Sirolimus Attenuates Renal Ischemia-reperfusion Injury And Subsequent Regeneration Of Tubular Epithelial Cells In A Rat Model

In kidney transplantation, ischemia-reperfusion injury can significantly increase the incidence of acute rejection, delayed graft function caused by recovery,thus affecting kidney transplant recipients and long-term survival. Tubular cell culture models, animal experiments and clinical studies have prompted renal ischemia-reperfusion injury led to renal tubular epithelial cells, renal tissue structural damage, affect renal function. With advanced renal tubular epithelial cell proliferation gradually start, impaired renal function gradually recovered part. How to take effective measures to check the renal tubular epithelial cell apoptosis, reduce renal ischemia-reperfusion injury in order to protect renal function, to reduce delayed graft function recovery and the incidence of acute rejection and prolong graft and recipient of survival has important clinical significance.Sirolimus (SRL) target molecules mammals (mammalian target of rapamycin, mTOR) inhibitors, animal experiments and years of clinical application of results to SRL-based and CNI-free immunosuppressive regimen and to CNI as based immunosuppressive regimen compared to the former almost no renal toxicity, liver toxicity is low, and the1-year graft survival of patients, the incidence of acute rejection was no significant difference, and renal function was significantly better than the CNI group (creatinine clearance:82.1mL/min Vs61.1mL/min, p=0.004).For patients with chronic allograft nephropathy has occurred.Timely SRL conversion therapy can still be significantly improved graft function and prolong graft survival.Currently, the SRL role of ischemia-reperfusion injury remains controversial. SRL was found possible to make direct access by a non-activated T cell apoptosis, blocking the dendritic cells (DC) presenting antigen and inhibit the maturation, reduce the secretion of cytokines, preventing DC migration gathered in the kidney tissue, can significantly alleviation and improvement of ischemia-reperfusion injury in kidney tissue pathology and renal function.Thursday Gui treated with SRL by hypoxia in vascular endothelial cells by inhibiting ROS, JNK, NF-κB signaling pathway, inhibition of vascular endothelial cells and neutrophil adhesion.Yang B found that the use of SRL intervention renal ischemia-reperfusion injury, mainly activated24KD Caspase-3, to reduce the incidence of apoptosis, while reducing the expression of Fas distal convoluted small tube, Fas signaling pathway by reducing the damage caused by the immune, play a role in protecting the ischemic kidney reperfusion injury.Bohmova R such as renal hypertension in transgenic ischemia-reperfusion injury in rats observed that small doses of SRL can reduce excessive because the kidneys produce renin-induced reperfusion injury. But there are some scholars hold the opposite view, in vitro, SRL inhibit proliferation of rat proximal tubule cells in the lng/ml,while renal tubular cells by inhibiting protein kinase70kDa S6to play a catalytic role in apoptosis. SRL in vivo by inhibiting renal tubular cell regeneration and increased tubular cell apoptosis, inhibition of renal ischemia-reperfusion renal tubular regeneration, repair the damage impact, thereby delaying the recovery of renal function. Lui SL and other reports SRL in1day,3days reduced proliferating cell nuclear antigen (PCNA) expression, but the seventh day is no difference with the control group, indicating that the SRL will aggravate kidney damage mainly through the inhibition of renal tubular cell regeneration The delayed recovery of renal function, but not always inhibit the regeneration of renal tubular cells.In ischemia-reperfusion injury, different doses of SRL rat renal ischemia-reperfusion injury in different periods and apoptosis of renal tubular epithelial cells proliferation, repair is not clear. We speculate that tissue injury may have a protective effect of early ischemia-reperfusion injury, SRL anti-apoptotic role, but in the latter part of the repair process, the anti-proliferative effect may inhibit renal tubular epithelial cell repair, thereby affecting renal function recovery and there is a dose-dependent and reversible. We found that giving rats SRL3mg/(kg.d),its trough concentrations after kidney transplant early therapeutic concentrations, this dose is the standard we have established rat model of renal ischemia-reperfusion injury by detecting sensitive marker of kidney damage matter NGAL, IL-18; aspects apoptotic genes Fas, Bcl-2and PCNA protein expression in renal tubular epithelial cell repair factor HGF and BMP-7mRNA expression, etc.To study the renal ischemia-reperfusion injury and renal tubular epithelial cell repair the combined effect.Part OneSirolimus protecte the early renal ischemia-reperfusion injury in a rat modelObjective:To explore the sirolimus (SRL) for different periods of renal ischemia-reperfusion injury.Method:1.To choice of male SD (Sprague an Dawley) rats were90, were randomly divided into five groups evenly:sham, I/R, I/R+SRLlmg/(kg.d), I/R+SRL3mg/(kg.d), I/R+SRL5mg/(kg.d).2.Establishment of a rat model of ischemia-reperfusion injury.3.Three days before and after each group of patients the drug dose given daily correspondence SR1Lmg,3mg,5mg and10ml of normal saline to each observation date.4.The sham, I/R saline was given orally to each observation date.5.The day after1,3,7rats in each group were sacrificed six whole blood and kidney tissue, serum creatinine, β2-MG 6.ELISA assay neutrophil gelatinase-associated lipocalin (NGAL), IL-18.7.HE staining evaluation of histopathological changes.8.Immunohistochemical SABC method to detect kidney tissue expression of Fas, Bcl-2protein.9. Western-blot detection of Fas, Bcl-2protein expression intensity.10.Deoxynucleotidyl transferase-mediated labeling (TUNEL) method to detect kidney tissue cell apoptosis.Results:1.1、3days after the blood Cr, β2-MG, IL-18, Fas and Bcl-2levels, pathology score, Fas and Bcl-2protein expression, apoptosis index were increased, except for Bcl-outer two levels sham<I/R+SRL5mg/(kg.d)<I/R+SRL3mg/(kg.d)<I/R+SRLlmg/(kg.d)<I/R(P<0.05).2.Bcl-2levels:I/R+SRL5mg/(kg.d)>I/R+SRL3mg/(kg.d)>I/R+SRL1mg/(kg.d)>I/R>sham(P<0.05).3.After7days Fas, Bcl-2levels:I/R+SRL5mg/(kg.d) with sham and I/R+SRLlmg/(kg.d) compare differences(P<0.05).4.NGAL levels after one day higher than sham(P<0.05). Apoptotic index I/R+SRL5mg/(kg.d) compare differences (P<0.05) and more than four.Conclusion:1Successfully established rat model of ischemia-reperfusion injury.2.SRL early in ischemia-reperfusion injury can protect renal function, significantly inhibited apoptosis,3.SRL early in ischemia-reperfusion injury in renal protection in a dose-dependent manner.Part TwoSirolimus attenuates renal ischemia-reperfusion regeneration of tubular epithelial cells in a rat model Objective:To investigate sirolimus (SRL) on rat renal tubular epithelial cells in different stages of repair.Method:1.To choice of male SD (Sprague an Dawley) rats were90, were randomly divided into five groups evenly:sham, I/R, I/R+SRLlmg/(kg.d), I/R+SRL3mg/(kg.d), I/R+SRL5mg/(kg.d).2.Establishment of a rat model of ischemia-reperfusion injury.3.Three days before and after each group of patients the drug dose given daily correspondence SRL1mg,3mg,5mg and10ml of normal saline to each observation date.4.Sham, I/R saline was given orally to each observation date.5.The days after1,3,7rats in each group were sacrificed six whole blood and kidney tissue, serum creatinine.6.ELISA assay p2-MG, hepatocyte growth factor (HGF) levels.7.HE staining evaluation of histopathological changes.8.Immunohistochemical SABC method to detect kidney tissue of proliferating cell nuclear antigen (PCNA) protein expression.9.MRNA, bone morphogenetic RT-PCR technique to detect the occurrence of repair factor HGF protein-7(BMP-7) mRNA expression.Method:1.To choice of male SD (Sprague an Dawley) rats were90, were randomly divided into five groups evenly:sham, I/R, I/R+SRLlmg/(kg.d), I/R+SRL3mg/(kg.d), I/R+SRL5mg/(kg.d).2.Establishment of a rat model of ischemia-reperfusion injury.3.Three days before and after each group of patients the drug dose given daily correspondence SRLlmg,3mg,5mg and10ml of normal saline to each observation date.4.The group of Sham, I/R saline was given orally to each observation date. 5.The days after1,3,7rats in each group were sacrificed six whole blood and kidney tissue, serum creatinine.6.ELISA assay β2-MG, hepatocyte growth factor (HGF) levels;7.HE staining evaluation of histopathological changes.8.Tmmunohistochemical SABC method to detect kidney tissue of proliferating cell nuclear antigen (PCNA) protein expression.9.MRNA, bone morphogenetic RT-PCR technique to detect the occurrence of repair factor HGF protein-7(BMP-7) mRNA expression.Conclusion:1. Successfully established rat model of ischemia-reperfusion injury.2.SRL in ischemia-reperfusion injury hampered its anti-proliferative effect of renal tubular epithelial cells repair.3.SRL in ischemia-reperfusion injury in its anti-proliferative effect in a dose-dependent manner.