G Protein‐Coupled Receptor Kinase 4 (GRK4) Variant 65L‐Mediated Impairment of Dopamine D1 Receptor Function causes Hypertension

Abstract

Salt sensitivity is associated with impaired dopamine receptor function, which is due, in part, to its desensitization by constitutively active GRK4γ variants. While GRK4 is essential for the normal desensitization and resensitization of dopamine receptors, certain GRK4 polymorphisms desensitize and internalize the dopamine D1 (D1R), thus impairing its natriuretic function. Activating variants of the human GRK4 (65R>L, 142A>V, and 486A>V) are involved in the desensitization of the D1R and are associated with hypertension in several ethnic groups. Furthermore, transgenic mice expressing the GRK4 65L are hypertensive when fed a high salt diet. We have reported that global transgenic mice expressing hGRK4 65L have salt-sensitive hypertension. To avoid the effect of protein overexpression in these transgenic mice, we generated mice globally expressing hGRK4 65L by crispr-cas9-mediated genome editing in C57Bl/6 mice (hGRK4 65L GE). Systolic blood pressure (SBP) in these mice under pentobarbital anesthesia was higher (126±2 vs 103±1 mmHg, n=3/group, P<0.05) than in their littermates not expressing the human gene (GE WT). Renal D1R expression was slightly but not significantly lower (100 ± 2 vs 85 ± 4%, n=3/group) but D1R phosphorylation was higher in hGRK4 65L GE than GE WT (112±2 vs 100±1%, n=3/group, P<0.05). Renal ENaC and NHE3 expressions were similar in both genotypes. To determine the role of the kidney in the salt sensitivity of these mice, we generated mice with kidney-specific (KS) expression of the GRK4 65L (KS hGRK4 65L) by the bilateral ureteral infusion of adeno-associated virus (AAV) vectors carrying hGRK4 65L in GRK4 knockout mice. Mice infused with hGRK4 wild type served as controls (KS hGRK4 WT). SBP (under anesthesia) before AAV was similar in both groups. SBP post AAV increased in KS hGRK4 65L (93±1 vs 117±4 mm Hg, P<0.05, n=4) but not in KS hGRK4 WT (96±2 vs 105±6, n=5). Renal cortical expression of D1R (63±1 vs 100±4 % n=5, P<0.01) was lower but D1R phosphorylation was higher (170 ± 10 vs 100 ±15%; n=5, P<0.02) in KS hGRK4 65L than KS hGRK4 WT. In addition, the renal expression of ENaC (1.4±0.14 vs 1.0±0.11, P<0.05) was increased KS hGRK4 65L but NHE3 was similar in both groups. We also studied human renal proximal tubule cells (hRPTCs) endogenously carrying GRK4 wild type or GRK4 65L. In hRPTCs with GRK4 65L, relative to GRK4 WT, D1R expression (599±80 vs 100±8 % n=6/group; P<0.05), as well as D1R phosphorylation (204 ± 22 vs 100±24%, n=6/group; P<0.05), were increased. Our results show across mouse models and hRPTCs that the presence of the GRK4 65L results in increased D1R phosphorylation, which impairs D1R function for normal renal sodium handling and increases blood pressure in GRK4 65L mice. These results show the important role of renal D1R in the regulation of blood pressure.