Abstract 518: Loss of Caveolin-1 in Mouse, Rat and Human Proximal Tubule Cells Increases GRK4 Activity and Decreases D1-Like Receptor Cell Surface Levels

Abstract

Normal dopaminergic signaling in the renal proximal tubule under conditions of sodium loading leads to natriuresis without the need to increase blood pressure. Low caveolin-1 (CAV1) expression in the kidney is associated with dopaminergic signaling defects and salt sensitive hypertension in both mice and rats. We and others have shown that SNPs in human CAV1 are associated with hypertension. We hypothesize that low CAV1 expression causes increased GRK4 kinase activity due to lack of physical interaction and inhibition of GRK4; thus overactive GRK4 would inactivate the dopamine-1 receptor (D 1 R). We have previously shown that CAV1 reduction, knockdown or knockout in mouse, rat and human renal proximal tubule cells (RPTC) leads to decreased dopamine D 1 -like receptor binding at the proximal tubule cell surface and that re-expression of CAV1 alpha isoform is necessary to rescue this dopaminergic defect. Here we extend those studies by showing that defects in D 1 R cell surface expression due to low CAV1 expression can also be rescued by inhibiting GRK4 in human RPTC using GRK4 siRNA or by introducing heparin (1 μmol/L, 24 hrs) in mouse and rat RPTC. GRK4 siRNA lowered GRK4 expression by greater than 80% in all four human RPTC cell lines and partially restored fluorescent D 1 -like receptor antagonist bodipy630 SKF83566 cell surface binding (scrambled siRNA 66,231±7,286 RFU, GRK4 siRNA 96,206±11,035 RFU, P<0.05, N=28). In RPTC from CAV1 knockout mice, electroporation of heparin restored bodipy630 SKF83566 cell surface binding (vehicle 21,346±3,219 RFU, heparin 58,380±8,032 RFU, P<0.05, N=7). In rat SHR RPTC, CAV1 expression is very low, and the stable transfection of CAV1 partially restored their D 1 -like receptor surface expression. Electroporation of heparin in these CAV1 transfected cells (SHR CAV1) restored bodipy630 SKF83566 cell surface binding back to levels found in WKY RPTC (SHR CAV1 35,241±4,280 RFU, SHR CAV1 heparin 54,218±6,564 RFU, WKY 56,821± 3,546 RFU, P<0.05, N=7). In summary, we show that dopamine D 1 -like receptor defects caused by loss of CAV1 are likely due to increased GRK4 activity and can be restored by either increasing CAV1 expression or decreasing GRK4 expression.