PMCA4b inhibition abrogates renalase peptide mediated MAPK signaling and cytoprotection.

Abstract

<p>A, PMCA4b inhibition abrogates RP-220 mediated ERK and p38 signaling in HK-2 cells; caloxin1b = peptide inhibitor of PMCA4; <i>left panel</i>: RP-220 mediated ERK and p38 activation, phospho = phosphorylated, representative blot; <i>middle panel</i>: Inhibition of RP-220 mediated ERK and p38 activation by caloxin1b (100 μM); <i>right panel</i>: quantification of phosphorylated p38 (P-p38) and phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 3, * = P<0.05. B, siRNA mediated inhibition of PMCA4b expression downregulates RP-220 mediated MAPK signaling; <i>left panel</i>: RP-220 mediated ERK and p38 activation in HK-2 cells transfected with non-targeting siRNA, p = phosphorylated, representative immunoblot; <i>middle panel</i>: Inhibition of RP-220 mediated ERK and p38 activation in HK-2 cells transfected with PMCA4b siRNA, representative blot; <i>right panel</i>: quantification of phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 3, * = P<0.05. C, Lack of effect of siRNA mediated inhibition of PMCA4b expression on epidermal growth factor (EGF)- mediated MAPK signaling; <i>left panel</i>: EGF (100 ng/ml) mediated ERK, p38 activation and c-Jun N-Terminal Kinase (JNK) in HK-2 cells transfected with non-targeting siRNA, p = phosphorylated, representative blot; <i>right panel</i>: EGF-mediated ERK, p38 and JNK activation in HK-2 cells unaffected by transfection with PMCA4b siRNA and downregulation of PMCA4b expression; representative blot (n = 3). D, Inhibition of PMCA4b expression abrogates protective effect of renalase peptides for HK-2 cells exposed to cisplatin: HK-2 cells exposed to 20μM cisplatin for 24 hrs; cell survival is depicted as % change in survival compared to that in cisplatin-treated HK-2 cells without renalase peptides; cell survival measured by the WST-1 method, peptide concentration 15 μg/ml, indicated in top line; n = 4, * = p<0.05. E, Endogenous expression of PMCA4b in WT and renalase KO mice, western immunoblot using an anti-renalase monoclonal antibody; 10 μg protein loaded in each lane.</p