Apoptosis induction is associated with decreased NHE1 expression in neonatal unilateral ureteric obstruction

OBJECTIVE To examine the participation of NHE1 in the regulation of the apoptotic response in neonatal obstruction, as the ubiquitously expressed NHE1 isoform is an important component of regulatory volume increase. MATERIALS AND METHODS Rats had a unilateral ureteric obstruction (UUO) or a sham operation, and the kidneys were harvested 5 and 14 days afterward. Cellular apoptosis in proximal tubules (PT) and collecting ducts (CD) was assessed using a standard assay, and NHE1 expression in the renal cortex assessed using reverse transcription-polymerase chain reaction and Western blots. Mitochondrial apoptosis was evaluated by Bax/BcL2 expression, and caspase-3 expression and activity. In addition, we evaluated the in vivo administration of increasing doses of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on NHE1 inhibition associated with the induction of apoptosis. RESULTS After 14 days there were consistently more apoptotic cells in CD than in PT, associated with a lower expression of NHE1 at the mRNA and protein levels. There was increased expression of the Bax/BcL2 ratio, linked to decreased pro-caspase-3 protein levels and with increased caspase-3 activation. NHE1 inhibition by increasing doses of EIPA induced epithelial cell apoptosis and increased caspase-3 activity in a dose-dependent manner. After in vitro incubation with amiloride (100 mm) there was less NHE1 expression associated with reduced 32 kDa pro-caspase-3 protein levels. Kidneys obstructed for 5 days showed no changes in NHE1 expression or induction of apoptosis. CONCLUSION In neonatal obstruction, we suggest that the decreased NHE1 expression could be a signal-transduction event participating in the induction of epithelial tubular cell apoptosis, through the regulation of the BcL-2 gene family and activation of caspase-3.