Int Urol Nephrol DOI 10.1007/s11255-015-0997-x

UROLOGY - ORIGINAL PAPER

Ischemic postconditioning inhibits apoptosis of renal cells following reperfusion: a novel in vitro model Xiaodong Weng1 · Min Wang1 · Hui Chen1 · Zhiyuan Chen1 · Xiuheng Liu1 

Received: 11 March 2015 / Accepted: 21 April 2015 © Springer Science+Business Media Dordrecht 2015

Abstract  Purpose  The majority of renal ischemic/reperfusion (I/R) and ischemic postconditioning (IPO) studies have been based on animal models. To gain mechanistic insights into ischemic postconditioning-induced alterations at the cell level, a novel in vitro model of I/R and IPO is set up by using the rat proximal tubule cell line NRK-52E. Methods  Cells are incubated in 1 mL ischemic buffer under hypoxia conditions for 3 h to simulate the clinical condition of a cellular microenvironment representative of ischemia, including oxygen deprivation, carbon dioxide elevation, nutrient depletion, and waste accumulation. IPO model is established by exposing the cells to three cycles of ‘mimic reperfusion condition’ for 10 min and ischemic condition for 10 min after placing the cells in ischemic condition for 3 h. Flow cytometry and Hoechst are used to assessing apoptosis. The expression spot and protein levels of PDK, Akt, and ERK are also analyzed. Results  I/R results in severe injury in NRK-52E cells as evidenced by increased LDH leakage in the culture medium, as well as increased apoptotic index, which may be significantly attenuated by IPO treatment applied before the abrupt reperfusion (P < 0.05 vs. I/R group). Meanwhile, IPO, compared with I/R, increases phosphorylation levels of Akt and ERK (P < 0.05 vs. I/R group), which have been

Xiaodong Weng and Min Wang have contributed equally to this work and are co-first authors. * Xiuheng Liu [email protected] 1



Department of Urology, Renmin Hospital of Wuhan University, Wuhan University, Jiefang Road 238, Wuhan 430060, China

identified to play a vital role in the regulation of cell proliferation, survival, and metabolism. Conclusion  A new in vitro model of I/R and IPO is established successfully. These results offer evidence that 3 h of simulating ischemic/reperfusion injury may cause cell apoptosis, and IPO is effective to attenuate renal cell apoptosis and potentially mediate via activation of Akt and ERK signal. Keywords  Ischemic/reperfusion · Ischemic postconditioning · In vitro model · Akt · ERK

Introduction Renal ischemia is a major cause of acute renal failure (ARF), which is encountered in many urological clinical situations: kidney transplantation, partial nephrectomy, renal artery angioplasty, hydronephrosis, and elective urological operations. Although reperfusion can result in the recovery of normal function, there are evidences showing that reperfusion itself may cause additional injury [1, 2]. Ischemia/reperfusion injury (IRI) is associated with high morbidity and mortality. There are different ways to deal with IRI, such as ischemic preconditioning (IPC) and ischemic postconditioning (IPO). Although IPC is successful to attenuate I/R injury [3–8], its utilization as clinical strategy is obviously limited because it is difficult to predict the onset of ischemia. Recently, IPO is first reported in 2003 by Zhao et al. [9], which is defined as a series of rapid brief alternating periods of arterial reperfusion and reocclusions in the early phase of reperfusion. Our previous researches [10, 11] have demonstrated that IPO can attenuate renal damage, decrease apoptosis, and increase phosphorylation of Akt and ERK1/2 after I/R injury in vivo.

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When kidneys are exposed to I/R injury, the fate of the renal tubular epithelium is diverse. Some cells undergo necrosis and apoptosis, while others still survive. The fate of cells suffering from I/R injury is determined by the balance between survival signals and death signals [12, 13]. The phosphatidylinositol 3-kinase/Akt (PI3 K/ Akt) plays a critical role in cell proliferation, differentiation, and survival in many cell types [14, 15]. Moreover, our previous study [11] and several other studies [16–22] have reported that activation of Akt pathway is involved in ischemic injury and protection of kidney, heart, brain, and liver. In addition, the ERK pathway also mediates a number of cellular fates including growth, proliferation, and survival. At the same time, activation of ERK has been reported to be increased after renal epithelial cells suffering from ischemic injury in vivo [11, 23, 24]. So far, the vast majority of renal I/R and IPO studies are based on animal models. In order to eliminate the influence of various confounding factors in animal experiment, we attempt to set up a neoteric in vitro model of I/R and IPO using the rat proximal tubule cell line NRK-52E. The mechanism of protective effect of IPO is unknown, and this protective effect may be related with the phosphorylation levels of Akt and ERK1/2; we examine renal cell apoptosis rate and phosphorylation status of Akt and ERK at 3, 6, 12, and 24 h of reperfusion after IPO treatment.

Materials and methods Reagents and cell line Rat proximal tubule cell line NRK-52E is purchased from Cell resource center CAS Shanghai Institute of life science. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) are obtained from Hyclone Corporation (USA). High-purity nitrogen is purchased from Wuhan Nanyang company (China). Annexin-V/PI kit is purchased from Bender MedSystems (USA). Hoechst 33342/PI kit is obtained from Sigma (USA). Lactic dehydrogenase (LDH) detection kit is purchased from Yatai Corporation (China). P-PDK antibody, P-Akt SER473 antibody, T-Akt antibody, P-ERK antibody, and T-ERK antibody are purchased from Cell Signaling Technology (USA). β-Actin antibody is purchased from Santa Cruz Biotechnology (USA). Experimental protocol and cell treatments The cells are plated at a density of 1 × 105 cells/mL in 100mL culture flasks containing 5 mL DMEM supplemented with 10 % FBS, streptomycin 100 g/mL, and penicillin 100 U/mL and placed in a humidified incubator with 95 %

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air and 5 % CO2 at 37 °C. The cells are fed every 2–3 days and subcultured once reaching 80–90 % confluence. In order to synchronize the cells, the complete medium is replaced with serum-free DMEM 24 h before the experiment. Then, the cells are randomly divided into three treatment groups as follows: (1) Normal group: NRK-52E cells are first incubated in 1 mL control buffer (NaHCO3 24.0 mM, Na2HPO4 0.8 mM, NaH2PO4 0.2 mM, NaCl 86.5 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM, and HEPES 20 mM; pH adjustment to 7.4 with 1 N NaOH) under normoxic conditions (95 % air–5 % CO2) for 3 h and then are incubated in 2 mL fresh complete DMEM medium (DMEM, 10 % FBS, streptomycin 100 mg/mL, and penicillin 100 U/mL) under normoxic conditions (95 % air–5 % CO2) for 24 h; (2) Ischemic/reperfusion injury (I/R) group: NRK-52E cells are washed with 1 mL serum-free DMEM medium pH 7.4, following with 1 mL sugar-free DMEM medium pH 7.4 and then are incubated in 1 mL ischemic buffer (NaHCO3 4.5 mM, Na2HPO4 0.8 mM, NaH2PO4 0.2 mM, NaCl 106.0 mM, KCl 5.4 mM, CaCl2 1.2 mM, MgCl2 0.8 mM, MES 20 mM; pH adjustment to 6.6 with 1 N NaOH) under hypoxia conditions (0.5 % O2–5 % CO2–94.5 % N2) for 3 h. After this phase, the serum-free and sugar-free DMEM medium is replaced with complete DMEM medium to simulate reperfusion, followed by 3, 6, 12, and 24 h in normoxic conditions; (3) Ischemic postconditioning by “adding medium” group(IPO): NRK-52E cells are first incubated in 1 mL ischemic buffer under hypoxia conditions for 3 h. After this phase, the culture dishes are transferred to a normoxic condition incubator in a humidified atmosphere with adding another 0.5 mL fresh complete DMEM medium for 10 min. Subsequently, the cells are returned to hypoxia conditions for 10 min. The postconditioning cycle is repeated three times and is described as IPO, which is implemented in the initial phase of reperfusion. Finally, the cells are incubated in 2 mL fresh complete DMEM medium under normoxic conditions for 3, 6, 12, and 24 h. Fluorescein isothiocyanate (FITC)‑conjugated Annexin‑V‑propidium iodide (PI) dual staining The apoptotic cells are measured by flow cytometer using the Annexin-V/PI kit according to the manufacturer’s instructions. In brief, cells are collected, washed with calcium-free PBS, resuspended with binding buffer, and incubated with Annexin-V at room temperature in dark for 10 min. Then the cells are centrifuged and resuspended with binding buffer. Propidium iodide (PI) is added to the resuspended cells before they are analyzed by flow cytometry using a FACSCalibur system(Becton–Dickinson; USA). The apoptotic and live cells are able to bind

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FITC-conjugated Annexin-V and exclude PI, because their membranes are intact. On the contrary, necrotic cells are stained by both PI and FITC-conjugated Annexin-V because of the imperfection of their cellular membranes. At least 400 cells are counted in each field. Hoechst 33342 assay Exponentially growing cells are plated in six-well plates at a density of 5 × 105 cells/well and cultured for 72 h. Following simulated I/R and IPO procedures, cells are fixed for 8 min with the precooled (−20 °C) formaldehyde and acetone solution (1:1, v/v) and washed with PBS 3 × 3 min, followed by staining with Hoechst 33342 solution (50 mmol/L) at 37 °C in darkness for 5 min. Apoptotic cells are observed, and images are taken using fluorescence microscope (OlympusBX51, Tokyo, Japan), with excitation wavelength of 350 nm and emission wavelength of 460 nm. LDH assay The previous study has shown that LDH release is a reliable marker of hypoxia injury correlating well with cell death [25]. Activities of LDH released into the culture medium from the normal cells and injured cells after 24 h of reperfusion with or without IPO treatment are assayed, respectively, using a LDH detection kit. The absorbance of the reaction mixture at 340 nm is measured by a spectrophotometer (Analytical instrument factory of Shanghai, China) following instructions of the manufacturer. Western blot analysis The protein expression levels of P-PDK, P-Akt SER473, T-Akt, P-ERK, and T-ERK are examined by Western blotting. Briefly, proteins are extracted from NRK-52E cells, separated on 10 % SDS-PAGE gels (20 μg/lane), and then transferred to nitrocellulose membrane. The membranes are blocked with 5 % nonfat milk in TBST buffer (10 mmol/L Tris–HCl, 0.15 mol/L NaCl, and 0.05 % Tween 20, pH 7.2) and incubated with primary antibodies overnight at 4 °C. Primary antibodies used here are polyclonal rabbit antibodies against P-PDK, P-Akt SER473, T-Akt, P-ERK, and T-ERK (1:1000 dilution; Cell Signaling Technology, USA). After extensive washing with TBST buffer, the membranes are incubated with HRP-conjugated anti-rabbit secondary antibodies (Santa Cruz Biotechnology). The proteins are detected using an enhanced chemiluminescence system (ECL kit, Pierce Biotechnology, Beijing, China) and captured on light-sensitive X-ray film (Kodak, Shanghai, China). Optical densities are detected using ImageJ software.

Statistical analysis All data are presented as mean ± SEM. The means of the different groups are compared using one-way analysis of variance (ANOVA) and the Student–Newman–Keuls test. The Kruskal–Wallis ANOVA on ranks is used for non-normally distributed data. The level of statistical significance is set at P < 0.05.

Results Evaluation of apoptosis rate by fluorescein isothiocyanate (FITC)‑conjugated Annexin‑V‑propidium iodide (PI) dual staining Apoptosis rate is the most important marker after IRI, which determines the fate of diverse cells. Effects of postconditioning on the I/R-induced cell apoptosis are evaluated by fluorescein isothiocyanate (FITC)-conjugated Annexin-V-propidium iodide (PI) dual staining. The results show that: (1) in the I/R group, the cells are incubated in 1 mL serum-free and sugar-free DMEM medium under hypoxia conditions to “mimic ischemic” for 3 h and then are incubated in 2 mL complete DMEM medium in normoxic conditions to “mimic reperfusion” for 24 h. Compared with the normal group, it is obviously effective to induce cell apoptosis (P < 0.05 vs. normal group); (2) in the IPO group, the cells are first incubated in the same condition as the I/R group to “mimic ischemic” for 3 h, and then 0.5 mL fresh medium is added to the culture flask in each postconditioning cycle to mimic the slow process of removing metabolic waste and supplying nutrient. This approach is markedly able to reduce ischemicinduced apoptosis, the effect of which is more obvious with the increase in cycle. As shown in Fig. 1, I/R is obviously effective to induce cell apoptosis, but IPO is markedly able to reduce ischemic-induced apoptosis. Detection of apoptotic cells by Hoechst 33342 assay Effects of postconditioning on the I/R-induced cell apoptosis are also assayed by Hoechst 33342 staining. As shown in Fig. 2, few apoptotic cells are present in the normal group. As expected, there are many apoptotic (Hoechst 33342-positive) cells in the I/R group and in the IPO group. However, compared with the I/R group, fewer apoptotic cells are observed in the IPO group (P < 0.05 vs. I/R group). LDH release To further confirm the protection of IPO for ischemic cells against reperfusion injury, the amount of LDH released

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Fig. 1  Representative flow cytometric analysis of apoptosis rate of NRK-52E cell in N group, I/R group, and IPO group after 24 h. Apoptosis rate of I/R group and IPO group are obviously higher than

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that of N group, but IPO group significantly reduced apoptosis rate related to I/R group. (*P < 0.05 vs. normal group, #P < 0.05 vs. I/R group. n = 4)

Fig. 2  Representative images of Hoechst 33342 staining: the arrows represent the apoptotic cells

into the culture medium from the injured cells is measured after 24 h of reperfusion. As shown in Fig. 3, LDH leakage in the culture medium determined after I/R is significantly higher than normoxic controls (P < 0.05 vs. normal group). When the cells are treated with postconditioning, LDH content in the medium is reduced (P < 0.05 vs. I/R group).

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IPO promotes levels of P‑PDK, P‑Akt SER473, and P‑ERK Activation of Akt and ERK are thought to play an important role in the renal IRI and the renal protection of IPO. To investigate the different levels of protein expression, we measure P-PDK, P-Akt SER473, T-Akt, P-ERK, and

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Fig. 3  LDH levels after treatment of 24 h. LDH levels of I/R group and IPO group are both higher than that of N group, but IPO group significantly reduced LDH levels compared to I/R group. (*P < 0.05 vs. normal group, #P < 0.05 vs. I/R group. n = 5)

Fig. 5  Change in expression AKT and P-AKT protein determined by Western blot analysis in N group, I/R group, and IPO group after 3, 6, 12, and 24 h. (*P < 0.05 vs. normal group, #P < 0.05 vs. I/R group. n = 4)

Fig. 4  Change in expression P-PDK protein determined by Western blot analysis in N group, I/R group, and IPO group after 3, 6, 12, and 24 h. (*P < 0.05 vs. normal group, #P < 0.05 vs. I/R group. n = 4)

T-ERK by Western blot. As shown in Figs. 4, 5, 6, compared with the normal group, the I/R and IPO groups both induce a transient increase in phosphorylation of ERK and Akt, without any change in total ERK and Akt expression; meanwhile, the I/R and IPO groups induce a corresponding increase in phosphorylation of PDK, which is a upstream regulator and a crucial marker for activation of Akt. The Western blot analysis shows a significant increase in P-PDK and P-ERK in the IPO group compared with the I/R group at 3, 6, and 12 h of reperfusion after ischemic treatment. It also shows a significant increase in P-Akt SER473 in the IPO group compared

Fig. 6  Change in expression ERK and P-ERK protein determined by Western blot analysis in N group, I/R group, and IPO group after 3, 6, 12, and 24 h. (*P < 0.05 vs. normal group, #P < 0.05 vs. I/R group. n = 4)

with the I/R group at 6 and 12 h of reperfusion after ischemic treatment. However, there are no significant differences in the levels of P-PDK, P-Akt SER473, and P-ERK at 24 h of reperfusion after ischemic treatment. Overall, I/R and IPO both up-regulate the level of P-Akt, P-ERK, and PDK (an upstream regulator of Akt), but IPO increases the level of P-Akt, P-ERK, and PDK more obviously.

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Discussion Substantial studies have demonstrated that IPO is an effective therapy for IRI. The onset of reperfusion is predictable, which makes the intervention with IPO in clinical practice a promising, appropriate, and feasible option. The protective effects of IPO in heart, brain, liver, and renal have been widely verified in animal models. Although, these in vivo models closely simulate the clinical condition of renal IRI and IPO, they are limited by a number of factors, such as differential renal cell inflammatory mediator production and sensitivity to ischemia. So, it is difficult to estimate the mechanism of IRI and IPO. In order to eliminate these confounding variables, we and other researchers have attempted to build in vitro models of renal IRI and IPO, in which specific stimuli can be isolated, controlled, and tested to demonstrate their contribution to physiological or pathological events. A neoteric in vitro model of I/R and IPO is successfully set up by using the rat proximal tubule cell line NRK-52E, which is based on the model of Christoph Sauvant [25]. Investigators often incorporate hypoxia and glucose deprivation into their in vitro model merely, but do not take other important experimental variables into account. However, this in vitro model simulates the clinical condition of a cellular microenvironment representative of ischemia, including oxygen deprivation, carbon dioxide elevation, nutrient depletion, and waste accumulation. Meldrum et al. [26] have described an in vitro oil immersion model of ischemia, which mimics the cellular injury observed in animal models of renal ischemia/reperfusion successfully. However, that in vitro model cannot simulate the cellular microenvironment of ischemia. In addition, it is unable to investigate the protective effect of IPO. Kelly et al. [27] set up a novel method for studying hypoxia and ischemia in vitro by using glass coverslip, which is simple and represents the tissue environment of restricting nutrient supply and accumulating metabolites, ions, and gases. The same as the previous model, the model is restricted to implementing IPO. Several studies [28, 29] set up an in vitro ischemia and reperfusion model by simply using hypoxia and reoxygenation, and other groups [30, 31] attempt to improve this in vitro model by adding nutrient deprivation and restoration along with hypoxia and reoxygenation. However, these models fail to mimic the typical characteristic of carbon dioxide elevation and waste accumulation in the process of ischemia and reperfusion. In this in vitro model of I/R and IPO: (1) As we know, an O2 fraction below 1 % represents a pO2 of below 7.6 mmHg which is well below the critical pO2 for oxygen consumption in the kidney cortex [32]. So, cells are cultured in a hermetic chamber filled with gas consisting of 0.5 % O2 which can well represent the condition of hypoxia; (2)

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In fact, fetal bovine serum (FBS) contains approximately 1.20 ± 0.27 g glucose/L. This is much greater than physiological blood glucose (0.8–0.9 g/L or approximately 4–5 mM). In order to more accurately mimic nutrient depletion, cells are exposed to both sugar-free and serumfree medium; (3) cells are exposed to ischemic buffer (pH 6.6) which is described in the renal cortical tissue during ischemia [33]; (4) IPO treatment is implemented by retaining the original culture liquid, adding another 0.5 mL fresh complete medium, and being placed to normoxic conditions in each postconditioning cycle. The results of flow cytometry, Hoechst assay, and LDH release assay show that IPO by adding medium is more effective to reduce apoptotic rate than by changing medium (the date not shown). Renal apoptosis is an important factor in the development of ARF after I/R injury [34, 35]. Through previous studies, IPO has an anti-apoptotic effect in vivo, but it is still unclear how IPO inhibits apoptosis after renal I/R injury. As we know, the activation of Akt and ERK are involved in ischemic injury and protection of kidney and other organs. Full activation of Akt requires both translocation and phosphorylation at its residues. The phosphorylation of the residues is accomplished through phosphoinositide-dependent kinase (PDK) and thought to be the major activating event. This study indicates that IPO upregulate activity of Akt and ERK1/2 after renal I/R injury, which produces similar results as in vivo model. There are some limitations in this study. Firstly, whether the ischemic postconditioning plays its role in an ‘on–off’ style or a ‘dose-dependent’ one is not fully elucidated in this study. Postconditioning may play its role in a “dosedependent” manner, which is not fully elucidated in this study, and 10 min may not afford the maximal protective effect against I/R injury if postconditioning acts in a “dose-dependent” style. Thus, the exact number of optimal intervals and cycles may also need additional investigation. Secondly, we mimic tissue acidosis by ischemic buffer to adjust pH to 6.6, which is different from tissue acidosis resulting from hypercapnia. Carbon dioxide can influence cellular function via acidification as well as pH-independent mechanisms. Additionally, in our in vitro model, pH changes are modified by extracellular environment, but they first occur intracellularly then spread to the extracellular environment in vivo. Finally, our in vitro model does not incorporate three-dimensional structure- or neighboring cell-type interactions present in vivo. So, it may produce results that differ from in vivo experiments. In conclusion, an in vitro model of renal I/R and IPO is set up by using the rat proximal tubule cell line NRK52E. Moreover, this model simulates the clinical condition of a cellular microenvironment representative of ischemia, including oxygen deprivation, carbon dioxide elevation, nutrient depletion, and waste accumulation. Our results

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offer evidence that 3 h of simulating ischemic/reperfusion injury may cause cell apoptosis, and IPO is effective to attenuate renal cell apoptosis, potentially mediate via trigger the up-regulation of Akt and ERK signal. This model may be used to research the role of a specific protein in I/R and IPO and further to study the mechanism of I/R and IPO. Acknowledgments  This work was supported by the National Natural Science Foundation of China (Nos. 30901494 and 30901552), the Hubei Province Natural Science Foundation (No. 2012FFA096), and supported by “the Fundamental Research Funds for the Central Universities (No. 302-274231)”. Conflict of interest None.

References 1. Martins PN, Chandraker A, Tullius SG (2006) Modifying graft immunogenicity and immune response prior to transplantation: potential clinical applications of donor and graft treatment. Transpl Int 19:351–359 2. Sehirli O, Sener E, Cetinel S, Yüksel M, Gedik N, Sener G (2008) Alpha-lipoic acid protects against renal ischaemia-reperfusion injury in rats. Clin Exp Pharmacol Physiol 35:249–255 3. Zager RA, Iwata M, Burkhart KM, Schimpf BA (1994) Postischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia. Kidney Int 45:1760–1768 4. Park KM, Chen A, Bonventre JV (1999) Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J Biol Chem 276:11870–11876 5. Toosy N, McMorris EL, Grace PA (1999) Ischemic preconditioning protects the rat kidney from reperfusion injury. BJU Int 84:489–494 6. Jefayri MK, Grace PA, Mathie RT (2000) Attenuation of reperfusion injury by renal ischemic preconditioning: the role of nitric oxide. BJU Int 85:1007–1013 7. Lee HT, Emala CW (2001) Protein kinase C and Gi/o proteins are involved in adenosine and ischemic preconditioning-mediated renal protection. J Am Soc Nephrol 12:233–240 8. Torras J, Herrero-Fresneda I, Lloberas N, Riera M, Ma Cruzado J, Ma Grinyó J (2002) Promising effects of ischemic preconditioning in renal transplantation. Kidney In 61:2218–2227 9. Zhao ZQ, Vinten-Johansen J (2006) Postconditioning: reduction of reperfusion-induced injury. Cardiovasc Res 70:200–211 10. Liu X, Chen H, Zhan B, Xing B, Zhou J, Zhu H, Chen Z (2007) Attenuation of reperfusion injury by renal ischemic postconditioning: the role of NO. Biochem Biophys Res Commun 359:628–634 11. Chen H, Xing B, Liu X, Zhan B, Zhou J, Zhu H, Chen Z (2008) Ischemic post- conditioning inhibits apoptosis after renal ischemia/reperfusion injury in rat. Transpl Int 21:364–371 12. Toback FG (1992) Regeneration after acute tubular necrosis. Kidney Int 41:226–246 13. Safirstein R, DiMari J, Megyesi J, Price P (1998) Mechanisms of renal repair and survival following acute injury. Semin Nephrol 18:519–522 14. Aikawa R, Nawano M, Gu Y, Katagiri H, Asano T, Zhu W, Nagai R, Komuro I (2000) Insulin prevents cardiomyocytes from oxidative stress-induced apoptosis through activation of PI3 Kinase/ Akt. Circulation 102:2873–2879

15. Steelman LS, Stadelman KM, Chappell WH, Horn S, Bäsecke J, Cervello M, Nicoletti F, Libra M, Stivala F, Martelli AM, McCubrey JA (2008) Akt as a therapeutic target in cancer. Expert Opin Ther Targets 12:1139–1165 16. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, Force TL, Franke TF, Hajjar RJ, Rosenzweig A (2001) Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation 104:330–335 17. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM (2004) Postconditioning: a form of ‘‘modified reperfusion’’ protects the myocardium by activating the phosphatidylinositol 3-kinase-akt pathway. Circ Res 95:230–232 18. Galatou E, Carnická S, Nemcˇeková M, Ledvényiová V, Adameová A, Kelly T, Barlaka E, Galatou E, Khandelwal VK, Lazou A (2012) PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia-reperfusion injury: involvement of PI3 K-Akt. Can J Physiol Pharmacol 90:1135–1144 19. Shibata M, Yamawaki T, Sasaki T, Hattori H, Hamada J, Fukuuchi Y, Okano H, Miura M (2002) Upregulation of Akt phosphorylation at the early stage of middle cerebral artery occlusion in mice. Brain Res 942:1–10 20. Harada N, Hatano E, Koizumi N, Nitta T, Yoshida M, Yamamoto N, Brenner DA, Yamaoka Y (2004) Akt activation protects rat liver from ischemia/reperfusion injury. J Surg Res 121:159–170 21. Toledo-Pereyra LH, Lopez-Neblina F, Reuben JS, Toledo AH, Ward PA (2004) Selectin inhibition modulates Akt/MAPK signaling and chemokine expression after liver ischemia–reperfusion. J Invest Surg 17:303–313 22. Sano T, Izuishi K, Hossain MA, Kakinoki K, Okano K, Masaki T, Suzuki Y (2010) Protective effect of lipopolysaccharide preconditioning in hepatic ischemia reperfusion injury. HPB (Oxford) 12:538–545 23. Park KM, Chen A, Bonventre JV (2001) Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J Biol Chem 276:11870–11876 24. Park KM, Kramers C, Vayssier-Taussat M, Chen A, Bonventre JV (2002) Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J Biol Chem 277:2040–2049 25. Sauvant C, Schneider R, Holzinger H, Renker S, Wanner C, Gekle M (2009) Implementation of an in vitro model system for investigation of reperfusion damage after renal ischemia. Cell Physiol Biochem 24:567–576 26. Meldrum KK, Meldrum DR, Hile KL, Burnett AL, Harken AH (2001) A novel model of ischemia in renal tubular cells which closely parallels in vivo injury. J Surg Res 99:288–293 27. Pitts KR, Toomb CF (2004) Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro. Am J Physiol Heart Circ Physiol 287:H1801–H1812 28. Cavdar Z, Oktay G, Egrilmez MY, Genc S, Genc K, Altun Z, Islekel H, Guner G (2010) In vitro reoxygenation following hypoxia increases MMP-2 and TIMP-2 secretion by human umbilical vein endothelial cells. Acta Biochim Pol 57:69–73 29. Saenz-Morales D, Escribese MM, Stamatakis K, García-Martos M, Alegre L, Conde E, Pérez-Sala D, Mampaso F, García-Bermejo ML (2006) Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress. Exp Cell Res 312:3711–3727 30. Saenz-Morales D, Conde E, Escribese MM, García-Martos M, Alegre L, Blanco-Sánchez I, García-Bermejo ML (2009) ERK1/2 mediates cytoskeleton and focal adhesion impairment in proximal epithelial cells after renal ischemia. Cell Physiol Biochem 23:285–294

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31. Basnakian AG, Ueda N, Hong X, Galitovsky VE, Yin X, Shah SV (2005) Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxiareoxygenation. Am J Physiol Renal Physiol 288:F308–F314 32. Leichtweiss HP, Lübbers DW, Weiss C, Baumgärtl H, Reschke W (1969) The oxygen supply of the rat kidney: measurements of int4arenal pO2. Pflugers Arch 309:328–349 33. Holloway JC, Phifer T, Henderson R, Welbourne TC (1986) Renal acid-base metabolism after ischemia. Kidney Int 29:989–994

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Int Urol Nephrol 34. Bonegio R, Lieberthal W (2002) Role of apoptosis in the pathogenesis of acute renal failure. Curr Opin Nephrol Hypertens 11:301–308 35. Padanilam BJ (2003) Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 284:F608–F627