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Renalase Prevents AKI Independent of Amine Oxidase Activity

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Renalase Prevents AKI Independent of Amine Oxidase Activity BASIC RESEARCH www.jasn.org Renalase Prevents AKI Independent of Amine Oxidase Activity † ‡ Ling Wang,* Heino Velazquez,* Gilbert Moeckel, John Chang,* Ahrom Ham,§ H. Thomas Lee,§ Robert Safirstein,* and Gary V. Desir* *Department of Medicine, Veterans Affairs Connecticut Healthcare System, Yale University, New Haven, Connecticut; †Renal Division, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China; ‡Department of Pathology, Yale University, New Haven, Connecticut; and §Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York ABSTRACT AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide–induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20–amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein ki- nases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI. J Am Soc Nephrol 25: 1226–1235, 2014. doi: 10.1681/ASN.2013060665 AKI is a common clinical condition affecting up to oxidase activity of renalase.8,9 We had proposed 20% of hospitalized patients and is frequently asso- that, in contrast to the classic amine oxidases, renalase ciated with sepsis, surgery, and certain drugs. Epide- reacts with oxygen to generate superoxide anions miologic data indicate a positive association between and hydrogen peroxide, with subsequent oxidation the severity of AKI and in-hospital and long-term of catecholamines to their respective amino- mortality.1,2 Unfortunately, the development of effec- chromes. Recent results indicate that renalase func- tive therapy for AKI has been hampered by (1)an tions as an oxidase/anomerase, using molecular inherent delay in diagnosis, a consequence of relying oxygen to convert a-NAD(P)H to b-NAD+, with on serum creatinine, which only increases 48–72 hydrogen peroxide as reaction byproduct.10 Because hours after the original insult, and (2) the paucity of validated targets of therapy. There is an urgent Received June 26, 2013. Accepted October 16, 2013. need to identify novel therapeutic modalities. fl Published online ahead of print. Publication date available at Renalase is a novel secretory avoprotein with www.jasn.org. amine oxidase activity.3–5 In vitro, renalase metabo- Correspondence: Dr. Gary Desir, Section of Nephrology, De- lizes epinephrine, norepinephrine, and dopamine partment of Medicine, Yale School of Medicine, P.O. Box and also possesses significant intrinsic nicotinamide 208029, New Haven, CT 06520-8029. Email: gary.desir@yale. adenine dinucleotide (NADH) oxidase activity.6,7 edu Other investigators have questioned the amine Copyright © 2014 by the American Society of Nephrology 1226 ISSN : 1046-6673/2506-1226 JAmSocNephrol25: 1226–1235, 2014 www.jasn.org BASIC RESEARCH it was also shown to bind epinephrine, the authors pointed out intraperitoneal injection), plasma creatinine was significantly thatthehydrogenperoxide(H2O2) generated from the anomerase higher in renalase KO mice than in WT mice (1.8260.56 reaction will drive the oxidation of epinephrine to adreno- versus 0.7160.02 mg/dl, n=6; P=0.02) (Figure 1A). Renalase- chrome, albeit at a slower rate. Single-nucleotide polymorphisms deficient mice developed worse renal histologic injury com- present in the gene are associated with hypertension, cardiac pared with renalase WT mice (Figure 1B). Renalase KO mice disease, and diabetes.6,11–14 The administration of renalase in showed more severe acute tubular necrosis compared with WT wild-type (WT) mice lowers plasma catecholamines and sys- mice (injury scores: KO, 56.62%67.38%, n=6; WT, 17.07% temic BP.In contrast, the deficiency of renalase in renalase knock- 64.03%; n=5; P=0.002). The number of apoptotic renal cells out (KO) mice raises catecholamine levels and BP.15 Renalase also (reddish stain) was increased two-fold in cisplatin-treated KO modulates the severity of renal ischemia and reperfusion injury.16 mice compared with WT mice (Figure 1C). Likewise, renal In WTmice, the administration of recombinant human renalase macrophage infiltration was significantly increased (brown before induction of renal ischemia significantly blunts the sever- stain) in KO mice compared with WT mice (Figure 1D). ity of renal injury, with less renal tubular necrosis, inflammation, To elucidate the mechanism of renal protection by renalase, and apoptosis. In contrast, the lack of renalase in renalase KO we asked whether recombinant renalase could protect cells in mice exacerbates the renal damage after similar ischemic injury. culture. In HK-2 cells, recombinant renalase significantly AKI is characterized by an elevation in plasma catechol- reduced necrosis induced with 2 mM H2O2 compared with amine levels. It has been postulated that, in addition to causing vehicle-treated HK-2 cells (Figure 2A). HK-2 cells exposed to hypertension, excess catecholamines in AKI may produce an cisplatin for 24 hours showed decreased cell viability (Figure inflammatory response, aggravating tissue damage and con- 2B, left panel) and renalase expression (Figure 2B, right tributing to multiorgan dysfunction.17 Interestingly, renalase panel), both of which were reversed by the addition of exog- levels in the blood and kidneys of WT mice are reduced fol- enous renalase. Renalase treatment inhibited caspase-3 acti- lowing acute renal ischemia. Because the renalase in blood is vation (Figure 2C) and increased Bcl-2 expression (Figure secreted from the kidneys and is thought to metabolize circu- 2D). These in vitro cell data suggest that renalase’s in vivo pro- lating catecholamines, the excess catecholamines in AKI may tective effect may not be mediated solely by a reduction in be a direct consequence of the concurrent renalase deficiency. circulating catecholamines. Notably, the administration of renalase to WT mice before induction of ischemia, which greatly attenuates ischemic renal Renalase’s Protection against Cellular and Organ Injury injury, dampens the rise in blood catecholamine levels. On the Is Independent of Its Enzymatic Function basis of these findings, it is inviting to speculate that the renal There is a single renalase GENE (10 exons), which undergoes protective effect in ischemic injury of renalase and its hemo- alternative splicing to give rise to at least 6 isoforms (Renalase dynamic effect stem from its amine oxidase activity. 1–7) as documented by PCR of human tissue20 and gene se- The role of amine oxidase activity of renalase in mediating quencing data (GENBANK) (Figure 3A). The carboxy termi- its hemodynamic effect has been questioned. First, measure- nal half of exon 6 is present in all spliced isoforms detected to ment of the amine oxidase activity of renalase relies on the date. Examination of renalase’s crystal structure indicates that production of H2O2 as an indirect measure of oxidase activity. renalase peptide 220 (RP-220; Figure 3A) is partly solvent ex- 8 Because the measured rate of H2O2 synthesis is low, the puta- posed and located on the external surface of the protein. tive oxidase activity of renalase has been deemed unlikely to CCL-119 cells (CCF-MEC, acute lymphoblastic leukemic have physiologic significance.9 Second, the recombinant rena- cell line; American Type Culture Collection) divide rapidly lase synthesized in Escherichia coli with a histidine-tag pos- and express the highest level of renalase (approximately 3.8- sesses no detectable oxidase activity, and yet it markedly lowers fold over mean; microarray data from BioGPS.org) among the BP when injected into rats.8,18 cells making up the NCI-60 panel, which contains the most In this study, we sought to clarify the role of amine oxidase frequently studied human tumor cell lines in cancer research. activity in the renal protective effects of renalase and to explore Although the monoclonal antibody raised against RP-220 did an additional mechanism of action of renalase that is in- not alter the amine oxidase activity of renalase, the antibody dependent of its oxidase activity. was cytotoxic to CCL-119 cells (Figure 3B). These findings in- dicated that the epitope is accessible to the antibody and is likely located on the external surface of the protein. The cytotoxic RESULTS effect of the antibody in the absence of altered amine oxidase activity also suggested that the antibody might decrease cell
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