Differential resolution of inflammation and recovery after renal ischemia–reperfusion injury in Brown Norway compared with Sprague Dawley rats
2010; Elsevier BV; Volume: 77; Issue: 9 Linguagem: Inglês
10.1038/ki.2010.10
ISSN1523-1755
AutoresDavid Sáenz-Morales, Elisa Conde, Ignacio Blanco-Sánchez, Belén Ponte, Elia Aguado-Fraile, Gonzalo de las Casas, María Garcia Martos, Laura Alegre, María M. Escribese, Ana Molina, Carmen Santiuste, Fernando Liaño, Laura García‐Bermejo,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoTo investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia–reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-γ, IL-1β, and TNF-α were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFκB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia–reperfusion injury. To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia–reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-γ, IL-1β, and TNF-α were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFκB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia–reperfusion injury. Acute tubular necrosis (ATN) due to ischemia–reperfusion (I/R) is observed in important clinical settings, such as patients in intensive care units and those undergoing kidney transplantation.1.Ricci Z. Ronco C. Today's approach to the critically ill patient with acute kidney injury.Blood Purif. 2009; 27: 127-134Crossref PubMed Scopus (20) Google Scholar In this situation, ATN contributes to most cases of delayed graft function, is related to higher immunogenicity and is a predictive factor for poor allograft outcome.2.Ojo A.O. Wolfe R.A. Held P.J. et al.Delayed graft function: risk factors and implications for renal allograft survival.Transplantation. 1997; 63: 968-974Crossref PubMed Scopus (839) Google Scholar ATN is characterized by proximal epithelial cell shedding, cell death inflammation in renal parenchyma and kidney dysfunction.3.Kellum J.A. Acute kidney injury.Crit Care Med. 2008; 36: S141-S145Crossref PubMed Scopus (158) Google Scholar Mechanisms responsible for ATN development have been identified by experimental models, such as the widely used 45-min of bilateral renal ischemia in rats. In this model, analogous to humans, moderate damage occurring mainly in the proximal tubules and recovery after some days of reperfusion were observed.4.Lieberthal W. Nigam S.K. Acute renal failure. II. Experimental models of acute renal failure: imperfect but indispensable.Am J Physiol Renal Physiol. 2000; 278: F1-F12PubMed Google Scholar In vitro models have also shed light on the mechanisms underlying proximal tubule cell response to ischemia.5.Sáenz-Morales D. Escribese M.M. Stamatakis K. et al.Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress.Exp Cell Res. 2006; 312: 3711-3727Crossref PubMed Scopus (39) Google Scholar However, in spite of the advances in experimental models, not much progress has reached clinical practice. Genetically modified animals could be used to identify new targets to improve ATN outcome. Unfortunately, genetic modification in rats is difficult. Therefore, some rat strains have been selected for their different response or susceptibility to ischemia. Indeed, some reports have identified inbred Brown Norway rats as more resistant to cardiac ischemia than other rat strains,6.Baker J.E. Konorev E.A. Gross G.J. et al.Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?.Am J Physiol Heart Circ Physiol. 2000; 278: H1395-H1400PubMed Google Scholar,7.Shi Y. Hutchins W. Ogawa H. et al.Increased resistance to myocardial ischemia in the Brown Norway vs. Dahl S rat: role of nitric oxide synthase and Hsp90.J Mol Cell Cardiol. 2005; 38: 625-635Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar including the outbred Sprague Dawley rats. Resistance to the development of tubular damage after renal ischemia was also described in Brown Norway rats.8.Basile D.P. Donohoe D. Cao X. et al.Resistance to ischemic acute renal failure in the Brown Norway rat: a new model to study cytoprotection.Kidney Int. 2004; 65: 2201-2211Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,9.Nilakantan V. Hilton G. Maenpaa C. et al.Favorable balance of anti-oxidant/pro-oxidant systems and ablated oxidative stress in Brown Norway rats in renal ischemia-reperfusion injury.Mol Cell Biochem. 2007; 304: 1-11Crossref PubMed Scopus (29) Google Scholar These reports indicated higher levels of heat-shock proteins and ablated oxidative stress in Brown Norway rats as putative mechanisms contributing to this resistance. In this study, we used Brown Norway and Sprague Dawley rats, as well as primary cultures of their proximal epithelial cells to elucidate new mechanisms underlying the resistance/susceptibility to renal ischemia and to identify novel targets for experimental intervention in ATN. We demonstrate that Brown Norway rats recovered earlier after I/R, thus suggesting Brown Norway rats as a useful experimental model to design new therapeutic approaches for ATN resolution. We performed 45 min of bilateral ischemia and 24 h of reperfusion in male Brown Norway and Sprague Dawley rats obtained from our own breeding facility. Renal injury is maximal at this time point in this model.10.Forbes J.M. Hewitson T.D. Becker G.J. et al.Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat.Kidney Int. 2000; 57: 2375-2385Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar Sham-operated animals were used as controls. Renal function was estimated by urea and creatinine levels in serum. After periodic acid-Schiff (PAS) staining of paraffin-embedded renal sections, histological damage was scored according to the following criteria: proximal tubule cell morphology alterations, brush border loss, proximal tubular dilation and denudation, as well as the presence of casts and infiltrates. Sprague Dawley and Brown Norway rats showed similar high urea and creatinine levels at 24 h after ischemia (Figure 1a). Accordingly, histological examination after PAS staining also showed patched ATN in both Sprague Dawley and Brown Norway rats at 24 h of reperfusion (Figure 1b). In addition, phalloidin staining of renal tissue cryosections showed similar brush border loss and actin cytoskeleton disorganization (Figure 1b). Histopathological scoring confirming similar I/R injury is shown in Table 1.Table 1Histopathological scoreTubular alterationsPresence of castsPresence of infiltratesSD IR-24h2.86±0.142.57±0.301.43±0.20BN IR-24h2.91±0.012.73±0.031.91±0.03SD IR-3d2.55±0.352.47±0.603.25±0.02BN IR-3d2.05±0.262.07±0.432.80±0.04*P<0.05SD IR-5d2.46±0.32.00±0.352.14±0.24BN IR-5d1.95±0.28*P<0.052.50±0.501.76±0.12SD IR-7d2.55±0.252.05±0.501.97±0.07BN IR-7d1.86±0.3*P<0.051.56±0.351.14±0.24*P<0.05SD IR-15d0.96±0.210.31±0.170.73±0.09BN IR-15d0.55±0.07*P<0.050.04±0.02*P<0.050.25±0.04*P<0.05Abbreviations: ATN, acute tubular necrosis; BN, Brown Norway; IR, ischemia–reperfusion; SD, Sprague Dawley.Scoring indicates similar ATN development in SD and BN rats at 24 h of reperfusion, but accelerated ATN recovery in BN rats during reperfusion (IR-3, IR-5, IR-7, and IR-15 days). Data are expressed as means±s.e.m. Scoring criteria are described in the ‘Materials and methods’ section. Statistically significant differences were found in comparison with SD values* P<0.05 Open table in a new tab Abbreviations: ATN, acute tubular necrosis; BN, Brown Norway; IR, ischemia–reperfusion; SD, Sprague Dawley. Scoring indicates similar ATN development in SD and BN rats at 24 h of reperfusion, but accelerated ATN recovery in BN rats during reperfusion (IR-3, IR-5, IR-7, and IR-15 days). Data are expressed as means±s.e.m. Scoring criteria are described in the ‘Materials and methods’ section. Statistically significant differences were found in comparison with SD values To verify that this unexpected susceptibility of Brown Norway rats was not a feature of our own colony, we also studied Brown Norway rats obtained from two widely recognized commercial suppliers (Harlan Laboratories Models, Boxmeer, The Netherlands and Charles River Laboratories International, Margate, UK) (Figure 1c). Renal function at 24 h of reperfusion was similarly affected in Brown Norway rats from our colony and from the other two suppliers: BN/RijHsd (Harlan Laboratories Models) and BN/OrlCrl (Charles River Laboratories International). Accordingly, similar injury was observed in PAS-stained tissue sections of Brown Norway rats obtained from both suppliers (data not shown). These results demonstrate that both Sprague Dawley and Brown Norway rats develop similar acute renal injury after I/R. We further analyzed the response of Sprague Dawley and Brown Norway rats to I/R using primary cultures of their proximal tubule cells after hypoxia/reoxygenation. We previously demonstrated that this protocol provokes, in the rat proximal cell line NRK-52E, cytoskeleton alterations, focal adhesion disassembly, cell–cell adhesion impairment, and cell shedding, all features evidenced in the I/R rat model.5.Sáenz-Morales D. Escribese M.M. Stamatakis K. et al.Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress.Exp Cell Res. 2006; 312: 3711-3727Crossref PubMed Scopus (39) Google Scholar Primary cells under hypoxia/reoxygenation were stained with phalloidin and anti-zonula occludens-1 (ZO-1) antibody to observe actin cytoskeleton organization and determine cell–cell adhesion integrity, respectively. Hypoxia/reoxygenation induced marked actin cytoskeleton disorganization and depolymerization in primary cells from both strains. The lower signal for F-actin detectable at 3 h of reoxygenation in both cases, indicating actin depolymerization (Figure 2a) is noted. Cytokeratin immunostaining was used as an epithelial marker. In addition, ZO-1 internalization from the plasma membrane was evident early during reoxygenation in cells from Sprague Dawley and Brown Norway rats, indicating that in both cases, cell–cell adhesion is similarly affected in response to hypoxia/reoxygenation (Figure 2b). These results demonstrate that proximal tubular cells from Sprague Dawley and Brown Norway rats exhibit similar injury in response to hypoxia/reoxygenation and support our in vivo data. To analyze the renal response of both strains to ischemia, we extended the studies up to 15 days of reperfusion when recovery of renal function is observed in this in vivo model.10.Forbes J.M. Hewitson T.D. Becker G.J. et al.Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat.Kidney Int. 2000; 57: 2375-2385Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar We studied the activity of signaling pathways involved in cell survival and proliferation, oxidative stress response, and adaptation to hypoxia, such as the extracellular signal-regulated kinase (ERK1/2), serine–threonine protein kinase Akt (AKT), manganese superoxide dismutase (MnSOD), and the hypoxia-inducible factor-1 alpha (HIF-1α) target genes: prolyl-hydroxylase-3 (PHD-3), vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1). Western blot of total renal lysates showed clear differences in cell signaling triggered by I/R in both strains. As shown in Figure 3a, Brown Norway rats had a marked induction of p-AKT at 24 h and 5 days of reperfusion. Notably, p-ERK 1/2 was markedly induced after ischemia in Brown Norway rats. MnSOD levels were slightly higher in Brown Norway than in Sprague Dawley rats, in the whole I/R kinetic. Using real-time PCR, we assessed the expression of HIF-1α target genes, namely VEGF, TSP-1, and PHD-3 (Figure 3b). Both strains showed a similar expression at 24 h, but VEGF and PHD-3 remained higher in Brown Norway, later in reperfusion. In contrast, TSP-1, which has been described as a marker of renal ischemic damage,11.Thakar C.V. Zahedi K. Revelo M.P. et al.Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia.J Clin Invest. 2005; 115: 3451-3459Crossref PubMed Scopus (100) Google Scholar was higher in Sprague Dawley rats. Although I/R-induced injury is similar in both Brown Norway and Sprague Dawley rats, these results indicate that both strains exhibit a different response to ischemia and recovery mechanisms might be differently triggered in time. Therefore, a distinct outcome might be expected. Differences in gene expression and cell signaling could account for a different outcome of ischemic injury as suggested above. Thus, we studied renal function and kidney structure during I/R up to 15 days after ischemia. As presented in Figure 4, serum urea and creatinine values, as well as creatinine clearance, normalized in both rat strains after 5–7 days of reperfusion, consistent with reported data.10.Forbes J.M. Hewitson T.D. Becker G.J. et al.Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat.Kidney Int. 2000; 57: 2375-2385Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar However, the improvement in renal function measured by serum creatinine clearance was observed earlier in Brown Norway rats, that is, around day 3. This delay in renal function recovery of Sprague Dawley vs Brown Norway rats correlated with a delay in kidney size recovery in the Sprague Dawley strain. Indeed, the length of the major kidney diameter was longer in Sprague Dawley rats at the seventh day, whereas it was normal in Brown Norway rats: I/R at 24 h: Sprague Dawley, 15.0±1.15 mm and Brown Norway, 14.6±1.35 mm; I/R at day 7: Sprague Dawley, 17.1±2.7 mm and Brown Norway, 14.3±2.4 mm. Moreover, between 5 and 7 days of reperfusion, PAS staining showed that Sprague Dawley kidneys still exhibited ATN, dilated tubules, thinner brush border, more evident infiltrates, and overall more altered renal structure (Figure 5a). Brown Norway kidneys presented a much better preserved structure. Phalloidin staining confirmed the improved recovery of kidney structure in Brown Norway rats at 5–7 days, as evidenced by the well-organized brush border. Brush border absence and collapsed actin at the cell basement are still observed in several tubules in Sprague Dawley rats (Figure 5b). At 15 days of reperfusion, histopathology differences diminished, according to the functional parameter normalization observed in both strains. Scores for tissue damage also confirmed faster recovery in Brown Norway rats throughout reperfusion (Table 1). Supporting the different response, the HIF-dependent gene TSP-1 remained elevated at 7 days in Sprague Dawley rats, correlating with histological damage. Accordingly, 5-bromo-2-deoxyuridine (BrdU) and PCNA (proliferating cell nuclear antigen) staining in proximal tubules were much lower in Brown Norway rats at 5 days of reperfusion (Figure 6 and Table 2), probably due to the fact that proximal cells had already recovered. However, proliferation was higher in Brown Norway rats at 24 h, suggesting earlier triggering of recovery mechanisms in this strain.Table 2BrdU and PCNA quantificationShamIschemiaIR-24hIR-3dIR-5dIR-7dIR-15dBRDU SD7±2.0810±3.4622±3191±3218±2.3110±16±0.58 BN6±112±2.5260±4.04*P<0.05120±6.43*P<0.0512±1.158±2.086±1.53PCNA SD6±3.064±0.5838±8.14112±16.269±2.658±3.063±1.73 BN9±224±1*P<0.0560±3.51*P<0.0544±5.51*P<0.058±2.524±1.731±0.58Abbreviations: BN, Brown Norway; BrdU, 5-bromo-2-deoxyuridine; IR, ischemia–reperfusion; PCNA, proliferating cell nuclear antigen; SD, Sprague Dawley.Quantification of both BrdU and PCNA nuclear signals, in SD and BN rats during IR, expressed as means±s.e.m. of positive nuclei in 10 fields of each condition are shown. Statistically significant differences were found in comparison with SD values* P<0.05 Open table in a new tab Abbreviations: BN, Brown Norway; BrdU, 5-bromo-2-deoxyuridine; IR, ischemia–reperfusion; PCNA, proliferating cell nuclear antigen; SD, Sprague Dawley. Quantification of both BrdU and PCNA nuclear signals, in SD and BN rats during IR, expressed as means±s.e.m. of positive nuclei in 10 fields of each condition are shown. Statistically significant differences were found in comparison with SD values These data demonstrate that although Sprague Dawley and Brown Norway rats show similar I/R injury, Brown Norway rats show faster recovery, in agreement with the differential signaling found. As prominent cell infiltrates were still observed in Sprague Dawley rats at 7 days of reperfusion, and inflammatory response resolution affects renal recovery, using real-time PCR, we analyzed the proinflammatory gene expression, that is, MCP-1 (monocyte chemoattractant protein-1), interleukin IL-10, TNF (tumor necrosis factor)-α, IL-1β, INF (interferon)-γ, STAT-4 and STAT-6; nuclear factor NFκB activity and very late antigen-4/vascular cell adhesion molecule-1 (VLA-4/VCAM-1) pathway for leukocyte infiltration, in both strains. As presented in Figure 7a, a strong cytokine induction was observed in reperfusion, although no marked differences were found between strains at 24 h. However, Brown Norway rats recovered basal levels faster. In fact, some of these genes (MCP-1 and IL-1β) still remained elevated at 7 days of reperfusion in Sprague Dawley rats. STAT-4 and STAT-6 were higher in Sprague Dawley rats but they were not altered significantly in Brown Norway. Moreover, NFκB activation was shorter in nuclear extracts of Brown Norway rat kidneys, as shown in Figure 7b. Immunostaining for macrophages (CD68) and T lymphocytes (T-cell marker) in renal tissues obtained from Sprague Dawley and Brown Norway rats were performed to quantify cell infiltrates. Results in Figure 8c show similar macrophage infiltration in Sprague Dawley and Brown Norway rats at 24 h of reperfusion, although the number of T cells is significantly higher in Brown Norway. Both infiltrating cell populations diminished earlier in Brown Norway rats, in agreement with cytokine expression results and lesser infiltrates in PAS staining in comparison with Sprague Dawley, at 7 days of reperfusion. Thus, to analyze leukocyte activation, we measured, by flow cytometry, VLA-4 expression in peripheral blood leukocytes (PBLs) from both rat strains. As presented in Figure 8a, VLA-4 expression was similar in PBLs from Sprague Dawley and Brown Norway rats at 24 h of reperfusion, although this expression was reduced faster in Brown Norway. Accordingly, VCAM-1 expression in total kidney lysates was higher and maintained in Sprague Dawley rats (Figure 8b). Together, these results suggest a shorter proinflammatory response in Brown Norway rats during I/R that may lead to the accelerated recovery exhibited by this strain. Identification of mechanisms to accelerate recovery after I/R damage could be critical to reduce morbidity and mortality of some clinical situations, such as renal transplant or acute renal failure. The use of experimental animal models with different susceptibility to ischemic damage could shed light on this issue. In this study, we report the different outcomes after I/R in two rat strains, Brown Norway and Sprague Dawley. Both develop similar damage after I/R, but Brown Norway rats recover faster. A different regulation of signaling mechanisms and a faster resolution of inflammatory response contributes to this better recovery. Recently, the resistance of Brown Norway rats to renal ischemia was described.8.Basile D.P. Donohoe D. Cao X. et al.Resistance to ischemic acute renal failure in the Brown Norway rat: a new model to study cytoprotection.Kidney Int. 2004; 65: 2201-2211Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,9.Nilakantan V. Hilton G. Maenpaa C. et al.Favorable balance of anti-oxidant/pro-oxidant systems and ablated oxidative stress in Brown Norway rats in renal ischemia-reperfusion injury.Mol Cell Biochem. 2007; 304: 1-11Crossref PubMed Scopus (29) Google Scholar In contrast, Sprague Dawley rats have been extensively reported as a strain susceptible to develop renal ischemic injury. With the aim to unveil mechanisms responsible for this ischemia-resistant phenotype, in this study, we compared Sprague Dawley and Brown Norway responses with renal ischemia. Surprisingly, our results did not agree with those of earlier studies,8.Basile D.P. Donohoe D. Cao X. et al.Resistance to ischemic acute renal failure in the Brown Norway rat: a new model to study cytoprotection.Kidney Int. 2004; 65: 2201-2211Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar as we found similar renal dysfunction and histopathological damage at 24 h of reperfusion, suggesting that Brown Norway rats were not resistant to I/R. To ascertain whether the genetic background in our colony could be responsible for the Brown Norway susceptibility described in this study, Brown Norway rats from commercial suppliers Harlan and Charles River were also included in the study. These animals exhibited similar renal damage after I/R to our colony, thus confirming that the Brown Norway susceptibility observed is not due to local conditions. In any case, the uncharacterized degree of genetic variation remaining in an inbred population and the husbandry alterations between institutions should be taken into account when comparing results obtained from different laboratories. Similar damage after I/R was also shown using primary proximal tubule cells in an in vitro model of hypoxia/reoxygenation, which reproduces I/R stimuli in vivo.5.Sáenz-Morales D. Escribese M.M. Stamatakis K. et al.Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress.Exp Cell Res. 2006; 312: 3711-3727Crossref PubMed Scopus (39) Google Scholar No differences were found in primary cultures from both strains regarding cytoskeleton disorganization, ZO-1, and E-cadherin redistribution (data not shown), indicating that proximal tubular cells of both strains are equally affected by ischemia, and supporting the in vivo results. In spite of the similar renal damage observed, marked differences in the renal tissue response to ischemia were exhibited. This fact could be related to the different I/R outcomes reported in this study. ERK1/2 and AKT were differently activated in Brown Norway rats. Some studies have related them to a better response after renal ischemia by inducing cell adhesion or increased survival.12.Arany I. Megyesi J.K. 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Zheng X. Qin J. et al.Role of PI3-kinase/Akt signalling pathway in renal function and cell proliferation after renal ischaemia/reperfusion injury in mice.Nephrology (Carlton). 2006; 11: 207-212Crossref PubMed Scopus (35) Google Scholar Consequently, stronger AKT activation and earlier ERK1/2 induction in Brown Norway rats at 24 h might promote tubular cells proliferation, as the increase in BrdU incorporation and PCNA staining indicate. Thus, the triggering of repair mechanisms as soon as at 24 h of reperfusion could contribute to the fast recovery exhibited by Brown Norway rats later during reperfusion. Moreover, proximal tubule cell proliferation is delayed in Sprague Dawley rats, in accordance with the delay in renal recovery. Related to survival, high levels of MnSOD have been detected in Brown Norway rats that could ameliorate reactive oxygen species-induced damage,9.Nilakantan V. Hilton G. Maenpaa C. et al.Favorable balance of anti-oxidant/pro-oxidant systems and ablated oxidative stress in Brown Norway rats in renal ischemia-reperfusion injury.Mol Cell Biochem. 2007; 304: 1-11Crossref PubMed Scopus (29) Google Scholar as reactive oxygen species have been proposed as important mediator of renal damage during reperfusion.17.Plotnikov E.Y. Kazachenko A.V. Vyssokikh M.Y. et al.The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney.Kidney Int. 2007; 72: 1493-1502Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar Indeed, exogenous administration of SOD mimetics improves tubular damage after I/R in vivo18.Liang H.L. Hilton G. Mortensen J. et al.MnTMPyP, a cell-permeant SOD mimetic, reduces oxidative stress and apoptosis following renal ischemia-reperfusion.Am J Physiol Renal Physiol. 2009; 296: F266-F276Crossref PubMed Scopus (62) Google Scholar and hypoxia/reoxygenation in vitro.19.Maenpaa C.J. Shames B.D. Van Why S.K. et al.Oxidant-mediated apoptosis in proximal tubular epithelial cells following ATP depletion and recovery.Free Radic Biol Med. 2008; 44: 518-526Crossref PubMed Scopus (28) Google Scholar We also observed greater induction of nitric oxide synthase 2 in Brown Norway at 24 h of reperfusion (data not shown). The beneficial effects of nitric oxide have been described in ischemic preconditioning in several organs including the kidney, related to vascular tone and permeability.20.Huang S.S. Wei F.C. Hung L.M. Ischemic preconditioning attenuates postischemic leukocyte–endothelial cell interactions: role of nitric oxide and protein kinase C.Circ J. 2006; 70: 1070-1075Crossref PubMed Scopus (45) Google Scholar, 21.Wang W.Z. Fang X.H. Stepheson L.L. et al.NOS upregulation attenuates vascular endothelial dysfunction in the late phase of ischemic preconditioning in skeletal muscle.J Orthop Res. 2004; 22: 578-585Crossref PubMed Scopus (32) Google Scholar, 22.Ishida T. Yarimizu K. 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