Associate Professor Tulane University School of Medicine New Orleans, Louisiana, United States
Background: Our previous work demonstrated that targeted ablation of the prorenin receptor (PRR/Atp6ap2), an accessory subunit of the vacuolar proton pump V-ATPase, in the UB lineage in Hoxb7Cre/PRRflox/flox (PRRUB-/-) mice causes severe defects in UB branching, leading to decreased nephron endowment and reduced ability to acidify the urine (Song. PLoS ONE, 2013). Objective: We examined the role of autophagy, a process of protein digestion in lysosomes dependent on correct intravesical pH, in UB branching defects observed in PRRUB-/- mice. Design/Methods: The subcellular localization of PRR and V1b1 subunit of V-ATPase was determined in the UB of embryonic (E) day E15.5 PRRUB+/+ (wild type) metanephroi in vivo by immunofluorescence with anti-PRR (Santa Cruz) and -V1b1 subunit (Abnova) antibodies using confocal microscopy (Nikon A). The expression of lysosomal protein LAMP2 (a marker of late endosome/lysosome) was determined in the UB of E15.5 PRRUB-/- (mutant) and PRRUB+/+ metanephroi in vivo by immunofluorescence with anti-LAMP2 (Abcam) antibody. The expression of V0α4, V0b, V1a, V1g1, V1b1, V1f subunits of V-ATPase was determined by real-time qPCR in UB cells FACS-isolated from postnatal (P) day P0 PRRUB-/- (n=3) and PRRUB+/+ (n=3) kidneys. Results: To determine if PRR regulates V-ATPase activity via V-ATPase trafficking, we determined the subcellular co-localization of the PRR and V-ATPase in the UB. V-ATPase V1b1 subunit and PRR immunofluorescence was detected in the apical and basolateral plasma membrane with less punctate staining in the cytoplasm of UB cells, consistent with both V-ATPase and PRR protein localization in plasma membrane and intracellular organelles. Immunofluorescent staining for lysosomal protein LAMP2 was increased in the UB of E15.5 PRRUB-/- compared with PRRUB+/+ kidneys. The expression of V0α4, V0b, V1a, V1g1, V1b1, V1f V-ATPase subunits and of the PRR mRNA was decreased in the UB cells FACS-isolated from P0 PRRUB-/- compared with PRRUB+/+ kidneys.
Conclusion(s): Co-localization of V-ATPase and PRR proteins in the plasma membrane and intracellular organelles of UB cells suggests that UB cell PRR (Atp6ap2) may regulate function of V-ATPase present in the plasma and intracellular (e.g., autolysosome) membranes. Increased expression of LAMP2 in the UB of PRRUB-/- compared with PRRUB+/+ kidneys is consistent with autophagic defects in UB cells in mutant kidneys. Lack of UB PRR may decrease V-ATPase activity via reduced levels/assembly of the V-ATPase. Thus, endogenous UB PRR may direct normal UB branching morphogenesis by regulation of lysosomal pH and appropriate autophagic flux in UB cells.
