Abstract 88: Single Nucleotide Polymorphisms of the Dopamine D2 Receptor Increase Inflammation and Fibrosis in Human Renal Proximal Tubule Cells

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We have reported that lack or downregulation of dopamine D(2) receptor (D2R) function in mice increases the vulnerability to renal inflammation independent of blood pressure and that the D2R negatively regulates inflammation and prevents injury in mouse renal proximal tubule cells (RPTCs). Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277 and 1800497) in the human 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 markers and injury. We studied immortalized human RPTCs isolated from normal tissue of nephrectomies which were genotyped for the D2R SNPs; 10 cell lines were selected: wild type (WT) n=5; heterozygous for both rs6276 and 6277 n=3; heterozygous for both rs6276 and 1800497 n=2. All 5 cell lines expressing SNPs (hRPTC-SNP) had decreased D2R mRNA (0.52±0.03 vs. 1±0.02 (WT) fold; P<0.02 ) and protein (62±4 vs. 100±6%; P<0.04) and were pooled together for analysis. D2R function was also decreased as determined by the decreased ability of a D2R agonist to increase PMA-induced cAMP accumulation (hRPTC-SNP: 201±16; hRPTC-WT 270 ±5 %; n=4; P<0.05). The expression of the pro-inflammatory cytokines TNFα (2.5 ± 0.3 fold; P<0.04) and CD40L (2.3 ± 0.5 fold; P<0.05), as well as the inflammation marker CRP (1.5 ± 0.2 fold; P<0.04) was increased in hRPTC-SNP. The expression of the pro-fibrotic TGFβ1 (1.8 ± 0.4 fold; P<0.04) and its signaling targets PAI-1 (6.3± 0.5 fold; P<0.01), SMAD3 (2.8± 0.4 fold; P<0.02) and VEGFa (2.3± 0.6 fold; P<0.04) were also increased. Furthermore hRPTC-SNP showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins as shown by increased fibronectin -1 (3.1± 0.7 fold; P<0.03), col 1a (1.6± 0.2 fold; P<0.05), col 3a (2.2± 0.5 fold; P<0.05), MMP3 (5.6± 0.8 fold; P<0.04), and MMP9 (3.5± 0.5 fold; P<0.04). 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.

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