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, 119 (5), 1275-85

Regulation of Mitochondrial Dynamics in Acute Kidney Injury in Cell Culture and Rodent Models

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Regulation of Mitochondrial Dynamics in Acute Kidney Injury in Cell Culture and Rodent Models

Craig Brooks et al. J Clin Invest.

Abstract

The mechanism of mitochondrial damage, a key contributor to renal tubular cell death during acute kidney injury, remains largely unknown. Here, we have demonstrated a striking morphological change of mitochondria in experimental models of renal ischemia/reperfusion and cisplatin-induced nephrotoxicity. This change contributed to mitochondrial outer membrane permeabilization, release of apoptogenic factors, and consequent apoptosis. Following either ATP depletion or cisplatin treatment of rat renal tubular cells, mitochondrial fragmentation was observed prior to cytochrome c release and apoptosis. This mitochondrial fragmentation was inhibited by Bcl2 but not by caspase inhibitors. Dynamin-related protein 1 (Drp1), a critical mitochondrial fission protein, translocated to mitochondria early during tubular cell injury, and both siRNA knockdown of Drp1 and expression of a dominant-negative Drp1 attenuated mitochondrial fragmentation, cytochrome c release, caspase activation, and apoptosis. Further in vivo analysis revealed that mitochondrial fragmentation also occurred in proximal tubular cells in mice during renal ischemia/reperfusion and cisplatin-induced nephrotoxicity. Notably, both tubular cell apoptosis and acute kidney injury were attenuated by mdivi-1, a newly identified pharmacological inhibitor of Drp1. This study demonstrates a rapid regulation of mitochondrial dynamics during acute kidney injury and identifies mitochondrial fragmentation as what we believe to be a novel mechanism contributing to mitochondrial damage and apoptosis in vivo in mouse models of disease.

Figures

Figure 1
Figure 1. Mitochondrial fragmentation following ATP depletion and cisplatin treatment in RPTCs.
RPTCs were transfected with MitoRed to fluorescently label mitochondria. The cells were then incubated with 10 mM azide in glucose-free medium to induce ATP depletion or treated with 20 μM cisplatin in cell culture medium. Mitochondrial morphology in MitoRed-labeled cells was evaluated by fluorescence microscopy to determine the percentage of cells that fragmented mitochondria. Apoptosis was assessed in these cells by cellular and nuclear morphology. (A) Representative images of mitochondrial morphology. Left panel, an untreated control RPTC showing long filamentous mitochondria with a thread-like appearance. Right panel, an azide-treated (3 hours) cell showing shortened punctate mitochondria. Scale bars: 5 μm. (B) Time courses of mitochondrial fragmentation and apoptosis during azide-induced ATP depletion. (C) Time courses of mitochondrial fragmentation and apoptosis during cisplatin incubation. Data in B and C are presented as mean ± SD; n = 3.
Figure 2
Figure 2. Inhibition of mitochondrial fragmentation and membrane permeabilization by Bcl2 and not by VAD.
Wild-type and Bcl2-overexpressing RPTCs were transfected with MitoRed to label mitochondria. The cells were then treated with azide (10 mM, 3 hours) or cisplatin (20 μM, 16 hours) in the absence or presence of 100 μM VAD. Mitochondrial morphology in individual cells was evaluated by fluorescence microscopy to determine the percentage of cells with mitochondria fragmentation. (A) Mitochondrial fragmentation during azide-induced ATP depletion. (B) Mitochondrial fragmentation during cisplatin incubation. (C) Cytochrome c (Cyt. c) release during azide treatment. Cells were fractionated to collect cytosolic fraction for immunoblot analysis of cytochrome c. Data in A and B are presented as mean ± SD; n ≥ 3. *P < 0.05, significantly different from untreated control; #P < 0.05, significantly different from azide- or cisplatin-treated RPTCs. Results show that Bcl2 (but not caspase inhibitors) can suppress mitochondrial fragmentation and outer membrane permeabilization. Ctrl, control.
Figure 3
Figure 3. Drp1 translocation to mitochondria during azide-induced ATP depletion.
(A) Immunoblot analysis of Drp1 in mitochondrial and cytosolic fractions. RPTCs were subjected to 0 to 3 hours of 10 mM azide treatment for ATP depletion and then fractionated into mitochondrial and cytosolic fractions for immunoblot analysis of Drp1. (B) Dual immunofluorescence staining of Drp1 and Fis1 in azide-treated cells. After 3 hours of ATP depletion by azide treatment, RPTCs were fixed for immunofluorescence of Drp1 and Fis1. Drp1 and Fis1 signals were examined by confocal microscopy, and both separate and merged images are shown. The results show the translocation of a portion of Drp1 to mitochondria, overlapping with Fis1 during ATP depletion. Scale bars: 5 μm.
Figure 4
Figure 4. Suppression of mitochondrial fragmentation during RPTC injury by DN-Drp1.
RPTCs were cotransfected with MitoRed and DN-Drp1 or empty vector. Cells were then incubated with 10 mM azide for 3 hours or 20 μM cisplatin for 16 hours. (A) Effects of DN-Drp1 on mitochondrial fragmentation during azide-induced ATP depletion. (B) Effects of DN-Drp1 on mitochondrial fragmentation during cisplatin treatment. (C) Representative images of mitochondria. Left upper panel, untreated control cells showing long thread-like filamentous mitochondria; middle upper panel, fragmented mitochondria in vector-transfected cells after azide treatment; right upper panel, cells transfected with DN-Drp1 retaining filamentous mitochondria after azide treatment. Higher-magnification images of the framed areas are shown in the bottom panels. Scale bars: 5 μm (upper panels); 1 μm (lower panels). Data in A and B are presented as mean ± SD; n ≥ 3. *P < 0.05, significantly different from untreated control; #P < 0.05, significantly different from treated group transfected with empty vector.
Figure 5
Figure 5. Inhibition of cytochrome c release during ATP depletion by DN-Drp1.
RPTCs were cotransfected with MitoRed and DN-Drp1 or empty vector and then treated with 10 mM azide for 3 hours. The cells were fixed for immunofluorescence of cytochrome c or Bax. The examination was focused on the transfected (MitoRed labeled) cells to determine the effects of DN-Drp1. (A) Representative images of MitoRed and cytochrome c staining. The staining was examined by confocal microscopy in the same cells. In untreated cells (control), cytochrome c staining colocalized with MitoRed in filamentous mitochondria. Following azide treatment, mitochondria in vector-transfected cells became fragmented and cytochrome c was released into the cytosol. Cells transfected with DN-Drp1 retained their filamentous mitochondria, and cytochrome c was retained in mitochondria. Scale bars: 5 μm. (B) Quantification of the effects of DN-Drp1 on cytochrome c release. The localization of cytochrome c in MitoRed-transfected cells was evaluated to determine the percentage of cells that released cytochrome c into cytosol. (C) Quantification of the effects of DN-Drp1 on Bax translocation to mitochondria. The localization of Bax in MitoRed-transfected cells was evaluated to determine the percentage of cells that showed Bax accumulation in mitochondria. Data in B and C are presented as mean ± SD; n ≥ 3. *P < 0.05, significantly different from untreated control; #P < 0.05, significantly different from azide-treated cells that were transfected with empty vector.
Figure 6
Figure 6. Inhibition of ATP depletion–induced apoptosis by DN-Drp1.
(A) Effects of DN-Drp1 on azide-induced apoptosis. RPTCs were cotransfected with GFP and DN-Drp1 or empty vector. Cells were then treated with 10 mM azide for 3 hours followed by 2 hours recovery. After treatment, cells were subjected to TUNEL assay. Cells were examined by fluorescence microscopy to determine the percentage of apoptosis (TUNEL positive) in transfected (GFP labeled) cells. (B) Representative cell morphology. RPTCs were transfected with wild-type or DN-Drp1 and then subjected to 3 hours of azide treatment followed by 2 hours recovery. Cells were stained with Hoechst 33342 and examined by fluorescence microscopy. Scale bars: 5 μm. (C) Effects of DN-Drp1 on cisplatin-induced apoptosis. RPTCs were cotransfected with GFP and DN-Drp1 or empty vector and then incubated with 20 μM cisplatin for 16 hours. Cells were stained with Hoechst 33342 for examination by fluorescence microscopy to determine the percentage of apoptosis in transfected (GFP labeled) cells. Data in A and C are presented as mean ± SD; n ≥ 3. *P < 0.05, significantly different from untreated control; #P < 0.05, significantly different from vector-transfected cells treated with azide or cisplatin.
Figure 7
Figure 7. siRNA knockdown of Drp1 inhibits mitochondrial fragmentation, cytochrome c release, and apoptosis following ATP depletion in RPTCs.
(A) RPTCs were transfected with Drp1 short hairpin siRNA to select 2 stable cell lines: R3 and R24. Knockdown of Drp1 in R3 and R24 cells was verified by immunoblot analysis. (B) RPTCs, R3 cells, and R24 cells were transfected with MitoRed and then incubated with 10 mM azide for 3 hours. Cells were examined by fluorescence microscopy to enable counting of cells with mitochondrial fragmentation. (C) RPTCs, R3 cells, and R24 cells were incubated with 10 mM azide for 3 hours followed by 2 hours recovery to evaluate apoptosis by morphological criteria. (D) RPTCs, R3 cells, and R24 cells were incubated with 10 mM azide for 3 hours. Cells were then fractionated to collect the cytosolic fraction for immunoblot analysis of cytochrome c. Data in B and C are presented as mean ± SD; n ≥ 3. *P < 0.05, significantly different from untreated control RPTCs; #P < 0.05, significantly different from azide-treated RPTCs.
Figure 8
Figure 8. Mitochondrial fragmentation and its inhibition by DN-Drp1 in primary cultures of proximal tubular cells.
Proximal tubular cells were isolated from renal cortex of male C57BL/6 mice for primary culture. Primary cells were cotransfected with MitoRed and DN-Drp1 or empty vector. Cells were then incubated with 50 μM cisplatin for 24 hours. Mitochondrial morphology was examined by fluorescence microscopy. Apoptosis in transfected cells was evaluated by morphological criteria. (A) Representative mitochondrial morphology. Scale bars: 5 μm. Insets show higher magnification of the framed areas. (B) Effects of DN-Drp1 on mitochondrial fragmentation. (C) Effects of DN-Drp1 on apoptosis. Data in B and C are presented as mean ± SD; n ≥ 3. *P < 0.05, significantly different from untreated control; #P < 0.05, significantly different from cisplatin-treated vector-transfected cells.
Figure 9
Figure 9. 2D EM analysis of mitochondrial fragmentation in kidney tissues.
C57BL/6 mice (male, ~8 weeks) were subjected to 30 minutes of bilateral renal ischemia followed by 15 minutes of reperfusion (Ischemia) or control sham operation (Ctrl). Kidneys were fixed in situ via vascular perfusion and processed for EM. (A) EM micrographs of a control and an ischemically injured proximal tubular cell. Scale bars: 1 μm. Asterisks indicate elongated (>2 μm) mitochondria. (B) Quantification of mitochondrial fragmentation. Mitochondrial length was measured in individual tubular cells to determine the percentage of cells that showed filamentous mitochondria less than 1% long (>2 μm). A total of 90 cells in control and 160 cells in ischemic kidneys from 4 animals were evaluated.
Figure 10
Figure 10. 3D image of mitochondria in control and ischemically injured tubular cells.
C57BL/6 mice (male, ~8 weeks) were subjected to 30 minutes of bilateral renal ischemia followed by 15 minutes of reperfusion or control sham operation. Kidneys were fixed in situ via vascular perfusion and processed to collect 100 serial sections of a representative region at 45 nm/section for EM. EM micrographs of serial section no. 50 were shown for 2D image. For 3D image, EM images of the 100 serial sections were aligned for 3D reconstruction using the Reconstruct software. (A) 2D EM image of a control tubular cell. (B) 3D EM image of the same control cell as shown in A. (C) 2D EM image of an ischemically injured tubular cell. (D) 3D EM image of the same ischemic cell as shown in C. Note: The numbered mitochondria shown in A and B correspond respectively with those in C and D. In addition, some numbered mitochondria in 2D images are masked in the 3D images.
Figure 11
Figure 11. Amelioration of ischemic renal injury and tubular apoptosis by mdivi-1, a pharmacological inhibitor of Drp1.
C57BL/6 mice were injected with 50 mg/kg mdivi-1 or vehicle solution for 1 hour and then subjected to 30 minutes of bilateral renal ischemia followed by 48 hours of reperfusion. Control animals were subjected to sham operation without renal ischemia. Blood samples and renal tissues were collected for analysis. (A) Serum creatinine. (B) BUN. (C) Representative renal histology. Insets show higher magnification. Scale bars: 80 μm; 20 μm (insets). (D) Quantification of tubular damage. The percentage of damaged renal tubules was determined for each animal for histology scoring as described in Methods. (E) Tubular apoptosis. Renal tissues were subjected to TUNEL assay to enable counting positive cells to indicate apoptosis. Data are presented as mean ± SD; n ≥ 5. *P < 0.05, significantly different from sham control. #P < 0.05, significantly different from ischemic group injected with vehicle solution.

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