Research For The Role And The Relations Of Renal Cell Apoptosis And TLR4/9 In Septic AKI

Acute kidney injury (AKI) is a common complication of critical illness and an independent risk factor for poor prognosis. The development of AKI increases patient morbidity, predicts higher mortality, and consumes considerable health resources. The cause of AKI in critically ill patients is often multifactorial. Sepsis and septic shock remain the most important cause of ARF in critically ill patients, and account for more than 50% of cases of AKI in the ICU. The mortality of septic AKI is higher than non-septic AKI. Despite our increasing ability to support vital organs and resuscitate patients, the incidence and mortality of septic ARF remain high. A possible explanation of why, despite treatment, AKI is so common in severe sepsis and septic shock and why mortality has remained high might relate to our minimal understanding of septic AKI and its pathogenesis.The traditional paradigm for septic AKI is haemodynamic changes that AKI is due to renal ischaemia/underperfusion and its major pathophysiology is renal tubular epithelial cell necrosis by renal ischaemia/underperfusion. Led by the paradigm, clinicians try to restore renal blood flow by filling the circulation and using vasoactive agents to increase mean arterial pressure in order to improve renal function. But this paradigm remains controversial because these animal models have been mostly based on ischemia/reperfusion injury or drug-induced injury. Neither model is relevant to clinical septic AKI and no random control tests exist to support it. More and more studies have demonstrated that global renal blood flow remains stable or even increases and the distribution of medullary and cortical blood flow is not changed. So, renal hypoperfusion might be important in hypodynamic states but is unlikely to play a key role in the development of ARF during hyperdynamic sepsis (the state seen in the vast majority of critically ill, septic patients with severe AKI). And the majority of studies including animal expriments and clinical studies reported normal histology or only mild, nonspecific changes. Acute tubular necrosis (ATN) was relatively uncommon. Recently, reseachers found that renal cell apoptosis maybe the major pathophysiology of septic AKI. There is now strong evidence to show that human renal tubular cells die by apoptosis as well as necrosis in experimental models of acute ischemic and toxic renal injury, such as glycerol-induced AKI (Gly-AKI), ischemia/reperfusion induced AKI (I/R-AKI) and lipopolysaccharide (LPS)-induced AKI (LPS-I-AKI). But studies about the role of renal apoptosis in the pathophysiology of polymicrobial septic AKI seen in the vast majority of critical illness are still scarce.The course is complex that how pathogenic microorganism invade into our bodies, how induce renal cells apoptosis and AKI. In that, the most important problem is which receptors recognize these bacteria and how to initiate inflammation then sustain it, and how to induce apoptosis. Recently, the discovery of Toll-like receptors (TLRs) revolutionized our understanding of it. Indeed, these pattern-recognition molecules, positioned on cells of the innate and adaptive immune systems, provide the interface between pathogen and organism. The unravelling of their signalling pathways as well as their intricate regulation has shed important light on the complex systemic manifestations of sepsis. Recently, the identification of TLRs on organs and tissues, such as heart, kidney, and liver so on, outside the immune system has added an unexpected level of complexity to our understanding of the role of these molecules. This was further confounded by the fact that TLRs can interact with nonmicrobial endogenous substances. The role of TLRs in septic AKI remains controversial and there are little reports about it in the literature both at home and abroad. Recent studies have documented that RNA interference (RNAi) is an accurate and potent gene-silencing technique.Here, based on the problems above, we prepared to use murine model of cecal ligation and puncture (CLP) to induce abdominal polymicrobial septic AKI. In the serial study, we prepared to use animal and cell experiments, pathomorphology, biochemistry, enzyme linked immunosorbent assay, flow cytometry, molecular biology and so on to explore septic AKI murine model prepared, and use specific caspase-3 inhibitor and TLR4/9 siRNA prophylacticly and respectively to observe the effects of them on renal cell apoptosis, inflammatory response, renal function and prognosis. The study was to discuse the role of renal cell apoptosis, inflammatory response, TLR4 and TLR9 in septic AKI and study the pathogenesis of septic AKI in order to provide animal experiment bases and theoretical evidence for septic AKI prophylaxis and treatment.The main contents of this article are as follows:Part one:Prepare septic AKI murine modelObjective:To explore an easy to prepare and reproducible model which can closely replicate the nature and course of clinical sepsis in patients.Methods:Sixty-eight healthy male C57BL/6 mice were randomly assigned into two equal groups:sham operation group and cecal ligation and puncture group (CLP group, model group). Intra-abdominal infection was induced by CLP. According to the experimental requirements, each group mice were randomly divided into three time point (6h,12h,24h after operation) groups (n=8). The blood samples were collected and the kidneys were harvested at three time points from eight mice in each group. Then the mice were sacrificed. The levels of blood plasma endotoxin, blood bacteria and serum creafinine (Cr) and urea nitrogen (BUN) were detected. Pathological changes in renal tissues were observed with light microscope and electron microscope. The biological behaviour changes (including consciousness, color and luster of fur, food and drink, activity and so on) and the 4-day and 7-day survival rates of two groups of mice were observed (n=10).Data were presented as mean±SD. Statistical analysis was performed using the Student's t-test between two groups. Multiple groups were analyzed by analysis of variance (ANOVA) followed by a LSD multiple comparison test. Survival analyses were performed by Kaplan-Meier analysis and survival curves were compared with a log-rank test. A significant difference was presumed at a P value of<0.05. Data were analyzed using the statistical program SPSS version 13.0.Results:1 Compared with sham group, the levels of blood plasma endotoxin in CLP group mice were significantly higher (P<0.05). Bacterial cultures were negative in sham group mice blood. Escherichia coli, Bacillus proteus, Pseudomonas and a small quantity of Enterococcus were isolated from CLP group mice blood.2 Changes of biological behaviours:The biological behaviour of sham group mice snaped back to normal, while CLP group mice showed early septic behaviour at 6h after CLP and sustained to 48-72h.3 Changes of renal function:Compared with sham group, the concentrations of serum Cr were increased significantly at 6 hours (P<0.05) and the concentrations of serum BUN were increased at all time points (all P<0.05).4 Changes of histopathological injuries:Compared with sham group, there were significantly severe in CLP group mice at all time points (all P<0.05).5 Changes of survival rate:The 4-day and 7-day survival rates of CLP group were 50% and 80% respectively, while sham group mice all survived (all P<0.05).Summary:CLP induced abdominal polymicrobial infection accompanied with renal function impaired and renal histopathological injuried without ATN phenomenon, which showed the abdominal polymicrobial septic AKI model was made successful. The septic AKI model induced by CLP is easy and replicative.Part two:Study on the role of renal cell apoptosis in septic AKI mice and the prophylactic intervention effects of selective caspase-3 inhibitor on septic AKI miceObjects:To investigate the role of renal cell apoptosis in AKI induced by abdominal polymicrobial sepsis in mice and to explore the prophylactic intervention effects of specific caspase-3 inhibitor on it.Methods:One hundred and two male C57BL/6 mice were randomly assigned into three equal groups:sham operation group, cecal ligation and puncture group (CLP group, model group) and caspase-3 inhibitor (CI) group. Thirty minutes before CLP, Ac-DEVD-CH0(4μg/g) was injected subcutaneously in CI group. According to the experimental requirements, each group mice were randomly divided into three time point (6h,12h,24h after operation) groups (n=8). The blood samples were collected and the kidneys were harvested at three time points from eight mice in each group. Then the mice were sacrificed. The concentrations of serum creafinine (Cr) and urea nitrogen (BUN) were detected, the renal cell apoptosis was determined by flow cytometry and terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and the expressions of caspase-3 were determined by real time quantitative reverse transcription-polymerase chain reaction. Pathological changes in renal tissues were observed with light microscope. The biological behaviour changes (including consciousness, color and luster of fur, food and drink, activity and so on) and the 4-day and 7-day survival rates of three group mice were observed (n=10).Data were presented as mean±SD. Multiple groups were analyzed by analysis of variance (ANOVA) followed by a LSD multiple comparison test. Survival analyses were performed by Kaplan-Meier analysis and survival curves were compared with a log-rank test. The Kruskal-Wallis nonparametric one-way analysis of variance by ranks was used, followed by post hoc pairwise comparisons using the Wilcoxon's two-sample test for all comparisons. A significant difference was presumed at a P value of<0.05. Data were analyzed using the statistical program SPSS version 13.0.Results:1 Changes of biological behaviours:The biological behaviours of sham group and CLP group mice were similarly as those in part one. The biological behaviours of CI group mice were more gently than CLP group mice.2 Changes of renal function:(1)Serum Cr:Compared with sham group, the concentrations of serum Cr were increased significantly at 6 hours in CLP group (P<0.05). The concentrations of serum Cr in CLP group were decreased from 12 hours, but still higher than that in sham group although without significant difference. Compared with CLP group, the concentrations of serum Cr were decreased significantly at 6 hours (P<0.05), and decreased continuously without significant differences among three groups at 12 and 24 hours(all P>0.05) in CI group.(all P>0.05). (2)Serum BUN:Compared with sham group, the concentrations of serum BUN were increased significantly at all time points in CLP group (all P<0.05). Compared with CLP group, the concentrations of serum BUN were decreased significantly at all time points(all P<0.05) in CI group and with no significant difference when compared to sham group(all P>0.05). There were no significant differences in the same group among all time points of three groups (all P>0.05).3 Changes of renal histopathological injuries:Compared with sham group, the histopathological injuries were more severe in CLP group at all time points (all P<0.05). Compared with CLP group, the histopathological injuries were gentle in CI group at 6 and 12 hours (all P<0.05). There was no significant difference in the same group among all time points of three groups(all P> 0.05).4 Changes of renal cell apoptosis rates:Compared with sham group, the renal cell apoptosis rates were increased significantly at all time points in CLP group (all P<0.05) [18.27±1.4 vs.6.17±.9,15.86±3.5 vs.5.60±.6 and 12.48±2.1 vs.5.64±.5 (%) respectively]. Compared with CLP group, the renal cell apoptosis rates were decreased significantly at all time points in CI group (all P<0.05)[13.88±3.2,10.48±3.6 and 8.45±1.8 (%)], but still significantly higher than sham group (all P<0.05). There was no significant difference in sham group among all time points (all P>0.05). Compared with 6 hours, the renal cell apoptosis rates were decreased progressively in CLP and CI groups and had significant difference at 24 hours (all P<0.05).5 Changes of caspase-3 expressions:(1) Compared with sham group, the renal tissue caspase-3 mRNA expression was increased significantly at all time points in CLP group (all P<0.05). Compared with CLP group, the caspase-3 mRNA expression was decreased significantly at all time points in CI group (all P<0.05). The inhibition rate of Ac-DEVD-CHO on the caspase-3 mRNA expression was about 53%. There were no significant differences in the same group among all time points in sham and CI group (all P>0.05). Compared with 6 hours, the caspase-3 mRNA expression was increased progressively in CLP group and had a significant difference at 24 hours (P<0.05). (2)The positive caspase-3 proteins which were mainly expressed in cytoplasm and nucleus of tubular cells and renal glomeruli vascular endothelial cells were rare in sham group. Compared with sham group, CLP increased renal caspase-3 protein expression without significant difference (P<0.05). Compared with CLP group, renal caspase-3 protein expression was reduced in CI group without significant difference (P<0.05).6 Changes of survival rates: The 4-day survival rate of sham, CLP and CI group was 100%,40% and 80 % respectively, and the 7-day survival rate was 100%,20% and 20% respectively. Compared with sham group, the 4-day and 7-day survival rates were decreased significantly in CLP and CI groups (all P<0.05). There was no significant difference between CLP and CI groups (all P>0.05).Summary:1 Renal cell apoptosis was the most frequent histopathological form of polymicrobial sptic AKI without ATN phenomenon. 2 With the renal tissue caspase-3 expression and renal cell apoptosis rate reduced significantly by Ac-DEVD-CHO, the histopathological injury was ameliorated and renal function was improved. Our results documented that renal cell apoptosis may be the major pathophysiology of polymicrobial septic AKI and specific caspase-3 inhibitor had a protective effect on polymicrobial septic AKI, which provided the theoretical evidences for further studies on septic AKI pathogenesis and using antiapoptosis therapy in clinic.3 Using Ac-DEVD-CHO did not improve the final prognosis that means there were multiple factors involved in the pathogenesis and need to be studied further.Part three:Study on the role of the inflammatory response in septic AKI mice and the prophylactic intervention effects of selective caspase-3 inhibitor on septic AKI miceObjective:To explore the role of the inflammatory response in acute kidney injury induced by abdominal polymicrobial infection in mice, and to discuse the association between inflammatory response and renal cell apoptosis and the prophylactic intervention effects of selective caspase-3 inhibitor on them. Methods:Experimental animals, groups and modle-making methods were the same as Part two. The concentrations of serum tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-10 were measured by enzyme linked immunosorbent assay (ELISA) at 6,12 and 24 hours after operation. Other main methods were the same as Part two. Statistical analysis was the same as Part two.Results:1 The biological behaviour changes of three group mice were similar as those in Part one.2 The renal function changes of three group mice were the same as Part two.3 The renal histopathological injury changes of three group mice were same as Part two.4 The changes about apoptosis of three group mice were the same as Part two.5 Compared with sham group, the concentrations of serum TNF-a, IL-6 and IL-10 were significantly higher at all time points in CLP group [TNF-α(μg/L) 6h:653.6±8.9 vs.29.5±19.0,12h: 579.7±137.1 vs.28.3±9.3,24h:551.0±119.8 vs.26.3±8.9; IL-6(μg/L) 6h: 1595.3±159.4 vs.38.2±8.5,12h:1330.7±249.8 vs.32.0±9.0,24h:815.3±572.7 vs.24.3±9.7; IL-10(μg/L) 6h:26.6±4.5 vs.5.1±1.2,12h:24.5±4.3 vs.4.9±1.0, 24h:18.2±1.6 vs.4.9±1.2, all P<0.05)]; compared with CLP group, in CI group, the concentrations of serum TNF-a and IL-6 were decreased significantly at all time points [TNF-α(μg/L):436.2±64.2,233.4±85.4, 151.0±90.3;IL-6(μg/L):1033.2±345.8,366.3±68.3,241.2±208.4, all P<0.05)] and those of IL-10 were elevated significantly at 12h and 24h (37.2±5.0, 38.3±5.5, all P<0.05).6 The survival rates of three group mice were the same as Part two.Summary:1 CLP induced significantly elevated serum TNF-α, IL-6 and IL-10 levels, especially the pro-inflammatory cytokines, about 22.13 times, 41.78 times and 5.218 times respectively compared with sham group at 6h after operation. And CLP increased renal cell apoptosis, decreased renal function and survival.2 Inhibition of tubular cell death by specific caspase-3 inhibitor led to balance the inflammatory response including pro-inflammatory cytokines reduced and anti-inflammatory cytokines elevated, which improved renal function and survival.3 Above results demonstrated that inflammatory response and renal cell apoptosis were the major pathogenesis of polymicrobial septic AKI which provided the theoretical evidences for further studies on septic AKI pathogenesis and using antiapoptosis therapy in clinic.Part four:Study on the role of TLR4/9 in septic AKI mice and the prophylactic intervention effects of TLR4/9 siRNA on septic AKI miceObjective:To observe the expressions of TLR4,9 in renal tissue in acute kidney injury induced by abdominal polymicrobial infection in mice and the effects of their small interfering RNA (siRNA) on renal cell apoptosis and septic AKI.Methods:1 In vitro:In this part RNA interference was used to achieve potent and specific silence of TLR4 and TLR9. Two siRNA aimed directly to TLR4 and TLR9 each respectively:S-T41, S-T42, S-T91 and S-T92. Mouse RAW264.7 cells were cultured. Cellular TLR4 and TLR9 expressions were analysised by Western blot and realtime quantitative PCR after transfection for 48 hours, then the potent and specific siRNA were selected as in vivo study. 2 In vivo:One hundred and seventy healthy male C57BL/6 mice were randomly assigned into five equal groups:sham operation group, blank control group (S-B group), empty plasmid control group (S-N group), TLR4 siRNA group (S-T4 group) and TLR9 siRNA group (S-T9 group). The polymicrobial septic AKI model was induced by cecal ligation and puncture (CLP) operation. Every mouse was injected with 1.5 ml TransIT-EE Hydrodynamic Delivery Solution via the tail vein by hydrodynamic injections within five seconds 24h before operation. In vivo delivery of siRNA was performed via the tail vein by hydrodynamic injections (50μg siRNA dissolved in above solution). The method of CLP was the same as Part one. According to the experimental requirements, each group mice were randomly divided into three time point (6h,12h,24h after operation) groups (n=8). The changes of serum Cr and BUN concentrations, the renal cell apoptosis, the caspase-3 and TLR4 expressions and the renal histopathological injuries were determined. The biological behaviour changes (including consciousness, color and luster of fur, food and drink, activity and so on) and the 4-day and 7-day survival rates of five group mice were observed (n=10). The detailed methods were the same as Part two. Statistical analysis was the same as Part two.Results:In vivo study, there were no significant differences in all results between S-B and S-N groups (all P<0.05) and there were no significant differences in all results except for the serum BUN concentrations and the expressions of TLR4 between S-T4 and S-T9 groups (all P<0.05).1 In vitro: The silence effects of S-T41 and S-T91 on cellular TLR4 and TLR9 expressions respectively were marked potent than those of S-T42 and S-T92 and the silence effects were specific without crossing silence effects. So, S-T41 and S-T91 were selected as in vivo study.2 Changes of biological behaviours:The biological behaviour changes in sham, S-B and S-N group mice were similar to those of sham and CLP group mice in Part two respectively. The biological behaviour changes of S-T4 and S-T9 group mice were more gently than those of S-B and S-N group mice.3 Changes of renal fnction:(1)Serum Cr concentrations:Compared with sham group, the concentrations of serum Cr in S-B and S-N groups were elevated significantly at 6h after operation (all P<0.05) and until to 24h but without significant difference (all P>0.05); Compared with S-B and S-N groups, the concentrations of serum Cr in S-T4 and S-T9 groups were decreased significantly at 6h after operation (all P<0.05), then restored to those of the sham group at 12 and 24 hours (all P>0.05). (2)Serum BUN concentrations: Compared with sham group, the concentrations of serum BUN in S-B and S-N groups were elevated significantly at all time points after operation(all P< 0.05) and those of the S-T4 and S-T9 groups were elevated significantly only at 6h (P<0.05). Compared with S-B and S-N groups, the concentrations of serum BUN in S-T4 and S-T9 groups were decreased significantly at all time points and that of S-T9 group was lower significantly than that of S-T4 group (P<0.05). There were not significant differences in the same group of sham, S-B, S-N and S-T4 groups (all P>0.05). The BUN concentrations in S-T9 group decreased progressively after operation (all P<0.05).4 Changes of renal histopathological injuries:Renal cell apoptosis was the main pathological feature with no cell necrosis in five groups. Compared with sham group, the renal histopathological injury scores were elevated significantly at all time points (all P<0.05) and that of S-T4 was elevated significantly at 6h only(P<0.05). Compared with S-B and S-N groups, the renal histopathological injury scores were elevated significantly at all time points in S-T9 group and at 12h,24h in S-T4 group (all P<0.05). There was a decreased trend in all five groups and there were significant differences between 6h and 24h especially in S-T4 and S-T9 groups (all P<0.05).5 Changes about renal cell apoptosis:(1)Compared with sham group, the renal cell apoptosis rates of S-B and S-N groups were increased significantly at all time points (all P<0.05), and those of S-T4 and S-T9 groups were decreased significantly compared with S-B and S-N groups (all P<0.05). In the same group, the apoptosis rates were decreased progressively in S-B and S-N group and had a significant decrease at 24h than those of other two time points (all P <0.05). The differences in the same group were not significant in the other groups (all P>0.05). (2)Terminal uridine deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining showed that renal apoptosis cells were much less in sham, S-T4 and S-T9 groups than in S-B and S-N groups and the apoptosis cells located mainly at renal tubular epithelial cells.6 Changes of renal caspase-3 expressions:(1) caspase-3 mRNA expressions:Compared with sham group, the renal caspase-3 mRNA expressions of S-B, S-N, S-T4, S-T9 groups were increased significantly at 24h after operation (increased 5.21times,5.03times,2.83times,2.69 times compared with S-N group respectively, all P<0.05). Compared with S-B and S-N groups, the renal caspase-3 mRNA expressions of S-T4 and S-T9 groups were decreased significantly (decreased 44% and 47% compared with S-N group respectively, all P<0.05). (2) caspase-3 protein:Immunohischemical staining for caspase-3 protein showed that the caspase-3 positive cells which were mainly expressed in cytoplasm and nucleus of tubular cells and renal glomeruli vascular endothelial cells were much less in sham, S-T4 and S-T9 groups than in S-B and S-N groups at 24h after operation (all P<0.05).7 Changes about renal tissue TLR4 expression:(1)Compared with sham group, the TLR4 mRNA expressions of S-B, S-N, S-T9 groups were upregulated significantly at 24h after operation (increased 4.28 times,4.35 times,2 times compared with S-N group respectively, all P<0.05) and those of S-T4 and S-T9 groups were reduced significantly than that of S-N group (reduced 89% and 54% compared with S-N group respectively, all P<0.05). (2) Immunohischemical staining for TLR4 protein showed that the TLR4 positive cells which were mainly expressed in cytoplasm and nucleus of tubular cells and renal glomeruli vascular endothelial cells were much less in sham, S-T4 and S-T9 groups than in S-B and S-N groups at 24h after operation (all P<0.05). Although there was no significant difference between S-T4 and S-T9 groups in TLR4 protein expressions, the TLR4 protein expression in S-T9 group was still higher obviously than that in S-T4 group.8 Changes of survival rates:The 4-day survival rates of sham, S-B, S-N, S-T4 and S-T9 group were 100,40,50,80 and 80(%) respectively, and those of 7-day were 100,20,30,70 and 80(%). Comared with sham group, the 4-day and 7-day survival rates of S-B and S-N group were decreased significantly(all P<0.05). Comared with S-B and S-N group, the 4-day survival rates of S-T4 and S-T9 group were decreased obviously but with no significant difference(all P>0.05); the 7-day survival rates of S-T9 were decreased significantly(P<0.05); there was a significant difference between S-B and S-T4 group only (P<0.05).Summary:1 In vitro experiment documented that TLR4/9 siRNA silenced their gene potently and specificly respectively.2 In vivo delivery of TLR4 siRNA was performed via the tail vein by hydrodynamic injections (siRNA dissolved in 1.5 ml TransIT-EE Hydrodynamic Delivery Solution) within five seconds 24h before operation could silence renal tissue TLR4 expressions effectively which demonstrated that this methods was effective. The fact that renal cell TLR4 expression was also silenced effectively by TLR9 siRNA may be related to endogenic ligands of TLR4 reduced by TLR9 siRNA.3 In vivo delivery of TLR4 siRNA could reduce renal cell apoptosis, ameliorate renal histopathological injuries and improve renal function and prognosis significantly may be related to the down-regulation of the TLR4 expressions of renal cells and immune cells.4 In vivo delivery of TLR9 siRNA could reduce renal cell apoptosis, ameliorate renal histopathological injuries and improve renal function and prognosis significantly may be related to the down-regulation of the TLR9 expressions of immune cells.5 These results demonstrated that TLR4,9 played key roles in polymicrobial septic AKI.Part five:Study on the prophylactic intervention effects of TLR4/9 siRNA on inflammatory response in septic AKI miceObjective:To explore the effects of small interference RNA of TLR4/9 on inflammation in acute kidney injury induced by abdominal polymicrobial infection in mice and to discussion the protective role of them in septic AKI.Methods:Experimental animals, groups, modle-making methods and other methods were the same as Part four. The concentrations of serum tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-10 were measured by enzyme linked immunosorbent assay (ELISA) at 6,12 and 24 hours after operation. Statistical analysis was the same as Part two.Results:1 The biological behaviour changes, the renal function changes, the renal histopathological injury changes, the renal apoptosis changes, the TLR4,9 expression changes and the survival rates of five group mice were the same as those in Part four.2 Changes of serum TNF-α, IL-6 and IL-10 among groups:The differences between S-B and S-N groups at all time points were not significant (all P>0.05). Compared with sham group, the concentrations of serum TNF-α, IL-6 and IL-10 were elevated significantly at all time points (all P<0.05). Compared with S-B and S-N groups, the concentrations of serum TNF-αwere decreased at all time points(all P<0.05) and the concentrations of serum IL-10 were further elevated significantly at 6h,12h time points(all P<0.05) in S-T4 and S-T9 groups; the concentrations of serum IL-6 were decreased significantly at all time points in S-T9 group (all P<0.05) and those in S-T4 group were decreased significantly at 12h and 24h (all P< 0.05). Compared with S-T4 group, the concentrations of serum TNF-αand IL-6 were reduced significantly at all time points in S-T9 group (all P<0.05). 3 Changes in the same group:(1)The serum TNF-a concentration changes of sham, S-B and S-N groups had not significant differences in the same group (all P>0.05) and those in S-T4 groups were decreased significantly at 12h and 24h than that at 6h (all P<0.05). The serum TNF-a concentrations of all time points were reduced significantly in S-T9 group (all P<0.05). (2)The trends of the serum IL-6 levels in all groups were decreased progressively. There were significant differences between 24h and other two time points in S-B and S-N groups (all P<0.05), and there were significant differences between 6h and other two time points in S-T4 group (all P<0.05) and among all time points in S-T9 group (all P<0.05). (3) The serum IL-10 levels in S-B and S-N groups were decreased significantly at 24h than those at other time points (all P<0.05) and those in S-T4 and S-T9 groups were elevated significantly at 12h and 24h than that at 6h (all P<0.05).Summary:1 The method that in vivo delivery of siRNA performed via the tail vein by hydrodynamic injections within five seconds 24h before operation led to balance the inflammation by down-regulating the pro-inflammatory cytokines and up-regulating the anti-inflammatory cytokines.2 The protective effects of TLR4 siRNA on polymicrobial septic AKI may be related to balance the inflammation by down-regulation the TLR4 expressions of renal cells and immune cells.3 The protective effects of TLR9 siRNA on polymicrobial septic AKI may be related to balance the inflammation by down-regulation the TLR9 expressions of immune cells.4 Above results demonstrated that TLR4,9 played key roles in polymicrobial septic AKI.Conclusions:1 Cecal ligation and puncture could induce abdominal polymicrobial septic AKI successfully.2 Renal cell apoptosis was the main pathological feature in CLP induced AKI; selective inhibitor of caspase-3 (4μg/g) injected subcutaneously could decrease renal cell apoptosis, improve renal function and prognosis, which demonstrated that renal cell apoptosis may play a key role in polymicrobial septic AKI. That provided the theoretical evidences for further studying septic AKI pathogenesis and using antiapoptosis therapy in clinic.3 The inflammation may be one of the main pathogenesis in CLP induced AKI. With the inhibition of renal cell apoptosis by Ac-DEVD-CHO, the selective inhibitor of caspase-3, inflammatory response was balanced which further provided the theoretical evidences for further studying septic AKI pathogenesis and using antiapoptosis therapy in clinic.4 CLP operation elevated the renal TLR4 expressions. In vivo delivery of TLR4 siRNA could reduce renal cell apoptosis, balance inflammatory response, ameliorate renal histopathological injuries and improve renal function and prognosis significantly may be related to the down-regulation of the TLR4 expressions of renal cells and immune cells. In vivo delivery of TLR9 siRNA could reduce renal cell apoptosis, balance inflammatory response, ameliorate renal histopathological injuries and improve renal function and prognosis significantly may be related to the down-regulation of the TLR9 expressions of immune cells. These demonstrated that TLR4 and TLR9 may play key roles in the pathogenesis of polymicrobial septic AKI and also documented that siRNA technique and hydrodynamic injection were effective to silence the renal tissue gene expression in mice, which provided pathophysiological rationale and experimental evidences for TLRs gene intervention therapy in future.