Complement C3 - Mediated Podocyte Injury Is Involved In Chronic Progression After Acute Kidney Injury

Objective:This study was designed to analyze the relationship between podocyte injury and complement C3 during chronic renal injury post AKI?Acute Kidney Disease?.We will articulate the effect and mechanism of C3 damaging podocyte in the process of the development of AKI into chronic kidney disease.We hope that through this re-search new ideas will be available for future researches on the prevention and treat-ment of post-AKI chronic kidney disease.Methods:1.Fifty BALB/C mice were randomly divided into control group,sham group,AKI20 group?left kidney ischemia 20 minutes reperfusion?,AKI 30 group?left kidney ischemia 30 minutes reperfusion?and AKI 40 groups?left kidney ischemia 40 minutes reperfusion?,and the right kidney was excised on the 8th day.On the 8th,9th,10th,14th and 28th days,the mice urine was collected for 24-hour and the blood was col-lected from the iliac veins in the eye.The 24-hour urinary protein,serum creatinine?Scr?,and blood urea nitrogen?BUN?were measured.On the 28th days,mice were sacrificed by cervical dislocation to obtain kidney specimens.HE staining and Mas-son staining were used to classify and grade pathological lesions.Electron microscopy was used to observe pathological changes of renal podocytes.The expression of Nephrin,CD2AP,Synaptopodin and TRPC6 mRNA and protein were detected by Q-PCR,Western Blot and immunohistochemistry.2.Thirty BALB/C mice were randomly divided into control group?WT-Control?,sham group?WT-sham?,and CKD post AKI group?WT-AKI-CKD,left kidney is-chemia 30 minutes reperfusion?;thirty complement C3 knockout mice were randomly divided into control group(C3^-/--Control),sham group(C3^-/--sham),and CKD post AKI group(C3^-/--AKI-CKD,left kidney ischemia 30 minutes reperfusion).The right kidney was excised on the 8th day in each AKI-CKD group.On the 28th day,the mice urine was collected for 24-hour and the blood was collected from the iliac veins in the eye.The 24-hour urinary protein,Scr,and BUN were measured.The mice were then sacrificed by cervical dislocation to obtain kidney specimens.HE staining and Masson staining were performed to classify and grade pathological lesions.Electron microscopy was employed to observe pathological changes of renal podocytes.The concentrations of interleukin-1?IL-1?,interleukin-6?IL-6?and TGF-?of renal tissue were measured by ELISA.The expression of Nephrin,CD2AP,Synaptopodin,TRPC6,complement C3,LXR?and TLR4 mRNA and protein were detected by Q-PCR,Western Blot and immunohistochemistry.3.Mice podocytes were cultured in vitro,establishment of glomerular podocyte hy-poxic environment by continuous nitrogen method,stimulation with different concen-trations of C3a,co-culture with TLR4 inhibitor?HCQ?,transduction of kidney LXR?SiRNA?small interfering RNA,siRNA?by AB method and transduction plas-mid LXR?DNA vector by liposome method.The morphology of glomerular podo-cytes was observed by HE staining.The levels of inflammatory cytokines IL1,IL6and TGF-?1 in glomerular podocytes were measured by ELISA.The expression of LXR?mRNA was detected by q-PCR technique.The glomerular podocyte LXR?/TLR4 and Synaptopodin,Nephrin,CD2AP,and TRPC6 protein expression levels were measured by Western Blot.Results:1.Compared with the control group,serum levels of Scr and BUN in AKI 20 group,AKI 30 group and AKI 40 group were significantly higher on the 9th day?P<0.05?.Compared with the 9th day,the Scr and BUN levels in the three groups on the 10th day decreased?P<0.05?.On the 14th day,Scr and BUN in AKI 30 group and AKI 40group significantly increased?P<0.05?,but there was no statistical difference between AKI 20 group,control group and sham group?P>0.05?.On the 28th day,Scr and BUN in AKI 30 and AKI 40 groups continued to increase?P<0.05?,but there was no significant difference in Scr and BUN between the two groups?P>0.05?.On the 9th day,the quantitation of urine protein in AKI 40 group increased significantly.?P<0.05?.On the 10th day,the quantification of urine protein in the AKI 40 group de-creased compared with the 9th day?P<0.05?.The urine protein levels in the AKI 40group on the 14th and 28th days were not significantly different from those in the control group?P>0.05?.On the 14th day,the urinary protein level in the AKI 30group significantly increased?P<0.05?,and the urinary protein in the AKI 30 group further increased on the 28th day?P<0.05?,while the urine protein level in the AKI 20group was no significant difference compared to the control group and the sham oper-ation group.?P>0.05?.HE staining showed that the fibrosis of the renal tissue was time-dependent and aggravated.There was no significant difference in the glomerular mesangial proliferation,glomerulosclerosis and tubulointerstitial scores between the AKI 20 group and the control and sham groups?P>0.05?.Injury aggravation of three aspects took place in the AKI 30 group?P<0.05?;AKI 40 group renal tubulointerstitial score,compared with AKI 30 group,was statistically significantly different?P<0.05?.Masson staining showed that the glomerular,renal tubules and interstitial collagen areas showed a time-dependent increase in ischemia.Transmission electron microscopy revealed that the podocytes in the AKI 20 group,the sham operation group,and the control group were intact,with clear structure,and no foot process fu-sion was observed;the foot cells of the AKI 30 group had extensive foot process fu-sion;and the AKI 40 group had extensive podocyte foot process stripping.GBM ex-posed.Synaptopodin,Nephrin,CD2AP mRNA and protein expression showed ische-mia time-dependent decrease,TRPC6 mRNA and protein expression ische-mia-dependently increased.Compared with the control group,mRNA and protein ex-pression levels of Synaptopodin,Nephrin,and CD2AP in AKI group significantly de-creased?P<0.01?,TRPC6 mRNA and protein expression levels significantly increased?P<0.01?.Compared with AKI 30 group,mRNA and protein expression levels of Synaptopodin,Nephrin,and CD2AP significantly decreased in AKI 40 group?P<0.05?,mRNA and protein expression levels of TRPC6 were significantly increased?P<0.05?.There was no significant difference in the above indexes between AKI 20group,sham operation group and control group?P>0.05?.2.Compared with the WT-AKI-CKD group,the Scr,BUN and urinary protein levels in the C3^-/--AKI-CKD group were significantly lower?P<0.05?;glomerular mesangial hyperplasia,glomerular sclerosis and kidney tubulointerstitial scores substantially de-creased?P<0.05?;the glomerular,renal tubule and renal interstitial collagen area decreased?P<0.05?;transmission electron microscopy showed that WT-Control group,WT-sham group,C3^-/--Control group and the C3^-/--sham group,the podocytes were intact,the structure was clear,and no foot process fusion was visible.Extensive po-docyte fusion was observed in the WT-AKI-CKD group,while the podocyte foot process fusion was observed in the C3^-/--AKI-CKD group.Compared with the WT-AKI-CKD group,the levels of IL1,IL6 and TGF-?1 in the C3^-/--AKI-CKD group significantly declined?P<0.05?;complement C3,TRPC6,LXR?The mRNA and pro-tein expression levels of TLR4 substantially decreased?P<0.05?,the mRNA and pro-tein expression levels of Synaptopodin,Nephrin and CD2AP were significantly rose?P<0.05?.3.Compared with Control group,the levels of IL1,IL6 and TGF-?1 in podocyte su-pernatants ascended significantly in C3a concentration?P<0.05?.Compared with Control group,the synthesis expression of podocyte protein TLR4 and LXR?in C3a group was much higher.The expression levels of podocyte function protein Synapto-podin,Nephrin and CD2AP significantly declined,and the expression of TRPC6 pro-tein significantly increased?P<0.01?.Compared with C3a group,the levels of IL1,IL6 and TGF-?1 of podocyte inflammatory factors in C3a+HCQ group substantially decreased?P<0.01?,the expressions of TLR4,TRPC6 protein significantly decreased,the expression of Synaptopodin,Nephrin and CD2AP significantly increased?P<0.01?,and the expression of LXR?protein was not statistically different?P<0.01?.P>0.01);glomerular podocytes were transfected with LXR?-specific SiRNA,compared with C3a+ScrambledRNA group,the levels of IL6 and TGF-?1 of glomerular podocyte supernatant inflammatory factor IL1 in C3a+LXR?SiRNA group significantly in-creased?P<0.01?.Compared with the Scramble group,LXR?SiRNA 12.5 nM group and 25 nM group had shown significantly lower LXR?mRNA and protein expression levels after transfection.P<0.01),25nM group The expression of LXR?mRNA and protein in glomerular podocytes of 12.5nM group was significantly decreased?P<0.05?,and the expression of TLR4,TRPC6 protein in LXR?SiRNA+C3a group substantially increased?P<0.05?.The expression of Synaptopodin,Nephrin and CD2AP significantly decreased?P<0.05?.The plasmid of LXR?was transfected into podocytes,and the expression of LXR?mRNA and protein in the podocytes trans-fected with LXR?was significantly higher than that of the empty plasmid group.?P<0.01?,compared with the empty plasmid group,the levels of inflammatory factors IL1,IL6 and TGF-?1 in the supernatant of glomerular podocytes of C3a+LXR?plasmid group obviously decreased?P<0.01?.The expression levels of TLR4,TRPC6protein in podocytes substantially decreased,the expression of Synaptopodin,Nephrin and CD2AP conspicuously increased?P<0.05?.Conclusion:1.In the process of mouse AKI,it can complicated with podocyte injury.2,Mice AKI process can induce complement C3 activation.Complement C3 gene knockout can improve the inflammatory reaction and podocyte damage of kidney tis-sue and reduce renal fibrosis in mice with chronic AKI.3.Uner anoxic condition,Complement C3a can promote podocyte inflammatory ef-fects and LXR?/TLR4 expression under hypoxic and ischemic conditions,and inhibi-tion of LXR?/TLR4 can down-regulate podocyte inflammatory response and injury.4.Complement C3 may regulate podocyte inflammatory injury through LXR?/TLR4signaling pathway,and serves as an important mechanism of chronic fibrosis in mice after AKI.