Vacuolar ATPase (V-ATPase) Regulates Ureteric Bud (UB) Branching Morphogenesis During Kidney Development

Background: Branching morphogenesis of the ureteric bud (UB) is a fundamental developmental process that directs organogenesis of the kidney. Physiological factors that regulate normal UB branching and kidney development are not fully understood. In previous studies, we established that ureteric bud (UB) prorenin receptor (PRR/ATP6AP2), an accessory subunit of the vacuolar H+-ATPase (V-ATPase), is critical for normal UB branching. Here, we tested the hypothesis that V-ATPase activity (an important H+ extruding mechanism), acidosis and UB cell intracellular pH (pHi) regulate UB branching morphogenesis during kidney development. Methods: Embryonic kidneys at day E12.5 were aseptically microdissected from timed-pregnant Hoxb7GFP+ mice. Isolated kidneys were cultured on polycarbonate Transwell filters and ex-vivo UB branching was monitored by time-lapse photomicroscopy. The effect of specific V-ATPase inhibitor Bafilomycin, hypercapnic (high CO2) and metabolic (low HCO3-) acidosis on UB branching were determined. The effect of Bafilomycin on UB cell migration in vitro was examined using transwell migration assay (n=3 wells/treatment group). The presence of functional V-ATPase and Na+-H+ exchanger (NHE) in UB cells was investigated by measurements of intracellular pH (pHi). The ability of UB cells to regulate cell pHiin vitro was determined by measurements of Na-dependent and Na-independent pHi recovery from acid loads. Results: We show that H+-ATPase (V-ATPase) and Na+-H+ exchanger are present in UB cells and can regulate pHi. The mean number of UB cells that migrated through membrane after 24h culture was reduced with Bafilomycin compared to control. Treatment with Bafilomycin, hypercapnic acidosis (induced by high CO2) or metabolic acidosis (induced by low HCO3- concentration) in the culture media caused a marked reduction in the number of UB tips compared to control. Conclusion: We conclude that intact V-ATPase activity is essential for normal UB branching during kidney development. V-ATPase-dependent reduction in UB cell pHi is likely a cause of decreasing UB branching by inhibiting directional movements of UB cells.