Identification of a Receptor for Extracellular Renalase

<div><p>Background</p><p>An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI) and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI) in wild type (WT) mice. Therefore, we sought to identity the receptor for extracellular renalase.</p><p>Methods and Results</p><p>RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.</p><p>Conclusions</p><p>PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.</p></div

RP-220 alters the pattern of MAPK activation in cisplatin treated cells.

<p>A, MAPK phosphorylation with cisplatin alone (CP) and with CP and RP-220, representative study, p-p38: phosphorylated p38 MAPK; p-ERK: phosphorylated ERK. B, Quantification of ERK activation, signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control; n = 3, * = P<0.05. C, Quantification of p38 activation, signals normalized to GAPDH loading control; n = 3, * = P<0.05.</p

Inhibition of p38 abrogates the protective effect of RP-220.

<p>A, Inhibition of either p38 (SB203580) or ERK (U0126) did not adversely affect the survival of HK-2 cells measured using the WST-1 method; cell survival is depicted as % change in survival compared to that of untreated HK-2 cells; n = 4, * = P<0.05. B, Inhibition of p38 (10 μM SB203580)abrogated the protective action of RP-220 for HK-2 cells exposed to 20 μM cisplatin (Cis) for 24 hrs; n = 4, * = P<0.05.</p

Renalase peptides demonstrate protective effects.

<p>A, amino acid sequence of renalase peptides: RP-Scr220: scrambled RP-220. B, Effect of renalase peptides on survival of 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, RP-A220: mutated RP-220, and RP-19 and RP-128: control peptides; peptide concentration (μg/ml) indicated in top line; n = 4, * = p<0.05. C, Comparison of protective effect of recombinant renalase, RP-224, RP-220, and RP-H220 on survival of 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; n = 4, * = p<0.05. D, Dose response curve for RP-220 and RP-H220; 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; n = 4, * = p<0.05.</p

PMCA4b inhibition abrogates renalase peptide mediated MAPK signaling and cytoprotection.

<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

Identification of plasma membrane calcium ATPase isoform PMCA4b as a renalase binding protein.

<p>A, HK-2 cells incubated with either labeled RP-Scr220 or RP-220, biotin-labeled proteins purified using streptavidin column, separated by SDS-PAGE and visualized by western blot using streptavidin-HRP; * = regions evaluated by mass spectrometry in samples labeled with either RP-Scr220 or RP-220; # = RP-220 band containing the plasma membrane calcium ATPase isoform PMCA4b. B, Endogenous expression of PMCA4b in HK-2 cells, western immunoblot using isoform specific monoclonal; CCL-119: human leukemic cell line; thyroid tumor = human thyroid tumor cell line (ATCC, CRL-1803) 10 μg protein loaded in each lane. C, co-immunolocalization of PMCA4b and renalase in HK-2 cells, images acquired using a Zeiss laser scanning confocal microscope, scale bar = 9 μm; arrow = plasma membrane. D, Co-Immunoprecipitation of PMCA4b and renalase from HK-2 cell lysates; renalase-Ab-beads = renalase antibody coated beads; PMCA4b-Ab-beads = PMCA4b antibody coated beads.</p

Molecular basis of renalase’s action: working model.

<p>PMCA4b: plasma membrane calcium ATPase isoform 4b; PM: plasma membrane; N: amino terminus; C: carboxy terminus; CaM-BD1: calmodulin binding domain 1; CaM-BD2: calmodulin binding domain 2; CaM-BD-IS: calmodulin binding domain interaction sites.</p

Time-adjusted survival curve for IL-6 and renalase among patients with COVID-19 disease (A) For composite outcome^a: High renalase + High IL-6; High renalase + Low IL-6; Low renalase + High IL-6; Low renalase + Low IL-6 and (B) For Mortality: High renalase + High IL-6; High renalase + Low IL-6; Low renalase + High IL-6; Low renalase + Low IL-6.

a For composite outcomes, patients with renalase samples drawn after intubution were excluded from survival analysis (n = 55).</p

Flow chart of study patients from all COVID-19 admissions to the institution from March to July 2020.

Flow chart of study patients from all COVID-19 admissions to the institution from March to July 2020.</p

Time adjusted survival curves for high vs. low renalase among patients with COVID-19 disease (using first quartile as cutoff).

Kaplan-Meir curves created and compared using log-rank test. (A) For composite outcomea: High renalase; Low renalase and (B) For Mortality: High renalase; Low renalase. a For composite outcomes, patients with renalase samples drawn after intubution were excluded from survival analysis (n = 55).</p