The Mechanism Of LncRNA AK044604 Inducing Podocytes Injury In Diabetic Nephropathy By Sirt1/GSK3β

Diabetic Nephropathy(DN)is a serious public health problem in the world and the incidence rate and mortality is increasing worldwide,especially the incidence rate of DN in China has become the main cause of chronic kidney disease(CKD),and about 40%of patients have developed to End Stage Renal Disease(ESRD)eventually.Therefore,in the context of global diabetic chronic kidney disease in China and the world,it is urgent to explore the pathogenesis of DN and new targets for diagnosis and treatment.DN is widely regarded as a "podocyte disease",which is characterized by the imbalance and dysfunction of podocyte homeostasis after the damage of glomerular filtration barrier,which further leads to proteinuria and progressive renal failure.Podocyte autophagy homeostasis plays a key protective role in maintaining podocyte function,thus podocyte autophagy is the key to decipher the mechanism of DN.Long-noncoding RNA(lncRNA)has been proved to play a key role in the pathophysiology of DN and participate in diabetic glomerulopathy and diabetic renal tubular disease.lnCRNA AK044604 is a newly discovered transcript with the function of Sirtl regulating insulin sensitivity and autophagy,also known as Risa(Regulator of insulin sensitivity and autophagy),and vivo and vitro experiments have confirmed that Risa could influence insulin function via Akt/GSK3β autophagy pathway.However,the regulatory role of Risa has not been reported in DN.Silent information regulator 2 proteins(Sirtl)and glycogen synthase kinase 3β(GSK3β)both have the biological function of regulating autophagy.In high glucosestimulated podocyte and diabetic nephropathy mouse model,the expression and activity of Sirt1 was decreased,and activation of GSK3β was increased,which lead to podocyte injury through impaired downstream autophagy flux of Beclin-1 and LC3B.However,the regulation function of Sirtl/GSK3β in the course of podocyte injury in DN has not been reported.Therefore,in this subject,we cultured immortalized mouse podocyte model in vitro and raised the diabetic nephropathy db/db mouse model in vivo,and used qRT-PCR,double luciferase reporter gene detection,Western blotting,immunofluorescence and other experimental methods to explore the mechanism of Risa/Sirt1/GSK3β in podocyte injury of DN.Part1 The mechanism of IncRNAAK044604 inducing podocyte injury by Sirtl/GSK3β in high glucose-cultured podocytesObjectiveTo explore the mechanism of lncRNA AK044604 inducing podocytes injury by Sirtl/GSK3β in high glucose-cultured immortalized mouse podocyte model.MethodsThe differentiated and mature immortalized mouse podocytes were divided into the following groups:Ⅰ)Normal glucose concentration group(NG);Ⅱ)High mannitol group(HM);Ⅲ)High glucose group(HG);Ⅳ)High glucose+lithium chloride group(HG+LiCI);Ⅵ)High glucose+lncRNA AK044604-shRNA group(HG+Risa-LV);Ⅵ)High glucose+lncRNA AK044604 empty vehicle group(HG+Risa-vehicle);Ⅶ)High glucose+lncRNA AK044604-LV+Sirt1 inhibitor EX-527 group(HG+Risa-LV+EX-527);Ⅷ)High glucose+lncRNAAK044604-shRNA+GSK3β overexpression lentivirus group(HG+Risa-LV+GSK3β-LV).1.Immortalized mouse podocytes were cultured and transfected with lncRNA AK044604 knockdown lentivirus.The transfection effects at different times and different viral transfection amounts were observed to explore the optimal conditions for lncRNA AK044604 knockdown lentivirus infection of podocytes.2.Western blotting and immunofluorescence were used to detect the expression levels of autophagy factors(p-GSK3βSer9,Beclin-1,LC3B and p62)and podocyte marker proteins(WT-1,Podocin and Desmin)in high glucose-stimulated podocytes under different time gradients,and explore the effect of stimulation time by high glucose on podocyte autophagy.3.The expression level of lncRNA AK044604 in each group was detected by qRT-PCR to verify the IncRNA AK044604 overexpression and knockdown podocyte models.4.The miRNAs bounding with IncRNAAK044604 and GSK3β were detected by double luciferase reporter gene.5.The changes of autophagy related indicators(p-Sirt1Ser27,p-GSK3βSer9,Beclin-1,LC3B and p62;autophagosome)and podocyte marker proteins(Nephrin,WT-1,Podocin and Desmin)were detected in lncRNA AK044604 overexpression and knockdown podocyte models by Western blotting,immunofluorescence and electron microscope.6.The levels of autophagy and podocyte marker proteins were detected in lncRNA AK044604 knockdown podocytes using Sirtl inhibitor EX-527.7.The autophagy function and expression level of podocyte marker were detected in lncRNA AK044604 knockdown podocytes using GSK3β overexpression lentivirus.8.The levels of autophagy and podocyte marker were detected in high glucosestimulated podocytes using LiCl(GSK3β inhibitor).Results1.The optimal condition for IncRNA AK044604 knockdown lentivirus infection of immortalized mouse podocytes was MOI 50,72 hours.2.At 0,12,24,48,72 and 96 h of differentiated and mature MPCs by high glucose stimulation,the expression levels of autophagy factors(p-GSK3βSer9,Beclin-1,LC3B and LC3B Ⅱ/Ⅰ ratio)and podocyte marker proteins(WT-1 and Podocin)were decreased gradually with the extension of high glucose stimulation time,and significantly decreased after 24 hours,while the opposite trend was showed for p62 and Desmin,indicating the characteristics of time-dependent reduction in podocyte autophagy by high glucose stimulation.3.The expression of lncRNA AK044604 in HG group was significantly higher than that in NG group and HM group,and the expression of lncRNA AK044604 in HG+Risa-LV group was significantly lower than that in HG group and HG+Risa-vehicle group.Risa overexpression and knockdown cell models were successfully constructed.4.The results of double luciferase reporter gene showed that mmu-miR-6380/mmu-miR-706/mmu-miR-1195/mmu-miR-3098-5p could significantly bind to lncRNA AK044604,but the miRNAs could not bind to GSK3β and regulate the expression of GSK3β.5.The expression levels of autophagy related indicators(p-sirtlSer27,p-GSK3βSer9,Beclin-1,LC3B and LC3B Ⅱ/Ⅰ ratio,number of autophagosome)and podocyte marker proteins(Nephrin,WT-1 and Podocin)were significantly lower in HG group than those in NG group and HM group.After using Risa-shRNA lentivirus,the expression levels of autophagy proteins,podocyte marker proteins and the number of autophagosomes were significantly improved.6.The expression levels of autophagy factors(p-sirtlSer27,p-GSK3βSer9,Beclin-1,LC3B and LC3B Ⅱ/Ⅰ ratio)and podocyte marker proteins(WT-1 and Podocin)were significantly lower than those in NG group,HM group and HG+Risa-LV group,while the opposite trend was observed for p62 and Desmin.7.The expression levels of autophagy factors(p-GSK3βSer9 and Beclin-1)and podocyte marker protein(Podocin)in HG+Risa-LV+GSK3β-LV group were significantly lower than those in NG group,HM group and HG+Risa-LV group,while the opposite trend was observed for p62 and Desmin.8.The expression levels of autophagy factors(p-GSK3βSer9,LC3B and LC3B Ⅱ/Ⅰratio)and podocyte marker protein(Podocin)in HG+LiCl group were significantly higher than those in HG group and HG+Risa-vehicle group.Conclusions1.In high glucose-cultivated podocytes,the expression of IncRNA AK044604 is up-regulated,which down-regulates the phosphorylation level of Sirtl and GSK3βand in turn attenuates autophagy and leads to podocyte damage.2.Knockdown of lncRNA AK044604 can significantly improve the phosphorylation level of Sirtl and GSK3β and alleviate podocyte autophagy injury caused by high glucose.3.EX-527 targeted inhibition of Sirtl can significantly decrease the phosphorylation level of GSK3β and aggravate podocyte autophagy damage caused by high glucose.4.Overexpression of GSK3β cause podocyte injury in diabetic nephropathy by attenuating autophagy.5.Inhibition of GSK3β can improve the podocyte autophagy injury caused by high glucose to some extent.Part2 The mechanism of lncRNA AK044604 inducing podocyte injury by Sirt1/GSK3β in diabetic nephropathy db/db miceObjectiveTo explore the mechanism of lncRNA AK044604 inducing podocyte injury and proteinuria by Sirtl/GSK3β in diabetic nephropathy db/db mice model.MethodsDiabetic nephropathy db/db mice were fed and randomly divided into db/db group,db/db+Risa-vehicle group,db/db+Risa-AAV group,db/db+Risa-AAV+EX-527 group and db/db+LiCl group.db/m mice were matched as healthy control group at the same age.The blood,urine and kidney tissue samples of mice were collected at different observation points,and the relevant indexes were detected by qRT-PCR,Western blotting,immunofluorescence,PAS staining and electron microscope.1.db/db mice were injected with Risa knockdown adeno-associated virus through caudal vein,and the expression of lncRNA AK044604 in the mice glomerulus in each group was detected by qRT-PCR to verify the success of lncRNA AK044604 overexpression and knockdown animal models.2.The changes of autophagy indexes(autophagy protein;number of autophagosome)and podocyte related indexes(podocyte marker protein;basement membrane thickness)and vivo parameters of mice(urinary ACR,blood creatinine.etc)were detected in the glomerulus of lncRNA AK044604 overexpression and knockdown db/db mice.3.The expression levels of autophagy protein and podocyte marker protein were detected in the glomerulus of lncRNA AK044604 knockdown db/db mice intraperitoneally injected with Sirtl inhibitor of EX-527.4.The expression levels of p-GSK3βSer9,LC3B and Podocin were detected in the glomerulus of db/db mice intraperitoneally injected with LiCl.Results1.The expression of lnRNA AK044604 in the mice glomerulus of db/db group was significantly higher than that of db/m group,and the expression of lncRNA AK044604 in db/db+Risa-AAV group was significantly lower than that of db/db group and db/db+/Risa-vehicle group.Risa overexpression and knockdown animal models were successfully constructed.2.The expression levels of autophagy related factors(p-Sirt1Ser27,p-GSK3βSer9,Beclin-1,LC3B,LC3B Ⅱ/Ⅰ ratio and number of autophagosome),podocyte marker proteins(Nephrin,WT-1 and Podocin)in the mice glomerulus of db/db group were significantly lower than those in db/m group,but the basement membrane thickness,urinary ACR and serum creatinine were significantly higher than those in db/m group.12 weeks after tail vein injection of Risa-shRNA adeno-associated virus,the expression levels of autophagy protein and podocyte marker protein and the number of autophagosomes were significantly improved,the podocyte basement membrane were repaired,and the levels of urinary ACR and blood creatinine were reduced.3.The expression levels of autophagy factors p-Sirt1Ser27,p-GSK3βSer9,LC3B and podocyte marker protein Podocin in the mice glomerulus of db/db+Risa-AAV+EX-527 group were significantly lower than those in db/m group and db/db+Risa-AAV group.4.The expression levels of autophagy factors p-GSK3βSer9,LC3B and podocyte marker protein Podocin in the mice glomerulus of db/db+LiCl group were significantly higher than those in db/db group.Conclusions1.In diabetic nephropathy db/db mice,the expression of lncRNA AK044604 is significantly up-regulated,which negatively reduces the phosphorylation level of Sirt1 and GSK3β and attenuates autophagy,further resulting in podocyte injury,proteinuria and kidney damage.2.Knockdown of lncRNA AK044604 can improve the activity of Sirtl and GSK3β,and alleviate podocyte autophagy damage,proteinuria and kidney damage.3.LncRNA AK044604 knockdown db/db mice intraperitoneally injected with EX-527 can inhibit the phosphorylation level of GSK3β,and aggravate the damage of podocyte autophagy in diabetic nephropathy.4.db/db mice intraperitoneally injected with LiCl can restore the level of podocyte autophagy and improve podocyte damage in diabetic nephropathy to some extent.Full-text Conclusions:1.Under the condition of diabetes,the expression of lncRNA AK044604 is significantly up-regulated,which significantly reduces the phosphorylation level of Sirt1 and GSK3β and attenuates autophagy,further resulting in podocyte injury,proteinuria and kidney damage.2.Knockdown of lncRNA AK044604 can improve the activity of Sirtl and GSK3β,and enhance autophagy,so as to exert the protective effect of podocytes and improve renal function.3.EX-527 targeted inhibition of Sirtl can down-regulate the phosphorylation level of GSK3β and aggravate the autophagy damage of podocytes in diabetic nephropathy;GSK3β inhibitor LiCl can restore the level of podocyte autophagy and improve podocyte damage in diabetic nephropathy to some extent.4.LncRNAAK044604 attenuates autophagy by Sirt1/GSK3β pathway and leads to podocyte damage in DN.