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. 2022 Oct 24;11(11):2096.
doi: 10.3390/antiox11112096.

Prevention of LPS-Induced Acute Kidney Injury in Mice by Bavachin and Its Potential Mechanisms

Ka-Yun Ban  1 , Ga-Young Nam  1 , Donghee Kim  2 , Yoon Sin Oh  3 , Hee-Sook Jun  1   2   4
Affiliations

Prevention of LPS-Induced Acute Kidney Injury in Mice by Bavachin and Its Potential Mechanisms

Ka-Yun Ban et al. Antioxidants (Basel). .

Abstract

Acute kidney injury (AKI) is a serious complication of sepsis with a rapid onset and high mortality rate. Bavachin, an active component of Psoralea corylifolia L., reportedly has antioxidant, anti-apoptotic, and anti-inflammatory effects; however, its beneficial effects on AKI remain undetermined. We investigated the protective effect of bavachin on lipopolysaccharide (LPS)-induced AKI in mice and elucidated the underlying mechanism in human renal tubular epithelial HK-2 cells. Increased serum creatinine and blood urea nitrogen levels were observed in LPS-injected mice; however, bavachin pretreatment significantly inhibited this increase. Bavachin improved the kidney injury score and decreased the expression level of tubular injury markers, such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), in both LPS-injected mice and LPS-treated HK-2 cells. LPS-induced oxidative stress via phosphorylated protein kinase C (PKC) β and upregulation of the NADPH oxidase (NOX) 4 pathway was also significantly decreased by treatment with bavachin. Moreover, bavachin treatment inhibited the phosphorylation of MAPKs (P38, ERK, and JNK) and nuclear factor (NF)-κB, as well as the increase in inflammatory cytokine levels in LPS-injected mice. Krüppel-like factor 5 (KLF5) expression was upregulated in the LPS-treated HK-2 cells and kidneys of LPS-injected mice. However, RNAi-mediated silencing of KLF5 inhibited the phosphorylation of NF-kB, consequently reversing LPS-induced KIM-1 and NGAL expression in HK-2 cells. Therefore, bavachin may ameliorate LPS-induced AKI by inhibiting oxidative stress and inflammation via the downregulation of the PKCβ/MAPK/KLF5 axis.

Keywords: acute kidney injury; bavachin; inflammatory response; lipopolysaccharide; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bavachin restores histological changes and kidney function in LPS-induced AKI mice. After 24 h of LPS injection, the mice were sacrificed and kidney tissue and blood were collected. (A) Representative images of hematoxylin and eosin (H & E) and periodic acid-Schiff (PAS) staining of the kidney tissues. Nuclei were counterstained with hematoxylin (original magnification, 200×; scale bars, 20 μm; n = 3 mice per group). The black arrow in the H & E-stained section indicates degeneration and vacuolization of renal tubular epithelial cells. (B) Tubular injury was scored based on the percentage of injured area as follows: 0, no damage; 1, injured area 1–10%; 2, injured area 11–25%; 3, injured area 26–75%; and 4, injured area 75% or more. (C) Serum levels of blood urea nitrogen (BUN) and creatinine were analyzed (n = 6 mice per group). Data are presented as mean ± standard error of the mean (SEM). ** p < 0.01, *** p < 0.005 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. vehicle + LPS.
Figure 2
Figure 2
Bavachin decreases the expression of NGAL and KIM-1 in LPS-induced AKI mice and LPS-treated HK-2 cells. (A–C) After 24 h of LPS injection, mice were sacrificed and kidney tissues and urine were collected. (A) The urine protein levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) were analyzed by Western blotting and quantified using ImageJ software (n = 3 mice per group). (B) Immunohistochemical detection of NGAL and KIM-1 was performed in kidney tissues using DAB. Nuclei were counterstained with hematoxylin (original magnification, 200×; scale bars, 20 μm; n = 3 mice per group). (C) The mRNA levels of NGAL and KIM-1 in the kidney tissues were analyzed by qRT-PCR (n = 5 mice per group). (D,E) HK-2 cells were pretreated with 0.1 μg/mL bavachin for 1 h and then treated with 1 μg/mL LPS for 15 h. (D) The mRNA levels of NGAL and KIM-1 were analyzed by qRT-PCR (n = 3). (E) Protein levels of NGAL and KIM-1 were analyzed by Western blotting, quantified using ImageJ software, and normalized to β-actin (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. vehicle + LPS or LPS.
Figure 3
Figure 3
Bavachin decreases ROS production in LPS-induced AKI mice and LPS-treated HK-2 cells. (A) Immunohistochemical detection of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), as markers of ROS generation, was performed in kidney tissues using DAB. Nuclei were counterstained with hematoxylin (original magnification, 200×; scale bars, 20 μm; n = 3 mice per group). Bottom: Relative expression densities of 4-HNE and MDA quantified using ImageJ software. (B) HK-2 cells were seeded in a 96-well black plate, pretreated with 0.1 μg/mL bavachin for 1 h, and then treated with 5 μg/mL LPS for 1.5 h. Intracellular ROS levels were measured using a VICTOR Nivo™ multimode plate reader using a fluorometric assay with DCFH-DA. The relative levels of ROS by DCF fluorescence intensity were compared to those in the control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. vehicle + LPS or LPS.
Figure 4
Figure 4
Bavachin decreases PKCβ activation and NOX4 expression in LPS-induced AKI mice and LPS-treated HK-2 cells. (A) Immunohistochemical detection of phospho-protein kinase C β (P-PKCβ) and NADPH oxidase 4 (NOX4) in kidney tissues using DAB. Nuclei were counterstained with hematoxylin (original magnification, 200×; scale bars, 20 μm; n = 3 mice per group). (B) HK-2 cells were pretreated with bavachin 0.1 μg/mL for 1 h and then treated with 1 μg/mL LPS for 1 h. The protein levels of p-PKCβ and NOX4 were analyzed by Western blotting, quantified using ImageJ software, and normalized to PCKβ and β-actin, respectively (n = 3). Data are presented as mean ± SEM. ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. LPS.
Figure 5
Figure 5
Bavachin decreases the expression of inflammatory cytokines by downregulating the MAPK/NF-κB pathway in LPS-induced AKI mice and LPS-treated HK-2 cells. (A) The mRNA levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in kidney tissues were analyzed by qRT-PCR (n = 5 mice per group). (B) Protein levels of IL-1β, IL-6, and TNF-α in serum were analyzed by ELISA (n = 5 mice per group). (C) HK-2 cells were pretreated with 0.1 μg/mL bavachin for 1 h and then treated with LPS for 15 h. The mRNA levels of IL-1β, IL-6, and TNF-α were analyzed using qRT-PCR (n = 4). (D,E) HK-2 cells were pretreated with 0.1 μg/mL bavachin for 1 h and then treated with 1 μg/mL LPS for 1.5 h. (D) The protein levels of phospho (P)-P38 (P-P38), P-ERK, P-JNK MAP kinase, and (E) phospho-nuclear factor kappa B (P-NF-κB) were analyzed by Western blotting; quantified using ImageJ software; and normalized to P38, ERK, JNK, and NF-κB, respectively (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. vehicle + LPS or LPS.
Figure 6
Figure 6
Bavachin decreases the expression level of kidney injury markers and NF-κB signaling via down-regulation of KLF5 in LPS-induced AKI mice and LPS-treated HK-2 cells. (A) Immunohistochemical detection of Krüppel-like factor 5 (KLF5) in kidney tissues using DAB. Nuclei were counterstained with hematoxylin (original magnification, 200×; scale bars, 20 μm; n = 3 mice per group). (B) The mRNA levels of KLF5 in kidney tissues were analyzed by qRT-PCR (n = 6 mice per group). (C) HK-2 cells were pretreated with 0.1 μg/mL bavachin for 1 h and then treated with LPS for 1.5 h. The protein level of KLF5 was analyzed by Western blotting, quantified using ImageJ software, and normalized to β-actin. (D–G) HK-2 cells were transfected with scrambled siRNA (siCon) and KLF5 siRNA (siKLF5) for 6 h and then treated with 1 μg/mL LPS for another 1.5 or 15 h. (D) The knockdown efficiencies of KLF5 siRNA in HK-2 cells were evaluated by qRT-PCR analysis. (E) The mRNA and (F) protein levels of NGAL and KIM-1 were analyzed using qRT-PCR and Western blotting, respectively. The bands were quantified using ImageJ software and normalized to β-actin (n = 3 or 4). (G) The protein levels of P-NF-κB were analyzed by Western blotting, quantified using ImageJ software, and normalized to NF-κB (n = 3). All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005 vs. control or siCon; # p < 0.05, ## p <0.01, ### p < 0.005 vs. vehicle + LPS or LPS or siCon + LPS.
Figure 7
Figure 7
Schematic of the mechanism by which bavachin ameliorated acute kidney injury induced by LPS through PKCβ/MAPK/KLF5 signaling.
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References

    1. Napolitano L.M. Sepsis 2018: Definitions and Guideline Changes. Surg. Infect. 2018;19:117–125. doi: 10.1089/sur.2017.278. - DOI - PubMed
    1. Zhang D., Lu H., Hou W., Bai Y., Wu X. Effect of miR-132-3p on sepsis-induced acute kidney injury in mice via regulating HAVCR1/KIM-1. Am. J. Transl. Res. 2021;13:7794–7803. - PMC - PubMed
    1. Singer M., Deutschman C.S., Seymour C.W., Shankar-Hari M., Annane D., Bauer M., Bellomo R., Bernard G.R., Chiche J.D., Coopersmith C.M., et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) JAMA. 2016;315:801–810. doi: 10.1001/jama.2016.0287. - DOI - PMC - PubMed
    1. Singbartl K., Kellum J.A. AKI in the ICU: Definition, epidemiology, risk stratification, and outcomes. Kidney Int. 2012;81:819–825. doi: 10.1038/ki.2011.339. - DOI - PubMed
    1. Wang F., Zhang G., Lu Z., Geurts A.M., Usa K., Jacob H.J., Cowley A.W., Wang N., Liang M. Antithrombin III/SerpinC1 insufficiency exacerbates renal ischemia/reperfusion injury. Kidney Int. 2015;88:796–803. doi: 10.1038/ki.2015.176. - DOI - PMC - PubMed
-