The Role Of High Glucose In The Mitophagy Of Renal Tubularcells And Its Mechanism(s)

BackgroundDiabetic kidney disease (DKD) is one of the microvascular complications of diabetes, leading the major cause of end-stage renal disease (ESRD). The pathogenesis of DKD is very complicated and not fully understood. It is of great significance to study the pathogenesis of DKD. The tubular injury is the early character of ESRD, which is tightly correlated with the progression of DKD. Over production of reactive oxygen species (ROS) induced by high glucose plays a key role in the tubular injury of DKD. Mitophagy is the process of selective removing damage or dsyfunction mitochondrial by autophagy mechanism, to maintain ROS balance, which is involved in the development of various diseases, such as anima, tumor and neurodegenerative diseases, etc. Previous study suggested that autophagy was found in the tubular cells of type2diabetic mice, however, whether mitophagy exist in renal tubular under diabetic condition is still unknown.ObjectiveThe aim of study is to observe the condition of mitophagy and the expression of Nix, a regulator of mitophagy, in the tissue of DKD patients and in the tubular cells induced by high glucose, which aimed to clarify mitophagy of tubular cells in DKD and possible mechanism(s)MethodsFirst, The expressions of mitochondrial autophagy related protein LC3, p62and mitochondrial regulator protein Nix were observed in Kidney tissues of DKD patients with immunohistochemical technology. Secondly, to observe the effect of high glucose on the mitophagy and the expression of Nix, HK-2cells, a cell line of proximal tubular was inverstigated. HK-2cells was exposed to different concentrations of D-glucose (15mM、30mM) and mannitol for24hours, then total protein and RNA were extracted. The expression of LC3、p62and Nix protein were detected by western blot assay. Real Time-PCR was used to detect expression of Nix mRNA. In addition, HK-2cells were also treated with30mM glucose, with or without autophagy inhibitor3-Methyladenine (3-MA,5mmol/L)) for24hours, then co-stained with anti-LC3or anti-p62antibody, and mitotracker, a special staining for mitochondria, detected by confocal image. The effect of3-MA on the expression of LC3, p62and Nix in HK-2cells was also observed with western blot and real Time-PCR.ResultsThe level of blood glucose was higher in DKD patients, as compared to that of minimal lesion disease patients (P<0.05). There is no significant difference of serum creatinine, BUN, albumin, cholesterol, urine red blood cell counts, the amount of24hours proteins between DKD and control group (P>0.05). HE staining showed that glomerular sclerosis, basement membrane thickening, increased mesangial matrix and mesangial cells, renal tubular atrophy in the kidney biopsy tissue of DKD patients. The expression of autophagy-related protein LC3, p62and mitochondrial autophagy regulator protein Nix significantly increased in the kidney tissue of DKD patients with immunohistochemical technology. In addition, expression of LC3、p62and Nix are up-regulated in HK-2cells exposed to high glucose, which is in a dose dependent manner (P<0.05). Real-time PCR also showed increased Nix mRNA expression in high glucose group as compared with control (P<0.05). Moreover, with co-staining with LC3、p62and mitotracker, increased intensity of LC3、 p62in mitochondria were found in HK-2cells with stimulate by30mM glucose, which was inhibited with autopghy inhibitor,3-MA, On the other hand,3-MA also decreased LC3、p62and Nix expression in HK-2cells induced by high glucose with western blot assay and Real-time PCR(P<0.05), indicating that high glucose induced mitophagy of tubular cells and Nix may be involved in this process.ConclusionFirstly, The expression of autophagy-related protein LC3, p62and mitochondrial regulator protein Nix were up-regulated in the kidney tissues of DKD patients, indicating that the mitophagy may exist in the tubule of DKD patients and Nix possibly involved in this process. Secondly, high glucose could up-regulate the expression of LC3, p62and Nix, and also increased theLevel of LC3, p62expression in the mitochondria, while3-MA inhibited this effect, indicating that high glucose could induce abnormal mitophagy in the tubular cells. Thirdly, high glucose could increase the expression of Nix in the tubular cells, which was decreased with3-MA, suggesting that Nix may be contributed the mitopaghy of tubular cells induced by high glucose.