Vascular adhesion protein-1 enhances neutrophil infiltration by generation of hydrogen peroxide in renal ischemia/reperfusion injury
2017; Elsevier BV; Volume: 92; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2017.01.014
ISSN1523-1755
AutoresShinji Tanaka, Tetsuhiro Tanaka, Takahisa Kawakami, Hideki Takano, Mai Sugahara, Hisako Saito, Yoshiki Higashijima, Junna Yamaguchi, Reiko Inagi, Masaomi Nangaku,
Tópico(s)Acute Kidney Injury Research
ResumoVascular adhesion protein-1 (VAP-1) is a unique molecule since it acts as an adhesion molecule as well as an ectoenzyme catalyzing oxidative deamination of primary amines and generates hydrogen peroxide in the extracellular space. While VAP-1 is implicated in various inflammatory diseases, its role in acute kidney injury is less characterized. Here we studied VAP-1 expression in the kidney and the effect of its inhibition in a rat model of renal ischemia/reperfusion injury. VAP-1 was predominantly expressed in pericytes, which released enzymatically active enzyme. In vivo, a specific VAP-1 inhibitor, RTU-1096, significantly ameliorated rat renal ischemia/reperfusion injury and decreased neutrophil infiltration measured 12 hours after injury without altering macrophage or T lymphocyte populations. The protective effect of VAP-1 inhibition was lost in neutrophil-depleted rats, suggesting its inhibition ameliorated renal ischemia/reperfusion injury by suppressing neutrophil infiltration. To investigate whether hydrogen peroxide generated by VAP-1 enzyme reaction enhances neutrophil infiltration, we conducted an under-agarose migration assay with purified human neutrophils. Recombinant human VAP-1 significantly induced neutrophil migration, which was almost completely inhibited by RTU-1096 or catalase. Thus, VAP-1 plays a critical role in the pathophysiology of renal ischemia/reperfusion injury by enhancement of neutrophil infiltration generating a local hydrogen peroxide gradient. Hence, VAP-1 inhibition may be a novel therapy in ischemic acute kidney injury. Vascular adhesion protein-1 (VAP-1) is a unique molecule since it acts as an adhesion molecule as well as an ectoenzyme catalyzing oxidative deamination of primary amines and generates hydrogen peroxide in the extracellular space. While VAP-1 is implicated in various inflammatory diseases, its role in acute kidney injury is less characterized. Here we studied VAP-1 expression in the kidney and the effect of its inhibition in a rat model of renal ischemia/reperfusion injury. VAP-1 was predominantly expressed in pericytes, which released enzymatically active enzyme. In vivo, a specific VAP-1 inhibitor, RTU-1096, significantly ameliorated rat renal ischemia/reperfusion injury and decreased neutrophil infiltration measured 12 hours after injury without altering macrophage or T lymphocyte populations. The protective effect of VAP-1 inhibition was lost in neutrophil-depleted rats, suggesting its inhibition ameliorated renal ischemia/reperfusion injury by suppressing neutrophil infiltration. To investigate whether hydrogen peroxide generated by VAP-1 enzyme reaction enhances neutrophil infiltration, we conducted an under-agarose migration assay with purified human neutrophils. Recombinant human VAP-1 significantly induced neutrophil migration, which was almost completely inhibited by RTU-1096 or catalase. Thus, VAP-1 plays a critical role in the pathophysiology of renal ischemia/reperfusion injury by enhancement of neutrophil infiltration generating a local hydrogen peroxide gradient. Hence, VAP-1 inhibition may be a novel therapy in ischemic acute kidney injury. Acute kidney injury (AKI) is an emerging clinical concern shown to be strongly associated with increased mortality,1Lassnigg A. Schmidlin D. Mouhieddine M. et al.Minimal changes of serum creatinine predict prognosis in patients after cardiothoracic surgery: a prospective cohort study.J Am Soc Nephrol. 2004; 15: 1597-1605Crossref PubMed Scopus (1090) Google Scholar, 2Srisawat N. Hoste E.E. Kellum J.A. Modern classification of acute kidney injury.Blood Purif. 2010; 29: 300-307Crossref PubMed Scopus (105) Google Scholar and AKI episodes can lead to chronic kidney disease.3Tanaka S. Tanaka T. Nangaku M. Hypoxia as a key player in the AKI-to-CKD transition.Am J Physiol Renal Physiol. 2014; 307: F1187-F1195Crossref PubMed Scopus (166) Google Scholar, 4Coca S.G. Singanamala S. Parikh C.R. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis.Kidney Int. 2012; 81: 442-448Abstract Full Text Full Text PDF PubMed Scopus (1369) Google Scholar However, no drug has been approved for AKI treatment in humans. There is an urgent need to develop a novel therapeutic strategy for AKI. The renal ischemia-reperfusion (IR) injury model is frequently used in AKI studies,5Eltzschig H.K. Eckle T. Ischemia and reperfusion–from mechanism to translation.Nat Med. 2011; 17: 1391-1401Crossref PubMed Scopus (2104) Google Scholar because ischemia is the most important clinical cause of human AKI.6Lameire N.H. Bagga A. Cruz D. et al.Acute kidney injury: an increasing global concern.Lancet. 2013; 382: 170-179Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar In renal IR injury, innate immunity plays a critical role in pathogenesis.7Li L. Okusa M.D. Macrophages, dendritic cells, and kidney ischemia-reperfusion injury.Semin Nephrol. 2010; 30: 268-277Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar Leukocyte infiltration into IR-injured kidneys starts with the interaction between leukocytes and various adhesion molecules in endothelial cells, such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and selectins. Blocking these molecules successfully ameliorated renal IR injury,8Singbartl K. Ley K. Leukocyte recruitment and acute renal failure.J Mol Med (Berl). 2004; 82: 91-101Crossref PubMed Scopus (65) Google Scholar, 9Rabb H. Mendiola C.C. Dietz J. et al.Role of CD11a and CD11b in ischemic acute renal failure in rats.Am J Physiol. 1994; 267: F1052-F1058PubMed Google Scholar, 10Kelly K.J. Williams W.W. Colvin R.B. et al.Antibody to intercellular adhesion molecule 1 protects the kidney against ischemic injury.Proc Natl Acad Sci U S A. 1994; 91: 812-816Crossref PubMed Scopus (460) Google Scholar, 11Rabb H. Mendiola C.C. Saba S.R. et al.Antibodies to ICAM-1 protect kidneys in severe ischemic reperfusion injury.Biochem Biophys Res Commun. 1995; 211: 67-73Crossref PubMed Scopus (137) Google Scholar, 12Nemoto T. Burne M.J. Daniels F. et al.Small molecule selectin ligand inhibition improves outcome in ischemic acute renal failure.Kidney Int. 2001; 60: 2205-2214Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar and efforts are under way to translate this therapeutic concept into clinical settings but without much success. For example, an anti–ICAM-1 antibody in renal transplant recipients failed to demonstrate decreased rates of delayed graft function or acute rejection in a randomized controlled trial.13Salmela K. Wramner L. Ekberg H. et al.A randomized multicenter trial of the anti-ICAM-1 monoclonal antibody (enlimomab) for the prevention of acute rejection and delayed onset of graft function in cadaveric renal transplantation: a report of the European Anti-ICAM-1 Renal Transplant Study Group.Transplantation. 1999; 67: 729-736Crossref PubMed Scopus (157) Google Scholar Additional strategies are eagerly sought to block leukocyte infiltration into injured kidneys. Vascular adhesion protein-1 (VAP-1) is another adhesion molecule expressed in endothelial cells and is regarded as a candidate therapeutic target.14Pannecoeck R. Serruys D. Benmeridja L. et al.Vascular adhesion protein-1: Role in human pathology and application as a biomarker.Crit Rev Clin Lab Sci. 2015; 52: 284-300Crossref PubMed Scopus (61) Google Scholar, 15Salmi M. Jalkanen S. VAP-1: an adhesin and an enzyme.Trends Immunol. 2001; 22: 211-216Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar It is a 170- to 180-kDa homodimeric transmembrane glycoprotein.16Kaitaniemi S. Grön K. Elovaara H. et al.Functional modulation of vascular adhesion protein-1 by a novel splice variant.PLoS One. 2013; 8: e54151Crossref PubMed Scopus (8) Google Scholar, 17Noonan T. Lukas S. Peet G.W. et al.The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.Am J Clin Exp Immunol. 2013; 2: 172-185PubMed Google Scholar In addition to the transmembrane form, a soluble form of VAP-1 (sVAP-1) is detected in circulation. sVAP-1 can be produced via shedding of the transmembrane form, but the function and regulation of this shedding are not yet determined. This molecule is unique because it also acts as an ectoenzyme catalyzing oxidative deamination of primary amines (e.g., benzylamine) in the extracellular space (R-CH2-NH2 + O2 + H2O → R-CHO + NH3 + H2O2). In initial studies, VAP-1 expression was confirmed in smooth muscle cells, pericytes, and adipocytes, as well as in endothelial cells in various organs,18Salmi M. Jalkanen S. A 90-kilodalton endothelial cell molecule mediating lymphocyte binding in humans.Science. 1992; 257: 1407-1409Crossref PubMed Scopus (266) Google Scholar, 19Salmi M. Kalimo K. Jalkanen S. Induction and function of vascular adhesion protein-1 at sites of inflammation.J Exp Med. 1993; 178: 2255-2260Crossref PubMed Scopus (201) Google Scholar, 20Jaakkola K. Kaunismäki K. Tohka S. et al.Human vascular adhesion protein-1 in smooth muscle cells.Am J Pathol. 1999; 155: 1953-1965Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Smith D.J. Vainio P.J. Targeting vascular adhesion protein-1 to treat autoimmune and inflammatory diseases.Ann N Y Acad Sci. 2007; 1110: 382-388Crossref PubMed Scopus (20) Google Scholar and most functional studies on VAP-1 were limited to endothelial cells. Interestingly, a recent study by Weston et al.22Weston C.J. Shepherd E.L. Claridge L.C. et al.Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.J Clin Invest. 2015; 125: 501-520Crossref PubMed Scopus (132) Google Scholar revealed that VAP-1 is predominantly expressed in stellate cells (pericytes) in the liver and that its enzyme activity enhances lymphocyte infiltration, resulting in inflammation and fibrosis. In the kidney, VAP-1 is expressed mainly in the interstitium,23Kurkijärvi R. Jalkanen S. Isoniemi H. et al.Vascular adhesion protein-1 (VAP-1) mediates lymphocyte-endothelial interactions in chronic kidney rejection.Eur J Immunol. 2001; 31: 2876-2884Crossref PubMed Scopus (25) Google Scholar with detailed cellular localization remaining obscure. Because VAP-1 enhances leukocyte infiltration into inflammatory tissues and its enzymatic activity plays a pivotal role,14Pannecoeck R. Serruys D. Benmeridja L. et al.Vascular adhesion protein-1: Role in human pathology and application as a biomarker.Crit Rev Clin Lab Sci. 2015; 52: 284-300Crossref PubMed Scopus (61) Google Scholar, 24Stolen C.M. Marttila-Ichihara F. Koskinen K. et al.Absence of the endothelial oxidase AOC3 leads to abnormal leukocyte traffic in vivo.Immunity. 2005; 22: 105-115Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar inhibition of its enzyme activity could be a novel anti-inflammation strategy. Inhibition of VAP-1 enzyme activity in animals resulted in decreased leukocyte infiltration and ameliorated inflammation in various organs, such as intestine,25Kiss J. Jalkanen S. Fülöp F. et al.Ischemia-reperfusion injury is attenuated in VAP-1-deficient mice and by VAP-1 inhibitors.Eur J Immunol. 2008; 38: 3041-3049Crossref PubMed Scopus (29) Google Scholar, 26Salter-Cid L.M. Wang E. O'Rourke A.M. et al.Anti-inflammatory effects of inhibiting the amine oxidase activity of semicarbazide-sensitive amine oxidase.J Pharmacol Exp Ther. 2005; 315: 553-562Crossref PubMed Scopus (64) Google Scholar liver,22Weston C.J. Shepherd E.L. Claridge L.C. et al.Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis.J Clin Invest. 2015; 125: 501-520Crossref PubMed Scopus (132) Google Scholar lung,25Kiss J. Jalkanen S. Fülöp F. et al.Ischemia-reperfusion injury is attenuated in VAP-1-deficient mice and by VAP-1 inhibitors.Eur J Immunol. 2008; 38: 3041-3049Crossref PubMed Scopus (29) Google Scholar, 27Schilter H.C. Collison A. Russo R.C. et al.Effects of an anti-inflammatory VAP-1/SSAO inhibitor, PXS-4728A, on pulmonary neutrophil migration.Respir Res. 2015; 16: 42Crossref PubMed Scopus (39) Google Scholar, 28O'Rourke A.M. Wang E.Y. Miller A. et al.Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity.J Pharmacol Exp Ther. 2008; 324: 867-875Crossref PubMed Scopus (51) Google Scholar eye,29Noda K. Miyahara S. Nakazawa T. et al.Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis.FASEB J. 2008; 22: 1094-1103Crossref PubMed Scopus (46) Google Scholar and brain.30Xu H. Testai F.D. Valyi-Nagy T. et al.VAP-1 blockade prevents subarachnoid hemorrhage-associated cerebrovascular dilating dysfunction via repression of a neutrophil recruitment-related mechanism.Brain Res. 2015; 1603: 141-149Crossref PubMed Scopus (26) Google Scholar, 31Xu H.L. Garcia M. Testai F. et al.Pharmacologic blockade of vascular adhesion protein-1 lessens neurologic dysfunction in rats subjected to subarachnoid hemorrhage.Brain Res. 2014; 1586: 83-89Crossref PubMed Scopus (23) Google Scholar, 32Xu H.L. Salter-Cid L. Linnik M.D. et al.Vascular adhesion protein-1 plays an important role in postischemic inflammation and neuropathology in diabetic, estrogen-treated ovariectomized female rats subjected to transient forebrain ischemia.J Pharmacol Exp Ther. 2006; 317: 19-29Crossref PubMed Scopus (47) Google Scholar, 33Watcharotayangul J. Mao L. Xu H. et al.Post-ischemic vascular adhesion protein-1 inhibition provides neuroprotection in a rat temporary middle cerebral artery occlusion model.J Neurochem. 2012; 123: 116-124Crossref PubMed Scopus (30) Google Scholar However, no study has addressed the effect of VAP-1 inhibition in AKI models, and the mechanism by which VAP-1 inhibition ameliorates kidney disease is unknown. Based on the hypothesis that inhibiting VAP-1 enzyme activity ameliorates renal IR injury via suppression of leukocyte infiltration, we investigated VAP-1 localization in the kidney, the effect of a VAP-1 inhibitor in a rat renal IR model, and the mechanism through which VAP-1 is associated with leukocyte infiltration in this model and how its inhibition ameliorates renal IR injury. In a pilot study, we compared kidney and plasma VAP-1 enzyme activity in healthy rats and mice with that in healthy humans to determine which animal species is most similar to humans (human kidney data were extrapolated from Boomsma et al.34Boomsma F. van Dijk J. Bhaggoe U.M. et al.Variation in semicarbazide-sensitive amine oxidase activity in plasma and tissues of mammals.Comp Biochem Physiol C Toxicol Pharmacol. 2000; 126: 69-78PubMed Google Scholar). The VAP-1 enzyme activities were in the following order: C57BL/6J mouse < Sprague-Dawley rat < human (Figure 1).34Boomsma F. van Dijk J. Bhaggoe U.M. et al.Variation in semicarbazide-sensitive amine oxidase activity in plasma and tissues of mammals.Comp Biochem Physiol C Toxicol Pharmacol. 2000; 126: 69-78PubMed Google Scholar Because BTBR wild-type mice and Wistar rats exhibited VAP-1 enzyme activity to a similar degree as C57BL/6J mice and Sprague-Dawley rats, respectively (data not shown), this tendency seems to be species specific. These results suggest that function and regulation of VAP-1 in humans are more similar to those in rats compared with mice; thus, we used rats in this study in view of a potential clinical application.35Okusa M.D. Rosner M.H. Kellum J.A. et al.Therapeutic targets of human AKI: harmonizing human and animal AKI.J Am Soc Nephrol. 2016; 27: 44-48Crossref PubMed Scopus (51) Google Scholar VAP-1 has been reported to be expressed mainly in the interstitium of human kidney,23Kurkijärvi R. Jalkanen S. Isoniemi H. et al.Vascular adhesion protein-1 (VAP-1) mediates lymphocyte-endothelial interactions in chronic kidney rejection.Eur J Immunol. 2001; 31: 2876-2884Crossref PubMed Scopus (25) Google Scholar but its detailed cellular localization remains obscure. Immunofluorescence analysis using sham-operated rat kidneys confirmed VAP-1 expression in the interstitium (corticomedullary junction > superficial cortex, medulla; Figure 2a). Merged images at higher magnification demonstrated that VAP-1 is expressed predominantly in pericytes rather than in endothelial cells (Figure 2b). It was difficult to obtain clear images of VAP-1 localization in injured kidneys owing to nonspecific signals from injured tubules and casts, which were excluded from further analysis. Reverse transcription polymerase chain reaction and immunoblotting also revealed robust VAP-1 expression in primary kidney pericytes (Supplementary Figure S1A).36Kida Y. Ieronimakis N. Schrimpf C. et al.EphrinB2 reverse signaling protects against capillary rarefaction and fibrosis after kidney injury.J Am Soc Nephrol. 2013; 24: 559-572Crossref PubMed Scopus (80) Google Scholar VAP-1 knockdown with small, interfering RNA in these pericytes (70% reduction in mRNA expression) resulted in diminished bands in immunoblotting, which supports the specificity of the anti–VAP-1 antibody we used (Supplementary Figure S1B and C). Furthermore, we investigated VAP-1 mRNA expression in vitro by using primary kidney pericytes, endothelial cells, and tubular epithelial cells (Supplementary Figure S1D). VAP-1 mRNA was readily detectable in primary kidney pericytes, but not in endothelial cells or tubular epithelial cells within the amplification range, which further indicates the predominant expression of VAP-1 in pericytes. To gain insight into the functional role of VAP-1 in ischemic AKI, a specific VAP-1 inhibitor, RTU-1096, was studied in a rat model of renal IR injury. Kidney VAP-1 enzyme activity, which did not change 6 hours after IR, tended to increase (nonsignificantly) at 48 hours (Figure 3a). In contrast, IR surgery significantly increased plasma VAP-1 enzyme activity at 6 hours, which returned to baseline at 48 hours (Figure 3b). RTU-1096 significantly reduced kidney and plasma VAP-1 enzyme activity at all time points. VAP-1 inhibition was associated with significantly lowered blood urea nitrogen and plasma creatinine levels at 24 and 48 hours (Figure 4a), which was corroborated by histological analysis and additional biochemical parameters. Tubular injury mitigation (Figure 4b) and decreased Kim-1 and Ngal mRNA expression in whole kidney (Figure 4c) were observed in VAP-1 inhibitor–treated rats 48 hours after IR. The effect of VAP-1 inhibition on the recovery phase was also investigated at day 7 (Supplementary Figure S2). VAP-1 inhibition was significantly associated with preserved renal function (Supplementary Figure S2A), decreased tubular vimentin staining (Supplementary Figure S2B), and decreased proliferating cell nuclear antigen (PCNA)-positive cells (Supplementary Figure S2C). These findings suggest that VAP-1 inhibition was not detrimental to the recovery phase of IR injury.Figure 4Renal ischemia-reperfusion (IR) injury was ameliorated by vascular adhesion protein-1 (VAP-1) inhibition. Rats underwent renal IR injury and were killed at 48 hours after IR. (a) Time course of blood urea nitrogen (BUN) and plasma creatinine (Cr). (b) Semiquantitative analysis of histological tubular injury (Jablonski scale) with representative images of the kidneys stained with periodic acid–Schiff. Bar = 200 μm. (c) Quantitative polymerase chain reaction of Kim-1 and Ngal mRNA in the whole kidney. Data are expressed as mean ± SEM; sham groups, n = 3; IR groups, n = 10; *P < 0.05, **P < 0.01, ***P < 0.001. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the role of VAP-1 in leukocyte infiltration into injured tissues, we quantitatively evaluated numbers of infiltrating leukocytes (neutrophils, macrophages, and T lymphocytes). VAP-1 inhibition did not affect the number of macrophages (Figure 5b) or T lymphocytes (Figure 5c and d) infiltrating into the corticomedullary junction. In contrast, the number of infiltrating neutrophils, which was not affected by VAP-1 inhibition 6 hours after IR, was significantly decreased by VAP-1 inhibition at 12 hours after IR (Figure 5a). Whole-kidney CXCL-1 and CXCL-2 expression, remarkably upregulated by IR injury, was not altered at 6 hours but was significantly suppressed at 48 hours by VAP-1 inhibition. In contrast, CCL-2 expression was not affected by VAP-1 inhibition at 6 or 48 hours (Supplementary Figure S3A). We also examined whether VAP-1 inhibition influenced expression of alternative adhesion molecules that possibly suppress neutrophil infiltration into IR-injured kidneys. VCAM-1, ICAM-1, E-selectin, or P-selectin expression in the whole kidney was not affected by VAP-1 inhibition at 6 or 48 hours after IR (Supplementary Figure S3B). To explore whether decreased neutrophil infiltration into IR-injured kidneys by VAP-1 inhibition is upstream or downstream of the ameliorated kidney injury, the effect of RTU-1096 was studied in neutrophil-depleted rats. Injection of anti-rat neutrophil antibody significantly decreased the number of neutrophils in blood, which was sustained until 48 hours (Figure 6a) without affecting the number of lymphocytes (Supplementary Figure S4A). Furthermore, anti–rat neutrophil antibody treatment did not affect kidney or plasma VAP-1 enzyme activity (data not shown). Neutrophil depletion significantly protected kidneys from IR injury as determined by plasma creatinine (Figure 6b) and blood urea nitrogen (Supplementary Figure S4B) levels and histological tubular injury (Figure 6c). RTU-1096 administration to neutrophil-depleted rats had no additional beneficial effect on IR injury. Neutrophil depletion significantly decreased the number of infiltrating neutrophils, which was not altered by VAP-1 inhibition (Figure 6d). In contrast, neutrophil depletion or VAP-1 inhibition did not affect the number of infiltrating macrophages or T lymphocytes (Supplementary Figure S4C). These results suggest that VAP-1 inhibition directly blocks neutrophil infiltration into IR-injured kidneys, which in turn ameliorates kidney injury. Primary kidney pericytes were used to gain further insight into VAP-1 expression in kidney pericytes. VAP-1 enzyme activity was confirmed by detection of benzaldehyde generation in response to benzylamine, which was significantly blocked by RTU-1096 (Supplementary Figure S5A). We also detected VAP-1 enzyme activity in conditioned media from primary kidney pericytes, which could be blocked by RTU-1096 (Supplementary Figure S5B), suggesting that kidney pericytes release enzymatically active sVAP-1. To investigate VAP-1 regulation in injured kidney pericytes, primary kidney pericytes were stimulated with platelet-derived growth factor (PDGF)-BB37Lin S.L. Chang F.C. Schrimpf C. et al.Targeting endothelium-pericyte cross talk by inhibiting VEGF receptor signaling attenuates kidney microvascular rarefaction and fibrosis.Am J Pathol. 2011; 178: 911-923Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar or hypoxia. PDGF-BB and hypoxia increased sVAP-1 release into medium (Supplementary Figure S5B), but not VAP-1 mRNA levels or VAP-1 enzyme activity in pericytes (data not shown). The extent of increase in sVAP-1 in medium by PDGF-BB and hypoxia was small, possibly because cultured pericytes are already in an activated state.38Kawakami T. Mimura I. Shoji K. et al.Hypoxia and fibrosis in chronic kidney disease: crossing at pericytes.Kidney Int Suppl (2011). 2014; 4: 107-112Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Hydrogen peroxide (H2O2) is a critical chemoattractant for neutrophils that is mainly studied in zebrafish with tail transection.39Niethammer P. Grabher C. Look A.T. et al.A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.Nature. 2009; 459: 996-999Crossref PubMed Scopus (1120) Google Scholar, 40Yoo S.K. Starnes T.W. Deng Q. et al.Lyn is a redox sensor that mediates leukocyte wound attraction in vivo.Nature. 2011; 480: 109-112Crossref PubMed Scopus (328) Google Scholar We hypothesized that H2O2, a product generated by VAP-1 enzyme reaction, attracts neutrophils into IR-injured kidneys. To test this, the under-agarose migration assay with freshly isolated human neutrophils was performed (Supplementary Figure S6). H2O2 facilitated neutrophil migration, confirming it as a neutrophil chemoattractant (Figure 7a). Because benzylamine is an efficient substrate for VAP-1, it was used in experiments with recombinant human VAP-1 protein. Addition of VAP-1 significantly induced neutrophil migration, which was almost completely abolished by treatment with RTU-1096 or catalase (Figure 7b). These results indicate that VAP-1 acts as a neutrophil chemotactic factor primarily via H2O2 generation. Because leukocyte infiltration plays an important role in the pathophysiology of ischemic AKI, many drugs have been tested in clinical trials but without much success. Recently, inhibition of VAP-1 enzyme activity in animals has been shown to decrease leukocyte infiltration and ameliorate inflammation in various organs, which led us to hypothesize that VAP-1 inhibition ameliorates renal IR injury via suppression of leukocyte infiltration. We demonstrated that VAP-1 is expressed predominantly in pericytes in the kidney and that VAP-1 inhibition ameliorates rat renal IR injury via suppression of neutrophil infiltration. Moreover, in vitro studies show that VAP-1 acts as a neutrophil chemotactic factor via generation of a local H2O2 gradient (Figure 8). A novel finding in this study is that VAP-1 is expressed predominantly in pericytes in the kidney. Immunofluorescence studies demonstrated colocalization of VAP-1–positive and PDGF receptor β (PDGFRβ)–positive cells in the interstitium, indicating that they are pericytes, which was corroborated by mRNA and protein expression of VAP-1 in primary kidney pericytes. VAP-1 expression was more robust in the corticomedullary junction than in other areas within the kidney, which suggests the importance of this molecule in the renal IR injury model. Kidney VAP-1 enzyme activity tended to increase after IR surgery, in accordance with previous results in other kidney disease models.41Wong M. Saad S. Zhang J. et al.Semicarbazide-sensitive amine oxidase (SSAO) inhibition ameliorates kidney fibrosis in a unilateral ureteral obstruction murine model.Am J Physiol Renal Physiol. 2014; 307: F908-F916Crossref PubMed Scopus (16) Google Scholar, 42Katagiri D. Hamasaki Y. Doi K. et al.Interstitial renal fibrosis due to multiple cisplatin treatments is ameliorated by semicarbazide-sensitive amine oxidase inhibition.Kidney Int. 2016; 89: 374-385Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Administration of RTU-1096, a specific VAP-1 inhibitor, successfully decreased kidney VAP-1 enzyme activity by approximately 90% and dramatically ameliorated rat renal IR injury. Mechanistically, neutrophil infiltration into IR-injured kidneys was significantly suppressed by VAP-1 inhibition from 12 hours after IR without affecting infiltration of macrophages or T lymphocytes. Awad et al.43Awad A.S. Rouse M. Huang L. et al.Compartmentalization of neutrophils in the kidney and lung following acute ischemic kidney injury.Kidney Int. 2009; 75: 689-698Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar demonstrated the time course of in vivo neutrophil trafficking in a mouse renal IR injury model. Although the number of marginated neutrophils (adhered to endothelial cells) peaked 2 hours after IR, the number of interstitial neutrophils did not increase at 2 hours and peaked at 24 and 48 hours. These results suggest that VAP-1 in kidney pericytes affects only the neutrophils that have passed through an endothelial cell layer, and helps them to migrate further. It is possible that suppressed neutrophil infiltration is secondary to ameliorated kidney injury. However, our results in neutrophil-depleted rats clearly indicate that VAP-1 inhibition ameliorated renal IR injury via suppression of neutrophil infiltration. Limited information is available regarding the mechanism by which VAP-1 in pericytes enhances leukocyte infiltration into inflamed tissues. VAP-1 inhibition did not alter the expression of other adhesion molecules in this study. We confirmed that primary kidney pericytes express and release enzymatically active VAP-1. Together with previous reports showing extracellular H2O2-mediated neutrophil chemotaxis in zebrafish with tail transection,39Niethammer P. Grabher C. Look A.T. et al.A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.Nature. 2009; 459: 996-999Crossref PubMed Scopus (1120) Google Scholar, 40Yoo S.K. Starnes T.W. Deng Q. et al.Lyn is a redox sensor that mediates leukocyte wound attraction in vivo.Nature. 2011; 480: 109-112Crossref PubMed Scopus (328) Google Scholar we assume that extracellular H2O2 generated by VAP-1 enzyme reaction forms a local H2O2 gradient and enhances neutrophil infiltration into IR-injured kidneys. IR surgery produces many reactive oxygen species, including H2O2, within the kidney, but we found no study investigating local extracellular H2O2 around pericytes and endothelial cells in IR-injured kidneys. In the under-agarose migration assay, recombinant human VAP-1 attracted human neutrophils, which was almost completely blocked by addition of RTU-1096 or catalase. These findings support our assumption that VAP-1 enhances neutrophil infiltration into IR-injured kidneys via generation of H2O2. The concentration of recombinant human VAP-1 protein used in this assa
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