| ObjectivesTo investigate the expression and mechanism of Metrnl in the renal tissues of diabetic kidney disease(DKD)patients and diabetes mellitus(DM)mice.To elucidate Metrnl effects on the imbalance of mitochondrial dynamics and lipid deposition in renal tubular epithelial cells,which provides an experimental basis for Metrnl as a target to treat DKD.Methods1.To observe the expression of Metrnl in the kidney of DKD patients and DM mice:(1)Metrnl protein and m RNA expression were detected in each tissue of C57BL/6 mice by WB and PCR;(2)The expression of Metrnl m RNA in proximal renal tubules of db/db mice captured by laser capture microdissection technique(LCM)was detected by q PCR;(3)The morphological changes of kidney in db/db mice and HFD/STZ combined-induced DM mice were observed by HE staining,the expression of Metrnl in the kidney tissues of DM mice and DKD patients were detected by IHC staining;and the Metrnl expression in kidney tissues and serum of DM mice was detected by WB,q PCR and ELISA.2.To explore the relationship between Metrnl and lipid metabolism in renal tubular epithelial cells:(1)Metrnl protein expression in NRK-52 E cells stimulated with different concentrations of palmitic acid(PA)and glucose for 48 h was detected by WB;(2)NRK-52 E cells treated with PA or PA+Metrnl recombinant protein(r Metrnl,200ng/m L)were performed RNA-Seq KEGG analysis;(3)The correlation analysis between Metrnl expression levels in renal tissues and serum creatinine,and triglyceride levels of db/db mice and DKD patients were performed;(4)Acorrding to GO and KEGG analysis of RNA-seq data in proximal renal tubular captured by LCM in db/db mice,genes expression of adipogenesis(Scd1),the fatty acid β-oxidation(PGC-1α,Cpt1α),oxidative phosphorylation(Atp5b,Ndufa10)were detected by q PCR;(5)In vitro,we constructed lentivirus-mediated over-expression Metrnl(OE-Metrnl)and short-hairpin-RNA Metrnl(sh-Metrnl)stable cells;in vivo,db/db mice were injected r Metnrl(3mg/kg)and AAV-Metrnl via tail vein,and renal in situ injection of AAV-Metrnl to prepare DM mouse models overexpressing Metrnl;the effects of Metrnl on lipid droplet protein Perilipin-2 and lipid droplet deposition were examined by IF,IHC,WB,q PCR,Oil-red and Nile red staining.The kidney tissues of db/db mice treated with r Metrnl were analyzed by lipidomics to quantify lipid species,and TG content in kidney tissues was determined.3.To investigate the relationship between Metrnl and mitochondrial function in renal tubular epithelial cells:(1)Mitochondrial morphological changes were observed by Mito tracker staining;mitochondrial membrane potential changes were observed by JC-1 staining;(2)The expression of proteins related to mitochondrial kinetic homeostasis(Drp1,Mfn1)and fatty acid metabolism(ACOX1,CPT1a)in the kidney tissues and cells of each group were detected by WB;(3)genes related to mitochondrial production(PGC-1α,TFAM,mt DNA/n DNA),β-oxidation(PPAR,CPT1-a,Acadl,PGC-1α),oxidative phosphorylation(ATP5b,Ndufa10)in OE-Mernl cells and kidney tissues were analyzed by q PCR.(4)Oxygen consumption rate(OCR)and ATP production were detected by sea-horse in OE-Metrnl NRK-52 E cells.4.To elucidate the molecular mechanisms by which Metrnl affects lipid deposition in the kidney:(1)Protein expression of Sirtuins,PGC-1α,UCP1,Perilipin-2,Mfn1,and Drp1 were detected by WB in the kidney tissues of DM mice and NRK-52 E cells;(2)The expression of Sirt3 in the kidney of DKD patients was detected by IHC staining and the correlation between Sirt3 and Metrnl was analysed;(3)In OE-Metrnl NRK-52 E cells with Sirt3 inhibition,lipid droplet deposition was observed by Oil red O staining,and the protein levels of Perilipin-2,UCP1,Mfn1,and Drp1 were detected by WB.(4)The effect of PGC-1α binding to the promoter region of Sirt3 was performed by Ch IP-q PCR.5.To observe the effect of Metrnl on renal injury in DM:(1)In vivo,the renal pathological changes in DM mice treated with r Metnrl,AAV-Metrnl and renal in situ injection of AAV-Metrnl were observed by HE and PAS staining;(2)The levels of blood urea nitrogen(BUN),serum creatinine(Scr),serum insulin and urine protein contents were measured with kits Results:1.Metrnl expression is gradually down-regulated with the progression of DKD.(1)Metrnl was widely expressed in tissues,especially in heart,liver,kidney and muscle;the m RNA level of Metrnl was significantly down-regulated in the proximal tubules of the kidney in db/db mice.(2)Metrnl significantly decreased in the proximal tubules of DKD patients with different pathological grades;focal or even lager tubular atrophy was observed in the kidney of db/db mice and HFD/STZ-induced DM mice,where the protein level of Metrnl was significantly reduced by IHC.Metrnl protein,m RNA,and serum are significantly reduced in the kidney tissues;in addition,Metrnl expression was significantly reduced in renal tubular epithelial cells treated with PA(0.25 m M).2.Metrnl reduces the lipid deposition in renal tubular epithelial cells under DM condition.(1)Metrnl related with mitochondria-related signaling pathways including fatty acid β-oxidation,oxidative phosphorylation,thermogenesis,and AMPK;The protein levels of Metrnl in the kidneys of DM mice and DKD patients are significant negative correlation with serum creatinine and triglyceride levels.Meanwhile,the genes related to oxidative phosphorylation,fatty acid β-oxidation,and thermogenic processes were differentially expressed in the proximal renal tubules of db/db mice.The expression of adipogenesis related gene Scd1 was significantly increased,the fatty acidβ-oxidation(PGC-1α,Cpt1α),and oxidative phosphorylation(Atp5b,Ndufa10)related genes were significantly down-regulated.(2)Perilipin-2 was mainly expressed in the renal tubules,and Perilipin-2 was gradually increased with disease progression in DKD patients.Large amount of lipid droplet deposition were observed in the renal tubular epithelium of DM mice,and Perilipin-2 protein expression was significantly upregulated in DM mice.However,Metrnl intervention reduced lipid droplet deposition,significantly decreased the protein level of Perilipin-2 and TG content in kidney tissues.3.Metrnl maintains mitochondrial dynamics imbalances and ameliorates lipid deposition.(1)Metrnl regulates the proteins levels of mitochondrial membranes,such as significantly elevating the expression of cardiolipin(CL),increasing the species of phosphatidylethanolamine(PE),and decreasing the expression of sphingomyelin(SM);Also,Metrnl significantly up-regulated the expression of genes about fatty acidβ-oxidation(Ppara,CPT1-a,Acadl,PGC-1α)and oxidative phosphorylation(ATP5b,Ndufa10).(2)Metrnl intervention can reverse the elevated protein levels of mitochondrial splitting protein(Drp1)and diglyceride(DG),improved the expression of mitochondrial fusion protein(Mfn1)and fatty acid metabolism-related proteins(ACOX1,CPT1a),also elevated the expression of mitochondrial nascent genes(PGC-1α and TFAM).Metrnl increased the ratio of mitochondrial DNA to nuclear DNA(mt DNA/n DNA),improved PA-induced mitochondrial overdivision and membrane potential,increased oxygen consumption rate(OCR),and promoted ATP production.4.Metrnl maintains mitochondrial dynamics homeostasis via PGC-1α-Sirt3,while upregulating UCP1 promotes fatty acid thermogenesis,thereby attenuates ectopic deposition of lipids in the renal tubules.(1)The protein level of Sirt3 in renal tissues of DKD patients was gradually decreased with disease progression,and mainly expressed in renal tubules;While Metrnl significantly upregulated Sirt3 expression and showed a significant positive correlation with Metrnl;Meanwhile,inhibition the expression of Sirt3 could partially block the regulatory effect of Metrnl on mitochondrial dynamics and lipid metabolism.(2)The protein level of UCP1 was significantly reduced in the kidney of DM mice,and UCP1 intervention significantly reduced lipid droplet deposition and the protein level of Perilipin-2;OE-Metrnl significantly elevated the protein of UCP1,while Sirt3 inhibition could partially block Metrnl’s regulatory of UCP1 and Perilipin-2.(3)Metrnl positively regulated the expression of PGC-1α,and PGC-1α could bind to the promoter region of Sirt3,while OE-Metrnl significantly increased this binding.5.Metrnl has a positive protective effect on renal injury in DM mice.Cell proliferation in the glomerular thylakoid region,basement membrane thickening and tubular injury were improved in db/db mice after Metrnl intervention.The serum insulin levels were significantly up-regulated,and blood urea nitrogen(BUN),serum creatinine(Scr)and urinary protein levels were significantly reduced.Conclusions:1.Metrnl is mainly expressed in renal tubular epithelial cells,and Metrnl expression significantly negatively correlated with the progression of DKD,which is an important regulatory factor of renal tubular injury caused by ectopic lipid deposition in DKD.2.Metrnl maintains mitochondrial function in renal tubules through PGC-1α-Sirt3 axis,also up-regulates the expression of coupled protein 1(UCP1)to promote pyregenesis,which reduces the lipid accumulation in renal tubules,improve renal tubular injury in DM and alleviate renal dysfunction and delays the progression of DKD. |
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