| Background:Diabetic nephropathy(DN)has become one of the most common and serious microvascular complications of diabetes,and an important factor in the death of diabetic patients.The incidence of DN is increasing continuously,causing a heavy economic burden.DN has surpassed glomerulonephritis as the leading cause of end-stage renal disease(ESRD),and there is still a lack of effective treatments.Therefore,early diagnosis and treatment can improve the quality of life and survival rate of patients.In the kidney-related damage caused by hyperglycemia,the structure and function damage and the decrease in the number of podocytes are important factors in the occurrence and progression of DN,which can cause renal dysfunction,lead to proteinuria and glomerular filtration rate(GFR)decline.Therefore,DN is also regarded as a kind of "podocytes disease".Long non-coding RNA(LncRNA)can act as regulatory elements in the cytoplasm and nucleus to play a regulatory role.LncRNA can bind with DNAs,RNAs and proteins to regulate gene expression in terms of epigenetics,transcriptional regulation and post-regulation.We measure the blood,urine and kidney tissue samples of patients with DN(determined by renal biopsy results)through high-throughput chips,and found that the expression of LncRNA EVF-2(the mouse gene evf-2)in the renal tissues of DN patients is significantly higher than that of normal control human.However,whether LncRNA evf-2 is involved in the process of inflammation and podocytes injury in the development of DN still needs further research.Methods:This study used immortalized human podocytes cells(HPC),control mice,EVF-2 knockout mice(KO)as the research objects.1.The environment of diabetic nephropathy is simulated in vitro,and HPC is divided into three groups:high glucose group:5.6mM normal glucose group(NG),high mannitol group(HM),30 mM high glucose group(HG);LncRNA evf-2 overexpression group:5.6mM normal glucose group(NG),5.6mM normal glucose+control lentivirus group(NG+CON),5.6mM normal glucose+overexpression lentivirus group(NG+OE);LncRNA evf-2 knockdown group:30 mM high glucose group(HG),30mM high glucose+control lentivirus(HG+CON),30 mM high glucose+shRNA lentivirus sequence 1(HG+shRNA1),30 mM high glucose+shRNA lentivirus sequence 2(HG+shRNA2),30 mM high glucose+shRNA lentivirus sequence 3(HG+shRNA3).2.The qRT-PCR method was used to detect the effect of high glucose,overexpression of LncRNA evf-2 and knockdown of LncRNA evf-2 on the expression of evf-2 in HPC.3.Western blot was used to detect the effects of high glucose,overexpression of LncRNA evf-2,and knockdown of LncRNA evf-2 on the expression of podocin,claudinl and inflammatory factors.4.Cellular immunofluorescence was used to detect the effect of high glucose on the morphology of HPC and the expression of podocin and claudin1.5.A mouse model of LncRNA evf-2 gene knockout(KO)was constructed to continuously monitor random blood glucose and body weight.The mouse glomerulus were isolated and primary mouse podocytes were culture in vitro.The methods of Fluorescence in situ hybridization(FISH)and qRT-PCR were used to locate and quantify the expression of glomerular LncRNA evf-2 in CON mice and KO mice respectively.6.Methods of immunofluorescence,Periodic Acid-Schiff stain(PAS)and transmission electron microscopy were used to detect the effect of LncRNA evf-2 gene knockout on the glomerular structure,the structure and number of podocytes.Western blot was used to detect the expression of podocin,claudinl and inflammatory factors after LncRNA evf-2 gene knockout compared with control groups.7.Type 1 DN mice were constructed by intraperitoneal injection of 5.5mg/ml STZ in CON mice and KO mice at a dose of 55mg/kg for 5 days,and random blood glucose and urine microalbumin/urine creatinine ratio were continuously monitored.8.Using methods of FISH and qRT-PCR to locate and quantify the glomerular expression of LncRNA evf-2 in CON mice and KO mice injected with STZ respectively.9.Immunofluorescence,PAS and transmission electron microscopy were used to observe glomerular structure,podocyte structure and number of the two groups of mice induced by STZ respectively.Western blot was performed to detect the expression of podocin,claudinl and inflammatory factors after LncRNA evf-2 gene knockout compared with control group.Results:1.Under high glucose conditions,the expression of podocin in HPC decreased,and the expression of claudinl and inflammatory factors increased,accompanied by an increase in the expression of LncRNA evf-2.The podocytes injury appeared after overexpression of LncRNA evf-2.Reducing the expression of LncRNA evf-2 under high glucose conditions can alleviate the podocyte injury caused by high glucose.2.LncRNA evf-2 gene knockout(KO)mice were constructed.Both FISH and qRT-PCR proved that LncRNA evf-2 was knocked down in podocytes of KO mice.3.The body weight and random blood glucose of KO mice were not significantly different from those of the control group,and the glomerular structure,the number of foot processes and the thickness of basement membrane were not significantly different from those of the control group.4.After the mouse podocytes were specifically knocked out of LncRNA evf-2,the expression of podocyte marker proteins Podocin,Claudinl and inflammatory factors were not significantly different from those of the control group.5.Both FISH and qRT-PCR proved that the expression of EVF-2 in KO mice was lower than that in normal control mice after induced by STZ.6.The random blood glucose of KO mice were not significantly different from those of control mice after induced by STZ,but the increase in u A/CR was lower than that of control mice.The podocyte-specific knockout of LncRNA evf-2 can alleviate STZ-induced damage of glomerular structure in mice,foot processes fusion and basement membrane thickening.7.The expression of podocin increased,while the expression of claudinl and inflammatory factors decreased in EVF-2 KO mice compared with normal control mice after induced by STZ.Conclusion:Elevated LncRNA evf-2 can cause podocyte damage in diabetic nephropathy,and podocyte-specific knockout of evf-2 can alleviate podocyte damage in diabetic nephropathy. |
