Post-treatment With Irisin Attenuates Acute Kidney Injury in Sepsis Mice Through Anti-Ferroptosis via the SIRT1/Nrf2 Pathway

Abstract

Kidney is one of the most vulnerable organs in sepsis, resulting in sepsis-associated acute kidney injury (SA-AKI), which brings about not only morbidity but also mortality of sepsis. Ferroptosis is a new kind of death type of cells elicited by iron-dependent lipid peroxidation, which participates in pathogenesis of sepsis. The aim of this study was to verify the occurrence of ferroptosis in the SA-AKI pathogenesis and demonstrate that post-treatment with irisin could restrain ferroptosis and alleviate SA-AKI via activating the SIRT1/Nrf2 signaling pathway. We established a SA-AKI model by cecal ligation and puncture (CLP) operation and an in vitro model in LPS-induced HK2 cells, respectively. Our result exhibited that irisin inhibited the level of ferroptosis and ameliorated kidney injury in CLP mice, as evidenced by reducing the ROS production, iron content, and MDA level and increasing the GSH level, as well as the alteration of ferroptosis-related protein (GPX4 and ACSL4) expressions in renal, which was consistent with the ferroptosis inhibitor ferrostatin-1 (Fer-1). Additionally, we consistently observed that irisin inhibited ROS accumulation, iron production, and ameliorated mitochondrial dysfunction in LPS-stimulated HK-2 cells. Furthermore, our result also revealed that irisin could activate SIRT1/Nrf2 signaling pathways both in vivo and vitro. However, the beneficial effects of irisin were weakened by EX527 (an inhibitor of SIRT1) in vivo and by SIRT1 siRNA in vitro. In conclusion, irisin could protect against SA-AKI through ferroptotic resistance via activating the SIRT1/Nrf2 signaling pathway.

Keywords: Nrf2; SIRT1; acute kidney injury; ferroptosis; irisin; sepsis.

FIGURE 1

Irisin attenuates CLP-induced kidney injury. Mice were given a sham operation or CLP model operation, with or without irisin (Ir), ferrostatin-1 (Fer-1), or/and Fe-citrate (Fe) (Fe + Ir) administration. (A) Representative images (200×) showing HE staining of renal sections (scale bar = 50 μm). (B) Representative fluorescent images of DHE staining (scale bar = 50 μm). (C) Acute tubular necrosis score (ATN score). (D) The quantification of the DHE fluorescence intensity. (E) BUN concentration in the plasma. (F) Creatinine (Cr) concentration in the plasma. (G) Plasma neutrophil gelatinase-associated lipocalin (NGAL) concentration. (H–J) The level of TNF-α, IL-6, and IL-1β in the kidney tissue. *p < 0.05 vs. Sham group, p < 0.05 vs. CLP group, P <0.05 vs. Fe group, and p > 0.05 vs CLP group. Data are presented as mean ± SD (n = 10).

FIGURE 2

Irisin alleviates the occurrence of ferroptosis in SA-AKI. (A) Representative TEM images (scale bar = 1 μm, upper panels). The red arrow indicates representative mitochondria in mouse kidney (n = 3 mice/group). The levels of MDA (B), GSH (C), and Fe2+ (D) in mouse kidney homogenates. (n = 10). (E) Western blot analysis of GPX4 and ACSL4 proteins in the kidney tissue. (n = 3). (F,G) Quantitation of results in (E). *p < 0.05 vs. Sham group, ^# p < 0.05 vs. CLP group, ^& p < 0.05 vs. Fe group. Data are presented as mean ± SD.

FIGURE 3

Irisin inhibits LPS-induced ferroptosis in HK-2 cells. (A) Images of intracellular ROS levels in HK-2 cells stained by DCFH-DA (10 μM) fluorescent probes (Bar = 50 μm). (B) Fluorescence images of Mito FerroGreen (5 μM)-stained HK-2 cells (Bar = 20 μm). (C) Quantitative results of ROS. (D) Quantitative results of mitochondrial ferrous iron. (E) Cell viability was determined by CCK-8 in HK-2 cells. (F) Western blot analysis of GPX4 and ACSL4 proteins in HK-2 cells. (G,H), Quantitation of results in (F). *p < 0.05 vs. Con group, ^# p < 0.05 vs. LPS group, ^& p < 0.05 vs. Fe group. Data are presented as mean ± SD (n = 3).

FIGURE 4

Irisin upregulates the SIRT1/Nrf-2 pathway to inhibit LPS-induced ferroptosis. HK-2 cells were transfected with SIRT1 siRNA (si-Sirt1) or negative control (si-con) followed by exposure to LPS and irisin. (A) The expression of SIRT1 protein in cells transfected with SIRT1 siRNA (si-Sirt1) or negative control (si-con) was detected using Western blot to validate the knockdown of SIRT1. (B) Western blots and quantitative analyses of GPX4 and ACSL4 proteins in cells. (C) Western blots and quantitative analyses of SIRT1 and Nrf2 proteins in cells. (D) Cell viability using the CCK-8 kit. The levels of MDA (E) and GSH (F) in cells were analyzed using a corresponding kit. *p < 0.05 vs. si-con group, ^# p < 0.05 vs. si-con + LPS group, ^& p < 0.05 vs. si-con + LPS + Ir group. Data are presented as mean ± SD (n = 3).

FIGURE 5

Irisin inhibits LPS-induced accumulation of ROS and lipid peroxidation. (A) Fluorescence images of 5 μM JC-1-stained HK-2 cells. Scale bars: 20 μm. The ratio of red and green fluorescence reflected changes of the mitochondrial membrane potential (MMP) (n = 3). (B) Quantitative results of MMP. (C) lipid ROS was analyzed using 2 μM BODIPY® 581/591 C11 by flow cytometry (n = 5). (D) Quantitative results of lipid ROS (n = 3). *p < 0.05 vs. si-con group, ^# p < 0.05 vs. si-con + LPS group, ^& p < 0.05 vs. si-con + LPS + Ir group. Data are presented as mean ± SD (n = 3).

FIGURE 6

Irisin attenuates CLP-induced kidney injury through the SIRT1/Nrf-2 pathway. Mice were given a sham operation or CLP model operation, with or without irisin (Ir) and Ex527 administration. (A) Representative images (200×) showing HE staining of renal sections (scale bar = 50 μm). (B) Representative fluorescent images of DHE Staining (scale bar = 50 μm). (C) Acute tubular necrosis score (ATN score). (D) The quantification of DHE fluorescence intensity. The level of TNF-α (E) and IL-6 (F) in the kidney tissue. (G) Western blot analysis of GPX4 and ACSL4 proteins in the kidney tissue. (n = 6). (H) Quantitation of results in (G). (I) Western blot analysis of SIRT1 and Nrf2 proteins in the kidney tissue. (J) Quantitation of results in (I). *p < 0.05 vs. Con group, ^# p < 0.05 vs. CLP group, ^& p < 0.05 vs. CLP + Ir group, ^$ p > 0.05 vs CLP group. Data are presented as mean ± SD (n = 6).

FIGURE 7

Scheme summarizing the protective effects of irisin post-treatment on sepsis-associated acute kidney injury (SA-AKI) via induction of the SIRT1/Nrf2 signal axis. SA-AKI leads to increased iron and lipid peroxidation, along with GSH depletion, which is implicated in the pathological process of SA-AKI. Irisin post-treatment effectively suppressed ferroptosis and alleviated SA-AKI and improved mitochondrial function via induction of the SIRT1/Nrf2 signal axis. The ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) and the ferroptosis inducer Fe-citrate (Fe) could attenuate or exacerbate ferroptosis in SA-AKI, respectively.