Abstract P180: Dopamine D2 Receptors Regulate Wnt Signaling and Apoptosis in Human Renal Proximal Tubule Cells

Previous work from our laboratory indicates that the dopamine D2 receptor (D2R) in the kidney has a direct role in regulating renal inflammation and injury and blood pressure. Some common single nucleotide polymorphisms (D2R SNPs; rs 6276, 6277, and 1800497) in the human DRD2 gene are associated with decreased D2R expression and function. Immortalized renal proximal tubule cells (RPTCs) from subjects carrying D2R SNPs (RPTC-D2R SNPs) express less D2Rs than RPTCs carrying no D2R SNPs (RPTC-D2R WT) (62±4 vs 100±6%; P<0.04) and a pro-inflammatory and pro-fibrotic phenotype with markers of epithelial mesenchymal transition. RPTC-D2R SNPs showed increased apoptosis compared with RPTC-D2R WT (11± 0.8 vs 2.3±0.4% TUNEL positive cells, P<0.01, n=5/group). We hypothesized that the D2R regulates renal cell survival through effects on Wnt signaling. We found that Wnt3 expression was increased in RPTC-D2R SNPs compared with RPTC-D2R WT (mRNA: 2.6±0.35 vs 1±0.11 fold; P<0.05; protein: 133±4 vs 100±5%; P<0.05). RPTC-D2R SNPs showed activated Wnt3/β-catenin signaling pathway demonstrated by decreased β-catenin phosphorylation (64±4 vs 100±8%; P<0.05) and increased expression of downstream pro-apoptotic factors Bax (136±4.6 vs 100±5%, P<0.05) and FasL (128±5.6 vs 100±6.5%, P<0.05). Silencing D2R in RPTC-D2R WT (siRNA; 0.30±0.02 vs 1±0.07 fold, P<0.05) increased Wnt 3 expression, decreased β-catenin phosphorylation, and increased expression of Bax and FasL. By contrast, treatment of RPTC-D2R WT with a D2R agonist (quinpirole,1μM, 24h) or transfection of RPTC-D2R SNPs with a DRD2 which restored D2R expression decreased Wnt3 expression, increased β-catenin phosphorylation, and decreased Bax and FasL expression. Moreover, Wnt3 silencing (siRNA) in RPTC-D2R SNPs increased β-catenin phosphorylation (132±5 vs100±9%, P<0.05), decreased Bax and FasL expression, and reduced the number of apoptotic cells (6±1.0 vs12± 0.9 % TUNEL positive cells, P<0.01). Our results indicate that D2R function is important in the regulation of the Wnt pathway and that the alterations in D2R function result in modifications in the pathway potentially leading to fibrosis, cell death, and hypertension. These results may have clinical relevance for subjects bearing D2R SNPs.