Fig. 6

Mitophagy and autophagic flux were improved following losartan therapy in HK-2 cells exposed to a high-glucose environment. A and B: HK-2 cells were exposed for 24 h to low glucose (LG, 5 mM), high glucose (HG, 30 mM), or losartan (HG, 30 mM + LST, 1 µM). In LG, mitochondria appeared as thin threads, but in HG, they transformed into short rods. HG treatment reduced the co localization intensities of LC3 (green) and Mitotracker Red. This effect was partially reversed by LST. Co staining with SQSTM1 (green) and Mitotracker Red showed the opposite outcome. C and D: Numerical evaluation showed a slight initial increase in autophagy, followed by a significant decrease after 6 h under high glucose (HG) conditions. Mitochondrial fragmentation due to HG was time-dependent. A partial reversal of HG-induced mitophagy and fragmented mitochondrial structure was achieved in HK-2 cell when losartan was administered. E: LC3II/I, Atg5, SQSTM1, TOMM20, Beclin, and VDAC1 protein expression analyzed by western-blot. F: After a 48-hour transfection with mCherry-GFP-LC3B adenovirus, the HK-2 cell then treated with LG, HG, or HG + LST for 24 h, and HG + CQ (40 µM) for 4 h. Using a laser confocal microscopy, GFP appeared green, mCherry red, autophagosome yellow, and autolysosome red (due to GFP quenching in acidic pH). Nuclei were stained blue with DAPI. Each experiment was repeated three times with at least 10 microscopy fields analyzed per experiment. The images show outcomes of three different experiments. Scale bar: 5 μm. Outcomes are showed as mean ± SE, *P < 0.05 vs. LG subset, #P < 0.05 vs. HG subset