| Objective: Chronic kidney disease(Chronic Kidney Disease,CKD)is one of the major diseases that threaten human health,and renal fibrosis is the common pathological basis of CKD.However,the pathophysiological process and related regulatory mechanisms of renal fibrosis remain to be explored.Leukemia inhibitory factor(LIF)is a multi-function cytokine,LIF functions through an autocrine or paracrine manner to bind to its receptor complex,LIF receptor(LIF-R)/gp130,and leads to the activation of many signaling pathways.Previous studies,including ours,have shown that LIF is abnormally elevated in renal fibrosis models caused by different causes,this study explores the role of LIF in renal fibrosis and related mechanisms,aiming to expand the understanding of the pathophysiological mechanism of renal fibrosis and provide new target for renal fibrosis treatment.Methods:1.To detect the expression of LIF protein in kidney of mice with unilateral ureteral obstruction(UUO).(1)The mice with ureteral obstruction caused by ligation of the left ureter(male C57BL/6J)were randomly divided into Sham operation(Sham)group,UUO1 d group,UUO3 d group,UUO7 d group and UUO10 d group.The mice were sacrificed at 1 day,3 days,7 days and 10 days after operation,and the basic morphological results were observed by H&E staining and collagen deposition by Masson staining and Sirius Red staining.The indexes related to fibrosis were detected by immunohistochemistry(IHC)and Western blotting(Western Blot,WB).(2)The expressions of LIF in kidney of UUO mice were detected by enzyme-linked immunosorbent assay(enzyme-linked immunosorbent assay,ELISA),real-time fluorescence quantitative PCR(quantitative real time polymerase chain reaction,q PCR)and WB.(3)NRK-49 F,HK-2 and THP-1 were studied in vitro.The cells were stimulated with TGF-β1,and the secretions of LIF were detected by ELISA.2.To detect the effects of LIF recombinant protein(rhLIF)and LIF neutralizing antibody(LIF nAb)on renal fibrosis in mice.(1)UUO mice were randomly divided into three groups: Sham group,UUO+PBS group,UUO+rhLIF group and UUO group.On the 1st and 3rd day after operation,PBS and rhLIF were injected intraperitoneally.On the 7th day,the kidneys were collected and the expressions of LIF were detected by WB and q PCR.The fibrosis related indexes were detected by H&E staining,Masson staining and Sirius Red staining.(2)UUO mice were randomly divided into three groups: Sham group,UUO+Ig G group,UUO+LIF nAb group and UUO group.PBS and LIF nAb were injected intraperitoneally on the 1st and 3rd day after operation,and the kidneys were collected on the 7th day.3.To detect the effects of LIF recombinant protein on rat fibroblast cell line NRK-49 F and human renal tubular epithelial cell line HK-2.(1)HK-2 was randomly divided into control group(control),TGF-βgroup,LIF group and TGF-β+LIF group.The protein was collected and the expression of α-SMA,E-cadherin was detected by WB.HK-2 was randomly divided into control group(control),10 ng/ml LIF group,25 ng/ml LIF group and 50 ng/ml LIF group.HK-2 was stimulated with different concentrations of rhLIF.The protein was collected and the expression of α-SMA,E-cadherin and p-STAT3 was detected by WB.(2)NRK-49 F was randomly divided into control group(control),2 ng/ml LIF group,5 ng/ml LIF group and 10 ng/ml LIF group.The cell proliferation was detected by NRK-49 F,CCK-8 stimulated by different concentrations of rhLIF.The protein was collected and the expression of α-SMA,COL-Ⅰ,COL-Ⅲ and p-ERK was detected by WB.Results:1.The expression of LIF is increased in UUO model,and many kinds of cells can secrete LIF.(1)The results of H&E staining showed that compared with Sham group,renal tubular dilatation and inflammatory infiltration could be seen in the early stage of UUO group,and some renal tubular atrophy could be seen in the later stage.The results of Masson and Sirius Red staining showed that with the progress of time,the deposition of collagen in the kidney of UUO group was obvious.The results of IHC staining showed that the expression of α-SMA,COL-Ⅰ,COL-Ⅲ,F4/80 in UUO group was up-regulated compared with Sham group.WB got the same result.(2)After homogenization of mouse kidney,ELISA results showed that LIF increased significantly in kidney tissue of UUO mice,and showed dynamic changes.It began to express in the early stage of fibrosis and reached the peak on the 7th day.The results of real time PCR showed the same trend.(3)Under the stimulation of TGF-β,fibroblasts,renal tubular epithelial cells and macrophages can synthesize and secrete LIF.2.Intraperitoneal injection of rhLIF can aggravate renal fibrosis in UUO mice,while injection of neutralizing antibody can delay the progression of renal fibrosis.(1)The effect of intraperitoneal injection of rhLIF was verified at first.The results of WB showed that the expression of LIF in the treatment group was more significant than that in the UUO group,and the results of q PCR also showed that rhLIF induced the up-regulation of endogenous LIF expression at the m RNA level.(2)The results of HE showed that rhLIF could aggravate the degree of renal tubular dilation and inflammatory infiltration.The results of Masson and Sirius Red staining showed that rhLIF could aggravate the collagen deposition in kidney.IHC results showed that rhLIF could up-regulate the expression ofα-SMA,COL-Ⅰ,COL-Ⅲ,F4/80 in the kidney of UUO mice.The same conclusion was obtained from WB results.(3)Then the effect of intraperitoneal injection of LIF nAb was verified.The results of WB showed that the expression of LIF in the treatment group was down-regulated compared with the UUO group.(4)The results of H&E staining showed that LIF nAb could reduce the degree of renal tubular dilatation and inflammatory infiltration.The results of Masson and Sirius Red staining showed that LIF nAb could reduce the collagen deposition in kidney.IHC results showed that LIF nAb could down-regulate the expression of α-SMA,COL-Ⅰ,COL-Ⅲ,F4/80 in the kidney of UUO mice.The same conclusion was obtained from WB results.3.LIF induce the activation of fibroblasts and the interstitial transformation of renal tubular epithelial cells.(1)The results of HK-2,WB stimulation with rhLIF showed that the expression of α-SMA in epithelial cells was down-regulated with the increase of stimulation concentration.Further analysis of its downstream signal pathway,WB results showed that the effect of LIF on epithelial cells is through the phosphorylation of STAT3 signal pathway.(2)The results of stimulating NRK-49 F,CCK-8 with rhLIF showed that LIF could stimulate the proliferation of fibroblasts.WB results showed that the expression of α-SMA,COL-Ⅰ,COL-Ⅲ in fibroblasts was up-regulated with the increase of stimulation concentration.Further analysis of its downstream signal pathway,WB results show that the effect of LIF on fibroblasts is through the phosphorylation of ERK signal pathway.Conclusions:In renal interstitial fibrosis,the expression of LIF is increased and secreted by a variety of cells.LIF mediates interstitial fibrosis by regulating fibroblast activation and epithelial cell phenotype switching. |
PDF Full Text Request