Ischemia-reperfusion is a common injury of clinical ischemic disease and surgical lesions. demonstrated increased expression of Bax, cleaved caspase-3 and caspase-8 at the end of reperfusion. However, Rabbit polyclonal to FBXO42 the levels of Bax, cleaved caspase-3 and caspase-8 were significantly attenuated in cells, which had undergone IPO. In conclusion, apoptosis was observed in cells subjected to 3 h of ischemia-reperfusion injury 21535-47-7 and IPO was able to inhibit this apoptosis. IPO inhibited apoptosis by inhibiting the caspase pathway thereby exerting protective effects. model, apoptosis, caspase cascade Introduction One of the major causes of acute renal failure (ARF) is ischemia, which occurs in kidney transplantation, partial nephrectomy, renal artery angioplasty, sepsis, accidental or iatrogenic trauma, hydronephrosis, elective urological operations, aortic bypass surgery, cardiopulmonary bypass, the use of 21535-47-7 vasoconstricting drugs and certain hypotensive states (1,2). ARF has a high incidence in intensive care units, representing an isolated prognostic factor in patients with multiple organ dysfunction syndrome (3). The clinical significance of ARF is due to its high mortality, which ranges between 30 and 70% (4). Thus, novel 21535-47-7 therapies are required to prevent or alleviate ischemic injury. Previous studies have demonstrated that ischemic preconditioning (IPR) and ischemic postconditioning (IPO) are two important mechanical methods, which are able to improve the ability of organs subjected to ischemia to tolerate injury (5,6). Although IPR is effective at reducing ischemia-reperfusion injury (IRI), its clinical application is limited as it must be initiated prior to the ischemic period, which is unreasonable in a clinical situation. IPO is a series of brief rapid intermittent cycles of ischemia applied at the onset of reperfusion in the previously ischemic tissue or organ (7). Several studies have demonstrated that IPO was able to cause a significant reduction in the systemic inflammatory response, inhibit the expression of apoptosis-associated molecules and activate endogenous protective molecules (8C10). In renal IPO studies, major studies were based on animal models, including our earlier studies using rat or canine models (11,12). However, to the best of our knowledge, an postconditioning model, which is able to effectively simulate the process of IPO against IRI in the kidney, has not yet been investigated. Based on a study using an model for 13), a novel IPO model, which simulates IPO in the kidney was developed in the present study using a rat proximal tubular cell line (NRK-52E cells). In addition, the molecular mechanism involved in IPO of renal tubular epithelial cells was analyzed. Materials and methods Cell culture The renal tubular epithelial cell line, NRK-52E, was purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured on culture dishes with 5% CO2 and maintained at pH 7.4 and 37C. The medium was changed once every 3 days and the cells were used for experiments at day 10 after seeding. Cells were cultured in serum-free medium for 24 h prior to the experiments. Cells were seeded on 6-well plates or culture dishes as appropriate. In vitro IPO model Prior to the experiment, the cells were placed in serum-free medium for 24 h. Subsequently, all cell culture dishes were randomly divided into nine groups (Fig. 1). For the normal group, the cells were cultured in complete medium under normal conditions (5% CO2, 21535-47-7 saturated humidity and 37C) and 3 h later fresh medium was added and cultured under the same conditions for 24 h. For the control group, the cells were cultured in 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, HEPES 20 mM and 5 mM glucose; pH adjustment to 7.4 with 1 N NaOH) (13) for 3 h and further cultured in complete medium for 24 h. The cells in the ischemia/reperfusion (I/R) group were washed with phosphate-buffered saline (PBS; Gibco Life Technologies, Carlsbad, CA, USA) and placed in 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 and morpholinoethanesulfonic acid.