Effect of Sirt1 on high glucose-induced exosome release from podocytes

Abstract

Objective To investigate the effect of nicotinamide adenine dinucleotide-dependent deacetylase 1 (Sirt1) on high glucose-induced exosome release from podocytes. Methods Immortalized mouse podocytes MPC5 were divided into six groups: normal glucose group (5.5 mmol/L glucose, group A), high mannitol group (5.5 mmol/L glucose+24.5 mmol/L mannitol, group B), high glucose group (30.0 mmol/L glucose, group C), high glucose+Sirt1-overexpressed lentivirus transfection group (Sirt1-overexpressed lentivirus transfection+30.0 mmol/L glucose, group D), high glucose+negative lentivirus transfection group (negative lentivirus transfection+30.0 mmol/L glucose, group E), and high glucose+exosome secretion inhibitor group (GW4869+30.0 mmol/L glucose, group F). Western blot was used to analyze the expression levels of Nephrin, Podocin, Sirt1, CD63, CD81, and Alix in each group. Real-time quantitative polymerase chain reaction was used to analyze the expression level of Sirt1 mRNA in D and E group. The morphology of podocyte exosomes was observed by a transmission electron microscope. The particle size and concentration of exosomes were determined by nanoparticle tracking analysis. Results The results of RT-qPCR showed that the relative expression of Sirt1 mRNA was significantly increased in group D compared with that in group E (t=14.580,P<0.01). The results of nanoparticle tracking analysis and Western blot showed that the relative expression of Sirt1, Nephrin, and Podocin proteins in podocytes among groups A to C was significantly different (F=49.84-106.40,P<0.01). Compared with group A, group C had significantly increased secretion of podocyte exosomes (t=14.550,P<0.01) and significantly reduced expression of Sirt1, Nephrin, and Podocin (t=7.446-15.110,P<0.01). Compared with group E, group D had significantly reduced release of podocyte exosomes (t=74.610,P<0.01) and significantly increased relative expression of Sirt1, Nephrin, and Podocin (t=4.657-32.860,P<0.05). Compared with group C, group F had significantly reduced release of podocyte exosomes (t=16.300,P<0.05) and significantly increased relative expression of Nephrin and Podocin (t=3.790,8.151,P<0.01), but showed no significant change in the expression level of Sirt1 (P>0.05). Conclusion Loss of Sirt1 in high glucose-treated podocytes promotes exosome secretion and podocyte injury.

Keywords

• diabetic nephropathies|blood glucose|podocytes|exosomes|sirtuin 1