Study On Role Of TOLL-like Receptor 4 In Mice With LPS-induced Acute Renal Failure

PartⅠThe expression of TOLL-like receptor 4 in mice with LPS- induced actue renal failureThesis 1: Correlation of Toll like receptor 4 with endotoxin-acute renal failure of miceObjective : To investigate the correlation of Toll like receptor 4(TLR4) with endotoxin-acute renal failure(ARF) .Methodology: Forty-eight health C57BL/6 male mice, eight-week-old, were divided into two groups. They were ARF group with lipopolysaccharide(LPS,15mg/kg) peritoneal injection to induce endotoxin-ARF model and normal control group(NG) which treated with saline peritoneal injection. Mice were sacrificed at the 12,24,36,48 hour respectively to collect the sample of blood and renal tissue. Serum creatinine (Cr) and blood urea nitrogen (BUN) were measured to judge renal function. Nomura score method was adopted to evaluate renal morphologic pathological changes.Immunohistochemistry (IHC) assay was applied to detect the distribution of TLR4 on kidney. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detecte the expression of TLR4 mRNA and TLR4 protein respectively. Terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling (TUNEL) was applied to monitor apoptosis of renal tissue.Results: After injecting LPS,the level of Cr and BUN were increased rapidly, up to the peak at 24 hour(BUN 28.14±2.55mmol/L,Cr 207.73±12.1μmol/L) ,then decrease slowly and regain to normal gradually. Comparison with NG mice, the light and medium pathological changes in the renal tissue of ARF mice were exhibited, the injury was mainly on renal tubule, but renal tissue textures were integrity and their renal functions were seriously worsened. There was significance difference in TLR4 protein optical density value (OD) between the NG and the ARF group. The level of renal TLR4 mRNA and protein were up-regulated in ARF group comparison with NG mice, which reach the top point at 24 hour in ARF group. In ARF group, the expression of TLR4 protein was positive correlated with Cr(r=0.907), BUN(r=0.926) and renal apoptotic body(r=0.954). The apoptotic body was detected on the renal tubule in the ARF group mice. There was no apoptotic body in the NG mice. The apoptotic body was positive correlated with Cr(r=0.932), BUN(r=0.951) Conclusion: TLR4 and renal apoptosis were positive correlated with the renal function of ARF mice; TLR4 was positive correlated with the renal apoptotic body in ARF group.TLR4 may play a role in endotoxin ARF via promoting renal apoptosis.Thesis 2: Role of Toll like receptor 4 in gene-deficient mice with LPS-induced actue renal failureObjective:To identify the expression and its role of Toll like receptor 4(TLR4) in endotoxin-acute renal failure(ARF) mice.Methods The TLR4-deficient mice(C3H/HeJ) and wild normal contrast mice(C3H/HeN) were injected by Lipopolysaccharide(LPS,15mg/kg) to establish the actue renal failure model. Mice were sacrificed at 24 hour to collect the sample of blood and renal tissue.The value of serum creatinine (Cr) and blood urea nitrogen (BUN) were measured to judge renal function. Nomura score method was adopted to evaluate renal morphologic pathological changes. Immunohistochemistry(IHC),RT-PCR and Western blot were appled to detected expression of TLR4 mRNA and TLR4 protein respectively. Terminal deoxynucleotidyltransferase mediated X-dUTP nick end labeling(TUNEL) was applied to monitor apoptosis of renal tissue.Results: The value of Cr,BUN of TLR4+(C3H/HeN) mice were higher than that of TLR4-(C3H/HeJ) mice respectively(Cr: 202.26±11.08umol/L vs 109.67±13.32umol/L,P<0.01 ; BUN: 20.36±1.52mmol/L vs 6.42±0.41mmol/L, P < 0.01) ; Comparison with TLR4-(C3H/HeJ) mice ,the level of renal TLR4 mRNA and protein expression was up-regulated in TLR4+(C3H/HeN) mice;The apoptotic body was mainly detected on the renal tubule. There was significance difference in apoptotic body optical density value (OD) between TLR4+(C3H/HeN) mice and TLR4-(C3H/HeJ) mice(0.117±0.008 vs 0.038±0.003,P<0.001)。Conclusion: TLR4 really play a role in LPS-induced mice acte renal failure.LPS may cause the over apoptosis of renal tubular cell via TLR4 signal transduct pathway, decrease the number of renal tubular cell, result in ARF.PartⅡStudy on the expression of TLR4 in human proximal renal tubular cell with stimulation of LPSThesis 1: LPS Increase the Expression of TLR4 in Human Proximal Renal Tubular CellObjective To investigate the effect of lipopolysaccharide(LPS) on Toll like receptor 4(TLR4) mRNA and protein expression in cultured human proximal renal tubular epithelial cells(HKC)Methods The cultured HKC were divided into two groups: LPS-RG which irritated by LPS with 50ng/ml for 24 hours and HKC normal control group(HKC-CG).The distribution levels of TLR4 in HKC were revealed by immunofluorescence staining under laser scanning confocal microscopy . The mRNA and protein expression of TLR4,β-actin were detected by semi-quantitative RT-PCR and Western blotting respectively.The rate of earlier period cell apoptosis of HKC were detected by Annexin V-FITC combining with flow cytometry。 Results The fluorescence intensity of TLR4 of LPS-RG were significantly higher than that in HKC-CG. The distribution of TLR4 was mainly locate on cell membrane and cytoplasmic domain. The expression of TLR4 mRNA in LPS-RG were apparently higher than that in HKC-CG(1.051±0.082 vs 0.38±0.036,P<0.01). The expression of TLR4 protein in LPS-RG were apparently higher than that in HKC-CG(0.371±0.033 vs 0.105±0.008,P<0.01).The HKC earlier period cell apoptosis rate of LPS-RG(41.29%) was higher than that in HKC-CG(2.36%).Conclusion LPS stimulated HKC increase the expression of TLR4 and promoted the rate of earlier period cell apoptosis of HKC;TLR4 may take some role in LPS-induced HKC apoptosis.PartⅢSpecific shRNA suppress the expression of TLR4 in HEK 293 cell and HKC cellThesis 1:Specific shRNA suppress the expression of Toll like receptor 4 in HEK 293 cellObjective To investigate the role of suppression on Toll like receptor (TLR4) in cultured HEK 293 cell with RNA interference(RNAi).Methods The short hairpin RNA(shRNA) which specifically targeting TLR4 mRNA was biologic synthesized with in vitro transcription, then transfected into cultured HEK 293 cell.HEK 293 cell were divided into three groups. Interference group( which transfected into HEK293cell with specificTLR4 shRNA),negative control group (which transfected into HEK293 cell with non-specificnegative control shRNA) and normal group( no transfection);Detected the shRNA transfection rate by flow cytometry;The distribution levels of TLR4 were revealed by immunofluorescence staining under laser scanning confocal microscopy. The mRNA and protein expression of TLR4,β-actin were detected by semi-quantitative RT-PCR and Western blotting respectively. Results The transfection rate of specific TLR4 shRNA in HEK293 was 82.47%;The fluorescence intensity of TLR4 was significantly lower than that in negative control group and normal group. The distribution of TLR4 was mainly locate on cell membrane and cytoplasmic domain.The optical density ratio of TLR4 mRNA in interference group was apparently lower than that in negative control group and normal group(0.35±0.003 vs 0.98±0.061 vs 0.955±0.058);The optical density ratio of TLR4 mRNA in interference group was apparently lower than that in negative control group and normal group (0.089±0.007 vs 0.153±0.011 vs 0.142±0.001).Conclusion The specific shRNA duplex molecule targeting TLR4 mRNA was successfully synthesized and efficiently transfected into HEK 293 cell. The expression of TLR4 of HKC was suppressed with RNAi.Thesis2: Specific shRNA suppress the role of TLR4 increasing cell apoptosis in HKC with stimulation of LPSObjective To investigate the role of specific shRNA which suppress the expression of Toll like receptor (TLR4) in cultured human renal tubular cell(HKC) and inhibitate the role of TLR4 increasing cell apoptosis in HKC with stimulation of lipopolysaccharide(LPS).Methods The short hairpin RNA(shRNA) which specifically targeting TLR4 mRNA was biologic synthesized with in vitro transcription, then transfected into cultured HKC .The cell was divided into four groups:LPS group( which stimulated by LPS with 50ng/ml ),LPS+interference group(which stimulated by LPS with 50ng/ml,then transfected the specific TLR4 shRNA into HKC),LPS+negative control group (which stimulated by LPS with 50ng/ml,then transfected the non-specificnegative control shRNA into HEK293 cell) and normal group( no LPS,no transfection);The distribution and levels of TLR4 were revealed by immunofluorescence staining under laser scanning confocal microscopy. The mRNA and protein expression of TLR4,β-actin were detected by semi-quantitative RT-PCR and Western blotting respectively. The rate of earlier period cell apoptosis of HKC was detected by Annexin V-FITC combining with flow cytometry.Results The fluorescence intensity of TLR4 of LPS+interference group was significantly lower than that in LPS group and LPS+negative control group. The optical density ratio of TLR4 mRNA of LPS+interference group(0.198±0.041) was apparently lower than that of LPS group(0.445±0.049,P<0.01) and LPS+ negative control group(0.438±0.052,P<0.01),no obviously difference with normal group(0.152±0.038,P>0.05);The optical density ratio of TLR4 protein of LPS+interference group(0.13±0.028) was apparently lower than that of LPS group(0.307±0.035, P < 0.01) and LPS+ negative control group(0.283±0.032,P<0.05),no obviously difference with normal group(0.098±0.018,P>0.05)Conclusion The specific shRNA duplex molecule targeting TLR4 mRNA was successfully synthesized and efficiently transfected into HKC. The expression of TLR4 of HKC stimulated by LPS and the role of TLR4 increasing HKC apoptosis were suppressed by the specific shRNA.