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, 299 (1), F199-206

Drp1 Dephosphorylation in ATP Depletion-Induced Mitochondrial Injury and Tubular Cell Apoptosis

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Drp1 Dephosphorylation in ATP Depletion-Induced Mitochondrial Injury and Tubular Cell Apoptosis

Sung-Gyu Cho et al. Am J Physiol Renal Physiol.

Abstract

Recent studies revealed a striking morphological change of mitochondria during apoptosis. Mitochondria become fragmented and notably, the fragmentation contributes to mitochondrial outer membrane permeabilization and consequent release of apoptotic factors. In renal tubular cells, mitochondrial fragmentation involves the activation of Drp1, a key mitochondrial fission protein. However, it is unclear how Drp1 is regulated during tubular cell apoptosis. In this study, we examined Drp1 regulation during tubular cell apoptosis following ATP depletion. Rat kidney proximal tubular cells (RPTC) were subjected to azide treatment or severe hypoxia in glucose-free medium to induce ATP depletion. During ATP depletion, Drp1 was shown to be dephosphorylated at serine-637. Drp1 dephosphorylation could be suppressed by cyclosporine A and FK506, two calcineurin inhibitors. Importantly, cyclosporine A and FK506 could also prevent mitochondrial fragmentation, Bax accumulation, cytochrome c release, and apoptosis following ATP depletion in RPTC. The results suggest that calcineurin-mediated serine-637 dephosphorylation is involved in Drp1 activation during ATP depletion in renal tubular cells. Upon activation, Drp1 contributes to mitochondrial fragmentation and outer membrane permeabilization, resulting in the release of apoptogenic factors and apoptosis.

Figures

Fig. 1.
Fig. 1.
Dephosphorylation of Drp1 at serine-637 during ATP depletion. Rat kidney proximal tubular cell line (RPTC) cells were subjected to 0–3 h of ATP depletion with 10 mM azide in glucose-free buffer. One group of the cells was returned to full culture medium for 2 h of recovery. Whole cell lysates were collected for immunoblot analysis of phosphorylated (serine-637) Drp1, total Drp1, and β-actin. The blots are representative of 3 experiments.
Fig. 2.
Fig. 2.
Inhibition of Drp1 dephosphorylation during ATP depletion by cyclosporin A (CsA) and FK506. RPTC cells were pretreated for 30 min without or with indicated concentrations (in μM) of CsA or FK506. The cells were then subjected to ATP depletion by severe hypoxia (A) or 10 mM azide treatment (B) in a glucose-free buffer with or without CsA or FK506. Whole cell lysates were collected for immunoblot analysis of phosphorylated (serine-637) Drp1, total Drp1, and β-actin. The blots are representative of 3–4 experiments. C: RPTC cells were treated with azide for 3 h with or without 10 μM CsA or FK506 to collect lysate to measure ATP and protein. Cell ATP was expressed as nmol per mg protein. Data are expressed as means ± SD, n = 4; *P < 0.01 vs. control.
Fig. 3.
Fig. 3.
Inhibition of mitochondrial fragmentation during ATP depletion by CsA and FK506. RPTC cells were transfected with MitoRed to fluorescently label mitochondria. The cells were then pretreated for 30 min with or without CsA or FK506. Finally, the cells were subjected to 3 h of ATP depletion with 10 mM azide in glucose-free buffer in the absence or presence of CsA or FK506. A: an untreated control RPTC cell showing long mitochondria with a rod- or worm-like appearance. B: an azide-treated cell showing fragmented or punctate mitochondria. C, D: effects of CsA and FK506 on mitochondrial fragmentation. The percentage of cells with fragmented mitochondria was determined by cell counting. Data are means ± SD, n = 3 (≥100 cells were counted in 3 independent experiments).
Fig. 4.
Fig. 4.
CsA and FK506 partially decrease Bax translocation during ATP depletion. After pretreatment with CsA or FK506 for 30 min, RPTC cells were subjected to 3 h of ATP depletion with 10 mM azide in the presence or absence of CsA or FK506. The cells were then fractionated into cytosolic fraction and membrane-bound organellar fraction for immunoblot analysis of Bax. A: immunoblot analysis of cytosolic and organellar Bax. B: results of densitometric analysis of Bax blots. Note: the experimental condition represented by each bar in B was the same as the aligned lane in A. Data are means ± SD, n = 4.
Fig. 5.
Fig. 5.
Effects of CsA and FK506 on cytochrome c (cyt.c) release during ATP depletion. RPTC cells pretreated with CsA and FK506 were subjected to 3 h of ATP depletion in the presence or absence of CsA or FK506. A: immunoblot analysis of cyt.c release. The cells were fractionated into cytosolic and membrane-bound organellar fraction for immunoblot analysis of cyt.c. B: results of densitometric analysis of cyt.c blots. Note: the experimental condition represented by each bar in B was the same as the aligned lane in A. Data are means ± SD, n = 4. C: immunofluorescence of cyt.c. Cells were fixed for immunofluorescence staining of cyt.c. The cells were also stained with Hoechest 33342 to reveal nuclei. Shown are merged images of cyt.c and nuclear staining. Insets: cells at higher magnification. D: quantification of the cells in immunofluorescence analysis that released cyt.c. The cells with cytosolic staining of cyt.c were counted to determine the percentage of cells with cyt.c release. Data are means ± SD (n = 3). #Significantly different from control. *Significantly different from azide-only treated group.
Fig. 6.
Fig. 6.
Effects of CsA and FK506 on RPTC apoptosis following ATP depletion. RPTC cells were pretreated with or without 10 μM CsA or FK506 for 30 min. The cells were then subjected to 3 h of ATP depletion with 10 mM azide in glucose-free buffer in the presence or absence of CsA or FK506, followed by 2 h of recovery in full culture medium. A: cell morphology recorded by phase contrast microscopy. B, C: quantification of the effects of CsA (B) and FK506 (C) on RPTC apoptosis. Cells with typical apoptotic morphology were counted to determine the percentage of apoptosis. D: caspase activity. Cell lysate was collected to determine caspase activity. E: long-term cell survival. After azide treatment, the cells were recovered for 24 h in full culture medium to determine cell viability by MTT assay. Data are expressed as means ± SD (n = 3). #Significantly different from control group without azide treatment. *Significantly different from azide-only treated group.

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