Kidney Week

Abstract: SA-PO446

Ureteric Bud (UB) Prorenin Receptor (PRR) Directs UB Branching Morphogenesis by Regulating V-ATPase Activity and Autophagy in UB Cells

Session Information

• Development and Regenerative Medicine
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 500 Development, Stem Cells, and Regenerative Medicine

Authors

• Yosypiv, Ihor V., Tulane University, New Orleans, Louisiana, United States

Ihor V. Yosypiv,

• Taylor, Renfang Song, Southwest Baptist University, Bolivar, Missouri, United States

Renfang Song Taylor,

• Nakhoul, Nazih L., Tulane Medical School, New Orleans, Louisiana, United States

Nazih L. Nakhoul,

Background

Targeted ablation of the PRR in the UB lineage in Hoxb7^Cre/PRR^flox/flox (PRR^UB-/-) mice causes severe defects in UB branching, leading to decreased nephron endowment and renal hypodysplasia. Since PRR is an accessory subunit of the vacuolar proton pump V-ATPase, we investigated the role of V-ATPase and autophagy in PRR-induced UB branching in mice.

Methods

UB cells were FACS-isolated from Hoxb7^Cre+/GFP+/PRR^flox/flox (PRR^UB-/-) and control (PRR^UB+/+) littermates at birth (P0). V-ATPase subunit expression in isolated cells was determined by microarray and validated by real-time RT-PCR. PRR knockdown in immortalized UB cells (iUBc) was achieved with adenovirus-driven short hairpin RNA (shPRR). Effect of PRR knockdown on cell pH and V-ATPase activity was determined by staining with Lysotracker (a lysosomal pH marker) and from measurements of Na-independent cell pH recovery rates after intracellular acidification with a NH₄Cl prepulse.

Results

Expression of V-ATPase subunits Atp6ap2 (PRR), V0a4, V0b, V1b1 and V1g1 was reduced in PRR^UB-/- compared to PRR^UB+/+ UB cells. shPRR decreased mRNA levels of PRR by 80-90% and of kidney-specific α4 V-ATPase subunit by 50% compared to control scrambled control PRR (scPRR) virus in iUBc. shPRR decreased Lysotracker fluorescence in iUBc compared to scPRR (p<0.001). Intracellular pH (pHi) measurements (by BCECF fluorescence) indicated slower recovery from acid loads in shPRR-treated cells due to reduced V-ATPase activity. Immunofluorescence of key autophagy protein LAMP2 was increased in the UB of PRR^UB-/- compared to PRR^UB+/+ kidneys on E14.5, consistent with autophagic defects in UB cells in PRR^UB-/- kidneys.

Conclusion

PRR knockdown decreases expression of α4 subunit of V-ATPase and suppresses V-ATPase activity in iUB cells in vitro, resulting in deacidification of intracellular vesicles. We propose that endogenous UB PRR regulates normal UB branching morphogenesis through stimulation of V-ATPase activity, control of lysosomal pH and appropriate autophagic flux in UB cells.