Kaempferol Reverses Acute Kidney Injury in Septic Model by Inhibiting NF-κ B/AKT Signaling Pathway

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Introduction
Acute kidney injury (AKI) is a clinical syndrome of rapidly declining kidney function and kidney injury caused by multiple factors, and infection is one of the most common causes of AKI [1].Acute kidney injury currently afects 13 million people worldwide each year and is one of the most common complications in hospitalized patients [2,3]; its adverse consequences include kidney failure, cardiovascular events, and increased mortality [4,5].In China, 29 million patients are hospitalized due to AKI; approximately 40% of them die of AKI, and most of them cannot fully recover [6].
Sepsis is a systemic immune response triggered by infection and is the main killer of patients in the ICU with AKI [7,8].Terefore, it is a challenge to prevent and control the occurrence of AKI and reduce the morbidity and mortality of septic patients.Pathophysiological mechanisms of AKI in sepsis are complex and multifactorial, posing signifcant obstacles to its treatment.Among the known mechanisms of AKI in sepsis, oxidative stress responses are associated with infammatory responses [9,10].Huang et al. showed that ultrasmall TWNDs have a very strong ability to eliminate a variety of ROS, which allows them to restore renal function in AKI at very low doses [11].Ren et al. showed that fesarone prevented AKI in sepsis by reducing renal infammation and apoptosis through inhibition of Src-mediated NF-κB p65 and MAPK signaling pathways, thereby preventing AKI in sepsis [12].Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, KAE) is a natural favonoid compound that is present in a variety of edible plants (e.g., legumes, leeks, tea) and commonly used drugs in traditional Chinese medicines (e.g., kaempferol, cat eye grass, white hair grass) [13].Kaempferol and its derivatives have been largely demonstrated to have anti-infammatory, antioxidant, antibacterial, and antitumor efects [14].Molitorisova et al. reported that kaempferol modulated allergic airway infammation and associated asthma characteristics [15].Similarly, Liu et al. demonstrated that kaempferol was able to reduce oxidative stress and infammatory responses through the downregulation of ROS-dependent MAPK/NF-κB and pyroptosis signaling pathways [16].However, kaempferol has not been reported to treat sepsis-induced AKI.It is a product of natural origin and has fewer toxic side efects and interactions with chemical agents and biological agents.Terefore, in this study, we established in vitro and in vivo AKI models of sepsis and investigated the efect of kaempferol on sepsis-induced AKI and its mechanism.

Construction and Grouping of Sepsis Mouse
Model.Eightweek-old male C57BL/6 mice (22-26 g, SFP grade) were purchased from Kevens Animal Laboratory Co., Ltd., Changzhou, China.Mice were maintained at room temperature (25 ± 2 °C, 55 ± 5% humidity) for 3 days with free access to food and water.All animal study procedures were performed following the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and the National Institutes of Health (NIH Publication No. 86-23, Revised 1996).Te animal protocol was approved by the Ethics Committee of Shanghai University.Sepsis models were established using cecal ligation and puncture (CLP) surgery [17].Forty mice were randomly divided into the following 5 groups (n � 8/group): control group, AKI group, KAE 25 mg/kg group, KAE 50 mg/kg group, and KAE 100 mg/kg group.All treatment reagents were injected through the tail vein, and treatment was continuously administered to the treatment groups for 3 weeks.At the end of the study, tissues were collected from euthanized mice after weighing and anaesthetization with ether (Sigma-Aldrich).

ELISA Experiments.
Tissue and cell samples were processed according to TNF-α and IL-1β ELISA kit instructions (MULTISCIENCES, Hangzhou, China), and the absorbance at 450 nm was measured using a microplate reader to draw a standard curve and calculate the concentration.

RT-PCR.
Total RNA was extracted using TRIzol according to the manufacturer's protocol, and recovered RNA was purifed in strict accordance with the RNA recovery kit's instructions.cDNA from a reverse transcription reaction was used, and we performed real-time PCR in triplicate using Power SYBR Green polymerase chain reaction (PCR) master mix and an ABI 7500 real-time PCR system.All procedures were performed according to the manufacturer's recommendations.All primers were synthesized by Sangon Biotech Co., Ltd.(Shanghai, China), and the primer sequences used are shown in Table 1, with GAPDH used as an internal control.Te relative expression of target genes was calculated based on the 2-Ct value.
2.6.Cell Culture.Te human tubular epithelial cell line HK-2 (FH0228, FuHeng Biology) was cultured in high glucose DMEM containing 10% fetal bovine serum at 37 °C and 5% CO 2 .HK-2 cells were treated with 10 µg/ml lipopolysaccharide (Sigma-Aldrich) for 24 h to induce a cellular model of sepsis acute kidney injury in vitro according to Sun et al. [18].

Cell Proliferation.
Cells from the experimental and control groups in the logarithmic growth phase were inoculated into 96well plates at 1 × 10 4 cells/well, and culture medium was added to a fnal volume of 100 µl in each well; the cells were cultured in 5% CO 2 at 37 °C, and 6 replicate wells and 2 control wells were included.CCK-8 reagent was added, and the absorbance at 450 nm was measured using a microplate reader.Cell growth curves were plotted with control zeroing, with time as the abscissa and absorbance as the ordinate.

Flow Apoptosis.
Apoptosis was detected by fow cytometry using an Annexin V-FITC Apoptosis Assay Kit (Beyotime, Shanghai, China).After removing the intervention culture medium from each group, trypsin was added to digest the cells, and DMEM complete culture medium was used to adjust the cell suspension concentration to 10 6 /mL.Ten, 75% ethanol was added up to 2 mL to fx the cells, and the cells were gently mixed after standing for 5 min and then placed in a lowtemperature centrifuge for centrifugation at 12,000 rpg for 10 min.After the end of centrifugation, the supernatant was removed, and PBS solution was added to the cell pellet to gently wash the cells three times for 5 min each time.Annexin V-FITC/PI double staining and fow cytometer onboard detection were performed according to the manufacturer's instructions.

Statistical Analysis.
Experimental data are presented as the means ± SD, and one-way analysis of variance (ANOVA) and correlation analysis were performed using GraphPad Prism statistical software 7.0.Diferences between the two groups were compared using Bonferroni post hoc comparisons.p < 0.05 was considered statistically signifcant.

KAE Improves
Sepsis-Induced AKI.KAE treatment of septic mice signifcantly increased body weight (Figure 1(a)), signifcantly decreased the renal index (kidney weight/body weight) (Figure 1(b)), and signifcantly increased serum creatinine and blood urea nitrogen (Figures 1(c) and 1(d)).Taken together, the results suggest that KAE ameliorates sepsis-induced acute kidney injury in mice.

KAE Ameliorates Pathological Changes in Mice with
Sepsis-Induced AKI.We performed HE staining to observe morphological changes in the kidney.As shown in Figure 2, septic mice showed marked changes in the renal tissue, such as glomerular necrosis and hypertrophy, which were signifcantly improved by KAE treatment.

Anti-infammatory Response to KAE in Sepsis-Induced AKI Mice.
Infammation is well known to contribute to the pathogenesis of AKI [19].In this study, we detected increased IL-1β and TNF-α protein levels in renal tissue during AKI and signifcantly decreased expression after administration of diferent doses of KAE (Figures 3(a) and 3(b)).We then detected IL-1β and TNF-α expression levels in kidney tissues from each group and found consistent changes in protein expression detected by ELISA (Figures 3(c) and 3(d)).
Tese results suggest that KAE plays an antiinfammatory role in AKI therapy.

Mechanism of KAE in Sepsis-Induced AKI in Mice.
To further investigate the potential mechanism of KAE in sepsis-induced AKI in mice, we examined changes in NF-κB/AKT pathway expression levels within renal tissue (Figure 4(a)).We found that KAE did not afect NF-κB p65 and AKT expression, whereas levels of NF-κB p65 and AKT phosphorylation were dose-dependently decreased (Figure 4(b)).Overall, our fndings suggest that KAE reverses sepsis-induced AKI by inhibiting NF-κB/AKT pathway phosphorylation.

KAE Inhibits LPS-Induced Proliferation of Glomerular
Mesangial Cells.To assess the toxicity of KAE to glomerular mesangial cells, we performed viability tests with diferent KAE concentrations.As KAE showed no signifcant inhibition of cell viability at concentrations below 25 µM (Figure 5(a)), we chose KAE doses of 5, 10, and 20 µM for in vitro experiments.In addition, LPS was found to inhibit glomerular mesangial cell proliferation, which was reversed by KAE (Figure 5(b)).In conclusion, KAE can promote mesangial cell proliferation in a dose-dependent manner.

KAE Suppresses the Secretion of Infammatory Cytokines.
Administration of KAE to LPS-treatedHK-2 cells signifcantly decreased the secretion of the infammatory cytokines TNF-α and IL-1β, as measured by ELISA, and the secretion of infammatory cytokines was signifcantly increased in LPS-treated cells (Figure 6(a)).Subsequently, we measured the mRNA expression levels of IL-1β and TNF-α in the cells of each group (Figure 6(b)), and the results were consistent with the ELISA results.Tese data suggest that KAE can reverse AKI injury by inhibiting the secretion of infammatory cytokines.

KAE Inhibits HK-2 Cell
Apoptosis.LPS promoted apoptosis in HK-2 cells, and KAE treatment signifcantly decreased the LPS-induced increase in apoptosis (Figure 7).Tese results suggest that KAE reverses AKI injury by inhibiting the apoptosis of HK-2 cells.

Discussion
AKI is an acute disease that seriously threatens human life and health, and the mortality rate of AKI caused by sepsis is signifcantly higher than that of AKI patients without sepsis [20].Studies have shown that AKI caused by sepsis is mostly manifested as injury caused by an infammatory response and accompanied by apoptosis [21].In our study, we frst demonstrated through small mouse experiments that KAE can improve renal index and renal histological damage in sepsis-induced AKI, i.e., decrease BUN and SCr concentrations and TNF-α and IL-1β expression.In vitro, we constructed a LPS-induced sepsis injury model in HK-2 cells.Te efects of KAE on cell proliferation, infammation, and apoptosis were examined to further investigate the cytoprotective efect of KAE on septic kidney injury.Te experimental results showed that KAE could reduce the expression of proinfammatory cytokines and increase the expression of anti-infammatory cytokines in mice with suppurative AKI, thereby reversing AKI kidney injury.In addition, we demonstrated that KAE could modulate proliferation, apoptosis, and infammation responses in LPS-treatedHK-2 cells.BUN and SCr are important indicators for judging the severity of the renal injury and are commonly used to evaluate renal function.Alshehri et al. found that kaempferol decreased concentrations of BUN and SCr in a rat model of CdCl 2-induced kidney injury [22].In this study, we found that kaempferol signifcantly improved the degree of kidney injury and decreased levels of injury parameters such as  kaempferol can further explain its protective efect by inhibiting the infammatory response caused by renal injury in sepsis.
In cellular experiments, Zhang et al. found that LPS can increase TNF-α and IL-1β expression in HK-2 cells [23].In this study, HK-2 cells were treated with 10 µg/ml LPS, and TNF-α and IL-1β expression was increased, confrming that LPS can induce infammatory damage in HK-2 cells.Subsequently, treatment with kaempferol at diferent concentrations was found to be efective in reducing infammatory cell levels, and in addition, we found that kaempferol treatment signifcantly reduced LPS-induced apoptosis of HK-2 cells by fow cytometry, suggesting that kaempferol could attenuate LPS-induced infammatory injury by inhibiting proinfammatory cytokine expression and inhibiting apoptosis.
NF-κB/AKT is an important regulator of cellular homeostasis [24].NF-κB is a transcription factor that has been implicated in infammation, tumors, apoptosis, and various autoimmune diseases, and it is recognized that NF-κB activation is caused by various external stimuli and is essential for the induction of various proinfammatory cytokines (e.g., IL-1β, TNF-α) [25,26].Inhibition of NF-κB signaling has been shown to attenuate acute kidney injury [27,28].AKT is a downstream target gene of NF-κB and an important functional protein in maintaining cell survival [29].It has been demonstrated that AKT inhibition improves infammatory responses to reverse AKI kidney injury [30].In this study, we clarifed that KAE reversed AKI kidney injury by inhibiting the NF-κB/AKT signaling pathway.
In our study, we frst demonstrated that KAE lowers the expression of proinfammatory cytokines and increases the expression of anti-infammatory cytokines in septic AKI mice, thereby reversing AKI kidney injury.Akhter N et al. reported that calcium dobesilate could reverse AKI injury in severely burned mice by inhibiting NF-κB/AKT expression [31], which is consistent with our study.Park et al. found that kaempferol was able to reduce LPS-induced infammatory mediators by downregulating NF-κB and AKT [32].Te above information provides an efective research strategy from our study to reverse acute kidney injury caused by sepsis.In this study, we demonstrate that kaempferol reverses sepsis-induced acute kidney injury by inhibiting the NF-κB/AKT signaling pathway.

Figure 4 :Figure 5 :Figure 6 Figure 6 :Figure 7 :Figure 8 :
Figure 4: Mechanism of KAE on sepsis-induced AKI in mice.(a) Western blot analysis for total NF-κB p65, P-NF-kB p65, AKT and P-AKT expression in diferent groups.(b) In relative protein expression statistics, all of the data are expressed as the means ± SD of the data obtained in three parallel experiments.* p < 0.05, * * p < 0.01 and * * * p < 0.001 versus AKI group.

Table 1 :
Primer sequences used for PCR.