The Role Of LncRNA AL139280.1 In Podocyte Injury Of Diabetic Nephropathy

Part 1:Verify the expression and clinical significance of lncRNA AL139280.1 in patients with diabetic nephropathyObjective:To verify the expression of AL139280.1 in patients with diabetic nephropathy,to explore the role of AL139280.1 in the production of proteinuria and the correlation between AL139280.1 and clinical indicators.Methods:1.Collection of human kidney tissue specimens:collect kidney tissue specimens from normal control group and diabetic nephropathy group.Among them,the kidney tissue specimens of the normal control group were derived from the normal kidney tissue surrounding the tumorous tissues of non-diabetic patients with renal tumors,and the diabetic nephropathy group was derived from renal tissue specimens of diabetic nephropathy patients diagnosed by renal biopsy.All specimens are obtained from Shandong Provincial Hospital Affiliated to Shandong University.(1)RNA fluorescence in situ hybridization(RNA-FISH)labeled AL139280.1 and immunofluorescence staining labeled podocin,to observe the expression and distribution of AL139280.1 in kidney tissues.(2)HE,PAS,Masson staining of kidney tissue to observe the pathological changes of kidney tissues.(3)Transmission electron microscope to observe the ultrastructure of podocytes.2.Collection of human plasma samples:blood samples of normal control group and blood samples of patients with diabetic nephropathy were collected and centrifugated to obtain plasma samples.All specimens are obtained from Shandong Provincial Hospital Affiliated to Shandong University.(1)Real-time PCR to detect the expression of AL139280.1 in plasma.(2)Collect systolic blood pressure,diastolic blood pressure,glycosylated hemoglobin,blood albumin,blood urea nitrogen,blood creatinine,blood uric acid,blood lipids,ACR,and glomerular filtration rate of normal healthy controls and patients with diabetic nephropathy,and observe the correlation between the relative expression level of plasma AL139280.1 and clinical indicators.Results:1.RNA-FISH results showed that the expression of AL139280.1 was significantly increased in the glomeruli of patients with diabetic nephropathy.Co-staining with the podocyte marker protein Podocin showed that AL139280.1 co-localizes with podocytes,and the expression of AL139280.1 in podocytes was up-regulated when podocytes were injured.2.HE,PAS,Masson staining showed that glomerular basement membrane was thickened,and mesangial matrix was increased in diabetic nephropathy.3.Transmission electron microscopy results showed that the glomerular basement membrane was thickened,the mesangial matrix was increased,and the podocyte foot processes were fused.4.The expression of AL139280.1 in the plasma of patients with diabetic nephropathy was significantly up-regulated,and the expression of AL139280.1 in plasma of patients with macroalbuminuria was significantly higher than that of patients with microalbuminuria.Spearman’s correlation analysis showed that the relative expression level of plasma AL139280.1 was correlated with ACR(rs=0.546,P<0.001).Conclusions:The expression of LncRNA AL139280.1 is significantly up-regulated in kidney tissues and plasma of patients with diabetic nephropathy,and its expression is positively correlated with ACR.It may lead to renal damage of diabetic nephropathy by damaging podocytes.Part 2:The role of AL139280.1 in podocyte injury induced by high glucoseObjective:To explore whether AL139280.1 is involved in podocyte injury induced by high glucose stimulation,and the role of AL 139280.1 in podocyte injury induced by high glucose stimulation.Methods:1.The immortalized human podocytes were divided into:low glucose group(LG 5.5 mmol/L glucose),high glucose group(HG:30 mmol/L glucose),hypertonic control group(HO:5.5 mmol/L glucose+24.5 mmol/L Mannitol),after 48 hours,real-time PCR was used to detect the expression of AL139280.1;RNA-FISH was used to detect the expression and location of AL 139280.1;real-time PCR,Western Blot and immunofluorescence staining were used to detect the expression and distribution of podocyte injury markers Desmin and ZO-1;phalloidin-FITC staining was used to observe the changes of the podocyte cytoskeleton.2.Construct AL 139280.1 overexpression and knockdown vector,and further divide the podocytes into:low glucose group(LG),high glucose group(HG),high glucose+knockdown control group(HG+si-NC),high glucose+ AL139280.1 knockdown group(HG+si-AL139280.1),low glucose+empty carrier group(LG+OE-NC),low glucose+AL 139280.1 overexpression group(LG+OE-AL139280.1).Real-time PCR was used to detect the overexpression and knockdown efficiency of AL139280.1;real-time PCR,Western Blot,and immunofluorescence staining were used to detect the expression and distribution of podocyte injury markers Desmin and ZO-1;phalloidin-FITC staining was used to observe the changes of the podocyte cytoskeleton.Result:1.After 48 h of high-glucose culture of immortalized human podocytes,the expression of AL139280.1 was significantly up-regulated(P<0.05).The results of RNA-FISH showed that AL139280.1 was predominantly distributed in the nucleus.2.After culturing immortalized human podocytes for 48 h under high glucose,real-time PCR,Western Blot and immunofluorescence staining showed that the expression of podocyte injury marker Desmin was significantly up-regulated,and ZO-1 was significantly down-regulated(P<0.05);phalloidin-FITC staining showed that high glucose leads to cytoskeleton rearrangement of podocyte.3.Real-time PCR,Western Blot and immunofluorescence results showed that knocking down AL139180.1 reversed the up-regulation of Desmin and down-regulation of ZO-1 induced by high glucose,alleviating podocyte injury,while overexpression of AL139280.1 promoted the occurrence of podocyte injury.Phalloidin-FITC staining showed that knocking down AL139280.1 inhibited cytoskeleton rearrangement of podocyte under high glucose conditions,while overexpression of AL139280.1 promoted cytoskeleton rearrangement.Conclusions:LncRNA AL 139280.1 was up-regulated in podocytes induced by high glucose,and regulation of AL139280.1 could affect podocytes injury and cytoskeleton rearrangement induced by high glucose,suggesting that AL139280.1 played an important role in podocyte injury of diabetic nephropathy.