Klotho Inhibits Renal Tubulointerstitial Fibrosis By Regulating Basic Fibroblast Growth Factor-2Signaling

Renal tubulointerstitial fibrosis is a common pathway in chronic kidney disease (CKD) that leads toend-stage renal failure. Its two prominent pathological characteristics are excessive deposition of extracellularmatrix (ECM) components and accumulation of interstitial fibroblasts. Previous studies suggest that themyofibroblast differentiated from fibroblast is the main source of ECM and that tubuloepithelial cell remodelingcan result in a myofibroblast phenotype and the synthesis of matrix proteins, such as type I and III collagens.Thus, inhibiting fibroblast proliferation and differentiation and tubuloepithelial cell remodeling is an importantway to reduce the number of myofibroblasts and ECM synthesis, and the studies on the underlying mechanismscan provide a novel strategy and target for prevention and treatment of renal interstitial fibrosis.Basic fibroblast growth factor-2(FGF2) is involved in proliferation and differentiation of many kinds ofcells, and also plays an important role in renal development. The expression of FGF2can be detected inglomerular endothelial, mesangial, and tubuloepithelial cells, and renal interstitium. Recent research indicatesthat FGF2is closely related to renal interstitial fibrosis, which can induce a myofibroblast phenotype oftubuloepithelial cells, increase expression of α-smooth muscle actin (α-SMA) and vimentin, release matrixmetalloproteinases MMP-9and MMP-2, and inhibit expression of epithelial cell marker cytokeratin andE-cadherin. In addition, it is shown that transforming growth factor β1(TGF-β1) can promote FGF2generationin fibroblasts. FGF2executes its function by binding to and activating FGF receptors (FGFR1-FGFR4),particularly FGFR1, which is highly expressed in kidney. However, the role of FGF2in renal interstitial fibrosisand the underlying mechanism have not been completely elucidated, and the regulation of FGF2/FGFRsignaling pathway will be an important way to inhibit renal interstitial fibrosis.Klotho is predominately expressed in renal tubulothelial cells, and its secreted protein is a hormone-likesubstance, which can act as a cytokine to regulate target cells. The Klotho knock-out mouse showed aphenotype similar to that of the patients with chronic renal failure and obvious renal fibrosis. Recently, therelationship between Klotho and renal diseases arouses the concern of researchers, and many studies suggestthat Klotho can protect renal function and inhibit renal aging. Klotho expression is markedly reduced in CKDpatients. Noticeably, Klotho is closely related to the FGF family, which can bind to FGFR1-4to regulate theFGF signaling. The binding of Klotho to FGFR can significantly promote the binding of FGF23, FGF19andFGF21to FGFR to enhance their activity. Klotho can compete with FGF2for binding to FGFR to inhibit FGF2activity. Normally a balance between Klotho and FGF2can be maintained, but in CKD, decreased expression ofrenal Klotho can attenuate its inhibition of FGF2signaling pathway, and the increased FGF2signal can induce changes in the phenotype of tubuloepithelial cells, proliferation and differentiation of fibroblast, excessivesynthesis of ECM and finally renal fibrosis. Therefore, the regulation of FGF2by Klotho can play an importantrole in renal fibrosis, and the elucidating the underlying mechanisms can explain the pathogenesis of renalfibrosis and provide theoretical evidence for effective prevention and treatment measures.In this study, we used renal interstitium cells (HK-2) and renal fibroblasts (NRK-49F) as in vitro researchobjects, and employed FGF2knock-out and C57BL/6mice to construct the animal model of unilateral ureteralobstruction (UUO) to study the regulation of FGF2/FGFR by Klotho in renal interstitium fibrosis from threeaspects:(1) Firstly we verified weather Klotho can regulate FGF2and the interaction between the two,(2) andevaluated the expression of Klotho and renal fibrosis in FGF2konck-out mice to confirm the role of FGF2inrenal fibrosis,(3) and constructed a mouse model of UUO, and injected exogenous Klotho to the mouse, tostudy the inhibition of FGF2/FGFR and renal fibrosis by Klotho. The results and conclusions are listed asfollows:1. The remodeling of tubuloepithelial cells by FGF2was accompanied by the decreased expression ofKlotho.HK-2cells were used as a model of proximal tubular epithelial cells. Cells treated with recombinanthuman FGF-2for48h underwent a series of phenotypic changes, such as elongation, branching, and loss oftheir cobblestone appearance in a dose-responsive manner. Western blotting revealed that FGF2dosedependently reduced E-cadherin expression (a tubular epithelial marker) and increased α-SMA andfibroblast-specific protein-1(FSP-1) expression, indicating that the cells were undergoing a transition from atubular epithelial phenotype to a pro-fibrotic phenotype. Interestingly, Klotho expression at both mRNA andprotein levels declined in HK-2cells treated with FGF2, indicating that FGF2has a potent inhibitive effect onthe expression of Klotho in association with the described phenotypic changes.2. FGF2-induced tubuloepithelial cell remodeling was mediated by ERK1/2signaling and repressed byKlotho protein.FGF2has been previously shown to induce phosphorylation of the FGF receptor substrate2(FRS2α) andstimulate ERK1/2activity downstream of its receptor, but the signaling of FGF2has not been confirmed inrenal tubular epithelial cells. FGF2stimulated phosphorylation of FRS2α and ERK1/2in HK-2cells within5-10min after FGF2treatment. Pretreatment with20μM U0126(ERK1/2inhibitor) significantly abated theup-regulation of α-SMA and FSP-1and the down-regulation of E-cadherin induced by FGF2. These resultssuggest that the activation of the ERK1/2signaling pathway plays an important role in FGF2-inducedtubuloepithelial cell remodeling.Interestingly, preincubation with Klotho protein for30min dose dependently inhibited FGF2-inducedactivation of FRS2α and ERK1/2. Moreover, Klotho pretreatment significantly prevented the down-regulationof E-cadherin and the up-regulation of α-SMA and FSP-1induced by FGF2. Immunofluorescence stainingrevealed that Klotho protein also inhibited the decreased expression of cytokeratin (another differentiated epithelial marker) and the increased expression of vimentin (another mesenchymal marker) induced by FGF2.3. Klotho competed with FGF2to bind to FGFR1in HK-2cells.To determine the underlying mechanism of the inhibitory effect of Klotho on FGF2, we examined thechanges in FGF2expression in Klotho-treated HK-2cells, and found that Klotho treatment did not inhibit FGF2expression directly, but it could suppress the increased expression of FGF2induced by TGF-β1. To determine ifTGF-β1signaling blocking contributes to the inhibitory effect of Klotho on FGF2-induced tubuloepithelial cellremodeling, the cells were preincubated with LY-364947, an inhibitor of TGF-β1type I receptor kinase (ALK5),before the treatment with Klotho and FGF2. We found that pretreatment with10μM LY-364947did not affectthe inhibitory effect of Klotho on the FGF2-induced changes in the expression of E-cadherin, α-SMA and FSP-1,suggesting that the inhibitory effect of Klotho on FGF2-induced tubuloepithelial cell remodeling was notmediated via interference with TGF-β receptor signaling.We then explored the direct effect of Klotho on the binding of FGF2to FGFR1in HK-2cells. Usingimmunoprecipitation with antibody to the FGFR1, we found that treatment with Klotho protein weakened theability of FGF2to bind FGFR1, although Klotho facilitated the binding of FGF23to FGFR1. The presence ofFGF2also significantly reduced the ability of Klotho to bind to FGFR1. As FGF23was markedly increased inCKD, we also investigated the effect of Klotho on the ability of FGF2to bind to FGFR1in the presence ofFGF23. The result showed that FGF23slightly enhanced the ability of Klotho to compete with FGF2forbinding to FGFR1. Hence, we presume that competition of Klotho with FGF2for binding to FGFR1mayexplain its potent ability to inhibit FGF2signaling.4. Klotho inhibited FGF2-induced fibroblast proliferation and differentiation into myofibroblastsAs fibroblast proliferation is likely to be involved in renal fibrosis, we observed the effect of Klotho onFGF2-induced fibroblast proliferation. FGF2significantly induced the proliferation of NRK-49F renalfibroblast cells, which can be almost entirely prevented by perincubation with Klotho. Meanwhile,FGF2-induced transformation of fibroblasts into myofibroblasts was also significantly inhibited by Klothoprotein, which can be reflected by the decreased expression of α-SMAand fibronectin.5. FGF2partially mediated the decreased expression of Klotho in UUO-induced renal fibrosisTo determine whether FGF2regulates Klotho in vivo during the development of renal fibrosis, we studiedFGF2knockout (FGF2-/-) mice. The obstructed kidney of FGF2-/-UUO mice exhibited significantly higherKlotho levels than those of wild-type (WT) UUO mice on the14th day after surgery. However, the activation ofERK1/2signaling and the expansion of interstitial area were alleviated in FGF2-/-UUO mice compared withWT UUO mice. We found a marked decrease in the expression of E-cadherin in tubules, an obvious increase inthe number of interstitial cells stained with α-SMA and Ki67, and an increase in the expression of fibronectin inthe obstructed kidney in WT UUO mice. But all of these were attenuated in FGF2-/-UUO mice. These datademonstrate that FGF2contributes to the reduced expression of Klotho and the development of renal fibrosis.6. FGF2was significantly increased in the obstructed kidney.Klotho treatment significantly mitigated renal fibrosis, and inhibited FGF2signaling in UUO miceTo investigate the interaction between Klotho and FGF2in the progression of renal fibrosis, we examinedthe changes in the expression of Klotho and FGF2during the progression of renal fibrosis using the wild-typemouse model of UUO. The expression of Klotho was reduced progressively with maximal reduction on the14thday following UUO. FGF2was significantly increased in the obstructed kidney on the7th and14thpost-surgical days, suggesting reciprocal inhibition between Klotho and FGF2in the progression of renalfibrosis.To demonstrate that Klotho may have reno-protective effect after UUO, we treated UUO mice withdifferent doses of recombinant Klotho protein (0.01mg/kg/48h and0.02mg/kg/48h) immediately after surgery.We found a significant increase in the expression of α-SMA and fibronectin (an extracellular matrix protein) inthe obstructed kidney at the mRNA level. Both doses of Klotho protein can suppress α-SMA and fibronectinmRNA expression in UUO mice, and the injection of0.02mg/kg/48h showed a slight stronger effect. Inaddition, treatment with0.01mg/kg/48h Klotho significantly attenuated renal fibrosis in the obstructed kidney,as revealed by H&E and Masson staining. Meanwhile, Klotho treatment significantly inhibited the decrease inE-cadherin expression in tubules, and the increase in the number of interstitial cells stained with α-SMA andKi67(a proliferation marker) and in the fibronectin expression in interstitium of the obstructed kidney,indicating that both the tubuloepithelia cell remodeling and fibroblast proliferation and activation can besuppressed by Klotho treatment. The expression level of FGF2in the obstructed kidney in Klotho-treated UUOmice was much lower than that of the control, and the activation of FRS2α and ERK1/2was significantlyattenuated.In conclusion, our results suggested that FGF2could induce tubuloepithelial cells remodeling andproliferation of fibroblast to promote renal fibrosis, which could be related to ERK1/2activation; and Klothocould inhibit renal fibrosis by completively binding FGFR with FGF2to inhibit FGF2signal. In the process ofrenal fibrosis, the expression of Klotho decreased and externally supplement of Klotho could significantlyinhibit development of renal fibrosis.