NIH Public Access Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
NIH-PA Author Manuscript
Published in final edited form as: Hypertension. 2014 March ; 63(3): e74–e80. doi:10.1161/HYPERTENSIONAHA.113.02569.
CHBPR: SINGLE NUCLEOTIDE POLYMORPHISMS OF THE DOPAMINE D2 RECEPTOR INCREASE INFLAMMATION AND FIBROSIS IN HUMAN RENAL PROXIMAL TUBULE CELLS Xiaoliang Jiang1,4, Prasad Konkalmatt1, Yu Yang1, John Gildea2, John E. Jones1, Santiago Cuevas1, Robin A. Felder2, Pedro A. Jose1, and Ines Armando1 1Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 2Department
of Pathology, The University of Virginia School of Medicine, Charlottesville, VA
22908 3Department
of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201
NIH-PA Author Manuscript
4Institute
of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) & Comparative Medicine Centre, Peking Union Medical Collage (PUMC). 5 Pan Jia Yuan Nan Li, Chaoyang District, Beijing 100021, P.R. China
Abstract
NIH-PA Author Manuscript
The dopamine D2 receptor (D2R) negatively regulates inflammation in mouse renal proximal tubule cells (RPTCs) and lack or downregulation of the receptor in mice increases the vulnerability to renal inflammation independent of blood pressure. Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277, and 1800497) in the human (h) DRD2 gene are associated with decreased D2R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs expressing these SNPs have increased expression of inflammatory and injury markers. We studied immortalized hRPTCs carrying D2R SNPs and compared them with cells carrying no D2R SNPs. RPTCs with D2R SNPs had decreased D2R expression and function. The expressions of the pro-inflammatory TNFα and the pro-fibrotic TGFβ1 and its signaling targets Smad3 and Snail1 were increased in hRPTC with D2R SNPs. These cells also showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins, assessed by increased vimentin, fibronectin -1, and Col 1a. To test the specificity of these D2R SNP effects, hRPTC with D2R SNPs were transfected with a plasmid encoding wild-type DRD2. D2R expression was increased and those of TGFβ1, Smad3, Snail1, vimentin, fibronecti-1 and Col 1a were decreased in hRPTC with D2R SNPs transfected with wildtype DRD2 compared to hRPTC-D2R SNP transfected with empty vector. These data support the hypothesis that D2R function has protective effects in human RPTCs and suggest that carriers of these SNPs may be prone to chronic renal disease and high blood pressure.
Keywords dopamine 2 receptor; D2R; kidney; renal injury
Corresponding author: Ines Armando, PhD, University of Maryland School of Medicine, Department of Medicine, Division of Nephrology, 20 Penn Street- HSFII Suite S003C, Baltimore, MD 21201, Phone: 410-706-6013, Fax: 410-706-6048, [email protected]. Conflict of Interest None
Jiang et al.
Page 2
INTRODUCTION NIH-PA Author Manuscript
Dopamine synthesized in the kidney is necessary for the maintenance of normal renal function and blood pressure.1 Dopamine and dopaminergic drugs have also been shown to regulate the immune response and the inflammatory reaction.2 Mice with intrarenal dopamine deficiency have increased oxidative stress and infiltration of inflammatory cells3 and decreased renal dopamine production aggravates angiotensin II-mediated renal injury4. The anti-inflammatory effects of dopamine are mediated, at least in part, by the dopamine D2 receptor (D2R).1, 5. Previous work from our laboratory has indicated that the D2R in the kidney has a direct and significant role in regulating the mechanisms involved in the development of renal inflammation and injury, as well as in blood pressure control.6,7. Mice with lack or reduced level of D2R expression and function in the kidney have increased renal expression of proinflammatory and decreased expression of anti-inflammatory cytokines/chemokines, as well as histological and functional evidence of renal inflammation and injury, suggesting that the D2R has protective effects in the kidney by limiting the inflammatory reaction and that impaired function of the D2R results in renal inflammation and organ damage.8
NIH-PA Author Manuscript
Deficient renal D2R function may be of clinical relevance. Polymorphisms of the D2R gene are commonly observed in humans and some of them have been associated with elevated blood pressure and even hypertension.9,10 Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277, and 1800497) in the non-coding region of the human DRD2 gene are associated with decreased D2R expression and function.11–14 We hypothesized that the presence of the DRD2 variants in human renal proximal tubule cells (RPTCs) is associated with decreased D2R protective effects and increased expression of inflammatory and injury markers. To test this hypothesis we studied (RPTCs) from subjects with and without D2R SNPs. rs 6276, 6277, and 1800497.
MATERIAL AND METHODS Cell culture RPTCs were isolated from human kidney specimens from patients who had unilateral nephrectomy due to renal carcinoma or trauma. Only the visually and histologically normal pole, distal from the affected part of the kidney, was used to isolate RPTCs. Cell were immortalized, as previously described.15. A University of Virginia institutional review board–approved protocol was used according to the Declaration of Helsinki.
NIH-PA Author Manuscript
The phenotypic characteristics of the subjects from whom the cells lines were derived are shown in Supplemental Table S1. The RPTCs were genotyped for the presence of the rs6276, rs6277 and rs1800497 variants which are in the non-coding region. Ten cell lines were studied, 5 of them from subjects not bearing SNPs, 3 from subjects being heterozygous for both rs6276 and rs6277 and 2 from subjects being heterozygous for both rs6276 and rs1800497. The cells were cultured in non-polarizing conditions as described in Supplemental Methods. Immunoblotting RPTC lysates were subjected to immunoblotting as described previously8. The primary antibodies used were rabbit polyclonal D2R (Millipore, Billerica, MA), rabbit polyclonal TNFα (Abcam, Cambridge, MA), mouse monoclonal fibronectin (R&D, Minneapolis, MN), mouse anti-human TGF-β (R&D) and polyclonal anti-GAPDH (Sigma). The densitometry values were corrected by the expression of GAPDH and are shown as percentage of the mean density of the wild- type group. Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 3
Cyclic AMP accumulation assay
NIH-PA Author Manuscript
RPTCs were grown to confluence in 6-well plates and pretreated with 1 μM phorbol 12myristate 13-acetate (PMA) for 5 min in the presence of the phosphodiesterase inhibitor IBMX; (1 μM) before treatment with the D2R/D3R agonist quinpirole (1 nM-1 μM/20min; Sigma). Cell lysates were prepared to determine the protein concentration using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) and cAMP concentration using the Cyclic AMP chemiluminescent immunoassay kit (Arbor Assays, Ann Arbor, Michigan), following the manufacturer’s procedures. RNA Extraction and cDNA Preparation Total RNA was purified using the RNeasy RNA Extraction Mini kit (Qiagen, Valencia, CA). RNA samples were converted into first strand cDNA using an RT2 First Strand kit, following the manufacturer’s protocol (SABiosciences-Qiagen). Quantitative Real-time PCR
NIH-PA Author Manuscript
Quantitative gene expression was analyzed by real-time PCR (ABI Prism 7900 HT, Applied Biosystems, Foster City, CA).8 The assay used gene specific primers (SABiosciencesQiagen) and SYBR Green real-time PCR detection method, and was performed as described in the manufacturer’s manual. The primers used are described in Supplemental Table S2. Data were analyzed using the ΔΔ Ct method.16 Immunofluorescence and confocal microscopy RPTCs, grown on poly-D-lysine-coated coverslips to 50% confluence, were immunostained using a mouse anti-TGF-β antibody (R&D), followed by Alexa Fluor 488 goat anti-mouse IgG antibody (Molecular Probes, Grand Island, NY). FN-1 was visualized using a mouse monoclonal antibody (Millipore) followed by Alexa Fluor 557 goat anti-mouse IgG antibody (Molecular Probes). Snail was visualized with an anti-human Snail antibody conjugated with a NL557 fluorochrome (R&D Systems). Vimentin was visualized with an antihuman Vimentin antibody conjugated with a NL493 fluorochrome (R&D Systems). DAPI was used to visualize the nuclei. Transient transfection with a DRD2 plasmid
NIH-PA Author Manuscript
RPTCs carrying SNPs were transfected with an empty vector or plasmid harboring wildtype human DRD2 under the control of CMV promoter (RC202476, Origene Technologies, Inc.), as described in Supplemental Methods. Three days following transfection, the RPTCs were harvested for protein extraction and immunoblotting, total RNA preparation and RTqPCR or processed for immune-fluorescence staining. Treatment with TGFβ RPTCs were grown on poly-D-lysine-coated coverslips to 50% confluence. After TGFβ(10ng/ml; R&D) treatment for 30 hours, cells were immunostained for Snail 1, FN1 and vimentin as described above and subjected to confocal microscopy. Statistical analysis The data are expressed as means±SEM. Comparisons between 2 groups used the Student’s t test. One-way ANOVA followed by Newman–Keuls was used to assess significant differences in more than two groups. PT) in exon7, in both in vitro and in vivo studies.11–13 These polymorphisms are commonly observed with allele frequencies of 0.401 for rs 1800497, 0.422 for rs 6276 and 0.229 for rs 6277 across several populations.28 These three SNPs occur within and adjacent to the DRD2 gene. Due to their close proximity, the SNPs are likely in linkage disequilibrium. This, coupled with the small number of human immortalized RPTCs available, resulted in our being unable to identify cell lines that were homozygous for only one DRD2 variant, limitations that have to be acknowledged. Other limitation of this study is the use of immortalized cell lines rather than primary cultures however this limitation is minimized by the fact that transformed RPTCs retain many of the functional and morphological characteristics of RPTCs in primary culture.15
PERSPECTIVES The prevalence of chronic kidney disease is rising sharply worldwide and affects 13.1% of the population in the US.29 Patients with chronic kidney disease represent a population not only at risk of progression to end organ failure but are also at higher risk for cardiovascular disease.30
NIH-PA Author Manuscript
Hypertension is one of the major causes of renal injury and together with diabetes account for up to 75% of the end-stage renal disease in the US.31 Inflammation and oxidative stress are major mediators in the development and progression of renal disease Low-grade inflammation is associated with cardiovascular disease. Infiltration of inflammatory cells32 and increased expression of pro-inflammatory factors are crucial in the development of renal injury, as well as in the induction and maintenance of hypertension.33,34 There is increasing evidence that genetic factors contribute to the susceptibility to renal disease associated with essential hypertension and it has been suggested that hypertension may cause progressive kidney disease only in genetically susceptible individuals.35,36 The genetic predisposition to chronic kidney disease is polygenic but so far only a few genes have been shown to be contributory.37–39 Renal susceptibility genes may determine the occurrence and severity of hypertension-induced progressive renal damage. Much of the research in this field has been directed to determining the detrimental factors involved in disease progression. However, the role of factors that prevent inflammation and slow the progression of renal disease has received much less attention. Our results show that individuals carrying D2R polymorphisms that result in decreased D2R expression and function could be more vulnerable to renal injury when challenged with an insult such as, elevated blood pressure. Genetic testing could identify the individuals at risk and pharmacological treatment tailored to ameliorate the insult which should result in decreased prevalence of renal injury and chronic kidney disease.
Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 7
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
NIH-PA Author Manuscript
Acknowledgments Source of funding The work was funded by grants from the US National Institutes of Health, P01HL074940, P01HL068686, R01HL092196, R37HL023081and R01DK039308.
References
NIH-PA Author Manuscript NIH-PA Author Manuscript
1. Armando I, Villar VA, Jose PA. Dopamine and renal function and blood pressure regulation. Compr Physiol. 2011; 1:1075–117. [PubMed: 23733636] 2. Bendele AM, Spaethe SM, Benslay DN, Bryant HU. Anti-inflammatory activity of pergolide, a dopamine receptor agonist. J Pharmacol Exp Ther. 1991; 259:169–175. [PubMed: 1681083] 3. Zhang MZ, Yao B, Wang S, Fan X, Wu G, Yang H, Yin H, Yang S, Harris RC. Intrarenal dopamine deficiency leads to hypertension and decreased longevity in mice. J Clin Invest. 2011; 121:2845– 2854. [PubMed: 21701066] 4. Yang S, Yao B, Zhou Y, Yin H, Zhang MZ, Harris RC. Intrarenal dopamine modulates progressive angiotensin II-mediated renal injury. Am J Physiol Renal Physiol. 2012; 302:F742–F749. [PubMed: 22169008] 5. McMurray RW. Bromocriptine in rheumatic and autoimmune diseases. Semin Arthritis Rheum. 2001; 31:21–32. [PubMed: 11503136] 6. Li XX, Bek M, Asico LD, Yang Z, Grandy DK, Goldstein DS, Rubinstein M, Eisner GM, Jose PA. Adrenergic and endothelin B receptor-dependent hypertension in dopamine receptor type-2 knockout mice. Hypertension. 2001; 38:303–308. [PubMed: 11566895] 7. Armando I, Wang X, Villar VA, Jones JE, Asico LD, Escano C, Jose PA. Reactive oxygen species dependent hypertension in dopamine D2 receptor-deficient mice. Hypertension. 2007; 49:672–678. [PubMed: 17190875] 8. Zhang Y, Cuevas S, Asico LD, Escano C, Yang Y, Pascua AM, Wang X, Jones JE, Grandy D, Eisner G, Jose PA, Armando I. Deficient dopamine D2 receptor function causes renal inflammation independently of high blood pressure. PLoS One. 2012; 7:e38745. [PubMed: 22719934] 9. Fang YJ, Thomas GN, Xu ZL, Fang JQ, Critchley JA, Tomlinson B. An affected pedigree member analysis of linkage between the dopamine D2 receptor gene TaqI polymorphism and obesity and hypertension. Int J Cardiol. 2005; 102:111–116. [PubMed: 15939106] 10. Thomas GN, Tomlinson B, Critchley JA. Modulation of blood pressure and obesity with the dopamine D2 receptor gene TaqI polymorphism. Hypertension. 2000; 36:177–182. [PubMed: 10948074] 11. Duan J, Wainwright MS, Comeron JM, Saitou N, Sanders AR, Gelernter J, Gejman P. Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Hum Mol Genet. 2003; 12:205–216. [PubMed: 12554675] 12. Noble EP, Blum K, Ritchie T, Montgomery A, Sheridan PJ. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism. Arch Gen Psychiatry. 1991; 48:648–654. [PubMed: 2069496] 13. Thompson J, Thomas N, Singleton A, Piggott M, Lloyd S, Perry EK, Ferrier IN. Court JAl. D2 dopamine receptor gene (DRD2) Taq1A polymorphism: reduced dopamine D2 receptor binding in the human striatum associated with the A1 allele. Pharmacogenetics. 1997; 7:479–484. [PubMed: 9429233] 14. Jönsson EG, Nöthen MM, Grünhage F, Farde L, Nakashima Y, Propping P, Sedvall GC. Polymorphisms in the dopamine D2 receptor gene and their relationships to striatal dopamine receptor density of healthy volunteers. Mol Psychiatry. 1999; 4:290–296. [PubMed: 10395223]
Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 8
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
15. Gildea JJ, McGrath HE, Van Sciver RE, Wang DB, Felder RA. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods. Methods Mol Biol. 2013; 945:329–345. [PubMed: 23097116] 16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 22 DD CT method. Methods. 2001; 25:402–408. [PubMed: 11846609] 17. Conley JM, Watts VJ. Differential effects of AGS3 expression on D(2L) dopamine receptormediated adenylyl cyclase signaling. Cell Mol Neurobiol. 2013; 33:551–558. [PubMed: 23504261] 18. Therrien FJ, Agharazii M, Lebel M, Larivière R. Neutralization of tumor necrosis factor-alpha reduces renal fibrosis and hypertension in rats with renal failure. Am J Nephrol. 2012; 36:151– 161. [PubMed: 22813949] 19. Deelman L, Sharma K. Mechanisms of kidney fibrosis and the role of antifibrotic therapies. Curr Opin Nephrol Hypertens. 2009; 18:85–90. [PubMed: 19077695] 20. Massage J, Gomis RR. The logic of TGFbeta signaling. FEBS Lett. 2006; 580:2811–2820. [PubMed: 16678165] 21. Böttinger EP, Bitzer M. TGF-beta signaling in renal disease. J Am Soc Nephrol. 2002; 13:2600– 2610. [PubMed: 12239251] 22. Peinado H, Quintanilla M, Cano A. Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Biol Chem. 2003; 278:21113–211123. [PubMed: 12665527] 23. Huang WY, Li ZG, Rus H, Wang X, Jose PA, Chen SY. RGC-32 mediates transforming growth factor-beta-induced epithelial-mesenchymal transition in human renal proximal tubular cells. J Biol Chem. 2009; 284:9426–9432. [PubMed: 19158077] 24. Guo X, Jose PA, Chen SY. Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells. Am J Physiol Cell Physiol. 2011; 300:C1415–1421. [PubMed: 21307346] 25. Li Y, Tan X, Dai C, Stolz DB, Wang D, Liu Y. Inhibition of integrin-linked kinase attenuates renal interstitial fibrosis. J Am Soc Nephrol. 2009; 20:1907–1918. [PubMed: 19541809] 26. Ibarra FR, Armando I, Nowicki S, Carranza A, Sarobe VDL, Arrizurieta EE, Barontini M. Dopamine is metabolized by different enzymes along the rat nephron. Pflugers Arch- Eur J Physiol. 2005; 450:185–191. [PubMed: 15864503] 27. Kozell LB, Neve KA. Constitutive activity of a chimeric D2/D1 dopamine receptor. Mol Pharmacol. 1997; 52:1137–1149. [PubMed: 9396784] 28. Rajeevan H, Soundararajan U, Kidd JR, Pakstis AJ, Kidd KK. ALFRED: an allele frequency resource for research and teaching. Nucleic Acids Res. 2012; 40:D1010–1015. [PubMed: 22039151] 29. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, Van Lente F, Levey AS. Prevalence of chronic kidney disease in the United States. JAMA. 2007; 298:2038–2047. [PubMed: 17986697] 30. Schiffrin EL, Lipman ML, Mann JF. Chronic kidney disease: effects on the cardiovascular system. Circulation. 2007; 116:85–97. [PubMed: 17606856] 31. US Renal Data System: USRDS. US Census Data 2008. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; Bethesda, MD: 2008 Annual data report: atlas of chronic kidney disease and end-stage renal disease in the United States. www.usrds.org 32. Harrison DG, Guzik TJ, Lob HE, Madhur MS, Marvar PJ, Thabet S, Vinh A, Weyand C. Inflammation, immunity, and hypertension. Hypertension. 2011; 57:132–140. [PubMed: 21149826] 33. Massy ZA, Stenvinkel P, Drueke TB. The role of oxidative stress in chronic kidney disease. Semin Dial. 2009; 22:405–408. [PubMed: 19708991] 34. Vaziri ND, Rodríguez-Iturbe B. Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol. 2006; 2:582–593. [PubMed: 17003837] 35. McKnight AJ, Currie D, Maxwell AP. Unravelling the genetic basis of renal diseases; from single gene to multifactorial disorders. J Pathol. 2010; 220:198–216. [PubMed: 19882676]
Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 9
NIH-PA Author Manuscript
36. Garrett MR, Pezzolesi MG, Korstanje R. Integrating human and rodent data to identify the genetic factors involved in chronic kidney disease. J Am Soc Nephrol. 2010; 21:398–405. [PubMed: 20133484] 37. Ma RC, Tam CH, Wang Y, Luk AO, Hu C, Yang X, Lam V, Chan AW, Ho JS, Chow CC, Tong PC, Jia W, Ng MC, So WY, Chan JC. Genetic variants in the PRKCB1 gene were independently associated withdevelopment of ESRD in Chinese patients with type 2 diabetes. JAMA. 2010; 304:881–889. [PubMed: 20736472] 38. Xia Y, Entman ML, Wang Y. Critical Role of CXCL16 in Hypertensive Kidney Injury and Fibrosis. Hypertension. 2013 In Press. 39. Kottgen A, Hwang SJ, Rampersaud E, Coresh J, North KE, Pankow JS, Meigs JB, Florez JC, Parsa A, Levy D, Boerwinkle E, Shuldiner AR, Fox CS, Kao WH. TCF7L2 variants associate with CKD progression and renal function in population-based cohorts. J Am Soc Nephrol. 2008; 19:1989– 1999. [PubMed: 18650481]
NIH-PA Author Manuscript NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 10
Novelty and Significance
NIH-PA Author Manuscript
1) What is new? We show that renal proximal tubule cells from subjects carrying common polymorphisms in the DRD2 gene that result in decreased expression of the D2R have increased levels of pro-inflammatory factors, extracellular matrix components, and express mesenchymal markers. 2) What is relevant? Individuals carrying DRD2 polymorphisms may be more vulnerable to renal injury when challenged with an insult such as, elevated blood pressure. Genetic testing can be used to identify the individuals at risk and pharmacological treatment tailored to ameliorate the insult which should result in decreased prevalence of renal injury and chronic kidney disease
NIH-PA Author Manuscript NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 11
Summary
NIH-PA Author Manuscript
These results show that renal proximal tubule cells from human subjects carrying D2R polymorphisms that result in decreased D2R mRNA and protein express a proinflammatory and pro-fibrotic phenotype as well as markers of epithelial mesenchymal transition. Individuals carrying these D2R polymorphisms could be more vulnerable to renal injury.
NIH-PA Author Manuscript NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 12
NIH-PA Author Manuscript Figure 1. D2R expression and function in renal proximal tubular cells (RPTCs) from subjects carrying D2R wild-type (WT) or D2R SNPs
NIH-PA Author Manuscript
(A) Expression of D2R mRNA in RPTCs from subjects carrying D2R WT or D2R SNPs was quantified by qRT-PCR; GAPDH mRNA was used for normalization of the data, n = 5/3/2. *= P< 0.05 vs. WT, one way ANOVA plus Neuman Keuls test.. (B) RPTC homogenates were immunoblotted for D2R; GAPDH protein was used for normalization of the data, n = 5/3/2. *= P< 0.05, t-test, (C) The accumulation of cAMP in response to D2R simulation by quinpirole in the presence of PMA was measured. n=5/group. *=P< 0.05, one way ANOVA plus Neuman Keuls test.
NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 13
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2. Expression of pro-inflammatory factors in RPTCs from subjects carrying D2R wildtype (WT) or D2R SNPs
(A) mRNA expressions of TNFα, C-reactive protein (CRP), and the chemokines MCP3 and MIP1β, NFkB1A and the cytokine IL-10 mRNA in RPTCs from subjects carrying D2R WT or D2R SNPs were quantified by qRT-PCR; results and were corrected for expression of GAPDH mRNA, n=5/group. *=P
NIH-PA Author Manuscript
Published in final edited form as: Hypertension. 2014 March ; 63(3): e74–e80. doi:10.1161/HYPERTENSIONAHA.113.02569.
CHBPR: SINGLE NUCLEOTIDE POLYMORPHISMS OF THE DOPAMINE D2 RECEPTOR INCREASE INFLAMMATION AND FIBROSIS IN HUMAN RENAL PROXIMAL TUBULE CELLS Xiaoliang Jiang1,4, Prasad Konkalmatt1, Yu Yang1, John Gildea2, John E. Jones1, Santiago Cuevas1, Robin A. Felder2, Pedro A. Jose1, and Ines Armando1 1Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 2Department
of Pathology, The University of Virginia School of Medicine, Charlottesville, VA
22908 3Department
of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201
NIH-PA Author Manuscript
4Institute
of Laboratory Animal Science, Chinese Academy of Medical Sciences (CAMS) & Comparative Medicine Centre, Peking Union Medical Collage (PUMC). 5 Pan Jia Yuan Nan Li, Chaoyang District, Beijing 100021, P.R. China
Abstract
NIH-PA Author Manuscript
The dopamine D2 receptor (D2R) negatively regulates inflammation in mouse renal proximal tubule cells (RPTCs) and lack or downregulation of the receptor in mice increases the vulnerability to renal inflammation independent of blood pressure. Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277, and 1800497) in the human (h) DRD2 gene are associated with decreased D2R expression and function, as well as high blood pressure. We tested the hypothesis that human RPTCs expressing these SNPs have increased expression of inflammatory and injury markers. We studied immortalized hRPTCs carrying D2R SNPs and compared them with cells carrying no D2R SNPs. RPTCs with D2R SNPs had decreased D2R expression and function. The expressions of the pro-inflammatory TNFα and the pro-fibrotic TGFβ1 and its signaling targets Smad3 and Snail1 were increased in hRPTC with D2R SNPs. These cells also showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins, assessed by increased vimentin, fibronectin -1, and Col 1a. To test the specificity of these D2R SNP effects, hRPTC with D2R SNPs were transfected with a plasmid encoding wild-type DRD2. D2R expression was increased and those of TGFβ1, Smad3, Snail1, vimentin, fibronecti-1 and Col 1a were decreased in hRPTC with D2R SNPs transfected with wildtype DRD2 compared to hRPTC-D2R SNP transfected with empty vector. These data support the hypothesis that D2R function has protective effects in human RPTCs and suggest that carriers of these SNPs may be prone to chronic renal disease and high blood pressure.
Keywords dopamine 2 receptor; D2R; kidney; renal injury
Corresponding author: Ines Armando, PhD, University of Maryland School of Medicine, Department of Medicine, Division of Nephrology, 20 Penn Street- HSFII Suite S003C, Baltimore, MD 21201, Phone: 410-706-6013, Fax: 410-706-6048, [email protected]. Conflict of Interest None
Jiang et al.
Page 2
INTRODUCTION NIH-PA Author Manuscript
Dopamine synthesized in the kidney is necessary for the maintenance of normal renal function and blood pressure.1 Dopamine and dopaminergic drugs have also been shown to regulate the immune response and the inflammatory reaction.2 Mice with intrarenal dopamine deficiency have increased oxidative stress and infiltration of inflammatory cells3 and decreased renal dopamine production aggravates angiotensin II-mediated renal injury4. The anti-inflammatory effects of dopamine are mediated, at least in part, by the dopamine D2 receptor (D2R).1, 5. Previous work from our laboratory has indicated that the D2R in the kidney has a direct and significant role in regulating the mechanisms involved in the development of renal inflammation and injury, as well as in blood pressure control.6,7. Mice with lack or reduced level of D2R expression and function in the kidney have increased renal expression of proinflammatory and decreased expression of anti-inflammatory cytokines/chemokines, as well as histological and functional evidence of renal inflammation and injury, suggesting that the D2R has protective effects in the kidney by limiting the inflammatory reaction and that impaired function of the D2R results in renal inflammation and organ damage.8
NIH-PA Author Manuscript
Deficient renal D2R function may be of clinical relevance. Polymorphisms of the D2R gene are commonly observed in humans and some of them have been associated with elevated blood pressure and even hypertension.9,10 Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277, and 1800497) in the non-coding region of the human DRD2 gene are associated with decreased D2R expression and function.11–14 We hypothesized that the presence of the DRD2 variants in human renal proximal tubule cells (RPTCs) is associated with decreased D2R protective effects and increased expression of inflammatory and injury markers. To test this hypothesis we studied (RPTCs) from subjects with and without D2R SNPs. rs 6276, 6277, and 1800497.
MATERIAL AND METHODS Cell culture RPTCs were isolated from human kidney specimens from patients who had unilateral nephrectomy due to renal carcinoma or trauma. Only the visually and histologically normal pole, distal from the affected part of the kidney, was used to isolate RPTCs. Cell were immortalized, as previously described.15. A University of Virginia institutional review board–approved protocol was used according to the Declaration of Helsinki.
NIH-PA Author Manuscript
The phenotypic characteristics of the subjects from whom the cells lines were derived are shown in Supplemental Table S1. The RPTCs were genotyped for the presence of the rs6276, rs6277 and rs1800497 variants which are in the non-coding region. Ten cell lines were studied, 5 of them from subjects not bearing SNPs, 3 from subjects being heterozygous for both rs6276 and rs6277 and 2 from subjects being heterozygous for both rs6276 and rs1800497. The cells were cultured in non-polarizing conditions as described in Supplemental Methods. Immunoblotting RPTC lysates were subjected to immunoblotting as described previously8. The primary antibodies used were rabbit polyclonal D2R (Millipore, Billerica, MA), rabbit polyclonal TNFα (Abcam, Cambridge, MA), mouse monoclonal fibronectin (R&D, Minneapolis, MN), mouse anti-human TGF-β (R&D) and polyclonal anti-GAPDH (Sigma). The densitometry values were corrected by the expression of GAPDH and are shown as percentage of the mean density of the wild- type group. Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 3
Cyclic AMP accumulation assay
NIH-PA Author Manuscript
RPTCs were grown to confluence in 6-well plates and pretreated with 1 μM phorbol 12myristate 13-acetate (PMA) for 5 min in the presence of the phosphodiesterase inhibitor IBMX; (1 μM) before treatment with the D2R/D3R agonist quinpirole (1 nM-1 μM/20min; Sigma). Cell lysates were prepared to determine the protein concentration using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) and cAMP concentration using the Cyclic AMP chemiluminescent immunoassay kit (Arbor Assays, Ann Arbor, Michigan), following the manufacturer’s procedures. RNA Extraction and cDNA Preparation Total RNA was purified using the RNeasy RNA Extraction Mini kit (Qiagen, Valencia, CA). RNA samples were converted into first strand cDNA using an RT2 First Strand kit, following the manufacturer’s protocol (SABiosciences-Qiagen). Quantitative Real-time PCR
NIH-PA Author Manuscript
Quantitative gene expression was analyzed by real-time PCR (ABI Prism 7900 HT, Applied Biosystems, Foster City, CA).8 The assay used gene specific primers (SABiosciencesQiagen) and SYBR Green real-time PCR detection method, and was performed as described in the manufacturer’s manual. The primers used are described in Supplemental Table S2. Data were analyzed using the ΔΔ Ct method.16 Immunofluorescence and confocal microscopy RPTCs, grown on poly-D-lysine-coated coverslips to 50% confluence, were immunostained using a mouse anti-TGF-β antibody (R&D), followed by Alexa Fluor 488 goat anti-mouse IgG antibody (Molecular Probes, Grand Island, NY). FN-1 was visualized using a mouse monoclonal antibody (Millipore) followed by Alexa Fluor 557 goat anti-mouse IgG antibody (Molecular Probes). Snail was visualized with an anti-human Snail antibody conjugated with a NL557 fluorochrome (R&D Systems). Vimentin was visualized with an antihuman Vimentin antibody conjugated with a NL493 fluorochrome (R&D Systems). DAPI was used to visualize the nuclei. Transient transfection with a DRD2 plasmid
NIH-PA Author Manuscript
RPTCs carrying SNPs were transfected with an empty vector or plasmid harboring wildtype human DRD2 under the control of CMV promoter (RC202476, Origene Technologies, Inc.), as described in Supplemental Methods. Three days following transfection, the RPTCs were harvested for protein extraction and immunoblotting, total RNA preparation and RTqPCR or processed for immune-fluorescence staining. Treatment with TGFβ RPTCs were grown on poly-D-lysine-coated coverslips to 50% confluence. After TGFβ(10ng/ml; R&D) treatment for 30 hours, cells were immunostained for Snail 1, FN1 and vimentin as described above and subjected to confocal microscopy. Statistical analysis The data are expressed as means±SEM. Comparisons between 2 groups used the Student’s t test. One-way ANOVA followed by Newman–Keuls was used to assess significant differences in more than two groups. PT) in exon7, in both in vitro and in vivo studies.11–13 These polymorphisms are commonly observed with allele frequencies of 0.401 for rs 1800497, 0.422 for rs 6276 and 0.229 for rs 6277 across several populations.28 These three SNPs occur within and adjacent to the DRD2 gene. Due to their close proximity, the SNPs are likely in linkage disequilibrium. This, coupled with the small number of human immortalized RPTCs available, resulted in our being unable to identify cell lines that were homozygous for only one DRD2 variant, limitations that have to be acknowledged. Other limitation of this study is the use of immortalized cell lines rather than primary cultures however this limitation is minimized by the fact that transformed RPTCs retain many of the functional and morphological characteristics of RPTCs in primary culture.15
PERSPECTIVES The prevalence of chronic kidney disease is rising sharply worldwide and affects 13.1% of the population in the US.29 Patients with chronic kidney disease represent a population not only at risk of progression to end organ failure but are also at higher risk for cardiovascular disease.30
NIH-PA Author Manuscript
Hypertension is one of the major causes of renal injury and together with diabetes account for up to 75% of the end-stage renal disease in the US.31 Inflammation and oxidative stress are major mediators in the development and progression of renal disease Low-grade inflammation is associated with cardiovascular disease. Infiltration of inflammatory cells32 and increased expression of pro-inflammatory factors are crucial in the development of renal injury, as well as in the induction and maintenance of hypertension.33,34 There is increasing evidence that genetic factors contribute to the susceptibility to renal disease associated with essential hypertension and it has been suggested that hypertension may cause progressive kidney disease only in genetically susceptible individuals.35,36 The genetic predisposition to chronic kidney disease is polygenic but so far only a few genes have been shown to be contributory.37–39 Renal susceptibility genes may determine the occurrence and severity of hypertension-induced progressive renal damage. Much of the research in this field has been directed to determining the detrimental factors involved in disease progression. However, the role of factors that prevent inflammation and slow the progression of renal disease has received much less attention. Our results show that individuals carrying D2R polymorphisms that result in decreased D2R expression and function could be more vulnerable to renal injury when challenged with an insult such as, elevated blood pressure. Genetic testing could identify the individuals at risk and pharmacological treatment tailored to ameliorate the insult which should result in decreased prevalence of renal injury and chronic kidney disease.
Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 7
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
NIH-PA Author Manuscript
Acknowledgments Source of funding The work was funded by grants from the US National Institutes of Health, P01HL074940, P01HL068686, R01HL092196, R37HL023081and R01DK039308.
References
NIH-PA Author Manuscript NIH-PA Author Manuscript
1. Armando I, Villar VA, Jose PA. Dopamine and renal function and blood pressure regulation. Compr Physiol. 2011; 1:1075–117. [PubMed: 23733636] 2. Bendele AM, Spaethe SM, Benslay DN, Bryant HU. Anti-inflammatory activity of pergolide, a dopamine receptor agonist. J Pharmacol Exp Ther. 1991; 259:169–175. [PubMed: 1681083] 3. Zhang MZ, Yao B, Wang S, Fan X, Wu G, Yang H, Yin H, Yang S, Harris RC. Intrarenal dopamine deficiency leads to hypertension and decreased longevity in mice. J Clin Invest. 2011; 121:2845– 2854. [PubMed: 21701066] 4. Yang S, Yao B, Zhou Y, Yin H, Zhang MZ, Harris RC. Intrarenal dopamine modulates progressive angiotensin II-mediated renal injury. Am J Physiol Renal Physiol. 2012; 302:F742–F749. [PubMed: 22169008] 5. McMurray RW. Bromocriptine in rheumatic and autoimmune diseases. Semin Arthritis Rheum. 2001; 31:21–32. [PubMed: 11503136] 6. Li XX, Bek M, Asico LD, Yang Z, Grandy DK, Goldstein DS, Rubinstein M, Eisner GM, Jose PA. Adrenergic and endothelin B receptor-dependent hypertension in dopamine receptor type-2 knockout mice. Hypertension. 2001; 38:303–308. [PubMed: 11566895] 7. Armando I, Wang X, Villar VA, Jones JE, Asico LD, Escano C, Jose PA. Reactive oxygen species dependent hypertension in dopamine D2 receptor-deficient mice. Hypertension. 2007; 49:672–678. [PubMed: 17190875] 8. Zhang Y, Cuevas S, Asico LD, Escano C, Yang Y, Pascua AM, Wang X, Jones JE, Grandy D, Eisner G, Jose PA, Armando I. Deficient dopamine D2 receptor function causes renal inflammation independently of high blood pressure. PLoS One. 2012; 7:e38745. [PubMed: 22719934] 9. Fang YJ, Thomas GN, Xu ZL, Fang JQ, Critchley JA, Tomlinson B. An affected pedigree member analysis of linkage between the dopamine D2 receptor gene TaqI polymorphism and obesity and hypertension. Int J Cardiol. 2005; 102:111–116. [PubMed: 15939106] 10. Thomas GN, Tomlinson B, Critchley JA. Modulation of blood pressure and obesity with the dopamine D2 receptor gene TaqI polymorphism. Hypertension. 2000; 36:177–182. [PubMed: 10948074] 11. Duan J, Wainwright MS, Comeron JM, Saitou N, Sanders AR, Gelernter J, Gejman P. Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Hum Mol Genet. 2003; 12:205–216. [PubMed: 12554675] 12. Noble EP, Blum K, Ritchie T, Montgomery A, Sheridan PJ. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism. Arch Gen Psychiatry. 1991; 48:648–654. [PubMed: 2069496] 13. Thompson J, Thomas N, Singleton A, Piggott M, Lloyd S, Perry EK, Ferrier IN. Court JAl. D2 dopamine receptor gene (DRD2) Taq1A polymorphism: reduced dopamine D2 receptor binding in the human striatum associated with the A1 allele. Pharmacogenetics. 1997; 7:479–484. [PubMed: 9429233] 14. Jönsson EG, Nöthen MM, Grünhage F, Farde L, Nakashima Y, Propping P, Sedvall GC. Polymorphisms in the dopamine D2 receptor gene and their relationships to striatal dopamine receptor density of healthy volunteers. Mol Psychiatry. 1999; 4:290–296. [PubMed: 10395223]
Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 8
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
15. Gildea JJ, McGrath HE, Van Sciver RE, Wang DB, Felder RA. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods. Methods Mol Biol. 2013; 945:329–345. [PubMed: 23097116] 16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 22 DD CT method. Methods. 2001; 25:402–408. [PubMed: 11846609] 17. Conley JM, Watts VJ. Differential effects of AGS3 expression on D(2L) dopamine receptormediated adenylyl cyclase signaling. Cell Mol Neurobiol. 2013; 33:551–558. [PubMed: 23504261] 18. Therrien FJ, Agharazii M, Lebel M, Larivière R. Neutralization of tumor necrosis factor-alpha reduces renal fibrosis and hypertension in rats with renal failure. Am J Nephrol. 2012; 36:151– 161. [PubMed: 22813949] 19. Deelman L, Sharma K. Mechanisms of kidney fibrosis and the role of antifibrotic therapies. Curr Opin Nephrol Hypertens. 2009; 18:85–90. [PubMed: 19077695] 20. Massage J, Gomis RR. The logic of TGFbeta signaling. FEBS Lett. 2006; 580:2811–2820. [PubMed: 16678165] 21. Böttinger EP, Bitzer M. TGF-beta signaling in renal disease. J Am Soc Nephrol. 2002; 13:2600– 2610. [PubMed: 12239251] 22. Peinado H, Quintanilla M, Cano A. Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Biol Chem. 2003; 278:21113–211123. [PubMed: 12665527] 23. Huang WY, Li ZG, Rus H, Wang X, Jose PA, Chen SY. RGC-32 mediates transforming growth factor-beta-induced epithelial-mesenchymal transition in human renal proximal tubular cells. J Biol Chem. 2009; 284:9426–9432. [PubMed: 19158077] 24. Guo X, Jose PA, Chen SY. Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells. Am J Physiol Cell Physiol. 2011; 300:C1415–1421. [PubMed: 21307346] 25. Li Y, Tan X, Dai C, Stolz DB, Wang D, Liu Y. Inhibition of integrin-linked kinase attenuates renal interstitial fibrosis. J Am Soc Nephrol. 2009; 20:1907–1918. [PubMed: 19541809] 26. Ibarra FR, Armando I, Nowicki S, Carranza A, Sarobe VDL, Arrizurieta EE, Barontini M. Dopamine is metabolized by different enzymes along the rat nephron. Pflugers Arch- Eur J Physiol. 2005; 450:185–191. [PubMed: 15864503] 27. Kozell LB, Neve KA. Constitutive activity of a chimeric D2/D1 dopamine receptor. Mol Pharmacol. 1997; 52:1137–1149. [PubMed: 9396784] 28. Rajeevan H, Soundararajan U, Kidd JR, Pakstis AJ, Kidd KK. ALFRED: an allele frequency resource for research and teaching. Nucleic Acids Res. 2012; 40:D1010–1015. [PubMed: 22039151] 29. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, Van Lente F, Levey AS. Prevalence of chronic kidney disease in the United States. JAMA. 2007; 298:2038–2047. [PubMed: 17986697] 30. Schiffrin EL, Lipman ML, Mann JF. Chronic kidney disease: effects on the cardiovascular system. Circulation. 2007; 116:85–97. [PubMed: 17606856] 31. US Renal Data System: USRDS. US Census Data 2008. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases; Bethesda, MD: 2008 Annual data report: atlas of chronic kidney disease and end-stage renal disease in the United States. www.usrds.org 32. Harrison DG, Guzik TJ, Lob HE, Madhur MS, Marvar PJ, Thabet S, Vinh A, Weyand C. Inflammation, immunity, and hypertension. Hypertension. 2011; 57:132–140. [PubMed: 21149826] 33. Massy ZA, Stenvinkel P, Drueke TB. The role of oxidative stress in chronic kidney disease. Semin Dial. 2009; 22:405–408. [PubMed: 19708991] 34. Vaziri ND, Rodríguez-Iturbe B. Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol. 2006; 2:582–593. [PubMed: 17003837] 35. McKnight AJ, Currie D, Maxwell AP. Unravelling the genetic basis of renal diseases; from single gene to multifactorial disorders. J Pathol. 2010; 220:198–216. [PubMed: 19882676]
Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 9
NIH-PA Author Manuscript
36. Garrett MR, Pezzolesi MG, Korstanje R. Integrating human and rodent data to identify the genetic factors involved in chronic kidney disease. J Am Soc Nephrol. 2010; 21:398–405. [PubMed: 20133484] 37. Ma RC, Tam CH, Wang Y, Luk AO, Hu C, Yang X, Lam V, Chan AW, Ho JS, Chow CC, Tong PC, Jia W, Ng MC, So WY, Chan JC. Genetic variants in the PRKCB1 gene were independently associated withdevelopment of ESRD in Chinese patients with type 2 diabetes. JAMA. 2010; 304:881–889. [PubMed: 20736472] 38. Xia Y, Entman ML, Wang Y. Critical Role of CXCL16 in Hypertensive Kidney Injury and Fibrosis. Hypertension. 2013 In Press. 39. Kottgen A, Hwang SJ, Rampersaud E, Coresh J, North KE, Pankow JS, Meigs JB, Florez JC, Parsa A, Levy D, Boerwinkle E, Shuldiner AR, Fox CS, Kao WH. TCF7L2 variants associate with CKD progression and renal function in population-based cohorts. J Am Soc Nephrol. 2008; 19:1989– 1999. [PubMed: 18650481]
NIH-PA Author Manuscript NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 10
Novelty and Significance
NIH-PA Author Manuscript
1) What is new? We show that renal proximal tubule cells from subjects carrying common polymorphisms in the DRD2 gene that result in decreased expression of the D2R have increased levels of pro-inflammatory factors, extracellular matrix components, and express mesenchymal markers. 2) What is relevant? Individuals carrying DRD2 polymorphisms may be more vulnerable to renal injury when challenged with an insult such as, elevated blood pressure. Genetic testing can be used to identify the individuals at risk and pharmacological treatment tailored to ameliorate the insult which should result in decreased prevalence of renal injury and chronic kidney disease
NIH-PA Author Manuscript NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 11
Summary
NIH-PA Author Manuscript
These results show that renal proximal tubule cells from human subjects carrying D2R polymorphisms that result in decreased D2R mRNA and protein express a proinflammatory and pro-fibrotic phenotype as well as markers of epithelial mesenchymal transition. Individuals carrying these D2R polymorphisms could be more vulnerable to renal injury.
NIH-PA Author Manuscript NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 12
NIH-PA Author Manuscript Figure 1. D2R expression and function in renal proximal tubular cells (RPTCs) from subjects carrying D2R wild-type (WT) or D2R SNPs
NIH-PA Author Manuscript
(A) Expression of D2R mRNA in RPTCs from subjects carrying D2R WT or D2R SNPs was quantified by qRT-PCR; GAPDH mRNA was used for normalization of the data, n = 5/3/2. *= P< 0.05 vs. WT, one way ANOVA plus Neuman Keuls test.. (B) RPTC homogenates were immunoblotted for D2R; GAPDH protein was used for normalization of the data, n = 5/3/2. *= P< 0.05, t-test, (C) The accumulation of cAMP in response to D2R simulation by quinpirole in the presence of PMA was measured. n=5/group. *=P< 0.05, one way ANOVA plus Neuman Keuls test.
NIH-PA Author Manuscript Hypertension. Author manuscript; available in PMC 2015 March 01.
Jiang et al.
Page 13
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2. Expression of pro-inflammatory factors in RPTCs from subjects carrying D2R wildtype (WT) or D2R SNPs
(A) mRNA expressions of TNFα, C-reactive protein (CRP), and the chemokines MCP3 and MIP1β, NFkB1A and the cytokine IL-10 mRNA in RPTCs from subjects carrying D2R WT or D2R SNPs were quantified by qRT-PCR; results and were corrected for expression of GAPDH mRNA, n=5/group. *=P
