Abstract
The molecular signaling mechanisms of Coenzyme Q10 (CoQ10) in diabetic nephropathy (DN) remain poorly understood. We verified that mitochondrial abnormalities, like defective mitophagy, the generation of mitochondrial reactive oxygen species (mtROS) and the reduction of mitochondrial membrane potential, occurred in the glomerulus of db/db mice, accompanied by reduced PINK and parkin expression and increased apoptosis. These changes were partially reversed following oral administration of CoQ10. In inner fenestrated murine glomerular endothelial cells (mGECs), high glucose (HG) also resulted in deficient mitophagy, mitochondrial dysfunction and apoptosis, which were reversed by CoQ10. Mitophagy suppression mediated by Mdivi-1 or siPINK abrogated the renoprotective effects exerted by CoQ10, suggesting a beneficial role for CoQ10-restored mitophagy in DN. Mechanistically, CoQ10 restored the expression, activity and nuclear translocation of Nrf2 in HG-cultured mGECs. In addition, the reduced PINK and parkin expression observed in HG-cultured mGECs were partially elevated by CoQ10. CoQ10-mediated renoprotective effects were abrogated by the Nrf2 inhibitor ML385. When ML385 abolished mitophagy and the renoprotective effects exerted by CoQ10, mGECs could be rescued by treatment with mitoTEMPO, which is a mtROS-targeted antioxidant. These results suggest that CoQ10, as an effective antioxidant in mitochondria, exerts beneficial effects in DN via mitophagy by restoring Nrf2/ARE signaling. In summary, CoQ10-mediated mitophagy activation positively regulates DN through a mechanism involving mtROS, which influences the activation of the Nrf2/ARE pathway.
Introduction
Diabetic nephropathy (DN), referred to chronic kidney disease (CKD) caused by diabetes mellitus (Möllsten et al. 2007), affects approximately 30% of diabetic patients (Parving et al. 2006, Thomas et al. 2006, Nathan et al. 2009). Patients with DN carry a greater risk for comorbidities, like cardiovascular disease (Gilbertson et al. 2005), and indeed, renal dysfunction is a central predictor of all-cause mortality (Borch-Johnsen & Kreiner 1987).
In DN, the glomerular filtration barrier, which consists of an inner fenestrated glomerular endothelial cell layer, a glomerular basement membrane and an outer layer of podocytes with interdigitated foot processes that enwrap the glomerular capillaries, is injured (Haraldsson & Nyström 2012). Diabetes induces lesions in glomeruli characterized by mesangial expansion and podocyte loss, among others. Loss of podocytes is a strong predictor of glomerular progression of DN in diabetic patients (Meyer et al. 1999) and murine models of diabetes (Susztak et al. 2006). Interestingly, in models of glomerulosclerosis and in human DKD, endothelial dysfunction has been recently recognized to play a critical role in the development and progression of glomerular disease (Haraldsson et al. 2008, Sun et al. 2013). mGECs are highly specialized cells with fenestrae and a luminal glycocalyx layer (Ballermann 2007, Fogo & Kon 2010), which contribute to the filtration barrier (Haraldsson & Nyström 2012). Currently, the mechanisms and manifestations of glomerular endothelial cell injury in diabetes remain poorly understood.
In fact, the kidneys are second only to the heart in oxygen consumption and mitochondrial abundance. Studies in animal models indicate that disturbances in mitochondrial homeostasis are central to the pathogenesis of DN, but the dissection of the mechanistic nature of mitochondrial dysfunction in the development of DN remains elusive (Galvan et al. 2017). Among several theories linking mitochondrial dysfunction to DN, the mitochondrial dysfunction theory of diabetic complications, also known as the unifying hypothesis, deserves special attention because it has provided significant insights into the potential role of mtROS in mitochondrial dysfunction and DN development in diabetic patients (Brownlee 2005). Moreover, many studies have identified enhanced generation of mtROS in vitro and in kidneys of diabetic mice in vivo (Brownlee 2005, Dieter et al. 2015, Ayanga et al. 2016).
Therapies thought to improve mitochondrial function, including CoQ10 (Persson et al. 2012, Sourris et al. 2012, Dugan et al. 2013) and SS-31 treatment, have beneficial effects on kidney function and fibrosis in experimental models of diabetes (Hou et al. 2016) and obesity (Szeto et al. 2016). CoQ10 is an important component of the mitochondrial electron transport chain, which has an intermittent function in ATP production as well as an antioxidant property and provides protection of lipids in membranes and blood circulation against peroxidation (Littarru & Tiano 2007). In addition, a few studies have reported that CoQ10 levels in diabetic subjects are decreased compared with those in healthy subjects (Ates et al. 2013, Suksomboon et al. 2015). Furthermore, CoQ10 decreases oxidative stress and participates in endothelial metabolism (Lim et al. 2006).
Given an association of DN pathogenesis with increased mitochondrial dysfunction, oxidative stress and apoptosis, as well as the evidence suggesting the importance of CoQ10 supplementation in DN patients, we aimed to evaluate the molecular mechanism of CoQ10 supplementation for treating type 2 diabetes–associated DN. Herein, we describe that CoQ10, as an effective antioxidant in mitochondria, exerts beneficial effects in DN via mitophagy through restoration of Nrf2/ARE signaling. Thus, a novel role of CoQ10 in alleviating DN-induced mitochondrial dysfunction by modulating mitophagy is identified.
Materials and methods
Animal procedures
Twelve-week-old male C57BL/6 J db/db and C57BL/6 J db/m mice were used for animal experiments. These mice were purchased from the Model Animal Research Center of Nanjing University. They were organized into three groups for the animal experiments: db/m mice, and db/db mice with or without CoQ10 treatment. Food (standard mouse chow R70, Labfor, Lantmännen, Sweden) and water were provided ad libitum throughout the study. Treatment with CoQ10 (Native CoQ10 was from Kaneka, Japan) (0.1% in the food) was started 7 weeks before measurements of physiological features and renal function (Persson et al. 2012). For pharmacological manipulation of mitophagy, mice received an i.p. injection of mitophagy inhibitor Mdivi-1 (25 mg/kg/day, Sigma-Aldrich, M0199) (Givvimani et al. 2012) or the mitophagy agonist Torin 1 (2 mg/kg/day, Santa Cruz Biotechnology, sc-396760) (Ito et al. 2015) dissolved in DMSO. MitoTEMPO (3 mg/kg/day, Sigma-Aldrich, SML0737) (Qi et al. 2017) was used to scavenge the mtROS, and the antagonist ML385 (30 mg/kg/day, MedChem Express, HY-100523) (Singh et al. 2016) was used to inhibit Nrf2. Vehicle control mice received an i.p. injection of DMSO every days.
Left kidney weights were measured, and glucose levels in blood samples obtained from the cut tip of the tail were detected using a blood glucose monitor (Boehringer Mannheim). Urinary albumin and creatinine were measured using mouse albumin-specific ELISA and creatinine companion kits (Exocell Laboratories, Philadelphia, PA, USA), and serum creatinine levels were tested using the QuantiChrom Creatinine Assay Kit (BioAssay Systems, USA), according to the manufacturer’s protocol. Plasma insulin levels were determined using the mouse insulin ELISA kit (Linco Research, St. Charles, MO, USA).
All animal experiments and methods performed in this study followed ethical guidelines for animal studies and were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University, China.
Cell culture
Conditionally immortalized mGECs were purchased from Lonza Walkersville, Inc. and cultured in RPMI-1640 containing 10% fetal bovine serum (FBS, Gibco, 16010159). The mGECs were exposed to RPMI-1640 containing either NG (5.5 mM) or HG (33 mM) in the presence or the absence of CoQ10 (10 μM) for 72 h to observe the effects of CoQ10 on mitophagy, mitochondrial function and apoptosis. The culture media were changed every 24 h to ensure stable glucose levels. The CoQ10 concentration used in this study was estimated as that approximating a peak plasma concentration in clinical use (Gholnari et al. 2018).
For pharmacological manipulation of mitophagy, PINK siRNA was transfected into the mGECs using Lipofectamine 2000 reagent (Life Technologies) and was used for our in vitro study. In some experiments, mGECs were treated with the mitophagy inhibitor Mdivi-1 (1 μM), or the mitophagy agonist Torin 1 (250 nM). mitoTEMPO (200 μM, Sigma-Aldrich, SML0737) was added for 2 h before glucose treatment to scavenge the mtROS (Chen et al. 2018). In addition, pretreatment with the antagonist ML385 (10 μM, Abcam, ab120843) (Tang et al. 2018) was done for 2 h every day before CoQ10 administration to inhibit Nrf2.
Western blotting
Immunoblotting was performed as described previously (Kato et al. 2009, 2013). Cells or kidney tissue were lysed in Laemmli’s sample buffer. Lysates were fractionated on 11.5% SDS-polyacrylamide gels (Bio-Rad) and transferred to PVDF membrane. Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with primary antibodies overnight at 4°C. Membranes were washed three times for 5 min with TBST and incubated in either HRP-goat-anti-mouse (Abcam, ab6789) or HRP-goat-anti-rabbit (Abcam, ab6721) secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using Pierce ECL Plus western blotting substrate (Thermo Scientific, 32132) and quantified with Quantity One software (Bio-Rad). Antibodies used were cleaved-Caspase-3 (Merck KGaA, PC679), Bax (Cell Signaling Technology, 2772), Bcl-2 (Abcam, ab692), Nrf2 (Cell Signaling Technology, 12721), HO-1 (Abcam, ab13243), NQO-1 (Abcam, ab28947), LC3 (Abcam, ab48394), p62 (Abcam, ab56416), PINK (Abcam, ab23707), parkin (Cell Signaling Technology, 2132), Cytochrome c (Abcam, ab13575), COX IV (Cell Signaling Technology, 4844), α-tubulin (Cell Signaling Technology, 2125), Lamin-b (Cell Signaling Technology, 13435) and GAPDH (Abcam, ab9485). Antibodies were used at 1:1000 dilution.
Periodic acid-Schiff (PAS) staining
Morphological changes in the kidney sections were assessed using periodic acid-schiff (PAS) and hematoxylin and eosin (H&E) staining according to routine procedures. PAS staining was performed to analyze ECM deposition. The sections were imaged with a Nikon Eclipse Ti-SR epifluorescence microscope (Nikon) at 200× magnification in five randomly selected fields from each of the six mice. The Image-Pro Plus, version 6.0 software (Media Cybernetics) was used to quantify staining.
Immunofluorescence
Frozen 4 mm sections of kidneys were incubated with anti-LC3 monoclonal antibody (Abcam, ab51520) and anti-TOM20 antibody (Abcam, ab186734) to delineate the mitophagy in the glomerulus. Finally, sections were incubated with Alexa Fluor 647-conjugated donkey anti-rabbit antibody (Abcam, ab150075) and Alexa Fluor 488-conjugated donkey anti-mouse antibody (Abcam, ab150109) for 1 h, and nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI, Invitrogen) for 15 min. In parallel, we performed immunofluorescence co-staining using mitotracker and LC3 antibody, following with secondary antibodies to monitor mitophagy in vitro. Then, the cells were counterstained with DAPI. Images were analyzed using a Leica TCS SP5 confocal microscope (Leica).
TUNEL staining
DNA fragmentation was detected in situ with the use of TUNEL according to the manufacturer’s instructions (Roche Applied Science). Images were analyzed using a Leica TCS SP5 confocal microscope (Leica), and the number of TUNEL-positive nuclei was counted by examining the entire section with the same power objective using the Image-Pro Plus software (version 6.0, Media Cybernetics).
Luciferase assays of promoter activities for Nrf2/ARE, SIRT1 and PGC-1α
Luciferase assays were performed as described elsewhere (Lee et al. 2014, Li et al. 2015). ARE-bla construct was used for Nrf2/ARE reporter assay (Li et al. 2015); and the PGC-1α-2kb (the Renilla luciferase-expressing plasmid/pRL-TK used as an internal control) was used for PGC-1α promoter activity detection (Lee et al. 2014). SIRT1 promoter activity was detected by a pTA-Luc SIRT1 promoter (Son et al. 2013). In brief, plasmids were transfected into mGECs for 24 h, and cells were then exposed to RPMI-1640 containing either NG (5.5 mM) or HG (33 mM) in the presence or the absence of CoQ10 (10 μM) for 72 h. Luciferase activity was measured using a Dual-Luciferase-Reporter System (Promega), and luciferase activity for each well was normalized to the internal Renilla luciferase activity.
Subcellular fractionation
Mitochondrial and cytosolic fractions of mice kidneys were purified by a previously described procedure (Nemoto et al. 2004). Briefly, isolated mice kidneys were homogenized in ten volumes of ice-cold buffer A (200 mM mannitol, 50 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM Hepes-KOH (pH 7.4), 0.1% BSA and a mixture of protease inhibitors). Homogenates were centrifuged at 600 g for 5 min at 4°C. Supernatants were then centrifuged at 3500 g for 15 min at 4°C. The pellets were resuspended in buffer A and centrifuged at 1500 g for 5 min. The supernatants were centrifuged at 5500 g for 10 min at 4°C, and then the pellets were suspended as the mitochondrial fraction in PBS containing protease inhibitors. The supernatant was further centrifuged at 100,000 g for 60 min, and the resultant pellet and supernatant were used as microsomal and cytosolic fractions, respectively.
Mitochondrial and cytosolic fractions of mGECs were purified as follows. Cells were suspended in 500 μL fractionation buffer (250 mM Sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES, pH 7.4) containing protease inhibitor cocktail and passed through a 26-gauge needle 20 times using a 1 mL syringe, followed by 20-min incubation on ice. Unbroken cells and nuclei were removed by centrifugation at 800 g for 5 min at 4°C. Supernatants were transferred to fresh tubes and centrifuged at 10,000 g for 10 min at 4°C. Subsequent supernatants were collected as the cytosolic fractions, while mitochondrial pellets were washed once in fractionation buffer and then centrifuged at 10,000 g for 10 min at 4°C. Finally, mitochondrial pellets were suspended in standard lysis buffer for 30 min on ice and the supernatant lysate was obtained after centrifugation at 10,000 g for 10 min at 4°C.
For nuclear Nrf2 accumulation assays, mGECs and mice kidneys were harvested and lysed to obtain cytoplasmic and nuclear lysates using the Keygen Nuclear-Cytosol Protein Extraction Kit from Nanjing KeyGen Biotech. Co., Ltd. (China).
ATP production assay
The mitochondrial fraction of mouse kidneys and mGECs were prepared as described earlier. ATP production was measured with the ATP Bioluminescent Assay kit (Sigma). Twenty-five micrograms of mitochondria were incubated with ATP assay mix and MSH buffer containing 625 μM ADP and substrate (10 mM pyruvate and 10 mM malate).
Measurement of ROS production
MtROS production was analyzed by MitoSOX Red staining according to the manufacturer’s instructions. The staining was evaluated with Nikon Eclipse Ti-SR epifluorescence microscope (Nikon).
Assessment of mitochondrial membrane potential and relative mtDNA copy numbers
To evaluate mitochondrial membrane potential/integrity, cultured mGECs were stained with tetramethylrhodamine ethyl ester (TMRE) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) using MitoPT TMRE and MitoPT JC-1 (ImmunoChemistry Technologies), respectively, according to the manufacturer’s instructions (Montaigne et al. 2014).
MtDNA was extracted and measured as previously described (Gurevich et al. 2001). Briefly, mtDNA was extracted from mGECs and kidneys using a commercial kit (Qiagen) and measured using real-time PCR with a SYBR Green Kit (Pierce).
Mitochondrial respiration assay
Mitochondrial oxygen consumption rate (OCR) was assessed with a Seahorse XF24 Analyzer 9. In brief, mGECs (8000–10,000 cells per well) were cultured in 24-well plates and culture medium was changed 1 h before the assay to unbuffered DMEM medium (pH 7.4) supplemented with 1 mM pyruvate (Thermo Fisher), 10 mM D-glucose. Oligomycin (1 μM), FCCP (2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile) (1 μM) and rotenone (1 μM) combined with antimycin (1 μM) were injected sequentially through ports in the Seahorse Flux Park cartridges as previously reported (Gurevich et al. 2001). First, the basal OCR (basal respiration) was measured. Oligomycin inhibited ATP synthase activity, which led to the development of a proton gradient that inhibited electron flux and revealed the state of the coupling efficiency. FCCP uncoupled the respiratory chain and revealed the maximal capacity for reducing oxygen. The spare respiratory capacity was calculated by subtracting the basal respiration from the maximal respiration. Finally, rotenone combined with antimycin A was injected to inhibit the flux of electrons through complexes I and III; the remaining OCR was primarily due to non-mitochondrial respiration. After OCR measurement, the cells were fixed and stained with the Hoechst 33258 nuclear dye. The plates were scanned to quantify cell numbers using a Cellomics ArrayScan VTI HCS Reader (Thermo Scientific). OCR was normalized to cell number per respective well.
Statistical analyses
The results are expressed as the mean ± s.e.m. Statistical differences were assessed with the unpaired two-tailed Student’s t-test for two experimental groups and the one-way ANOVA for multiple groups with SPSS software. Bonferroni’s post hoc tests were employed after ANOVA to test for significant differences between groups. A two-tailed P value of less than 0.05 was considered statistically significant. Statistical analyses were done using the GraphPad Prism (GraphPad Software).
Results
CoQ10 restores renal function impaired by DN
Db/db mice exhibited a higher left kidney weight, increased serum creatinine, an increased albumin-to-creatinine ratio, higher blood glucose and more hyperinsulinemic than db/m mice (Fig. 1D, E, G, H and Tables 1, 2). Obviously, the levels of serum creatinine, albumin–to-creatinine ratio and blood glucose were all decreased in db/db mice after CoQ10 administration. Also, a slight reduction in blood glucose levels was observed in db/db mice treated with CoQ10, as well as the left kidney weights. PAS and H&E staining analyses showed obvious morphological changes in db/db mice kidneys, covering the tubular epithelial disruption, increases in the mesangial matrix and glomerular hypertrophy. Notably, CoQ10 treatment of db/db mice reversed the changes (Fig. 1A and B).
CoQ10 restores the renal function impaired by diabetic nephropathy. (A) PAS staining in glomeruli from db/m + vehicle, db/db + vehicle and db/db + CoQ10 mice. (B) H&E staining in glomeruli from db/m + vehicle, db/db + vehicle and db/db + CoQ10 mice. (C) TUNEL assay in glomeruli from db/m + vehicle, db/db + vehicle and db/db + CoQ10 mice. The apoptotic cells were labeled with green, and nuclei were stained with DAPI (blue). (D) Left kidney weight changes in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (E) Serum creatinine levels in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (F) The quantitative analysis of TUNEL+ cells in (C) in at least six separate fields, values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (G) Urinary Albumin to creatinine levels in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (H) Blood glucose concentrations in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (I) Tissue lysates of glomeruli were used to detect the c-Caspase 3, Bax and Bcl-2 protein levels by immunoblotting in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. (J) The quantitative analysis of each immunoblots in (I). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (K) Cell lysates of each group (CON, HG, HG + CoQ10) were used to detect the c-Caspase 3, Bax and Bcl-2 protein levels by immunoblotting. (L) The quantitative analysis of each immunoblots in (K). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (M) Tissue lysates of glomeruli were used to detect the release of Cytochrome c from mitochondria into cytosol in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. (N) Cell lysates of each group (CON, HG, HG + CoQ10) were used to detect the release of Cytochrome c from mitochondria into cytosol by immunoblotting. (O) The quantitative analysis of each immunoblots in (M). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (P) The quantitative analysis of each immunoblots in (N). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
Biochemical and physiological parameters in mice.
| Parameter | db/m | db/db | |
|---|---|---|---|
| Vehicle | Vehicle | CoQ10 | |
| Body weight (g) | 23 ± 3 | 55 ± 5a | 54 ± 6a |
| Food intake (g/24 h) | 3.1 ± 0.7 | 2.7 ± 1.3 | 3.3 ± 1.1 |
| Insulin (μIU/mL) | 50.32 ± 2.14 | 520.13 ± 21.42a | 72.51 ± 8.32 |
All data are expressed as means ± s.d.
a P < 0.05 vs db/m + vehicle.
Plasma insulin concentrations in mice at various ages.
| Insulin (μIU/mL) | 4 weeks | 8 weeks | 12 weeks | 16 weeks | 20 weeks |
|---|---|---|---|---|---|
| db/m | 47.21 ± 3.81 | 51.54 ± 4.52 | 57.32 ± 7.33 | 52.41 ± 8.31 | 53.63 ± 7.96 |
| db/db | 300.06 ± 20.96a | 450.43 ± 24.81a | 560.30 ± 22.83a | 680.34 ± 43.78a | 890.22 ± 49.51a |
All data are expressed as means ± s.d.
a P < 0.05 vs db/m.
An increase in apoptosis, assessed by the TUNEL procedure, was observed in the glomeruli of db/db mice (Fig. 1C and F). In addition, an increase in the protein levels of c-Caspase 3, Bax/Bcl-2 and cytosol Cytochrome c further confirmed the occurrence of apoptosis (Fig. 1I, J, M and O).
To establish whether the anti-apoptotic effects of CoQ10 observed in the in vivo studies involved direct actions on mGECs, cells were cultured in HG (33 mM) medium in the presence or the absence of CoQ10 (10 μM). The cells cultured in HG medium showed dramatically increased apoptosis compared with cells cultured in NG medium, which was inhibited by CoQ10 co-treatment (Fig. 1K, L, N and P). Thus, a direct protective role of CoQ10 against HG-induced DN was confirmed.
CoQ10 ameliorates DN-induced mitochondrial dysfunction both in vivo and in vitro
Mitochondrial dysfunction observed early in the development of experimental DN (Kaneda et al. 1992, Coughlan et al. 2016) has been identified as a major contributor to disease progression both in preclinical models (Chacko et al. 2010, Sourris et al. 2012, Coughlan et al. 2016) and in humans with DN (Huang et al. 2006, Sharma et al. 2013, Czajka et al. 2015). There is also evidence of mitochondrial dysfunction in other CKDs (Hall & Unwin 2007, Che et al. 2014, Forbes 2016). Therefore, we assessed whether CoQ10 could play a role in mitochondrial dysfunction. We first assessed the mGECs mitochondrial OCR. A mitochondrial respiratory reserve capacity (maximal OCR over baseline OCR) was significantly reduced in HG-treated mGECs as compared to the CON cells. In contrast, a reverse respiratory capacity was found in HG-treated cells with CoQ10 addition (Fig. 2D). An accumulation of oxidative mtDNA lesions and reduced ATP production is associated with oxidative stress and mitochondrial dysfunction (Czajka et al. 2015). In our study, the relative mtDNA content was dramatically increased in db/db mice, and CoQ10 largely decreased the extent of the mtDNA content. Additionally, relative ATP production was significantly impaired in db/db mice compared with db/m mice, but it was greatly enhanced by CoQ10 treatment (Fig. 2A). Furthermore, we assessed the mtROS with MitoSOX probe, which showed that the level of DN-induced mtROS was decreased by treatment with CoQ10 (Fig. 2B and C).
CoQ10 ameliorates DN-induced mitochondrial dysfunction both in vivo and in vitro. (A) mtDNA content and ATP production in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (B) Effects of CoQ10 on mitoSOX in glomeruli. (C) The quantitative analysis of fluorescence intensity in (B), values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (D) SeaHorse XF24 Flux Analyses of OCR during a maximal electron flow test in mGECs of each group (CON, HG, HG + CoQ10). For electron flow, all data are expressed as median ± interquartile range. (E) Relative mtDNA content and ATP production in mGECs of each group (CON, HG, HG + CoQ10). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (F) Representative images of JC-1 fluorescence in mGECs of each group (CON, HG, HG + CoQ10). (G) Effects of CoQ10 on mitoSOX in mGECs. (H) The quantitative analysis of fluorescence intensity in (F), values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (I) Effects of CoQ10 on mitochondrial membrane potential in mGECs of each group (CON, HG, HG + CoQ10) measured using TMREfluorescence. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
Similar to the results obtained in glomerulus, the relative mtDNA content and mtROS were also significantly increased in mGECs exposed to HG, compared with mGECs maintained in NG, but they were greatly decreased by CoQ10 co-treatment (Figs 2E, left and G, H). In addition, CoQ10 treatment restored the impaired ATP production induced by HG in mGECs (Fig. 2E, right). Furthermore, JC-1 fluorescence detection exhibited a decrease in red fluorescence that accumulated in the mitochondria and an increase in green fluorescence distributed in the cytoplasm of mGECs incubated with HG, suggesting that HG treatment reduced the mitochondrial membrane potential (Fig. 2F). Importantly, co-incubation with CoQ10 restored the mitochondrial membrane potential impaired by HG. These results were confirmed with tetramethyl rhodamine ethyl ester (TMRM) staining (Fig. 2I).
Taken together, these results indicate that CoQ10 restores the DN-induced mitochondrial dysfunction.
CoQ10 increases mitophagy in DN
A deficiency in mitophagy induces oxidative stress in kidneys, as mitophagy blockage leads to the accumulation of damaged mitochondria. Early in diabetes, the kidney activates mitophagy to clear dysfunctional mitochondria, but as DN progresses, this process becomes overwhelmed, leading to an impairment of mitophagy, accumulation of fragmented mitochondria and cell death (Higgins & Coughlan 2014). Having proved that CoQ10 effectively restores DN-induced mitochondrial dysfunction, we further ascertained the role of CoQ10 in mitophagy. In the present study, we found a decreased accumulation of LC3 puncta in glomeruli of db/db mice compared with the corresponding control littermates. However, CoQ10-treated db/db mice manifested more LC3 puncta when compared with the vehicle-treated group (Fig. 3A). Moreover, an increase in the expression levels of LC3-II, PINK and parkin were also observed in isolated mitochondria from CoQ10-treated db/db kidney and HG-treated mGECs (Fig. 3B, C, D and E). These results suggest that CoQ10 positively regulates mitophagy in DN.
CoQ10 increases mitophagy in DN. (A) Representative images of LC3 (red) staining in glomeruli from db/m + vehicle, db/db + vehicle and db/db + CoQ10 mice. Glomeruli mitochondrial is labeled with TOM20 (green). (B) Glomerular endothelial mitochondrial lysates were used to detect the LC3, PINK, and parkin protein levels by immunoblotting in db/m, db/db and db/db mice receiving CoQ10 treatment for 8 weeks. (C) The quantitative analysis of each immunoblots in (B). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (D) mGECs mitochondrial lysates were used to detect the LC3, PINK, and parkin protein levels by immunoblotting of each group (CON, HG, HG + CoQ10). (E) The quantitative analysis of each immunoblots in (D). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
The renoprotection exerted by CoQ10 requires PINK-related mitophagy
To confirm the role of CoQ10-upregulated mitophagy in the protection against DN, the commonly used mitophagy inhibitor Mdivi-1 was used to affect CoQ10-upregulated mitophagy. After reinhibition of mitophagy by Mdivi-1 pretreatment, CoQ10 could no longer exert its beneficial renoprotective role against HG impairment, as reflected by left kidney weights (Fig. 4E), serum creatinine (Fig. 4F), albumin-to-creatinine ratio (Fig. 4G) and TUNEL-positive cells (Fig. 4C and H). Additionally, the morphological changes, including tubular epithelial disruption, increases in mesangial matrix and glomerular hypertrophy, were not ameliorated without mitophagy (Fig. 5A and B). Also, the CoQ10 effects on glomerular mtROS and mitochondrial homeostasis were also abolished by Mdivi-1 pretreatment as measured by the relative mtDNA content and ATP production (Fig. 4I and J), accompanied by unchanged mtROS detected by MitoSOX staining (Fig. 4D and K).
The renoprotection in vivo exerted by CoQ10 requires PINK-related mitophagy. (A) PAS staining in glomeruli from the indicated groups. (B) H&E staining in glomeruli from the indicated groups. (C) TUNEL assay in glomeruli from the indicated groups. The apoptotic cells were labeled with green, and nuclei were stained with DAPI (blue). (D) MitoSOX staining in glomeruli from the indicated groups. (E) Left kidney weight changes in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. (F) Serum creatinine levels in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. (G) Albumin to creatinine levels in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. (H) The quantitative analysis of TUNEL+ cells in (C) in at least six separate fields, values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. (I) Relative mtDNA content in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. (J) Relative ATP production in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. (K) The quantitative analysis of fluorescence intensity in (D), values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
The renoprotection in vitro exerted by CoQ10 requires PINK-related mitophagy. (A) Representative images of JC-1 fluorescence in mGECs of each group (CON, HG, HG + CoQ10, HG + siPINK, HG + CoQ10 + siPINK). (B) MitoSOX staining in mGECs from the indicated groups. (C) TMRE fluorescence staining in mGECs from the indicated groups. (D) Relative mtDNA content in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10. (E) Relative ATP production in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10. (F) The quantitative analysis of fluorescence intensity in (B), values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10. (G) Cell lysates of the indicated groups were used to detect the c-Caspase 3, Bax and Bcl-2 protein levels by immunoblotting. (H) The quantitative analysis of each immunoblots in (G). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10. (I) Tissue lysates of glomeruli from the indicated groups were used to detect the c-Caspase 3, Bax and Bcl-2 protein levels by immunoblotting. (J) The quantitative analysis of each immunoblots in (I). Values displayed are means ± s.e.m. of six independent experiments. *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
Although the results obtained with Mdivi-1 suggested a positive role of mitophagy in DN, a more direct proof was still needed. Thus, we further investigated whether activation of mitophagy could provide a renoprotective effect similar to CoQ10. For this, a commonly used mitophagy activator Torin 1 was employed. Torin 1 positively improved the morphological changes of glomeruli of db/db mice, which were measured by PAS and H&E staining (Fig. 4A and B). An increased apoptosis in glomeruli of db/db mice was also alleviated by Torin 1, as reflected by the downregulated number of TUNEL-positive cells (Fig. 4C and H), and the reduced protein levels of c-Caspase 3, Bax/Bcl-2 and cytosol Cytochrome c, further confirming the decrease in apoptosis (Fig. 5I and J).
In addition, we used PINK siRNA for genetic downregulation of autophagy in mGECs. In PINK-deficient mGECs, CoQ10 did not affect the relative mtDNA content or ATP production in HG mGECs (Fig. 5D and E). In addition, CoQ10 could no longer exert its beneficial role in mitochondrial homeostasis or protect against HG impairment of mitochondrial membrane potential, as detected by JC-1 and TMRE staining assays (Fig. 5A, C and F). Additionally, the inhibitory effects of CoQ10 on mtROS and cellular apoptosis in mGECs under HG medium were not apparent due to PINK siRNA (Fig. 5B, G and H).
These observations suggest that the effects of CoQ10 on HG-impaired mitochondrial dysfunction might be mainly attributed to its role in activating mitophagy.
CoQ10 activates mitophagy through the Nrf2/ARE pathway
We next determined the mechanism by which CoQ10 induced mitophagy. Here, we first assessed whether CoQ10 affected the promoter activities of several transcriptional activators, including the longevity and autophagy-regulating protein SIRT1, the master regulator of mitochondrial biogenesis PGC-1α and the antioxidant nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) (Hariharan et al. 2010, Cheng et al. 2012, Son et al. 2013, Fang et al. 2014), which can regulate mitochondrial homeostasis. In mGECs, exposure to HG decreased the promoter activities of SIRT1, PGC-1α and Nrf2/ARE (Fig. 6A, B and C). Of note, CoQ10 did not affect SIRT1 or PGC-1α promoter activities but induced an obvious increase in Nrf2/ARE reporter activity in mGECs exposed to HG (Fig. 6A, B and C). Since CoQ10 increased HG-induced Nrf2/ARE reporter activity, we next determined the levels of several proteins in mGECs encoded by Nrf2-responsive genes by immunoblot analysis. The translocation of Nrf2 from the cytoplasm to the nucleus is a feature of ROS activation. In the nucleus, Nrf2 binds to the transcriptionally active genes of ARE regions, than the related genes encode antioxidant proteins NAD(P)H quinone oxidoreductase 1 (NQO-1) and heme oxygenase 1 (HO-1) (Johnson et al. 2008, Dinkova-Kostova & Abramov 2015). HG-treated mGECs showed decreased amounts of nuclear Nrf2, as well as the decreased levels of NQO-1 and HO-1 (Fig. 6D and K), suggesting that HG inhibits the Nrf2/ARE pathway. However, HG-induced inhibition of the Nrf2/ARE pathway in mGECs was significantly abolished by CoQ10 co-incubation (Fig. 6D and K). Similarly, CoQ10 treatment of db/db mice resulted in the upregulation of the Nrf2/ARE reporter activity in glomeruli of mice compared to the db/db group, as demonstrated by the raised amounts of NQO-1, HO-1 and nuclear Nrf2 (Fig. 6G and J).
CoQ10 induces mitophagy by activating the Nrf2/ARE pathway. (A) Assessment of Nrf2/ARE reporter activity in mGECs from the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (B) Assessment of PGC-1α reporter activity in mGECs from the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (C) Assessment of SIRT1 reporter activity in mGECs from the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (D) Cell lysates of the indicated groups were used to detect the Nrf2, NQO-1 and HO-1 protein levels by immunoblotting. (E) The quantitative analysis of each immunoblots in (D). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (F) The quantitative analysis of the Nrf2 in (J). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (G) Tissue lysates of glomeruli were used to detect the Nrf2, NQO-1 and HO-1 protein levels by immunoblotting. (H) The quantitative analysis of each immunoblots in (G). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle. (I) The quantitative analysis of the Nrf2 in (K). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG. (J) Subcellular localization of endogenous Nrf2 in glomerulus from the indicated groups was measured by immunoblotting. (K) Subcellular localization of endogenous Nrf2 in mGECs from the indicated groups was measured by immunoblotting.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
The importance of Nrf2/ARE pathway in CoQ10-upregulated mitophagy in DN was further demonstrated by the specific NRF2 inhibitor ML385 (Singh et al. 2016). ML385 was sufficient to inhibit mitophagy in glomeruli of db/db mice, as revealed by the lessening of yellow puncta and decreased protein levels of LC3-II, PINK and parkin. Furthermore, ML385 disrupted CoQ10-promoted mitophagy in DN (Fig. 7A, B and E). Consistently, we confirmed the necessity of the Nrf2/ARE pathway in CoQ10-upregulated mitophagy in mGECs treated with HG (Fig. 7C and D).
Nrf2/ARE is necessary for CoQ10-mediated mitophagy activation. (A) Mice glomerulus mitochondrial lysates were used to detect the LC3, PINK, and parkin protein levels from each group. (B) The quantitative analysis of each immunoblots in (A). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (C) mGECs mitochondrial lysates were used to detect the LC3, PINK, and parkin protein levels by immunoblotting of each group. (D) The quantitative analysis of each immunoblots in (C). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10; @ P < 0.05 vs HG + CoQ10 + siNrf2. (E) Representative images of LC3 (red) staining in glomeruli from the indicated groups. Glomeruli mitochondrial is labeled with TOM20 (green). A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
In addition, a specific mtROS-targeted antioxidant, mitoTEMPO, activated mitophagy and increased the expression of LC3-II, PINK and parkin, which was impaired in glomeruli of the DN mice (Fig. 7A, B and E). Moreover, mitoTEMPO restored the mitophagy after HG treatment in the presence of ML385 and CoQ10 (Fig. 7C and D), indicating that the role of the Nrf2/ARE pathway in CoQ10-upregulated mitophagy in DN is mediated by mtROS.
We also verified the effects of CoQ10 treatment on mitophagy and Nrf2 signaling in db/m mice and the control mGECs as shown in Fig. 8. We found that there were no significant effects on mitophagy and Nrf2 signaling either in db/m mice or in control mGECs treated with the indicated dosages of CoQ10.
CoQ10 treatment has no effect on mitophagy and Nrf2 signaling under basal conditions. (A) Representative images of LC3 (red) staining in glomeruli from the indicated groups. Glomeruli mitochondrial is labeled with TOM20 (green). (B) Glomerular endothelial mitochondrial lysates were used to detect the LC3, PINK, and parkin protein levels by immunoblotting in db/m mice receiving vehicle or CoQ10 treatment. (C) mGECs mitochondrial lysates were used to detect the LC3, PINK, and parkin protein levels by immunoblotting of each group (CON, CON + CoQ10). (D) Tissue lysates of glomeruli were used to detect the Nrf2, NQO-1 and HO-1 protein levels by immunoblotting. (E) Cell lysates of the indicated groups were used to detect the Nrf2, NQO-1 and HO-1 protein levels by immunoblotting. (F) Subcellular localization of endogenous Nrf2 in glomerulus from the indicated groups was measured by immunoblotting. (G) Subcellular localization of endogenous Nrf2 in mGECs from the indicated groups was measured by immunoblotting. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
CoQ10 restores renal function and morphological changes via Nrf2/ARE
We next confirmed the necessity of the Nrf2/ARE pathway in CoQ10-mediated renoprotection. After inhibiting Nrf2 by the specific Nrf2 inhibitor ML385, CoQ10 could no longer exert its beneficial role in renoprotection against HG impairment, as reflected by left kidney weights, serum creatinine, albumin-to-creatinine ratio and blood glucose level (Fig. 9E, F and G). In addition, the morphological changes including tubular epithelial disruption, increases in mesangial matrix and glomerular hypertrophy were not ameliorated, as detected by PAS and H&E staining (Fig. 9A and B). Moreover, the protein levels of c-Caspase 3 and Bax/Bcl-2 further confirmed the loss of CoQ10 function without Nrf2 (Fig. 10F). In addition, the CoQ10 effects on glomerular mtROS and mitochondrial homeostasis were also abolished by ML385 co-treatment measured by the relative mtDNA content and ATP production (Fig. 9I and J) and MitoSOX staining assay (Fig. 9D and K).
CoQ10 restores renal function and morphological changes via Nrf2/ARE. (A) PAS staining in glomeruli from the indicated groups. (B) H&E staining in glomeruli from the indicated groups. (C) TUNEL assay in glomeruli from the indicated groups. The apoptotic cells were labeled with green, and nuclei were stained with DAPI (blue). (D) MitoSOX staining in glomeruli from the indicated groups. (E) Left kidney weight changes in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (F) Serum creatinine levels in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (G) Albumin to creatinine levels in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (H) The quantitative analysis of TUNEL+ cells in (C) in at least six separate fields, values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (I) Relative mtDNA content in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (J) Relative ATP production in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (K) The quantitative analysis of fluorescence intensity in (D), values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
CoQ10 restores renal function and morphological changes via Nrf2/ARE. (A) Representative images of JC-1 fluorescence in mGECs of each group. (B) MitoSOX staining in mGECs from the indicated groups. (C) TMRE fluorescence staining in mGECs from the indicated groups. (D)The quantitative analysis of fluorescence intensity in (B), values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10; @ P < 0.05 vs HG + CoQ10 + siNrf2. (E) Cell lysates of the indicated groups were used to detect the c-Caspase 3, Bax and Bcl-2 protein levels by immunoblotting. (F) Tissue lysates of glomeruli from the indicated groups were used to detect the c-Caspase 3, Bax and Bcl-2 protein levels by immunoblotting. (G) Relative mtDNA content in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10; @ P < 0.05 vs HG + CoQ10 + siNrf2. (H) Relative ATP production in the indicated groups. Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. (I) The quantitative analysis of each immunoblots in (E). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs CON; *P < 0.05 vs HG; & P < 0.05 vs HG + CoQ10; @ P < 0.05 vs HG + CoQ10 + siNrf2. (J) The quantitative analysis of each immunoblots in (F). Values displayed are means ± s.e.m. of six independent experiments. # P < 0.05 vs db/m + vehicle; *P < 0.05 vs db/db + vehicle; & P < 0.05 vs db/db + CoQ10 + vehicle; @ P < 0.05 vs db/db + CoQ10 + ML385. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0578.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0578
Then, we inspected whether the role of the Nrf2/ARE pathway in CoQ10-upregulated mitophagy was mediated by mtROS. For this, a commonly used mtROS-targeted antioxidant mitoTEMPO was employed to inhibit mtROS. In db/db mice pretreated with ML385, mitoTEMPO improved the morphological changes of glomerulus treated with CoQ10, which were measured by PAS and H&E staining (Fig. 9A and B). An increased apoptosis in glomerulus was also alleviated by mitoTEMPO, as reflected by the decreased TUNEL-positive cells (Fig. 9C and H), accompanied by the reduced protein levels of c-Caspase 3, Bax/Bcl-2 and cytosol Cytochrome c (Fig. 10F).
To establish whether the CoQ10 effects observed in in vivo studies involved a direct action on mGECs, we used Nrf2 siRNA for genetic downregulation of Nrf2 in mGECs. Nrf2 deficiency reversed the beneficial role of CoQ10 in mitochondrial homeostasis and maintenance of mitochondrial membrane potential against HG impairment, as reflected by the relative mtDNA content and ATP production (Fig. 10G and H), JC-1 staining and TMRE staining assays (Fig. 10A and C). Additionally, the inhibitory effects of CoQ10 on mtROS (Fig. 10B) and cellular apoptosis (Fig. 10E) of mGECs in HG medium were not apparent due to Nrf2 siRNA. Altogether, these results indicate that CoQ10 exerts renoprotective effects by decreasing mtROS, which activates mitophagy through the Nrf2/ARE pathway.
Discussion
Although there is ample evidence suggesting the disturbance of mitochondrial bioenergetics in DN, studies characterizing the role of mitophagy in this disease process are lacking. How the process of mitophagy is changed in DN and whether this change is beneficial or detrimental to kidney function still remains to be fully understood (Higgins & Coughlan 2014). Until recently, CoQ10 was known only as a component of the mitochondrial electron transport chain, but newer data have revealed that the reduced form of CoQ10 is also a potent antioxidant (Ernster & Dallner 1995, Thomas et al. 1996). CoQ10 exerts its antioxidant effect at the beginning of lipid peroxidation, and its physiological concentration is related to its activity level (Ernster & Dallner 1995). In the present study, oral supplementation of CoQ10 was investigated because no other forms of CoQ10 can currently be used in human subjects (Matthews et al. 1998, Kwong et al. 2002, Hwang et al. 2015). We demonstrated that CoQ10 exerts its renoprotective effects, at least in part, by reactivating DN-induced inhibition of mitophagy, and this process is mediated via mtROS, which affects the activation of the Nrf2/ARE pathway.
Mitochondrial dysfunction occurs following inhibition of oxidative phosphorylation (OXPHOS), which results in decreased ATP production and a loss of mitochondrial membrane potential, and can lead to changes in cationic gradients, increased ROS generation at various sites of the electron transport chain and, ultimately, the redistribution of mitochondrial cell death proteins. Whereas much attention has been given to the molecular events that govern mitochondrial homeostasis in cancer (Wallace 2012) and neurodegeneration, such as Parkinson’s disease and Alzheimer’s disease (Higgins et al. 2010, Lezi & Swerdlow 2012). Limited investigations have been undertaken on these events in DN. In this study, we have observed mitochondrial dysfunction in the glomeruli of db/db type 2 diabetic mice, as well as in HG-cultured mGECs, exhibited by increased mtDNA content and mtROS, accompanied by decreased ATP production, a decreased mitochondrial respiratory reserve capacity and a decreased mitochondrial membrane potential. In particular, the HG-impaired mitochondrial homeostasis is ameliorated by CoQ10 supplementation in db/db mice and mGECs exposed to HG medium, suggesting a positive role for CoQ10 in mitochondrial homeostasis.
The role of mitophagy in DN is of particular interest, given that accumulation of damaged mitochondria has been observed in the kidney, raising the possibility that an impairment in the mitophagy system may occur (Higgins & Coughlan 2014). In the present study, we found that the decreased mitophagy levels in the glomeruli of db/db mice, and the HG-impaired kidney function and mitochondrial homeostasis are ameliorated by mitophagy activator Torin 1, suggesting the detrimental role of decreased mitophagy in DN. This finding is consistent with previous observations obtained in STZ-induced diabetic rat kidney (Kitada et al. 2011, Lezi & Swerdlow 2012, Vallon et al. 2013). Interestingly, CoQ10 apparently increased mitophagy in the glomeruli of db/db mice, along with the improved kidney function and mitochondrial homeostasis. In addition, CoQ10 was prevented from exerting its beneficial effect in renoprotection against HG impairment by the mitophagy inhibitor Mdivi-1 co-treatment. In parallel, we obtained consistent results in mGECs, which suggests that the CoQ10 effects in DN are mainly related to its role in activating mitophagy.
Our findings suggest that under hyperglycemic conditions CoQ10 may protect kidney function by reactivating mitophagy through the Nrf2/ARE pathway. Most importantly, mitophagy is indispensable for CoQ10-generated renoprotection. In particular, the specific Nrf2 inhibitor ML385 disrupted CoQ10-promoted mitophagy in DN, accompanied by the impaired kidney function, suggesting that CoQ10-induced upregulation of mitophagy is partly dependent on the Nrf2/ARE pathway. Actually, it is still possible that CoQ10 could affect SIRT1 or PGC-1α activities at the post-translational levels, directly or indirectly. Additionally, other mechanistic pathways may also act a role in CoQ10-induced mitophagy. For instance, p62, an essential mitophagy receptor (Fang et al. 2013), may participate in CoQ10-dependent stimulation of mitophagy considering the fact that PMI (a synthetic p62-mediated mitophagy inducer) can activate mitophagy through an induction of p62 via the ARE-Nrf2 pathways (Bjørkøy et al. 2009, Jain et al. 2010, East et al. 2014). Altogether, our findings confirm that CoQ10 increases mitophagy through the Nrf2/ARE pathway, which is necessary for mitochondrial homeostasis.
Enhanced mtROS have been implicated in numerous processes ranging from aging to cancer and, for decades, maintained a center stage as key mediators of kidney damage. A large body of evidence obtained from both experimental models and patients has suggested that the generation of ROS is significantly increased in failing kidneys. Of particular interest is the free-radical theory of diabetic microvascular complications, also known as the unifying hypothesis that links mtROS to mitochondrial dysfunction and organ damage in kidneys. The unifying hypothesis proposes that overproduction of mtROS is associated with mitochondrial dysfunction, which ultimately causes cellular damage and progression of kidney disease. Corresponding with these studies, we have determined at first that CoQ10 alleviated DN by improving mitochondrial homeostasis, including decreasing mtROS. In addition, our present study provides strong evidence that CoQ10 restores renal function via the Nrf2/ARE pathway. Since the Nrf2 activation is directly mediated by ROS (Jain et al. 2010), we further investigated whether the role of Nrf2/ARE pathway in CoQ10-upregulated mitophagy in DN was mediated by mtROS. As expected, mtROS-targeted antioxidant mitoTEMPO not only restored the mitophagy but also alleviated kidney function in glomeruli of db/db mice co-treated with ML385 and CoQ10, indicating the participation of mtROS in CoQ10-induced renoprotection.
In summary, we have shown that CoQ10-mediated mitophagy activation positively regulates DN through a mechanism involving mtROS, which influences the activation of the Nrf2/ARE pathway. Importantly, the results from the current study establish a novel role of CoQ10 in renoprotection through the mitophagy machinery mediated by Nrf2/ARE, a finding that may have implications for the pathogenesis and treatment of diabetes-associated kidney complications.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by WenZhou Science & Technology Bureau Foundation (Grant #Y20150039).
Author contributivon statement
Jia Sun was the primary investigators in this study and wrote the first draft and participated in editing of the manuscript. Haiping Zhu and Xiaorong Wang contributed to the in vivo data. Qiuqi Gao and Zhuoying Li participated in the cell experiments. Huiya Huang conceived, designed, supervised the study, contributed to data analysis, interpretation and editing of the paper.
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