| Background:Renal interstitial fibrosis is the main pathological change in chronic kidney disease?CKD?.Once it occurs,it will continue to progress,and eventually lead to the occurrence of end-stage renal disease?ESRD?.However,the occurrence and development mechanism of renal interstitial fibrosis has not been fully clarified yet,and the treatment strategy of anti-renal fibrosis has not achieved good expectations.Therefore,it is of great clinical significance to explore the mechanism of renal interstitial fibrosis and find effective intervention measures.Inflammation is an important initiating factor in the occurrence and development of renal fibrosis.Interferon regulatory factor-1?IRF-1?,a major member of the IRFs family,is expressed in a variety of adult cells.As a transcriptional factor,it regulates the transcription of a series target genes and participates in a variety of physiological and pathological processes,such as inflammatory injury,immune response,tumor immune surveillance,virus infection and so on.It is well known that immune abnormalities and inflammation are the key pathogenic mechanisms of many kidney diseases.In recent years,some studies have found that ROS aggravates ischemic acute renal injury by inducing the expression of IRF-1 in damaged renal tubular epithelial cells,which leads to up-regulation of pro-inflammatory genes.In the neonatal rat UUO model,the expression of IRF-1 was significantly increased in the obstructive kidney.It is worth noting that IRF-1 and Klotho participate in the physiological process of calcium and phosphorus metabolism in kidney by reverse effect.The increase of IRF-1 expression caused by JAK3?Janus kinase 3?deficiency promoted the expression of Cyp27b1 and the production of 1,25?OH?2D3,while Klotho down-regulated the expression of Cyp27b1 and decreased the synthesis of 1,25?OH?2D3.In addition,in the kidneys of jak3-/-mice,the up-regulation of IRF-1 and the decrease of Klotho suggest that there may be a negative regulatory relationship between IRF-1 and Klotho.Klotho is mainly high expressed in renal tubular epithelial cells,including membrane and secreted proteins,while the extracellular segment of membrane Klotho protein and secreted Klotho protein can enter the blood circulation,which is called soluble Klotho protein.Soluble Klotho protein can protect many organs and cells.During recent years,Klotho has been proved to be an important factor to inhibit renal fibrosis,which can significantly inhibit the activation of TGF-?,Wnt/?-catenin signal pathway and RAAS system,thus improving renal interstitial fibrosis in unilateral ureteral obstruction?UUO?or adriamycin nephropathy model.We have previously demonstrated that Klotho can inhibit the activation of FGF2 signal pathway by competing with FGF2 to bind FGFR receptors,reduce renal tubular epithelial cell transdifferentiation and fibroblast proliferation and activation,and then improve renal interstitial fibrosis.At the same time,a study found that the expression of Klotho in the kidney decreased significantly during the occurrence of CKD and renal interstitial fibrosis.Therefore,the decrease of Klotho expression is an important link between the occurrence and development of renal interstitial fibrosis.So far,people have not clarified why the expression of Klotho is significantly decreased in CKD,and the key molecular mechanism leading to the down-regulation of Klotho expression in CKD may be an important breakthrough to explore effective intervention measures for renal interstitial fibrosis.Accordingly,we speculate that IRF-1 may induce renal tubular epithelial cells to secrete profibrotic factors by inhibiting the expression of Klotho in CKD,which eventually leads to the occurrence and development of renal interstitial fibrosis.In order to verify the above inference,we first observed the expression of IRF-1 in renal tubular epithelial cells of patients with CKD and mice with unilateral ureteral obstruction?UUO?,and the correlation between IRF-1 expression and the degree of renal fibrosis.Then,in vitro study,the effect of IRF-1 on the expression of fibrosis markers in human renal tubular epithelial cells?HK-2?and human embryonic kidney cells 293?HEK293?was studied.Whether IRF-1 effects the regulation of Klotho expression,and the upstream and downstream molecular mechanisms was elucidated.Finally,IRF-1 knockout(IRF-1-/-)mice and C57BL/6mice were used to establish UUO models.UUO models made by C57BL/6 mice were injected with TNF-?neutralizing antibody through tail vein to clarify the role of knockout or inhibition of IRF-1 in improving renal interstitial fibrosis by regulating Klotho.Methods and Results:Part one:The relationship between IRF-1 and renal interstitial fibrosis1.The expression of IRF-1 in renal biopsy specimens of patients with CKD increased,which was positively correlated with the degree of renal interstitial fibrosis.The specimen of CKD patients with no obvious renal pathological changes and renal fibrosis were selected from the biological specimen library in Department of Nephrology,Xinqiao Hospital,Army Medical University.Masson staining and IRF-1immunohistochemical staining were performed to analyze the degree of renal interstitial fibrosis and the expression level of IRF-1.The results showed that compared with normal renal tissue,the expression of IRF-1 in renal tubular epithelial cells was significantly increased in CKD patients.The expression level of IRF-1 was positively correlated with the area of renal interstitial fibrosis and negatively correlated with eGFR.These results suggest that the expression of IRF-1 in renal tissue of patients with CKD may be closely related to the severity of renal interstitial fibrosis.2.The expression of IRF-1 in renal tubular epithelial cells increased in UUO model and adriamycin nephropathy model?ADR?mice.The wild-type male C57BL/6 mice?n=32?were used to prepare UUO model and ADR model at the age of 10 to 12 weeks.14 days after the establishment of the model,the mice in each experimental group were killed and the kidneys were taken.Renal injury,renal fibrosis and IRF-1 expression were detected by HE?Hematoxylin-eosin?,Masson,immunohistochemical staining and Western blot?WB?.The results showed that the kidneys from UUO and ADR model mice showed significant injury and fibrosis,and the expression of IRF-1 in renal proximal tubular epithelial cells increased significantly.Therefore,we also found that IRF-1 may be closely related to renal interstitial fibrosis in both kinds of the renal fibrotic animal models.3.Overexpression of IRF-1 significantly promoted the expression of fibrosis markers in renal tubular epithelial cells.In vitro,human IRF-1 plasmid was constructed and transfected into human renal tubular epithelial cells?HK-2?and rat renal tubular epithelial cells?NRK52E?.The expression of IRF-1,?-SMA and fibronectin was detected by Western blot.The results showed that under normal conditions,there was only weak IRF-1 expression in HK-2 cells and NRK52E cells,while IRF-1 overexpression induced by IRF-1 plasmid transfection could significantly up-regulate the expression of fibrotic markers,for example,?-SMA and fibronectin.These results suggested that IRF-1 overexpression can significantly promote the expression of fibrotic markers in renal tubular epithelial cells.Part two:the role and mechanism of IRF-1 in promoting the expression of fibrotic markers by regulating Klotho in renal tubular epithelial cells.1.The expression of Klotho in kidney tissue of patients with CKD and UUO mice decreased,which was negatively correlated with the expression of IRF-1.The first part renal biopsy specimens of CKD patients without obvious renal pathological changes and with renal fibrosis were stained with immunohistochemistry to detect the expression of Klotho.The results showed that the expression of Klotho in renal tubular epithelial cells of CKD patients was significantly lower than that of normal renal tissues,and it was negatively correlated with the expression of IRF-1,which indicated that the increased expression of IRF-1 in renal tissues of patients with CKD might be closely related to the decreased expression of Klotho.Wild-type male C57BL/6 mice?n=32?aged 10 to 12 weeks were used to establish UUO model.Kidney samples were taken at 3,7 and 14 days after UUO surgery.The expression of IRF-1 and Klotho in mouse kidney,the co-localization of IRF-1 and Klotho in renal tubular epithelial cells were detected by Western blot,Real-time PCR and double staining of immunofluorescence.The results showed that in the UUO model,the expression of IRF-1 in renal tubular epithelial cells of the obstructied kidneys was increased and the expression of Klotho was reduced.The above results suggest that the high expression of IRF-1 is accompanied by the significant decreased Klotho expression in renal interstitial fibrosis,and whether there is a regulatory relationship between them is worthy for further discussion.2.IRF-1 promotes the expression of fibrotic markers by down-regulating Klotho in HK-2cells.After transfection with IRF-1 plasmid into HK-2 cells,the expression of Klotho was detected by Western blot.The results showed that the overexpression of IRF-1 inhibited the expression of Klotho in a dose-dependent manner.If both of IRF-1 and Klotho plasmids were transfected at the same time,and the expression of fibrotic markers??-SMA and fibronectin?were detected by Western blot.It was found that the overexpression of Klotho significantly reversed the expression of?-SMA and fibronectin which were induced by IRF-1 plasmid transfection,suggesting that IRF-1 promoted the expression of fibrotic markers by inhibiting Klotho in renal tubular epithelial cells.3.IRF-1 inhibits Klotho gene transcription.Human embryonic kidney cells?HEK293?were cultured in vitro,and the expression of IRF-1 was detected by Western blot.The results showed that under normal conditions,there was only weak expression of IRF-1 in HEK293 cells.The changes of luciferase activity in Klotho promoter were detected by Dual Luciferase Assay kit after simultaneous transfection of IRF-1 plasmid and Klotho promoter luciferase reporter plasmid.It was found that IRF-1 overexpression inhibited luciferase activity driven by Klotho?KL?promoter in a dose-dependent manner.Thirteen potential binding sites of transcription factor IRF-1 in human Klotho promoter?including the upstream 4000bp of Klotho transcription initiation site?were predicted by JASPAR database.Primers were designed and chromatin immunoprecipitation?ChIP?was used to determine whether IRF-1 directly binds to the Klotho promoter.The results showed that there were no obvious positive bands of PCR products.The results suggested that IRF-1 inhibit the transcription of Klotho gene,but not directly bind to the promoter region of Klotho to regulate its transcription.4.IRF-1 down-regulates the expression of Klotho in HK-2 cells by interfering with the activation of C/EBP-?.In order to explore the mechanism of IRF-1 inhibiting Klotho transcription,we used three bioinformatic databases?PROMO,Genecards and JASPAR?to analyze the human Klotho gene promoter and predicted 11 transcription factors that may bind to the Klotho promoter region?relative score more than 80 points?,of which nine transcription factors were reported to be involved in renal fibrosis.Furthermore,in order to identify the transcription factors that mediate the regulation of Klotho by IRF-1,after IRF-1 plasmid was transfected into HK-2 cells,the expression of 9 transcription factors,namely C/EBP-?,PPAR-?,p53,SP1,YY1,USF2,C/EBP-?,GATA3 and ATF3 mRNA,was detected by Real-time PCR.The results showed that IRF-1 overexpression significantly inhibited the expression of C/EBP-?in HK-2 cells?down to 1/3?.Western blot detection showed that IRF-1 overexpression inhibited the C/EBP-?protein expression in a dose-dependent manner.Both IRF-1 and C/EBP-?plasmids were transfected into HK-2 cells at the same time,and Western blot was used to detect the expression of Klotho,fibrotic markers?-SMA and fibronectin.It was found that overexpression of C/EBP-?significantly reversed the Klotho reduction and the increasion of ?-SMA and fibronectin induced by IRF-1 plasmid transfection.These results suggest that IRF-1 down-regulates Klotho expression by interfering with the activation of C/EBP-?,and then promote the expression of fibrotic markers in renal tubular epithelial cells.5.C/EBP-?regulates its transcription by directly binding to the region of Klotho promoter.A series of Klotho promoter luciferase reporter plasmids KL5?-2000bp?+100bp?,KL4?-1705bp?+100bp?,KL3?-1530bp?+100bp?,KL2?-1220bp?+100bp?and KL1?-1000bp?+100bp?were constructed and were transfected into HEK293 cells with C/EBP-?plasmid,respectively.The change of luciferase activity of truncated Klotho promoter was detected by Dual Luciferase Assay kit.The results showed that C/EBP-?overexpression significantly enhanced the luciferase activity driven by KL5,KL4,KL3 and KL2,but had no effect on the luciferase activity driven by KL1 and PGL3-Basic.A site-specific mutation was carried out on the second C/EBP-?binding site in the Klotho promoter?KL2?.Using Dual Luciferase Assay,it was found that C/EBP-?overexpression had no effect on the activity of luciferase driven by mutant KL2 truncated plasmid.The chromatin immunoprecipitation assay?Ch IP?showed that C/EBP-?directly binds to the dominant region of the second potential binding site in Klotho promoter.These results further suggested that C/EBP-?could directly bind to the region?-1039?-1029?of Klotho promoter to regulate its transcription.6.TNF-?induced increased expression of IRF-1 in HK-2 cells.HK-2 cells were incubated with TNF-?,then the expression of C/EBP-?,Klotho,?-SMA and fibronectin were detected by Western blot.The results showed that TNF-?induced the expression of IRF-1,?-SMA and fibronectin in a dose-dependent manner,accompanied by down-regulation of C/EBP-?and Klotho expression.IRF-1 siRNA was transfected into HK-2cells and then treated with TNF-?,and the expression of Klotho,?-SMA and fibronectin was detected by Western blot.The results showed that siRNA interference with IRF-1 significantly reversed the effect of TNF-?on the expression of Klotho,?-SMA and fibronectin,suggestingthat TNF-?promote the expression of fibrotic markers in renal tubular epithelial cells by inducing IRF-1 to down-regulate Klotho.Similarly,HK-2 cells were treated with TNF-?after transfection with C/EBP-?plasmid,and the expression of Klotho,?-SMA and fibronectin was detected by Western blot.The results suggested that the overexpression of C/EBP-?significantly reversed TNF-?induced Klotho reduction and the increasion of?-SMA and fibronectin.These results suggested that TNF-?induce IRF-1 expression,and then down-regulate the expression of Klotho by inhibiting the activation of C/EBP-?,which may promote renal fibrosis.Part three:knockout or inhibition of IRF-1 significantly improves renal interstitial fibrosis.1.Renal fibrosis in UUO model made by IRF-1 knockout(IRF-1-/-)mice was significantly reduced.Sixteen IRF-1-/-mice and sixteen wild-type?WT?mice aged 10 to 12 weeks,eight were selected respectively from the two groups for UUO models.After 14 days,mice in each group were killed and kidney samples were taken.The degree of renal injury and fibrosis was evaluated by HE and Masson staining,the results showed that the degree of renal injury and fibrosis in IRF-1-/-mice was significantly less than that in WT mice,and the expression of?-SMA and fibronectin in IRF-1-/-mice was significantly lower than that of WT mice.The results suggest that knockout of IRF-1 significantly reduce renal fibrosis in UUO model mice.Then the expression of inflammatory cytokines TNF-?,IL-1?and IL-6 was detected by Real-time PCR.It was found that the expression of inflammatory cytokines TNF-?,IL-1?and IL-6 in the kidney of IRF-1-/-mice was significantly lower than that of WT mice,suggesting that knockout of IRF-1 significantly reduce the inflammatory response in the kidneys of UUO mice.In order to verify the mechanism of IRF-1,Real-time PCR,Western blot and immunostaining were used to detect the expression of C/EBP-?and Klotho.The results showed that compared with WT mice,IRF-1 gene knockout could partially reverse the down-regulated expression of C/EBP-?and Klotho in kidneys and renal tubular epithelial cells in the obstructed kidneys.These results suggest that knockout of IRF-1 significantly reduce the down-regulation of C/EBP-?and Klotho expression and improve renal interstitial fibrosis.Therefore,the increased expression of IRF-1 caused by UUO and the subsequent down-regulation of C/EBP-?and Klotho may be one of the important mechanisms in the occurrence and development of renal interstitial fibrosis.2.Intravenous injection of anti-TNF-?neutralizing antibody significantly inhibited the down-regulation of Klotho expression and renal interstitial fibrosis in UUO model mice.Thirty-two male C57BL/6 mice aged 10 to 12 weeks were randomly selected to establish UUO model.Eight mice in sham group and UUO model group were injected with anti-TNF-?neutralizing antibody through tail vein,and the other eight mice were injected with the same volume of PBS solution.The mice in each group were killed 14 days after surgery and kidney samples were taken.HE and Masson staining were used to evaluate the degree of renal injury and fibrosis.The results showed that the degree of renal injury and fibrosis in mice injected with anti-TNF-?neutralization antibody was significantly less than that in wild mice.The expression of IRF-1,Klotho,?-SMA and fibronectin in renal tissue was detected by Western blot and the expression of IRF-1 and Klotho in renal tubular epithelial cells was detected by immunofluorescence double staining.The results showed that injection of anti-TNF-?neutralizing antibody significantly inhibited the increased expression of IRF-1 and decreased expression of Klotho in kidneys and tubular epithelial cells of UUO model mice compared with mice injected with PBS solution.These results suggest that anti-TNF-?neutralizing antibody can improve renal interstitial fibrosis by inhibiting IRF-1.Conclusion:In renal biopsy specimens of CKD patients,UUO model and ADR model mice,the expression of IRF-1 in renal tissue and renal tubular epithelial cells was significantly increased,and was positively correlated with the degree of renal fibrosis.IRF-1overexpression significantly decreased the expression of renal fibrosis inhibitor Klotho and induced the expression of fibrotic markers in renal tubular epithelial cells,which was related to the down-regulation of the expression of Klotho transcriptional regulator C/EBP-?.Further studies showed that TNF-?was the main reason for the high expression of IRF-1 in renal tubular epithelial cells induced by UUO.Knockout or inhibition of IRF-1 by anti-TNF-?neutralization antibody significantly improve renal interstitial fibrosis.Therefore,the up-regulation of IRF-1 expression induced by inflammatory factors resulting in the lack of Klotho in renal tubular epithelial cells may be one of the important mechanisms for the occurrence and development of renal fibrosis,and the interaction between inflammation and IRF-1/Klotho also promote the continuous progress of renal fibrosis. |
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