BASIC RESEARCH www.jasn.org Activated Protein C Ameliorates Renal Ischemia- Reperfusion Injury by Restricting Y-Box Binding Protein-1 Ubiquitination † ‡ Wei Dong,* Hongjie Wang,* Khurrum Shahzad,* Fabian Bock,* Moh’d Mohanad Al-Dabet,* Satish Ranjan,* Juliane Wolter,* Shrey Kohli,* Juliane Hoffmann,* Vishnu Mukund Dhople,§ | | Cheng Zhu, Jonathan A. Lindquist, Charles T. Esmon,¶ Elisabeth Gröne,** Herman-Josef | †† Gröne,** Thati Madhusudhan,* Peter R. Mertens, Dirk Schlüter, and Berend Isermann* *Institute of Clinical Chemistry and Pathobiochemistry, Medical Faculty, |Department of Nephrology and Hypertension, Diabetes and Endocrinology, and ††Institute of Microbiology, Medical Faculty, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; †Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; ‡Department of Molecular Genetics, University of Health Sciences, Khayaban-e-Jamia Punjab, Lahore, Pakistan; §Department of Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany; ¶Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma; and **Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany ABSTRACT Ischemia-reperfusion injury (IRI) is the leading cause of ARF. A pathophysiologic role of the coagulation system in renal IRI has been established, but the functional relevance of thrombomodulin (TM)-dependent activated protein C (aPC) generation and the intracellular targets of aPC remain undefined. Here, we investigated the role of TM-dependent aPC generation and therapeutic aPC application in a murine renal IRI model and in an in vitro hypoxia and reoxygenation (HR) model using proximal tubular cells. In renal IRI, endogenous aPC levels were reduced. Genetic or therapeutic reconstitution of aPC efficiently ameliorated renal IRI independently of its anticoagulant properties. In tubular cells, cytoprotective aPC signaling was mediated through protease activated receptor-1- and endothelial protein C receptor-dependent regulation of the cold-shock protein Y-box binding protein-1 (YB-1). The mature 50 kD form of YB-1 was required for the nephro- and cytoprotective effects of aPC in vivo and in vitro, respectively. Reduction of mature YB-1 and K48-linked ubiquitination of YB-1 was prevented by aPC after renal IRI or tubular HR injury. aPC preserved the interaction of YB-1 with the deubiquitinating enzyme otubain-1 and maintained expression of otubain-1, which was required to reduce K48-linked YB-1 ubiquitination and to stabilize the 50 kD form of YB-1 after renal IRI and tubular HR injury. These data link the cyto- and nephroprotective effects of aPC with the ubiquitin-proteasome system and identify YB-1 as a novel intracellular target of aPC. These insights may provide new impetus for translational efforts aiming to restrict renal IRI. J Am Soc Nephrol 26: 2789–2799, 2015. doi: 10.1681/ASN.2014080846 Ischemia-reperfusion injury (IRI) is the leading cause Received August 31, 2014. Accepted January 6, 2015. ofARF.TheconsequencesofrenalIRIcanbedramatic, W.D., H.W., K.S., P.R.M., D.S., and B.I. contributed equally to this resulting in a marked decline of renal function and work. high mortality rates. A number of candidate mech- Published online ahead of print. Publication date available at anisms involved in renal IRI have been identified in www.jasn.org. preclinical studies, including mechanisms such as Correspondence: Dr. Berend Isermann, Institute of Clinical protein ubiquitination, endothelial dysfunction, or Chemistry and Pathobiochemistry, Otto-von-Guericke-University coagulation activation.1–5 Whether and how these Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany. Email: intra- and extracellular signaling mechanisms may [email protected] or [email protected] interact in renal IRI remains unknown. Copyright © 2015 by the American Society of Nephrology J Am Soc Nephrol 26: 2789–2799, 2015 ISSN : 1046-6673/2611-2789 2789 BASIC RESEARCH www.jasn.org Tissue factor-dependent coagulationandthrombinactivation system and YB-1. First, we established that renal IRI impairs in aggravate tubular injury through protease activated receptor-1 vivo PC activation (Figure 1A), reflecting impairment of the (PAR1)–dependent signaling.5 Unlike thrombin, the anticoagulant endogenous TM-PC system in this model. To evaluate the serine protease activated protein C (aPC) is nephroprotective, mechanistic relevance of altered endogenous PC activation, we ameliorating chronic6,7 and acute8,9 renal injury. The opposing ef- used genetically modified mice with impaired TM-dependent fects of thrombin and aPC are largely controlled by thrombomodulin PC activation (TMPro/Pro mice), resulting in low blood levels of (TM).10 TM is a type 1 transmembrane receptor which binds aPC, or mice expressing a hyperactivatable PC mutant, resulting thrombin, directing its activity toward protein C (PC) activation, in high blood levels of aPC (APChigh mice).6,24 Although no dif- therefore providing a functional switch between the serine proteases ferences were observed in sham-operated mice, BUN, creatinine, thrombin and aPC.10 In preclinical studies, therapeutic application tubular injury, and expression of the kidney injury marker of recombinant soluble TM ameliorates IRI in rats.11,12 However, kidney-injury-molecule 1 (KIM1) were significantly induced whether TM-dependent PC activation and signaling through aPC’s inwild-type(WT)miceandfurtherincreasedinTMPro/Pro IRI pivotal receptors PAR1 and endothelial protein C receptor (EPCR) mice (Figure 1, B–G, Supplemental Figure 1A). Of note, these are required for TM-dependent nephroprotection after IRI remains markers were markedly reduced in APChigh IRI mice in compar- unknown.13,14 In addition, although aPC mediated cytoprotection ison with WT IRI mice (Figure 1, B–G). The expression of YB-1 after hepatic IRI has been demonstrated,15 the role of aPC in renal was likewise significantly altered in mice with IRI. YB-1 levels de- IRI or aPC’s intracellular targets in IRI remains unknown. clined in WT IRI and to an even larger extent in TMPro/Pro IRI One potential intracellular target of coagulation proteases in mice, but they were preserved in APChigh mice (Figure 1, F and H). renal IRI is the cold-shock protein Y-box binding protein-1 (YB-1) To determine the therapeutic potential of aPC and the because (1) YB-1 activity is regulated by thrombin, (2) YB-1 is relevance of aPC’s anticoagulant function, we next treated regulated in cardiac IRI, and (3) YB-1 has an established role in mice with exogenous aPC (Supplemental Figure 1B). A subgroup renal diseases other than IRI.16–19 YB-1 is a highly conserved of mice received aPC preincubated with an antibody HAPC1573, protein involved in the regulation of inflammatory processes, which specifically inhibits aPC’s anticoagulant, but not its cyto- including sterile inflammation in renal diseases.19 YB-1 conveys protective effects.25 Both aPC and the aPC-HAPC1573 complex multiple functions through its interaction with DNA, mRNA, and were equally protective in the IRI model (Supplemental Figure 1, proteins.20,21 The diverse functions of YB-1 are in part regulated by its subcellular locali- zation and post-translational modifications, including ubiquitination.20,21 Subcellular lo- calization of YB-1 is furthermore regulated by the coagulation protease thrombin, which induces a partial degradation of YB-1 and nuclear translocation of the N-terminal YB- 1fragmentinendothelialcells.22,23 However, it remains unknown which receptors mediate these effects and whether other coagulation proteases regulate YB-1. Furthermore, the mechanisms through which coagulation pro- teases modulate partial YB-1 degradation (e.g., relevance of the proteasome or ubiquitination) remain unknown. We speculated that TM, coagulation proteases, and their receptors control renal IRI injury by modulating YB-1 function and that the partial degradation of YB-1 is controlled by the ubiquitin-proteasome system. Hence, we evaluated the effect of TM-dependent aPC generation on YB-1 in Figure 1. Protection from renal IRI by TM-dependent PC activation is associated with renal IRI in this study. sustained YB-1 expression. (A) Plasma levels of aPC are reduced in WT mice after renal IRI (black bars) compared with control (sham, open bars) mice. BUN (B) and creatinine (Crea) (C) in control (Sham) (open bars) and experimental (IRI) (black bars) WT, TMPro/Pro,andAPChigh mice. RESULTS Exemplary images of H&E-stained kidney section from WT, TMPro/Pro,andAPChigh mice without(Sham)orwithIRI(D)andabargraphsummarizing results of pathologic scores (E). Activated PC Maintains Renal YB-1 TM-dependent PC activation modulates KIM1 and YB-1 expression during renal IRI in vivo; Levels during IRI representative immunoblots (F) and bar graphs summarizing results [(G) and (H)]. Mean6SD Renal IRI was induced in mice to evaluate a values of at least six mice per group [(A)–(C), (E), (G), and (H)]; size bar: 20 mm(D);*P,0.05; possible interaction between the TM-PC **P,0.01 [(A): t test;(B),(C),(E),(G),and(H):ANOVA].H&E,hematoxylinandeosin. 2790 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 2789–2799, 2015 www.jasn.org BASIC RESEARCH C–G), and both maintained YB-1 levels (Supplemental Figure 1, challenged by hypoxia and reoxygenation (HR). Pretreatment