Inhibiting NLRP3 inflammasome attenuates apoptosis in contrast-induced acute kidney injury through the upregulation of HIF1A and BNIP3-mediated mitophagy

Abstract

The pathogenetic mechanism of contrast-induced acute kidney injury (CI-AKI), which is the third most common cause of hospital-acquired AKI, has not been elucidated. Previously, we demonstrated that renal injury and cell apoptosis were attenuated in nlrp3 knockout CI-AKI mice. Here, we investigated the mechanism underlying NLRP3 inhibition-mediated attenuation of apoptosis in CI-AKI. The RNA sequencing analysis of renal cortex revealed that the nlrp3 or casp1 knockout CI-AKI mice exhibited upregulated cellular response to hypoxia, mitochondrial oxidation, and autophagy when compared with the wild-type (WT) CI-AKI mice, which indicated that NLRP3 inflammasome inhibition resulted in the upregulation of hypoxia signaling pathway and mitophagy. The nlrp3 or casp1 knockout CI-AKI mice and iohexol-treated HK-2 cells with MCC950 pretreatment exhibited upregulated levels of HIF1A, BECN1, BNIP3, and LC3B-II, as well as enhanced colocalization of LC3B with BNIP3 and mitochondria, and colocalization of mitochondria with lysosomes. Additionally, roxadustat, a HIF prolyl-hydroxylase inhibitor, protected the renal tubular epithelial cells against iohexol-induced injury through stabilization of HIF1A and activation of downstream BNIP3-mediated mitophagy in vivo and in vitro. Moreover, BNIP3 deficiency markedly decreased mitophagy, and also significantly exacerbated apoptosis and renal injury. This suggested the protective function of BNIP3-mediated mitophagy in CI-AKI. This study elucidated a novel mechanism in which NLRP3 inflammasome inhibition attenuated apoptosis and upregulated HIF1A and BNIP3-mediated mitophagy in CI-AKI. Additionally, this study demonstrated the potential applications of MCC950 and roxadustat in clinical CI-AKI treatment.Abbreviations: BNIP3: BCL2/adenovirus E1B interacting protein 3; Ctrl: control; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EGLN2/PHD1: egl-9 family hypoxia-inducible factor 2; HIF1A: hypoxia inducible factor 1, alpha subunit; H-E: hematoxylin and eosin; IL18: interleukin 18; IL1B: interleukin 1 beta; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mRNA: messenger RNA; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NLRP3: NLR family, pyrin domain containing 3; NS: normal saline; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RNA: ribonucleic acid; SEM: standard error of the mean; siRNA: small interfering RNA; TEM: transmission electron microscopy; TUBA/α-tubulin: tubulin, alpha; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VDAC: voltage-dependent anion channel; WT: wild-type.

Keywords: Acute kidney injury; NLRP3 inflammasome; contrast media; hypoxia inducible factor; mitophagy.

Figure 1.

NLRP3 or CASP1 deficiency alleviated iohexol-induced renal injury and apoptosis. Unilateral nephrectomy, dehydration, furosemide (10 mL/kg, tail vein) were used to establish mice model. CI-AKI mice were established by iohexol (10 mL/kg bodyweight; injected through the tail vein, Model+Iohexol), and negative control group was injected with the same volume of normal saline (Model). (A and B) Immunoblotting analysis and quantification of NLRP3, CASP1 p20, IL1B p17. (C) Diagrammatic representation of Ctrl and CI-AKI in different groups of mice: WT(C57BL/6), nlrp3 and casp1 knockout. (D) The renal function was evaluated by serum creatinine. (E and F) Representative histology and pathological tubular injury score in the renal cortex by H-E staining. The tubular injury was indicated by arrows. Scale bar: 50 μm. (G and H) Immunoblotting analysis and quantification of cleaved CASP3. (I and J) Apoptosis was also evaluated by TUNEL staining and quantification of TUNEL-positive cells. Scale bar: 50 μm. Data were presented as mean ± SEM. n = 3–5. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 2.

Renal cortex RNA-sequencing analysis of the WT, nlrp3^−/− and casp1^−/- CI-AKI mice. (A) PCA of RNA-sequencing results comparing nlrp3^−/− or casp1^−/- CI-AKI mice with WT CI-AKI mice. (B and C) KEGG analysis in nlrp3^−/− versus WT CI-AKI and casp1^−/- versus WT CI-AKI groups. GO set enrichment analysis on CC (D and E) and BP (F and G) comparing nlrp3^−/− or casp1^−/- with WT CI-AKI mice. (H and I) GO enrichment map of genes with nlrp3^−/− versus WT CI-AKI, casp1^−/- CI-AKI versus WT CI-AKI. n = 3

Figure 3.

Iohexol promoted mitophagy in the renal tubular epithelial cells through HIF1A and BNIP3. (A) Representative TEM images of a mitophagosome (arrow) in renal tubular epithelial cells in CI-AKI. Scale bar: 500 nm. (B-E) The colocalization of BNIP3 and autophagic flux was showed by immunofluorescence and quantification of BNIP3 and LC3B (autophagy marker). Mitophagosomes and Mitolysosomes were evaluated by LC3B and VDAC (mitochondrial marker), VDAC and LAMP1 (lysosomal marker), respectively. Scale bar: 20 μm. (F and G) Immunoblotting analysis and quantification of HIF1A, BECN1, BNIP3, LC3B-II. Data were presented as mean ± SEM. n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 4.

NLRP3 or CASP1 deficiency attenuated mitochondrial injury in the CI-AKI mice through HIF1A and BNIP3-mediated mitophagy. (A and B) Immunoblotting analysis and quantification of HIF1A, BECN1, BNIP3, LC3B-II. (C-F) Representative images and quantification of immunofluorescence double-labeling of LC3B and BNIP3, LC3B and VDAC, VDAC and LAMP1. Scale bar: 20 μm. (G) Representative TEM images of mitochondrial morphology in renal tubular epithelial cells of Ctrl and CI-AKI groups of WT, nlrp3^−/−, casp1^−/- mice. Mitophagosomes were indicated by arrows. Scale bar: 500 nm. (H and I) Immunoblotting analysis and quantification of EGLN2. Data were presented as mean ± SEM. n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 5.

MCC950 upregulated HIF1A and BNIP3-mediated mitophagy, and attenuated apoptosis in the HK-2 cells in response to iohexol treatment. HK-2 cells were incubated in DMEM/F-12 media with iohexol (20 mg I/mL) for 72 h. MCC950 (10 μM) pretreated cells 4 h before iohexol treatment. (A) Cell viability of iohexol-treated HK-2 cells with MCC950 was evaluated by CCK-8. (B and C) Immunoblotting analysis and quantification of HIF1A, BECN1, BNIP3, LC3B-II, VDAC. (D-F) Representative images and quantification immunofluorescence double-labeling LC3B and BNIP3, LC3B and MitoTracker, MitoTracker and LysoTracker in MCC950-pretreated HK-2 cells. Scale bar: 20 μm. (G and H) Immunoblotting analysis and quantification of cleaved CASP3. (I) Representative images of cell apoptosis by flow cytometry. Data were presented as mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 6.

Roxadustat activated HIF1A-BNIP3 signaling pathway and attenuated apoptosis in the CI-AKI mice. (A) Diagrammatic representation of roxadustat to Ctrl and CI-AKI mice. Briefly, the mice were intraperitoneally injected with roxadustat (10 mg/kg bodyweight/day) for 5 days. (B-D) The role of roxadustat on Ctrl and CI-AKI mice was evaluated by serum creatinine, H-E staining and tubular injury score. The tubular injury was indicated by arrows. Scale bar: 50 μm. (E and F) Immunoblotting analysis and quantification of HIF1A, BECN1, BNIP3, LC3B-II. (G-J) Representative images and quantification of immunofluorescence double-labeling LC3B and BNIP3, LC3B and VDAC, VDAC and LAMP1. Scale bar: 20 μm. (K) Representative TEM images of mitochondrial morphology in renal tubular epithelial cells. Scale bar: 500 nm. (L-O) Apoptosis was evaluated by immunoblotting analysis of cleaved CASP3 and TUNEL staining. Scale bar: 50 μm. (P) DEGs in renal cortex of nlrp3^−/−, casp1^−/- CI-AKI and roxadustat-treated CI-AKI in a Venn diagram. The number of overlapping genes was shown in the overlapping regions. n = 3–5. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 7.

Roxadustat protected the HK-2 cells against iohexol-induced apoptosis in vitro through the upregulation of mitophagy. Roxadustat (10 μM) pretreated cells 4 h before iohexol treatment. (A) Cell viability of iohexol-treated HK-2 cells with roxadustat was evaluated by CCK-8. (B and C) Immunoblotting analysis and quantification of HIF1A, BECN1, BNIP3, LC3B-II, VDAC. (D-G) Representative images and quantification of immunofluorescence double-labeling LC3B and BNIP3, LC3B and MitoTracker, MitoTracker and LysoTracker. Scale bar: 20 μm. (G and H) 3-MA (5 mM) with roxadustat pretreated HK-2 cells 4 h before iohexol treatment. Immunoblotting analysis of LC3B-II, VDAC and cleaved CASP3. (I) Representative images of cell apoptosis by flow cytometry. Data were presented as mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 8.

BNIP3 deficiency exacerbated mitochondrial damage and apoptosis in the CI-AKI mice. (A) Survival of WT CI-AKI and bnip3^−/− CI-AKI mice with iohexol treatment (10 mL/kg bodyweight) after 24 h (n = 10). (B) Diagrammatic representation of modified WT and bnip3^−/− CI-AKI models. Briefly, the dose of iohexol reduced to 5 mL/kg bodyweight. (C) The renal function was evaluated by serum creatinine. (D and E) Representative histology and pathological tubular injury score in the renal cortex by H-E staining. The tubular injury was indicated by arrows. Scale bar: 50 μm. (F and G) Representative IHC images and quantification of BNIP3 in kidney cortex. Scale bar: 50 μm. (H and I) Immunoblotting analysis and quantification of BNIP3 and LC3B-II. (J-L) Representative images and quantification of immunofluorescence double-labeling LC3B and VDAC, VDAC and LAMP1. Scale bar: 20 μm. (M) Representative TEM images of mitochondrial morphology in renal tubular epithelial cells of WT and bnip3^−/− mice. Scale bar: 500 nm. (N-Q) Apoptosis was measured by immunoblotting analysis of cleaved CASP3 and TUNEL staining of renal tubular epithelial cells of WT and bnip3^−/− CI-AKI mice. Scale bar: 50 μm. n = 3–5. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 9.

Silencing BNIP3 downregulated mitophagy in the HK-2 cells and exacerbated apoptosis. (A) Cell viability was evaluated by CCK-8. (B and C) Immunoblotting analysis and quantification of BNIP3, LC3B-II, VDAC. (D and E) Representative images and quantification of immunofluorescence double-labeling LC3B and MitoTracker. Scale bar: 20 μm. (F and G) Representative images and quantification of MitoTracker and LysoTracker. Scale bar: 20 μm. (H and I) Immunoblotting analysis and quantification of cleaved CASP3. (J) Representative images of cell apoptosis by flow cytometry. Data were presented as mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 10.

Schematic representation of NLRP3 inflammasome, HIF1A and BNIP3 mediated mitophagy, and apoptosis in CI-AKI. Contrast media (iohexol) activated NLRP3 inflammasome and increased HIF1A expression. HIF1A and BNIP3-mediated mitophagy further were upregulated by inhibiting NLRP3 inflammasome in vivo and in vitro. Additionally, roxadustat, a HIF prolyl-hydroxylase inhibitor, enhanced HIF1A-BNIP3-mediated mitophagy to alleviate iohexol-induced apoptosis. Both inhibition of NLRP3 inflammasome and stabilization of HIF1A, protected the renal tubular epithelial cells against apoptosis through BNIP3-mediated mitophagy in CI-AKI