| Background Renal failure is a major clinical problem without effective therapy and contributes greatly to patient morbidity and mortality. Transplantation is considered to be one of the most important replacement therapy. However, renal ischemia reperfusion injury (RIRI) is unavoidable in renal transplantation, which firstly lead to acute tubular necrosis and then causes interstitial fibrosis. Fibrosis would finally result in graft dysfunction. Therefore, to alleviate the renal injury in early ischemia-reperfusion (IR) is of great important significance in renal transplantation.The transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein characterized by ion channel (e.g. Ca2+and Mg2+) and kinase activitiy. Like most of calcium channel, TRPM7 activity might be promoted by ischemia and consequently lead to overload of free and total intracellular calcium, which results in reactive oxygen species (ROS) production and cell death. However, the action of TRPM7 is still effective even if other known calcium influx pathways are pharmacologically blocked. Studies showed that TRPM7 up-regulation in cells increases levels of ROS and nitric oxide (NO) and stimulates p38 mitogen-activated protein kinase (p38 MAPK) and Jun N-terminal kinase (JNK). Another investigation showed role of Mg2+ in regulation of TRPM7 on intracellular ROS levels during cell stress is similar to that of calcium [14]. The MAPKs (p38, ERK-1/2 and JNK) are a family of protein kinases playing an important role in apoptosis and survival signaling, and the ROS and calcium are the most important impact factors in the ischemia reperfusion. ROS up-regulation can damage cellular components such as proteins, lipids, and DNA. Intracellular Ca2+ overload is also involved in cell death during IR. TRPM7 has been reported to be involved in neurons cell death during ischemia reperfusion via increasing intracellular Ca2+overload and oxidative stress. Suppression of hippocampal TRPM7 can provide protection against brain ischemia.However, we don't know how and where TRPM7 changed before and after renal ischemia reperfusion. And we also have no idea about the mechanism. If mice renal function in renal transplantation could be protected by down-regulated level of TRPM7? In part three, we aim to find whether suppression of TRPM7 by M7shRNA located under renal cortex, could protect mice renal function in renal transplantation or not.Part I The expression levels and location of TRPM7 during renal ischemia reperfusion Objective To investigate the expression levels and location of TRPM7 during renal ischemia reperfusion.Methods The Sprague Dawley rats were anesthetized with sodium pentobarbital. Bilateral flank incisions were made and the right kidney was removed. After the right kidney was removed, the rats underwent 45 minutes of left renal arteries occlusion followed by reperfusion for different time points (1,12,24,48,72 and 96 hours). Then the expression of trpm7 mRNA was measured by real-time RT-PCR, protein was measured by western blot and location was measured by quantum dots based immunofluorescence technology.Results Compared with control group, the expression of trpm.7 mRNA was significantly up-regulated in 24 hours after ischemia reperfusion. We set the expression of trpm7 mRNA of control group as standard (1.0), the 1h,12h,24h,48h,72h and 96h group was 2.83,3.16,3.95, 0.31,0.82 and 1.13. (n= 7, P< 0.05) The TRPM7 protein showed the same trends with mRNA. The main change of the TRPM7 protein was located in tubular cell.Conclusions The expression of trpm7 mRNA was obviously up-regulated in 24 hours after renal ischemia and it was also shown in protein level. The main change of the TRPM7 protein was located in tubular cell.Part ? The role of TRPM7 in cell hypoxia/reperfusion mode and mechanism Objective To investigate the role of TRPM7 in cell hypoxia/reperfusion mode and mechanism.Methods M7shRNA were used to decrease the expression of TRPM7 in NRK-52e cells. Then the effects of M7shRNA were measured by fluorescent probe for reactive oxygen species (ROS), intracellular calcium and magnesium; western blot was applied for p38-MAPKs and Bax expression in cell studies. And cell vitality was measured by cck-8.Results In cell model of hypoxia, the level of ROS in NRK-52e-M7shRNA was significantly lower than that in both NRK-52e and NRK-52e-Control cells while the activation of p38-MAPKs was limited. The level of calcium and magnesium was also lower in M7shRNA group. The result of cck-8 showed that vitality of NRK-52e-M7shRNA cell was better than others after hypoxia.Conclusion Suppression of TRPM7 could alleviate cell hypoxia reperfusion injury. The mechanism may by alleviate cell stress with inhibition of intracellular Ca2+, Mg2+ and ROS.Part ? The role of TRPM7 in syngeneic renal transplantation in vivo and mechanismObjective To investigate the role of TRPM7 in syngeneic renal transplantation in vivo and mechanism.Methods M7shRNA were used to decrease the expression of TRPM7 in NRK-52e cells. The mice were subjected to renal intra-parenchymal injection with lentivirus containing M7shRNA to produce hypo-expression of TRPM7 in renal cortex. In vivo studies, mice were sacrificed 24 h,48 h,72 h,7 d and 21 d, respectively after transplantation and the kidneys were dissected. Serum creatinine was measured and the H&E, Masson's trichrome staining, TUNEL, Kim-1, and a-SMA were used to evaluate pathological change.Results In renal transplanted mice, level of Scr on M7shRNA group was conspicuously lower than PBS and Vector-Control treated group. The result of the H&E and immunohistochemical staining for Kim-1 showed us that renal tubule injury was lower in M7shRNA treated group compared with PBS and Vector-Control group. The number of positive TUNEL renal tubule cells per field of view was larger than that in PBS and Vector-Control treated group than M7shRNA treated group. M7shRNA treated group mice showed decreased expression of a-SMA which was associated with interstitial damage and fibrosis at 7 days after transplantation. And the presence of collagen deposition, measured with Masson's trichrome staining, was also reduced in the M7shRNA group at 21 days after transplantation.Conclusion Suppression of renal cortex TRPM7 could alleviate kidney injury induced by transplantation in mice. The mechanism may involve reducing renal fibrosis. |
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