Human Umbilical Cord Mesenchymal Stem Cells Cultured In Decellularized Kidney Scaffold Attenuate Renal Fibrosis By Reducing Epithelial-mesenchymal Transition Via The TGF-β/Smad Signaling Pathway

Objective: To optimize the preparation of decellularized kidney scaffold(DKS),and to explore the effect of the hucMSCs cultured in DKSs on renal fibrosis and its underlying mechanism.Methods: 1.The kidneys of SD rats were perfused with SDS(S group),Triton X-100 combined with SDS(TS group),and Triton X-100 combined with SDS after repeated freeze and thawing(FTS group)in different flow velocity.He staining,DAPI staining and DNA quantitation were used to detect the degree of decellularization.Masson staining,PAS staining and immunohistochemical staining were used to detect the preservation of main components and structural integrity.Scanning electron microscopy(SEM)was used to detect the ultrastructure,MTT assay was used to detect the cytotoxicity,and ELISA assay was used to detect the content of growth factors.2.hucMSCs were perfused through renal artery and ureter to recellularize the DKSs.HE staining was used to detect the distribution of hucMSCs,and immunofluorescence staining was used to detect the expression of PCNA.SD rats were randomly divided into 4 groups to operate sham surgical incision(Sham group),subtotal(5/6th)nephrectomy(STN group),transplant the DKS on STN rats(DKST group),and transplant the hucMSC-recellularized kidney scaffold(RKS)on STN rats(RKST group).After 8 weeks,serum creatinine(SCr)and blood urea nitrogen(BUN)were quantified,HE staining and Masson staining were used to detect inflammation and renal fibrosis,immunofluorescence staining was used to detect the epithelial-mesenchymal transition(EMT),and western blot was used to detect the expression of TGF-β/Smad signaling pathway related proteins,such as TGF-β1,p-smad2/3,Smad7,and EMT markers,E-cadhein and α-SMA.Results: 1.The decellularization time of FTS group was shorter.HE and DAPI staining showed there were no cells residing.The content of DNA was lower than 50 ng/mg.Masson and PAS staining showed the collagen and polysaccharides in ECM.Immunohistochemical staining showed the expression of Collagen Ⅰ,Collagen Ⅳ,Fibronectin and Laminin.SEM showed the DKS was smooth and honeycomb-shaped.The cytotoxicity of DKS was level 1.ELISA assay showed that the content of VEGF,EGF,IGF-1 and PDGF-BB in FTS group was significantly higher than that in S group and TS group.2.HE staining showed that there was cellular distribution in glomerulus and tubular areas of the RKSs,and the tubular-like structure was formed.Immunofluorescence staining showed PCNA expression of some hucMSCs.Compared with STN group,We found that SCr and BUN in RKST group were decreased,inflammation and fibrosis were reduced,expression of E-cadherin was increased,expression of α-SMA was decreased,the co-expression of E-cadherin and α-SMA was decreased,and the expression of TGF-β1,p-smad2/3 was decreased,expression of Smad7 was increased.There were no significant differences in renal function,renal fibrosis,EMT and expression of TGF-β/Smad signaling pathway between DKST and STN groups.Conclusion: 1.Freeze-thawing combined with perfusion can prepare the DKSs with a lower detergent concentration and a higher efficiency.The DKSs have no cells.Collagen,polysaccharides and major proteins in ECM can be retained without obvious cytotoxicity.The structure is honeycomb-shaped and more cytokines in FTS group are retained.It provides favorable conditions for cells.2.hucMSCs cultured in DKSs can attenuate renal fibrosis by reducing EMT via TGF-β/Smad signaling pathway and hucMSCs may play a key role.