CD133 Expression Characteristics And Its Protective Potential In Proximal Tubular Cells Of Diabetic Kidney Disease

BackgroundDiabetic kidney disease(DKD)is the most common microvascular complication of diabetes and the main cause of end-stage renal disease.Many risk factors are associated with the development of DKD,and hyperglycemia is considered to be the most important risk factor.Hyperglycemia causes metabolic and hemodynamic alterations,and participates in the pathological process of DKD at multiple cellular levels.The high demands of proximal renal tubular cells(PTCs)for energy and aerobic metabolism make them highly susceptible to diabetic disorders.Therefore,PTCs are considered as the driving force and key therapeutic target of DKD.Hyperglycemia could induce cell apoptosis and epithelial-mesenchymal transition(EMT)of PTCs,eventually leading to renal fibrosis.However,PTCs have strong regenerative proliferative potential.In acute kidney injury,some residual PTCs undergo dedifferentiation and re-differentiate into mature PTCs during renal repair,and promote restoration of renal function.These dedifferentiated PTCs usually express progenitor cell markers such as CD 133 and CD24.In DKD,PTCs undergo EMT which make PTCs lose the epithelial phenotype and regain the mesenchymal phenotype.Previous studies have shown that PTCs expressing CD 133 in acute kidney injury also expressed mesenchymal markers such as vimentin,while PTCs in chronic kidney injury also re-expressed the progenitor marker PAX2 and other kidney development related genes.The dedifferentiation behavior of PTCs between acute and chronic kidney disease has a certain commonality.CD 133 is a single-chain transmembrane glycoprotein expressed in immature cells.In the kidney,renal progenitor cells are characterized by the expression of CD 133,and its expression is considered to be a marker of renal repair.In non-malignant kidney pathology,CD 133 itself has physiological functions of promoting proliferation and inhibiting aging.At present,most of the studies on the dedifferentiation of PTCs and the expression of CD 133 after dedifferentiation focus on acute kidney injury,and there is no report in DKD.Aims1.Single-cell RNA sequencing data analysis was used to reveal the expression of CD 133 in different kidney cells of DKD,and DKD rat models with different disease courses were constructed to explore the expression characteristics of CD 133 in PTCs.2.Exploring the relationship between the dedifferentiation behavior of PTCs characterized by the expression of CD133 and high glucose-induced EMT.3.Clarifying the molecular function of CD133 under the intervention of high glucose by regulating the expression of CD 133,and the Nephroseq database was used to analyze the clinical significance of CD 133 in DKD.Methods1.Data mining analysis of human early DKD single-cell RNA sequencing.The raw data of human DKD single-cell sequencing was downloaded from the GEO database,and the"Seurat" software package of R was used to define the kidney cell population,and analyze the expression of CD 133.Then we used the pearson test to analyze the genes positively correlated with CD 133.Enrichment analysis was used to predict the protein functions of CD133.2.Construction of DKD rat models with different disease course.In order to construct DKD at different stages of disease at 4 weeks,8 weeks and 12 weeks,male SD rats aged 6-7 weeks were selected for unilateral right nephrectomy combined with streptozocin(STZ)injection at a dose of 45 mg/kg.The DKD rats were divided into three groups in each disease course:sham operation(Sham)group,unilateral nephrectomy(Unx)group,and DKD group.Body weight,blood glucose,urine protein,and urine microalbumin were recorded every two weeks.Rats were sacrificed at the specified time,and the pathological changes of DKD in different stages of the disease were detected by PAS staining,blood urea nitrogen(BUN)and serum creatinine(Scr).3.Analysis of CD133 expression characteristics.Western blot and immunohistochemistry were used to detect the change of CD 133 expression in PTCs with the progression of DKD.Immunofluorescence of CD133 and AQP1,PCNA,vimentin,CD24 and KIM-1 was used to verify the expression characteristic and localization of CD 133 in PTCs.4.Mechanism of high glucose(HG)-induced expression of progenitor cells and mesenchymal markers in PTCs.The proximal renal tubular cell lines(human HK-2 cells and rat NRK-52E cells)were induced by HG for 48 h,and the expressions of progenitor cell markers CD133,PAX2 and the mesenchymal markers vimentin,snaill and the epithelial markers CK-18,ZO-1,claudin-2 were detected by western blot and RT-qPCR.The co-expression of CD24,CK-18,vimentin and PCNA with CD 133 was verified by immunofluorescence.Furthermore,the number of CD 133 and CD24 co-expressing cells induced by HG was confirmed by flow cytometry.In addition,TGF-β and Methacycline hydrochloride(Met)was used to induce and inhibit the EMT process.Experiments were divided into 5 groups:normal glucose(NG),NG+TGF-β(10 ng/mL),NG+TGF-β+Met(10 μm),high glucose(HG),HG+Met group.The above indicators were verified by western blot,RT-qPCR and flow cytometry to confirm whether the differentiation process of PTCs expressing CD 133 induced by HG is mediated by EMT.5.Knockdown and overexpression of CD133.The CD133 siRNA and CD133 overexpression plasmids were transferred into HK-2 cells.RT-qPCR was used to detect the knockdown and overexpression efficiency.Western blot was used to detection the expression of CD 133,vimentin,PCNA,P53,and the apoptosis proteins caspase-3,cleaved caspase-3 and BAX.Cell proliferation was detected by EdU assay and real-time cell proliferation monitoring system,and cell apoptosis was detected by flow cytometry.6.Analysis of the clinical significance of CD 133 expression in DKD from Nephroseq database.Relevant studies on transcriptome sequencing of renal tissue of human DKD patients were retrieved from Nephroseq database.Raw data and corresponding clinical data of patients were obtained for statistical analysis.Results1.The results of single-cell sequencing data mining analysis showed that the expression of CD 133 increased in human DKD kidney tissue,especially in the PTC cluster,and there was a large differential expression between the control group and DKD group in PTCs.Enrichment analysis predicted that CD 133 was associated with the biological process of cell proliferation,apoptosis and cell phenotype maintenance.In addition,the gene characteristics and functions of CD 133-positive cells in PTCs was altered in the DKD group.2.The results of DKD rats at different stages of disease showed that the expression of CD 133 in the PTCs of DKD increased significantly compared with the Sham group and the Unx group,and the expression gradually increased with the progression of DKD.The immunofluorescence results of CD133/CD24/KIM-1 indicated that PTCs expressing CD 133 may present a state of dedifferentiated cell.With the progression of DKD,the expression of vimentin is positively correlated with CD133,and CD133-positive cells in PTCs have a vimentin-indicated mesenchymal phenotype.Furthermore,CD 133 was also co-expressed with the proliferation marker PCNA.3.HG could induce the increased expression of progenitor cell markers CD133,PAX2,and mesenchymal markers vimentin and snail 1 in HK-2 cells and NRK-52E cells,and down-regulated the expression of epithelial markers CK-18,ZO-1 and claudin-2,indicating that HG could induce cells undergoing a dedifferentiated process in which progenitor and mesenchymal markers coexist.Activation of the canonical EMT process did not upregulate CD 133 expression,and inhibition of HG-induced EMT did not significantly alter CD 133 expression.4.CD 133 knockdown significantly inhibited the proliferation of HK-2 cells and the expression of PCNA,whereas the expression of vimentin was not significantly changed.The expression of P53 was further increased compared with the HG group.Flow cytometry results indicated that down-regulation of CD 133 could significantly increase cell apoptosis,and the expressions of apoptosis proteins caspase-3,cleaved caspase-3 and BAX were further up-regulated compared with the HG group.Overexpression of CD 133 had no effect on cell proliferation,but could alleviate the apoptosis of HK-2 cells induced by HG.5.Nephroseq database mining analysis showed that the expression of CD 133 in the tubulointerstitium of DKD was significantly upregulated than that in the glomerulus;compared with other types of chronic kidney disease,the expression level of CD 133 increased most significantly in the tubulointerstitium of DKD,and its expression level was negatively correlated with glomerular filtration rate(GFR).Conclusions:1.The expression of CD133 is increased in PTCs in DKD.PTCs expressing CD133 were in a damaged dedifferentiated cell state.This dedifferentiation mechanism was not mediated by the classical EMT pathway,but a unique dedifferentiation mechanism under high glucose intervention.2.The expression of CD 133 in PTCs under high glucose intervention is a self-protective response of cells.CD 133 has a certain anti-injury function.However,with the progression of the disease,persistent high expression of CD 133 may be involved in the pathological process of DKD.Therefore,the role of CD 133 in PTCs in different stages of DKD has two sides.