Protective Role Of Sirt1on Sepsis Induced Acute Kidney Injury

BACKGROUNDSepsis is one of the serious complications of trauma, shock, infection and major surgey and is the important cause of septic shock and multiple organ dysfunction syndrome. Sepsis induced acute kidney injury is an independent risk factor for poor prognosis. According to epidemiology data, there are30to50%sepsis induced AKI, account for50%of cases of AKI. Although with the development of early diagnosis and renal replacement,the prognosis of AKI gets little improvemen, the mortality of septic AKI is higher than non-septic AKI and remains50to70%.A possible explanation of why mortality of septic AKI has remained high might relate to our minimal understanding of its pathogenesis.The early research showed that renal hemodynamic changes due to renal ischaemia-hypoperfusion induced renal tubular necrosis was the main pathophysiology of septic AKI. But more and more evidences proved that renal hypoperfusion is not the core mechanism of septic AKI.The pathogenesis of septic AKI involves multiple factors, among which renal cell apoptosis is a key one. Apoptosis can be triggered by a variety of stressors such as inflammatory cytokine, ischemia, oxidative stress. Among the multiple mediators of the complex process of apoptosis, the caspase enzyme cascade plays a central role and caspase-3is one of key enzymes in the cysteine proteases. Researches have demonstrated that using caspase inhibitor improve the different cause renal injury, suchas glycerol-induced AKI, ischemia/reperfusion induced AKI and lipoply-saeeharide-induced AKI.As a NAD+-dependent deacetylase, Sirtl (silent information regulator1)impacts potentially on different important biological processes, such as genetic silence,genetic group stability, cell life and metabolic control. Early studies focused on ageassociated disease, such as obesity, diabetes, heart disease, neuro-degenerative diseases and tumor. Recently researches target at the effect of Sirtl on renal diseases. Evidence has revealed that Sirtl played a renal protective effect through anti-oxidation, mitochondria regeneration, reducing apoptosis and other mechanisms in oxidative stress, cisplatin and renal ischemia/perfusion induced acute renal injury. But so far, there are no reports on the effect of Sirtl in septic AKI.Here, based on the problems above, this study was to investigate the role of Sirtl on sepsis induced acute renal injury. For this, a murine model of septic AKI induced by cecal ligation and puncture (CLP) was established. Changes of Sirtl genes and expression of protein was measured by real-time PCR and Western blot. Renal apop-tosis and the expression of caspase-3was evaluated by TUNLE and immunohistochemistry stain. EX-527, an inhibitor of Sirtl, were used to septic AKI model to observe the changes of kidney histopathology, renal function, renal injury biomarks, Sirtl genes and expression of protein, renal cell apoptosis and caspase-3expression and to investigate whether Sirtl rescued renal insults. We also attempted to explore the possible mechanism of Sirtl protective effect in septic AKI. The study was to provide a theoretical basis for Sirtl on role of sepsis induced AKI.The main contents of this article are as follows:Part one:Prepare Septic AKI Murine ModleObjective:To establish an easy and clinically relevant septic AKI murine model.Methods:62healthy male C57BL/6mice (16-20weeks) were randomly assigned into three groups:Normal control group(normal group):(n=6) only collected blood by digging the eyeball. Sham operation group(sham group)(n=28), the cecum was isolated but neither ligated nor punctured. CLP group (n=28), according to Miyaji T, perform CLP surgery.Sham group and CLP group were devided into three time point(6h,12h,24h after operation). At three time points6mice were in each group. Bloodwas collected by digging the eyeball to detect the concentrations of serum creatinine, blood urea nitrogen(BUN). Urine was collected through metobolism cage to detect NGAL and KIM-1.Renal pathology was observed with light microscopy.Thebiological behaviour changes and the4-day and7-day survival rates of sham group and CLP group were observed(n=10).Data were presented as mean±SD. One-way ANOVA was used to analyze changes among multiple groups and followed by a LSD-test. Survival analyses were performed by Kaplan-Meier analysis and survival curves were compared with a log-rank test. P<0.05was considered significant.Results:1. Changes of renal function among different groupsThere was no difference between sham group and normal group (P>0.05). Compared with sham group, the levels of serum Cr were increased significantly at12hours and24hours(P<0.01) and the levels of BUN were increased significantly at all time points (P<0.01).2. Changes of urine renal injury biomarks among different groupsThe levels of urine NGAL and KIM-1were increased at6hours, but compared with normal group there is nosignificant difference. Compared with sham group, the levels of urine NGAL and KIM-1were increased significantly at all time points after operation (P<0.01).3. Histological changes and renal pathology scores among different groupsHistological changes:nothing abnormal were detected in normal and sham group. CLP group, glomerular and tubular hyperemia, tubular epithelial swelling and vacuolar degeneration.Renal pathology scores:compared with sham group, the CLP group’s renal pathology score was increased significantly at all time points after operation (P<0.01).4. comparisons of survival rates between sham and CLP groupThe4-day and7-day survival rates·of CLP group were60%and30%respectively,while sham group mice all survived(P<0.01).Summary:1. Our study successfully created clinically relevant septic AKI model induced by CLP.2The septic AKI model induced by CLP is easy and clinically relevant through controlling the variety, age of mice and standardization of operation.Part TworThe effect of Sirtl on septic AKIObjective:To investigate the effect of Sirtl on septic AKI.Methods:48healthy male C57BL/6mice were randomly assigned into two groups: Sham operation group(sham group)(n=24):the cecum was isolated but neither ligated nor punctured. Septic AKI group (n=24):according to Miyaji T, perform CLP surgery. Each group was devided into four time point(1d,3d,7d and14d after operation). There were6mice in each group at four time points and samples were collected.SirtlmRNA and protein expressionwas measured by real-time PCR and Western blot.T test was used to analyze changes between two groups.P<0.05was considered significant.Results:1. Comparison of renal function between two groups Compared with Sham group, the levels of serum Cr and BUN in septic AKI group were increased significantly at1d and3d (P<0.01),while there was no significant difference at7d and14d (P>0.05).2. Comparison of urine renal injury biomarks between two groups Compared with Sham group,the levels of urine NGAL and KIM-1weresignificantly higher in septic AKI group at1d,3d and7d after operation(P<0.01), while there was no significant difference at14d (P>0.05).3. Comparison of renal pathology scores between two groupsCompared with sham group, the renal pathology scores were significantly higher in septic AKI group at Id,3d and7d after operation(P<0.01), while there was no significant difference at14d (P>0.05).4. Comparion of SirtlmRNA and protein expression in renal cortex between two groupsCompared with sham group, the levels of SirtlmRNA and Sirtlprotein expression in renal cortex were significantly higher in septic AKI group at1d,3d and7d after operation(P<0.01). The levels of Sirtl mRNA and protein expression in renal cortex at14d decreased to the same levels of Sham group (P>0.05).Summary:1. SirtlmRNA and protein expression in renal cortex were significantly increased early after CLP operation, which showed that Sirtl gene was intrinsic activated early on septic AKI.2. Expressionof SirtlmRNA and protein peaked while renal injury wasalleviated, which presumed that Sirtl gene intrinsic activationmightplay a protective effecton septic AKI.Part Three:The protective mechasim of Sirtl on septic AKIObjective:Toexplore the protective mechanism of Sirtl on septic AKI.Methods:70healthy male C57BL/6mice were randomly assigned into three groups: Sham operation group(sham group):(n=10),the cecum was isolated but neither ligated nor punctured. Septic AKI group:(n=30),perform CLP surgery. EX527group:(n=30) thirty minutes after CLP, EX527(10mg/kg) was injected via caudal vein, once a day for7days, while Septic AKI group was injected equal ammount normal saline. After7days samples were collected as Part Two. The renal cell apoptosis was determined by terminal uridinedeoxy nucleotidy ltransferased UTP nick-end labeling(TUNEL) and the expressions of caspase-3were determined by immunohistochemistry stain. Statistical analysis was the same as Part One and correlation analysis was performed on some datas.Results:1. Comparison of renal function among different groupsThere was no difference between Sham group and Septic AKI group on the levels of serum Cr and BUN (P>0.05).The levels of serum BUN in EX527group were significantly higher than those in septic AKI group (P<0.05), while there wasno significant difference of the levels of serum Cr between Septic AKI group and EX527group.(P>0.05).2. Comparison of urine renal injury biomarks among different groupsCompared with Sham group,the levels of urine NGAL and KIM-1were significantly higher in septic AKI group and EX527group (P<0.05). Compared with septic AKI group, the levels of urine NGAL and KIM-1were dramatically higher in EX527group (P<0.01).3. Comparison of histological changes among different groupsCompared with septic AKI group, the histopathological injuries were more severe in EX527group and the renal pathology scores were significantly increased(P<0.01).4. Comparion of levels of Sirtl genes and Sirtl protein expression in renal cortexCompared with sham group, the levels of Sirtl mRNA and Sirtlprotein expression in renal cortex were significantly higher in Septic AKI group (P<0.01). Compared with Septic AKI group, SirtlmRNA and protein expression in renal cortex were dramatically lower in EX527group (P<0.01).5. Comparison of renal cell apoptosis rates among different groupsCompared with sham group, the renal cell apoptosis rates were significantly higher in septic AKI group at7d after operationn(9.15±2.27vs1.87±0.29)(P<0.01). Compared with septic AKI group, the renal cell apoptosis rates were dramatically higher in EX527group (12.55±3.89)(P<0.05).6. Comparison of the expression of renal caspase-3among different groupsThe expressions of capase-3in sham group were little (2.05±0.31); higher expression in septic group (7.48±1.43)(P<0.01). Compared with septic AKI group, The expressions of capase-3were significantly increased in EX527group (9.4±2.12)(P <0.05).7. Comparisons of survival rates among different groupsSham group mice all survived.The7-day survival rates of Septic AKI group and EX527group were36.67%and23.33%respectively. There was no significant differences between septic AKI group and EX527group(P>0.05).8. Correlation analysisCorrelation analysis showed that the renal cell apoptosis and caspase-3expression had a positive correlation with urine NGAL, KIM-1and renal pathology scores. SirtlmRNA got a negative correlation with renal cell apoptosis rate in EX527group.Summary:1. EX527inhibited the SirtlmRNA and proteinexpression, meanwhileworsened renal impairment during septic AKI.2. EX527inhibited the renal apoptosis and caspase-3expression, meanwhile the histopathological injury and renal injury biomarkers were exacerbated. It demonstrated that the inhibitor of Sirtl gene might increase the renal apoptosis through caspase-3, thus, worsen renal impairment during septic AKI, but the caspase-3was not the only pathway which affected renal apoptosis.Conclusions:1. The septic AKI model induced by CLP was easy and clinically relevant through controlling the variety, age of mice and standardization of operation. 2. Intrinsic activation of Sirtl gene probably played a protective effect on septic AKI.3. Intrinsic activation of Sirtl gene paly a protective role on septic AKI by decreasing the renal apotosis through caspase-3which is not the only pathway.