MECHANICAL DEFORMATION INDUCES APOPTOTIC INDUCTION IN RESPONSE TO NA+/H+ EXCHANGER NHE1 ISOFORM DOWNREGULATION FOLLOWING ERK ACTIVATION AND P38 INHIBITION

.BOCANEGRA VICTORIA; GIL LORENZO ANDREA; CACCIAMANI VALERIA; BENARDON MARIA EUGENIA; VALLES PATRICIA

Edimburgo

Congreso; Twelfth International Workshop on Developmental Nephrology; 2013

Introduction: Mechanical deformation following congenital ureteral obstruction is traduced into biochemical signals that represent a key step in the understanding of the mechanisms that leads to the tubular atrophy during obstructive nephropathy.The study included the analysis of Na+/H+ isoform 1 exchanger, NHE1, on apoptosis regulation and the signaling pathways associated with NHE1 regulation. Material and Methods: For cell mechanical deformation,HK-2 (Human Kidney-2) cells were subjected to mechanical stretch which included 15, 60, 90 and 180 minutes of cyclic exposure. Apoptosis was examined with flow cytometry (Annexin V) and protein expression with western blot that included Caspase 3,Bcl2,NHE1,RhoA,ERK 1/2 and p38.NHE1 expression inhibition was assessed with NHE1-siRNA. Results:when mechanical stretch was applied to HK-2 cells, we observed an apoptosis induction in a time dependent manner with an increase in Annexin V+ cells, a decrease in Bcl2 and an increase in caspase 3 expression.Apoptotic response to mechanical stretch was associated with a progressive decrease of NHE1.NHE1 expression inhibition with siRNA lead to a subsequent decreased Bcl2 expression and increased Caspase 3 activation.HK-2 cells subjected to mechanical stretch showed progressive RhoA activation,with early activation of ERK 1/2 associated to an opposite p38 expression effect.Treatment with specific MAPKinasas signaling pathway inhibitors increased NHE1 expression by inhibiting p38 pathway with absence of caspase 3 expression,whereas persistent lower NHE1 protein levels and increased Caspase 3 expression were shown after ERK 1/2 inhibition.Conclusions:Renal tubular apoptosis following mechanical deformation is associated with decreased NHE1 protein expression. Different signaling pathways participate in NHE1 regulation including both RhoA GTPase activation and the opposing actions of the ERK and -p38 MAP kinase signaling pathways as events involved in tubular cell apoptosis induction in response to mechanical injury.