The Investigation On Bcl-2 In Facilitating The Recovering Process Of Initial Renal Tubular Epithelium Injury In Culture And Acute Renal Failure

BACKGROUND: The mortality in patients with acute renal failure has remained unchanged since the mid-1960s and remains high at approximately 50%. It is unlikely that the high mortality rate of the disease will be reduced until we have a better understanding of the cellular and molecular mechanisms of renal tubular cell injury and recovery. It has long been recognized that in acute renal failure, renal tubule cells die in a catastrophic breakdown of regulated cellular homeostasis, known as necrosis. Over the last decade there is an accumulating evidence suggesting the renal epithelial cell apoptosis contributes significantly to the pathogenesis of acute renal failure. A better understanding of the mechanisms of apoptosis could lead to safer and more specific therapeutic interventions for acute renal failure. In this study,we present evidence to support the hypothesis that apoptosis is an important pathogenic mechanism of acute renal failure in which the renal tubular epithelial cell is the primary target of ischemic and toxic injury.Bcl-2 is a putative anti-apoptosis oncogene which inhibits apoptosis happening. But there is different point of view about its effects on the renal epithelial cells apoptosis induced by different forms of injuries. Up to now, there is limited data for its effect on the apoptotic renal epithelial cells after ischemia and toxins induced ARF. We investigated whether the overexpression of Bcl-2 protein had the anti-apoptosis function in renal epithelial cells in vivo and in vitro. Apoptosis was evaluated by TUNEL and annexin V-staining. The expression of Bcl-2 and Bax proteins was determined by immunohistochemistry and Western blotting.We cloning Bcl-2 cDNA into the replication defective retroviral vector pLXSN, A recombinant retroviral vector PLXBcl-2SN was constructed and transfected to packaging cell line PA317 by lipofectine. The G418 resistant colonies were selected, and the supernatants of the colony cultures were used to infect the human kidney epithelial cell line HK-2. Cells in G418 resistant HK-2 colonies were characterized by PCR and In situ hybridization,which were named as HK-2Bcl-2.-2Bd~2Hydrogen peroxide (H2O2) was applied to HK-2 and HK-2Bcl-2 cells for inducing apoptosis which mimicked the apoptosis after I/R and toxins on kidney. To explore the apoptosis-inducing effect of H2O2 on the renal epithelial cells (HK-2) and establish the model of H2O2-induced apoptosis in HK-2 cells. Cell viability was assessed by measuring LDH,The cytotoxic effect of 5mM H2O2 on HK-2 was detected by thiazolyl-blue(MTT) assay. Then apoptosis in HK-2 cells was induced with 5mM H2O2 for 8h, 1d and 3d. The changes of apoptotic cells number were assessed by flow cytometry.A model of acute renal failure (ARF) was done by injecting glycerin into the muscle with or without renal artery Bcl-2 retrovirus perfusion of Wistar rats. The changes of apoptotic cells number were detected by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.Bcl-2,Bax,IGF-l and PCNA expression was detected by immunohistochemistry.The main results are as follows:Western blot revealed that the Bcl-2 protein of the PLBcl-2SN transfected HK-2Bc1-2 cells increased remarkbly while there are no change in control one (p<0.05).lmM H2O2 cause a significant mitogenic response of HK-2 cells. 5mM H2O2 induced HK-2 cell morphology changes (cell shrinkage and rounding up) and part of cells loss. 20mM H2O2 induced the almost all HK-2 cell loss and detachment. LDH leakage test shows after treatment with H2O2 (5mM for8 h) caused significant increase in the release of LDH (283.48 24.24 vs control 32.64 5.22, p< 0.05). LDH released level in the HK-2Bc1-2 group was much lower compared to that of in the controls (173.85 25.32 vs 283.48 24.24 p< 0.05).We have also compared with trypan blue stain for assessing cell death and found them to be equivalent.H2O2 induced apoptosis on HK-2 cells was estabished successfully and the concentration of 5mM H2O2 can induce it's apoptosis.The higher t...