidney. Kidneys and purified CD4(+) T cells from "T cell knockout" mice lacking AT(1) receptors on T lymphocytes had augmented expression of Th1-associated cytokines including interferon-γ and tumor necrosis factor-α. Within T lymphocytes, the transcription factors T-bet and GATA-3 promote differentiation toward the Th1 and Th2 lineages, respectively, and AT(1) receptor-deficient CD4(+) T cells had enhanced T-bet/GATA-3 expression ratios favoring induction of the Th1 response. Inversely, mice that were unable to mount a Th1 response due to T-bet deficiency were protected from kidney injury in our hypertension model. CONCLUSIONS: The current studies identify an unexpected role for AT(1) receptors on T lymphocytes to protect the kidney in the setting of hypertension by favorably modulating CD4(+) T helper cell differentiation.

Authors
Zhang, J-D; Patel, MB; Song, Y-S; Griffiths, R; Burchette, J; Ruiz, P; Sparks, MA; Yan, M; Howell, DN; Gomez, JA; Spurney, RF; Coffman, TM; Crowley, SD
MLA Citation
Zhang, J-D, Patel, MB, Song, Y-S, Griffiths, R, Burchette, J, Ruiz, P, Sparks, MA, Yan, M, Howell, DN, Gomez, JA, Spurney, RF, Coffman, TM, and Crowley, SD. "A novel role for type 1 angiotensin receptors on T lymphocytes to limit target organ damage in hypertension." Circ Res 110.12 (June 8, 2012): 1604-1617.
PMID
22534490
Source
pubmed
Published In
Circulation Research
Volume
110
Issue
12
Publish Date
2012
Start Page
1604
End Page
1617
DOI
10.1161/CIRCRESAHA.111.261768

Mechanisms of the proteinuria induced by Rho GTPases.

Podocytes are highly differentiated cells that play an important role in maintaining glomerular filtration barrier integrity; a function regulated by small GTPase proteins of the Rho family. To investigate the role of Rho A in podocyte biology, we created transgenic mice expressing doxycycline-inducible constitutively active (V14 Rho) or dominant-negative Rho A (N19 Rho) in podocytes. Specific induction of either Rho A construct in podocytes caused albuminuria and foot process effacement along with disruption of the actin cytoskeleton as evidenced by decreased expression of the actin-associated protein synaptopodin. The mechanisms of these adverse effects, however, appeared to be different. Active V14 Rho enhanced actin polymerization, caused a reduction in nephrin mRNA and protein levels, promoted podocyte apoptosis, and decreased endogenous Rho A levels. In contrast, the dominant-negative N19 Rho caused a loss of podocyte stress fibers, did not alter the expression of either nephrin or Rho A, and did not cause podocyte apoptosis. Thus, our findings suggest that Rho A plays an important role in maintaining the integrity of the glomerular filtration barrier under basal conditions, but enhancement of Rho A activity above basal levels promotes podocyte injury.

Authors
Wang, L; Ellis, MJ; Gomez, JA; Eisner, W; Fennell, W; Howell, DN; Ruiz, P; Fields, TA; Spurney, RF
MLA Citation
Wang, L, Ellis, MJ, Gomez, JA, Eisner, W, Fennell, W, Howell, DN, Ruiz, P, Fields, TA, and Spurney, RF. "Mechanisms of the proteinuria induced by Rho GTPases." Kidney Int 81.11 (June 2012): 1075-1085.
PMID
22278020
Source
pubmed
Published In
Kidney international
Volume
81
Issue
11
Publish Date
2012
Start Page
1075
End Page
1085
DOI
10.1038/ki.2011.472

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion.

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.

Authors
Chang, J-H; Paik, S-Y; Mao, L; Eisner, W; Flannery, PJ; Wang, L; Tang, Y; Mattocks, N; Hadjadj, S; Goujon, J-M; Ruiz, P; Gurley, SB; Spurney, RF
MLA Citation
Chang, J-H, Paik, S-Y, Mao, L, Eisner, W, Flannery, PJ, Wang, L, Tang, Y, Mattocks, N, Hadjadj, S, Goujon, J-M, Ruiz, P, Gurley, SB, and Spurney, RF. "Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion." PLoS One 7.4 (2012): e33942-.
PMID
22496773
Source
pubmed
Published In
PloS one
Volume
7
Issue
4
Publish Date
2012
Start Page
e33942
DOI
10.1371/journal.pone.0033942

A novel mouse model of podocyte depletion.

AIM: The goal of this study was to examine the capacity for glomerular repair after a podocyte-depleting injury. METHODS: We created transgenic (TG) mice expressing the yeast enzyme cytosine deaminase specifically in glomerular podocytes. In these TG animals, the prodrug 5-flucytosine (5-FC) is converted to 5-fluorouracil and promotes cell death. RESULTS: Treatment with increasing dosages of 5-FC caused graded increases in proteinuria 1-2 weeks after treatment, which returned to control levels by the 10-week time point. Light microscopic examination revealed minimal pathology at the 2-week time point, but electron microscopy revealed found foot process effacement as well as focal areas of glomerular basement membrane duplication, and immunohistochemical studies detected podocyte apoptosis and a decrease in the number of Wilms' tumor protein 1 (WT1)-positive cells. By the 10-week time point, however, the number of WT1-positive cells was similar to controls and a few mice had developed focal areas of glomerulosclerosis. Consistent with the effects of 5-FC on podocyte number, expression of the podocyte mRNAs for nephrin, podocin, synaptopodin and podocalyxin were altered in a similar temporal fashion. CONCLUSION: The glomerulus has a significant capacity for repair after a podocyte-depleting injury.

Authors
Wang, L; Tang, Y; Howell, DN; Ruiz, P; Spurney, RF
MLA Citation
Wang, L, Tang, Y, Howell, DN, Ruiz, P, and Spurney, RF. "A novel mouse model of podocyte depletion." Nephron Exp Nephrol 121.1-2 (2012): e10-e22.
PMID
23095233
Source
pubmed
Published In
Nephron - Experimental Nephrology
Volume
121
Issue
1-2
Publish Date
2012
Start Page
e10
End Page
e22
DOI
10.1159/000342369

Calcineurin (CN) activation promotes apoptosis of glomerular podocytes both in vitro and in vivo.

To determine the role of Gq signaling and calcineurin (CN) activation in promoting apoptosis of glomerular podocytes, constitutively active Gq [Gq(+)] or CN [CN(+)] proteins were introduced into cultured podocytes using protein transduction by tagging the proteins with the transactivator of transcription peptide. To investigate the role of CN in promoting podocyte apoptosis in vivo, a genetic model of type 1 diabetes mellitus (Akita mice) was treated with the CN inhibitor FK506. In cultured podocytes, Gq(+) stimulated nuclear translocation of nuclear factor of activated T cells (NFAT) family members, activated an NFAT reporter construct, and enhanced podocyte apoptosis in a CN-dependent fashion. CN(+) similarly promoted podocyte apoptosis, and apoptosis induced by either angiotensin II or endothelin-1 was blocked by FK506. Induction of apoptosis required NFAT-induced gene transcription because apoptosis induced by either Gq(+) or CN(+) was blocked by an inhibitor that prevented CN-dependent NFAT activation without affecting CN phosphatase activity. Podocyte apoptosis was mediated, in part, by the NFAT-responsive gene cyclooxygenase 2 (COX2) and prostaglandin E(2) generation because apoptosis induced by Gq(+) was attenuated by either COX2 inhibition or blockade of the Gq-coupled E-series prostaglandins receptor. The findings appeared relevant to podocyte apoptosis in diabetic nephropathy because apoptosis was significantly reduced in Akita mice by treatment with FK506. These data suggest that Gq stimulates CN and promotes podocyte apoptosis both in vitro and in vivo. Apoptosis requires NFAT-dependent gene transcription and is mediated, in part, by CN-dependent COX2 induction, prostaglandin E(2) generation, and autocrine activation of the Gq-coupled E-series prostaglandins receptor.

Authors
Wang, L; Chang, J-H; Paik, S-Y; Tang, Y; Eisner, W; Spurney, RF
MLA Citation
Wang, L, Chang, J-H, Paik, S-Y, Tang, Y, Eisner, W, and Spurney, RF. "Calcineurin (CN) activation promotes apoptosis of glomerular podocytes both in vitro and in vivo." Mol Endocrinol 25.8 (August 2011): 1376-1386.
PMID
21622531
Source
pubmed
Published In
Molecular endocrinology (Baltimore, Md.)
Volume
25
Issue
8
Publish Date
2011
Start Page
1376
End Page
1386
DOI
10.1210/me.2011-0029

Regulator of G protein signaling 5 is highly expressed in parathyroid tumors and inhibits signaling by the calcium-sensing receptor.

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.

Authors
Koh, J; Dar, M; Untch, BR; Dixit, D; Shi, Y; Yang, Z; Adam, MA; Dressman, H; Wang, X; Gesty-Palmer, D; Marks, JR; Spurney, R; Druey, KM; Olson, JA
MLA Citation
Koh, J, Dar, M, Untch, BR, Dixit, D, Shi, Y, Yang, Z, Adam, MA, Dressman, H, Wang, X, Gesty-Palmer, D, Marks, JR, Spurney, R, Druey, KM, and Olson, JA. "Regulator of G protein signaling 5 is highly expressed in parathyroid tumors and inhibits signaling by the calcium-sensing receptor." Mol Endocrinol 25.5 (May 2011): 867-876.
PMID
21393447
Source
pubmed
Published In
Molecular endocrinology (Baltimore, Md.)
Volume
25
Issue
5
Publish Date
2011
Start Page
867
End Page
876
DOI
10.1210/me.2010-0277

A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation.

About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through beta-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (D-Trp(12),Tyr(34))-PTH(7-34) (PTH-betaarr), which activates beta-arrestin but not classic G protein signaling. In mice, PTH-betaarr induces anabolic bone formation, as does the nonselective agonist PTH(1-34), which activates both mechanisms. In beta-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-betaarr is ablated. The beta-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the beta-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.

Authors
Gesty-Palmer, D; Flannery, P; Yuan, L; Corsino, L; Spurney, R; Lefkowitz, RJ; Luttrell, LM
MLA Citation
Gesty-Palmer, D, Flannery, P, Yuan, L, Corsino, L, Spurney, R, Lefkowitz, RJ, and Luttrell, LM. "A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation." Sci Transl Med 1.1 (October 7, 2009): 1ra1-.
PMID
20368153
Source
pubmed
Published In
Science Translational Medicine
Volume
1
Issue
1
Publish Date
2009
Start Page
1ra1
DOI
10.1126/scitranslmed.3000071

Inhibition of WNT signaling by G protein-coupled receptor (GPCR) kinase 2 (GRK2).

Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator beta-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axin, we determined whether GRK2 regulated canonical signaling. We found that GRK2 inhibited Wnt1-induced activation of a reporter construct as well as reduced Wnt3a-dependent stabilization and nuclear translocation of beta-catenin. GRK2 enzymatic activity was required for this negative regulatory effect, and depletion of endogenous GRK2 using small interfering RNA enhanced canonical signaling. GRK2-dependent inhibition of canonical signaling is relevant to osteoblast (OB) biology because overexpression of GRK2 attenuated Wnt/beta-catenin signaling in calvarial OBs. Coimmunoprecipitation studies found that: 1) GRK2 bound APC; 2) The GRK2-APC interaction was promoted by GRK2 enzymatic activity; and 3) Deletion of the RGS domain in GRK2 prevented both the GRK2-APC interaction and GRK2-dependent inhibition of canonical signaling. These data suggest that: 1) GRK2 negatively regulates Wnt signaling; 2) GRK2-dependent inhibition of canonical signaling requires a protein-protein interaction between the RGS domain in GRK2 and APC; and 3) Enzymatic activity promotes the GRK2-APC interaction and is required for the negative regulatory effect on canonical signaling. We speculate that inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.

Authors
Wang, L; Gesty-Palmer, D; Fields, TA; Spurney, RF
MLA Citation
Wang, L, Gesty-Palmer, D, Fields, TA, and Spurney, RF. "Inhibition of WNT signaling by G protein-coupled receptor (GPCR) kinase 2 (GRK2)." Mol Endocrinol 23.9 (September 2009): 1455-1465.
PMID
19556343
Source
pubmed
Published In
Molecular endocrinology (Baltimore, Md.)
Volume
23
Issue
9
Publish Date
2009
Start Page
1455
End Page
1465
DOI
10.1210/me.2009-0084

Gq-dependent signaling upregulates COX2 in glomerular podocytes.

Accumulating evidence suggests that upregulation of cyclooxygenase 2 (COX2) in glomerular podocytes promotes podocyte injury. Because Gq signaling activates calcineurin and calcineurin-dependent mechanisms are known to mediate COX2 expression, this study investigated the role of Gqalpha in promoting COX2 expression in podocytes. A constitutively active Gq alpha subunit tagged with the TAT HIV protein sequence was introduced into an immortalized podocyte cell line by protein transduction. This stimulated inositol trisphosphate production, activated an nuclear factor of activated T cells-responsive reporter construct, and enhanced levels of both COX2 mRNA and protein compared with cells treated with a Gq protein lacking the TAT sequence. Induction of COX2 was associated with increased prostaglandin E(2) production and podocyte death, both of which were attenuated by selective COX2 inhibition. In vivo, levels of COX2 mRNA and protein were significantly enhanced in podocytes from transgenic mice that expressed podocyte-targeted constitutively active Gqalpha compared with nontransgenic littermates. These data suggest that Gq-dependent signaling cascades stimulate calcineurin and, in turn, upregulate COX2 mRNA and protein, increase eicosanoid production, and cause podocyte injury.

Authors
Wang, L; Flannery, PJ; Rosenberg, PB; Fields, TA; Spurney, RF
MLA Citation
Wang, L, Flannery, PJ, Rosenberg, PB, Fields, TA, and Spurney, RF. "Gq-dependent signaling upregulates COX2 in glomerular podocytes." J Am Soc Nephrol 19.11 (November 2008): 2108-2118.
PMID
18667730
Source
pubmed
Published In
Journal of the American Society of Nephrology : JASN
Volume
19
Issue
11
Publish Date
2008
Start Page
2108
End Page
2118
DOI
10.1681/ASN.2008010113

Stressed-out podocytes in diabetes?

Authors
Spurney, RF; Coffman, TM
MLA Citation
Spurney, RF, and Coffman, TM. "Stressed-out podocytes in diabetes?." J Am Soc Nephrol 19.11 (November 2008): 2035-2037.
PMID
18842987
Source
pubmed
Published In
Journal of the American Society of Nephrology : JASN
Volume
19
Issue
11
Publish Date
2008
Start Page
2035
End Page
2037
DOI
10.1681/ASN.2008090955

Beneficial effects of the Rho kinase inhibitor Y27632 in murine puromycin aminonucleoside nephrosis.

BACKGROUND AND AIMS: Rho kinase (ROCK) inhibition reduces systemic blood pressure (BP) and decreases renal damage in animal models of kidney disease. The aim of this study was to determine if ROCK inhibition might have beneficial effects in glomerular disease processes that are independent of systemic BP. METHODS: We investigated the effects of the ROCK inhibitor Y27632 and hydralazine in murine puromycin aminonucleoside (PAN) nephrosis. RESULTS: Treatment with either Y27632 or hydralazine similarly reduced systolic BP compared to vehicle-treated controls. Seven days after treatment with PAN, albuminuria, proteinuria and effacement of podocyte foot processes were significantly reduced in Y27632- and hydralazine-treated mice compared to vehicle-treated animals. Treatment with PAN significantly reduced expression of the podocyte proteins nephrin and Neph1, and the loss of glomerular nephrin was attenuated by treatment with Y27632 but not by treatment with hydralazine. In cultured podocytes, PAN potently activated both Rho and ROCK, and PAN-induced ROCK activation was prevented by Y27632. CONCLUSIONS: The ROCK inhibitor Y27632 attenuated glomerular nephrin loss in murine PAN nephrosis independent of its effects on systemic BP.

Authors
Wang, L; Ellis, MJ; Fields, TA; Howell, DN; Spurney, RF
MLA Citation
Wang, L, Ellis, MJ, Fields, TA, Howell, DN, and Spurney, RF. "Beneficial effects of the Rho kinase inhibitor Y27632 in murine puromycin aminonucleoside nephrosis." Kidney Blood Press Res 31.2 (2008): 111-121.
PMID
18367845
Source
pubmed
Published In
Kidney & blood pressure research
Volume
31
Issue
2
Publish Date
2008
Start Page
111
End Page
121
DOI
10.1159/000121531

Galphaq-dependent signaling cascades stimulate water-seeking behavior.

We used the mouse nephrin promoter to express a constitutively active Galphaq [Galphaq(Q>L)] transgene in mice. As previously reported, the transgene was expressed in kidney, pancreas, and brain, and the kidney phenotype was characterized by albuminuria and reduced nephron mass. Additional studies revealed a second phenotype characterized by polyuria and polydipsia. The polyuric phenotype was not caused by abnormal glucose metabolism or hypercalcemia but was accompanied by reduced urinary concentrating ability. Additional studies found that 1) water restriction was associated with an appropriate increase in serum vasopressin levels in transgenic (TG) mice; 2) the urinary concentrating defect was not corrected by administration of desamino-d-arginine vasopressin (DDAVP); and 3) papillary length was similar in TG and non-TG mice. To examine the renal response to DDAVP at the molecular level, we monitored aquaporin 2 (AQP2) and vasopressin V2 receptor (V2R) mRNA levels in mouse kidney. Consistent with the known effects of vasopressin, administration of DDAVP caused a decrease in V2R mRNA levels and an increase in AQP2 mRNA levels in both TG and non-TG animals, suggesting an appropriate renal response to DDAVP in the TG mice. To determine whether the urine concentrating abnormality was the result of primary polydipsia, water intake by TG mice was restricted to the amount ingested by non-TG animals. After 5 days, urinary concentrating ability was similar in TG mice and non-TG littermate controls. These data are consistent with the notion that expression of the Galphaq(Q>L) transgene in the brain induced primary polydipsia in the TG mice.

Authors
Wang, L; Flannery, PJ; Athirakul, K; Dunn, SR; Kourany, WM; Spurney, RF
MLA Citation
Wang, L, Flannery, PJ, Athirakul, K, Dunn, SR, Kourany, WM, and Spurney, RF. "Galphaq-dependent signaling cascades stimulate water-seeking behavior." Am J Physiol Renal Physiol 291.4 (October 2006): F781-F789.
PMID
16609148
Source
pubmed
Published In
American Journal of Physiology: Renal Physiology
Volume
291
Issue
4
Publish Date
2006
Start Page
F781
End Page
F789
DOI
10.1152/ajprenal.00401.2005

Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation.

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Authors
Gesty-Palmer, D; Chen, M; Reiter, E; Ahn, S; Nelson, CD; Wang, S; Eckhardt, AE; Cowan, CL; Spurney, RF; Luttrell, LM; Lefkowitz, RJ
MLA Citation
Gesty-Palmer, D, Chen, M, Reiter, E, Ahn, S, Nelson, CD, Wang, S, Eckhardt, AE, Cowan, CL, Spurney, RF, Luttrell, LM, and Lefkowitz, RJ. "Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation." J Biol Chem 281.16 (April 21, 2006): 10856-10864.
PMID
16492667
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
16
Publish Date
2006
Start Page
10856
End Page
10864
DOI
10.1074/jbc.M513380200

Unexpected role of TRPC6 channel in familial nephrotic syndrome: does it have clinical implications?

Authors
Winn, MP; Daskalakis, N; Spurney, RF; Middleton, JP
MLA Citation
Winn, MP, Daskalakis, N, Spurney, RF, and Middleton, JP. "Unexpected role of TRPC6 channel in familial nephrotic syndrome: does it have clinical implications?." J Am Soc Nephrol 17.2 (February 2006): 378-387. (Review)
PMID
16396961
Source
pubmed
Published In
Journal of the American Society of Nephrology : JASN
Volume
17
Issue
2
Publish Date
2006
Start Page
378
End Page
387
DOI
10.1681/ASN.2005090962

Transactivation of the epidermal growth factor receptor by angiotensin II in glomerular podocytes.

BACKGROUND/AIMS: Activation of angiotensin II (ANG2) receptors stimulates extracellular signal-regulated kinases (ERKs) that, in some cell systems, are mediated by transactivating the epidermal growth factor (EGF) receptor (EGFR) through mechanisms involving matrix metalloprotease (MMP)-stimulated processing of heparin-binding EGF (HB-EGF) from its precursor. METHODS: The signaling pathways linked to ANG2-dependent ERK activation were determined in an immortalized mouse podocyte cell line by monitoring ANG2-stimulated phosphorylation of ERK1/2. RESULTS: ANG2 induced transient ERK phosphorylation that was maximal at 5 min and then rapidly dissipated. ANG2-dependent ERK activation was inhibited by: (1) the type-1 ANG2-selective antagonist losartan; (2) the type-2 ANG2-selective antagonist PD123319; (3) an inhibitor of MMP2/9; (4) the EGFR kinase inhibitor AG1478, and (5) the HB-EGF antagonists CRM197 and heparin. ANG2-dependent ERK activation was mediated by both protein kinase C (PKC)- and calcium-dependent mechanisms and was associated with tyrosine phosphorylation of EGFR. To determine if ANG2-dependent HB-EGF release could act in a paracrine fashion on adjacent cells, HEK293 cells were stably transfected with green fluorescent protein-tagged ERK2 (GFP-ERK2). In stably transfected HEK293 cells, EGF stimulated phosphorylation of endogenous ERK1/2 as well as GFP-ERK2. In contrast, ANG2 had no effect on ERK phosphorylation in stably transfected HEK293 cells. When podocytes were co-cultured with stably transfected HEK293 cells, however, treatment with ANG2 rapidly stimulated GFP-ERK2 phosphorylation. Both the MMP2/9 inhibitor and AG1478 attenuated ANG2-dependent phosphorylation of GFP-ERK2 in the co-culture system. CONCLUSIONS: These data indicate that ERK activation is induced by ANG2 in podocytes by mechanisms involving ANG2-dependent release of HB-EGF which, in turn, may act in an autocrine and paracrine fashion to stimulate ERK activity.

Authors
Flannery, PJ; Spurney, RF
MLA Citation
Flannery, PJ, and Spurney, RF. "Transactivation of the epidermal growth factor receptor by angiotensin II in glomerular podocytes." Nephron Exp Nephrol 103.3 (2006): e109-e118.
PMID
16554661
Source
pubmed
Published In
Nephron - Experimental Nephrology
Volume
103
Issue
3
Publish Date
2006
Start Page
e109
End Page
e118
DOI
10.1159/000092196

Lambda light chain deposition disease in a renal allograft.

Light chain deposition disease (LCDD) of the kidney is characterized by deposition of monoclonal light chains predominantly in glomeruli and in tubular basement membranes. The disease is frequently associated with a lymphoproliferative disorder, and the majority of cases are caused by deposition of kappa light chains. Although the occurrence of de novo multiple myeloma after renal transplantation is uncommon, there are several reports of LCDD involving renal allografts, either de novo or in patients with a diagnosis of LCDD prior to transplantation. To the best of our knowledge, all previously described cases in allografts have been in patients with kappa chain deposition. The relative importance of intrinsic properties of the kidney in predisposing to either kappa or lambda light chain deposition is not known. We present a case of LCDD caused by deposition of lambda light chains in a patient who received a cadaveric renal transplant.

Authors
Tanenbaum, ND; Howell, DN; Middleton, JP; Spurney, RF
MLA Citation
Tanenbaum, ND, Howell, DN, Middleton, JP, and Spurney, RF. "Lambda light chain deposition disease in a renal allograft." Transplant Proc 37.10 (December 2005): 4289-4292.
PMID
16387099
Source
pubmed
Published In
Transplantation Proceedings
Volume
37
Issue
10
Publish Date
2005
Start Page
4289
End Page
4292
DOI
10.1016/j.transproceed.2005.10.030

Activation of Galpha q-coupled signaling pathways in glomerular podocytes promotes renal injury.

The glomerular podocyte plays a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by activation of cell surface G protein-coupled receptors (GPCR). Studies suggest that podocytes express GPCR that are implicated in the pathogenesis of glomerular diseases. Common to these GPCR systems is activation of phospholipase C through the Gq alpha-subunit (Galpha q). For investigating the role of Galpha q-coupled signaling pathways in promoting renal injury in podocytes, a constitutively active Galpha q subunit (Galpha qQ > L) was expressed in glomerular podocytes using the mouse nephrin promoter. Transgenic (TG) mice demonstrated albuminuria as well as a decrease in both kidney mass and nephron number. By light microscopy, a portion of the TG mice had glomerular abnormalities, including focal to diffuse hypercellularity and segmental sclerosis. Consistent with injury-promoting effects of Galpha qQ > L, there was a significant reduction in podocalyxin mRNA as well as nephrin mRNA and protein levels in glomeruli from TG mice compared with non-TG controls. Expression of the transgene also seemed to increase susceptibility to glomerular injury, because treatment with puromycin aminonucleoside enhanced proteinuria in TG mice compared with non-TG littermate controls (4.2 +/- 1.0 [TG] versus 1.6 +/- 0.3 [non-TG] mg/24 h; P = 0.0161). Thus, activation of Galpha q in glomerular podocytes caused alterations in glomerular histomorphology, albuminuria, decreased nephron mass, and reduced glomerular expression of both nephrin and podocalyxin as well as enhanced susceptibility to glomerular damage induced by puromycin aminonucleoside. It is speculated that Galpha q-coupled signaling cascades may be important effector pathways mediating renal injury.

Authors
Wang, L; Fields, TA; Pazmino, K; Dai, Q; Burchette, JL; Howell, DN; Coffman, TM; Spurney, RF
MLA Citation
Wang, L, Fields, TA, Pazmino, K, Dai, Q, Burchette, JL, Howell, DN, Coffman, TM, and Spurney, RF. "Activation of Galpha q-coupled signaling pathways in glomerular podocytes promotes renal injury." J Am Soc Nephrol 16.12 (December 2005): 3611-3622.
PMID
16267159
Source
pubmed
Published In
Journal of the American Society of Nephrology : JASN
Volume
16
Issue
12
Publish Date
2005
Start Page
3611
End Page
3622
DOI
10.1681/ASN.2005020167

Role of thromboxane A2 in the induction of apoptosis of immature thymocytes by lipopolysaccharide.

Lipopolysaccharide (LPS) causes apoptotic deletion of CD4(+) CD8(+) thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4(+) CD8(+) thymocytes and in vitro evidence that thromboxane A(2) (TXA(2)) causes apoptosis of these cells, we tested whether enhanced generation of TXA(2) plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 micro LPS intraperitoneally displayed a marked increase in generation of TXA(2) and prostaglandin E(2) in the thymus as well as apoptotic deletion of CD4(+) CD8(+) thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA(2) mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.

Authors
Rocha, PN; Plumb, TJ; Robinson, LA; Spurney, R; Pisetsky, D; Koller, BH; Coffman, TM
MLA Citation
Rocha, PN, Plumb, TJ, Robinson, LA, Spurney, R, Pisetsky, D, Koller, BH, and Coffman, TM. "Role of thromboxane A2 in the induction of apoptosis of immature thymocytes by lipopolysaccharide." Clin Diagn Lab Immunol 12.8 (August 2005): 896-903.
PMID
16085905
Source
pubmed
Published In
Clinical and diagnostic laboratory immunology
Volume
12
Issue
8
Publish Date
2005
Start Page
896
End Page
903
DOI
10.1128/CDLI.12.8.896-903.2005

Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss.

To investigate the role of G protein-coupled receptor kinases (GRKs) in regulating bone formation in vivo, we overexpressed the potent G protein-coupled receptor (GPCR) regulator GRK2 in osteoblasts, using the osteocalcin gene-2 promoter to target expression to osteoblastic cells. Using the parathyroid hormone (PTH) receptor as a model system, we found that overexpression of GRK2 in osteoblasts attenuated PTH-induced cAMP generation by mouse calvaria ex vivo. This decrease in GPCR responsiveness was associated with a reduction in bone mineral density (BMD) in transgenic (TG) mice compared with non-TG littermate controls. The decrease in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. Quantitative computed tomography indicated that the loss of trabecular bone was due to a decrease in trabecular thickness, with little change in trabecular number. Histomorphometric analyses confirmed the decrease in trabecular bone volume and demonstrated reduced bone remodeling, as evidenced by a decrease in osteoblast numbers and osteoblast-mediated bone formation. Osteoclastic activity also appeared to be reduced because urinary excretion of the osteoclastic activity marker deoxypyridinoline was decreased in TG mice compared with control animals. Consistent with reduced coupling of osteoblast-mediated bone formation to osteoclastic bone resorption, mRNA levels of both osteoprotegrin and receptor activator of NF-kappaB ligand were altered in calvaria of TG mice in a pattern that would promote a low rate of bone remodeling. Taken together, these data suggest that enhancing GRK2 activity and consequently reducing GPCR activity in osteoblasts produces a low bone-turnover state that reduces bone mass.

Authors
Wang, L; Liu, S; Quarles, LD; Spurney, RF
MLA Citation
Wang, L, Liu, S, Quarles, LD, and Spurney, RF. "Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss." Am J Physiol Endocrinol Metab 288.4 (April 2005): E826-E834.
PMID
15585587
Source
pubmed
Published In
American journal of physiology. Endocrinology and metabolism
Volume
288
Issue
4
Publish Date
2005
Start Page
E826
End Page
E834
DOI
10.1152/ajpendo.00422.2004

Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system.

Angiotensin II, acting through type 1 angiotensin (AT(1)) receptors, has potent effects that alter renal excretory mechanisms. Control of sodium excretion by the kidney has been suggested to be the critical mechanism for blood pressure regulation by the renin-angiotensin system (RAS). However, since AT(1) receptors are ubiquitously expressed, precisely dissecting their physiological actions in individual tissue compartments including the kidney with conventional pharmacological or gene targeting experiments has been difficult. Here, we used a cross-transplantation strategy and AT(1A) receptor-deficient mice to demonstrate distinct and virtually equivalent contributions of AT(1) receptor actions in the kidney and in extrarenal tissues to determining the level of blood pressure. We demonstrate that regulation of blood pressure by extrarenal AT(1A) receptors cannot be explained by altered aldosterone generation, which suggests that AT(1) receptor actions in systemic tissues such as the vascular and/or the central nervous systems make nonredundant contributions to blood pressure regulation. We also show that interruption of the AT(1) receptor-mediated short-loop feedback in the kidney is not sufficient to explain the marked stimulation of renin production induced by global AT(1) receptor deficiency or by receptor blockade. Instead, the renin response seems to be primarily determined by renal baroreceptor mechanisms triggered by reduced blood pressure. Thus, the regulation of blood pressure by the RAS is mediated by AT(1) receptors both within and outside the kidney.

Authors
Crowley, SD; Gurley, SB; Oliverio, MI; Pazmino, AK; Griffiths, R; Flannery, PJ; Spurney, RF; Kim, H-S; Smithies, O; Le, TH; Coffman, TM
MLA Citation
Crowley, SD, Gurley, SB, Oliverio, MI, Pazmino, AK, Griffiths, R, Flannery, PJ, Spurney, RF, Kim, H-S, Smithies, O, Le, TH, and Coffman, TM. "Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system." J Clin Invest 115.4 (April 2005): 1092-1099.
PMID
15841186
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
115
Issue
4
Publish Date
2005
Start Page
1092
End Page
1099
DOI
10.1172/JCI23378

Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization.

Extracellular calcium rapidly controls PTH secretion through binding to the G protein-coupled calcium-sensing receptor (CASR) expressed in parathyroid glands. Very little is known about the regulatory proteins involved in desensitization of CASR. G protein receptor kinases (GRK) and beta-arrestins are important regulators of agonist-dependent desensitization of G protein-coupled receptors. In the present study, we investigated their role in mediating agonist-dependent desensitization of CASR. In heterologous cell culture models, we found that the transfection of GRK4 inhibits CASR signaling by enhancing receptor phosphorylation and beta-arrestin translocation to the CASR. In contrast, we found that overexpression of GRK2 desensitizes CASR by classical mechanisms as well as through phosphorylation-independent mechanisms involving disruption of Galphaq signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in beta-arrestin2 null mice, suggesting a functional role of beta-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and beta-arrestins play key roles in regulating CASR responsiveness in parathyroid glands.

Authors
Pi, M; Oakley, RH; Gesty-Palmer, D; Cruickshank, RD; Spurney, RF; Luttrell, LM; Quarles, LD
MLA Citation
Pi, M, Oakley, RH, Gesty-Palmer, D, Cruickshank, RD, Spurney, RF, Luttrell, LM, and Quarles, LD. "Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization." Mol Endocrinol 19.4 (April 2005): 1078-1087.
PMID
15637145
Source
pubmed
Published In
Molecular endocrinology (Baltimore, Md.)
Volume
19
Issue
4
Publish Date
2005
Start Page
1078
End Page
1087
DOI
10.1210/me.2004-0450

Unmasking the osteoinductive effects of a G-protein-coupled receptor (GPCR) kinase (GRK) inhibitor by treatment with PTH(1-34).

UNLABELLED: The effects of GPCR systems in bone are regulated by a family of enzymes termed GRKs. We found that (1) GRK inhibition in osteoblasts has age-dependent effects on bone mass, and (2) the anabolic actions of GRK inhibition are revealed by treatment with PTH(1-34). INTRODUCTION: The effects of G-protein-coupled receptor (GPCR) systems in bone are modulated by a family of enzymes termed GPCR kinases (GRKs). These enzymes directly phosphorylate GPCR substrate and desensitize receptor signaling. We previously found that expression of a GRK inhibitor in osteoblasts using transgenic (TG) technologies enhanced bone remodeling, and in turn, increased BMD in 6-week-old TG mice compared with non-TG littermate controls, presumably because of enhanced GPCR function. The aim of this study was to determine the age-dependent effects of the transgene. MATERIALS AND METHODS: BMD was monitored in TG mice and in controls at 6-week, 3-month, and 6-month time-points. To determine if the transgene enhanced responsiveness of bone to parathyroid hormone (PTH), we measured cyclic adenosine monophosphate (cAMP) generation by mouse calvaria ex vivo as well as the effects of treatment with PTH(1-34) on BMD, bone histomorphometry, and expression of the PTH-responsive gene RANKL in both TG mice and non-TG controls. RESULTS: Consistent with our previous findings, we found that BMD was increased in TG mice compared with controls at 6 weeks of age. The increase in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. In contrast to younger animals, however, BMD in older TG mice was not statistically different compared with non-TG mice at 3 months of age and was similar to non-TG animals at 6 months of age. The GRK inhibitor seemed to promote GPCR activation in older mice, however, because (1) PTH-induced cAMP generation by mouse calvaria ex vivo was enhanced in TG mice compared with controls, (2) GRK inhibition increased responsiveness of lumbar spine to the osteoinductive actions of PTH(1-34), and (3) the enhanced anabolic effect of PTH(1-34) was associated with increased expression of the PTH-responsive gene RANKL in calvaria of the TG animals. Bone histomorphometry confirmed that PTH(1-34) increased trabecular bone volume in TG mice and found that this increase in bone mass was caused by enhanced bone formation, predominantly as a result of an increase in the mineral apposition rate (MAR). CONCLUSIONS: These data suggest that the anabolic effects of GRK inhibition are age dependent. The osteoinductive actions of the GRK inhibitor are, however, unmasked by treatment with PTH(1-34).

Authors
Wang, L; Quarles, LD; Spurney, RF
MLA Citation
Wang, L, Quarles, LD, and Spurney, RF. "Unmasking the osteoinductive effects of a G-protein-coupled receptor (GPCR) kinase (GRK) inhibitor by treatment with PTH(1-34)." J Bone Miner Res 19.10 (October 2004): 1661-1670.
PMID
15355561
Source
pubmed
Published In
Journal of Bone and Mineral Research
Volume
19
Issue
10
Publish Date
2004
Start Page
1661
End Page
1670
DOI
10.1359/JBMR.040708

Proinflammatory actions of thromboxane receptors to enhance cellular immune responses.

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE(2), in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A(2) synthesis. To study the immunoregulatory actions of thromboxane A(2), we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP(-/-) spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.

Authors
Thomas, DW; Rocha, PN; Nataraj, C; Robinson, LA; Spurney, RF; Koller, BH; Coffman, TM
MLA Citation
Thomas, DW, Rocha, PN, Nataraj, C, Robinson, LA, Spurney, RF, Koller, BH, and Coffman, TM. "Proinflammatory actions of thromboxane receptors to enhance cellular immune responses." J Immunol 171.12 (December 15, 2003): 6389-6395.
PMID
14662837
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
171
Issue
12
Publish Date
2003
Start Page
6389
End Page
6395

Characterization of angiotensin II-receptor subtypes in podocytes.

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125-labeled Ang II to monitor Ang II-receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant approximately 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II-receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P<.005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (approximately 75%) with lesser amounts of AT2 (approximately 25%). Up-regulation of Ang II-receptor number was associated with increased Ang II-receptor responsiveness, as evidenced by enhanced Ang II-stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.

Authors
Wang, L; Flannery, PJ; Spurney, RF
MLA Citation
Wang, L, Flannery, PJ, and Spurney, RF. "Characterization of angiotensin II-receptor subtypes in podocytes." J Lab Clin Med 142.5 (November 2003): 313-321.
PMID
14647035
Source
pubmed
Published In
Journal of Laboratory and Clinical Medicine
Volume
142
Issue
5
Publish Date
2003
Start Page
313
End Page
321
DOI
10.1016/S0022-2143(03)00139-2

Physiological impact of increased expression of the AT1 angiotensin receptor.

To test the effect of increased AT1 receptor expression on blood pressure, we used gene targeting to generate mouse lines with a tandem duplication of the AT1A receptor gene locus (Agtr1a) along with >10 kb of 5' flanking DNA. By successive breeding, we generated mice with 3 and 4 copies of the Agtr1a gene locus on an inbred 129/Sv background. AT1A mRNA expression and AT1-specific binding of 125I-angiotensin II were increased in proportion to Agtr1a gene copy number. These animals survived in expected numbers, and their body, heart, and kidney weights were similar to wild-type, 2-copy control mice. Pressor responses to angiotensin II were blunted in the 4-copy mice compared with control mice. In male mice, there was no correlation between resting blood pressure and Agtr1a gene copy number or AT1A mRNA levels. However, in female mice, there was a highly significant positive correlation between blood pressure and AT1A receptor expression, paralleled by significant increases in aldosterone synthase expression with increase in gene copy number. Furthermore, in female but not male mice, there was a positive correlation between kallikrein and AT1A receptor mRNA levels and an inverse correlation between renin mRNA and Agtr1a copy number. Thus, in female but not male mice, genetic variants that increase expression of AT1 receptors affect blood pressure and gene expression programs. The impact of enhanced AT1 receptor expression on blood pressure may be blunted by systemic compensatory responses and altered signal-effector coupling in the vasculature.

Authors
Le, TH; Kim, H-S; Allen, AM; Spurney, RF; Smithies, O; Coffman, TM
MLA Citation
Le, TH, Kim, H-S, Allen, AM, Spurney, RF, Smithies, O, and Coffman, TM. "Physiological impact of increased expression of the AT1 angiotensin receptor." Hypertension 42.4 (October 2003): 507-514.
PMID
12963678
Source
pubmed
Published In
Hypertension
Volume
42
Issue
4
Publish Date
2003
Start Page
507
End Page
514
DOI
10.1161/01.HYP.0000092000.07559.57

Regulated expression of G protein-coupled receptor kinases (GRK's) and beta-arrestins in osteoblasts.

Desensitization of G-protein coupled receptors (GPCR's) is largely mediated by a family of enzymes and protein co-factors termed GRKs and arrestins, respectively. In the present studies, we investigated expression of GRKs and arrestins in osteoblastic cell lines concentrating on the enzymes (GRK2 and GRK3) and protein co-factors (beta-arrestint 1 and beta-arrestin 2) that play dominant roles in regulating GPCR responsiveness in most tissues and cell types. We found that osteoblastic cells express similar amounts of GRK2 with either undetectable or lesser amounts of GRK3. In contrast, expression of beta-arrestin 1 and beta-arrestin 2 by osteoblastic cells varied between cell lines. To determine if GRK2 or beta-arrestin expression is modulated during osteoblast development, we assessed expression of GRK2 and beta-arrestin proteins during differentiation of the mouse osteoblastic cell line MC3T3-E1 cells over a 21-day period. We found that expression of GRK2 and beta-arrestin 2 increased to maximal levels by day 7 and then decreased 4-fold by day 21. In contrast, expression of beta-arrestin 1 increased to maximal levels by day 14 and then decreased 2-fold by day 21. Over this same time period (days 7-21), PTH/PTHrP receptor number decreased to a greater extent than the decrease in PTH(1-34)-induced cAMP generation, suggesting that responsiveness of individual PTH/PTHrP receptors was enhanced in differentiated cells. We conclude that (1) osteoblastic cell lines differentially express the enzymes and protein co-factors that modulate GPCR responsiveness and (2) expression of both GRK2 and beta-arrestins is temporally regulated during osteoblast development. These data are consistent with the notion that GPCR responsiveness may be differentially regulated in osteoblastic cell lines and during osteoblast development.

Authors
Spurney, RF
MLA Citation
Spurney, RF. "Regulated expression of G protein-coupled receptor kinases (GRK's) and beta-arrestins in osteoblasts." Calcif Tissue Int 73.2 (August 2003): 153-160.
PMID
14565597
Source
pubmed
Published In
Calcified Tissue International
Volume
73
Issue
2
Publish Date
2003
Start Page
153
End Page
160
DOI
10.1007/s00223-002-1018-5

Rho kinase promotes alloimmune responses by regulating the proliferation and structure of T cells.

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling.

Authors
Tharaux, P-L; Bukoski, RC; Rocha, PN; Crowley, SD; Ruiz, P; Nataraj, C; Howell, DN; Kaibuchi, K; Spurney, RF; Coffman, TM
MLA Citation
Tharaux, P-L, Bukoski, RC, Rocha, PN, Crowley, SD, Ruiz, P, Nataraj, C, Howell, DN, Kaibuchi, K, Spurney, RF, and Coffman, TM. "Rho kinase promotes alloimmune responses by regulating the proliferation and structure of T cells." J Immunol 171.1 (July 1, 2003): 96-105.
PMID
12816987
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
171
Issue
1
Publish Date
2003
Start Page
96
End Page
105

Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor.

We investigated the role of Galphaq, filamin, Rho, the RhoGEF Lbc, and the C terminus of calcium-sensing receptor (CasR) in CasR signaling. We found that Ca(2+), Mg(2+), or the calcimimetic R isomer of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS-R568) stimulated serum response element (SRE) activity human embryonic kidney 293 cells transfected with CasR and an SRE-luciferase reporter construct. Coexpression of either the dominant negative Galphaq(305-359) minigene, regulators of G protein signaling (RGS)2 or RGS4, inhibited CasR-stimulated SRE activity, consistent with CasR activation of Galphaq. The cytoskeletal associated Rho protein is involved CasR activation of SRE, as evidenced by CasR-mediated increase in membrane-associated Rho A and by the ability of Clostridium botulinum C3 (C3) exoenzyme to inhibit both CasR and GalphaqQL-stimulated SRE activity. Overexpression of the RhoGEF Lbc, lacking either the Dbl-homology or Pleckstrin homology domain, as well as the filamin peptide (1530-1875) inhibited CasR-mediated activation of SRE. A carboxyl-terminal CasR minigene, CasR(906-980), encoding a filamin binding region, also blocked CasR- and GalphaqQL-stimulated SRE activity. Potential interactions between CasR, RhoGEF Lbc, Rho A, Galphaq, and filamin were demonstrated by reciprocal coimmunoprecipitation studies. Our results suggest that the C terminus of CasR may interact with filamin to create a cytoskeletal scaffold necessary for the spatial organization of Galphaq, RhoGEF Lbc, and Rho signaling pathways upstream of SRE activation.

Authors
Pi, M; Spurney, RF; Tu, Q; Hinson, T; Quarles, LD
MLA Citation
Pi, M, Spurney, RF, Tu, Q, Hinson, T, and Quarles, LD. "Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor." Endocrinology 143.10 (October 2002): 3830-3838.
PMID
12239094
Source
pubmed
Published In
Endocrinology
Volume
143
Issue
10
Publish Date
2002
Start Page
3830
End Page
3838
DOI
10.1210/en.2002-220240

Desensitization of the mouse thromboxane A2 receptor (TP) by G protein-coupled receptor kinases (Grks).

GRKs play a key role in regulating G protein-coupled receptor (GPCR) responsiveness. To investigate the role of GRKs in desensitization of TP, we replaced threonines with favorable phosphorylation motifs for GRKs (positions 226 and 230) with alanine. Mutant and wild-type receptors were expressed in cell culture models and clones expressing similar numbers of receptors were studied. We found that: (1) affinity and specificity of thromboxane A2 (TxA2) binding to mutant TP were identical to the wild-type, (2) replacement of threonines 226 and 230 with alanines delayed the onset of agonist-induced desensitization, and (3) inhibition of endogenous GRK activity with a dominant-negative construct inhibited agonist-induced phosphorylation and enhanced responsiveness of wild-type TP but had little effect on responsiveness of the receptor mutant. These data are consistent with the notion that GRKs contribute to desensitization of TP.

Authors
Flannery, PJ; Spurney, RF
MLA Citation
Flannery, PJ, and Spurney, RF. "Desensitization of the mouse thromboxane A2 receptor (TP) by G protein-coupled receptor kinases (Grks)." Prostaglandins Other Lipid Mediat 70.1-2 (September 2002): 79-90.
PMID
12428680
Source
pubmed
Published In
Prostaglandins & Other Lipid Mediators
Volume
70
Issue
1-2
Publish Date
2002
Start Page
79
End Page
90

Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts.

G protein-coupled receptors (GPCRs) play a key role in regulating bone remodeling. Whether GPCRs exert anabolic or catabolic osseous effects may be determined by the rate of receptor desensitization in osteoblasts. Receptor desensitization is largely mediated by direct phosphorylation of GPCR proteins by a family of enzymes termed GPCR kinases (GRKs). We have selectively manipulated GRK activity in osteoblasts in vitro and in vivo by overexpressing a GRK inhibitor. We found that expression of a GRK inhibitor enhanced parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor-stimulated cAMP generation and inhibited agonist-induced phosphorylation of this receptor in cell culture systems, consistent with attenuation of receptor desensitization. To determine the effect of GRK inhibition on bone formation in vivo, we targeted the expression of a GRK inhibitor to mature osteoblasts using the mouse osteocalcin gene 2 (OG2) promoter. Transgenic mice demonstrated enhanced bone remodeling as well as enhanced urinary excretion of the osteoclastic activity marker dexoypyridinoline. Both osteoprotegrin and OPG ligand mRNA levels were altered in calvaria of transgenic mice in a pattern that would promote osteoclast activation. The predominant effect of the transgene, however, was anabolic, as evidenced by an increase in bone density and trabecular bone volume in the transgenic mice compared with nontransgenic littermate controls.

Authors
Spurney, RF; Flannery, PJ; Garner, SC; Athirakul, K; Liu, S; Guilak, F; Quarles, LD
MLA Citation
Spurney, RF, Flannery, PJ, Garner, SC, Athirakul, K, Liu, S, Guilak, F, and Quarles, LD. "Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts." J Clin Invest 109.10 (May 2002): 1361-1371.
PMID
12021252
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
109
Issue
10
Publish Date
2002
Start Page
1361
End Page
1371
DOI
10.1172/JCI14663

Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs).

To investigate the domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group-containing amino acids at the C-terminus. In mutant #2, we replaced threonines 392 and 399 in the third intracellular loop with glycines. Co-transfection of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhibition of PTH receptor signaling was associated with enhanced phosphorylation receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cAMP generation by the wild-type receptor and mutant #1, and caused phosphorylation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agonist-induced phosphorylation of mutant #2 compared with that of either the wild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was associated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 failed to cause receptor desensitization and inhibited receptor internalization. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intracellular loop are not required for agonist-induced phosphorylation of PTH receptors, but are critical for both agonist-induced internalization of PTH receptors and GRK2-mediated regulation of PTH receptor signaling.

Authors
Flannery, PJ; Spurney, RF
MLA Citation
Flannery, PJ, and Spurney, RF. "Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs)." Biochem Pharmacol 62.8 (October 15, 2001): 1047-1058.
PMID
11597573
Source
pubmed
Published In
Biochemical Pharmacology
Volume
62
Issue
8
Publish Date
2001
Start Page
1047
End Page
1058

Analysis of recombinant Phex: an endopeptidase in search of a substrate.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3'M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3'M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

Authors
Guo, R; Liu, S; Spurney, RF; Quarles, LD
MLA Citation
Guo, R, Liu, S, Spurney, RF, and Quarles, LD. "Analysis of recombinant Phex: an endopeptidase in search of a substrate." Am J Physiol Endocrinol Metab 281.4 (October 2001): E837-E847.
PMID
11551862
Source
pubmed
Published In
American journal of physiology. Endocrinology and metabolism
Volume
281
Issue
4
Publish Date
2001
Start Page
E837
End Page
E847

Regulation of thromboxane receptor (TP) phosphorylation by protein phosphatase 1 (PP1) and PP2A.

To investigate the protein phosphatases that dephosphorylate TP, human embryonic kidney cells (HEK293 cells) stably transfected with 12CA5-tagged TP were treated with TP agonist, washed, and then allowed to recover in the presence or absence of the cell-permeable PP1 and PP2A inhibitors calyculin or okadaic acid (OKA). After recovery, cells were rechallenged with TP agonist and TP responsiveness was assessed by measuring inositol trisphosphate generation. TP responsiveness recovered over a 20-min time period. Recovery of TP responsiveness was inhibited by calyculin and OKA and was associated with dephosphorylation of receptor proteins. To further identify the TP phosphatase, TP phosphorylated in the intact cell were isolated by immunoprecipitation and were used as substrate for protein phosphatases prepared from HEK293 cells. TP were dephosphorylated by whole-cell homogenates. Dephosphorylation of TP was completely inhibited by the PP1 and PP2A inhibitors calyculin and microcystin-LR, suggesting that the decrease in TP phosphorylation was not due to receptor degradation. TP phosphatase activity was partially blocked by 1) inhibitor 2, a specific protein inhibitor of PP1; and 2) OKA at concentrations (1 nM) that specifically inhibit PP2A. TP phosphatase activity did not have an absolute requirement for divalent cations and was found primarily in cytosolic fractions of the cell. These data suggest that PP1- and PP2A-like protein phosphatases dephosphorylate TP. By regulating the phosphorylation state of TP, protein phosphatases may modulate tissue responsiveness to thromboxane.

Authors
Spurney, RF
MLA Citation
Spurney, RF. "Regulation of thromboxane receptor (TP) phosphorylation by protein phosphatase 1 (PP1) and PP2A." J Pharmacol Exp Ther 296.2 (February 2001): 592-599.
PMID
11160648
Source
pubmed
Published In
The Journal of pharmacology and experimental therapeutics
Volume
296
Issue
2
Publish Date
2001
Start Page
592
End Page
599

Analysis of recombinant Phex: An endopeptidase in search of a substrate

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

Authors
Guo, R; Liu, S; Spurney, RF; Quarles, LD
MLA Citation
Guo, R, Liu, S, Spurney, RF, and Quarles, LD. "Analysis of recombinant Phex: An endopeptidase in search of a substrate." American Journal of Physiology - Endocrinology and Metabolism 281.4 44-4 (2001): E837-E847.
Source
scival
Published In
American journal of physiology. Endocrinology and metabolism
Volume
281
Issue
4 44-4
Publish Date
2001
Start Page
E837
End Page
E847

A role for leukotrienes in cyclosporine nephrotoxicity.

BACKGROUND: Nephrotoxicity associated with cyclosporine A (CsA) administration is characterized by marked renal vasoconstriction, interstitial fibrosis, and arteriolar hypertrophy. While the molecular mechanisms of CsA toxicity are not well characterized, previous studies have demonstrated that altered arachidonic acid (AA) metabolism plays a role its pathogenesis. Using a rat renal transplant model, the purpose of this study was to examine the effects of CsA on the 5-lipoxygenase (5-LO) pathway of AA metabolism. METHODS: The PVG (RT1c) strain of rats underwent kidney transplantation, and recipients of nonrejecting kidney transplants were treated with either 50 mg/kg/day CsA or vehicle (N = 24). To determine the physiologic significance of increased leukotriene (LT) production, the peptidoleukotriene receptor antagonist SKF 106203 was administered to CsA-treated animals for six days. RESULTS: CsA caused a substantial reduction in glomerular filtration rate (GFR) in the transplanted rats compared with the vehicle-treated controls (1.5 +/- 0.6 vs. 4.1 +/- 0.8 mL/min/kg, P < 0.05). The reduction in renal function was associated with enhanced urinary excretion of the peptidoleukotriene metabolites LTE4 (1431 +/- 207 vs. 953 +/- 125 pg/24 h, P < 0.05) and N-acetyl-LTE4 (4411 +/- 848 vs. 463 +/- 70 pg/24 h, P < 0.001). LT receptor blockade had a significant protective effect on renal transplant function in CsA-treated animals (GFR, 4.8 +/- 1.1 vs. 1.7 +/- 0.9 mL/min/kg, P < 0.05), such that CsA-treated animals that received SKF106203 maintained GFR at levels similar to controls that never received CsA (4.1 +/- 0.8 mL/min/kg). Peptidoleukotriene receptor blockade also prevented the histomorphological abnormalities caused by CsA, including tubular vacuolization. CONCLUSIONS: These studies identify a critical role for LTs in the pathophysiology of CsA nephrotoxicity and suggest that LT antagonists may be useful in preventing CsA-associated kidney toxicity.

Authors
Butterly, DW; Spurney, RF; Ruiz, P; Griffiths, R; Albrightson, C; Coffman, TM
MLA Citation
Butterly, DW, Spurney, RF, Ruiz, P, Griffiths, R, Albrightson, C, and Coffman, TM. "A role for leukotrienes in cyclosporine nephrotoxicity." Kidney Int 57.6 (June 2000): 2586-2593.
PMID
10844628
Source
pubmed
Published In
Kidney international
Volume
57
Issue
6
Publish Date
2000
Start Page
2586
End Page
2593
DOI
10.1046/j.1523-1755.2000.00118.x

Sensing of extracellular cations in CasR-deficient osteoblasts. Evidence for a novel cation-sensing mechanism.

We isolated osteoblastic cell lines from wild-type (CasR(+/+)) and receptor null (CasR(-/-)) mice to investigate whether CasR is present in osteoblasts and accounts for their responses to extracellular cations. Osteoblasts from both CasR(+/+) and CasR(-/-) mice displayed an initial period of cell replication followed by a culture duration-dependent increase in alkaline phosphatase activity, expression of osteocalcin, and mineralization of extracellular matrix. In addition, a panel of extracellular cations, including aluminum and the CasR agonists gadolinium and calcium, stimulated DNA synthesis, activated a transfected serum response element-luciferase reporter construct, and inhibited agonist-induced cAMP in CasR(-/-) osteoblasts. The functional responses to these cations were identical in CasR(+/+) and CasR(-/-) osteoblasts. Thus, the absence of CasR alters neither the maturational profile of isolated osteoblast cultures nor their in vitro responses to extracellular cations. In addition, CasR transcripts could not be detected by reverse transcription-polymerase chain reaction with mouse specific primers in either CasR(+/+) or CasR(-/-) osteoblasts, and immunoblot analysis with a CasR-specific antibody was negative for CasR protein expression in osteoblasts. The presence of a cation-sensing response in osteoblasts from CasR(-/-) mice indicates the existence of a novel osteoblastic extracellular cation-sensing mechanism.

Authors
Pi, M; Garner, SC; Flannery, P; Spurney, RF; Quarles, LD
MLA Citation
Pi, M, Garner, SC, Flannery, P, Spurney, RF, and Quarles, LD. "Sensing of extracellular cations in CasR-deficient osteoblasts. Evidence for a novel cation-sensing mechanism." J Biol Chem 275.5 (February 4, 2000): 3256-3263.
PMID
10652312
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
5
Publish Date
2000
Start Page
3256
End Page
3263

G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction.

The Na(+)/H(+) exchanger regulatory factor (NHERF) is constitutively phosphorylated in cells, but the site(s) of this phosphorylation and the kinase(s) responsible for it have not been identified. We show here that the primary site of constitutive NHERF phosphorylation in human embryonic kidney 293 (HEK-293) cells is Ser(289), and that the stoichiometry of phosphorylation is near 1 mol/mol. NHERF contains two PDZ domains that recognize the sequence S/T-X-L at the carboxyl terminus of target proteins, and thus we examined the possibility that kinases containing this motif might associate with and phosphorylate NHERF. Overlay experiments and co-immunoprecipitation studies revealed that NHERF binds with high affinity to a splice variant of the G protein-coupled receptor kinase 6, GRK6A, which terminates in the motif T-R-L. NHERF does not associate with GRK6B or GRK6C, alternatively spliced variants that differ from GRK6A at their extreme carboxyl termini. GRK6A phosphorylates NHERF efficiently on Ser(289) in vitro, whereas GRK6B, GRK6C, and GRK2 do not. Furthermore, the endogenous "NHERF kinase" activity in HEK-293 cell lysates is sensitive to treatments that alter the activity of GRK6A. These data suggest that GRK6A phosphorylates NHERF via a PDZ domain-mediated interaction and that GRK6A is the kinase in HEK-293 cells responsible for the constitutive phosphorylation of NHERF.

Authors
Hall, RA; Spurney, RF; Premont, RT; Rahman, N; Blitzer, JT; Pitcher, JA; Lefkowitz, RJ
MLA Citation
Hall, RA, Spurney, RF, Premont, RT, Rahman, N, Blitzer, JT, Pitcher, JA, and Lefkowitz, RJ. "G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domain-mediated interaction." J Biol Chem 274.34 (August 20, 1999): 24328-24334.
PMID
10446210
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
34
Publish Date
1999
Start Page
24328
End Page
24334

Deficiency of 5-lipoxygenase abolishes sex-related survival differences in MRL-lpr/lpr mice.

Leukotrienes, the 5-lipoxygenase (5LO) products of arachidonic acid metabolism, have many proinflammatory actions that have been implicated in the pathogenesis of a variety of inflammatory diseases. To investigate the role of LTs in autoimmune disease, we generated an MRL-lpr/lpr mouse line with a targeted disruption of the 5lo gene. MRL-lpr/lpr mice spontaneously develop autoimmune disease that has many features resembling human systemic lupus erythematosus, including sex-related survival differences; female MRL-lpr/lpr mice experience significant early mortality compared with males. Unexpectedly, we found that mortality was accelerated in male 5LO-deficient MRL-lpr/lpr mice compared with male wild-type MRL-lpr/lpr animals. In contrast, the 5lo mutation had no effect on survival in females. Mortality was also accelerated in male MRL-lpr/lpr mice that were treated chronically with a pharmacological inhibitor of LT synthesis. Furthermore, LT-dependent inflammatory responses are enhanced in male MRL-lpr/lpr mice compared with females, and the 5lo mutation has greater impact on these responses in males. Because immune complex-mediated glomerulonephritis is the major cause of death in MRL-lpr/lpr mice and has been related to arachidonic acid metabolites, we also assessed kidney function and histopathology. In male MRL-lpr/lpr mice, renal plasma flow was significantly reduced in the 5lo-/- compared with the 5lo+/+ group, although there were no differences in the severity of renal histopathology, lymphoid hyperplasia, or arthritis between the groups. These findings suggest that the presence of a functional 5lo gene confers a survival advantage on male MRL-lpr/lpr mice and that, when 5LO function is inhibited, either genetically or pharmacologically, this advantage is abolished.

Authors
Goulet, JL; Griffiths, RC; Ruiz, P; Spurney, RF; Pisetsky, DS; Koller, BH; Coffman, TM
MLA Citation
Goulet, JL, Griffiths, RC, Ruiz, P, Spurney, RF, Pisetsky, DS, Koller, BH, and Coffman, TM. "Deficiency of 5-lipoxygenase abolishes sex-related survival differences in MRL-lpr/lpr mice." J Immunol 163.1 (July 1, 1999): 359-366.
PMID
10384136
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
163
Issue
1
Publish Date
1999
Start Page
359
End Page
366

Aluminum is a weak agonist for the calcium-sensing receptor.

BACKGROUND: Aluminum (Al3+) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al3+, may transduce some of the biological effects of Al3+. METHODS: To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca2+]i) levels and inositol monophosphate (IP1) generation following stimulation with Al3+ and a panel of CaSR agonists. RESULTS: The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca2+]i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca2+), magnesium (Mg2+), gadolinium (Gd3+), and neomycin only in cells transfected with CaSR. To determine if Al3+ activated CaSR, we stimulated cells transfected with rat CaSR with 10 microM to 1 mM concentrations of Al3+. Concentrations of Al3+ in the range of 10 microM to 100 microM had no effect on [Ca2+]i levels or IP1 generation. In contrast, 1 mM Al3+ induced small but significant increases in both parameters. Whereas Gd3+ antagonized calcium-mediated activation of CaSR, pretreatment with Al3+ failed to block subsequent activation of rat CaSR by Ca2+, suggesting a distinct mechanism of Al3+ action. CONCLUSION: Al3+ is not a potent agonist for CaSR. Because Al3+ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al3+.

Authors
Spurney, RF; Pi, M; Flannery, P; Quarles, LD
MLA Citation
Spurney, RF, Pi, M, Flannery, P, and Quarles, LD. "Aluminum is a weak agonist for the calcium-sensing receptor." Kidney Int 55.5 (May 1999): 1750-1758.
PMID
10231437
Source
pubmed
Published In
Kidney international
Volume
55
Issue
5
Publish Date
1999
Start Page
1750
End Page
1758
DOI
10.1046/j.1523-1755.1999.00432.x

Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor.

To investigate the role of C-terminal hydroxyamino acids in desensitization of the receptor for thromboxane A2 (TxA2), we created a mutant TxA2 receptor (TP receptor) in which serines at positions 321, 322, and 328 were replaced with either alanine or glycine. Mutant and wild type receptors were expressed in a mesangial cell line, and clones expressing similar numbers of receptors were studied. Affinity and specificity of TxA2 binding to the mutant receptor were identical to wild type receptors. In contrast, TxA2-induced inositol trisphosphate generation by the mutant receptor was enhanced compared with the wild type. Prior treatment with the TxA2 agonist U46619 reduced subsequent U46619-induced increases in inositol trisphosphate generation by both receptors; however, the extent of desensitization was significantly reduced in the receptor mutant. Protein kinase C (PKC) inhibitors attenuated TxA2-induced desensitization of wild type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with the phorbol ester phorbol 12, 13-dybutyrate (PDBu) (100 nM) decreased subsequent responsiveness of wild type but not mutant TP receptors. -induced desensitization of wild type receptors was associated with enhanced phosphorylation of receptor proteins. This agonist-specific phosphorylation of the TP receptor was largely prevented by inhibitors of PKC. Treatment with 100 nM PDBu increased phosphorylation of both wild type and mutant TP receptors, but the extent of phosphorylation of the receptor mutant was reduced compared with the wild type. Increasing the concentration of PDBu from 100 nM to 1 microM PDBu reduced responsiveness of both mutant and wild type receptors without enhancing phosphorylation of either of the receptor proteins. These data suggest that 1) phosphorylation of C-terminal serines contributes to agonist-specific desensitization of the TP receptor, 2) PKC-induced desensitization of TP receptors is caused, in part, by phosphorylation of C-terminal serines, and 3) desensitization of TP receptors by PKC is complex and involves mechanisms that may not require direct phosphorylation of receptor proteins.

Authors
Spurney, RF
MLA Citation
Spurney, RF. "Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor." J Biol Chem 273.43 (October 23, 1998): 28496-28503.
PMID
9774479
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
43
Publish Date
1998
Start Page
28496
End Page
28503

Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells.

We examined the effects of thromboxane A2 (TxA2) on the activities of the plasminogen-plasmin system in glomerular mesangial cells. When mesangial cells are exposed to the TxA2 agonist U-46619, a substantial increase in production of plasminogen activator inhibitor-1 (PAI-1) protein is observed that is significantly greater than that induced by 10% serum alone. This increase in PAI-1 protein production is accompanied by an increase in steady-state levels of PAI-1 mRNA. This stimulation is specifically mediated by TxA2 (thromboxane prostanoid, TP) receptors, since U-46619 also stimulates PAI-1 expression in cells that are transfected with TP receptors, and this stimulation of PAI-1 production is completely blocked by the TxA2 receptor antagonist, SQ-29,548. Despite the increase in PAI-1 production, there was net stimulation of plasmin activity in the medium of mesangial cells that had been exposed to U-46619. Furthermore, U-46619 also caused an increase in tissue plasminogen activator (tPA) mRNA levels. Thus TxA2 stimulates the production of PAI-1 and plasminogen activators by mesangial cells through a receptor-dependent mechanism. In inflammatory renal diseases, the balance of these effects may modulate glomerular thrombosis and renal fibrosis.

Authors
Coffman, TM; Spurney, RF; Mannon, RB; Levenson, R
MLA Citation
Coffman, TM, Spurney, RF, Mannon, RB, and Levenson, R. "Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells." Am J Physiol 275.2 Pt 2 (August 1998): F262-F269.
PMID
9691017
Source
pubmed
Published In
The American journal of physiology
Volume
275
Issue
2 Pt 2
Publish Date
1998
Start Page
F262
End Page
F269

Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF).

Inhibition of the renal brush border membrane (BBM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recently cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-RF). As deduced from the cDNA of this 358-amino acid protein, amino acids 11-101 and amino acids 150-241 of the NHE-RF protein share 74% overall homology suggesting duplication of these PDZ containing domains. The serine residues at amino acid position 289 and 340 are considered to be the most likely sites for PKA mediated phosphorylation. To study the structure- function relation between NHE-RF and PKA mediated inhibition of the rabbit BBM Na/H exchanger, the effect of recombinant proteins representing full-length NHE-RF as well as truncated and mutant forms of NHE-RF were determined using a reconstitution assay. The reconstitution assay employed a fraction of rabbit BBM proteins that contains Na/H exchanger activity that is not regulated by PKA. NHE-RF in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a significant left shift in the dose-response relation such that 10(-12) M NHE-RF inhibited Na/H exchange transport by 30% in the presence but not in the absence of PKA. A recombinant polypeptide representing amino acids 1-151 (Domain I) did not affect Na/H exchange transport in the presence or absence of PKA. A polypeptide representing amino acids 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a left shift in the dose-response relation. 10(-12) M of Domain II polypeptide inhibited transport by 18% in the presence but not in the absence of PKA. Mutation of serine residues 287, 289, and 290 to alanine did not affect the inhibitory effect in the absence of PKA but abolished the left shift in the dose-response relation elicited by PKA. Mutation of serine residues 339 and 340 to alanine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit renal BBM Na/H exchange activity-an effect which is augmented by PKA. The amino acid sequences in the polypeptide containing only the NH2-terminal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor but appears to be required for the full-length NHE-RF to express its full inhibitory effect on the BBM Na/H exchanger. One or more of the serine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the physiologic effect of cAMP on the renal BBM Na/H exchanger.

Authors
Weinman, EJ; Steplock, D; Tate, K; Hall, RA; Spurney, RF; Shenolikar, S
MLA Citation
Weinman, EJ, Steplock, D, Tate, K, Hall, RA, Spurney, RF, and Shenolikar, S. "Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF)." J Clin Invest 101.10 (May 15, 1998): 2199-2206.
PMID
9593775
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
101
Issue
10
Publish Date
1998
Start Page
2199
End Page
2206
DOI
10.1172/JCI204

Effect of receptor number on desensitization of the mouse thromboxane receptor.

Desensitization of G-protein coupled receptors limits the physiologic effects of an agonist. Short-term desensitization mechanisms are critically dependent on receptor phosphorylation by protein kinases. The effectiveness of these regulatory mechanisms might be limited by substrate (receptor) availability. To investigate the role of receptor number in the desensitization of G-protein coupled receptors, we transfected a mouse mesangial cell line with a genomic clone encoding the mouse thromboxane A2 (TxA2) receptor and obtained cell lines that expressed low (approximately 250-500 fmol/mg protein) or high (2500-4000 fmol/mg protein) levels of TxA2 receptors. Activation of TxA2 receptors stimulated phosphoinositide (PI) hydrolysis and increased intracellular calcium ([Ca2+]i) levels. Prior exposure to the TxA2 agonist (15S)-hydroxy-11alpha,9alpha-(epoxymethano)prosta-5Z,+ ++13E-dienoic acid (U46619) reduced subsequent (15S)-hydroxy- 11alpha,9alpha-(epoxymethano)prosta-5Z,13E-dieno ic acid-induced increases in inositol trisphosphates and intracellular calcium levels by approximately 50% in clones expressing low numbers of TxA2 receptors, but had little effect on TxA2 receptor responsiveness in clones expressing high receptor numbers. Failure of TxA2 receptors to desensitize caused sustained increases in intracellular calcium levels and phosphoinositide hydrolysis. Thus, homologous desensitization of TxA2 receptors is attenuated in cells expressing high levels of receptors for TxA2. These data suggest that receptor number plays a key role in the short-term regulation of G-protein coupled receptors.

Authors
Spurney, RF
MLA Citation
Spurney, RF. "Effect of receptor number on desensitization of the mouse thromboxane receptor." Biochem Pharmacol 55.8 (April 15, 1998): 1271-1281.
PMID
9719483
Source
pubmed
Published In
Biochemical Pharmacology
Volume
55
Issue
8
Publish Date
1998
Start Page
1271
End Page
1281

Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells

We examined the effects of thromboxane A2 (TxA2) on the activities of the plasminogen-plasmin system in glomerular mesangial cells. When mesangial cells are exposed to the TxA2 agonist U-46619, a substantial increase in production of plasminogen activator inhibitor-1 (PAI-1) protein is observed that is significantly greater than that induced by 10% serum alone. This increase in PAI-1 protein production is accompanied by an increase in steady- state levels of PAI-1 mRNA. This stimulation is specifically mediated by TxA2 (thromboxane prostanoid, TP) receptors, since U-46619 also stimulates PAI-1 expression in cells that are transfected with TP receptors, and this stimulation of PAI-1 production is completely blocked by the TxA2 receptor antagonist, SQ-29,548. Despite the increase in PAI-1 production, there was net stimulation of plasmin activity in the medium of mesangial cells that had been exposed to U-46619. Furthermore, U-46619 also caused an increase in tissue plasminogen activator (tPA) mRNA levels. Thus TxA2 stimulates the production of PAI-1 and plasminogen activators by mesangial cells through a receptor-dependent mechanism. In inflammatory renal diseases, the balance of these effects may modulate glomerular thrombosis and renal fibrosis.

Authors
Coffman, TM; Spurney, RF; Mannon, RB; Levenson, R
MLA Citation
Coffman, TM, Spurney, RF, Mannon, RB, and Levenson, R. "Thromboxane A2 modulates the fibrinolytic system in glomerular mesangial cells." American Journal of Physiology - Renal Physiology 275.2 44-2 (1998): F262-F269.
Source
scival
Published In
American Journal of Physiology - Renal Physiology
Volume
275
Issue
2 44-2
Publish Date
1998
Start Page
F262
End Page
F269

The C-terminus of the thromboxane receptor contributes to coupling and desensitization in a mouse mesangial cell line.

To investigate regulatory domains of the thromboxane A2 (TxA2) receptor, we constructed a truncated form of the mouse TxA2 receptor and expressed it in a mesangial cell line. The mutant receptor lacked 22 amino acids in the C-terminus including four potential phosphorylation sites. Ligand binding of mutant receptors was identical with the wild type. Stimulation with TxA2 agonist induced increases in inositol trisphosphate (IP3) generation and [Ca++]i by both wild-type and mutant receptors. However, the initial increase in IP3 generation by the mutant receptor was only approximately 50% of that seen in the wild type. Exposure of wild-type receptors to TxA2 agonist caused desensitization of IP3 and calcium responses. Pretreatment with TxA2 agonist caused some desensitization of mutant receptors, but the extent of desensitization was reduced compared with the wild type. The protein kinase C inhibitor staurosporine attenuated TxA2-induced desensitization of wild-type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with low concentrations of the phorbol ester, phorbol 12,13-dibutyrate (100 nM), reduced subsequent responsiveness of wild-type but not mutant TxA2 receptors. In contrast, high-dose phorbol 12,13-dibutyrate (1 microM) produced a similar degree of desensitization of both receptor types. These data suggest that: 1) the C-terminus participates in coupling of the TxA2 receptor to its effector systems; 2) the C-terminus contributes to agonist-specific desensitization of the TxA2 receptor; and 3) protein kinase C-induced desensitization of the TxA2 receptor is complex and depends, in part, on C-terminal domains of the TxA2 receptor.

Authors
Spurney, RF; Coffman, TM
MLA Citation
Spurney, RF, and Coffman, TM. "The C-terminus of the thromboxane receptor contributes to coupling and desensitization in a mouse mesangial cell line." J Pharmacol Exp Ther 283.1 (October 1997): 207-215.
PMID
9336326
Source
pubmed
Published In
The Journal of pharmacology and experimental therapeutics
Volume
283
Issue
1
Publish Date
1997
Start Page
207
End Page
215

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

Authors
Hartle, JE; Prpic, V; Siddhanti, SR; Spurney, RF; Quarles, LD
MLA Citation
Hartle, JE, Prpic, V, Siddhanti, SR, Spurney, RF, and Quarles, LD. "Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts." J Bone Miner Res 11.6 (June 1996): 789-799.
PMID
8725176
Source
pubmed
Published In
Journal of Bone and Mineral Research
Volume
11
Issue
6
Publish Date
1996
Start Page
789
End Page
799
DOI
10.1002/jbmr.5650110610

The Prostaglandin E(1) (PGE(1)) Analog Misoprostol Ameliorates Autoimmune Disease and Depletes T Lymphocytes in MRL-lpr/lpr Mice.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease similar to human systemic lupus erythematosus (SLE). Disease manifestations include anti-DNA autoantibody production, arthritis, vasculitis, and an immune-complex glomerulonephritis. The development of autoimmune disease is associated with massive, generalized lymphadenopathy caused by accumulation of an abnormal T-cell population in peripheral lymphoid organs. In MRL-lpr/lpr mice, treatment with E-series prostaglandins ameliorates renal disease and reduces peripheral lymphadenopathy. Little is known about mechanisms of action of E-series prostaglandins in murine lupus or the effects of these agents on other clinically important manifestations of SLE, such as arthritis and vasculitis. To further investigate the effects of an E-series prostaglandin in murine SLE, we administered the prostaglandin, E(1) (PGE(1)) analog misoprostol (1 mg kg(minus sign1) day(minus sign1)) to MRL-lpr/lpr mice for 8 weeks by twice daily subcutaneous injection. At 20 weeks of age, treatment with misoprostol reduced the severity of renal disease and arthritis but did not affect the extent or severity of vasculitis. The beneficial effects of misoprostol on arthritis and renal disease were associated with a significant decrease in splenic and lymph node weight in mice given the PGE(1) analog. This decrease in lymphoproliferation resulted primarily from a generalized reduction in the number of T cells in peripheral lymphoid organs. Thus, T-cell depletion was associated with beneficial effects on arthritis and nephritis in MRL-lpr/lpr mice, supporting a role for T lymphocytes in these disease processes. The ability of E-series prostaglandins to favorably modify autoimmune disease in this murine model suggests that misoprostol may be a useful adjunct to current therapies for the treatment of patients with SLE.

Authors
Best, V; Ruiz, P; Spurney, RF
MLA Citation
Best, V, Ruiz, P, and Spurney, RF. "The Prostaglandin E(1) (PGE(1)) Analog Misoprostol Ameliorates Autoimmune Disease and Depletes T Lymphocytes in MRL-lpr/lpr Mice." Am J Ther 2.12 (December 1995): 943-948.
PMID
11854812
Source
pubmed
Published In
American Journal of Therapeutics
Volume
2
Issue
12
Publish Date
1995
Start Page
943
End Page
948

Role of platelet activating factor in kidney transplant rejection in the rat.

Platelet activating factor (PAF) is a potent lipid mediator with a broad range of biologic activities. Experimental evidence suggests that PAF plays a role in the pathogenesis of a variety of inflammatory processes including allograft rejection. In this study, we evaluated the effects of the PAF antagonist BN52021 on the course of renal allograft rejection in a rat model. Kidneys from ACI (RT1a) rats were transplanted into fully allogeneic PVG (RT1c) rat recipients. Animals received 60 mg/kg/day of the PAF antagonist or vehicle beginning immediately prior to the transplantation procedure. In rats treated with the PAF antagonist, allograft GFR and plasma flow were maintained at levels that were significantly greater than controls. Despite the improvement in renal allograft function, BN52021 had no effect on allograft histomorphology and both groups manifested intense inflammatory cell infiltration consistent with acute cellular rejection. PAF antagonism reduced urinary excretion of thromboxane metabolites and decreased thromboxane production by homogenates prepared from kidney allografts. The PAF antagonist had no effect on urinary excretion of peptidoleukotriene metabolites or on the production of LTB4 by allografts. These data support a role for PAF in the pathophysiology of acute renal allograft rejection, and they suggest that the hemodynamic effects of PAF during rejection may be mediated through stimulation of thromboxane A2. In view of the beneficial effects of PAF blockade in rejection as well as recent reports describing efficacy in models of cyclosporine nephrotoxicity, PAF antagonists may have clinical applications in human renal allograft recipients.

Authors
Butterly, DW; Spurney, RF; Ruiz, P; Pirotzky, E; Braquet, P; Coffman, TM
MLA Citation
Butterly, DW, Spurney, RF, Ruiz, P, Pirotzky, E, Braquet, P, and Coffman, TM. "Role of platelet activating factor in kidney transplant rejection in the rat." Kidney Int 48.2 (August 1995): 337-343.
PMID
7564100
Source
pubmed
Published In
Kidney international
Volume
48
Issue
2
Publish Date
1995
Start Page
337
End Page
343

5-Hydroxytryptamine2A receptors expressed in rat renal mesangial cells inhibit cyclic AMP accumulation.

Second messenger coupling of the 5-hydroxytryptamine (5-HT)2A receptor endogenous to cultured rat glomerular mesangial cells was studied. 5-HT induced an increase in total inositol phosphate levels (EC50 = 265 +/- 55 nM, maximum stimulation = 150 +/- 23%). That effect was sensitive to antagonists of the 5-HT2A receptor and was insensitive to pertussis toxin at doses that eliminated detectable pertussis toxin substrate, as determined by membrane ADP-ribosylation. Surprisingly, 5-HT also induced an inhibition of forskolin-stimulated cAMP accumulation (55 +/- 6%, IC50 = 5 +/- 3 nM). This effect was competitively antagonized by the 5-HT2A receptor antagonists ketanserin, ritanserin, and spiperone and could be produced by the 5-HT2 receptor agonists alpha-methyl-5-HT (66 +/- 13%, IC50 = 23 +/- 14 nM) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (65 +/- 4%, IC50 = 14 +/- 7 nM). The inhibition of cAMP accumulation occurred in the presence of a number of agents that either stimulate or inhibit protein kinase C activity, arachidonic acid metabolism, or Ca2+ mobilization. In isolated membranes, 5-HT induced a 36 +/- 5% inhibition of adenylyl cyclase activity (IC50 = 8 +/- 4 nM). Inhibition of cAMP accumulation in intact cells and of adenylyl cyclase activity in washed membranes was (> 50%) sensitive to pertussis toxin, implicating Gi alpha or Go alpha subunits in the inhibitory signal. These data suggest that the 5-HT2A receptor can be permissive in its coupling to G proteins and second messengers.

Authors
Garnovskaya, MN; Nebigil, CG; Arthur, JM; Spurney, RF; Raymond, JR
MLA Citation
Garnovskaya, MN, Nebigil, CG, Arthur, JM, Spurney, RF, and Raymond, JR. "5-Hydroxytryptamine2A receptors expressed in rat renal mesangial cells inhibit cyclic AMP accumulation." Mol Pharmacol 48.2 (August 1995): 230-237.
PMID
7651356
Source
pubmed
Published In
Molecular pharmacology
Volume
48
Issue
2
Publish Date
1995
Start Page
230
End Page
237

The effects of short-term treatment with the prostaglandin E1 (PGE1) analog misoprostol on inflammatory mediator production in murine lupus nephritis.

MRL-lpr/lpr mice spontaneously develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus. In these animals, treatment with E-series prostaglandins ameliorates renal disease and prolongs survival, perhaps by modulating production of cytokines or eicosanoids. To further define the mechanisms of action of E-series prostaglandins in established murine lupus nephritis, we administered the prostaglandin E1 (PGE1) analog misoprostol to 20-week-old MRL-lpr/lpr mice by twice-daily subcutaneous injection. After 2 days of treatment, misoprostol reduced renal cortical interleukin-1 beta (IL-1 beta) mRNA levels compared to vehicle-treated controls (0.19 +/- 0.06 (misoprostol) vs 0.50 +/- 0.04 (vehicle) densitometry units; P < 0.005). A similar reduction in cortical IL-1 beta mRNA levels was found in left kidneys harvested from MRL-lpr/lpr mice following 2 days of treatment with misoprostol compared to right kidneys harvested from the same animal prior to the first dose of PGE1 analog (0.12 +/- .05 (left) vs 0.39 +/- 0.18 (right) densitometry units; P < 0.05). This reduction in cortical IL-1 beta mRNA levels was not associated with alterations in renal production of thromboxane B2, PGE2, or leukotriene B4 or with significant changes in the severity of renal inflammatory cell infiltrates. Time-course studies indicated that IL-1 beta mRNA levels were decreased within 24 hr of initiating misoprostol therapy. This reduction in IL-1 beta mRNA levels was transient because levels were not reduced after 1 week of treatment with the PGE1 analog.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Fan, PY; Ruiz, P; Pisetsky, DS; Spurney, RF
MLA Citation
Fan, PY, Ruiz, P, Pisetsky, DS, and Spurney, RF. "The effects of short-term treatment with the prostaglandin E1 (PGE1) analog misoprostol on inflammatory mediator production in murine lupus nephritis." Clin Immunol Immunopathol 75.2 (May 1995): 125-130.
PMID
7704969
Source
pubmed
Published In
Clinical Immunology and Immunopathology
Volume
75
Issue
2
Publish Date
1995
Start Page
125
End Page
130

Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor.

Previous studies have demonstrated the presence of mitogenic serotonin [i.e., 5-hydroxytryptamine (5-HT)] receptors on glomerular mesangial cells and have linked those receptors to a complicated array of intracellular and autocrine/paracrine signaling pathways [T. Knauss and H. E. Abboud. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F844-F850, 1986; and N. Takuwa, M. Ganz, Y. Takuwa, R. B. Sterzel, and H. Rasmussen. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F431-F439, 1989]. Those studies suggested that the mesangial subtype of 5-HT receptor is a member of the 5-HT2 receptor family, which consists of three known members, designated as subtypes A, B, and C. The purpose of the current study was to identify the subtype of 5-HT2 receptor present on mesangial cells. Northern blot showed detectable mRNA for a putative 5-HT2A receptor, but not for 5-HT1A or 5-HT2C receptors. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides derived from the putative third and sixth transmembrane domains of cloned 5-HT2 receptors yielded a 580-nucleotide (nt) fragment. RT-PCR with primers highly specific for the 5-HT2A receptor and designed to amplify > 95% of its coding block yielded a product of 1,320 nt. Nested PCR reactions yielded products of the predicted sizes for the 5-HT2A receptor. Partial sequence information was obtained, and the sequence corresponded exactly (627/627 nt) to that published for the cloned rat brain 5-HT2A receptor. These studies identify the mesangial cell mitogenic 5-HT receptor as a 5-HT2A receptor subtype.

Authors
Nebigil, CG; Garnovskaya, MN; Spurney, RF; Raymond, JR
MLA Citation
Nebigil, CG, Garnovskaya, MN, Spurney, RF, and Raymond, JR. "Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor." Am J Physiol 268.1 Pt 2 (January 1995): F122-F127.
PMID
7840237
Source
pubmed
Published In
The American journal of physiology
Volume
268
Issue
1 Pt 2
Publish Date
1995
Start Page
F122
End Page
F127

Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor

Previous studies have demonstrated the presence of mitogenic serotonin [i.e., 5-hydroxytryptamine (5-HT)] receptors on glomerular mesangial cells and have linked those receptors to a complicated array of intracellular and autocrine/ paracrine signaling pathways [T. Knauss and H. E. Abboud. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F844-F850, 1986; and N. Takuwa, M. Ganz, Y. Takuwa, R. B. Sterzel, and H. Rasmussen. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F431-F439, 1989]. Those studies suggested that the mesangial subtype of 5-HT receptor is a member of the 5-HT2 receptor family, which consists of three known members, designated as subtypes A, B, and C. The purpose of the current study was to identify the subtype of 5-HT2 receptor present on mesangial cells. Northern blot showed detectable mRNA for a putative 5-HT2A receptor, but not for 5-HT1A or 5-HT2C receptors. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides derived from the putative third and sixth transmembrane domains of cloned 5-HT2 receptors yielded a 580-nucleotide (nt) fragment. RT-PCR with primers highly specific for the 5-HT2A receptor and designed to amplify >95% of its coding block yielded a product of 1,320 nt. Nested PCR reactions yielded products of the predicted sizes for the 5-HT2A receptor. Partial sequence information was obtained, and the sequence corresponded exactly (627/627 nt) to that published for the cloned rat brain 5-HT2A receptor. These studies identify the mesangial cell mitogenic 5-HT receptor as a 5-HT2A receptor subtype. © 1995 the American Physiological Society.

Authors
Nebigil, CG; Garnovskaya, MN; Spurney, RF; Raymond, JR
MLA Citation
Nebigil, CG, Garnovskaya, MN, Spurney, RF, and Raymond, JR. "Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor." American Journal of Physiology - Renal Fluid and Electrolyte Physiology 268.1 37-1 (1995): F122-F127.
Source
scival
Published In
American Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume
268
Issue
1 37-1
Publish Date
1995
Start Page
F122
End Page
F127

Fish oil feeding modulates leukotriene production in murine lupus nephritis.

Diets enriched with fish oil (FO) ameliorate kidney disease in the MRL-lpr/lpr murine model of lupus nephritis. Although the mechanisms of this effect are not known, FO is rich in the polyunsaturated fatty acid eicosapentaenoic acid (EPA) which may have profound effects on eicosanoid metabolism. In MRL-lpr/lpr mice, FO feeding reduces renal production of cyclooxygenase metabolites. However, EPA may also affect the metabolism of arachidonate by the 5-lipoxygenase (5-LO) pathway and enhanced production of 5-LO metabolites has been implicated in the pathogenesis of kidney disease in MRL-lpr/lpr mice. We therefore investigated the effects of FO feeding on production of 5-LO metabolites in 20 week old MRL-lpr/lpr mice. After 8 weeks of dietary supplementation with FO, both renal hemodynamic function and glomerular histology were improved compared to safflower oil (SO) controls. Amelioration of kidney disease was associated with alterations in the pattern of leukotriene production by macrophages and kidneys from FO fed mice. There was a significant decrease in the production of leukotriene B4 (LTB4) and tetraene peptidoleukotrienes by peritoneal macrophages isolated from mice given FO compared to control animals. Similarly, dietary supplementation with FO decreased renal production of LTB4. Reduced production of tetraene leukotrienes was accompanied by a modest increase in the production of pentaene leukotrienes by macrophages from FO fed mice. We speculate that this modulation of leukotriene production by FO feeding may have beneficial effects on renal disease in autoimmune nephritis.

Authors
Spurney, RF; Ruiz, P; Albrightson, CR; Pisetsky, DS; Coffman, TM
MLA Citation
Spurney, RF, Ruiz, P, Albrightson, CR, Pisetsky, DS, and Coffman, TM. "Fish oil feeding modulates leukotriene production in murine lupus nephritis." Prostaglandins 48.5 (November 1994): 331-348.
PMID
7855311
Source
pubmed
Published In
Prostaglandins
Volume
48
Issue
5
Publish Date
1994
Start Page
331
End Page
348

Modulation of thromboxane receptor activation in rat glomerular mesangial cells.

Rat glomerular mesangial cells were used to investigate mechanisms of thromboxane A2 (TxA2) receptor regulation in the kidney. Exposure of mesangial cells to the TxA2 agonist U-46619 for 10 min reduced subsequent TxA2-induced increases in inositol phosphates and intracellular Ca2+ levels by approximately 70%. This loss of receptor responsiveness could be blocked by the TxA2 receptor antagonist SQ-29548 and was reversible after removal of agonist from the incubation medium. Radioligand binding studies using the TxA2 agonist [125I]BOP suggested that exposure of mesangial cells to U-46619 for 10 min reduced TxA2 receptor responsiveness without a loss of receptor sites from plasma membrane fractions of the cell, although the density of mesangial cell TxA2 receptors was decreased by approximately 60% after more prolonged exposure of mesangial cells to thromboxane agonists. Both desensitization to U-46619 and loss of TxA2 binding sites could be attenuated by the protein kinase C (PKC) inhibitors staurosporine, sphingosine, or H-7, and TxA2 receptor responsiveness was reduced in cells incubated with phorbol esters before stimulation with thromboxane agonists. We conclude that 1) agonist-specific decreases in TxA2 receptor responsiveness may involve initial uncoupling of the receptor from its effector systems, followed by a loss of TxA2 receptor sites from plasma membrane fractions of the cell, and 2) PKC may be involved in these processes.

Authors
Spurney, RF; Middleton, JP; Raymond, JR; Coffman, TM
MLA Citation
Spurney, RF, Middleton, JP, Raymond, JR, and Coffman, TM. "Modulation of thromboxane receptor activation in rat glomerular mesangial cells." Am J Physiol 267.3 Pt 2 (September 1994): F467-F478.
PMID
8092261
Source
pubmed
Published In
The American journal of physiology
Volume
267
Issue
3 Pt 2
Publish Date
1994
Start Page
F467
End Page
F478

Leukotrienes in renal transplant rejection in rats. Distinct roles for leukotriene B4 and peptidoleukotrienes in the pathogenesis of allograft injury.

To investigate the role of leukotrienes in renal allograft rejection, we studied the effects of specific leukotriene inhibitors in a rat kidney transplant model. The enhanced renal production of leukotrienes observed in allograft recipients was reduced in a dose-dependent manner by the specific 5-lipoxygenase inhibitor MK886. This suppression of leukotriene production caused a substantial improvement in renal function. Inhibition of 5-lipoxygenase also reduced the severity of vascular inflammation and endothelial injury in allografts, and profoundly inhibited expression of donor MHC class II Ag on kidney cells. Survival of renal allograft recipients was prolonged from 10 +/- 1 days in controls to 16 +/- 1 days in animals that received a 6-day course of MK886 (p < 0.05). To investigate the relative roles of LTB4 compared to peptidoleukotrienes in these processes, we treated a separate group of animals with the specific peptidoleukotriene receptor antagonist SKF106203. This agent inhibits the interaction of peptidoleukotrienes with their receptor(s) but does not affect the biologic actions of LTB4. In these studies, SKF106203 caused a modest improvement in renal allograft function that was of lesser magnitude than that seen with the 5-lipoxygenase inhibitor. SKF106203 also reduced vascular inflammation in allografts, but had no effect on expression of MHC class II Ag. We conclude that leukotrienes play a key role in the pathogenesis of renal allograft rejection. Furthermore, the detrimental effects of leukotrienes in rejection are mediated by distinct actions of LTB4 and peptidoleukotrienes.

Authors
Spurney, RF; Ibrahim, S; Butterly, D; Klotman, PE; Sanfilippo, F; Coffman, TM
MLA Citation
Spurney, RF, Ibrahim, S, Butterly, D, Klotman, PE, Sanfilippo, F, and Coffman, TM. "Leukotrienes in renal transplant rejection in rats. Distinct roles for leukotriene B4 and peptidoleukotrienes in the pathogenesis of allograft injury." J Immunol 152.2 (January 15, 1994): 867-876.
PMID
8283057
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
152
Issue
2
Publish Date
1994
Start Page
867
End Page
876

Modulation of thromboxane receptor activation in rat glomerular mesangial cells

Authors
Spurney, RF; Middleton, JP; Raymond, JR; Coffman, TM
MLA Citation
Spurney, RF, Middleton, JP, Raymond, JR, and Coffman, TM. "Modulation of thromboxane receptor activation in rat glomerular mesangial cells." AM.J.PHYSIOL. 267.3 part 2 (1994): F467-F478.
Source
scival
Published In
AM.J.PHYSIOL.
Volume
267
Issue
3 part 2
Publish Date
1994
Start Page
F467
End Page
F478

Modulation of thromboxane receptor activation in rat glomerular mesangial cells

Rat glomerular mesangial cells were used to investigate mechanisms of thromboxane A2 (TxA2) receptor regulation in the kidney. Exposure of mesangial cells to the TxA2 agonist U-46619 for 10 min reduced subsequent TxA2-induced increases in inositol phosphates and intracellular Ca2+ levels by ∼70%. This loss of receptor responsiveness could be blocked by the TxA2 receptor antagonist SQ-29548 and was reversible after removal of agonist from the incubation medium. Radioligand binding studies using the TxA2 agonist [125I]BOP suggested that exposure of mesangial cells to U-46619 for 10 min reduced TxA2 receptor responsiveness without a loss of receptor sites from plasma membrane fractions of the cell, although the density of mesangial cell TxA2 receptors was decreased by ∼60% after more prolonged exposure of mesangial cells to thromboxane agonists. Both desensitization to U-46619 and loss of TxA2 binding sites could be attenuated by the protein kinase C (PKC) inhibitors staurosporine, sphingosine, or H-7, and TxA2 receptor responsiveness was reduced in cells incubated with phorbol esters before stimulation with thromboxane agonists. We conclude that 1) agonist-specific decreases in TxA2 receptor responsiveness may involve initial uncoupling of the receptor from its effector systems, followed by a loss of TxA2 receptor sites from plasma membrane fractions of the cell, and 2) PKC may be involved in these processes. © the American Physiological Society.

Authors
Spurney, RF; Middleton, JP; Raymond, JR; Coffman, TM
MLA Citation
Spurney, RF, Middleton, JP, Raymond, JR, and Coffman, TM. "Modulation of thromboxane receptor activation in rat glomerular mesangial cells." American Journal of Physiology - Renal Fluid and Electrolyte Physiology 267.3 36-3 (1994): F467-F478.
Source
scival
Published In
American Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume
267
Issue
3 36-3
Publish Date
1994
Start Page
F467
End Page
F478

Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor

Previous studies have demonstrated the prèsence of mitogenic serotonin [i.e., 5-hydroxytryptamine (5-HT)] receptors on glomerular mesangial cells and have linked those receptors to a complicated array of intracellular and autocrine/ paracrine signaling pathways [T. Knauss and H. E. Abboud. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20): F844-F850, 1986; and N. Takuwa, M. Ganz, Y. Takuwa, R. B. Sterzel, and H. Rasmussen. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F431-F439, 1989]. Those studies suggested that the mesangial subtype of 5-HT receptor is a member of the 5-HT2 receptor family, which consists of three known members, designated as subtypes A, B, and C. The purpose of the current study was to identify the subtype of 5-HT2 receptor present on mesangial cells. Northern blot showed detectable mRNA for a putative 5-HT2A receptor, but not for 5-HT1A or 5-HT2c receptors. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligo-nucleotides derived from the putative third and sixth trans-membrane domains of cloned 5-HT2 receptors yielded a 580-nucleotide (nt) fragment. RT-PCR with primers highly specific for the 5-HT2A receptor and designed to amplify >95% of its coding block yielded a product of 1,320 nt. Nested PCR reactions yielded products of the predicted sizes for the 5-HT2A receptor. Partial sequence information was obtained, and the sequence corresponded exactly (627/627 nt) to that published for the cloned rat brain 5-HT2A receptor. These studies identify the mesangial cell mitogenic 5-HT receptor as a 5-HT2A receptor subtype. Copyright © 1995 the American Physiological Society.

Authors
Nebigil, CG; Garnovskaya, MN; Spurney, RF; Raymond, JR
MLA Citation
Nebigil, CG, Garnovskaya, MN, Spurney, RF, and Raymond, JR. "Identification of a rat glomerular mesangial cell mitogenic 5-HT2A receptor." American Journal of Physiology - Renal Physiology 268.1 (1994): F122-F127.
Source
scival
Published In
American Journal of Physiology - Renal Physiology
Volume
268
Issue
1
Publish Date
1994
Start Page
F122
End Page
F127

Thromboxane binding and signal transduction in rat glomerular mesangial cells.

Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Spurney, RF; Onorato, JJ; Albers, FJ; Coffman, TM
MLA Citation
Spurney, RF, Onorato, JJ, Albers, FJ, and Coffman, TM. "Thromboxane binding and signal transduction in rat glomerular mesangial cells." Am J Physiol 264.2 Pt 2 (February 1993): F292-F299.
PMID
8447439
Source
pubmed
Published In
The American journal of physiology
Volume
264
Issue
2 Pt 2
Publish Date
1993
Start Page
F292
End Page
F299

Characterization of glomerular thromboxane receptors in murine lupus nephritis.

Renal thromboxane (Tx) production is increased in the MRL-lpr murine model of lupus nephritis. To investigate the relationship between increased Tx production and number and affinity of Tx receptors, we measured binding of the Tx receptor antagonist [3H][SQ295481S-1 alpha,2 beta(5Z),3 beta,4 alpha]-7-(3-((2-((phenyl- amino)-carbonyl)hydrozino)methyl)-7-oxabicyclo-(2.2.1)heptan -2-yl)-5-heptenoic acid in glomerular preparations from MRL-lpr mice and both MRL(-)+/+ and LG/J controls. Renal Tx binding was first characterized in normal LG/J mice. In these animals, glomerular binding was specific, saturable and reversible. Scatchard analysis revealed a single class of high-affinity binding sites. We next evaluated Tx production and binding in 12- and 16-week-old MRL-lpr mice and MRL(-)+/+ controls. To assess renal Tx production, excretion of TxB2 was measured in urine. Urinary TxB2 was increased in MRL-lpr mice at 16 weeks of age. This increase in urinary TxB2 was associated with a reduction in density of glomerular Tx binding sites compared to either 12-week-old MRL-lpr mice or MRL(-)+/+ controls. Ligand binding affinity was similar in all groups. To investigate if this alteration in binding was specific for Tx, glomerular binding of [3H]angiotensin II was measured. In MRL-lpr mice, the number and affinity of glomerular angiotensin binding sites were similar at 12 and 16 weeks of age. Thus, in this murine model of lupus nephritis, enhanced renal Tx production is temporally associated with a decrease in glomerular Tx binding sites without a change in receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Authors
Spurney, RF; Onorato, JJ; Ruiz, P; Pisetsky, DS; Coffman, TM
MLA Citation
Spurney, RF, Onorato, JJ, Ruiz, P, Pisetsky, DS, and Coffman, TM. "Characterization of glomerular thromboxane receptors in murine lupus nephritis." J Pharmacol Exp Ther 264.2 (February 1993): 584-590.
PMID
8437109
Source
pubmed
Published In
The Journal of pharmacology and experimental therapeutics
Volume
264
Issue
2
Publish Date
1993
Start Page
584
End Page
590

Thromboxane binding and signal transduction in rat glomerular mesangial cells

Thromboxane A, (TxA2) stimulates contraction of glo-mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist {1S-[1α,2β(5Z),3α(lE,3S)4α]}-7-{3-[3-hydroxy-4-(p- iodophenoxy)-l-butenyl]7-oxabicyclo[2.2.1]hept-2-yl}-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11α,9α-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and {1R-[1α(Z),2β,3β,5α]}-(+)-7-{5-[(1,1′-biphenyl)-4- ylmethoxy] -3 -hydroxy-2- (1-piperidinyl)cyclopentyl} -4 -heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 μM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 μM), were ̃ 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C. These results suggest that functional, high-affinity receptor sites for TxA2 are present on rat glomerular mesangial cells, and that these receptors are linked to phospholipase C activation.

Authors
Spurney, RF; Onorato, JJ; Albers, FJ; Coffman, TM
MLA Citation
Spurney, RF, Onorato, JJ, Albers, FJ, and Coffman, TM. "Thromboxane binding and signal transduction in rat glomerular mesangial cells." American Journal of Physiology - Renal Fluid and Electrolyte Physiology 264.2 33-2 (1993): F292-F299.
Source
scival
Published In
American Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume
264
Issue
2 33-2
Publish Date
1993
Start Page
F292
End Page
F299

Improved renal function in mouse kidney allografts lacking MHC class I antigens

The immunological responses that lead to rejection of organ and tissue transplants are triggered by the recognition of proteins encoded within the MHC. The relative contributions of responses directed toward MHC class I compared with class II in the loss of functional integrity of vascularized organ grafts have been difficult to define. The recent development of technologies which allow the generation of mice in which specific genes have been altered by gene targeting offers a new approach to addressing this question. We examine here the rejection of kidney allografts from mice lacking native MHC class I Ag. These mice were obtained from embryonic stem cells in which the β2 microglobulin (β2m) gene had been disrupted by homologous recombination. We found a significant improvement in function of renal allografts from MHC class I-deficient donors compared with allografts from donors with normal MHC class I expression. Surprisingly, the improved function of the MHC class I-deficient grafts was not associated with differences in mononuclear inflammatory cell infiltration of these grafts nor in differences in alloreactive proliferative or cytotoxic T cell responses. However, we did find differences in alloantibody response between the groups. Recipients of control allografts produced antibodies against both donor MHC class I and II, whereas recipients of MHC class I-deficient grafts formed alloantibodies primarily against donor MHC class II Ag. These studies confirm that immune responses directed toward donor MHC class I alloantigens contribute to kidney transplant dysfunction in this model. Also, these findings suggest that, at least for renal transplants, genetic manipulations which reduce MHC class I expression may be effective in overcoming some of the effects of MHC incompatibility.

Authors
Coffman, T; Geier, S; Ibrahim, S; Griffiths, R; Spurney, R; Smithies, O; Koller, B; Sanfilippo, F
MLA Citation
Coffman, T, Geier, S, Ibrahim, S, Griffiths, R, Spurney, R, Smithies, O, Koller, B, and Sanfilippo, F. "Improved renal function in mouse kidney allografts lacking MHC class I antigens." Journal of Immunology 151.1 (1993): 425-435.
PMID
8326135
Source
scival
Published In
Journal of Immunology
Volume
151
Issue
1
Publish Date
1993
Start Page
425
End Page
435

Effect of anti-CD4 antibody treatment on inflammatory arthritis in MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop an inflammatory arthritis in association with other manifestations of autoimmunity. Although a variety of immune cell disturbances have been described in these mice, the relationship of these abnormalities to the pathogenesis of arthritis has not yet been determined; the role of T cells is especially unclear since synovial hypertrophy and joint erosions have been noted in some studies in the absence of a significant T cell infiltrate. Therefore, to determine if T cells are required for arthritis in MRL-lpr/lpr mice, we evaluated the effects of prolonged treatment with a monoclonal anti-CD4 antibody. Knee joints from treated mice had markedly reduced arthritis compared to saline-treated control animals as measured by the degree of synovial hypertrophy and inflammation. Nephritis in these mice was concomitantly reduced. In contrast, rheumatoid factor levels were not affected by CD4+ cell depletion, despite significant effects on anti-DNA. These results indicate that in MRL-lpr/lpr mice anti-CD4 therapy can inhibit arthritis, suggesting an important role of T cells in the pathogenesis of this lesion.

Authors
Gilkeson, GS; Spurney, R; Coffman, TM; Kurlander, R; Ruiz, P; Pisetsky, DS
MLA Citation
Gilkeson, GS, Spurney, R, Coffman, TM, Kurlander, R, Ruiz, P, and Pisetsky, DS. "Effect of anti-CD4 antibody treatment on inflammatory arthritis in MRL-lpr/lpr mice." Clin Immunol Immunopathol 64.2 (August 1992): 166-172.
PMID
1353712
Source
pubmed
Published In
Clinical Immunology and Immunopathology
Volume
64
Issue
2
Publish Date
1992
Start Page
166
End Page
172

Thromboxane receptor blockade reduces renal injury in murine lupus nephritis.

To investigate the role of thromboxane A2 (TxA2) in murine lupus, we assessed the effects of the specific thromboxane receptor antagonist GR32191 on immune complex glomerulonephritis in MRL-lpr/lpr mice. Forty mg/kg/day GR32191 was given by twice daily subcutaneous injection for eight weeks beginning at 12 weeks of age. This dose completely blocked the renal vasoconstriction produced by the thromboxane agonist U46619. After eight weeks of treatment, both glomerular filtration rate (GFR) (8.9 +/- 0.6 vs. 6.8 +/- 1.1 ml/min/kg; P less than 0.05) and PAH clearance (CPAH) (37.4 +/- 2.5 vs. 29.9 +/- 3.3 ml/min/kg; P less than 0.05) were significantly higher in mice given GR32191 compared to vehicle treated animals. Administration of GR32191 also reduced proteinuria from 18.1 +/- 11.6 to 3.7 +/- 1.3 mg/24 hours (P less than 0.05). In GR32191 treated MRL-lpr/lpr mice, renal hemodynamic function and proteinuria were not significantly different from congenic MRL-+/+ controls. Thromboxane receptor blockade had striking affects on renal histomorphology reducing both hyaline thrombi in glomeruli (P = 0.022) and interstitial inflammation (P = 0.006). Glomerular crescents and severity of vasculitis also tended to be reduced in mice receiving the thromboxane receptor antagonist. The overall histopathologic score in mice given GR32191 was significantly lower than vehicle treated animals (4.7 +/- 0.5 vs. 8.4 +/- 1.5; P = 0.016). These effects of GR32191 were associated with decreased excretion of thromboxane B2 (TxB2) in urine (292 +/- 37 vs. 747 +/- 155 pg/24 hr; P less than 0.005) as well as a modest reduction in glomerular deposits of IgG (semiquantitative score 2.6 +/- 0.2 vs. 3.5 +/- 0.2; P less than 0.02). Thus, chronic thromboxane receptor blockade markedly altered the course of renal disease in MRL-lpr/lpr mice, suggesting that TxA2 is an important mediator of renal dysfunction and injury in this murine model of lupus nephritis.

Authors
Spurney, RF; Fan, PY; Ruiz, P; Sanfilippo, F; Pisetsky, DS; Coffman, TM
MLA Citation
Spurney, RF, Fan, PY, Ruiz, P, Sanfilippo, F, Pisetsky, DS, and Coffman, TM. "Thromboxane receptor blockade reduces renal injury in murine lupus nephritis." Kidney Int 41.4 (April 1992): 973-982.
PMID
1387435
Source
pubmed
Published In
Kidney international
Volume
41
Issue
4
Publish Date
1992
Start Page
973
End Page
982

Thromboxane augmentation of alloreactive T cell function

Thromboxane (Tx) plays a vital role in the dysfunction and ultimate rejection of MHC-disparate renal allografts. In addition to its potent vasoconstrictory properties, in vivo studies have implied that Tx is capable of promoting immune cytotoxic T cell function within transplants. In this study, we have examined the in vitro effect of Tx inhibition on alloreactive immune cells using MHC-disparate mouse strain combinations. Coculture of either Tx-synthetase or Tx-receptor inhibitors modified the response of unprimed mouse lymphoid populations in a primary MLR, implying that Tx inhibition and not endoperoxide shunting was responsible for the modulatory effects seen. For example, B10.S lymphoid cells displayed decreased proliferation to H-2 disparate B10.A cells with Tx inhibitors present during the MLR, at pharmacologically active drug concentrations. Moreover, in vitro addition of TxA2 had an augmentory effect on the response in the primary and secondary MLR. Interleukin 2 production and percentages of T cell populations in the primary MLR were not affected by the presence of these compounds, although CD4 and CD8 expression was often increased in the treated populations. Finally, alloreactive primed effector cells also displayed reduced proliferation to specific alloantigen in a secondary MLR when Tx inhibitors were also present, although responses to IL-2 by T cells were not influenced by thromboxane inhibition. These data imply that thromboxane is an important immunoregulatory mediator capable of potentiating the function of naive and primed alloreactive immune T cell populations crucial to the rejection of the transplant.

Authors
Ruiz, P; Rey, L; Spurney, R; Coffman, T; Viciana, A
MLA Citation
Ruiz, P, Rey, L, Spurney, R, Coffman, T, and Viciana, A. "Thromboxane augmentation of alloreactive T cell function." Transplantation 54.3 (1992): 498-505.
PMID
1412730
Source
scival
Published In
Transplantation
Volume
54
Issue
3
Publish Date
1992
Start Page
498
End Page
505

Physiologic role for enhanced renal thromboxane production in murine lupus nephritis.

To investigate the physiologic significance of enhanced renal thromboxane production in murine lupus nephritis, we measured renal hemodynamics and eicosanoid production in MRL-lpr/lpr mice from 8 to 20 weeks of age. Over this age range, MRL-lpr/lpr mice develop an autoimmune disease with nephritis similar to human systemic lupus erythematosus (SLE). In these studies, glomerular filtration rate (GFR) and PAH clearance (CPAH) decreased progressively with age in MRL-lpr/lpr mice, but not in controls. This impairment of renal hemodynamics was associated with increased renal thromboxane production, as well as increased excretion of both thromboxane B2 (TxB2) and 2,3-dinor TxB2 in urine. There was an inverse correlation between renal thromboxane production in MRL-lpr/lpr mice and both GFR and CPAH. Furthermore, there were positive correlations between thromboxane production by the kidney and both the severity of renal histopathology and serum anti-DNA antibody levels measured in individual animals. Enhanced urinary excretion of TxB2 and the development of renal dysfunction also coincided temporally with the appearance of increased levels of interleukin 1 beta (IL-1 beta) mRNA in renal cortex. Acute administration of the specific thromboxane receptor antagonist GR32191 to MRL-lpr/lpr mice restored GFR to normal in early stages of the autoimmune disease. However, in animals with more advanced nephritis, the effect of acute thromboxane receptor blockade on renal hemodynamics was less marked. We conclude that thromboxane A2 is an important mediator of reversible renal hemodynamic impairment in murine lupus, especially in the early phase of disease.

Authors
Spurney, RF; Bernstein, RJ; Ruiz, P; Pisetsky, DS; Coffman, TM
MLA Citation
Spurney, RF, Bernstein, RJ, Ruiz, P, Pisetsky, DS, and Coffman, TM. "Physiologic role for enhanced renal thromboxane production in murine lupus nephritis." Prostaglandins 42.1 (July 1991): 15-28.
PMID
1771236
Source
pubmed
Published In
Prostaglandins
Volume
42
Issue
1
Publish Date
1991
Start Page
15
End Page
28

Acute renal failure in critically ill patients: prognosis for recovery of kidney function after prolonged dialysis support.

OBJECTIVE: To clarify the prognosis for eventual recovery of kidney function in patients who experience prolonged dialysis dependence after acute renal failure (ARF). DESIGN: Retrospective, chart review. SETTING: Inpatients of a large, referral-based hospital. PATIENTS: Twenty-six consecutive survivors of ARF who required greater than 4 wk of dialysis support. RESULTS: All 26 patients were critically ill and developed ARF during treatment in an ICU. The clinical course of these patients was characterized by multiple episodes of renal ischemia or nephrotoxin exposure during dialysis dependence. However, despite multiple renal insults and prolonged dialysis support (mean duration 8.4 +/- 0.7 wk), 23 (88%) of the 26 patients recovered sufficient kidney function to discontinue dialysis. Preexisting renal impairment was associated with a greater risk of irreversible renal failure, and, in patients able to discontinue dialysis, renal recovery was often incomplete. CONCLUSIONS: Despite some renal damage, most critically ill patients who survive ARF requiring prolonged dialysis support recover life-sustaining kidney function.

Authors
Spurney, RF; Fulkerson, WJ; Schwab, SJ
MLA Citation
Spurney, RF, Fulkerson, WJ, and Schwab, SJ. "Acute renal failure in critically ill patients: prognosis for recovery of kidney function after prolonged dialysis support." Crit Care Med 19.1 (January 1991): 8-11.
PMID
1986894
Source
pubmed
Published In
Critical Care Medicine
Volume
19
Issue
1
Publish Date
1991
Start Page
8
End Page
11

Enhanced renal leukotriene production in murine lupus: role of lipoxygenase metabolites.

To investigate the potential role of leukotrienes in murine lupus, we measured renal hemodynamics and renal leukotriene production in MRL-lpr/lpr mice at 12 and 20 weeks of age. Over this age range, these animals develop overt manifestations of autoimmune disease with nephritis similar to human SLE. In the current study, we demonstrated that glomerular filtration rate (GFR) and PAH clearance (CPAH) deteriorated with age in MRL-lpr/lpr mice, but not in MRL(-)+/+ controls. Impaired renal hemodynamic function in MRL-lpr/lpr mice was associated with enhanced ionophore-stimulated production of both leukotriene B4 (LTB4) and leukotriene C4 (LTC4) by preparations of renal cortex. There was a significant inverse correlation between GFR and in vitro production of both LTC4 and LTB4 in kidneys from MRL-lpr/lpr mice, but not in control animals. In addition, in vitro LTC4 production was correlated with the severity of renal histomorphologic abnormalities. Administration of the specific peptidoleukotriene receptor antagonist SKF104353 to 20 week old MRL-lpr/lpr mice significantly improved both GFR and CPAH, whereas this agent had no effect of renal hemodynamics in MRL(-)+/+ controls. These results suggest that renal production of LTC4 and LTB4 is increased in MRL-lpr/lpr mice with nephritis, and that enhanced production of peptidoleukotrienes causes reversible renal dysfunction. Increased leukotriene production within the kidney may therefore be important in the pathogenesis of lupus nephritis.

Authors
Spurney, RF; Ruiz, P; Pisetsky, DS; Coffman, TM
MLA Citation
Spurney, RF, Ruiz, P, Pisetsky, DS, and Coffman, TM. "Enhanced renal leukotriene production in murine lupus: role of lipoxygenase metabolites." Kidney Int 39.1 (January 1991): 95-102.
PMID
1848329
Source
pubmed
Published In
Kidney international
Volume
39
Issue
1
Publish Date
1991
Start Page
95
End Page
102

Thromboxane receptor blockade improves cyclosporine nephrotoxicity in rats.

Cyclosporine A (CyA) nephrotoxicity is associated with impaired renal hemodynamic function and increased production of the vasoconstrictor eicosanoid thromboxane A2 (TxA2). In CyA toxic rats, renal dysfunction can be partially reversed by inhibitors of thromboxane synthase. However, interpretation of these results is complicated since inhibition of thromboxane synthase may cause accumulation of prostaglandin endoperoxides that can act as partial agonists at the TxA2 receptor and may blunt the efficacy of treatment. Furthermore, these endoperoxides may be used as substrate for production of vasodilator prostaglandins causing beneficial effects on hemodynamics which are independent of thromboxane inhibition. To more specifically examine the role of TxA2 in CyA toxicity, we investigated the effects of the thromboxane receptor antagonist GR32191 on renal hemodynamics in a rat model of CyA nephrotoxicity. In this model, administration of CyA resulted in a significant decrease in glomerular filtration rate (GFR) (2.85 +/- 0.26 [CyA] vs 6.82 +/- 0.96 ml/min/kg [vehicle]; p less than 0.0005) and renal blood flow (RBF) (21.65 +/- 2.31 [CyA] vs 31.87 +/- 3.60 ml/min/kg [vehicle]; p less than 0.025). Renal vascular resistance (RVR) was significantly higher in rats given CyA compared to animals treated with CyA vehicle (5.32 +/- 0.55 [CyA] vs. 3.54 +/- 0.24 mm Hg/min/ml/kg [vehicle]; p less than 0.05). These renal hemodynamic alterations were associated with a significant increase in urinary excretion of unmetabolized, "native" thromboxane B2 (TxB2) (103 +/- 18 [CyA] vs 60 +/- 16 pg/hour [vehicle]; p less than 0.05). Only minimal histomorphologic changes were apparent by light microscopic examination of kidneys from both CyA and vehicle treated animals. However, with immunoperoxidase staining, a significantly greater number of cells expressing the rat common leukocyte antigen was found in the renal interstitium of rats given CyA. There was no detectable increase in monocytes/macrophages in the kidneys of CyA toxic animals. In rats treated with CyA, intraarterial infusion of GR32191 at maximally tolerated doses significantly increased GFR and RBF, and decreased RVR. Although both RBF and RVR were restored to levels not different from controls, GFR remained significantly reduced following administration of GR32191. These data suggest that the potent vasoconstrictor TxA2 plays an important role in mediating renal dysfunction in CyA nephrotoxicity. However, other factors may be important in producing nephrotoxicity associated with CyA.

Authors
Spurney, RF; Mayros, SD; Collins, D; Ruiz, P; Klotman, PE; Coffman, T
MLA Citation
Spurney, RF, Mayros, SD, Collins, D, Ruiz, P, Klotman, PE, and Coffman, T. "Thromboxane receptor blockade improves cyclosporine nephrotoxicity in rats." Prostaglandins 39.2 (February 1990): 135-146.
PMID
2138344
Source
pubmed
Published In
Prostaglandins
Volume
39
Issue
2
Publish Date
1990
Start Page
135
End Page
146

Immature omental teratoma.

Immature omental teratomas are extremely rare neoplasms. To our knowledge, only two cases have been reported in the literature. In some anatomic locations, the malignant potential of immature teratomas correlates with the presence and quantity of neuroectoderm within the tumor mass. We describe the first immature omental teratoma with prominent neuroectodermal differentiation.

Authors
Spurney, RF; McCormack, KM
MLA Citation
Spurney, RF, and McCormack, KM. "Immature omental teratoma." Arch Pathol Lab Med 111.8 (August 1987): 762-764.
PMID
3632294
Source
pubmed
Published In
Archives of Pathology and Laboratory Medicine
Volume
111
Issue
8
Publish Date
1987
Start Page
762
End Page
764
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