| BackgroundRenal fibrosis,including glomerulosclerosis and tubulointerstitial fibrosis,is a common feature of chronic kidney disease.Many pathological factors,such as chronic persistent infection,hypoxia,poorly controlled hypertension and diabetes,can lead to progressive and irreversible renal fibrosis.Among them,diabetic nephropathy(DN)is mainly characterized by glomerulosclerosis,and its pathogenesis is not yet fully clear.Hyperglycemia,genetic background,abnormal hemodynamics and other factors can cause metabolic changes and abnormal vasoactive substances,which can not accurately analyze the occurrence and development of renal fibrosis.Therefore,actively exploring the pathogenesis of renal fibrosis and developing new therapeutic strategies accordingly have been the goal and direction of researchers,in which hypoxia is considered to be a key factor.Recent studies have highlighted intrarenal hypoxia as a potential mechanism for chronic kidney disease.In this study,we successfully constructed a three-dimensional(3D)hypoxic microenvironment in vitro using single cell layer-by-layer assembly(LBL)technology to study the role of miR-382 and autophagy in renal fibrosis.Objective1.Construction of mesangial cells 3D hypoxic microenvironment culture system based on single-cell LBL technology platform.2.To determine the effect of hypoxic microenvironment on miR-382 and related signaling pathways in mesangial cells.3.To clarify the mechanism of renal fibrosis induced by miR-382.Contents and methods1.A 3D hypoxic microenvironment culture system of mesangial cells was constructed by single-cell layer-by-layer assembly(LBL)tissue engineering.2.The effects of hypoxic microenvironment on the expression of miR-382 in mesangial cells and related signaling pathways were studied by qRT-PCR and immunofluorescence analysis in 2D and 3D cultures.3.Immunofluorescence and Western blotting were used to compare the effects of hypoxic stimulation on mesangial cell fibrosis in 2D and 3D cultures,and the role of miR-382 in renal fibrosis.4.Statistical analysis.The results of statistical analysis using SPSS20.0 statistical software were expressed with mean standard deviation(Mean±SD).A single factor analysis of variance(One-way ANOVA)was used in the comparison between the groups.The Tukey method was used to compare the difference between groups.When the variance was not equal,the Dunnett,s T3 method was used between the groups.P<0.05 thought there was a significant difference.Results1.3D hypoxic microenvironment culture system of mesangial cells was successfully constructed by single-cell LBL tissue engineering and simple trypsin digestion.2.Compared to 2D culture system,3D hypoxic microenvironment culture system induced high expression of transforming growth factor-beta 1(TGF-β1)and miR-382,which resulted in glomerulosclerosis by down-regulating the expression of phosphatase and tensin homologous gene(PTEN)deleted on chromosome ten.3.Cell autophagy was detected in 3D cells(cells in 3D spheroid)and 3DM cells(cells migrated from the 3D spheroid).Unlike 3D cells,the expression of TGF-β1 and fibronectin(Fn)decreased in 3DM cells,which demonstrated the renoprotective role of autophagy in renal fibrosis.4.The proportion of CD24 + cells increased in 3DM cells.We speculate that some characteristics of progenitor cells may be acquired by dedifferentiation of cells surviving autophagy.ConclusionsIn this paper,HA-EDA with positive charge was successfully synthesized by modification of hyaluronic acid(HA)and crosslinking of ethylenediamine(EDA)and EDC.We used positive charged HA-EDA and negative charged HA to form ultrathin films on the surface of a single Renal Mesangial cell(RMC)through LBL.These single cells assembled layer by layer were treated with trypsin to form 3D RMCs multicellular spheres.Relevant experimental results show that we have successfully constructed a 3D hypoxic microenvironment irn vitro by using this LBL/trypsin treatment method.At the same time,we found that the hypoxic environment in 3D spheroid induced the high expression of TGF-pl and miR-382 in the RMCs,and then subsequently resulted in glomerulosclerosis by down-regulating the expression of PTEN.This LBL assembly spheroid provides us with a flexible and practical method to construct hypoxic microenvironment to study the effect and mechanism of hypoxia on renal fibrosis.Based on the 3D RMCs spheroid we developed,we were also surprised to discover that some cells survived through autophagy and hypoxic microenvironment experienced dedifferentiation and acquired some progenitor cell characteristics. |
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