The mitochondria-targeted antioxidant MitoQ ameliorated tubular injury mediated by mitophagy in diabetic kidney disease via Nrf2/PINK1 - PubMed (original) (raw)
doi: 10.1016/j.redox.2016.12.022. Epub 2016 Dec 21.
Xiaoxuan Xu 2, Fan Zhang 2, Ming Wang 2, Yan Xu 2, Dan Tang 2, Jiahui Wang 2, Yan Qin 2, Yu Liu 2, Chengyuan Tang 2, Liyu He 2, Anna Greka 3, Zhiguang Zhou 4, Fuyou Liu 2, Zheng Dong 5, Lin Sun 2
Affiliations
- PMID: 28033563
- PMCID: PMC5196243
- DOI: 10.1016/j.redox.2016.12.022
The mitochondria-targeted antioxidant MitoQ ameliorated tubular injury mediated by mitophagy in diabetic kidney disease via Nrf2/PINK1
Li Xiao et al. Redox Biol. 2017 Apr.
Abstract
Mitochondria play a crucial role in tubular injury in diabetic kidney disease (DKD). MitoQ is a mitochondria-targeted antioxidant that exerts protective effects in diabetic mice, but the mechanism underlying these effects is not clear. We demonstrated that mitochondrial abnormalities, such as defective mitophagy, mitochondrial reactive oxygen species (ROS) overexpression and mitochondrial fragmentation, occurred in the tubular cells of db/db mice, accompanied by reduced PINK and Parkin expression and increased apoptosis. These changes were partially reversed following an intraperitoneal injection of mitoQ. High glucose (HG) also induces deficient mitophagy, mitochondrial dysfunction and apoptosis in HK-2 cells, changes that were reversed by mitoQ. Moreover, mitoQ restored the expression, activity and translocation of HG-induced NF-E2-related factor 2 (Nrf2) and inhibited the expression of Kelch-like ECH-associated protein (Keap1), as well as the interaction between Nrf2 and Keap1. The reduced PINK and Parkin expression noted in HK-2 cells subjected to HG exposure was partially restored by mitoQ. This effect was abolished by Nrf2 siRNA and augmented by Keap1 siRNA. Transfection with Nrf2 siRNA or PINK siRNA in HK-2 cells exposed to HG conditions partially blocked the effects of mitoQ on mitophagy and tubular damage. These results suggest that mitoQ exerts beneficial effects on tubular injury in DKD via mitophagy and that mitochondrial quality control is mediated by Nrf2/PINK.
Keywords: Diabetic kidney disease; Mitophagy; Mitoq; Tubular.
Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
Figures
Fig. 1
Effect of mitoQ on renal functional and morphological characteristics in db/db mice. A: Body weight changes in db/m, db/db and db/db mice receiving mitoQ treatment for 8–16 weeks. B: Blood glucose concentrations in each group. C: Serum creatinine levels. D: Urinary ACRs. E and F: Urinary excretion of 8-OHdG and urine β-NAG levels. G: PAS staining (top panels) and EM (bottom panels) showing notable deformations in the tubules and glomeruli of db/db mice compared to those of db/m mice (Gb vs. Ga and Ge vs. Gd). These changes were dramatically ameliorated by mitoQ administration (Gc vs. Gb and Gf vs. Ge). H and I: Quantitative analysis of mesangial scores and tubular damage in each group. Values are the mean±SE, *P<0.05 vs. db/m; #P<0.05 vs. db/db mice. n=6.
Fig. 2
Restoration of mitochondrial dynamic-related protein expression, oxidative stress, mitochondrial membrane potential and apoptosis in the tubules of db/db mice after mitoQ administration. A: Profission protein Drp1 (a–c) and profusion protein Mfn2 (_d_-f) expression levels were assessed using IHC in db/m, db/db and db/db mice treated with mitoQ (magnification ×400). Oxidative stress and apoptosis in kidney tubular cells were assessed using DHE (_g_-i) and TUNEL-H staining (j-l). B: Semiquantification of IHC staining of Drp1 and Mfn2. C and D: Quantification of tissues stained with DHE and TUNEL-H. E: Western blotting assay showing that Drp1 (top panels) and cleaved caspase-3 (bottom panels) expression levels increased in the kidneys of db/db mice compared to those of db/m mice. These effects were reversed by mitoQ treatment. MitoQ also restored Mfn2 expression (middle panels) in db/db mice. F1-F3: Quantification of the average band densities calculated from different Western blots. G and H: Bar graphs depicting mitochondrial voltage potential (△Ψm) and mitochondrial DNA (mtDNA) copy numbers in the tubular cells of the kidneys in the three groups. Values are the mean±SE. *P<0.05 vs. db/m; #P<0.05 vs. db/db mice. n=6.
Fig. 3
Restoration of abnormal mitophagy in the proximal tubules in db/db mice treated with mitoQ. A: Kidney sections from db/m mice, db/db mice and db/db mice subjected to mitoQ treatment were stained with LC3 (column 1), p62 (column 2), TOM20 (column 3) and VDAC (column 4) antibodies for IHC analysis (magnification ×400). B: Using a TEM, we observed autophagic vacuoles observed in db/m mice. We observed less vacuoles in the tubular cells of db/db mice than in those of other mice (top panels, Bb vs. Ba). These vacuoles were restored in mice treated with mitoQ (Bc vs. Bb). Higher magnification TEM revealed mitophagy, which was visualized as autophagosome-containing mitochondria in db/m mice and db/db mice administered mitoQ (bottom panels). C and D: The bar graph represents quantification of the numbers of autophagic vacuoles in each tubular cell and fragmented mitochondria. E: Immunofluorescence demonstrating LC3 and VDAC colocalization in the kidney tubules of db/m mice stained with LC3 (green) and VDAC (red) antibodies (top panels). LC3 intensity decreased in the tubular cells of kidneys in db/db mice compared to those in db/m mice (Ef vs. Eb). This intensity was restored by mitoQ administration (Ej vs. Ef). Contrasting intensity changes were observed with respect to VDAC expression (column 1). F: Western blot analysis of LC3, p62, TOM20 and VDAC expression. G1-G4: Quantification of average Western blot band intensities. Values are the mean±SE. *P<0.01 vs. db/m; #P<0.01 vs. db/db. n=6.
Fig. 4
The effect of mitoQ on the expression of the mitophagy-associated proteins PINK and Parkin and the oxidative stress adaptors Keap1 and Nrf2 in db/db mice. A: IHC, kidney sections in db/m mice, db/db mice and db/db mice treated with mitoQ were stained with Keap1 (column 1), Nrf2 (column2), PINK (column3) and Parkin (column4) antibodies. PINK, Parkin, and Nrf2 intensity decreased in the tubules in the kidneys of db/db mice compared to those of control mice (middle panels vs. top panels). These intensities were restored by mitoQ treatment (bottom panels vs. middle panels). Conversely, Keap1 intensity was increased in db/db mice, but this increase was partially inhibited after mitoQ treatment. B1-B4: Semiquantification of IHC staining of Keap1, Nrf2, PINK and Parkin in kidney tissues. C: Western blot analysis of Keap1, Nrf2, PINK, Parkin and phosphorylated-Parkin (_p_-Parkin) expression. D1-D5: Quantification of average Western blot band intensity. Values are the mean±SE. *P<0.01 vs. db/m; #P<0.01 vs. db/db. n=6.
Fig. 5
Protective effects of mitoQ on mitochondrial damage in HK-2 cells subjected to HG exposure. A: Immunoblot assay for LC3II, PINK and nuclear Nrf2 expression in HK2 cells subjected to various concentrations of HG (0–45 mM) for 48 h. Dose dependent decreases in the expression of these proteins were observed in HK-2 cells treated with HG. B: LC3II, PINK and nuclear Nrf2 expression, as determined by Western blotting, in HK2 cells subjected to 30 mM HG exposure for different times (0–48 h), which induced a time-dependent decrease in protein expression. A1 and B1: Quantification of average band intensities using Western blotting. C: Confocal microscopic images revealing the levels of mitochondrial ROS (top panels), intracellular ROS (middle panels) and apoptosis (bottom panels) in HK-2 cells under low glucose (5 mM, LG) and HG exposure (30 mM) with or without mitoQ. D1-D3: Quantification of intracellular ROS, mitochondrial ROS and apoptotic cells, which was performed using H2-DCFDC, MitoSOX and TUNEL-F assays. E: Immunoblot assays of Drp1 (top panels), Mfn2 (middle panels) and cleaved caspase-3 (bottom panels) expression in HK-2 cells subjected to HG exposure. E1-E3: Quantification of average Western blot band intensities. F and G: Bar graphs revealing the mitochondrial membrane potential and ATP activity of HK-2 cells incubated under HG conditions. Values are the mean±SE. *P<0.01 vs. LG; #P<0.01 vs. HG. n=3.
Fig. 6
The effect of mitoQ on defective mitophagy in HK-2 cells subjected to HG exposure. A: Cellular immunofluorescence showing that the colocalization intensity of LC3 and mitochondria (staining with MitoRed) was reduced in HK-2 cells incubated under HG conditions (left middle panels vs. top panels). This effect was partially reversed by mitoQ treatment (left bottom panels vs. middle panels). The opposite result was observed with respect to co-staining with a p62 antibody and MitoRed. B: Immunofluorescence staining with a PINK antibody and MitoRed revealing decreased colocalization intensity in HK-2 cells subjected to HG exposure (middle panels vs. top panels). Colocalization intensity was restored by mitoQ treatment (bottom panels vs. middle panels). C: Immunoblotting assay for LC3II, TOM20, PINK, Parkin and p-Parkin expression. D1-D5: Quantification of average Western blot band intensity. E. Percentage (%) of tubular cells with fragmented mitochondria. *P<0.01 vs. LG; #P<0.01 vs. HG, n=3.
Fig. 7
The restorative effect of mitoQ on PINK and Parkin expression was mediated in part by Nrf2/Keap1. A: Immunoblotting assay for Keap1 and Nrf2 expression, which showed that mitoQ inhibited Keap1 and cytoplasmic Nrf2 expression in HK-2 cells exposed to HG and restored nuclear Nrf2 expression. A1-A3: Quantification of average Western blot band intensity. B: Immunofluorescence assay for Nrf2 showing decreased antibody staining intensity in HK-2 cells after incubation under HG conditions. MitoQ up-regulated the inhibition of Nrf2 intensity in HG-exposed cells. C: Bar graphs depicting Nrf2 binding to the ARE. D: The interaction between Keap1 and Nrf2 in HK-2 cells subjected to HG exposure with or without mitoQ was assessed using IP. D1 and D2: Quantification of the intensity of the interaction between Keap1 and Nrf2 using IP. E: Western blot analysis revealed that mitoQ restored PINK and Parkin expression in HK-2 cells exposed to HG, an effect that was partially abolished by transfection with Keap1 siRNA or Nrf2 siRNA. E1: Quantification of average Western blot band intensity. F: Similar results were observed for PINK mRNA expression, as demonstrated by real-time PCR. *P<0.01 vs. LG; #P<0.01 vs. HG, $P<0.05 compared to HG+mitoQ. &P<0.05 compared to HG+mitoQ+Nrf2 siRNA. n=3.
Fig. 8
Restorative effect of mitoQ on mitophagy, ROS production and apoptosis via Nrf2 and PINK. A: Confocal microscopic image showing that mitoQ ameliorated intracellular ROS production, mitochondrial ROS production and apoptosis in HK-2 cells subjected to HG exposure, as demonstrated by H2-DCFDA, MitoSOX and TUNEL assays, respectively. These effects were partially blocked by Nrf2 siRNA or PINK-siRNA. Transfection with Nrf2 siRNA or PINK-siRNA also attenuated LC3 and mitochondria colocalization intensity in cells treated with mitoQ under HG conditions. B–D: Bar graphs represent intracellular and mitochondrial ROS and apoptosis levels, as demonstrated using H2-DCFDA, MitoSOX and TUNEL assays. E: The effect of Nrf2 siRNA or PINK-siRNA on LC3II, p62, Drp1, and TOM20 expression in HK-2 cells subjected to HG exposure with or without mitoQ using immunoblot assays. E1-E5: Quantification of average Western blot band intensities. *P<0.05 compared to LG; # P<0.05 compared to HG. $P<0.05 compared to HG+mitoQ.
Fig. 9
Schematic diagram depicting the possible molecular mechanisms by which mitoQ prevents renal tubular cell injury by maintaining mitochondrial integrity under HG conditions. Under HG conditions, nuclear Nrf2 expression and activity are down-regulated in tubular cells, which decreases PINK1 transcription and subsequent Parkin phosphorylations and then leads to defective mitophagy. This insufficient mitophagy results in ROS overproduction and aberrant mitochondrial dynamics characterized by Drp1 activation and Mfn2 suppression, changes accompanied by mitochondrial dysfunction, fragmented mitochondria accumulation and mitochondrial apoptotic pathway activation, i.e., caspase 3 release, which eventually leads to cell apoptosis. Interestingly, mitoQ treatment increases the dissociation of Nrf2 from Keap1, and the nuclear location of the former up-regulates the transcription of PINK and restores mitophagy, which maintains mitochondrial quality control, thereby attenuating hyperglycemia-induced tubular injury and apoptosis.
References
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