Vacuolar (V) ATPase (V-ATPase) regulates ureteric bud (UB) branching morphogenesis during kidney development

Our studies have 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, acidosis and UB cell intracellular pH (pH i ) regulates UB branching morphogenesis during kidney development. The effect of specific V-ATPase inhibitor Bafilomycin, hypercapnic (high CO 2 ) and metabolic (low HCO 3 - ) acidosis on UB branching were determined in whole intact E12.5 Hoxb7 GFP+ mouse kidneys grown ex-vivo by time-lapse photomicroscopy. The effect of Bafilomycin on UB cell migration in vitro was examined using transwell migration assay (n=3 wells/treatment group). The presence of V-ATPase and Na + -H + exchanger (NHE) activity in UB cells was investigated by measurements of intracellular pH (pH i ). The ability of UB cells to regulate cell pH i in vitro was determined by measurements of Na-dependent and Na-independent pH i recovery from acid loads. 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 CO 2 ) or metabolic acidosis (induced by low HCO 3 - concentration) in the culture media caused a marked reduction in the number of UB tips compared to control. We conclude that intact V-ATPase activity is essential for normal UB branching during kidney development. V-ATPase-dependent reduction in UB cell pH i is likely a cause of decreasing UB branching by inhibiting directional movements of UB cells.