Effects Of Kidney And Myocardium After Renal Sympathetic Denervation On Renal Ischemia-Reperfusion Injury

Objective:Previous researches have studied the relationship between sympathetic interaction of kidney and renal ischemia-reperfusion injury with controversial conclusions. To further explore this issue, renal sympathetic denervation model was established to investigate the effects of kidney and heart on renal ischemia-reperfusion.Methods:Adult male Sprague-Dawley(SD) rats(weight 200-280g) were randomly divided into several groups: normal group(N group), Sham-operated group(Sham group), renal sympathetic denervation group(RSD group), renal ischemia-reperfusion group(I/R group), and Renal sympathetic denervation and renal ischemia-reperfusion(R+I group). According to renal sympathetic time points, RSD groups were divided into 24 hours group(R1), 48 hours Group(R2) and 72 hours group(R3). Sham group was only separated bilateral kidney vessels, RSD group was separated bilateral kidney arteries and veins, bilateral arteries were wrapped by no bacteria cotton dipped 10% phenol ethanol solution for 20 minutes, I/R group was separated bilateral kidney arteries and veins, bilateral arteries were occluded by non-trauma arterial clips for 45 minutes then reperfusion, R+I Group was operated I/R after RSD by the time point according to R1 group, R2 group and R3 group. Twenty-four hours after operation except the rats of N group which were directly underwent laparotomy, molecular biology and histology studies were described as follows:(1) To determine kidney function, plasma creatinine, urea nitrogen, Cystatin-C were detected by automatic biochemical analyzer, plasma NGAL were measured by ELISA, as well as the kidney staining with Hematoxylin eosin(HE). Plasma creatine kinase isoenzyme MB(CK-MB) detected by automatic biochemical analyzer and myocardial HE staining were taken to identify the potential myocardial damage.(2) Norepinephrine concentration in kidney and myocardium were measured by high performance liquid chromatography(HPLC) to evaluate the state of sympathetic nerve.(3) To assess the level of oxidative stress, expression of nuclear factor erythroid 2-related factor2(Nrf2) and heme oxygenase-1(HO-1) in kidney and myocardium were detected by western-blot, malondialdehyde(MDA) and superoxide gasification enzyme(SOD) concentration were also tested.(4) Expression of microtubule-associated protein1 light chain 3(LC3) and Beclin1 in kidney and myocardium were detected by Western-blot to evaluate the state of autophagy. Expression of Caspase3 was assayed by immunohistochemistry and western-blot, TUNEL method was estimated apoptosis in kidney and myocardium.Results:Determination of renal noradrenaline levels according to HPLC, 24 hours after renal sympathetic denervation was chosen for the optimum time point. Compared with the I/R group, R+I group of creatinine, urea nitrogen, Cystatin C, plasma levels of NGAL and CK-MB increased(P<0.05). HE staining of kidney showed cell degeneration, disintegration, detachment and lumen dilated renal interstitial edema in renal tubular cell at the junction of cortex and medulla. HE staining of myocardium presented abnormal sarcomeres, a loss of contractile materials and the intra-cellular space also appeared abnormal. Oxidative stress was found that enhanced in kidney and myocardium of group R+I with MDA increased and SOD decreased, the expression of Nrf2 and HO-1were higher than that group of I/R. In R+I group, the levels of autophagy was lower than I/R group with LC3 and Beclin1 decreased. Expression of Caspase3 and the number of TUNEL-positive cells were significantly increased in R+I group(P <0.05). In addition, noradrenaline levels according to HPLC in group R+I were higher than I/R group(P <0.05), both of them increased than Sham group(P <0.05).Conclusion:Our research has established the rat model of renal sympathetic denervation and explored the influence of renal sympathetic denervation on renal ischemia-reperfusion involving several aspects of organ function, oxidative stress, autophagy and apoptosis level in kidney and myocardium. Our study showed that renal sympathetic denervation worsened the deleterious effect of ischemia-reperfusion in kidney and myocardium. Considering the reasons as follows:(1) Renal sympathetic denervation by phenol was not enough to resist the sympathetic activity induced by renal ischemia reperfusion.(2) To determine optimum time point of renal sympathetic denervation, norepinephrine levels were detected after renal sympathetic denervation 24 hours, 48 hours and 72 hours. Renal sympathetic denervation 24 hours was taken as optimum time point. However, just because of it, the rats in R+I group were suffered laparotomy for two times,which may lead to oxidation stress injury and aggravate further injury of cardiac and renal function.(3) Whether there was residual renal sympathetic nerve and whether the rest of the compensatory mechanism or systemic sympathetic nervous feedback mechanisms lead to local and remote organs after kidney ischemia-reperfusion damage was unclear. Current relevant literatures were limited and controversial with the differences of research model, time of ischemia and reperfusion. More and further studies are urgent to clarify this issue in the future.