Kindlin-2 Promotes Renal Fibrosis Through Facilitating TGF-β-Smad Signaling

BackgroundChronic kidney disease (CKD) has become one of the major diseases which threaten worldwide public health and brought about enormous suffering and economic burden to the patients。The prevalence of chronic kidney disease has reached epidemic proportions with 10-13% of the populations in Taiwan, Iran, Japan, China, Canada, India and the USA. More than 100 million people around the world rely on dialysis to survive, and the ratio increases at an average annual growth rate of 8%. More and more young men suffer from CKD, even the youngest one is less than 10 years of age, how shocking! There is no time to delay in reducing damage to kidney and protecting kidney from all kinds of kidney disease.Renal fibrosis is inevitable outcome of a variety of progressive kidney diseases. Multiple cellular events and molecular mediators participate and interact in concert in renal fibrogenesis. Among the many fibrogenic factors that regulate renal fibrotic process, TGF-βhas long been considered as a central mediator of the fibrotic Response. TGF-P exerts its biological functions mainly through its downstream Smads signalling molecules. The TGF-β-Smad3 signal is transduced through its cell membrane typeⅠand typeⅡserine/threonine kinase receptors. TGF-β1 binds to its typeⅡreceptor, the complex then forms a dimmer with the typeⅠreceptor, resulting in the phosphorylation and activation of the type I receptor. The activated type I receptor phosphorylates Smad2 and/or 3, which then heterodimerizes with Smad4 and subsequently translocate into the nuclei, where they regulat the transcription of TGF-β-target genes. Some adaptor proteins including SARA, Disabled-2 ELF spectrin, Endofin and so on have been reported to facilitate TGF-βsignaling by linking Smad2/Smad3 to the receptor complex.Tubulointerstitial fibrosis is characterized by excess accumulation of extracellular matrix (ECM) in the renal interstitium. The major source of excessive ECM production in CKD appears to be the interstitial myofibroblast (MFB). Epithelial-mesenchymal transition (EMT) is considered as one important sourece of MFB. Epithelial-to-mesenchymal transition is a biologic process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. A growing number of researches strongly confirmed the important role of EMT in different types of kidney disease and the reversal of EMT could improve renal fibrosis.Although numerous studies have revealed a variety of initial factors, key mediators and signaling pathways of EMT, TGF-β1 appears to be the most potent inducer of such changes, and is able to both initiate and complete this process. TGF-β1-induced EMT mainly relies on the integrity of its Smads signaling pathway.Kindlin-2 is one type of Kindlin protein family, which is novel adaptor proteins, regulating the activation of integrin. At the molecular level, cell-ECM adhesion is mediated primarily by integrins. Kindlin-2-integrin interaction enhances talin-mediated integrin activation and Kindlin-2 is also required for actin polarization, cells spreading, and ILK localization into FAs. Two groups independently generated Kindlin-2-deficient mice and in both cases the knockout animals died at or before embryonic age 7.5 days due to severe detachment of the epiblast and endoderm resulting in peri-implantation lethality. We can understand the significance of Kindlin-2 in cell-ECM adhesion and embryonic development.At present, there is rare report about the role of Kindlin-2 in kidney. Only one research proved that Kindlin-2 regulated podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and TGF-P markly increased Kindlin-2 expression. Our group has previously demonstrated that Kindlin-2 could promote breast cancer metastasis by inducing the EMT of breast tumor cells. We draw a hypothesis that Kindlin-2 could promote renal fibrosis by inducing the EMT of renal tubular epithelial cells. If true, what is the molecular mechanism? We utilize the modal of human renal tubular epithelial cells in vitro, mice unilateral ureteral obstruction (UUO) modal in vivo, combined with Clinical specimens of patients with CKD to determine the role of Kindlin-2 in renal fibrosis, and discuss the detailed and innovative molecular mechanisms.ChapterⅠKindlin-2 is required for TGF-β1-induced EMT in human kidney tubular epithelial cellsObjectiveTo investigate the role of Kindlin-2 in EMT of HKC, observe the effect of TGF-β1 on Kindlin-2 expression in HKCs and to explore the role of Kindlin-2 in TGF-β1-induced EMT of HKCs.Methods1. HKCs were treated with various dose of TGF-β1 (0,1,2,5 and lOng/ml) for different times (0,12,24,48 and 72 hours). The expression of Kindlin-2 was determined by Western blot and Real-Time PCR. HKCs were treated with 5ng/ml TGF-β1 for various time (0,12,24 and 48 hours), the expression of E-cadherin, a-SMA and FN was determined by Western blot and Real-Time PCR.2. HKCs were cultured in regular medium and transfected with Flag vector or Kindlin-2 plasmid for 48 hours, the expression of E-cadherin,α-SMA and FN was examined using Western blot and Real-Time PCR.3. The stable cell lines of HKCs transfected with Flag Kindlin-2 or Flag were established and the expression of E-cadherin,α-SMA and fibronectin was detected by Western blot, Real-Time PCR and immunofluorescence.4. HKCs were treated with Control siRNA or Kindlin-2 siRNA for 24 hours followed by stimulation of TGF-(31 for 48 hours. The expression of E-cadherin,α-SMA and FN was checked by Western blot and Real-Time PCR.Results1. TGF-β1 upregulated the expression ofα-SMA protein (F=4.546, P=0.039) and mRNA (F=18.537, P=0.001), increased FN protein (F=5.784, P=0.015) and mRNA (F=20.468,P=0.002) and downregulated the expression of E-cadherin protein (F=7.457, P=0.011) and mRNA (F= 19.246, P=0.001) in a time-dependent manner.2. TGF-β1 upregulated the protein (F=4.275, P=0.028) and mRNA expression (F=16.909, P=0.000) of Kindlin-2 in a time-dependent manner, and also in dose-dependent manner.3. Overexpression of HKCs with Kindlin-2 for 48 hours resulted in significant increase in expression of FN and a-SMA protein (t=-2.905, P=0.044) and mRNA (t=-4.069, P=0.015) and significant reduce in expression of E-cadherin protein (t=3.135, P=0.035) and mRNA (t=4.546, P=0.010).4. Compared to control cell line, in stable HKCs cell line transfected with Kindlin-2, the cell shape was altered from Cobblestone-like to spindle-like. Stable overexpression of Kindlin-2 in HKCs ignificantly upregulated the expression of a-SMA and Fibronectin and downregulated the expression of E-cadherin.5. Kindlin-2 siRNA Group (Kindlin-2 siRNA intervenes at 24h, then TGF-β15 ng/ml intervenes for 48hs) could significantly reduce the expression of FN and a-SMA mRNA (F=16.663, P=0.001) and protein (F=5.774, P=0.021) and increase the expression of E-cadherin mRNA (F=7.218, P=0.012) and protein (F=12.054, P=0.002) compared to the intervention only by TGF-β15 ng/ml on HKCs cells.Conclusions1. TGF-β1 upregulates the expression of Kindlin-2 in a time-dependent and dose-dependent manner.2. Kindlin-2 promotes EMT of HKCs.3. Kindlin-2 is required for TGF-β1-induced EMT on HKCs.ChapterⅡKindlin-2 is involved in TGF-β1-Smad3 signal pathway ObjectiveTo observe the role of Kindlin-2 in Smad3 activation; To investigate the association of Kindlin-2 with Smad3 and TGF-β1 receptor and the role of Kindlin-2 in TGF-β1-Smad3 signal pathway.Methods1. HKCs were divided into four groups:transfection with Flag, transfection with Flag-Kindlin-2, transfection with Flag or Flag-Kindlin-2 followed by incubation with TGF-β1. Phosphorylation of Smad3 was detected by Western blot. 2. HKCs were divided into four groups:Control siRNA group, Kindlin-2 siRNA group, Control siRNA+TGF-β1 group and Kindlin-2 siRNA+TGF-β1 group. HKCs total protein and nuclei protein were extracted and phosphorylation of Smad3 was detected by Western blot.3. HKCs were divided into four groups:co-transfection with Control siRNA and Flag, co-transfection with Control siRNA or Kindlin-2 siRNA and Flag followed by treatment with TGF-β1, co-transfection with Kindlin-2 siRNA and Kindlin-2 siRNA resistant plasmid followed by being treated with TGF-β1, cell total protein were extracted and phosphorylation of Smad3 was determined by Western blot.4. HKCs were transfected with Flag-Kindlin-2, the association of Kindlin-2 with Smad3 was detected by co-immunoprecipitation.5. HKCs were transfected with Flag-Kindlin-2-N or Flag-Kindlin-2-C, mapping of the Kindlin-2 binding site with Smad3 was confirmed by GST-pull down.6. HKCs were co-transfected with Flag-Kindlin-2 and HA-TβRⅠor Flag-Kindlin-2 and HA-TβRⅡ, the interaction of Kindlin-2 with TβRⅠor TβRⅡwere detected by co-immunoprecipitation.7. Colocalization of Kindlin-2 with Smad3 or TβRⅠand TβRⅡwere observed by confocal microscope.8. Knockdown of Kindlin-2 in HKCs by Kindlin-2 siRNA, the association of TβRⅠwith Smad3 was measured by coimmunoprecipitation.Results1. Kindlin-2 couldn't activate Smad3 as the Flag group. When HKCs were treated with TGF-β1 for 30min after transfection with Flag or Flag-Kindlin-2, Kindlin-2 increased phosphorylation of Smad3 induced by TGF-β1.2. When HKCs were treated with TGF-β1 for 30min after transfection with Control siRNA or Kindlin-2 siRNA for 72 hours, Kindlin-2 significantly inhibited phosphorylation and nuclear transfer of Smad3 induced by TGF-β1 Kindlin-2 siRNA resistant plasmid could reverse the inhibition of phosphorylation of Smad3 by Kindlin-2 siRNA.3. Coimmunoprecipitation showed Kindlin-2 strongly interacted with Smad3.4. GST-pull down datas verified that N-terminal of Kindlin-2 interacted with MH1 domain of Smad3. While neither of N-terminal or C-terminal of Kindlin-2 had association with MH2 domain of Smad3.5. Coimmunoprecipitation results proved that Kindlin-2 interacted with both TβRⅠand TβRⅡ.6. HKCs were transfected with Flag-Kindlin-2, co-immunoprecipitation results showed that Kindlin-2 formed a complex with Smad3 and TGF-β1 receptor.7. Colocalization of endogenous Kindlin-2 and exogenous Smad3 and TGF-β1 receptor was observed at the membrane. HKCs cells were stained for Kindlin-2 by pAb (red), Smad3 by anti-Flag (green) and for TGF-β1 receptor by an anti-HA mAb (green). Nuclei were revealed by Hoechst stain (blue). When HKCs were treated with TGF-β1, Kindlin-2 had colocalization with Smad3 in nuclei.8. Knockdown of Kindlin-2 partially inhibited the combination of TGF-β1RI with Smad3.Conclusions1. Kindlin-2 can't activate Smad3, but has synergism with TGF-β1 in activating Smad3.2. Kindlin-2 is required for Smad3 activation induced by TGF-β1.3. Kindlin-2 interacts with Smad3, TβRⅠand TβRⅡand has colocalization with them.4. Konckdown of Kindlin-2 inhibits the combination of TβRⅠwith Smad3. ChapterⅢKindlin-2 plays an important role in renal fibrosisObjectiveTo explore the role of Kindlin-2 in renal fibrosis by animal experiments, examine the expression of Kindlin-2 in the tissues of clinical patients with CKD and reveal the role of Kindlin-2 in kidney disease.Methods1. ICR mice were randomly divided into three groups:Sham received Control siRNA, unilateral ureter obstruction (UUO) received Control siRNA and unilateral ureter obstruction (UUO) received Kindlin-2 siRNA. A unilateral ureter obstruction modal was induced in male mice by ligation of the left ureter as described. Mice were sacrificed at day 7 post-surgery and the obstructed kidney were harvested and used for immunohistochemistry, Western blot and Real-Time PCR respectively.2. Renal morphological changes were observed by HE staining. Collagen deposition was measured by masson staining. The location and expression of Kindlin-2, p-Smad3, a-SMA and Fibronectin was detected by immunohistochemistry.3. The protein expression of Kindlin-2, p-Smad3, Smad3,α-SMA and Fibronectin was examined by Western blot.4. The mRNA expression of Kindlin-2,α-SMA and Fibronectin was determined by Real-time PCR.5. The location and expression of Kindlin-2 and p-Smad3 in patient biopsies were detected by immunohistochemistry. Results1. HE staining demonstrated that varying degrees of tubular dilatation or atrophy, expansion of interstitial space, infiltration of inflammatory cells in interstitial areas were observed in the mice of UUO group under light microscopy compared to the sham-operated kidneys, while these symptoms were obviously improved in mice of UUO received Kindlin-2 siRNA.2. The results of masson staining showed that there was much more collagen deposition around renal tubule and in interstitial areas in UUO mice kidney than sham-operated kidneys. While in mice of UUO received Kindlin-2 siRNA, less collagen could be observed compared to UUO received Control siRNA mice.3. The results of immunohistochemistry displayed that the distribution of Kindlin-2 were mainly located in cytoplasm and some lied in nuclei, p-Smad3 only lied in the nuclei and a-SMA and Fibronectin were absolutely located in interstitium. Compared to the sham-operated kidneys, the expression of Kindlin-2, p-Smad3, a-SMA and Fibronectin was significantly increased in UUO received Control siRNA mice. And these four protein expression was decreased in mice of UUO received Kindlin-2 siRNA.4. The results of Western blot verified that the significantly increased expression of Kindlin-2 (F=18.540, P=0.003), p-Smad3 (F=14.735, P=0.005), a-SMA (F=27.928, P=0.001) and Fibronectin (F=56.200, P=0.000) was observed in UUO received Control siRNA mice compared to the sham-operated kidneys. And Kindlin-2, p-Smad3,α-SMA and Fibronectin protein expression was much less in mice of UUO received Kindlin-2 siRNA, but there was no change in Smad3 expression.5. Real-Time PCR results demonstrated that compared to the sham-operated kidneys, the mRNA expression of Kindlin-2 (F=9.913, P=0.013),α-SMA (F=8.175, P=0.019) and Fibronectin (F=80.396, P=0.000) were significantly upregulated in UUO received Control siRNA mice. And all mRNA expression was much less in mice of UUO received Kindlin-2 siRNA.6. The results of immunohistochemistry proved that the expression of Kindlin-2 and p-Smad3 was significantly elevated in patient tissues compared with normal kidney, and p-Smad3 expression was positively correlated to the expression of Kindlin-2.Conclusions1. The expression of Kindlin-2 is induced in fibrotic kidney of mice.2. Knockdown of Kindlin-2 alleviates kidney fibrosis induced by UUO.3. Kindlin-2 is highly expressed in the patient biopsies with renal fibrosis arid is positively related to the expression of p-Smad3.