The Study Of The Protective Effect Of Hypoxia-Preconditioning For The Apoptotic Injury Of Renal Tubular Epithelial Cell Induced By Anoxia/Reoxygenation

Objective:①During the renal ischemia, injury of tubular epithelial cell is the main course. To investigate the role of TEC in ischemia injury but not influenced by the other parts of the nephron, we need lots of pure TEC from cell culture for our study. For this purpose ,we should established a simple, effective and low-cost method of primary culture and subculture of TEC. ②Apoptosis and necrosis are central mechanisms leading to organ damage in the course of renal ischemia/reperfusion injury(I/R). which way cell to dead is associated with the severity and duration of initial injury. Renal ischemic preconditioning (IPC) is a phenomenon whereby a brief period of ischemia and reperfusion provides tolerance to subsequent periods of ischemia. IPC has been demonstrated to protect rodent kidneys during I/R. To investigate the apoptotic injury of TEC under the conditions of anoxia/reox-ygenation (A/R) and anoxia-precondition (APC) and to evaluate the protective effect of APC, we need to establish a experiment model of A/R and APC. The majority of research into this disorder has utilized animal models of renal ischemia-reperfusion injury. While these models closely simulate clinical renal IR injury, they are limited by differential renal cell inflammatory mediator production, sensitivity to ischemia, and mechanisms of injury. An in vitro model of simulated-ischemia may allow investigators to better elucidate the intracellular signaling mechanisms involved in renal IR injury. Based on previous work demonstrating that cellular immersion in mineral oil results in nutrient and oxygen deprivation, we developed an in vitro model of simulated anoxia using the cultured renal tubular epithelial cell. It has previously been shown in animal models that renal tubular cells are very susceptible to ischemic injury, undergoing apoptotic cell death. This study constitutes the initial demonstration that an in vitro oil immersion model of simulated anoxia/reoxygenation induces the apoptotic injury of the TEC and simulated APC reduces the apoptotic injury induced by A/R. Methods: ①Cell culture. The renal tubular segments were harvested by a mechanical grinding and enzymatic digesting (0.25% trypsin) from rat renal cortex and were cultured in medium RPMI1640 supplemented with 15% fetal bovine serum(FBS). The medium was refreshed after the first 72 hour and then was refreshed every 2-3 days. The cells were passaged by 0.25% trypsin following formation of a confluent monolayer every 7-10 days. ②Cell identify. The TEC was identified by dynamic observing with light microscope , cytochemistry (alkali-phosphatase AKP) staining and transmission electron microscope.③Simulated A/R and APC. The cells were grown until confluent. Following formation of an epithelial cell sheet, the cells were washed twice with PBS and the monolayer immersed in mineral oil. This immersion induced simulated anoxia by restricting cellular exposure to oxygen. And then the cells were reimmersed in the medium RPMI1640 supplemented with 15% FBS to simulate reoxygenation. ④Experimental groups. The specimens were divided into control(n=8), A/R(n=8) and APC groups(n=8), cells of the A/R groups were exposed to 1 h of simulated anoxia followed by 0, 24, 48 or 72h of simulated reoxygenat-ion(reimmersion in medium RPMI1640 15% FBS). Cells of APC groups were exposed to these procedure after exposed to simulated APC(three repeated procedure of 5 minutes of anoxia and 5 minutes of reoxygenation). ⑤Quantitation of apoptosis. The terminal-deox-ynucleotidyl transferase mediated nick and labeling (TUNEL) and the flowcytometry (FCM) techniques(FITC-AnnexinV Fluorescence) were taken to detect the occurrence of the apoptotic injury of TEC. ⑥Statisticalanalysis. Data are presented as mean values±standard deviation. Differences at the 95% confidence level were considered significant. The experimental groups were compared by SNK-q test using SPSS 11.5. Results: ①The TEC was successfully cultured in this experiment. The tubular segments began to anchor during the first 24-48h, and there were oval-shaped cells growing out around the anchored tubular segments during the 2nd or 3rd day. The cells turned into logarithmic growth phase from the 4th to 7th day. But the cells growth stopped and emerged aging from the 10th to 14th day. Moreover, the cells could be subcultured in the same medium up to about 10th passages , and then the cells division gradually slowed down and even stasised. ②The primary cultured TEC was all positive by alkali-phosphat-ase(AKP) staining and a great deal of microvilli were found by transmission electron microscope on the brush border of the TEC. ③The cellular apoptosis was successfully induced by immersing the cultured cells in mineral oil (paraffin)—anoxia for 60min and reoxygenate in 0,24h,48h and 72h. The highest rate of apoptosis was at 48h point(group). It can be seen that the degree of apoptosis was significantly decreased in every phase of APC(anoxia/precondition) group which was treated with the same method after the previous treatment of three short term(5 minutes) of repeated anoxia/reoxygenation compared with the corresponding group of AP. Apoptotic Index(AI) detected by TUNEL and FCM all showed that there was significant difference among each groups(p<0.05 or p<0.01 ). Conclusions: ①We can harvest relatively pure TEC(>90%) from the Wistar rat kidney by employing mechanical grinding and enzymatic digesting techniques.The TEC can be primary cultured and passaged to about 10 generations using medium RPMI1640 supplemented with 15% FBS and 0.25% trypsin. ②TEC can be identified by dynamic observing with light microscope,cytochemical staining and transmission electron microscope.③The TEC apoptosis can be successfully induced by simulated anoxia/reoxygenation by the means of immersing the cells in the mineral oil and reimmersing them in the medium RPMI1640 supplementedwith 15% FBS and can be detected by TUNEL and FCM. This in vitro model of renal ischemia-reperfusion injury is easy to put into practice and could be well servered for the study of renal ischemic-reperfusion in vitro. While this model is less representative of clinical renal ischemia/reperfusion injury than comparable animal models, it may serve as a means of investigating the more intricate intracellular mechanisms of this disorder.④This study has demonstrated that the degree of apoptosis of TEC in APC group is significantly reduced compared to the corresponding A/R group at the different time point. It therefore behoves us to understand this endogenous processes that the kidney has developed to protect itself against an ischemic insult. Armed with this understanding we can then attempt to mimic these processes and thereby prevent and treat ischemic renal diseases.