High Glucose Conditions Induce Epithelial-to-Mesenchymal Transition In Tubular Epithelial Cells:A Preliminary Study For Mechanism Of Calcium Oxalate Nephronlithiasis Formation

Objective:Metabolic factors are considered to be the main cause of most kidney stone disease and diabetes is a common metabolic disease with the higher risk of nephrolithiasis.In spite of the increased proportion of uric acid calculi in diabetes,overall the most common stone composition is still calcium oxalate.The reason for increased incidence of calcium oxalate nephrolithiasis while diabetes remains unclear.It is speculated that EMT might promote the onset of calcium oxalate kidney stone disease in some way.Our study has made preliminarily exploration in terms of these assumption.Methods:1.We prepared COM crystals,then labelled the crystals with FITC-conjugated Ig G.The crystal–cell interaction(adhesion and endocytosis)was observed by fluorescence microscopy,and quantitative analysis was made by flow cytometry.2.We proceed the cytology research of high glucose induced EMT on HK2 cells in vitro.The cells were divided into four groups: Low glucose control group;High glucose inducing EMT group;High glucose + metformin treatment group;Low glucose + TGFβ inducing EMT positive group.After the cells were incubated in these four different conditions,we observed cell morphology changes by inverted phase contrast microscope and detected EMT-related markers by Western Blot in order to verify cellular functional changes of EMT.3.We verifed that EMT enhanced adhesive capability and endocytosis of HK2 cells to COM crystals.Annexin V-FITC/PI analysis and quantitative analysis of cell proportion which had internalized COM crystals were carried out by flow cytometry.In zoological experiments we used STZ to establish renal EMT model in rats with diabetic nephropathy.Calcium oxalate kidney stones were induced by ethylene glycol method in different experimental groups.The promotion of kidney stone formation and the inhibitory effect of metformin were confirmed by imaging observation of kidney stone morphology and Western Blot detection of EMT-related markers.Results:1.COM crystals labeled with FITC-conjugated Ig G presented green monoclinic prismatic shape by fluorescence microscopy.The crystal-cell interaction was observed by fluorescence microscopy.It was observed by scanning electron microscopy that COM crystals were adhered to the cell surface by organic cord.And quantitative analysis could be made by flow cytometry for the HK2 cells which had internalized FITC fluorescence labeling COM crystals.2.Every group of HK2 cells presented different morphological changes by phase contrast microscopy.Low glucose group and metformin group presented cobblestone-like epithelial appearance.Some cells in high glucose group presented spindle-like fibroblastic shape.And most cells in TGFβ group presented spindle-like fibroblastic shape.Western Blot analysis showed that the expression of the cell epithelial marker(E-cadherin)increased and the expression of the mesenchymal markers(Vimentin and Snail1)decreased in low glucose group and the metformin group,but on the contrary in high glucose group and TGFβ group.3.The Annexin V-FITC/PI flow cytometry data of HK2 cells in different groups showed that high glucose and TGFβ could lead to an increase in the number of HK2 cells prone to death(apoptosis and necrosis),and the cell proportion was significantly higher than that of the low glucose group and the metformin group.Compared with TGFβ group,there was no significant difference in the proportion of apoptotic cells in high glucose group,and the proportion of necrotic cells in high glucose group was lower than that in TGFβ group.Flow cytometry quantitative analysis for HK2 cells which had internalized COM crystals in different groups showed that M2 value in high glucose group and TGFβ group were both higher than those in low glucose group and the metformin group.Zoological experiments confirmed that the incidence of kidney stones in EMT model rats was significantly higher than that in the control group and metformin treatment group.Conclusions:1.We labeled COM crystals with simple and highly efficient fluorescence labeling technology,and observed the interactions between COM crystals and HK2 cells by phase contrast microscopy and fluorescence microscopy.Flow cytometry was used to quantify the cell proportion of which had internalized COM crystals effectively.2.High glucose can induce EMT in tubular epithelial cells in a variety of ways.In this process,tubular cells undergo a series of morphological and functional changes.Drugs which can inhibit the process of EMT may be potential effective for the treatment of diabetic nephropathy.3.During the process of high-glucose-induced tubular cell EMT,ROS may play an important role.Tubular cells with a tendency of apoptosis or necrosis may enhance their adhesive capability and endocytosis to COM crystals,thereby promoting the pathogenesis of calcium oxalate kidney stones.Metformin can resist the role of EMT in promoting stone formation.