Kidney Week

Abstract: TH-PO209

SNX19 and p27 kip1 Regulate D1R Endocytosis in Renal Proximal Tubule Cells

Session Information

• Hypertension and CVD: Basic Research Findings
  October 24, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Hypertension and CVD

• 1601 Hypertension and CVD: Basic

Authors

• Amatya, Bibhas, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Bibhas Amatya

• Asico, Laureano D., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Laureano D. Asico, DVM

• Feranil, Jun, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Jun Feranil, MS, PhD, DVM

• Campisi Cadme, Raisha L., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Raisha L. Campisi Cadme, MSc

• Polzin, Jacob Quentin Mullins, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Jacob Quentin Mullins Polzin

• Armando, Ines, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Ines Armando, MS, PhD

• Felder, Robin Allen, University of Virginia, Charlottesville, Virginia, United States

Robin Allen Felder, PhD

• Jose, Pedro A., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Pedro A. Jose, MD, PhD

• Lee, Hewang, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Hewang Lee, MD, PhD

Background

SNX19, a member of the SNX-PXA-RGS-PXC subfamily of sorting nexin proteins, is localized in lipid raft microdomains and regulates dopamine D1 receptor (D₁R) endocytosis, renal sodium transport, and subsequently blood pressure. The interaction between SNX19 and p27^Kip1, a scaffold protein in lipid rafts, in the regulation of D₁R endocytosis is unknown.

Methods

Colocalization was observed by confocal immunofluorescence microscopy; protein-protein interaction was determined by co-immunoprecipitation. Blood pressure was measured in conscious C57BL/6 mice treated with a 7-day renal subcapsular infusion of Snx19 siRNA; mock siRNA served as the control.

Results

In human renal proximal tubule cells (RPTCs), SNX19 colocalized with caveolin-1 and flotillin-1. SNX19 and caveolin-1 or flotillin-1 also co-immunoprecipitated in human RPTCs. Fenoldopam (FEN, 25 nM, 30 mins, n=3), a D₁R/D₅R agonist, increased their co-immunoprecipitation. Interestingly, p27^Kip1, a canonical cyclin-dependent kinase inhibitor, colocalized with calveolin-1 and flotillin-1 in mouse and human RPTCs, confirming electron and confocal microscopic studies that p27^Kip1 is a lipid raft protein. Confocal immunofluorescence microscopy showed that in mouse RPTCs, FEN (25nM, 30 min) increased the colocalization of p27^Kip1 with α-tubulin which is essential in SNX19-mediated D₁R endocytosis. The D₁R/D₅R antagonist, Sch 39916 (SCH, 1 µM, 30 min), prevented the FEN-mediated increase in the colocalization of p27^Kip1 with α-tubulin. Moreover, nocodazole (10 µM, 1hr), a microtubule depolymerization inhibitor, interfered with the FEN-mediated increase in the colocalization of SNX19 with D₁R, confirming the importance of the microtubule in the SNX19-mediated D₁R endocytosis. The D₁R is downstream of SNX19 because silencing DRD1 in human RPTCs decreased D₁R but not SNX19 expression. SNX19 and D1R play a role in blood pressure regulation. In C57BL/6 mice on a normal salt diet (0.9% NaCl), renal subcapsular infusion of Snx19 siRNA (3 mg/day; 7 days) increased systolic BP (mock siRNA: 99.3 ± 1.52 mmHg, n=3; Snx19 siRNA: 114.1 ± 5.44 mmHg, n=3) and decreased renal D₁R expression.

Conclusion

SNX19 in the RPT interacts with p27^Kip1 in the lipid rafts to regulate SNX19-mediated D₁R endocytosis and subsequently, blood pressure.

Funding

• NIDDK Support