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. 2019 Sep:26:101254.
doi: 10.1016/j.redox.2019.101254. Epub 2019 Jun 11.

PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation

Qisheng Lin  1 , Shu Li  1 , Na Jiang  1 , Xinghua Shao  1 , Minfang Zhang  1 , Haijiao Jin  1 , Zhen Zhang  1 , Jianxiao Shen  1 , Yijun Zhou  1 , Wenyan Zhou  1 , Leyi Gu  1 , Renhua Lu  1 , Zhaohui Ni  2
Affiliations

PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation

Qisheng Lin et al. Redox Biol. 2019 Sep.

Abstract

Contrast-induced acute kidney injury (CI-AKI) occurs in more than 30% of patients after intravenous iodinated contrast media and causes serious complications, including renal failure and mortality. Recent research has demonstrated that routine antioxidant and alkaline therapy failed to show benefits in CI-AKI patients with high risk for renal complications. Mitophagy is a mechanism of selective autophagy, which controls mitochondrial quality and mitochondrial reactive oxygen species (ROS) through degradation of damaged mitochondria. The role of mitophagy and its regulation of apoptosis in CI-AKI are poorly understood. In this study, we demonstrated that mitophagy was induced in renal tubular epithelial cells (RTECs) during CI-AKI, both in vivo and in vitro. Meanwhile, contrast media-induced mitophagy was abolished when silencing PINK1 or PARK2 (Parkin), indicating a dominant role of the PINK1-Parkin pathway in mitophagy. Moreover, mitochondrial damage, mitochondrial ROS, RTEC apoptosis, and renal injury under contrast exposure were more severe in PINK1- or PARK2-deficient cells and mice than in wild-type groups. Functionally, PINK1-Parkin-mediated mitophagy prevented RTEC apoptosis and tissue damage in CI-AKI through reducing mitochondrial ROS and subsequent NLRP3 inflammasome activation. These results demonstrated that PINK1-Parkin-mediated mitophagy played a protective role in CI-AKI by reducing NLRP3 inflammasome activation.

Keywords: Acute kidney injury; Apoptosis; Contrast media; Mitochondrial ROS; Mitophagy; NLRP3 inflammasome.

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Figures

Fig. 1
Fig. 1
Mitophagy and activation of the NLRP3 inflammasome were induced in renal tubular epithelial cells in CI-AKI mice. (A) Diagrammatic representation of the inducible strategy in CI-AKI mice (Model + Iohexol, 10 μL/g), negative control mice (Model + NS, 10 μL/g), and control mice. (B–E) Immunoblot analysis and quantification of MFN1, DRP1, SQSTM1, LC3B I/II, and COX IV in the kidneys. (F, G) Representative images of immunofluorescence double-labelling autophagosomes (LC3B) and mitochondria outer membrane protein (VDAC) in WT mice. The percentage of renal tubules with mitophagosome formation was quantified. Scale bar: 50 μm. (H) Representative TEM images of a mitophagosome (red arrow) and a mitophagolysosome (blue arrow) in renal tubular epithelial cells after iohexol injection. Scale bar: 500 nm. (I, J) Fresh kidneys of WT mice were fractionated to collect cytosolic and mitochondrial fraction for immunoblot analysis of cytochrome c release. (K–N) Immunoblot analysis and quantification of NLRP3, caspase-1 p20, IL-1β p17, cleaved caspase-3, Bax, and Bcl-2 in the kidneys of WT mice. Data were presented as mean ± TEM. n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
PINK1 and Parkin mediated mitophagy and protected the aggravation of renal injury in CI-AKI mice. (A, B) Immunoblot analysis and quantification of PINK1 and Parkin in the kidneys. (C) Diagrammatic representation of CI-AKI in different groups of mice: WT, PINK1 knockout and PARK2 knockout. (D, E) Representative images and quantification of immunofluorescence double-labelling LC3B and VDAC in different groups of CI-AKI mice. Scale bar:50 μm. (F, G) PINK1-Parkin mediated mitophagy was showed by immunofluorescence and quantification of double-labelling LC3B and Parkin. Scale bar:50 μm. (H, I) Kidney injury was also measured by serum creatinine and kidney KIM-1 mRNA level of different groups of mice. (J, K) Representative histology and pathological score of tubular damage in kidney cortex by H-E staining. The tubular injury was indicated by arrows. Scale bar:50 μm. Data were presented as mean ± TEM. n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
PINK1 or PARK2 deficiency enhanced contrast-induced mitochondrial ROS production and NLRP3 inflammasome activation in renal tubular epithelial cells in vivo. (A, B) Representative TEM images of mitochondrial morphology in renal tubular epithelia cells. Data were shown as a dot plot of the percentage of damaged mitochondria from 10 images of each group. Scale bar: 1 μm. (C, D) Fresh kidneys of different groups of CI-AKI mice were fractionated to collect cytosolic and mitochondrial fraction for immunoblot analysis of cytochrome c release. (E) Relative mitochondrial DNA content (mtDNA: nDNA) of kidneys. (F) Mitochondrial ROS is assessed by MnSOD activity of fresh kidneys. (G, H) Representative images and quantification of 8-OHdG staining in kidney cortex. Scale bar: 50 μm. (I, J) Immunoblot analysis and quantification of NLRP3, caspase-1 p20, and IL-1β p17 in the kidneys of different groups of CI-AKI mice. (K–M) Representative images and quantification of immunofluorescence staining of caspase-1 and IL-1β in kidney tubules. Scale bar: 50 μm. Data were presented as mean ± TEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
PINK1 or PARK2 deficiency increased apoptosis of renal tubular epithelial cells in CI-AKI mice. (A, B) Immunoblot analysis and quantification of cleaved caspase-3, Bax, and Bcl-2 in the kidneys of different CI-AKI mice. (C, D) Apoptosis was accessed by TUNEL staining of kidney cortex and quantification of TUNEL-positive cells. Scale bar: 50 μm. Data were presented as mean ± TEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5
Fig. 5
Contrast media induced PINK1-Parkin–mediated mitophagy, NLRP3 inflammasome activation, and apoptosis in HK-2 cells. HK-2 cells are incubated in DMEM/F12 containing iohexol (20 mg I/ml) for 72 h. (A, B) Immunoblot analysis and quantification of MFN1 and DRP1. (C, D) Representative images and quantification of immunofluorescence double-labelling LC3B and mitochondrial marker (MitoTracker). Scale bar: 10 μm. (E, F) Representative TEM images of mitochondrial morphology and mitophagosomes (red arrow). Data were shown as a dot plot of the number of mitophagosomes from 10 images of each group. Scale bar: 500 nm. (G, H) Immunoblot analysis of PINK, Parkin, SQSTM1, LC3B I/II, and COX IV. (I–L) Representative images and quantification of immunofluorescence double-labelling PINK1/Parkin and MitoTracker. (M-P) Immunoblot analysis and quantification of NLRP3, caspase-1 p20, IL-1β p17, cleaved caspase-3, Bax, and Bcl-2. Data were presented as mean ± TEM. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6
Fig. 6
NLRP3 inflammasome and apoptosis increased in 3-MA–pretreated HK-2 cells in response to contrast media through inhibiting mitophagy. HK-2 cells are pretreated in 3-MA (5 nM) for 4 h and then incubated with iohexol (20 mg I/ml) for 72 h. (A, B) Immunoblot analysis and quantification of PINK1, Parkin, SQSTM1, LC3B I/II, and COX IV. (C–H) Immunoblot analysis and quantification of MnSOD, NLRP3, caspase-1 p20, IL-1β p17, cleaved caspase-3, Bax, and Bcl-2. (I, J) Representative images and quantification of TUNEL staining of HK-2 cells pretreated in 3-MA. Scale bar: 100 μm. Data were presented as mean ± TEM. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 7
Fig. 7
Silencing PINK1 or PARK2 decreased mitophagy in contrast-treated HK-2 cells. HK-2 cells are subjected to iohexol (20 mg I/ml) for 72 h at 24 h after transfection with negative control siRNA (SiNC), PINK1 siRNA (SiPINK1), or PARK2 siRNA (SiPARK2). (A, B) Immunoblot analysis of PINK1, Parkin, SQSTM1, LC3B I/II, and COX IV. (C, D) Representative images and quantification of immunofluorescence double-labelling LC3B and MitoTracker in SiNC, SiPINK1, or SiPARK2 cells treated with iohexol. Scale bar: 50 μm. (E, F) Representative images and quantification of double-labelling LC3B and Parkin. Scale bar: 50 μm. Data were presented as mean ± TEM. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8
Fig. 8
Silencing PINK1 or PARK2 increased mitochondrial ROS production and subsequently activated the NLRP3 inflammasome in contrast-treated HK-2 cells. HK-2 cells were transfected with negative control siRNA, PINK1 siRNA, or PARK2 siRNA. After transfection for 8 h, cells were pretreated with MitoTEMPO (100 μM) for 4 h and then cultured with iohexol for 72 h. (A–C) Representative images and quantification of mitochondrial ROS (MitoSOX) and 8-OHdG in HK-2 cells. Scale bar: 50 μm (MitoSOX) and 25 μm (8-OHdG). (D) Mitochondrial ROS was also assessed by MnSOD activity (U/mg protein) of HK-2 cells. (E, F) Immunoblot analysis and quantification of NLRP3, caspase-1 p20, and IL-1β p17 in SiNC, SiPINK1, and SiPARK2 cells with iohexol. (G, H). Representative images and quantification of immunofluorescence staining caspase-1 and IL-1β in HK-2cells. Scale bar: 50μm. (I–J) Immunoblot analysis and quantification of NLRP3, caspase-1 p20, and IL-1β p17 in HK-2 cells pretreated with or without MitoTEMPO, after silencing PINK1 or PARK2 in response to iohexol. Data were presented as mean ± TEM. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9
Fig. 9
Silencing PINK1 or PARK2 sensitised HK-2 cells to contrast-induced apoptosis, which was reduced by NLRP3 inflammasome inhibition. After transfection with SiNC, SiPINK1, or SiPARK2 for 8 h, HK-2 cells were pretreated with MCC950 (10 μM) for 4 h and then cultured with iohexol for 72 h. (A, B) Immunoblot analysis and quantification of cleaved caspase-3, Bax, and Bcl-2 in SiNC, SiPINK1, or SiPARK2 cells with iohexol. (C, D) Immunoblot analysis and quantification of cleaved caspase-3 in HK-2 cells pretreated with or without MCC950 after silencing PINK1 or PARK2 in response to iohexol. (E–G) Representative images and quantification of cell apoptosis by flow cytometry and TUNEL staining of HK-2 cells. Scale bar: 100 μm. Data were presented as mean ± TEM. *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 10
Fig. 10
Schematic representation of mitophagy, mitochondrial ROS, and NLRP3 inflammasome in CI-AKI. Contrast media (iohexol) causes the mitochondrial damage of renal tubular epithelial cells, which induces mitochondrial ROS and NLRP3 inflammasome activation. PINK1-Parkin–mediated mitophagy is also activated to repair the damaged mitochondria through reducing mitochondrial ROS production and NLRP3 inflammasome activation, which decreases apoptosis of renal tubular epithelial cells and kidney injury in CI-AKI. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Supplemental Figure legend
Supplemental Figure
(1) Immunoblot analysis and quantification of MnSOD in kidneys of Ctrl, Model, Model + Iohexol mice. (2) Immunoblot analysis of PINK1 and Parkin in PINK1-/- or PARK2-/- mice, respectively. (3) Immunoblot analysis and quantification of MnSOD in kidneys of WT, PINK1-/- and PARK2-/- CI-AKI mice. (4). Quantification of MitoSOX relative fluorescence units (RFU) after Iohexol intervention. (5) Immunoblot analysis and quantification of MnSOD in HK-2 cells treated with control medium, Mannitol and Iohexol. (6) Immunoblot analysis and quantification of MnSOD in HK-2 cells with Iohexol after transfection with SiNC, SiPINK1and SiPARK2. Data were presented as mean ± TEM. *p<0.05, **p<0.01, ***p<0.001.
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