A-054 Autolysosome formation involves a quality control mechanism of mitochondria and lysosomes in the renal cortex during the normoalbuminuric stage of diabetes mellitus

Abstract Background Oxidative stress during the normoalbuminuric stage of type 1 diabetes mellitus (DM) damages renal cortical mitochondria, which subsequently are degraded by mitochondria-selective autophagy (mitophagy) involving lysosomes. Any lysosomes damaged in DM are degraded by lysosome-selective autophagy (lysophagy) to allow mitophagy to reach fruition. Oxidative stress-related mitochondrial and lysosomal quality control mechanisms exist in the renal cortex during the normoalbuminuric stage of diabetes. Fusion of autophagosomes with lysosomes is the main reaction inducing autophagy and is important for autolysosome formation. This study aimed to determine if oxidative stress in DM triggers renal cortical autolysosome formation Methods Four groups of rats (n=5 per group) received one of the following treatments: 1) STZ group: rats with DM induced by streptozotocin injection (STZ, 65 mg/kg, i.p.); 2) Sham group: rats receiving the STZ vehicle; 3) STZ+TLM group: STZ rats treated with telmisartan (TLM, an angiotensin receptor blocker; 10 mg/kg/day in chow); and 4) Sham+TLM group: TLM-treated Sham rats. Two weeks later, blood glucose, blood pressure, glomerular filtration rate (GFR), and urinary excretion of albumin and N-acetyl-ß-D-glucosaminidase (NAG) were measured. The oxidative stress marker, 3-nitrotyrosine (3-NT), was detected by HPLC. Western blot was used to quantify proteins related to mitophagy (PINK1, BNIP3, LC3-II, p62), lysosomal and lysophagy (LAMP2, FBXO27). Proteins related to inducing autolysosome formation (syntaxin 17, Stx17) and inhibiting its formation (rubicon) were measured by Western blot analysis and localized by immunohistochemistry. Results Blood glucose levels were higher in STZ rats than in Sham rats (P<0.05) and were unaffected by TLM. Blood pressure, albumin excretion, and NAG excretion were unaltered by STZ or TLM. GFR and renal cortical 3-NT levels were increased in STZ rats (P<0.05), with both changes prevented by TLM (P<0.05). STZ rats had increased renal cortical LC3-II, PINK1, and FBXO27 protein levels compared with Sham (P<0.05), with these effects prevented by TLM (P<0.05). BNIP3, p62 and LAMP2 levels did not differ among groups. Stx17 and rubicon protein levels in the renal cortex did not differ among the 4 groups. However, immunoreactivity for the 2 proteins was evident in proximal tubules, most prominently in STZ rats. At least in the case of Stx17, TLM appeared to blunt proximal tubular immunoreactivity. Conclusions: During the normoalbuminuric stage of DM, renal cortical autolysosome formation was most prominent in the proximal tubule. This effect was blunted by TLM in association with its antioxidant effect, suggesting mitophagy and lysophagy-related proximal tubular protection mechanism triggered by oxidative damage. Furthermore, it was suggested that Stx17 and rubicon regulation is important for the onset and progression of diabetic nephropathy.