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, 42 (12), 893-905

Augmenter of Liver Regeneration Alleviates Renal Hypoxia-Reoxygenation Injury by Regulating Mitochondrial Dynamics in Renal Tubular Epithelial Cells

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Augmenter of Liver Regeneration Alleviates Renal Hypoxia-Reoxygenation Injury by Regulating Mitochondrial Dynamics in Renal Tubular Epithelial Cells

Rui-Ting Long et al. Mol Cells.

Abstract

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion processes that closely related to their function. Disruption of mitochondrial dynamics has been demonstrated in acute kidney injury (AKI), which could eventually result in cell injury and death. Previously, we reported that augmenter of liver regeneration (ALR) alleviates renal tubular epithelial cell injury. Here, we gained further insights into whether the renoprotective roles of ALR are associated with mitochondrial dynamics. Changes in mitochondrial dynamics were examined in experimental models of renal ischemia-reperfusion (IR). In a model of hypoxia-reoxygenation (HR) injury in vitro , dynamin-related protein 1 (Drp1) and mitochondrial fission process protein 1 (MTFP1), two key proteins of mitochondrial fission, were downregulated in the Lv-ALR + HR group. ALR overexpression additionally had an impact on phosphorylation of Drp1 Ser637 during AKI. The inner membrane fusion protein, Optic Atrophy 1 (OPA1), was significantly increased whereas levels of outer membrane fusion proteins Mitofusin-1 and -2 (Mfn1, Mfn2) were not affected in the Lv-ALR + HR group, compared with the control group. Furthermore, the mTOR/4E-BP1 signaling pathway was highly activated in the Lv-ALR + HR group. ALR overexpression led to suppression of HR-induced apoptosis. Our collective findings indicate that ALR gene transfection alleviates mitochondrial injury, possibly through inhibiting fission and promoting fusion of the mitochondrial inner membrane, both of which contribute to reduction of HK-2 cell apoptosis. Additionally, fission processes are potentially mediated by promoting tubular cell survival through activating the mTOR/4E-BP1 signaling pathway.

Keywords: acute kidney injury; augmenter of liver regeneration; hypoxia-reoxygenation; ischemia-reperfusion; mitochondrial dynamics.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. Renal dysfunction, histologic alterations and morphological changes in mitochondria in vivo following renal IR injury
(A and B) Mice were subjected to IR or sham operation, and BUN and Scr levels measured 24 h after reperfusion (n = 3–4 per group). Parametric data were analyzed with the Student’s t-test and presented as the mean ± SD (*P < 0.05, **P < 0.01, compared to the sham group). (C) Representative PAS-stained renal sections from sham and IR groups (×400 magnification, scale bars = 100 μm). (D) Electron microscopic analysis of mitochondrial morphology in sham and IR groups (scale bars = 2 μm). Asterisks indicate elongated (> 2 μm) mitochondria.
Fig. 2
Fig. 2. Expression of mitochondrial dynamic proteins in kidney
(A) Drp1 immunohistochemistry in sham-operated mice and 24 h after reperfusion in IR mice. (B–D) Representative images of immunohistochemical staining of OPA1, Mfn1, and Mfn2 in sections of kidney tissues from sham-operated and IR mice (×200 magnification). Average optical quantitative values for immunohistochemical images. Parametric data were analyzed with the Student’s t-test. Data are presented as the mean ± SD (*P < 0.05, compared to the sham group).
Fig. 3
Fig. 3. HR treatment induces mitochondrial fission in vitro
Western blot analysis of Drp1 expression in HK-2 cells. Parametric data were analyzed with ANOVA followed by Tukey’s post hoc test. Bar graphs represent the mean ± SD (**P < 0.01 vs the normal group).
Fig. 4
Fig. 4. Effects of ALR gene transfection on ALR expression
(A and B) Western blot analysis of expression of exogenously delivered ALR in HK-2 cells using an anti-Flag antibody. Parametric data were analyzed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (** P < 0.01, vs Lv-vector or Lv-vector + HR group).
Fig. 5
Fig. 5. Overexpression of ALR inhibits mitochondrial fission in renal HR injury in vitro
(A) Schematic representation of the MTFP1 target protein interaction network and confidence interaction scores of potential functionally associated proteins. The protein interaction network was constructed using STRING v10. (B) Western blot analysis of Drp1 and p-Drp1 (Ser637) levels in HK-2 cells. (C and D) Drp1 expression was normalized to that of β-actin and the p-Drp1: total Drp1 ratio quantified based on immunoblot images. (E and F) MTFP1 expression was verified via western blots and normalized to that of β-actin. Parametric data were analyzed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, compared to the normal group; #P < 0.05, ##P < 0.01, compared to the Lv-vector + HR group).
Fig. 6
Fig. 6. Potential mechanisms underlying ALR-mediated control of mitochondrial dynamics
(A) Representative images from immunoblot assays against phospho-mTOR (Ser2448), mTOR, phospho-4E-BP1 (Ser65), 4E-BP1, and β-actin. (B and C) p-mTOR: total mTOR and p-4E-BP1: total 4E-BP1 ratios were quantified based on immunoblot images. Parametric data were analyzed with ANOVA followed by Tukey’s post hoc test. Bar graphs represent the mean ± SD (*P < 0.05, **P < 0.01 vs the normal group; #P < 0.05, ##P < 0.01, compared to the Lv-vector + HR group).
Fig. 7
Fig. 7. Effect of ALR overexpression on mitochondrial fusion
(A) OPA1 expression was determined using western blots and normalized to that of β-actin. Parametric data were obtained with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (*P < 0.05, **P < 0.01, vs the normal group; #P < 0.05, ##P < 0.01, compared to the Lv-vector + HR group). (B) Representative images from immunoblot assays against Mfn1, Mfn2, and β-actin. (C and D) Mfn1 and Mfn2 levels were normalized to that of β-actin. Parametric data were analyzed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (**P < 0.01, vs the normal group).
Fig. 8
Fig. 8. Overexpression of ALR suppresses HR-induced apoptosis of HK-2 cells
(A) Annexin V-APC/DAPI staining for detection of apoptotic HK-2 cells. (B) Mean percentage of positively stained cells. Parametric data were analyzed with ANOVA followed by Tukey’s post hoc test. Data are presented as the mean ± SD (**P < 0.01, ##P < 0.01).

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