| Background:Most renal stones are calcium oxalate stones,and the formation of calcium oxalate kidney stones is closely related to Randall’s plaques(RP),which are originated from the calcium phosphate crystal deposition under the tubular basement membrane of Henle loops,and of which the formation mechanism is similar to osteogenesis.Renal interstitial fibroblasts(h RIFs)have the potential for osteogenic differentiation and are closely related to the formation of Randall’s plaques.It’s shown that Klotho(KL),an anti-aging gene is highly expressed in the kidney,especially renal tubular epithelial cells(HK-2),polymorphisms of which are associated with the risk of calcium oxalate stones.Encoded by Klotho gene,Klotho protein(α-KL),can act as an endocrine hormone involved in the regulation of mineral metabolism.The deficiency of circulating α-KL protein can induce ectopic calcification in soft tissues,such as vascular calcification in patients with chronic kidney disease.The Wnt/β-catenin signaling pathway is known to play an important role in osteogenic differentiation,and the soluble α-KL proteins in circulation can block this signaling pathway to inhibit vascular calcification due to osteogenic differentiation of vascular smooth muscle cells.Therefore,we speculate that α-KL protein secreted by HK-2 cells may participate in the regulation of the osteogenic differentiation process of h RIFs,and ultimately affect the formation of Randall’s plaques and calcium oxalate kidney stones,via affecting the activity of Wnt/β-catenin signaling pathway.To verify this speculation,we performed the following three-part experiments.Part 1 The relationship between α-KL protein and RPPurpose: Exploring the role of α-KL protein in the formation of RP by determining the expression of α-KL protein in RP.Methods: In the collected samples of RP,the m RNA transcript of KL gene was measured by RT-q PCR and the protein expression of α-KL and osteogenic markers(Runx2,MSX2,OCN)was measured by WB and IHC.In the urine samples collected from patients with calcium oxalate kidney stones,the urinary α-KL-to-creatinine ratio(UKCR)was measured by ELISA.Results: In RP,both the m RNA transcript of KL gene and the protein expression of α-KL were down-regulated,while the protein expression osteogenic markers(Runx2,MSX2,OCN)was up-regulated.The mean value of UKCR in patients with calcium oxalate kidney stones was lower than that in the health controls,but here were no statistically significant differences.Conclusion: Down-regulation of α-KL protein is a pathological feature of RP,and the α-KL protein might be associated with the progression of renal stromal biological mineralization leading to RP formation.Part 2 Roles of α-KL protein in the osteogenic differentiation of h RIFs and HK-2 cellsPurpose: To verify the osteogenic potential of h RIFs and HK-2 cells,and to explore the roles of α-KL protein in the osteogenic differentiation of h RIFs and HK-2 cells,as well as the effect of HK-2 cells on h RIFs.Methods:(1)In vitro via induction with osteogenic medium,the calcium deposits were measured by alizarin red staining and the protein expression of α-KL and osteogenic markers(Runx2,MSX2,OCN)was measured by WB,to verify the osteogenic potential of h RIFs.(2)The effects of endogenous and exogenous α-KL proteins on the osteogenic differentiation of h RIFs was explored by silencing or overexpression of KL gene and co-culture of recombinant human KL protein(r-KL).(3)The effect of α-KL protein on the osteogenic differentiation of h RIFs in vivo was explored by subcutaneous implantation of h RIFs with overexpressing KL gene in the back of nude mice.(4)In vitro via induction with osteogenic medium,the calcium deposits were measured by alizarin red staining and the protein expression of α-KL and osteogenic markers(Runx2,MSX2,OCN)was measured by WB,to verify the osteogenic potential of HK-2 cells.(5)The effect of α-KL protein secreted by HK-2 cells on osteogenic differentiation of h RIFs was explored by co-culture of h RIFs with HK-2cells of which the expression of KL gene is normal,excessive or silent.Results:(1)In osteogenic induction of h RIFs,the calcium deposits were increased obviously,and the protein expression of α-KL were downregulated,while the protein expression osteogenic markers(Runx2,MSX2,OCN)was up-regulated.(2)In vitro,both overexpression of KL and co-culture of r-KL protein inhibited the osteogenic phenotype of h RIFs,and the silence of KL enhanced the osteogenic phenotype,but the enhancement could be antagonized by the r-KL protein.(3)In vivo,overexpression of KL can down-regulate the levels of collagen fiber in h RIFs.(4)In osteogenic induction of HK-2 cells,the calcium deposits were increased,and the protein expression of α-KL were down-regulated,while the protein expression osteogenic markers(MSX2,OCN)was up-regulated.However,the osteogenic phenotype in HK-2 cells was much lower than that in h RIFs.(5)Co-culture with normal HK-2 cells inhibited the osteogenic phenotype in h RIFs,and co-culture with KL overexpressing HK-2 cells could enhance the inhibition due to up-regulated secretion of α-KL protein.Co-culture with KL silencing HK-2 cells relieved the osteogenic inhibition due to down-regulated secretion of α-KL protein.Conclusion: Both endogenous and exogenous α-KL proteins could inhibit osteogenic differentiation of h RIFs,and the deficiency of endogenous α-KL protein could be rescued by exogenous one.The potential for osteogenic differentiation of h RIFs was significantly stronger than that of HK-2 cells,but α-KL protein secreted by HK-2 cells could inhibit osteogenic differentiation of h RIFs.Part 3 Molecular mechanism of HK-2 cells inhibiting the osteogenic differentiation of h RIFsPurpose: To explore the effect of α-KL protein secreted by HK-2 cells on Wnt/β-catenin signaling pathway in h RIFs,leading to the regulation of osteogenic differentiation of h RIFs.Methods:(1)The effect of exogenous α-KL protein on the Wnt/β-catenin signaling in h RIFs was explored by co-culture of h RIFs cells respectively with r-KL protein and HK-2 cells.(2)The expression of all Wnt ligands was measured by RT-q PCR and WB to explore which Wnt ligand might affect the osteogenic differentiation of h RIFs,and later,which Wnt ligand possibly was bound to α-KL protein in h RIFs were explored by Co-IP and multiplex immunofluorescence.(3)To explore the effects of α-KL protein binding to Wnt 2 ligand on the Wnt/β-catenin signaling pathway and osteogenic differentiation of h RIFs cells via the co-culture of Wnt2 overexpressing h RIFs with r-KL protein.(4)After co-culture of h RIFs with r-KL protein,the expression levels of Wnt ligand inhibitors DKK1,SOST,SFRP1 and SFRP4 were determined by RT-q PCR and WB,to explore which Wnt ligand inhibitor might be affected by exogenous α-KL protein.(5)The effects of SFRP1/SFRP4 on Wnt/β-catenin signaling pathway and osteogenic differentiation of h RIFs were explored via the SFRP1/SFRP4 gene silencing by si RNA.(6)The effects of α-KL protein on SFRP1,Wnt/β-catenin signaling pathway and osteogenic differentiation of h RIFs were explored via coculture of SFRP1 silencing h RIFs with r-KL protein.Results:(1)Both exogenous r-KL protein and α-KL protein secreted by HK-2 cells could up-regulate the phosphorylation of β-catenin protein in h RIFs.(2)In osteogenic differentiation of h RIFs,Wnt 2,Wnt 9 a and Wnt 5were up-regulated,in which Wnt 2 ligand could bind to α-KL protein and was colocalized in the cytoplasm.(3)Overexpression of Wnt 2 ligand could downregulate the phosphorylation of β-catenin protein and enhance the osteogenic phenotype of h RIFs and supplementation of r-KL protein could antagonize the overexpression of Wnt ligand,up-regulate the phosphorylation of β-catenin protein and inhibit the osteogenic phenotype.(4)In osteogenesis induction of h RIFs,SFRP1 and SFRP4 were affected by r-KL protein while SOST and DKK1 were not affected.(5)Silent expression of SFRP1 down-regulated the phosphorylation of β-catenin protein and enhanced the osteogenic phenotype,while SFRP4 had no obvious effect on β-catenin protein phosphorylation or osteogenic phenotype.(6)Silent expression of SFRP1 could antagonize r-KL protein,upregulate the phosphorylation of β-catenin protein,and relieve the osteogenic inhibition of h RIFs.Conclusion: The α-KL protein secreted by HK-2 cells,could block the Wnt/β-catenin signaling pathway and then inhibit osteogenic differentiation of h RIFs cell,not only by directly binding of Wnt 2 ligand but also by up-regulating the Wnt inhibitor SFRP1.Conclusion & Outlook: The deficiency α-KL protein was identified as a pathological feature of Randall’s plaque,HK-2 cells can inhibit the osteogenic differentiation of h RIFs cells by secreting α-KL proteins to directly bind Wnt ligands as well as up-regulating SFRP1,to block the Wnt/β-catenin signaling pathway.In this study,not only the role of α-KL protein in renal interstitial biomineralization was confirmed,but also the interaction between HK-2 cells and h RIFs was revealed,which provided new insights into the mechanism of Randall’s plaque formation. |
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