Remote Ischemic Preconditioning Attenuates Mitochondrial Dysfunction and Ferroptosis of Tubular Epithelial Cells by Inhibiting NOX4-ROS Signaling in Acute Kidney Injury

Abstract

Acute kidney injury (AKI) is a worldwide clinical burden associated with high morbidity and mortality. Remote ischemic preconditioning (rIPC), a brief nonlethal ischemia and reperfusion (IR) in remote tissues or limbs, has been used in an attempt to protect against AKI, but its underlying signaling pathways has not been elucidated. In the present study, rIPC protected kidney function and pathological injury and mitigated NADPH oxidase 4 (NOX4) upregulation in different AKI models (cisplatin, LPS and IRI). Furthermore, rIPC significantly attenuated mitochondrial dysfunction and ameliorated tubular epithelial ferroptosis during AKI. Mechanistically, in wild-type AKI mice and TCMK-1 cells, rIPC effectively decreased kidney ROS production, preserved mitochondrial dynamics and mitophagy, and ameliorated tubular epithelial ferroptosis. Notably, these protective effects of rIPC were further enhanced by NOX4 knockout or silencing and mitigated by NOX4 overexpression. Our study showed that rIPC may attenuate mitochondrial dysfunction and ferroptosis in tubular epithelial cells in AKI by inhibiting NOX4-ROS signaling. NOX4 might be used as a biomarker for monitoring the biological effects of rIPC to optimize the rIPC protocol and facilitate future translational studies.

Keywords: NADPH oxidase 4; acute kidney injury; ferroptosis; mitochondria; remote ischemic preconditioning.

Figure 1

rIPC protects renal function and renal tubule injury in mice with AKI. (A) rIPC intervention in CP/LPS/IRI-induced AKI mouse models. (B) sCr and BUN levels in difference groups of CP-AKI mice. (C) Havcr1 and Lcn2 expression measured by RT-qPCR in CP-AKI mice kidney. (D) Representative image of hematoxylin and eosin (H&E) staining in CP-AKI mice kidney (200x, scale bar = 50μm; 400x, scale bar = 20 μm). (E) sCr and BUN levels in difference groups of LPS-AKI mice. (F) Havcr1 and Lcn2 expression measured by RT-qPCR in LPS-AKI mice kidney. (G) Representative image of hematoxylin and eosin (H&E) staining in LPS-AKI mice kidney (200x, scale bar = 50μm; 400x, scale bar = 20 μm). (H) sCr and BUN levels in difference groups of IRI-AKI mice. (I) Havcr1 and Lcn2 expression measured by RT-qPCR in IRI-AKI mice kidney. (J) Representative image of hematoxylin and eosin (H&E) staining in IRI-AKI mice kidney (200x, scale bar = 50μm; 400x, scale bar = 20 μm). Data are presented as mean ± SD, n = 6. Ripc: remote ischemic preconditioning, CP: cisplatin, LPS: lipopolysaccharides, IRI: ischemia/reperfusion injury, sCr: serum creatinine, BUN: blood urea nitrogen, Lcn2: (neutrophil gelatinase-associated lipocalin, NGAL) and Havcr1: (kidney injury molecule 1, KIM1). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.

Figure 2

rIPC attenuates inflammation, oxidative stress, mitochondrial malfunction and ferroptosis in AKI mice. (A) IL-6 and TNF-α expression by RT-qPCR and western blot in CP-AKI mouse kidney. (B) The morphology of mitochondria under transmission electron microscope of CP-AKI mouse kidney (8000x, scale bar = 2μm). (C) ROS assessed by DHE staining in CP-AKI mouse kidney (200x, scale bar = 100 μm). (D) Mitochondrial dynamic regulatory molecules (DRP-1, OPA-1 and MFN-2) measured by RT-qPCR, western blot and quantified by densitometry in CP-AKI mouse kidney. (E) Mitophagy level (PINK1, p62/SQSTM1 and LC3B) measured by RT-qPCR, western blot and quantified by densitometry in CP-AKI mouse kidney. (F) Ferroptosis-regulatory molecules (ACSL4 and GPX4) measured by RT-qPCR, western bolt and quantified by densitometry in CP-AKI mouse kidney. (G) Representative image of immunofluorescence staining of GPX4 in CP-AKI mouse kidney (200x, scale bar = 10 μm). (H) The levels of GSH in CP-AKI mouse kidney tissue. (I) The levels of MDA in CP-AKI mouse kidney tissue. (J) The morphological characteristic of ferroptosis under transmission electron microscope of CP-AKI mouse kidney (8000x, scale bar = 2μm, 30000x, scale bar = 500nm). Data are presented as mean ± SD, n = 6. rIPC: remote ischemic preconditioning, CP: cisplatin, ROS: reactive oxygen species, GSH: glutathione. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.

Figure 3

rIPC attenuates inflammation, oxidative stress, mitochondrial malfunction and ferroptosis in TCMK-1 cells. (A) rIPC intervention in cisplatin stimulated TCMK-1 cells. (B) Havcr1 and Lcn2 expression measured by RT-qPCR. (C) IL-6 and TNF-α expression analyzed by RT-qPCR, western blot and quantified by densitometry. (D) The ROS production in TCMK-1 cells was assessed by DCFH-DA staining (100x, scale bar = 100 μm). (E) Mitochondrial dynamic regulatory molecules (DRP-1, OPA-1 and MFN-2) assessed by RT-qPCR, western blot and quantified by densitometry. (F) Mitophagy level (PINK1, p62/SQSTM1 and LC3B) measured by RT-qPCR, western blot and quantified by densitometry in TCMK-1 cells. (G) Ferroptosis-regulatory molecules (ACSL4 and GPX4) measured by RT-qPCR, western blot and quantified by densitometry. Data are presented as mean ± SD, n = 3. CP: cisplatin, CCCP: carbonyl cyanide 3-chlorophenylhydrazone, ROS: reactive oxygen species, Lcn2: (neutrophil gelatinase-associated lipocalin, NGAL) and Havcr1: (kidney injury molecule 1, KIM1). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.

Figure 4

rIPC reverses the upregulation of NOX4 in AKI. (A) Representative heatmap of differentially expressed genes in the kidneys of CP-AKI mice (n=3). (B) Comparable analysis between cisplatin and cisplatin + rIPC group using Gene Set Enrichment Analysis (GSEA). (C) NOX4 expression assessed by Western blot in cisplatin, LPS and IRI-induced AKI. (D) Representative image of immunochemistry staining of NOX4 in kidney tissue sections (200x, scale bar = 50μm). (E) rIPC reverses the upregulation of NOX4 assessed by RT-qPCR, western blot and quantified by densitometry in mice kidney (n=6). (F) Representative image of immunofluorescence staining of NOX4 in CP-AKI mouse kidney (200x, scale bar = 10μm). (G) rIPC reverses the upregulation of NOX4 assessed by RT-qPCR, western blot and quantified by densitometry in TCMK-1 (n=3). (H) NOX4 inhibitor GKT137831 improved the pathological injury and tubular damage score in CP-induced AKI mice under hematoxylin and eosin (H&E) staining (200x, scale bar = 50μm; 400x, scale bar = 20 μm). Data are presented as mean ± SD, n = 6. rIPC: remote ischemic preconditioning, CP: cisplatin, CCCP: carbonyl cyanide 3-chlorophenylhydrazone, LPS: lipopolysaccharides, IRI: ischemia/reperfusion injury. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001.

Figure 5

The protective effects of rIPC on TCMK-1 cells are mitigated by NOX4 overexpression. (A) TCMK-1 cells were transfected with negative control (NC) Ad-RNA or Ad-NOX4 for 24 h and then treated with 2 μg/ml cisplatin for 24 h. The overexpression efficiency of NOX4 in TCMK-1 cells was evaluated by RT-qPCR analysis, western blot analysis and quantified by densitometry. (B) NOX4 expression evaluated by RT-qPCR analysis, western blot analysis and quantified by densitometry. (C) The ROS production in TCMK-1 cells was assessed by DCFH-DA staining (100x, scale bar = 100 μm). (D) Mitochondrial dynamic regulatory molecules (DRP-1, OPA-1 and MFN-2) evaluated by RT-qPCR analysis and western blot analysis. (E) Mitophagy level (PINK1, p62/SQSTM1 and LC3B) assessed by RT-qPCR analysis and western blot analysis. (F) Ferroptosis-regulatory molecules (ACSL4 and GPX4) assessed by RT-qPCR analysis, western blot analysis and quantified by densitometry. Data are presented as mean ± SD, n = 3. CCCP: carbonyl cyanide 3-chlorophenylhydrazone, CP: cisplatin, ROS: reactive oxygen species. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, ns: no significant.

Figure 6

The protective effects of rIPC on TCMK-1 cells are enhanced by NOX4 silencing. (A) TCMK-1 cells were transfected with negative control (NC) siRNA or NOX4 siRNA for 6 h and then treated with 2 μg/ml cisplatin for 24 h. The knockdown efficiency of NOX4 siRNA in TCMK-1 cells was evaluated by RT-qPCR analysis and western blot analysis. (B) NOX4 expression evaluated by RT-qPCR analysis, western blot analysis and quantified by densitometry. (C) The ROS production in TCMK-1 cells was assessed by DCFH-DA staining (100x, scale bar = 100 μm). (D) Mitochondrial dynamic regulatory molecules (DRP-1, OPA-1 and MFN-2) evaluated by RT-qPCR analysis, western blot analysis and quantified by densitometry. (E) Mitophagy level (PINK1, p62/SQSTM1 and LC3B) measured by RT-qPCR analysis, western blot analysis and quantified by densitometry. (F) Ferroptosis-regulatory molecules (ACSL4 and GPX4) assessed by RT-qPCR analysis, western blot analysis and quantified by densitometry. Data are presented as mean ± SD, n = 3. CCCP: carbonyl cyanide 3-chlorophenylhydrazone, CP: cisplatin, ROS: reactive oxygen species. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 7

The protective effects of rIPC on AKI mice are enhanced by NOX4 knockout. (A) sCr and BUN levels in difference groups of CP-AKI mice. (B) Havcr1 and Lcn2 expression measured by RT-qPCR in CP-AKI mouse kidney. (C) Representative image of hematoxylin and eosin (H&E) staining in CP-AKI mouse kidney (200x, scale bar = 50μm; 400x, scale bar = 20 μm). (D) ROS assessed by DHE staining in CP-AKI mouse kidney (200x, scale bar = 100 μm). (E) Mitochondrial dynamic regulatory molecules (DRP-1, OPA-1 and MFN-2) analyzed by RT-qPCR, western blot and quantified by densitometry in CP-AKI mouse kidney. (F) Mitophagy level (PINK1, p62/SQSTM1 and LC3B) analyzed by RT-qPCR, western blot and quantified by densitometry in CP-AKI mouse kidney. (G) Ferroptosis-regulatory molecules (ACSL4 and GPX4) assessed by RT-qPCR, western bolt and quantified by densitometry in CP-AKI mouse kidney. Data are presented as mean ± SD, n = 6. rIPC: remote ischemic preconditioning, CP: cisplatin, Lcn2: (neutrophil gelatinase-associated lipocalin, NGAL) and Havcr1: (kidney injury molecule 1, KIM1), ROS: reactive oxygen species. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, ns: no significant.

Figure 8

Mechanism of remote ischemic preconditioning in the protection against acute kidney injury. rIPC: remote ischemic preconditioning, CP: cisplatin, IRI: ischemia/reperfusion injury, AKI: acute kidney injury.