Dopamine D4 receptor inhibit sodium chloride cotransporter in renal distal convoluted tubules
Authors: Zhang Mingzhuo , Liu Mingda , Wang Weiwan , Wang Xiaoyan 
Published: May 2023

Background: Dopamine D4 receptor (D4R) is expressed in the distal convoluted tubule (DCT) and mice lacking the D4R have kidney-related hypertension. Hypothesis: The D4R negatively regulates the sodium chloride cotransporter (NCC) in vitro and in vivo. Methods: NCC activities were assessed in vivo on urinary sodium excretion and systolic blood pressures (SBP) responded to NCC inhibitor hydrochlorothiazide (HCTZ, 30mg/kg/day, IP) in Drd4 null ( Drd4-/-) mice and wild-type ( Drd4+/+) littermates (female 20 weeks old). Total and phosphorylation/ubiquitination of renal NCC were determined. The protein abundance of NCC was measured in the cultured immortalized mouse renal distal-convoluted-tubule cells (mDCT) treated by the D4R agonist PD168077 (PD), D4R antagonist L-745,870(L) or Drd4 siRNA respectively. D4R-related interaction, internalization, ubiquitination, degradation and sodium transport of NCC were also determined in vitro. T-test was used for 2 group comparisons while one-way ANOVA, Holm-Sidak test for multi-group comparisons (P<0.05 was considered significant). Results: HCTZ produced a stronger natriuretic effect in Drd4-/- mice than in WT littermates in initial 6-hrs (16.3±2.2 vs 13.1±3.1μmol/mg of Cr, n=8). SBP were higher in Drd4-/- mice than Drd4+/+ littermates at baseline (117.8±5.2 vs 104.1±6.3 mmHg, n=8, tail-cuff) but normalized by HCTZ on days 4-7. The renal protein abundances of NCC by immunoblotting (230.2±51.12, % of control, n=4-5) and immunofluorescence were greater but ubiquitinated-NCC levels (65.8±12.8, %, n=4) were lower in KO than in WT mice and mRNA levels of NCC (88.5±23.8, %) and phosphorylation-NCC (87.7±10.6, %) were not altered. In the mDCT cells, inhibition of D4R by its siRNA (1.5nM, 48 hrs) increased the protein expression of NCC (151.9±33.6, %, n=4). PD (24 hrs) decreased NCC protein abundance concentration-dependently blocked by L. Total NCC (72.1±18.1,%) was decreased but ubiquitinated-NCC was increased remarkably (265.8±114.1, %) by PD. There was a co-immunoprecipitation of D4R with NCC that was increased by PD. Colocalization of them was seen with confocal microscopy and confirmed by Fret. Within 1 hr, PD internalized NCC by membrane biotinlaytion-method and increased the colocalization of NCC with lysosomal-associated membrane protein 1. PD-induced NCC decrease was reversed by lysosomal inhibitor chloroquine. 1 hr PD also decreased total NCC and NCC-dependent-intracellular sodium transport (74±1, % of Veh, n=7), which was blocked by L. PD decreased NCC under CHX-chase time-dependently (33.7±21.3, %, 8 hrs, n=5) suggesting a reduced NCC half-life. Conclusion: D4R inhibits NaCl transport in renal DCT by reducing NCC activity and protein abundance and shortening NCC half-life with promoting its internalization, ubiquitination and consequent lysosomal degradation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.