Abstract: TH-OR89
Dopamine D4 Receptor Regulates Sodium Chloride Cotransporter in Renal Distal Convoluted Tubules
Session Information
- Mechanisms of Hypertension and Cardiorenal Disease: From the Vasculature to the Gut
November 02, 2023 | Location: Room 108, Pennsylvania Convention Center
Abstract Time: 05:42 PM - 05:51 PM
Category: Hypertension and CVD
- 1601 Hypertension and CVD: Basic
Authors
- Zhang, Mingzhuo, Nanjing BenQ Medical Center, Nanjing, Jiangsu, China
- Liu, Mingda, Nanjing BenQ Medical Center, Nanjing, Jiangsu, China
- Wang, Weiwan, Nanjing BenQ Medical Center, Nanjing, Jiangsu, China
- Ren, Zhiyun, Nanjing BenQ Medical Center, Nanjing, Jiangsu, China
- Wang, Xiaoyan, Nanjing BenQ Medical Center, Nanjing, Jiangsu, China
Background
Dopamine D4 receptor (D4R) is expressed in the distal convoluted tubule (DCT) and the mice lacking D4R have kidney-related hypertension.
Methods
The regulation and mechanisms of D4R on NCC were determined in D4R null mice (20 weeks-old, mixed sex) and cultured mouse distal convoluted tubule (mDCT) cells.
Results
NCC activities, measured by response to NCC inhibitor hydrothiazide (30mg/kg/day, IP, 7d), were greater in Drd4-/- mice than Drd4+/+ littermates in initial 6-hrs (UNa: 513±121 vs 635±87μmol/mg of Cr, n=8) with SBP (118±5 vs 104±6 mmHg, n=8, tail-cuff) normalized on days 4-7. The renal NCC protein abundance by immunoblotting (230±51, % of control, n=4-5, same as below) and immunofluorescence was greater but ubiquitinated-NCC levels (66±13) were lower in KO than in WT mice. mRNA levels of NCC by Q-PCR and phosphorylation-NCC were not altered. NCC abundance (121±9) in KO mice remained higher than WT under the infusion of dopamine via osmotic mini-pump (1μg/kg/min, 1wk). In the mDCT cells, the immunoprecipitation of D4R with NCC was increased by PD168077. Colocalization and interaction of them was seen with confocal microscopy and confirmed by Fret.D4R-siRNA (1.5nM, 48 hrs) increased the protein expression of NCC (152±34, n=4); D4R agonist PD168077 (0-10μM, 24 hrs) decreased NCC protein abundance concentration-dependently; D4R antagonist L-745,870 (10μM, 24 hrs) had no effect but blocked the D4R agonist-mediated inhibition of NCC abundance. Total NCC (72±18) was decreased but ubiquitinated-NCC was increased remarkably (266±114) by PD168077 (10μM). PD168077 internalized NCC by membrane-biotinlaytion-method and increased the NCC colocalization with lysosomal-associated membrane protein 1. The PD168077-induced NCC decrease was reversed by lysosomal inhibitor chloroquine (20μM). 1 hr PD168077 also decreased NCC-dependent-intracellular sodium transport (74±1, n=7), which was blocked by D4R antagonist L-745,870. AT1R protein abundances (96±8, n=4), Ncc mRNA levels (116±13, n=9)of were not altered in D4 agonist treated cells at 24 hrs.
Conclusion
D4R inhibits NaCl transport in renal DCT by reducing NCC activity and protein abundance with promoting its internalization, ubiquitination and consequent lysosomal degradation.