| ObjectDuring the last decades,the incidence rate of chronic renal disease has grown enormously.End-stage renal disease(ESRD)has become a serious public health problem worldwide.When the disease progress,patients require renal replacement therapy or hemodialysis for the maintenance of life.Hemodialysis can only compensate for part of the kidney function,and there are many complications related to hemodialysis.The long term survival of patients and quality of life with hemodialysis remains poor.The high cost of dialysis treatment also brings a huge economic burden to individuals and society.Kidney transplantation is the best choice for patients with end-stage kidney disease because it ensures higher quality of life and longer survival rates than other choices.However,shortage of kidney source and immune rejection after transplantation seriously restrict its wide application.In China,there are 1.2 million patients on transplant waiting lists for kidney transplantation with an increase of more than 100000 every year.Only 5000 patients have the opportunity to receive kidney transplantation,and a large number of patients suffer from pain or death while on the waiting list.Therefore,renal regeneration technology may develop new therapeutic strategies for ESRD patients.In recent years,the research on reprogramming and transdifferentiation of adult cells has developed rapidly.Somatic cells can be induced to differentiate into different human tissues or organs under certain conditions.This method avoids the ethical problems of human embryonic stem cells and the tumorigenicity induced by pluripotent stem cells in the differentiation process of i PSCs,and retains the epigenetic imprint of the original cells,so it has a good application prospect.The objective of this study was to find renal cell fate-inducing transcription factors to convert mouse and human fibroblasts into induced renal tubular epithelial cells(named mi REC and hi REC respectively).We also used the epigenetic compound library to screen for small molecules that can Simulate the renal development microenvironment and induce its further maturation.These technologies may provide important mechanistic insights into kidney development and disease,drug screening,regenerative medicine and individualized treatment.Methods1.We crossed ACTB-td Tomato,-EGFP mice and Cdh16-cre mice and obtained their embryos with Ksp-Cre;m TOM/m GFP genotype.Emx2,Pax8,HNF4a and HNF1b were transfected into mouse embryo fibroblasts(MEFs)through retroviral vectors.Quantitative real time polymerase chain reaction(q RT-PCR)and WB(Western blot)were used to verify the overexpression of genes.GFP positive cells(mi REC)were isolated by flow cytometry.2.The sorted mi RECs were cultured,enriched and identified for their phenotype,including cell morphology,colony forming ability,immunofluorescence staining of epithelial and renal specific marker genes,m RNA levels of kidney related genes,tube forming ability and morphological structure immunofluorescence staining.3.Functional verification of renal tubular epithelial cells:albumin reabsorption test,and renal toxicity drug detection of cisplatin,gentamicin and Tacrolimus.4.The epigenetic compound library was used for high-throughput screening of small molecule compounds and the detection was performed by automatic living cell monitor CBM to search for epigenetic molecular pathways that promote renal tubular epithelial transdifferentiation.5.The human CDH16 promoter sequence was cloned from the human genome,and the fluorescent reporter vector of human CDH16-promoter-GFP was constructed.The lentivirus was packaged and transfected into human renal carcinoma cell line 786-O and human embryonic renal cell line 293T for the validation of the promoter function.6.Human primary fibroblasts were obtained and constructed with CDH16-promoter-GFP fluorescence reporter gene.Renal-specific transcription factors EMX2,PAX8,HNF4αand HNF1βwere transfected into human primary fibroblasts by lentiviral vector.After the expression was verified by q PCR and WB,the fluorescence of GFP was observed,and the GFP~+cells were sorted by flow cytometry.7.The sorted hi RECs were cultured,enriched and phenotypic identified:cell morphological observation and comparison,immunofluorescence staining of epithelial and renal specific marker genes,q PCR detection of renal related gene m RNA levels,detection of cell 3D culture into tubes and immunofluorescence staining of morphological structure.Results1.The fluorescence reporting system of renal tubule transdifferentiation contained in Ksp-Cre;m TOM/m GFP mouse embryonic fibroblasts has no fluorescence leakage phenomenon.The system has good resolution and classification effect for the reprogrammed cells,which is suitable for the research tool of renal transdifferentiation in mice.2.MEFs can be transdifferentiated into mi RECs through overexpression of transcription factors EMX2,PAX8,HNF4α,and HNF1β,which have characteristic epithelial"paving stone"morphology and corresponding clonogenic ability.The m RNA levels of epithelial cell adhesion molecules such as CDH1,CDH6,and kidney-specific CDH16 were increased in mi RECs.Immunofluorescence staining showed that the tight junction molecule ZO-1,E-cadherin,and epithelial cell adhesion molecule Ep CAM were all expressed on the membrane of mi RECs.3.Except for vitamin D receptor VDR,mi RECs significantly expressed marker genes related to kidney,such as lineage factors HNF1A,PAX2,CDH16,and ion channel transporter-related genes GGT1,LPR2,and SLC families.At the same time,the interstitial related genes of mi RECs were significantly decreased.4.Immunofluorescence staining showed obvious polarity markers(ZO-1)and basolateral markers(β-catenin).However,no interstitial gene Vimentin was found in the renal tubules.5.Through albumin reabsorption experiment,we found that the fluorescence intensity of mi RECs was significantly higher than that of MEFs,indicating the endotic function of mi RECs.In the nephrotoxic drug trial,for all three nephrotoxic drugs cisplatin,gentamicin and tacrolimus,the mortality rate of mi RECs cells was significantly higher than that of MEFs cells,indicating the sensitivity of mi RECs cells to nephrotoxic drugs.6.Through high-throughput screening of epigenetic compound library,we found that small molecules with antioxidant effects,Resveratrol and 6-gingerol,could increase transdifferentiation efficiency by 3-5 times.7.Through flow detection and fluorescence microscopy,the fluorescent reporter gene CDH16promoter-GFP could be normally activated in human embryonic kidney 293T and renal carcinoma 786-O cell lines,which could help to conduct the first screening of human renal epithelial transdifferentiation.8.Human primary fibroblasts induced by transcription factors EMX2,PAX8,HNF1βand HNF4αcontained successfully transdifferentiated hi RECs,which were obtained by three criteria of CDH16 positive,Ep CAM positive and Thy-1 negative screening.9.After renal specific transcription factors were transferred into human primary fibroblasts,with the progress of induction,q PCR results showed that the expression of renal related genes increased continuously and reached the peak at 2 weeks,while the level of interstitial genes decreased gradually.After transdifferentiation of hi RECs,the expression level of renal related genes of hi RECs was similar to that of human kidney tissue,while the interstitial genes were significantly down-regulated or even silenced.10.Immunofluorescence staining showed that the tight junction molecule ZO-1,E-cadherin,and epithelial cell adhesion molecule Ep CAM were all expressed on the cell membrane of hi REC,while these epithelial genes were negative in primary fibroblasts.Just like mi REC,hi REC also forms lumen-like structures with molecular genetic markers on the epithelial surface.ConclusionSomatic cells of higher mammals still carry most of the genetic information and thus remain plasticity.We transdifferentiated mouse embryonic fibroblasts and human primary fibroblasts into renal epithelioid cells(mi RECs and hi RECs)by overexpressing renal lineage transcription factors EMX2,PAX8,HNF4α,and HNF1β.Both mi RECs and hi RECs cells have similar gene expression profiles with renal epithelial cell morphology and renal parenchymal cells,can form lumen like structures in 3D culture,and have partial renal tubule reabsorption function and sensitivity to nephrotoxic drugs in vitro.In addition,we found a class of compounds Resveratrol and 6-gingerol that can promote renal tubules transdifferentiation through high-throughput screening of epigenetic small molecule compounds.Transdifferentiated renal epithelial cells can provide a new model for the study of renal development and disease,and can be used in personalized therapy,drug screening and regenerative medicine. |
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