4-Octyl itaconate attenuates LPS-induced acute kidney injury by activating Nrf2 and inhibiting STAT3 signaling

Abstract

Background: Septic acute kidney injury (S-AKI) is the leading form of acute kidney failure among hospitalized patients, and the inflammatory response is involved in this process. 4-octyl itaconate (4-OI) is a multi-target itaconate derivative with potent anti-inflammatory action. However, it remains elusive whether and how 4-OI contributes to the regulation of S-AKI.

Methods: We employed a lipopolysaccharide (LPS)-induced AKI murine model and explored the potential renoprotective effect of 4-OI in vivo. In vitro experiments, BUMPT cells, a murine renal tubular cell line, were conducted to examine the effects of 4-OI on inflammation, oxidative stress, and mitophagy. Moreover, STAT3 plasmid was transfected in BUMPT cells to investigate the role of STAT3 signaling in the 4-OI-administrated state.

Results: We demonstrate that 4-OI protects against S-AKI through suppressing inflammation and oxidative stress and enhancing mitophagy. 4-OI significantly reduced the levels of Scr, BUN, Ngal as well as the tubular injury in LPS-induced AKI mice. 4-OI restrained inflammation by reducing macrophage infiltration and suppressing the expression of IL-1β and NLRP3 in the septic kidney. 4-OI also reduced ROS levels, as well as cleaved caspase-3 and boosted antioxidants such as HO-1, and NQO1 in mice. In addition, the 4-OI treatment significantly promoted mitophagy. Mechanistically, 4-OI activated Nrf2 signaling and suppressed phosphorylated STAT3 in vivo and vitro. Molecular docking revealed the binding affinity of 4-OI towards STAT3. ML385, a specific Nrf2 inhibitor, partially repressed the anti-inflammatory and anti-oxidative effects of 4-OI and partially restricted the mitophagy induced by 4-OI in vivo and in vitro. Transfected with STAT3 plasmid partially suppressed mitophagy and the anti-inflammatory effect provoked by 4-OI in vitro.

Conclusion: These data suggest that 4-OI ameliorates LPS-induced AKI by suppressing inflammation and oxidative stress and enhancing mitophagy through the overactivation of the Nrf2 signaling pathway, and inactivation of STAT3. Our study identifies 4-OI as a promising pharmacologic for S-AKI.

Keywords: 4-octyl itaconate; Acute kidney injury; Inflammation; Mitophagy; Nrf2; Oxidative stress; STAT3; sepsis.

Fig. 1

4-OI decreases LPS-induced nephrotoxicity in vivo and apoptosis in vitro. Scr (A) and BUN (B) levels of different groups of mice exposed to LPS or 4-OI (n = 6). (C) qPCR analysis of mRNA of Lcn2  in renal tissues (n = 6). (D) Representative images of hematoxylin and eosin (H&E) staining of the renal cortex. Vacuolization and loss of brush border in tubular cells are pointed to by the arrows. Scale bar: 50 μm. (E) Tubular injury is evaluated by figuring out the renal tubules with signs of injury (n = 6). (F) Representative western blot bands of BAX, cleaved caspase-3, BCL2, and β-actin (protein loading control) in renal cortex tissues. (G) Relative band density of BAX, cleaved caspase-3 and BCL2 signals (n = 6). (H) In Hoechst 33258 staining images, nuclear condensation, fragmentation, and apoptotic bodies were observed. Scale bar = 100 μm. (I) The apoptotic index (AI) was calculated as the percentage of apoptotic nuclei per total nuclei number per field. Representative western blot bands (J) and relative band intensity (K) of BAX, cleaved caspase-3 and BCL2 in BUMPT cells treated with different doses of 4-OI under saline or LPS conditions (n = 4). Each symbol (circle) represents an independent experiment. Data are presented as means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. LPS, lipopolysaccharide; 4-OI, 4-octyl itaconate; Scr, serum creatinine; BUN, blood urea nitrogen; qPCR, Real-time polymerase chain reaction

Fig. 2

4-OI activates Nrf2 and alleviates inflammation and oxidative stress in LPS-induced AKI in vivo and in vitro. (A) Representative IHC staining images of Nrf2 (pointed to by the arrowhead) of renal tissues. Scale bar: 50 μm. (B) Representative western blot bands of Nrf2, HO-1, NQO1, and NLRP3 in renal cortex. (C) Relative band density of Nrf2, HO-1, NQO1 and NLRP3 in kidney cortex (n = 6). (D) Representative DHE staining images and IF staining images of F4/80. Scale bar: 50 μm. (E) Representative western blot bands of Nrf2, HO-1, NQO1, and IL-1β in BUMPT cells. (F) Relative band density of Nrf2, HO-1, NQO1, and IL-1β in BUMPT cells (n = 4). (G) qPCR analysis of mRNA of Il6 in BUMPT cells (n = 4). (H) Representative images of MitoSOX staining in BUMPT cells. Scale bar: 20 μm. (I) Semi-quantification analysis of MitoSOX fluorescence intensity. Each symbol (circle) represents an independent experiment. Data are presented as means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. IHC, Immunohistochemistry; IF, immunofluorescence; DHE, dihydroethidium; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase-1; NQO1, NAD(P)H quinone oxidoreductase 1

Fig. 3

4-OI ameliorates renal mitochondrial injury and improves mitophagy in LPS-induced AKI in mice and tubular cells. (A) Mitochondria in proximal tubules were examined by TEM. Scale bar: 2 μm. (B) Representative TEM images of autophagosomes (pointed to by the arrowhead in the left two panels) and mitophagosomes (pointed to by the arrowhead in the right two panels) in renal proximal tubule cells. Scale bar: 1 μm. (C) Representative immunofluorescence images of LC3B (green) and TOMM20 (red) in kidney tissues from each group. The nuclei were counterstained by DAPI (blue) (n = 6). Scale bar: 50 μm. (D) Colocalization of LC3B and TOMM20 determined by Pearson’s correlation coefficient (n = 6). (E) Representative western blot bands of LC3B-II, p62, TIMM23, and TOMM20 in kidney cortices (n = 6). (F) Relative band intensity of LC3B-II, p62, TIMM23, and TOMM20 in kidneys (n = 6). (G) BUMPT cells were immunostained with LC3B (green) and TOMM20 (red) to show mitophagy (n = 4). Scale bar: 20 μm. (H) Colocalization of LC3B and TOMM20 determined by Pearson’s correlation coefficient (n = 4). (I) Representative western blot bands of LC3B-II, p62, TIMM23, and TOMM20 in BUNPT cells (n = 4). (J) Relative band intensity of LC3B-II, p62, TIMM23, and TOMM20 (n = 4). Each symbol (circle) represents an independent experiment. Data are presented as means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. TEM, transmission electron microscopy.

Fig. 4

4-OI attenuates LPS-induced mitochondrial ROS generation, and inflammation enhances mitophagy and subsequent apoptosis, which were partially abolished by the Nrf2 inhibitor. (A) Representative western blot bands of Nrf2, HO-1, NQO1, NLRP3, and cleaved caspase-3 in BUMPT cells. (B) Relative band intensity of Nrf2, HO-1, NQO1, NLRP3 and cleaved caspase-3 in BUMPT cells (n = 4). (C) Representative IF staining images of Nrf2 (red) in BUMPT cells. Scale bar: 20 μm. (D) Representative images of MitoSOX staining of BUMPT cells. Scale bar: 20 μm. (E) Representative western blot bands of Nrf2, HO-1, NQO1, IL-1β, and cleaved caspase-3 in murine kidneys. (F) Relative band intensity HO-1, NQO1, IL-1β and cleaved caspase-3 in murine kidneys (n = 6). (G) qPCR analysis of mRNA of Il6  in renal tissues (n = 6). (H) qPCR analysis of mRNA of Lcn2  in renal tissues (n = 6). (I) Representative western blot bands of LC3B-II, p62, TIMM23, and TOMM20 in BUMPT cells (n = 4). (J) Representative western blot bands of LC3B-II, p62, TIMM23, and TOMM20 in renal tissues. (K) Relative band intensity of LC3B-II, p62, TIMM23, and TOMM20 in BUMPT cells (n = 4). (L) Relative band intensity of LC3B-II, p62, TIMM23, and TOMM20 in renal tissues (n = 6). Each symbol (circle) represents an independent experiment. Data are presented as means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant

Fig. 5

Identified STAT3 as a potential target of 4-OI which was suppressed by 4-OI in LPS-induces AKI models. (A) Venn diagram composed of the number of proteins modified by the specific bioorthogonal probe, PharmMapper database retrieved 4-OI targets, SEA database retrieved 4-OI targets and SwissTargetPrediction database retrieved 4-OI targets. (B) Molecular docking of 4-OI with STAT3. The pink structures in the panel represent screened macromolecular receptor structures, while the yellow sticks represent the interacted residuals, and the yellow dotted line represents intermolecular hydrogen bonding. (C) Representative western blot bands of p-STAT3 (Y705), p-STAT3 (S727), and total STAT3 in BUMPT cells. (D) Relative band intensity of p-STAT3 (Y705), p-STAT3 (S727), and total STAT3 in BUMPT cells (n = 4). (E) Real-time polymerase chain reaction analysis of mRNA of Haptoglobin, SAA1, and SAA3 in BUMPT cells (n = 4). (F) Representative western blot bands of p-STAT3 (Y705), p-STAT3 (S727), and total STAT3 in renal tissues (n = 6). (G) Relative band intensity of p-STAT3 (Y705), p-STAT3 (S727), and total STAT3 in renal tissues (n = 6). (H) Representative IHC staining images of p-STAT3 (Y705) and IF staining images of p-STAT3 (S727) (red) in renal tissues. Scale bar: 50 μm. Each symbol (circle) represents an independent experiment. Data are presented as means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. SEA, Similarity ensemble approach

Fig. 6

Transfection with STAT3 plasmid attenuated 4-OI-induced anti-inflammatory effect and mitophagy in BUMPT cells treated with LPS. (A) Representative western blot bands of p-STAT3 (Y705), p-STAT3 (S727), total STAT3, HO-1, NQO1, IL-1β, LC3B-II, p62, TOMM20, TIMM23, and cleaved caspase-3 in BUMPT cells (n = 4). (B) Relative band intensity of p-STAT3 (Y705), p-STAT3 (S727), total STAT3, NLRP3, HO-1, NQO1, p62, TOMM20, TIMM23, LC3-II and cleaved caspase-3 in BUMPT cells (n = 4). (C) qPCR analysis of mRNA of Il6  in BUMPT cells (n = 4). (D) BUMPT cells were immunostained with LC3B (green) and TOMM20 (red) to show mitophagy (n = 4). Scale bar: 20 μm. (E) Colocalization of LC3B and TOMM20 determined by Pearson’s correlation coefficient (n = 4). Each symbol (circle) represents an independent experiment. Data are presented as means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. EV, empty vector; STAT3 OE, STAT3 overexpression

Fig. 7

A schematic diagram illustrating the protective effects of 4-OI against LPS-induced acute kidney injury. 4-OI prevented LPS-induced acute kidney injury by over-activating Nrf2 and inactivating STAT3 signaling, attenuating oxidative stress, and inflammation, promoting mitophagy, and moderating apoptosis