Identification of a Receptor for Extracellular Renalase

Background 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. Methods and Results 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. Conclusions PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

Introduction patents application for the discovery and use of renalase. The other authors have declared no competing interests exist. Note that Yale University has entered into a license agreement with Bessor Pharma to develop renalase as a potential diagnostic and therapeutic agent. This agreement does not preclude the publication of findings by my laboratory, and has no impact on the current manuscript. GVD confirms that these competing interests do not alter the authors' adherence to all PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
instructions. Conjugation was achieved through the sulfhydryl-reactive methanethiosulfonate (Mts) moiety, and the spacer arm between Mts, and the photoactivatable tetrafluorophenyl azide (Atf) moiety was 11 Å. Coupling efficiency was estimated by dot blot using streptavidin-HRP to measure biotin incorporation (#21130, Pierce Biotechnology, Rockford, IL). The labeled probes were protected from light and stored in 50 μl aliquots, at -80°C until use.
HK-2 cells, obtained from American Type Culture Collection (ATCC, Manassas, VA) were grown at 37°C to 80% confluence in 150 mm dishes in DMEM/F12 media supplemented with glutamine, 10%FBS, antibiotics, and 5% CO2. Cells were cooled to 4°C to prevent probe internalization, and then incubated for 16 hrs with 50 μg of either Mts-Atf-Biotin labeled RP220 or RP-Scr220. Cross-linking of the probes was initiated by exposing the cells to ultraviolet light for five minutes using a Stratalinker 2500 (Stratagene, Agilent Technologies, Santa Clara, CA). Renalase peptides demonstrate protective effects. 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.
The cells were suspended in phosphate buffered saline (PBS) with protease inhibitors (Complete Ultra # 05892953001, Roche Diagnostics, Indianapolis, IN) and subjected to 3 cycles of freeze-thawing. The membrane fraction was collected by centrifugation (180,000g for 1 hour) and solubilized in RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na 3 VO 4 , 1μg/ml leupeptin) (Cell Signaling Technologies, Danvers, MA) for 4 hours at 4°C The biotinylated proteins were purified using streptavidin agarose resin (Pierce Biotechnology, Rockford, IL) according to the manufacturer's instructions. The proteins were separated by SDS PAGE using a 4-20% gradient gel (Bio-Rad, Hercules, CA) and stained by Westernblotting using streptavidin-HRP to identify proteins bands found predominantly cross-linked to the renalase bait RP-220. These bands and the corresponding bands from the samples crosslinked to the control bait RP-Scr220 were cut out of coomassie blue stained gels, and individual proteins were identified by mass spectrometry (Yale Keck Biotechnology Resource Laboratory, S1 Methods). Plasma membrane protein(s) consistently present in the samples cross-linked to RP-220, and absent from those cross-linked to the control bait RP-Scr220 were evaluated further.
Detection of endogenous co-expression of PMCA4b and renalase HK-2 cells, which highly express renalase and PMCA4b endogenously, were fixed, permeabilized, and incubated with a goat polyclonal anti-renalase antibody (# AF5350, R&D Systems, Minneapolis, MN), and an anti-PMCA4 mouse monoclonal antibody (#H00000493-M07, Novus Biologicals, Littleton, CO) for 2 hours at room temperature. Following the application of two labeled secondary antibodies (Alexa488-rabbit anti-goat to detect renalase and Alex-a555-goat anti-mouse to detect PMCA4, Molecular Probes, Life Technologies Grand Island NY), cells were then examined using a Zeiss LSM 510 confocal imaging system.

Co-Immunoprecipitation of endogenous PMCA4b and renalase
HK-2 cells were lysed using ice-cold RIPA buffer (Cell Signaling Technologies, Danvers, MA), and the lysate was centrifuged 14,000 g for 15 min. The supernatant was collected and incubated with protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) to reduce nonspecific binding. Immunoprecipitation was carried out using A/G agarose beads and either goat polyclonal anti-renalase antibody or anti-PMCA4 mouse monoclonal antibody, and proteins were visualized by western blotting.

Gene expression analysis
Total RNA from tissue samples was extracted using RNeasy Plus kit (Qiagen, Valencia, CA) and converted into cDNA using an Omniscript RT kit (Qiagen, Valencia, CA). PMCA4b mRNA in kidney was assessed in renalase KO mice by real-time PCR and normalized to the corresponding levels in WT samples (defined as 1.0). Target cDNA was amplified using Qiagen Dr_atp2b4_1_SG QuantiTect primer assay and Platinum SYBR Green qPCR superMix-UDG. Standard cycling conditions were run with a Step-One-Plus real time PCR system (Applied Biosystems), and the resulting Ct values analyzed using the 2-ΔΔCT method.
In vitro model of cisplatin toxicity HK-2 cells obtained from ATTC (Manassas, VA USA) were cultured in DMEM/F12 supplemented with glutamine, 10%FBS and antibiotics, and were maintained at 37°C in 5% CO 2 . Cells were exposed to cisplatin (20 μM) in the presence or absence of recombinant renalase or renalase peptides for 24 hrs, and cell viability was assessed by the WST1 method (Roche Applied Science, Germany).

Statistical analysis
When appropriate, the Kruskal-Wallis one-way analysis of variance by ranks was used to evaluate statistical significance. When the Kruskal-Wallis test revealed statistical significance, the Mann-Whitney test was used for pairwise comparisons. All data are mean ± SEM, and values of P<0.05 were accepted as a statistically significant difference. Statistical analysis was carried out using GraphPad Prism (GraphPad Software, Inc.).

Protection by RP-220 against cisplatin toxicity linked to p38 MAPK activation
We have previously shown that in renalase KO mice renalase deficiency worsens ischemic AKI, while the administration of recombinant renalase significantly attenuates AKI [12]. Additional studies indicate that the protective effect of renalase against ischemic and cisplatin AKI does not depend on the enzymatic activity of renalase, but rather is mediated by the interaction of renalase or short renalase peptides (RP-224, RP-220 and RP-H220, Fig 1A) with a receptor (s). A scrambled renalase peptide, RP-Scr220 did not activate MAPK signaling, and failed to protect cells [13]. The protective effects of renalase peptides corresponded to the activation of intracellular signaling that promotes cell survival [13].
We previously showed that both hRenalase1 and RP-220 treatments rapidly increased ERK and p38 phosphorylation in HK-2 cells [13]. To test if protection of HK-2 cells against cisplatin toxicity by RP-220 was linked to MAPK activation, we examined the patterns of MAPK signaling at early time points (1-60 min) in HK-2 cells exposed to cisplatin with and without RP-220. Cisplatin alone modestly increased ERK and p38 phosphorylation (Fig 2A-2C). This effect on p38 phosphorylation was markedly enhanced (5 fold) by the addition of RP-220 ( Fig 2C). However, only a slight increase in ERK activation (0.5 fold) was noted with RP-220 over that elicited by cisplatin alone (Fig 2B). These results suggest that RP-220's cytoprotective action may be due to its activation of p38 and not ERK.
To determine the relative importance of ERK and p38 as mediators of RP-220's protective effect, their activities were chemically inhibited. Reducing the activity of ERK1/2 (U0126), or p38α-β (SB203580) was examined in HK-2 cells under control conditions. In control studies, ERK and p38 inhibition had no deleterious effect and not found to reduce HK-2 cell survival ( Fig 3A). In HK-2 cells treated with both cisplatin and RP-220, ERK inhibition did not diminish the protection conferred by RP-H220 (Fig 3B). In marked contrast, p38 inhibition completely abrogated the RP-220's protection (Fig 3B), thus providing strong support for the hypothesis that p38 is a key mediator of RP-220's defense against cisplatin toxicity.

Extracellular renalase binds to the plasma membrane Ca ++ -ATPase isoform PMCA4b
RP-220 was used as a probe to identify a plasma membrane protein(s) that interact with extracellular renalase. The biotin label transfer method was used with the label transfer reagent Mts-Atf-Biotin, which was linked to the single cysteine located at the N terminus of RP-220. Labeled RP-220 was incubated with HK-2 cells for 24 hrs at 4°C to minimize internalization, and was cross-linked to the interacting protein(s) by exposure to UV light. The biotin-labeled proteins were purified using a streptavidin column and identified by mass spectrometry. The plasma membrane calcium-ATPase isoform, PMCA4b, was reproducibly cross-linked to RP-220 ( Fig 4A). PMCA4b is abundantly expressed in HK-2 cells (Fig 4B) and co-localizes with renalase at the plasma membrane (arrows), and within the cytoplasm of HK-2 cells (Fig 4C).
In vitro interaction between endogenously expressed PMCA4b and RP-220 was evaluated by co-immunoprecipitation. Agarose beads coated with either PMCA4b or renalase antibody depleted whole cell lysates of renalase (Fig 4D, upper blot, lanes 1 vs 2 and 3), and renalase could be eluted from both sets of beads (Fig 4D, upper blot, lanes 4-5).). The blot was reprobed with an anti-PMCA4b antibody, and likewise, PMCA4b could be eluted from both sets of beads (Fig 4D, bottom blot, lanes 4-5). Of note, endogenous expression of PMCA4b is significantly higher than that of renalase (Fig 4D, upper and lower blots, lanes 1), and as a consequence less PMCA4b protein is eluted from beads coated with the renalase antibody than from those coated with the PMCA4b antibody. (Fig 4D, upper and lower blots, lanes 4 vs 5). In addition, PMCA4b coated beads completely depleted renalase from the supernatant suggesting that extracellular renalase is largely bound to PMCA4b.

PMCA4b mediates renalase's action on cell signaling and cytoprotection
We next sought to determine if inhibition of PMCA4b activity modulated renalase mediated MAPK signaling. Caloxin1b, a peptide inhibitor of PMCA4b [15], abrogated renalase-mediated ERK and p38 MAPKs phosphorylation in HK-2 cells (Fig 5A). Since Caloxin1b is reported to also inhibit PMCA1, additional evidence regarding PMCA4b's role in renalase mediated signaling was obtained by specifically down-regulating PMCA4b expression using siRNA. In control studies, non-targeting siRNAs affected neither PMCA4b expression nor RP-220 mediated p38 (Fig 5B, left panel). In contrast, PMCA4b-targeting siRNAs decreased protein expression by more than 90% and prevented RP-220 mediated p38 phosphorylation (Fig 5B, middle and  right panels). The specificity of the interaction between RP-220 and PMCA4b was examined by testing if PMCA4b downregulation also affected epidermal growth factor (EGF) mediated MAPK activation. As shown in Fig 5C, inhibition of PMCA4b expression had no effect on EGF dependent ERK, p38 and JNK phosphorylation.
Down-regulation of PMCA4b expression abrogated the protective effect of RP-220 and RP-H220 against cisplatin cytotoxicity (Fig 5D). We previously showed that in marked contrast to what's observed in WT mice, neither renalase nor RP-220 is effective at activating MAPK signaling and at protecting renalase KO mice against mild or severe ischemic AKI [13]. Based on these results, we hypothesized that the interaction of RP-220 with its cognate receptor was disrupted in the renalase KO, perhaps due to a decrease in receptor gene or protein expression brought about by deletion of the renalase gene. PMCA4b gene expression in the renalase KO mouse was measured by quantitative PCR, and found to be 11.4 fold lower than in WT control (n = 6, p<0.03). Expression of PMCA4b protein in WT and renalase KO kidneys was evaluated by western immunobloting. As shown in Fig 5E (representative blot), PMCA4b expression in the renalase KO was 63.5±7.5% lower than in WT (n = 6, p<0.03). These data provide strong support for the hypothesis that PMCA4b functions as a renalase receptor, and is critical in mediating the signaling and cytoprotective effects of renalase.

Discussion
We have identified the plasma membrane calcium ATPase isoform, PMCA4b as a receptor for extracellular renalase. Endogenously expressed PMCA4b and renalase are colocalized at the plasma membrane of HK-2 cells, and the renalase peptide RP-220 can be cross-linked to PMCA4b. Inhibition of PMCA4b enzymatic activity by caloxin1b and down-regulation of PMCA4b expression with siRNA completely abrogated renalase and RP-220-mediated MAPK signaling and cytoprotection.
The molecular events that are set in motion by the binding of extracellular renalase to PMCA4b ultimately lead to MAPK activation, and since the data indicate that renalase activates PMCA4b, we propose the working model shown in Fig 6. PMCA4b has previously been characterized as a plasma membrane ATPase involved in cell signaling and cardiac hypertrophy [16]. It ejects Ca2+ from the cytosol of a eukaryotic cell to the external environment using one molecule of ATP in transporting one molecule of Ca2+. It is a P-type ATPase encoded by four genes (ATP2B1-4), the transcripts of which undergo different types of alternative splicing. It appears to regulate local rather than bulk calcium concentration in both excitable and nonexcitable cells and to participate in a macromolecular complex that signals through Ras and the MAPKs (24-26). For instance, it modulates bone mass, cardiac contractility and hypertrophy by regulation of NF-kB, neuronal nitric oxide synthase (nNOS), and calcineurin, respectively [17][18][19][20]. That these processes are calcium-dependent provides support for the hypothesis that . 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. PMCA4b regulates local calcium concentration, thereby altering the activity of its many interacting partners. For example, PMCA4b modulates Ras signaling and ERK activation through its interaction with the tumor suppressor RASSF1, a Ras effector protein involved in H-Rasmediated signaling [21]. Extracellular renalase and renalase peptides (RP-220) activate the Ras-Raf-MEK-ERK (MAPK) pathway, and perhaps RASSF1 may play a role in RP-220 dependent MAPK signaling.
Our data indicate that the early activation of the stress kinase p38 pathway, and not ERK pathway, is crucial in mediating the protection of renalase against cisplatin cytotoxicity. Stress signals activate MAPKs, which in turn coordinate the cellular responses leading to cell death or survival [22]. While ERK activation by extracellular growth factors tends to promote cell survival, this effect has been shown to be cell and stimuli dependent. The p38 and JNK pathways, which are generally linked to cell death, are activated by a variety of stresses, including oxidants, UV irradiation, and inflammatory cytokines [22]. Cisplatin activates p38, ERK, and JNK in renal epithelial cells.
An unexpected finding in our study is that cells treated with cisplatin and renalase peptide, chemical inhibition of p38 completely eliminated the peptide's protective effect. Since renalase peptide is associated with early activation of p38, the most likely explanation relates to the biphasic nature of p38 activation by stressful stimuli. For instance, in the presence of tumor necrosis factor (TNF), p38 and JNK are activated twice, first before apoptosis and later at the time of apoptosis [23]. Inhibition of the early JNK and p38 activation, by chemical means or by expression of dominant negative mutants, increases TNF-induced apoptosis, suggesting that early activation of JNK and p38 is prosurvival.
We have presented evidence that PMCA4b functions as a renalase receptor, and mediates renalase dependent MAPK signaling and cytoprotection. In addition to providing insight into the mechanism of action of extracellular renalase, these findings reveal a previously unrecognized mode of regulation of PMCA4b. They provide additional evidence for the hypothesis that PMCA4b functions as a signaling molecule, and given that PMCA4b modulates the development of cardiac hypertrophy, its interaction with renalase may have particular clinical relevance for patients with hypertension and chronic kidney disease.