Increased AT₁ receptor expression mediates vasoconstriction leading to hypertension in Snx1^-/- mice

Angiotensin II type 1 receptor (AT₁R) is a vital therapeutic target for hypertension. Sorting nexin 1 (SNX1) participates in the sorting and trafficking of the renal dopamine D₅ receptor, while angiotensin and dopamine are counterregulatory factors in the regulation of blood pressure. The effect of SNX1 on AT₁R is not known. We hypothesized that SNX1, through arterial AT₁R sorting and trafficking, is involved in blood pressure regulation. CRISPR/Cas9 system-generated SNX1^-/- mice showed dramatic elevations in blood pressure compared to their wild-type littermates. The angiotensin II-mediated contractile reactivity of the mesenteric arteries and AT₁R expression in the aortas were also increased. Moreover, immunofluorescence and immunoprecipitation analyses revealed that SNX1 and AT₁R were colocalized and interacted in the aortas of wild-type mice. In vitro studies revealed that AT₁R protein levels and downstream calcium signaling were upregulated in A10 cells treated with SNX1 siRNA. This may have resulted from decreased AT₁R protein degradation since the AT₁R mRNA levels showed no changes. AT₁R protein was less degraded when SNX1 was downregulated, as reflected by a cycloheximide chase assay. Furthermore, proteasomal rather than lysosomal inhibition increased AT₁R protein content, and this effect was accompanied by decayed binding of ubiquitin and AT₁R after SNX1 knockdown. Confocal microscopy revealed that AT₁R colocalized with PSMD6, a proteasomal marker, and the colocalization was reduced after SNX1 knockdown. These findings suggest that SNX1 sorts AT₁R for proteasomal degradation and that SNX1 impairment increases arterial AT₁R expression, leading to increased vasoconstriction and blood pressure.