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• W2766157021 abstract "Ischemia/reperfusion is a common cause of acute kidney injury (AKI). However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we investigated the role of peptidyl arginine deiminase-4 (PAD4), which converts arginine to citrulline and plays a role in epigenetic regulation and inflammation, in renal ischemia/reperfusion injury. PAD4 expression was highly induced in infiltrating leukocytes 24 hours following renal ischemia and reperfusion. This induction was accompanied by citrullination of histone H3 and formation of neutrophil extracellular traps in kidneys of wild-type mice. By contrast, PAD4-deficient mice did not form neutrophil extracellular traps, expressed lower levels of pro-inflammatory cytokines and were partially protected from renal ischemia/reperfusion-induced AKI. Furthermore, PAD4-deficient mice recovered kidney function 48 hours after ischemia/reperfusion, whereas kidney function in the wild-type mice progressively worsened. Administration of DNase I, which degrades neutrophil extracellular traps or the PAD-specific inhibitor YW3-56 before ischemia, partially prevented renal ischemia/reperfusion-induced AKI. Notably, transfer of neutrophils from wild-type, but not from PAD4-deficient mice, was sufficient to restore renal neutrophil extracellular trap formation and impair kidney function following renal ischemia/reperfusion. Thus, neutrophil PAD4 plays a pivotal role in renal ischemia/reperfusion-induced AKI. Ischemia/reperfusion is a common cause of acute kidney injury (AKI). However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we investigated the role of peptidyl arginine deiminase-4 (PAD4), which converts arginine to citrulline and plays a role in epigenetic regulation and inflammation, in renal ischemia/reperfusion injury. PAD4 expression was highly induced in infiltrating leukocytes 24 hours following renal ischemia and reperfusion. This induction was accompanied by citrullination of histone H3 and formation of neutrophil extracellular traps in kidneys of wild-type mice. By contrast, PAD4-deficient mice did not form neutrophil extracellular traps, expressed lower levels of pro-inflammatory cytokines and were partially protected from renal ischemia/reperfusion-induced AKI. Furthermore, PAD4-deficient mice recovered kidney function 48 hours after ischemia/reperfusion, whereas kidney function in the wild-type mice progressively worsened. Administration of DNase I, which degrades neutrophil extracellular traps or the PAD-specific inhibitor YW3-56 before ischemia, partially prevented renal ischemia/reperfusion-induced AKI. Notably, transfer of neutrophils from wild-type, but not from PAD4-deficient mice, was sufficient to restore renal neutrophil extracellular trap formation and impair kidney function following renal ischemia/reperfusion. Thus, neutrophil PAD4 plays a pivotal role in renal ischemia/reperfusion-induced AKI. Acute kidney injury (AKI) results in high morbidity and mortality but has no effective treatment beyond supportive care.1National Institute for Health and Care Excellence (NICE). Acute kidney injury: prevention, detection and management up to the point of renal replacement therapy. In: National Clinical Guideline Centre, ed. NICE Clinical Guidelines, No. 169. London, UK: Royal College of Physicians. Available at: https://www.nice.org.uk/guidance/cg169. Accessed January 28, 2017.Google Scholar, 2Schrier R.W. Wang W. Poole B. et al.Acute renal failure: definitions, diagnosis, pathogenesis, and therapy.J Clin Invest. 2004; 114: 5-14Crossref PubMed Scopus (631) Google Scholar AKI induced by ischemia/reperfusion (I/R), the most common cause of AKI,3Kanagasundaram N.S. Pathophysiology of ischaemic acute kidney injury.Ann Clin Biochem. 2015; 52: 193-205Crossref PubMed Scopus (45) Google Scholar is characterized by injury to tubular epithelial cells4Thadhani R. Pascual M. Bonventre J.V. Acute renal failure.N Engl J Med. 1996; 334: 1448-1460Crossref PubMed Scopus (1511) Google Scholar, 5Lieberthal W. Biology of acute renal failure: therapeutic implications.Kidney Int. 1997; 52: 1102-1115Abstract Full Text PDF PubMed Scopus (63) Google Scholar and vascular endothelial cells,6Basile D.P. The endothelial cell in ischemic acute kidney injury: implications for acute and chronic function.Kidney Int. 2007; 72: 151-156Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 7Rabelink T.J. de Boer H.C. van Zonneveld A.J. Endothelial activation and circulating markers of endothelial activation in kidney disease.Nat Rev Nephrol. 2010; 6: 404-414Crossref PubMed Scopus (108) Google Scholar and robust inflammatory responses, including leukocyte infiltration and upregulation of chemokines and cytokines in the kidney.8Jang H.R. Rabb H. The innate immune response in ischemic acute kidney injury.Clin Immunol. 2009; 130: 41-50Crossref PubMed Scopus (266) Google Scholar, 9Linas S.L. Shanley P.F. Whittenburg D. et al.Neutrophils accentuate ischemia-reperfusion injury in isolated perfused rat kidneys.Am J Physiol. 1988; 255: F728-F735PubMed Google Scholar, 10Willinger C.C. Schramek H. Pfaller K. Pfaller W. Tissue distribution of neutrophils in postischemic acute renal failure.Virchows Arch B Cell Pathol Incl Mol Pathol. 1992; 62: 237-243Crossref PubMed Scopus (66) Google Scholar Although inflammatory responses to viral and bacterial infection are vital to host defense, tissue, and wound repair, I/R-triggered “sterile” inflammation may lead to tissue injury.11Chen G.Y. Nunez G. Sterile inflammation: sensing and reacting to damage.Nat Rev Immunol. 2010; 10: 826-837Crossref PubMed Scopus (2046) Google Scholar, 12Kalogeris T. Baines C.P. Krenz M. et al.Cell biology of ischemia/reperfusion injury.Int Rev Cell Mol Biol. 2012; 298: 229-317Crossref PubMed Scopus (1298) Google Scholar A relatively recently described pathway of neutrophil-induced injury involves the formation of neutrophil extracellular traps (NETs), which involves the decondensation of chromatin and subsequent extravasation of DNA into the extracellular space.13Cooper P.R. Palmer L.J. Chapple I.L. Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?.Periodontol 2000. 2013; 63: 165-197Crossref PubMed Scopus (114) Google Scholar Formation of NETs may aid clearance of bacteria during infection but also has been implicated in a growing list of autoimmune and inflammatory conditions,13Cooper P.R. Palmer L.J. Chapple I.L. Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?.Periodontol 2000. 2013; 63: 165-197Crossref PubMed Scopus (114) Google Scholar including ischemic injury to the heart,14Ge L. Zhou X. Ji W.J. et al.Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy.Am J Physiol Heart Circ Physiol. 2015; 308: H500-H509Crossref PubMed Scopus (129) Google Scholar liver,15Huang H. Tohme S. Al-Khafaji A.B. et al.Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury.Hepatology. 2015; 62: 600-614Crossref PubMed Scopus (281) Google Scholar muscle,16Oklu R. Albadawi H. Jones J.E. et al.Reduced hind limb ischemia-reperfusion injury in Toll-like receptor-4 mutant mice is associated with decreased neutrophil extracellular traps.J Vasc Surg. 2013; 58: 1627-1636Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar brain,17Enzmann G. Mysiorek C. Gorina R. et al.The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury.Acta Neuropathol. 2013; 125: 395-412Crossref PubMed Scopus (152) Google Scholar, 18Allen C. Thornton P. Denes A. et al.Neutrophil cerebrovascular transmigration triggers rapid neurotoxicity through release of proteases associated with decondensed DNA.J Immunol. 2012; 189: 381-392Crossref PubMed Scopus (143) Google Scholar and kidney.19Jansen M.P. Emal D. Teske G.J. et al.Release of extracellular DNA influences renal ischemia reperfusion injury by platelet activation and formation of neutrophil extracellular traps.Kidney Int. 2017; 91: 352-364Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 20Nakazawa D. Kumar S.V. Marschner J. et al.Histones and neutrophil extracellular traps enhance tubular necrosis and remote organ injury in ischemic AKI.J Am Soc Nephrol. 2017; 28: 1753-1768Crossref PubMed Scopus (160) Google Scholar The peptidyl arginine deiminase (PAD) family is comprised of 5 closely related proteins (PADs 1–4 and PAD6) that posttranslationally convert arginine to citrulline in proteins, thereby mediating signal transduction for diverse stimuli and affecting many biological processes.21Jones J.E. Causey C.P. Knuckley B. et al.Protein arginine deiminase 4 (PAD4): current understanding and future therapeutic potential.Curr Opin Drug Discov Devel. 2009; 12: 616-627PubMed Google Scholar Although PADs 1–3 and PAD6 are cytoplasmic enzymes,21Jones J.E. Causey C.P. Knuckley B. et al.Protein arginine deiminase 4 (PAD4): current understanding and future therapeutic potential.Curr Opin Drug Discov Devel. 2009; 12: 616-627PubMed Google Scholar PAD4 occurs in both the cytoplasm and the nucleus22Vossenaar E.R. Zendman A.J. van Venrooij W.J. et al.PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease.Bioessays. 2003; 25: 1106-1118Crossref PubMed Scopus (765) Google Scholar, 23Nakashima K. Hagiwara T. Yamada M. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes.J Biol Chem. 2002; 277: 49562-49568Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar and is primarily expressed in leukocytes, particularly in granulocytes.21Jones J.E. Causey C.P. Knuckley B. et al.Protein arginine deiminase 4 (PAD4): current understanding and future therapeutic potential.Curr Opin Drug Discov Devel. 2009; 12: 616-627PubMed Google Scholar The conversion of histone arginines to citrullines by PAD4 reduces the overall positive charges of histones and weakens histone-DNA binding, thereby disrupting the nuleosomes and triggering nuclear DNA release and formation of NETs.21Jones J.E. Causey C.P. Knuckley B. et al.Protein arginine deiminase 4 (PAD4): current understanding and future therapeutic potential.Curr Opin Drug Discov Devel. 2009; 12: 616-627PubMed Google Scholar Thus, PAD4 may be involved in signal transduction, leading to the inflammatory responses after I/R injury. Indeed, Rabadi et al.24Rabadi M. Kim M. D'Agati V. et al.Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion.Am J Physiol Renal Physiol. 2016; 311: F437-F449Crossref PubMed Scopus (37) Google Scholar reported that renal tubular cell PAD4 is highly induced by I/R injury and that PAD4-deficient mice are protected against I/R-induced kidney injury. Although neutrophil-PAD4 is implicated in renal I/R injury,20Nakazawa D. Kumar S.V. Marschner J. et al.Histones and neutrophil extracellular traps enhance tubular necrosis and remote organ injury in ischemic AKI.J Am Soc Nephrol. 2017; 28: 1753-1768Crossref PubMed Scopus (160) Google Scholar, 24Rabadi M. Kim M. D'Agati V. et al.Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion.Am J Physiol Renal Physiol. 2016; 311: F437-F449Crossref PubMed Scopus (37) Google Scholar nothing is known regarding its specific role in this pathologic context. Neutrophil PAD4 is especially relevant because it is essential to the formation of NETs, which evokes most aspects of inflammatory responses during autoimmunity and thrombosis.13Cooper P.R. Palmer L.J. Chapple I.L. Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?.Periodontol 2000. 2013; 63: 165-197Crossref PubMed Scopus (114) Google Scholar, 25Sørensen O.E. Borregaard N. Neutrophil extracellular traps—the dark side of neutrophils.J Clin Invest. 2016; 126: 1612-1620Crossref PubMed Scopus (268) Google Scholar Here, we tested the hypothesis that neutrophil PAD4 and NET formation are mechanistically linked to renal I/R-induced AKI. We found, in wild-type (WT) mice, the induction of PAD4 after renal I/R. PAD4 promoted inflammation after renal I/R injury and was essential for NET formation. DNase I treatment before renal I/R suppressed NET formation and partially protected mice from I/R-induced renal injury. A PAD-specific inhibitor, YW3-56, potently suppressed I/R-induced kidney injury in mice. In PAD4-deficient mice, transfer of neutrophils derived from WT mice was sufficient to promote NET formation in the kidney and loss of kidney function after I/R. By contrast, PAD4-knockout mice receiving neutrophils from PAD4-deficient mice showed no detectable NETs in the kidney and were protected from I/R-induced renal injury. To establish the biological role for PAD4 in renal I/R injury, we first assessed the expression and distribution of PAD4 and one of its products, citrullinated histone H3 (Cit-H3), in the kidney after ischemia. Relative to sham controls, PAD4 mRNA levels measured by quantitative reverse transcription–polymerase chain reaction (RT-PCR) were 3-fold higher 24 hours after I/R injury (n = 4; P < 0.01; Figure 1a). Citrullination of histone-H3 was not detectable in mice that underwent sham surgery and was markedly increased in mice 24 hours after I/R (Figure 1b). Thus, renal I/R induces PAD4 expression and activity. Although PAD4 is expressed mainly in hematopoietic cells,21Jones J.E. Causey C.P. Knuckley B. et al.Protein arginine deiminase 4 (PAD4): current understanding and future therapeutic potential.Curr Opin Drug Discov Devel. 2009; 12: 616-627PubMed Google Scholar it also has been described in renal epithelial cells.24Rabadi M. Kim M. D'Agati V. et al.Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion.Am J Physiol Renal Physiol. 2016; 311: F437-F449Crossref PubMed Scopus (37) Google Scholar We performed immunolocalization of PAD4 and Cit-H3 in kidneys after ischemia to determine the sites of PAD4 expression and activity. Immunofluorescence microscopy showed few detectable PAD4 (Figure 1c) or Cit-H3 (Figure 1d) signals co-localizing with nuclear DNA in kidneys from WT and PAD4 knock-out (PAD4KO) mice after sham surgery. By contrast, 24 hours after I/R injury, PAD4 (Figure 1c) and Cit-H3 (Figure 1d) were dramatically increased in the kidneys of WT mice, but they were barely detectable in the PAD4KO mice. Double labeling of PAD4 or Cit-H3 with a neutrophil marker, Ly6G, revealed that most of the PAD4- and Cit-H3-expressing cells in the ischemic kidneys were neutrophils (Figure 1, c and d). Although faint PAD4 signals were observed in renal epithelial cells from sham WT kidneys, they did not increase after I/R. Thus, renal I/R injury induces PAD4 expression and triggers Cit-H3 production mainly in infiltrating neutrophils. To determine whether PAD4 is involved in renal I/R injury, we assessed kidney function 24 hours and 48 hours after I/R in WT and PAD4KO mice. As expected, WT mice developed severe kidney dysfunction, as reflected by elevated plasma creatinine (Figure 2a) and blood urea nitrogen (BUN; Figure 2b) concentrations. In contrast, BUN (P < 0.001) and creatinine (P < 0.001) levels were significantly lower in PAD4KO mice, compared with WT mice 24 hours after I/R. Indeed, 48 hours after I/R injury, levels of both creatinine and BUN returned to the normal ranges in the PAD4KO mice, but they continued to climb in the WT mice (Figure 2, a and b). Likewise, mRNA levels of kidney injury molecule-1 (KIM-1), an established biomarker for renal proximal tubule injury,26Devarajan P. Biomarkers for the early detection of acute kidney injury.Curr Opin Pediatr. 2011; 23: 194-200Crossref PubMed Scopus (202) Google Scholar were barely detectable in the kidneys of both WT and PAD4KO mice that underwent sham surgery but increased dramatically 24 hours after renal I/R injury in WT mice (Figure 2c). The induction of KIM-1 expression after I/R was lower in PAD4KO mice (P < 0.001; Figure 2c). Finally, histologic evidence of injury was assessed in kidneys from WT and PAD4KO mice subjected to I/R (Figure 2d). Kidneys from sham-operated mice of either genotype displayed normal morphology, with well-preserved brush border membranes and no loss of tubular epithelial cells. Kidneys from WT mice subjected to I/R showed marked tubular injury as reflected by loss of brush border, cast formation, and extensive loss of tubular epithelial cells, tubular dilation, and intra-tubular debris (Figure 2d). Kidneys from PAD4KO mice subjected to I/R also showed evidence of injury, but less than that seen in WT mice (Figure 2d). Semiquantitative assessment of kidney tissue injury yielded tubular necrosis scores of 0.07 ± 0.01 and 0.09 ± 0.04, respectively, in WT and PADK4KO mice that received sham surgery, and 3.4 ± 0.06 and 2.1 ± 0.4 (P < 0.05) in WT and PAD4KO mice 24 hours after I/R injury (Figure 2e). These data support the conclusion that PAD4 mediates renal I/R-induced injury. Infiltration of inflammatory cells into the renal parenchyma occurs early in the course of renal I/R injury.10Willinger C.C. Schramek H. Pfaller K. Pfaller W. Tissue distribution of neutrophils in postischemic acute renal failure.Virchows Arch B Cell Pathol Incl Mol Pathol. 1992; 62: 237-243Crossref PubMed Scopus (66) Google Scholar, 27Ysebaert D.K. De Greef K.E. Vercauteren S.R. et al.Identification and kinetics of leukocytes after severe ischaemia/reperfusion renal injury.Nephrol Dial Transplant. 2000; 15: 1562-1574Crossref PubMed Scopus (307) Google Scholar Neutrophils are key effectors of the inflammatory cascade in a variety of kidney injury models, including I/R.10Willinger C.C. Schramek H. Pfaller K. Pfaller W. Tissue distribution of neutrophils in postischemic acute renal failure.Virchows Arch B Cell Pathol Incl Mol Pathol. 1992; 62: 237-243Crossref PubMed Scopus (66) Google Scholar, 27Ysebaert D.K. De Greef K.E. Vercauteren S.R. et al.Identification and kinetics of leukocytes after severe ischaemia/reperfusion renal injury.Nephrol Dial Transplant. 2000; 15: 1562-1574Crossref PubMed Scopus (307) Google Scholar We therefore assessed the role of PAD4 in renal I/R-triggered inflammatory responses and neutrophil infiltration. As shown in Figure 3a, neutrophil elastase (NE)–positive neutrophils were hardly detectable in the kidneys of WT and PAD4KO mice subjected to sham surgery. I/R induced significant neutrophil infiltration in kidneys of WT mice, and to a lesser extent, in PAD4KO mice 24 hours after I/R (P < 0.01; Figure 3a). To test whether PAD4 deficiency affects neutrophil mobility, we performed a Boyden trans-well migration assay using 10% fetal bovine serum as a chemo-attractant. Supplementary Figure S1 shows that neutrophil migration rates were similar in WT and PAD4KO mice. In addition, no difference in total number of white blood cells in the peripheral blood was observed (Supplementary Figure S2A). Next, we tested whether PAD4 deficiency reduced the circulating platelet number, which could impair neutrophil infiltration after I/R injury.19Jansen M.P. Emal D. Teske G.J. et al.Release of extracellular DNA influences renal ischemia reperfusion injury by platelet activation and formation of neutrophil extracellular traps.Kidney Int. 2017; 91: 352-364Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Supplementary Figure S2B shows that total platelet count was similar in WT and PAD4KO mice. To identify the role of PAD4 in pro- and anti-inflammatory cytokine production, we performed quantitative RT-PCR analyses 24 hours after renal I/R injury. Basal expressions of tumor necrosis factor–-α, interleukin (IL)-6, IL-8, and IL-10 were not affected by PAD4 deficiency (Figure 3b). The expression of tumor necrosis factor–α, IL-6, and IL-8 increased in WT mice 24 hours after I/R injury, but these increases were significantly blunted in PAD4-deficient mice (Figure 3b). In contrast, the expression of the anti-inflammatory cytokine IL-10 was significantly enhanced in the absence of PAD4 (Figure 3b). These data support the hypothesis that PAD4 mediates renal I/R-induced upregulation of pro-inflammatory cytokines, and that endogenous PAD4 activity may also suppress the expression of anti-inflammatory cytokines. Whereas neutrophil extracellular traps (NETs) mediate certain forms of tissue injury,28Hakkim A. Fürnrohr B.G. Amann K. et al.Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis.Proc Natl Acad Sci U S A. 2010; 107: 9813-9818Crossref PubMed Scopus (994) Google Scholar, 29Liu F.C. Chuang Y.H. Tsai Y.F. et al.Role of neutrophil extracellular traps following injury.Shock. 2014; 41: 491-498Crossref PubMed Scopus (52) Google Scholar their role in renal I/R injury is not well defined. To determine whether infiltrating neutrophils form NETs after I/R-induced injury, we examined kidney sections by confocal microscopy using 4′,6-diamidino-2-phenylindole (DAPI) and anti-NE antibody to test for co-localization of DNA and neutrophil granule proteins. Among intact neutrophils, NE staining revealed granular cytoplasmic patterns surrounding clearly defined multi-lobulated nuclei (Figure 4a, top). In WT mice, 24 hours after I/R injury, DAPI staining revealed occasional sheets of extracellular DNA co-localizing with diffuse NE signals, signifying NET formation (Figure 4a, bottom). We next determined whether PAD4 activity is required for renal I/R–induced NET formation. First, we confirmed that PAD4 activity is essential for histone citrullination, a prerequisite for NET formation; Figure 4b reveals that I/R-induced histone citrullination was completely abolished by PAD4 deficiency, demonstrating that PAD4 activity is essential for histone citrullination in response to I/R. Quantification of NETs revealed that kidneys of WT and PADKO mice that underwent sham surgery contained no detectable NETs, whereas those of WT mice 24 hours after I/R injury contained NETs, albeit involving only a small fraction of the total amount of neutrophils (Figure 4c). Kidney sections from PAD4KO mice contained fewer NETs compared with WT mice (P < 0.001), corroborating the Cit-H3 immunoblot data (Figure 4b). Taken together, these findings are consistent with previous evidence supporting the hypothesis that PAD4 is essential for efficient NET formation.30Wang Y. Li M. Stadler S. et al.Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.J Cell Biol. 2009; 184: 205-213Crossref PubMed Scopus (941) Google Scholar More broadly, we conclude that NETs are formed in the kidney after I/R in a PAD4-dependent fashion. Concentrations of circulating cell-free DNA (cfDNA) are a potential marker of NET formation31Lande R. Ganguly D. Facchinetti V. et al.Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.Sci Transl Med. 2011; 3: 73ra19Crossref PubMed Scopus (922) Google Scholar and may be mechanistically linked to inflammation through activation of toll-like receptors.32Nishimoto S. Fukuda D. Higashikuni Y. et al.Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.Sci Adv. 2016; 2e1501332Crossref PubMed Scopus (152) Google Scholar, 33Atamaniuk J. Kopecky C. Skoupy S. et al.Apoptotic cell-free DNA promotes inflammation in haemodialysis patients.Nephrol Dial Transplant. 2012; 27: 902-905Crossref PubMed Scopus (76) Google Scholar, 34Scharfe-Nugent A. Corr S.C. Carpenter S.B. et al.TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia.J Immunol. 2012; 188: 5706-5712Crossref PubMed Scopus (137) Google Scholar, 35Barrat F.J. Meeker T. Gregorio J. et al.Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus.J Exp Med. 2005; 202: 1131-1139Crossref PubMed Scopus (751) Google Scholar Twenty-four hours after I/R, circulating cfDNA concentrations increased in WT mice compared with WT mice that underwent sham surgery (P < 0.0002; Figure 5a). By contrast, PAD4KO deficiency blunted, but did not completely prevent, the renal I/R–triggered increase in circulating cfDNA (P < 0.005; Figure 5a). Thus, PAD4 at least partially mediates renal I/R–induced cfDNA release. Our findings reveal that NETs are formed in the kidney after I/R. Given that NETs contain extracellular DNA, we tested the therapeutic potential of DNase I administration for renal I/R–induced NET formation, cfDNA release, and kidney dysfunction. I.v. injection of DNase I suppressed the increase in plasma cfDNA 24 hours after renal I/R injury (P < 0.05; Figure 5b) with a concomitant reduction of kidney NETs, compared with I/R WT mice that received saline (P < 0.001; Figure 5c). Injection of DNase I also reduced plasma creatinine (P < 0.05; Figure 5d) and BUN levels (P < 0.01; Figure 5e) compared with the levels in saline-treated mice. These results further support the conclusion that NET formation and release of extracellular DNA are pathogenic components of renal I/R. Conversely, to determine whether inhibition of PAD4 activity is sufficient to prevent I/R-induced NET formation and renal injury, we treated WT mice with either vehicle or YW3-56 (6-dimethylaminonaph-2-yl-{N-S-[1-benzylcarba-moyl-4-(2-chloroacetamidobutyl)]-carboxamide), a PAD-specific inhibitor,36Wang Y. Li P. Wang S. et al.Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.J Biol Chem. 2012; 287: 25941-25953Crossref PubMed Scopus (119) Google Scholar 30 minutes before renal I/R. Twenty-four hours after I/R injury, plasma concentrations of creatinine (P < 0.01; Figure 5f) and BUN (P < 0.001; Figure 5g) were both markedly lower in mice that received YW3-56, compared with those that received vehicle, indicating that PAD4 inhibition protects mice from I/R-induced kidney injury. PAD4 is expressed at high levels in neutrophils.21Jones J.E. Causey C.P. Knuckley B. et al.Protein arginine deiminase 4 (PAD4): current understanding and future therapeutic potential.Curr Opin Drug Discov Devel. 2009; 12: 616-627PubMed Google Scholar However, other cells, such as monocytes,37Vossenaar E.R. Radstake T.R. van der Heijden A. et al.Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages.Ann Rheum Dis. 2004; 63: 373-381Crossref PubMed Scopus (349) Google Scholar macrophages,37Vossenaar E.R. Radstake T.R. van der Heijden A. et al.Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages.Ann Rheum Dis. 2004; 63: 373-381Crossref PubMed Scopus (349) Google Scholar and renal epithelial cells,24Rabadi M. Kim M. D'Agati V. et al.Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion.Am J Physiol Renal Physiol. 2016; 311: F437-F449Crossref PubMed Scopus (37) Google Scholar have been reported to express PAD4. To determine if neutrophil PAD4 mediates renal I/R–induced kidney injury, we performed neutrophil transfer experiments. Specifically, we isolated neutrophils from either WT or PAD4KO mice and transferred them to PAD4KO mice 30 minutes before performing renal I/R. Immunofluorescence microscopy of tissue obtained 24 hours after renal I/R revealed that the number of infiltrating neutrophils in the kidneys of PAD4KO mice receiving either WT or PAD4-deficient neutrophils was similar (Figure 6a). However, large numbers of NETs were present in the kidneys of PAD4KO mice that received WT neutrophils, whereas PAD4KO mice that received PAD4-deficient neutrophils did not reveal detectable NETs in the kidney (P < 0.001; Figure 6b). Notably, PAD4-deficient mice receiving WT neutrophils developed substantially more severe renal dysfunction 24 hours after renal I/R, as evidenced by increased creatinine (1.83 ± 0.17 vs. 0.44 ± 0.04, P < 0.0001; Figure 6c) and BUN (136 ± 8 vs. 63 ± 10, P < 0.001; Figure 6d) levels than did PAD4-deficient mice receiving PAD4-deficient neutrophils (Figure 6, c and d). These results strongly support an important role for neutrophil-PAD4 in renal I/R injury and NET formation. Although neutrophils have been implicated in kidney I/R injury,38Bolisetty S. Agarwal A. Neutrophils in acute kidney injury: not neutral any more.Kidney Int. 2009; 75: 674-676Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar the mechanisms whereby neutrophils contribute to injury remain unclear. The release of nuclear constituents, such as DNA, histones, and high-mobility group box-1 (HMGB1), triggers inflammatory pathways to exacerbate tissue ischemia and injury. Although NETs, formed by the decondensation and extrusion of nuclear DNA,13Cooper P.R. Palmer L.J. Chapple I.L. Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?.Periodontol 2000. 2013; 63: 165-197Crossref PubMed Scopus (114) Google Scholar, 25Sørensen O.E. Borregaard N. Neutrophil extracellular traps—the dark side of neutrophils.J Clin Invest. 2016; 126: 1612-1620Crossref PubMed Scopus (268) Google Scholar may protect against bacterial infection, recent work indicates that NETs may cause tissue injury during sterile inflammation, such as I/R injury.11Chen G.Y. Nunez G. Sterile inflammation: sensing and reacting to damage.Nat Rev Immunol. 2010; 10: 826-837Crossref PubMed Scopus (2046) Google Scholar, 12Kal" @default.
• W2766157021 created "2017-11-10" @default.
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• W2766157021 date "2018-02-01" @default.
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• W2766157021 title "Neutrophil peptidyl arginine deiminase-4 has a pivotal role in ischemia/reperfusion-induced acute kidney injury" @default.
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