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

MANUCHA W; CARRIZO L; RUETE C; VALLES P

BRITISH JOURNAL OF UROLOGY

Año: 2007 vol. 100 p. 191 - 198

0007-1331

OBJECTIVETo examine the participation of NHE1 in theregulation of the apoptotic response inneonatal obstruction, as the ubiquitouslyexpressed NHE1 isoform is an importantcomponent of regulatory volume increase.MATERIALS AND METHODSRats had a unilateral ureteric obstruction(UUO) or a sham operation, and the kidneyswere harvested 5 and 14 days afterward.Cellular apoptosis in proximal tubules (PT) andcollecting ducts (CD) was assessed using astandard assay, and NHE1 expression inthe renal cortex assessed using reversetranscription-polymerase chain reaction andWestern blots. Mitochondrial apoptosis wasevaluated by Bax/BcL2 expression, and caspase-3 expression and activity. In addition,we evaluated the in vivo administration ofincreasing doses of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on NHE1 inhibitionassociated with the induction of apoptosis.RESULTSAfter 14 days there were consistently moreapoptotic cells in CD than in PT, associatedwith a lower expression of NHE1 at the mRNAand protein levels. There was increasedexpression of the Bax/BcL2 ratio, linked todecreased pro-caspase-3 protein levels andwith increased caspase-3 activation. NHE1inhibition by increasing doses of EIPA inducedepithelial cell apoptosis and increasedcaspase-3 activity in a dose-dependentmanner. After in vitro incubation withamiloride (100 mM) there was less NHE1expression associated with reduced 32 kDapro-caspase-3 protein levels. Kidneysobstructed for 5 days showed no changes inNHE1 expression or induction of apoptosis.CONCLUSIONIn neonatal obstruction, we suggest that thedecreased NHE1 expression could be a signal-transduction event participating in theinduction of epithelial tubular cell apoptosis,through the regulation of the BcL-2 genefamily and activation of caspase-3.