Coenzyme Q10 alleviates tacrolimus-induced mitochondrial dysfunction in kidney

Abstract

The major side effect of tacrolimus (Tac) is nephrotoxicity. We studied whether supplementation of coenzyme Q10, (CoQ10) a potent antioxidant, can reduce Tac-induced nephrotoxicity via improving mitochondrial function. In an in vitro study, CoQ10 reduced the production of Tac-induced mitochondrial reactive oxygen species and abolished the loss of mitochondrial membrane potential in proximal tubular cell line. Assessment of mitochondrial function revealed that CoQ10 decreased oxygen consumption and mitochondrial respiration rate increased by Tac, suggesting improvement of mitochondrial function to synthesize ATP with CoQ10 treatment. The effect of the CoQ10in vitro study was observed in an experimental model of chronic Tac-induced nephropathy. CoQ10 attenuated Tac-induced oxidative stress and was accompanied by function and histologic improvement. On electron microscopy, addition of CoQ10 increased not only the number but also the volume of mitochondria compared with Tac treatment only. Our data indicate that CoQ10 improves Tac-induced mitochondrial dysfunction in kidney. Supplementary CoQ10 treatment may be a promising approach to reduce Tac-induced nephrotoxicity.-Yu, J. H., Lim, S. W., Luo, K., Cui, S., Quan, Y., Shin, Y. J., Lee, K. E., Kim, H. L., Ko, E. J., Chung, B. H., Kim, J. H., Chung, S. J., Yang, C. W. Coenzyme Q10 alleviates tacrolimus-induced mitochondrial dysfunction in kidney.

Keywords: 3D reconstruction; nephrotoxicity; reactive oxygen species.

Conflict of interest statement

This work was supported by the Korean Health Technology Research and Development Project, Ministry for Health and Welfare (HI14C3417, HI16C1641), and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, Information and Communications Technology (ICT) and Future Planning (NRF-2018R1D1A1A02043014). The authors declare no conflicts of interest.

Figures

Figure 1

Protective effects of CoQ10 against Tac-induced injury in HK-2 cells. Cells were seeded in plates at 90% confluence; the next day, they were treated with Tac (50 µg/ml) in the absence or presence of 1 pg/ml–10 μg/ml CoQ10 for 12 h. A) Cell Counting Kit-8 solution was added to each well for 2 h to evaluate cell viability. B, C) Annexin V solution was added to each well for 15 min to measure apoptosis, which was detected by flow cytometry. Data are presented as means ± se and are representative of ≥4 independent experiments. One-way ANOVA was used to analyze the data. &P < 0.05 vs. control group; $P < 0.05 vs. CoQ10 group; #P < 0.05 vs. Tac group.

Figure 2

Effects of CoQ10 on mitochondrial ROS production during Tac-induced HK-2 cells injury. Cells were seeded in culture plates at 90% confluence; the next day, they were treated with Tac (50 µg/ml) in the absence or presence of 1 ng/ml CoQ10 for 12 h. Incidence of mitochondrial O2−-induced apoptotic cells was measured by double staining with MitoSox Red (A) and Annexin V using flow cytometry (B). Data are presented as means ± se and are representative of ≥4 independent experiments. One-way ANOVA was used to analyze the data. &P < 0.05 vs. control group; $P < 0.05 vs. CoQ10 group; #P < 0.05 vs. Tac group.

Figure 3

Effect of CoQ10 on MMP of HK-2 cells with Tac-induced injury. Cells were seeded in culture plates at 90% confluence; the next day, they were treated with Tac (50 µg/ml) in the absence or presence of 1 ng/ml CoQ10 for 12 h, then labeled with JC-1 and then analyzed by flow cytometry to evaluate MMP. Flow cytometry plots (A) and quantitation of JC-1 labeling (B). Data are presented as means ± se and are representative of ≥4 independent experiments. One-way ANOVA was used to analyze the data. &P < 0.05 vs. control group; $P < 0.05 vs. CoQ10 group; #P < 0.05 vs. Tac group.

Figure 4

Effect of CoQ10 on mitochondrial function in HK-2 cells with Tac-induced injury. Cells were seeded in culture plates at 90% confluence; the next day, they were treated with Tac (50 µg/ml) in the absence or presence of 1 ng/ml CoQ10 for 12 h, then cultured in a non-CO2 incubator for 1 h. The ATP synthase inhibitor oligomycin, respiratory chain uncoupler carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, or respiratory chain complex I and III inhibitor rotenone/antimycin A were added to the culture medium as indicated. A) Areas under the curve for basal respiration. B) Basal respiration, ATP production, maximal respiration, proton leakage, and nonmitochondrial respiration were calculated from the OCR. Data represent means ± se and are representative of ≥4 independent experiments. One-way ANOVA was used to analyze the data. &P < 0.05 vs. control group; $P < 0.05 vs. CoQ10 group; #P < 0.05 vs. Tac group.

Figure 5

Effect of CoQ10 on a rat model of Tac-induced renal injury. CoQ10 concentration was determined in the plasma (A) and renal cortex (B) of rats treated with Tac or CoQ10 by oral administration after 4 wk. Total CoQ10 levels in both plasma and kidney tissue was higher in the Tac + CoQ10 than in the Tac group. Data represent means ± se (n = 8). One-way ANOVA was used to analyze the data. &P < 0.05 vs. Vh group; $P < 0.05 vs. CoQ10 group; #P < 0.05 vs. Tac group.

Figure 6

Effect of CoQ10 on Tac-induced renal tubulointerstitial fibrosis in a rat model. Histologic analysis of renal cortex tissue from rats treated with Tac for 4 wk (A); striped tubulointerstitial fibrosis, mononuclear cell infiltration, and tubular atrophy were observed (B). Renal tissue damage was reduced by CoQ10 treatment. Scale bars, 100 µm. Data represent means ± se (n = 8). One-way ANOVA was used to analyze the data. @P < 0.05 vs. Tac group.

Figure 7

Effect of CoQ10 on Tac-induced oxidative stress and apoptosis in a rat model. Representative images and quantification of immunopositivity for 8-OHdG (A–D, N) and 4-HHE (E–H, O) and results of the TUNEL assay (I–L, P) in rat kidney tissue. The high 8-OHdG and 4-HHE immunoreactivity induced by Tac was decreased by coadministration of CoQ10. M) Daily urinary 8-OHdG excretion. Tac-induced 8-OHdG excretion was reduced by CoQ10 coadministration. Arrowheads indicate TUNEL-positive cells. Scale bars, 50 µm. Data represent means ± se (n = 8). One-way ANOVA was used to analyze the data. &P < 0.05 vs. Vh group; $P < 0.05 vs. CoQ10 groups; #P < 0.05 vs. Tac group.

Figure 8

Transmission electron microscopy analysis of the proximal tubule in rat kidneys. A) Representative transmission electron micrographs of mitochondrial ultrastructure in proximal tubules. Asterisks indicate mitochondria. Scale bars, 1 µm. B, C) Quantitative analysis of mitochondrial area and number of mitochondria. BB, brush border. Data represent means ± se. &P < 0.05 vs. Vh group; $P < 0.05 vs. Tac groups.

Figure 9

3D reconstruction of mitochondria in proximal tubules. A) Representative transmission electron micrographs and 3D reconstruction of mitochondria, revealing mitochondrial morphology. The reconstruction was based on a series of about 20 sections cut using ultramicrotome. Scale bars, 200 nm. B, C) Quantitative analysis of mitochondrial volume (B) and surface area (C). SA, surface; V, volume. Data represent means ± se. &P < 0.05 vs. Vh group; $P < 0.05 vs. Tac groups.