The Role And Mechanism Of LncRNAENSG00000254561.4 In Podocyte Injury Of Diabetic Kidney Disease

Diabetic kidney disease(DKD)is one of the most common complications of diabetes mellitus(DM)and a major cause of end-stage renal disease(ESRD),mainly characterized by proteinuria,hypertension and progressive decline in renal function.As the disease progresses,serious complications of multiple organs such as retina,heart and nerves were observed,for which there is no effective treatment at present.It is imperative to understand the exact mechanism of diabetic kidney disease and explore new therapeutic targets.It has been reported that injury and loss of podocytes are key factors in the progression of diabetic kidney disease.Glomerular epithelial cells,also known as podocytes,are terminally differentiated cells and important functional cells in the glomerulus.Loss of podocytes is an irreversible event that leads to decrease in glomerular filtration barrier function,accelerating the progression of diabetic kidney disease.Long non-coding RNA(lncRNA)is a kind of non-coding RNA(ncRNA)with a length of more than 200 nucleotides lacking protein-coding capacity,which is essential in many biological processes such as chromatin modification,transcriptional regulation,posttranscriptional regulation,cell proliferation,differentiation and apoptosis.Although more and more lncRNAs are being discovered and studied,their roles and potential mechanisms in diseases such as diabetic kidney disease remain unclear.Our previous study found that lncRNA ENSG00000254561.4 was significantly changed in diabetic kidney disease.Therefore,this study using samples from diabetic kidney disease patients and human podocytes to verify the expression changes of lncRNA ENSG00000254561.4 and its clinical significance as well as to explore the role and mechanism of lncRNA ENSG00000254561.4 in high glucose-induced podocyte injury,which will provide a new target for the early diagnosis and clinical treatment of diabetic nephropathy.Part 1:Determine the expression level and clinical significance of lncRNA ENSG00000254561.4 in diabetic kidney disease patientsObjective:To clarify the expression of lncRNA ENSG00000254561.4 in patients with diabetic kidney disease and to explore the correlation between lncRNA ENSG00000254561.4 and clinical indicators.Methods:1.The normal renal tissues near cancer of non-diabetic kidney disease patients and renal tissues of diabetic kidney disease patients diagnosed by renal biopsy were collected.①PAS,HE and Masson staining were used to assess the pathological changes of renal tissues.②Transmission electron microscope was used to observed the morphological and structural changes of renal tissues and podocytes.③Immunofluorescence and RNA-fluorescence in situ hybridization were performed to detect the expression and distribution of lncRNA ENSG00000254561.4 in renal tissues and podocytes.2.Blood samples from control group and diabetic kidney disease group were collected,then plasma samples were collected after centrifugation and stored at-80℃.The samples of control group were obtained from the Health Examination Center of Shandong Provincial Hospital and diabetic kidney disease group were collected from the Department of Nephrology of Shandong Provincial Hospital.①Real-time PCR was performed to detect the expression level of lncRNA ENSG00000254561.4 in plasma.②Statistical methods were used to analyze the correlation between the expression level of lncRNAENSG00000254561.4 in plasma and clinical indicators such as patients’age,gender,serum creatinine,blood urea nitrogen,glomerular filtration rate,blood lipid,urinary albumin creatinine ratio.Results:1.PAS,HE and Masson staining revealed that glomerular basement membrane was thickened diffusely and mesangial matrix was widened significantly in diabetic kidney disease.Transmission electron microscopy showed that the foot processes of podocyte fused and lost,and thickening of glomerular basement membrane with increased mesangial matrix.2.The results of immunofluorescence and RNA-fluorescence in situ hybridization showed that IncRNA ENSG00000254561.4 had staining overlap with podocin,a podocyte marker protein and the expression of lncRNA ENSG00000254561.4 was significantly lower in renal tissues and podocytes of patients with diabetic kidney disease compared with the control group.3.Real-time PCR results showed that lncRNA ENSG00000254561.4 was significantly reduced in plasma of diabetic kidney disease patients compared with control group.Spearman’s correlation analysis showed that lncRNA ENSG00000254561.4 expression levels were negatively correlated with cystatin,blood urea nitrogen,serum creatinine and urinary albumin creatinine ratio,and positively correlated with glomerular filtration rate.Conclusions:The expression levels of lncRNAENSG00000254561.4 were decreased in the plasma and renal tissues of patients with diabetic kidney disease and correlated with the levels of cystatin,blood urea nitrogen,serum creatinine,glomerular filtration rate and urinary albumin creatinine ratio,suggesting that lncRNA ENSG00000254561.4 is involved in the occurrence and development of diabetic kidney disease as well as played an important role.Part 2:The role and mechanism of lncRNAENSG00000254561.4 in high glucose-induced podocyte injuryObjective:To explore the expression level of lncRNA ENSG00000254561.4 in human podocyte,and clarify the role and mechanism of lncRNA ENSG00000254561.4 in podocyte injury induced by high glucose.Methods:1.Human podocytes were cultured in low-glucose,high-glucose and high-osmotic medium for 48h.①RNA-FISH assay was performed to detect the expression and localization of lncRNA ENSG00000254561.4.②Real-time PCR was used to detect RNA expression levels.2.Construct specific lncRNA ENSG00000254561.4 overexpression plasmid,divide podocytes into:LG group(low glucose stimulation),HG group(high glucose stimulation),HG+OE-NC group(high glucose stimulation+empty plasmid)and HG+OE-254561.4 group(high glucose stimulation+lncRNA ENSG00000254561.4 overexpression plasmid).①Real-time PCR was used to detect overexpression efficiency of lncRNA ENSG00000254561.4.②Western Blot,Real-time PCR and immunofluorescence were used to detect the expression levels of ZO-1,Desmin,Drp1 and MFN2.③Podocyte cytoskeleton was observed by phalloidin-FITC staining.④Transwell assay was used to detect the change of podocytes migration.⑤MitoSOX staining was used to observe the change of mitochondrial ROS expression in podocyte.⑥Mito-tracker Red staining was used to observe morphological changes of mitochondria.3.Specific lncRNA ENSG00000254561.4 knockdown plasmid was constructed and podocytes were divided into:LG group(low glucose stimulation),HG group(high glucose stimulation),HG+OE-NC group(high glucose stimulation+empty plasmid)and LG+sh-NC group(low glucose stimulation+empty plasmid)and LG+sh-254561.4 group(low glucose stimulation+lncRNAENSG00000254561.4 knockdown plasmid).①Real-time PCR was used to detect overexpression efficiency of lncRNA ENSG00000254561.4.②Western Blot,Real-time PCR and immunofluorescence were used to detect the expression levels of ZO-1,Desmin,Drp1 and MFN2.③Podocyte cytoskeleton was observed by phalloidin-FITC staining.④Transwell assay was used to detect the change of podocytes migration.⑤MitoSOX staining was used to observe the change of mitochondrial ROS expression in podocyte.⑥Mito-tracker Red staining was used to observe morphological changes of mitochondria.Results:1.LncRNA ENSG00000254561.4 was significantly down-regulated in podocytes after high glucose stimulation for 48h.The results of RNA-FISH staining showed that lncRNA ENSG00000254561.4 was distributed in both cytoplasm and nucleus of podocytes,mainly in nucleus.2.Western Blot,Real-time PCR and immunofluorescence showed that the expressions of ZO-1 and MFN2 were down-regulated and the expressions of Desmin and Drpl were upregulated after 48h of high glucose stimulation compared with the low glucose group.Podocyte cytoskeleton fracture increased and concentrated to the cell edge were observed by FITC staining,Transwell assay showed that podocyte migration ability was enhanced.Meanwhile,ROS expression increased and mitochondrial morphology was spot-like.Compared with high glucose group,overexpression of lncRNA ENSG00000254561.4 up-regulated the expressions of ZO-1 and MFN2,and down-regulated the expressions of Desmin and Drpl,podocyte skeleton fracture was reduced and evenly distributed,podocyte migration ability was decreased.The expression of ROS in podocyte mitochondria was decreased and the morphology of mitochondria was mostly rod-shaped..3.Western Blot,Real-time PCR and immunofluorescence showed that the expressions of ZO-1 and MFN2 were down-regulated and the expressions of Desmin and Drpl were upregulated after lncRNA ENSG00000254561.4 knockdown compared with the low glucose group.Podocyte cytoskeleton fracture and rearrangement were observed by FITC staining and the migration ability of podocyte was enhanced by Transwell assay.ROS expression increased and mitochondrial morphology was spot-like.Conclusion:In this study,we found that lncRNA ENSG00000254561.4 was down-regulated in high glucose-induced podocytes and lncRNA ENSG00000254561.4 could be involved in the cytoskeletal rearrangement and podocyte injury induced by high glucose through regulating mitochondrial damage,providing an experimental basis for finding new targets for the treatment and prevention of diabetic kidney disease.