Role And Mechanism Of MGLL In Diabetic Nephropathy Induced By Lipotoxicity

Objective:Diabetic nephropathy（DN）is is the most common cause of chronic kidney disease（CKD）,renal tubular injury is a key factor in the progression of diabetic nephropathy,proximal tubular cell is the core site of renal lipid metabolism,and lipid metabolism imbalance is easy to cause lipid deposition,but the underlying molecular mechanism has not been elucidated.Monoacylglycerol lipase（mgll）is the rate limiting enzyme of lipolysis,which can regulate the level of free fatty acids and participate in a variety of pathogenic signaling pathways.The aim of this study was to investigate the effect of lipotoxicity on renal tubular injury in diabetic nephropathy mice and its possible mechanism.Methods:1.db/db mice and db/m mice were used as animal models.Blood lipid and urine biochemical levels were detected.Lipid deposition in renal tissue was observed by oil red O staining and immunofluorescence staining.HE,Masson and Sirius red staining were used to observe the morphological changes and evaluate the renal fibrosis.Immunohistochemistry and Western blot were used to detect the expression of key factors in renal tissue.RNA sequencing was used to determine the transcriptome gene level of mouse kidney.Bioinformatics methods were used to analyze the differentially expressed genes.Go and KEGG enrichment analysis were used to find the candidate genes and the biological processes and signaling pathways involved.RT-PCR and Western blot were used to verify the expression level of candidate genes.The specific staining of candidate genes was detected by immunohistochemistry.2.Human proximal renal tubular epithelial cells（HK-2）were used as cell model and they were divided into five groups:normal group（NC）,mannitol group（MA）,high glucose group（HG,25m M）,high palmitic acid group（HP,0.25m M）and high glucose and high palmitic acid group（HG+HP）.Lipid deposition was observed by oil red O staining and immunofluorescence staining.The accumulation of reactive oxygen species was detected.Western blot was used to detect lipid metabolism,oxidative stress,apoptosis and fibrosis related protein expression.After down-regulation and up-regulation of Mgll in vitro,lipid deposition was observed and lipid metabolism,oxidative stress,apoptosis and fibrosis related protein expression were detected.3.Mgll knockout animal model was established and they were divided into three groups:diabetic group（db/db）,diabetic+fenofibrate intervention group（db/db+Feno）and diabetic+MGLL gene knockout group(db/db+Sh-MGLL.The diabetic control group was given normal saline by gavage,the db/db+Feno group was given fenofibrate 30mg/kg/d by gavage,and the db/db+Sh-MGLL group was given tail vein injection of virus to inhibit Mgll.HE and Sirius red staining were used to observe the morphological changes the kidney in the mice.Lipid deposition was observed by oil red O staining and immunofluorescence staining.RT-PCR and Western blot were used to detect the expression of MGLL and lipid metabolism related factors.4.The clinical population was divided into normal control group（NDM）,diabetes mellitus group（DM）and diabetic nephropathy group（DN）.Blood lipid and urinary biochemical levels were detected.The content of MGLL in urine was determined by ELISA,and the relationship between MGLL and ACR,NAG,β₂MG.Finally,db/db mice of 8 weeks,10 weeks and 12 weeks old were used as animal models to detect the biochemical level of urine,the level of MGLL in urine and kidney tissue,and the lipid deposition in kidney.Results:1.The levels of blood glucose,body weight,urinary ACR,renal tubular injury markers（NAG andβ2MG）and biochemical indexes of lipid metabolism（TG and FFA）were significantly increased.In db/db group,there was obvious lipid deposition in the proximal tubular epithelial cells,and the renal tubule dilation,hypertrophy and fibrosis injury were increased.Meanwhile,the expression of fibrogenic factor FN and COL IV protein in the kidney of diabetic mice was significantly increased.The results of RNA Seq sequencing and analysis and in vivo verification confirmed that MGLLwas up-regulated in the kidney of db/db mice,and MGLL was specifically expressed in the renal tubules.Compared with db/m mice,The expression level of renal key factors of fatty acidβoxidation（PPARα,ACSL and CPT1A）was significantly decreased,while the expression of key factors of fatty acid uptake（CD36 and FABP4）was significantly increased.2.In HK-2 cells,MGLL expression was significantly up-regulated after high glucose and high fat induction,lipid deposition was increased in HK-2 cells,the expression level of renal key factors of fatty acidβoxidation was significantly decreased,while the expression of key factors of fatty acid uptake was significantly increased,the expression of oxidative stress markers（NOX4 and SOD2）,apoptosis markers（BAX,BCL-2 and Cleaved caspase3）and fibrosis markers（FN and COL IV）were significantly changed.Compared with HG+si-Ctl,transient transfection of si-mgll significantly reduced the lipid deposition in HK-2 cells,the expression level of renal key factors of fatty acidβoxidation was significantly increased,while the expression of key factors of fatty acid uptake was significantly decreased,the levels of oxidative stress,apoptosis and fibrosis were significantly decreased.In contrast,compared with vector,MGLL overexpression increased lipid deposition in HK-2 cells,the expression level of renal key factors of fatty acidβoxidation was significantly decreased,while the expression of key factors of fatty acid uptake was significantly increased,the levels of oxidative stress,apoptosis and fibrosis were significantly increased.3.Compared with db/db group and db/db+Feno group,the expression of MGLL protein in MGLL silenced db/db mice was significantly decreased.Compared with db/db group,ACR,NAG,β2MG,TG and FFA were significantly decreased,the lipid deposition in kidney were decreased,the expression level of renal key factors of fatty acidβoxidation was significantly increased in db/db+Feno group and db/db+sh-MGLL group.4.Compared with NDM group and DM group,the level of urinary MGLL in DN group was significantly higher.The level of urinary MGLL in patients with DN and urinary ACR,NAG andβ2MG was positively correlated.NAG andβ2MG in 10 and 12 weeks old diabetic mice were increased,ACR in 12weeks old db/db mice was increased.MGLL in urine of db/db mice began to increase at 10 weeks,and the excretion level of MGLL in urine gradually increased with the increase of age,accompanied by the increase of lipid deposition in kidney tissue.Conclusion:In diabetic environment,up-regulated MGLLaggravates lipid deposition by inhibiting PPARα/fatty acidβoxidative signaling pathway,which leads to renal tubular injury in diabetes mellitus.