Renalase Prevents AKI Independent of Amine Oxidase Activity

Ling Wang

Heino Velazquez

Gilbert Moeckel

John Chang

^{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}

^{Corresponding author.}

Correspondence: Dr. Gary Desir, Section of Nephrology, Department of Medicine, Yale School of Medicine, P.O. Box 208029, New Haven, CT 06520-8029. Email: ude.elay@rised.yrag

Received 2013 Jun 26; Accepted 2013 Oct 16.

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 kinases 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.

Abstract

AKI is a common clinical condition affecting up to 20% of hospitalized patients and is frequently associated with sepsis, surgery, and certain drugs. Epidemiologic data indicate a positive association between the severity of AKI and in-hospital and long-term mortality.¹,² Unfortunately, the development of effective therapy for AKI has been hampered by (1) an inherent delay in diagnosis, a consequence of relying on serum creatinine, which only increases 48–72 hours after the original insult, and (2) the paucity of validated targets of therapy. There is an urgent need to identify novel therapeutic modalities.

Renalase is a novel secretory flavoprotein with amine oxidase activity.³–⁵In vitro, renalase metabolizes epinephrine, norepinephrine, and dopamine and also possesses significant intrinsic nicotinamide adenine dinucleotide (NADH) oxidase activity.⁶,⁷ Other investigators have questioned the amine oxidase activity of renalase.⁸,⁹ We had proposed that, in contrast to the classic amine oxidases, renalase reacts with oxygen to generate superoxide anions and hydrogen peroxide, with subsequent oxidation of catecholamines to their respective aminochromes. Recent results indicate that renalase functions as an oxidase/anomerase, using molecular oxygen to convert α-NAD(P)H to β-NAD^{, with hydrogen peroxide as reaction byproduct.}¹⁰ Because it was also shown to bind epinephrine, the authors pointed out that the hydrogen peroxide (H₂O₂) generated from the anomerase reaction will drive the oxidation of epinephrine to adrenochrome, albeit at a slower rate. Single-nucleotide polymorphisms present in the gene are associated with hypertension, cardiac disease, and diabetes.⁶,¹¹–¹⁴ The administration of renalase in wild-type (WT) mice lowers plasma catecholamines and systemic BP. In contrast, the deficiency of renalase in renalase knockout (KO) mice raises catecholamine levels and BP.¹⁵ Renalase also modulates the severity of renal ischemia and reperfusion injury.¹⁶ In WT mice, the administration of recombinant human renalase before induction of renal ischemia significantly blunts the severity of renal injury, with less renal tubular necrosis, inflammation, and apoptosis. In contrast, the lack of renalase in renalase KO mice exacerbates the renal damage after similar ischemic injury.

AKI is characterized by an elevation in plasma catecholamine levels. It has been postulated that, in addition to causing hypertension, excess catecholamines in AKI may produce an inflammatory response, aggravating tissue damage and contributing to multiorgan dysfunction.¹⁷ Interestingly, renalase levels in the blood and kidneys of WT mice are reduced following acute renal ischemia. Because the renalase in blood is secreted from the kidneys and is thought to metabolize circulating catecholamines, the excess catecholamines in AKI may be a direct consequence of the concurrent renalase deficiency. Notably, the administration of renalase to WT mice before induction of ischemia, which greatly attenuates ischemic renal injury, dampens the rise in blood catecholamine levels. On the basis of these findings, it is inviting to speculate that the renal protective effect in ischemic injury of renalase and its hemodynamic effect stem from its amine oxidase activity.

The role of amine oxidase activity of renalase in mediating its hemodynamic effect has been questioned. First, measurement of the amine oxidase activity of renalase relies on the production of H₂O₂ as an indirect measure of oxidase activity. Because the measured rate of H₂O₂ synthesis is low, the putative oxidase activity of renalase has been deemed unlikely to have physiologic significance.⁹ Second, the recombinant renalase synthesized in Escherichia coli with a histidine-tag possesses no detectable oxidase activity, and yet it markedly lowers BP when injected into rats.⁸,¹⁸

In this study, we sought to clarify the role of amine oxidase activity in the renal protective effects of renalase and to explore an additional mechanism of action of renalase that is independent of its oxidase activity.

Acknowledgments

This work was supported in part by Veterans Affairs Connecticut Healthcare System (H.V., R.S., and G.V.D.); National Institutes of Health grants RC1DK086465;, RC1DK086402;, and R01DK081037 (G.V.D.); National Basic Research Program of China 973 Program 2012CB517600, 2012CB517602; Shanghai Health Bureau ZXSNXD-CC-ZDYJ002 (W.L.); and Department of Anesthesiology, Columbia University (H.T.L.)

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

Footnotes

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