1. INTRODUCTION
Sepsis is a life-threatening medical condition characterized by a dysregulated host response to infection or injury, as a systemic inflammatory response syndrome. Uncontrolled sepsis may cause organ failure, septic shock, and even death [1, 2]. Sepsis is a major global health problem because of its substantial morbidity and mortality. Approximately 30 million cases of sepsis occur worldwide each year, and its mortality rate is as high as 40% [3, 4]. Sepsis-associated acute kidney injury (S-AKI) is among the most serious and common complications of sepsis, and has an incidence of approximately 50–60% [5, 6]. S-AKI progresses rapidly and aggressively, and has relatively poor prognosis. Therefore, developing more effective strategies is critical for preventing the occurrence or slowing the progression of S-AKI [6].
Although S-AKI has attracted widespread attention, the detailed pathophysiological mechanisms of S-AKI remain complicated and poorly understood [7]. The major pathological manifestations of S-AKI are severe damage or even necrosis of renal tubules (particularly proximal convoluted tubules and distal tubules convoluted tubules) [8, 9]. In addition, extensive studies have reported various abnormal physiological processes in S-AKI, including impaired energy metabolism [10, 11], excess oxidative stress [12], apoptosis and necrosis of renal tubular epithelial cells [13], impaired renal microcirculation [14], activation of inflammatory cells [15], and inflammatory storms [16]. Moreover, because the kidneys are among the most energy-demanding organs in the body, energy metabolism is crucial for proper renal function. Mitochondrial damage and dysfunction are highly involved in tubular cell injury or death in acute kidney injury [17, 18]. Therefore, maintaining the structural and functional integrity of the mitochondria may prevent tubular cell apoptosis, thereby facilitating renal recovery from acute kidney injury. Furthermore, the macrophage polarization state and production of inflammatory mediators markedly affect the progression of S-AKI [19, 20]. For example, the release of pro-inflammatory cytokines or excessive oxidative stress from M1 macrophages can exacerbate renal injury [21]. Moreover, M2 macrophages releasing anti-inflammatory factors and growth factors have protective effects against kidney damage [22, 23]. Accordingly, renal mitochondria and macrophage polarization may be promising targets for the prevention and mitigation of S-AKI.
Cordyceps sinensis (CS) is a fungus with a long history of use in traditional Chinese medicine, owing to its anti-aging and anti-cancer properties [24]. The active ingredients of CS include cordycepin, polysaccharides, sterols, and phenolic compounds [25, 26]. Because of its broad physiological effects, including antioxidant, anti-fibrotic, and anti-inflammatory activity [25, 27], CS has been generally used for renal protection and therapy in patients with diabetic nephropathy, nephrotic syndrome, and lupus nephritis (LN) [28–30]. CS ameliorates renal triglyceride accumulation in diabetic rats by modulating the peroxisome proliferator activated receptor pathway [25]. However, the therapeutic effects and mechanism of actions of CS in S-AKI remain to be clarified, to provide a robust theoretical basis for the clinical application of CS.
In the present study, we first evaluated the protective and therapeutic effects of CS against LPS-induced AKI in mice via assays including histopathological staining, serum renal function indexes, and inflammatory cytokine analyses. To further determine the potential nephroprotective mechanisms of CS in mice, we performed high-throughput multi-omic analyses (transcriptomics and proteomics) on kidney tissues, thus revealing the molecular targets and pathways through which CS ameliorates S-AKI. Finally, we confirmed that CS protects the kidneys against S-AKI by synergistically reprogramming mitochondrial energy metabolism and macrophage polarization. This study provides new insights into the clinical application of CS for the prevention and treatment of S-AKI in patients with sepsis, and indicates that mitochondrial energy metabolism and macrophage polarization may be efficient targets for S-AKI therapy.
2. MATERIALS AND METHODS
2.1 Materials
Cordyceps sinensis powder in Bailing capsules was provided by Hangzhou Chinese-American Huadong Pharmaceutical Co., Ltd (Hangzhou, China). The powder encapsulated in CS was removed and then dissolved in sterile 0.5% sodium carboxymethylcellulose (Sinopharm Group Co., Ltd., China) for subsequent use. Dexamethasone (DEX) was purchased from Bide Pharmatech Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, Co., Ltd. (Missouri, USA). Enzyme-linked immunosorbent assay (ELISA) reagent kits for interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), blood urea nitrogen (BUN), creatinine (CRE), and cystatin C (Cys-C) were provided by Shanghai Enzyme-linked Biotechnology Co., Ltd (Shanghai, China).
2.2 Analysis of CS composition with UHPLC-LTQ-Orbitrap mass spectrometry
CS powder was weighted and added into 50 mL 70% methanol, and the mixture was filtered after ultrasonic treatment for 20 min. Subsequently, 25 mL of the filtrate was vacuum dried, and the dried powder was re-dissolved in 25 mL deionized water and was used as the test solution after an additional filtration step. Ingredient analysis was performed with an online LTQ Orbitrap Velos Pro (Thermo Fisher Scientific Inc.) coupled to a UHPLC instrument via an ESI interface. Liquid chromatography separation was performed on an ACQUITY UHPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column with a temperature of 30°C. The mobile phases were A (methanol) and B (0.1% formic acid in water) with the following gradient elution: 0–20 min, 1% A; 20–36 min, 1%–15% A; 36–45 min, 15%–60% A; 45–45.1 min, 60%–95% A; flow rate: 0.3 mL·min−1; sample volume: 10 μL for reference and 8 μL for test. The main ESI source parameters were set as follows: positive and negative ion scanning mode, capillary temperature 350°C, and spray voltage 3.5 KV in positive ion mode. The primary mass spectra of the samples were fully scanned in FT mode (resolution 30,000, m/z scanning range 50–2,000), and the secondary and tertiary mass spectra were collected in data-dependent mode. Data were collected and analyzed in Xcalibur, Metworks and Mass Frontier 7.0 software.
2.3 Animal experiments
All animal experimental procedures were approved by the China Animal Care and Use Committee, and the Care and Use of Laboratory Animals Committee of the China Academy of Chinese Medical Sciences (approval number 2022B160). C57BL/6 mice (male, 6 weeks old) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and adapted to standard conditions (constant temperature of 22°C; 50%–60% ambient humidity; 12-hour/12-hour light/dark cycle; and free access to water and standard rodent food) for 1 week before the experiments. A total of 60 mice were randomly divided into six groups: control (ctrl; sham treatment), model (LPS, 5 mg/kg), 1 g/kg CS + LPS (CS-L), 5 g/kg CS + LPS (CS-H), 5 mg/kg DEX + LPS (DEX), and 5 mg/kg DEX + 5 g/kg CS+ LPS (CS-H + DEX). The gavage doses of CS were determined on the basis of recommended doses from previous clinical reports [25, 31]. Mice in the CS-L, CS-H, and CS-H + DEX groups were pretreated with CS through gastric perfusion once a day for 14 days before the induction of systemic sepsis with LPS. The treatment duration was determined by comparison of treatment efficacy at days 7 and 14 in a preliminary examination. For the DEX and CS-H+DEX groups, DEX was delivered through gastric perfusion at 24 h before LPS administration to induce systemic sepsis in the mice. DEX was selected as a positive control drug because of its common clinical use in sepsis and its nephroprotective properties (Venkatesh et al. [32], Schirris et al., 2017, Auger et al. [33]). Mice in the ctrl group were treated with the same volume of sodium carboxymethylcellulose vehicle. Mice in all groups except the ctrl group were treated with intraperitoneal injection of LPS 12 h before euthanasia. Blood and kidney samples were collected after anesthesia. A portion of the kidney was fixed in 4% paraformaldehyde, and the remainder was flash-frozen in liquid nitrogen and stored at −80°C for the following tests.
2.4 Serum biochemical index assays
Early renal dysfunction in mice was reflected by the levels of relevant biochemical indicators in the serum. Serum levels of Cys-C, BUN, and CRE were detected according to the manufacturer’s protocols with the kits described above.
2.5 Enzyme-linked immunosorbent assays
The systemic inflammatory status in mice was indicated by the levels of relevant inflammatory factors in the serum. The levels of IL-6, TNF-α, and IL-1β were measured according to the manufacturer’s protocols with the ELISA reagent kits described above.
2.6 Immunohistochemistry analysis of kidney tissue
Kidney tissues were fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned into slices. Hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) staining were performed according to the manufacturer’s instructions for histological examination. Immunohistochemistry (IHC) staining was performed to evaluate the expression of IL-6, TGF-β, and IL-10 in kidney tissues with antibodies to IL-6 (Proteintech, China), TGF-β, and IL-10 (Servicebio, China). Sections were visualized under a light microscope (Nikon, Tokyo, Japan). Images were captured under an inverted fluorescence microscope (CKX53, Olympus, Japan) and analyzed in the configured MShot image analysis system.
2.7 Immunofluorescence staining
Paraffin sections of kidney tissue were deparaffinized and dehydrated, then subjected to antigen repair at 100°C for 5 minutes. Subsequently, the samples were permeabilized and blocked with bovine serum albumin in phosphate buffer saline (PBS) containing 0.1% Tween-20. The slices were then incubated with primary antibodies (anti-TOM20, anti-F4/80, anti-CD206, and anti-iNOS) at 4°C overnight. The corresponding fluorescent secondary antibodies and Hoechst dye were then incubated with the tissue sections for staining. Images were collected and recorded through confocal fluorescence microscopy (Leica TCS SP8 SR, Germany), and the fluorescence intensity of the images was quantified in ImageJ software (version 1.50i).
2.8 Transcriptomics and data analysis
Total messenger RNA (mRNA) was extracted from kidney samples (three samples per group) according to the manufacturer’s instructions with a Qiagen RNeasy Mini Kit. Isolated total mRNA was enriched with a poly(A) template and sequenced on a Novaseq 6000 sequencer (Illumina) with PE150 reads. The quality of raw sequencing data was controlled with fastp, as described in the literature [34]. Reads were then aligned to the mouse reference genome mm10 with STAR (version 2.2.1). Reads were quantified with featureCounts (version 1.5.0). The data were then subjected to statistical analysis and visualization in the R software package. Differentially expressed gene (DEG) analysis was performed with the limma R package (version 3.48.3). P values were generated from an empirical Bayesian test model and adjusted with the Benjamini-Hochberg method. Genes with an absolute fold change (FC) ≥ 2 and an adjusted P (FDR) < 0.05 were considered significant DEGs. Gene Ontology (GO) analysis was performed with the clusterprofiler R package (version 3.18.1). DEGs were further visualized with the ggplot2 R package (version 3.3.5) according to their log2 (FC) and −log10 (FDR) values. The downregulated genes in the model vs. ctrl comparison and upregulated genes in the CS vs. model comparison were subjected to Gene Set Enrichment Analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and GO functional enrichment analysis; the top ten enrichment terms were visualized.
2.9 Proteomics and data analysis
Kidney tissues (three samples for each group) were lysed with RIPA lysis buffer and ultrasonicated on ice. Supernatants were collected after samples were centrifuged (20,000 g at 4°C for 20 min), and the protein concentrations were measured with a BCA kit. Subsequently, 100 μg protein was reduced and alkylated with 20 mM dithiothreitol and 50 mM iodoacetamide sequentially. Samples were then precipitated with pre-chilled acetone at −20°C for 30 min, and the precipitate was collected after centrifugation (20,000 g at 4°C for 5 min) and dissolved in buffer (8 M urea, 100 mM triethylamine bicarbonate, pH 8.5). Each dissolved sample was digested with mass spectrometry grade trypsin (5 μg) in the presence of CaCl2 (1 mM) at 37°C for 17 h. Subsequently, the peptides were slowly loaded onto a C18 desalting column and washed with 0.1% formic acid (FA) buffer for desalination. The eluate for each sample was collected and lyophilized after elution (0.1% FA, 60% acetonitrile). Finally, the samples were re-dissolved in 0.1% formic acid and analyzed by liquid chromatography-mass spectrometry (Thermo Orbitrap Fusion Lumos, USA). Raw MS files were processed and analyzed in Proteome Discoverer 2.4 (Thermo Scientific). Proteins with absolute FC ≥ 1.5 and P value (FDR) < 0.05 were defined as differentially expressed proteins (DEPs). Data were visualized with the Bioladder website (https://www.bioladder.cn) and TBtools [35]. KEGG pathway enrichment analysis, GO analysis, and protein-protein interaction (PPI) analysis were performed with the bioinformatics platform http://www.bioinformatics.com.cn.
2.10 Western blotting analysis
Western blotting (WB) analysis was conducted to confirm the expression levels of proteins that significantly differed across groups in the proteomic analysis. Kidney samples were lysed with pre-chilled RIPA lysis buffer supplemented with protease inhibitors. Subsequently, the proteins were concentrated and separated with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Afterward, proteins in the gels were electro-transferred to polyvinylidene fluoride membranes at 120 V for 80 min. Next, the membranes were blocked with 5% bovine serum albumin in 1× TBST containing 0.1% Tween-20, and further incubated with the primary antibodies (anti-ND5, anti-SDHC, anti-CYTB, anti-MTCO2, anti-ATP6, anti-β-actin, anti-CD206, anti-Arg1, anti-iNOS, and anti-IL-6) overnight at 4°C, then with secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized with enzyme-linked chemiluminescence (Thermo Fisher, USA). The protein band intensity was semi-quantified in ImageJ software and normalized to β-actin expression.
2.11 Quantitative real-time polymerase chain reaction analysis
Quantitative real-time PCR (q-PCR) was used to measure the mRNA expression of target genes. Total RNA was isolated from kidney tissues with TranZol Up (TransGen Biotech, Beijing, China) according to the manufacturer’s guidelines. The concentration and purity of the total RNA were subsequently determined with a NanoDrop instrument (Thermo Scientific, USA). Reverse transcription was performed with 500 ng total RNA and a RevertAid First Strand cDNA Synthesis Kit (TransGen Biotech). The qPCR analysis was performed in the QuantStudio 5 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Relative mRNA levels were determined with the 2−ΔΔCt method, normalized to a housekeeping control, GAPDH. The primer sequences were as follows: for mouse ND5, forward: 5´-TTCCTAACAGGGTTCTACTC-3´, reverse: 5´-GGTCTGGGTCATTTTCGT-3´; for mouse SDHC, forward: 5´-ATAGCCTTGAGTGGAGGGGTC-3´, reverse: 5´-GTGAGTGGTACATGAGCGGG-3´; for mouse CYTB, forward: 5´-AGACAAAGCCACCTTGACCC-3´, reverse: 5´-GATTGCTAGGGCCGCGATAA-3´; for mouse MTCO2, forward: 5´-GCCGACTAAATCAAGCAACA-3´, reverse: 5´-CAATGGGCATAAAGCTATGG-3´; for mouse ATP6, forward: 5´-ACACCAAAAGGACGAACA-3´, reverse: 5´-AGGAAGTGGGCAAGTGAG-3´. The relative mRNA levels were normalized to GAPDH mRNA.
2.12 Cell culture and treatment
Human renal tubular epithelial cells (HK-2 cells) obtained from the Chinese Academy of Medical Sciences (Beijing, China) were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (Corning, USA). This medium was supplemented with 10% FBS (Corning, USA) and 1% (v/v) penicillin/streptomycin (Thermo Fisher, USA), and maintained at 37°C in a 5% CO2 atmosphere. Before stimulation, HK-2 cells were subjected to serum deprivation for 12 hours, then treated with 10 μg/mL LPS (Sigma-Aldrich, USA).
2.13 Cell viability assays
HK-2 cells were cultured in 96-well plates at a density of 1 × 104 cells/well. Cell viability was evaluated with a Cell Counting Kit (CCK)-8 (Meilun Biotechnology, China), according to the provided instructions. Absorbance was measured at 450 nm with a microplate reader (PerkinElmer, USA).
2.14 Mitochondrial function analysis
The mitochondrial membrane potential (MMP) in kidney tissue was detected instruction manual with the JC-1 kit (Beyotime, China). Briefly, fresh kidney tissue (100 mg) from each mouse was harvested (four samples per group), and mitochondria were isolated and purified as soon as possible with a Mitochondrial Isolation Kit (Beyotime, China) according to the manufacturer’s instructions. MMP was further detected according to red/green fluorescence intensity with a fluorescence microplate reader (PerkinElmer, USA). The content of ATP in fresh mouse kidney tissue was determined according to the manufacturer’s instructions with a commercial assay kit (mlbio, China).
2.15 Mitochondrial superoxide assays
Mitochondrial superoxide (MitoSOX) production was investigated with a commercial fluorogenic dye, MitoSOX. The assay was conducted according to the reagent vendor’s protocol, with minor adjustments. Briefly, cells were seeded in confocal dishes, incubated overnight, and subjected to various treatments for 24 hours. Cells were then washed with PBS at room temperature and stained with 5 μM MitoSOX in serum-free medium for 30 minutes in the dark. Concurrently, nuclei were stained with Hoechst dye. The cells were then rinsed three times with PBS, and images were captured with a confocal fluorescence microscope (Leica TCS SP8 SR).
2.16 Enzymatic assays for mitochondrial complex activity
First, mitochondria in fresh kidney tissue were isolated with a Mitochondrial Isolation Kit. Subsequently, the isolated mitochondria were subjected to evaluation of mitochondrial complex activity with commercial kits from Shanghai Enzyme-linked Biotechnology Co., Ltd.
2.17 Statistical analysis
All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis for several groups was performed with one-way ANOVA with Tukey’s post hoc test. For comparison of two groups, Student’s t-test (two-tailed) was used to calculate statistical significance. P values < 0.05 were considered statistically significant. All data were analyzed in GraphPad Prism software (version 8.0).
3. RESULTS
3.1 CS ameliorates renal dysfunction and inflammation in S-AKI mice
The extracted ion chromatograms of CS test solution in positive and negative ionization modes by UHPLC-LTQ-Orbitrap MS are shown in Figure S1 . A total of 58 compounds were identified, comprising 17 nucleosides, 15 amino acids, 5 purine alkaloids, 4 pyrimidine alkaloids, 8 organic acids, 2 nucleotides, 2 sugar alcohols, and 5 other compounds. The detailed mass spectrometry information is listed in Table S1 .
The nephroprotective effect of CS was first studied in a sepsis model induced by LPS in mice. As shown in the scheme in Figure 1A , mice were intragastrically administered vehicle or CS for 14 days before intraperitoneal injection of LPS. Subsequently, we evaluated the protective effect of CS at low (1 g/kg) and high doses (5 g/kg) on S-AKI in mice. Histopathological results based on H&E and PAS demonstrated that LPS induced clear pathological injury in the kidneys ( Figure 1B ), where most of the renal tubules were dilated and edematous, and the tubular epithelial cells were generally vacuolated, necrotic, detached, and displayed cast formation. As expected, treatment with CS and/or DEX, a classical corticosteroid with anti-inflammation activity, effectively ameliorated this pathological injury. Furthermore, IHC revealed that CS and/or DEX clearly suppressed the elevated expression of the inflammatory cytokine IL-6 elicited by LPS in the kidneys ( Figure 1B ). Additionally, the expression of Kidney Injury Molecule-1 (KIM-1), a transmembrane glycoprotein widely recognized as a marker of renal tubular injury [36], was assessed in kidney paraffin sections with IHC assays. Similarly, treatment with CS and/or DEX significantly decreased the elevated KIM-1 in the mouse model of kidney injury, thereby suggesting considerable attenuation of renal tubular injury by CS in S-AKI mice. Serum Cys-C, BUN, and CRE are essential indicators of renal function, and Cys-C is an effective marker of the glomerular filtration rate for early diagnosis of renal disease [37]. As shown in Figure 1C–E , LPS severely impaired renal function in mice, as indicated by increased serum levels of Cys-C, BUN, and CRE. As expected, CS and/or DEX effectively decreased the levels of these indicators in the serum in S-AKI mice, thus highlighting the fine nephroprotective effect of CS. Furthermore, CS and/or DEX treatment also significantly decreased the serum levels of the inflammatory factors IL-6, TNF-α, and IL-1β, thus suggesting anti-inflammatory activity of CS in S-AKI ( Figure 1F–H ). Notably, CS had similar nephroprotective effects to DEX, and their combination further enhanced the protective functions. Collectively, these results demonstrated that CS ameliorates renal dysfunction and inflammation in S-AKI mice, and thus may have potential medical value in treating renal injury.
CS ameliorates renal function and suppresses inflammation in S-AKI mice.
(A) Schematic illustration of the nephroprotective effects of CS in S-AKI mice. (B) Representative images of hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), IL-6 immunohistochemistry (IHC), and KIM-1 immunofluorescence staining in kidney tissues (400×, scale bar: 50 μm or100 μm). (C-E) Serum renal function indicators Cys-C (C), BUN (D), and CRE (E) in S-AKI mice after CS or/and DEX treatment. (F-H) Serum inflammatory cytokines IL-6 (F), TNF-α (G), and IL-1β (H) in S-AKI mice after CS or/and DEX treatment. All data are presented as mean ± SEM (n = 10). #### P < 0.0001 vs. ctrl; *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. model; & P < 0.05, && P < 0.01 vs. CS-H + DEX.
3.2 Transcriptomics reveals that CS reprograms mitochondrially mediated oxidative phosphorylation in the kidneys
To elucidate the potential mechanisms of CS’s nephroprotective activity in S-AKI mice, we performed transcriptomic analysis of kidney tissues from the vehicle (ctrl), S-AKI (model), and CS treatment (5 g/kg, CS) groups ( Figure S2 ). Violin plots ( Figure 2A ) and heatmaps ( Figure S3 ) showed the relative expression levels of genes in the transcriptomic dataset. The model group, compared with the ctrl group, displayed significantly upregulated gene expression, whereas CS partly reversed the change in gene expression induced by LPS, in line with pathological observations. DEGs with a fold change greater than 2 in the model vs. ctrl and the CS vs. model comparisons were further depicted and highlighted in the scatter plot ( Figure 2B ). The model group, as compared with the ctrl group, contained 1,807 up-regulated and 1,751 down-regulated genes. Meanwhile, 221 up-regulated and 206 down-regulated genes were identified in a comparison of the CS group vs. the model group. Furthermore, the intersection of the up- and down-regulated genes was analyzed and displayed in a Venn diagram ( Figure 2C ), wherein the expression of 89 and 37 genes was found to be respectively suppressed and activated, respectively, in the model vs. ctrl comparison, whereas opposite changes were observed in the CS vs. model comparison.
Transcriptomics reveals the involvement of CS reprogramming genes in mitochondrially mediated oxidative phosphorylation (OXPHOS) in S-AKI.
(A) Violin plot showing the expression of all genes in each group. (B) Differential analysis showing the fold changes in genes in the transcriptomics dataset. (C) Venn diagram showing the intersection of DEGs in each group. (D) Cluster analysis of DEGs in the ctrl, model, and CS groups, according to variation patterns. (E) GSEA enrichment results for the model vs. ctrl groups. (F) GSEA enrichment results for the CS vs. model groups. (G) KEGG enrichment results of downregulated genes in the model vs. ctrl groups and upregulated genes in the CS vs. model groups. (H) GO enrichment analysis of the DEGs of the model vs. ctrl and CS vs. model groups. (I) Heatmap of DEGs associated with the OXPHOS pathway.
To further clarify the potential transcriptional regulatory relationships within these genes, we divided the differential genes into five clusters according to their expression variation patterns ( Figure 2D ). Interestingly, for genes in clusters 3 and 5, CS could greatly neutralize their expression change caused by LPS, thus suggesting that these genes were highly likely to be involved in the pathways through which CS repairs renal injury. Therefore, GSEA was applied to these genes to enrich the molecular pathways associated with the protective effect of CS ( Figure 2E, F ). The GSEA results indicated that the oxidative phosphorylation (OXPHOS) process in the kidney was significantly downregulated in the model vs. ctrl analysis ( Figure 2E ), but upregulated in the CS vs. model analysis ( Figure 2F ). However, genes involved in the pathways of ribosome and protein processing in the endoplasmic reticulum showed an opposite variation tendency from the genes enriched in the OXPHOS pathway. Furthermore, KEGG analysis of DEGs indicated that OXPHOS and thermogenesis were the top two pathways in terms of enrichment scores ( Figure 2G ). Subsequently, functional enrichment analysis of DEGs was conducted, and the model vs. ctrl and CS vs. model groups were compared ( Figure 2H ). In agreement with the GSEA and KEGG results, OXPHOS, the electron respiratory chain, ATP biosynthesis, and other mitochondria-related energy metabolic processes were significantly down-regulated after LPS induction, whereas CS treatment rescued these effects in S-AKI. Furthermore, GO enrichment analysis of the DEGs regarding molecular function and cellular component classification identified enrichment in electron transfer activity and the respirasome, which were also closely associated with the mitochondria energy metabolic process ( Figure S4 ). The expression profiles of major genes associated with OXPHOS were further depicted in a heatmap ( Figure 2I ) showing that CS led to partial recovery in the expression of these genes down-regulated by LPS. Together the transcriptomics results suggested that CS may prevent and resist the development of S-AKI by repairing the perturbed mitochondrially mediated OXPHOS pathway.
3.3 Proteomic analysis reveals that CS promotes the expression of mitochondrial electron transport chain-related proteins in the kidneys in S-AKI mice
To profile and confirm the global effects of CS at the proteomic level, we performed label-free quantitative proteomics on kidney tissue ( Figure S5 ). A total of 5,302 proteins were identified across the ctrl, model, and CS (5 g/kg) groups, and cluster analysis, as depicted in heatmap plots of protein abundance, indicated that CS had a closer correlation to the ctrl than the model group ( Figure S6 ). Furthermore, correlation analysis among the three groups directly indicated that CS had a positive correlation coefficient with the ctrl group in scaled protein abundance, whereas the model group had a negative correlation coefficient with ctrl and CS ( Figure 3A ). These results indicated that CS treatment somewhat restored the proteomic composition in the kidney of S-AKI to a normal state. DEPs in the model vs. ctrl and CS vs. model comparisons are shown in volcano plots with a cutoff of fold change >2 and P-value < 0.05 ( Figure 3B, C ). A total of 177 down-regulated and 350 up-regulated proteins were found in the model vs. ctrl comparison, whereas 345 and 197, respectively, were identified respectively, in the CS vs. model comparison.
Proteomic analysis reveals that CS promotes the expression of mitochondrial electron transport chain (ETC)-related proteins in the kidneys in S-AKI mice.
(A) Correlogram of the scaled abundance of proteins across three groups, according to correlation analysis. (B) Volcano plot showing the DEPs in the model vs. ctrl groups. (C) Volcano plot showing the DEGs in the CS vs. model groups. (D) Sankey diagram showing the top 20 GO enrichment terms for upregulated proteins in the CS vs. model groups. (E) PPI analysis of the top 20 terms in GO enrichment analysis. (F) Cyclic heatmap showing expression changes in all quantified mitochondrial proteins.
To further confirm the protein expression of OXPHOS-associated genes in the transcriptomic analysis, the proteins that were down-regulated in the model vs. ctrl groups and up-regulated in the CS vs. model groups were annotated with protein names if they showed similar changes in transcriptomic data. Respiratory complex proteins including NADH-ubiquinone oxidoreductase chain (ND1, ND2, ND3, ND4, and ND5), cytochrome c oxidase subunit (COX1, COX2, and COX3), F-ATPase protein 6 (ATP6), and cytochrome b (CYTB) were highlighted. The abundance of these proteins decreased in S-AKI mice but was rescued after CS treatment, thereby indicating that CS might restore the expression of proteins associated with mitochondrial electron transport in the kidneys. GO enrichment analysis of DEPs was also conducted and visualized in Sankey bubble plots ( Figure 3D ). The pathway of OXPHOS was preferentially enriched, and other energy metabolism biological processes, including ATP synthesis, cellular respiration, mitochondrial respiratory chain complex, and the electron transport chain, also showed high enrichment. Moreover, most proteins involved in OXPHOS were associated with mitochondrial ETC function, thus suggesting that the mitochondrially mediated OXPHOS pathway was the critical factor through which CS protected against S-AKI. This result was further confirmed through more detailed GO ( Figure S7 ) and KEGG ( Figure S8 ) enrichment analyses of DEPs. DEPs were then subjected to PPI analysis to identify potential changes in protein networks during the model induction and CS treatment processes ( Figure 3E ). In particular, robust and close interactions were observed in mitochondrial proteins such as ND1, ND2, COX1. Therefore, we next analyzed the protein abundance of all quantified mitochondrial proteins and compared them in a circular heatmap ( Figure 3F ). Most mitochondrial proteins, particularly ETC-related proteins, were significantly down-regulated in the model vs. ctrl comparison, and their abundance was restored via CS administration. Overall, our proteomics and transcriptomics results collectively suggested that CS facilitates the expression of the suppressed proteins involved in OXPHOS and mitochondrial ETC in S-AKI, thus providing informative clues for illuminating the mechanisms of action of CS’s nephroprotective activities.
3.4 CS elevates the expression and activity of the mitochondrial complex
To further validate our findings on the nephroprotective mechanisms of CS from the transcriptomic and proteomic analyses, we used multiple biochemical approaches including WB, immunofluorescence, and enzyme activity assay kits to examine the expression and activity of mitochondrial complex proteins, as well as mitochondrial integrity in kidney tissues ( Figure 4 ). In agreement with the transcriptomic and proteomic results, LPS significantly suppressed the expression of mitochondrial protein complex subunit proteins, including complex I (CⅠ-ND5), complex II (CⅡ-SDHC), complex III (CⅢ-CYTB), complex IV (CⅣ-MTCO2), and complex V (CⅤ-ATP6), whereas CS restored the expression of all five mitochondrial complexes to varying degrees ( Figure 4A, B ). In line with the protein quantification results, the mRNA levels of the six genes assessed via qPCR displayed similar change trends across the three groups ( Figure 4C ). Consequently, we proposed that the maintenance of mitochondrial function via promoting the expression of mitochondrial complexes I, II, III, IV, and V is crucial for mice treated with CS to resist LPS-induced impairment of renal tubular energy metabolism. To further assess the number and integrity of mitochondria in the renal tubular region, we performed immunofluorescence staining on kidney tissue with the mitochondrial marker TOM20, as described in the literature [38]. The experimental results indicated a dramatically decreased number and distorted morphology of mitochondria in the proximal tubules in S-AKI mouse kidneys, whereas this effect was greatly ameliorated by CS treatment ( Figure 4D ), thereby further demonstrating that CS promoted mitochondrial biogenesis and maintained mitochondrial structural integrity.
CS elevates mitochondrial complex expression and activity.
(A, B) Protein expression of OXPHOS complexes in kidney tissues (n = 3) from the ctrl, model, and CS groups. (C) mRNA expression of ND5, SDHC, CYTB, MTCO2, and ATP6, determined by qPCR analysis in kidney tissues (n = 4). (D) Representative confocal immunofluorescence images of TOM20 (green) and nuclear counterstaining (Hoechst, blue) in kidney tissues from the ctrl, model, and CS groups. Scale bars, 20 μm. (E) MMP in purified mitochondria from kidney tissues (n = 4). (F) ATP levels in kidney tissues (n = 4). (G-I) Activity of mitochondrial complex I, Ⅱ, and Ⅳ in purified mitochondria from kidney tissues (n = 4). (J) Summary of the effects of CS on mitochondrial biogenesis and activity. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Because MMP is a key parameter in mitochondrial function [39], we performed JC-1 mitochondrial membrane potential assays to study MMP levels in kidney tissue, where mitochondrial dysfunction was determined according to a decreased ratio of JC-1 aggregates, indicated by red intensity, vs. JC-1 monomers, indicated by green intensity. As depicted in Figure 4E , LPS-induced S-AKI mice had lower MMP than ctrl mice, whereas CS significantly elevated the MMP beyond that observed in the model group, thus partly restoring the MMP decrease elicited by LPS. Meanwhile, the ATP level in kidney tissue showed similar changes to MMP across the three groups: CS rescued the ATP decrease in the S-AKI model ( Figure 4F ). Furthermore, the activity of mitochondrial complexes was further assessed after the mitochondria in kidney tissue were extracted. The activity of complexes I ( Figure 4G ), II ( Figure 4H ), and IV ( Figure 4I ) was suppressed after LPS induction but was restored to normal levels after CS treatment. Together, these findings demonstrated that CS elevated the expression and activity of proteins involved in mitochondrial ETC, and promoted subsequent mitochondrial biogenesis and OXPHOS, thereby resisting impaired energy metabolism in S-AKI mice ( Figure 4J ).
3.5 CS suppresses renal inflammation by regulating macrophage polarization in S-AKI mice
Previous studies have demonstrated that macrophages are largely responsible for inflammatory infiltration in various renal diseases, including S-AKI [40, 41]. As a highly heterogeneous cell type, macrophages can differentiate into morphologically and functionally distinct M1/M2 phenotypes depending on their microenvironment, and the disease type and stage [42]. Notably, emerging immunometabolism studies indicate that energy homeostasis of anti-inflammatory macrophages (M2) requires mitochondria-related OXPHOS, whereas that of pro-inflammatory macrophages (M1) relies more on glycolysis [43]. Furthermore, recent research has suggested that CS affects the polarization state of infiltrating macrophages in LN mice by changing the levels of typical protein markers participating in macrophage polarization [26]. Considering a that CS might enhance renal OXPHOS in mitochondria, thereby resisting LPS-induced injury, we questioned whether CS might also exert nephroprotective effects by regulating macrophage polarization in renal tissue. To this end, we performed immunofluorescence staining and WB assays to analyze the biomarkers of M1 and M2 macrophages. Inducible nitric oxide synthase (iNOS) and CD206 antigen are widely recognized as biomarkers for M1 and M2 type macrophages respectively, and ADGRE1/EMR1 (F4/80) is a protein marker commonly expressed in mammalian monocyte-macrophages [44].
As shown by double immunofluorescence staining ( Figure 5A, B ), CS effectively alleviated the macrophage infiltration caused by LPS, as demonstrated by a decrease in the F4/80 green fluorescence signal after pre-treatment of S-AKI mice with CS, in agreement with previous findings [26]. Moreover, a significantly greater number of CD206 + F4/80 macrophages and a markedly lower number of iNOS + F4/80 macrophages were detected in kidney tissue sections from the CS group than the model group, thus suggesting that CS might ameliorate renal inflammation by increasing the ratio of M2/M1 macrophages. To further validate the macrophage polarization state in the three groups, we examined the expression of additional biomarkers including arginase 1 (Arg1) and IL-6. The WB results revealed that LPS significantly activated M1 macrophages in the kidneys by simultaneously increasing the expression of the M1 macrophage markers iNOS and IL-6 and decreasing the expression of the M2 macrophage markers CD206 and Arg1. In contrast, CS administration promoted the transition from M1 macrophages to M2 macrophages by reversing the expression change in these biomarkers elicited by LPS, thereby supporting the anti-inflammatory capacity of CS by regulating macrophage polarization ( Figure 5C, D ). Additionally, the role of CS in regulating macrophage polarization was also demonstrated in an in vitro model of mouse mononuclear macrophages, RAW264.7 cells ( Figure S8 ). Finally, immunohistochemical staining verified that the expression of M2 macrophage-associated anti-inflammatory factors (transforming growth factor-beta (TGF-β) and IL-10) was greatly upregulated in the CS group, to levels higher than observed in the ctrl group ( Figure 5E–G ). The results of phenotypic macrophage-associated protein evaluation further demonstrated that CS promoted M2 macrophage polarization in S-AKI mice. Collectively, these results strongly indicated that CS suppresses renal inflammation by regulating macrophage polarization in S-AKI mice.
CS suppresses renal inflammation by regulating macrophage polarization in S-AKI mice.
(A, B) Immunofluorescence staining of classical macrophage markers (CD206 for M2 type and iNOS for M1 type) in kidney tissues (scale bar = 50 μm). Green fluorescence, F4/80; red fluorescence, CD206 or iNOS; blue fluorescence, nuclei stained with Hoechst. (C, D) Representative blots of M2 markers (CD206 and Arg1) and M1 markers (iNOS and IL-6) in kidney tissue (n = 3). Protein expression was normalized to β-actin expression and is represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Representative immunohistochemical images of TGF-β and IL-10 in kidney sections. Scale bar: 100 μm. (F) Quantitative analysis of immunohistochemical staining for TGF-β in kidney sections (ten fields from three mice, n = 3); ****P < 0.0001. (G) Quantitative analysis of immunohistochemical staining for IL-10 in kidney sections (ten fields from three mice, n = 3); ****P < 0.0001.
3.6 CS ameliorates mitochondrial dysfunction in LPS-induced HK-2 cells
To validate and extend our in vivo findings, we used human renal tubular epithelial cells (HK-2 cells) as an in vitro model. Morphological analysis revealed that after LPS stimulation, HK-2 cells exhibited cytoplasmic coagulation and lysis, resembling necrosis-like disruption. However, after intervention with CS, the morphology of HK-2 cells reverted to a state resembling normalcy, which was accompanied by a noticeable increase in cell proliferation ( Figure 6A ). Subsequently, we assessed the protective effect of CS in LPS-induced HK-2 cells, by using CCK8 assays. Interestingly, CS treatment not only enhanced the resistance of HK-2 cells to LPS-induced damage but also significantly promoted their proliferation ( Figure 6B ). In agreement with the outcomes observed in kidney tissues, the elevation in IL-6 levels induced by LPS in HK-2 cells was mitigated by CS treatment ( Figure 6C, D ).
CS ameliorates mitochondrial dysfunction in LPS-induced HK-2 cells.
(A) Representative images of HK-2 cells after LPS or/and CS treatment. (B) Cell viability of HK-2 cells after LPS and/or CS treatment. *P < 0.05, ***P < 0.001, ****P < 0.0001. (C, D) Representative blots showing mitochondrial regulatory proteins (PGC-1α and Drp1) and the inflammatory factor IL-6 in HK-2 cells (n = 3). Protein expression was normalized to β-actin expression and is represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (E) Mitochondrial morphology in HK-2 cells visualized by immunofluorescence staining with antibody to TOM-20. (F) Fluorescence images of HK-2 cells stained with JC-1 to assess mitochondrial membrane potential. (G) Mitochondrial superoxide production of HK-2 cells examined by MitoSOX.
Nephroprotective effect and underlying mechanism of CS against LPS-induced S-AKI.
CS treatment significantly alleviates LPS-induced renal dysfunction by facilitating mitochondrial biogenesis, increasing mitochondrial complex activity, improving renal energy metabolic reprogramming, and promoting M2 macrophage polarization. CS: Cordyceps sinensis.
Considering the potential renoprotective role of CS in regulating mitochondrial biogenesis, as inferred from transcriptomic and proteomic studies, we further investigated the effects of CS on mitochondrial biogenesis and function in LPS-induced HK-2 cells through in vitro experiments ( Figure 6C–G ). Our findings regarding mitochondrial regulatory proteins suggested that CS facilitated mitochondrial biogenesis and attenuated mitochondrial division, as evidenced by upregulation of PGC-1α protein expression and downregulation of DRP1 expression ( Figure 6C, D ). Additionally, antibody to TOM-20, a mitochondrial marker, was used for immunofluorescence staining to visualize mitochondria in HK-2 cells. After LPS stimulation, the mitochondria appeared fragmented and discontinuous; however, CS treatment effectively mitigated this damage, thereby restoring the mitochondria to an intact, continuous tubular network resembling that observed in normal cells ( Figure 6E ). Subsequently, JC-1 staining revealed that LPS treatment led to a more significant decrease in MMP than observed in the normal group. Similarly, this LPS-induced decline in MMP was counteracted by CS treatment ( Figure 6F ). Finally, mitochondrial ROS production was assessed. LPS treatment led to a marked elevation in mitochondrial ROS relative to that in the ctrl group, whereas this affect was effectively mitigated by CS intervention ( Figure 6G ). The outcomes were strikingly similar to those for MMP. In summary, these findings indicated that the therapeutic efficacy of CS on LPS-induced HK-2 cells is associated with the modulation of both mitochondrial dynamics and function.
4. DISCUSSION
Although understanding of S-AKI has; substantially improved, effective prevention or treatment strategies remain urgently needed [45, 46]. In the present study, we explored the potential protective effect of CS on LPS-induced S-AKI mice and the underlying mechanisms. CS exerted a beneficial effect in mice with LPS-induced S-AKI, and significantly prevented the deterioration of serum biochemical markers, inflammatory responses, and pathological changes in renal tissues. At the molecular level, CS facilitated mitochondrial biogenesis, restored MMP, and increased ATP levels, thereby promoting renal OXPHOS in the mitochondria, and achieving energy metabolic reprogramming in the kidneys. Furthermore, CS ameliorated inflammation by decreasing macrophage infiltration and regulating macrophage polarization. Integrated outcomes of both in vivo and in vitro studies suggested that CS may potentially help septic mice resist S-AKI by enhancing and sustaining mitochondrial biogenesis and function in kidney cells, as well as promoting the polarization of macrophages toward the M2 phenotype.
S-AKI is a widespread kidney disease with high prevalence worldwide that severely threatens people’s health. Currently, S-AKI is considered the leading cause of AKI in the intensive care unit setting, affecting almost 50% of critically ill patients with sepsis [47, 48]. In recent years, with increasing understanding of the potential mechanisms of S-AKI, cellular metabolic reprogramming, and M1-macrophage polarization might be considered important pathophysiological features of S-AKI, and are closely associated with disease progression [42, 45]. Thus, improvement in renal energy metabolism and regulation of macrophage polarization in the kidneys may be an effective strategy for the prevention and treatment of kidney injury. Bailing capsule is a pleiotropic Chinese traditional medicine made through low-temperature fermentation of CS [27]. Importantly, CS from Bailing capsule has demonstrated satisfactory nephroprotective effects in mice with diabetic nephropathy, through enhancing renal metabolism [25]. Moreover, CS has recently been reported to markedly decrease the infiltration of M1 macrophages in LN kidneys [26]. Accordingly, we believe that the potent nephroprotective effect of CS may advance the prevention and treatment of S-AKI.
Herein, CS effectively decreased the dysfunction, and the expression and secretion of inflammatory cytokines, in the kidneys of S-AKI mice. Of note, CS was administered to mice before LPS injection and exhibited favorable preventive effects against S-AKI. Subsequently, integrated multi-omic (transcriptomic and proteomic) analyses collectively indicated that enhanced mitochondrial-mediated OXPHOS is a critical mechanism through which CS protects mice against LPS-induced S-AKI. However, the differences observed between the transcriptomic and proteomic data suggested the involvement of post-transcriptional and post-translational regulatory mechanisms during CS treatment, which warrant further investigation. Sepsis predisposes people to mitochondrial damage and impairs energy metabolism, which also may be an important reason why sepsis easily progresses to multi-organ dysfunction syndrome [49]. As a highly energy-consuming organ enriched in mitochondria, the kidneys generate vast amounts ATP through OXPHOS, in which the electron transport chain is coupled with chemiosmosis in mitochondria. OXPHOS impairment is a critical indicator of the development of S-AKI [50]. In contrast, LPS substantially damages OXPHOS enzymatic reactions, thus resulting in a phenomenon known as cytopathic hypoxia [51]. Therefore, the restoration of mitochondrially mediated OXPHOS by CS in the kidneys might have greatly contributed to the improved ability of mice to resist LPS-induced S-AKI.
In later molecular mechanism experiments, we confirmed that CS treatment indeed increased the expression of mitochondrial complex-associated proteins, in line with the multi-omics results. Moreover, immunofluorescence results revealed that the structurally intact mitochondria were densely distributed in the kidneys in mice pretreated with CS. Furthermore, considering the results of in vitro experiments on LPS-induced HK-2 cells, specifically the effects of CS on the expression of the mitochondrial regulatory proteins PGC-1α and Drp-1, certain active components in CS might plausibly target mitochondrial kinetic homeostasis and stabilize mitochondrial biogenesis by inhibiting Drp1-mediated mitochondrial fission. Importantly, previous studies have reported that CS enhances the metabolic function of the kidneys in diabetic rats [25]; therefore, CS might influence mitochondrial gene expression and biogenesis by improving the metabolic function of the organism. Accordingly, CS probably exerted a stabilizing effect on energy metabolism by promoting mitochondrial biogenesis in the kidneys. The mitochondrial electron transport chain generates cellular ATP through a series of electron transfer reactions in OXPHOS, wherein mitochondrial complexes are an integral part of the electron transport chain [52]. Functional electron transport provides the necessary bioenergetic fuel to maintain the normal physiological activities of tissues and organs [52]. To further explore the mechanism through which CS promoted OXPHOS in the mitochondria, we examined mitochondrial functional indicators including MMP, ATP content, and the activity of the mitochondrial complexes I, II, and IV in the kidneys. CS treatment maintained mitochondrial energy metabolism in the kidneys by repairing the functional damage caused by LPS. These findings together suggested that enhancement of mitochondrial biogenesis and OXPHOS may be a potential mechanism through which CS acts against S-AKI.
Beyond the impairment in mitochondrially mediated energy metabolism in the kidneys, the inflammatory response also plays critical roles in the development and chronicity of kidney injury [53]. Macrophages in renal tissues are essential inflammatory cells involved in renal inflammation and repair, and precise regulation of macrophage activation is a potentially promising therapeutic strategy for S-AKI [40, 54]. More importantly, because energy metabolic status is closely associated with the activation of macrophages [43, 55], we also studied macrophage polarization in the kidneys. CS not only significantly decreased macrophage infiltration in the kidneys but also promoted the polarization of M1 macrophages into M2 macrophages, thereby suggesting that CS might also help mice resist sepsis-induced kidney injury by improving the infiltration and polarization state of macrophages in the kidney. Increasing evidence demonstrates that enhanced glycolysis and disruptions in the tricarboxylic acid cycle are highly associated with the polarization of pro-inflammatory M1-type macrophages, whereas anti-inflammatory M2-type macrophages are characterized by glutamine and FA-fueled mitochondrial respiration [43, 56, 57]. Notably, in this study, CS was found to strengthen mitochondrially mediated OXPHOS in the kidneys and simultaneously to facilitate M2 polarization of macrophages. Thus, CS-mediated energy metabolism reprogramming may serve as an effective intervention strategy to alleviate impaired energy metabolism and overactivation of M1 macrophages in the early stages of S-AKI. The relationship between mitochondrial energy metabolism and macrophage polarization phenotype during the occurrence and development of S-AKI is worthy of future study.
CS, a fungus with an extensive history in traditional Chinese medicine, exerts therapeutic effects through a diverse array of components, targets, and pathways. Our analysis identified 58 metabolites in CS, encompassing nucleosides, amino acids, purine alkaloids, pyrimidine alkaloids, organic acids, sugar alcohols, and other compounds. These bioactive constituents have a wide range of therapeutic properties, including anti-inflammatory, antifibrotic, and immunomodulatory activity [58]. Among these compounds, cordycepin, a nucleoside derived from Cordyceps, is a potential key active component of CS exerting multiple pharmacological effects. Extensive research has demonstrated that cordycepin has anti-inflammatory properties by modulating mitochondrial dynamics and enhancing mitochondrial function [59–62]. Recent studies have further indicated that cordycepin mitigates cisplatin-induced renal damage via GSK-3β-mediated ferroptosis [63]. Moreover, intraperitoneal injection of cordycepin after ischemia has been shown to protect renal tissues against oxidative stress in a rat model of renal ischemia/reperfusion injury [64]. In light of these findings, the cordycepin present in CS might potentially confer benefits in mice with S-AKI. However, despite these promising results, the primary active components of CS that regulate mitochondrial biogenesis against S-AKI remain unidentified and may be a potential avenue for future research endeavors.
Nonetheless, our study has several limitations. Mice were administered CS for 14 days before receiving high doses of LPS to induce shock. In clinical practice, patients are typically diagnosed with S-AKI before initiation of appropriate therapeutic interventions. Consequently, further basic research, including clinical trials, is necessary to elucidate the potential role of CS in treating S-AKI. Additionally, this study evaluated only the regulation of macrophage polarization by CS through serum inflammatory factor levels and macrophage marker expression in mouse renal tissues. A more comprehensive assessment of the overall status of inflammatory cells in mice may require a high-throughput single-cell sequencing approach, and might enhance understanding of the mechanisms underlying CS’s regulation of inflammatory cells.
5. CONCLUSION
In summary, our in vivo results revealed that CS treatment substantially ameliorated LPS-induced renal injury and the inflammatory response. Multi-omic analysis showed that the promotion of mitochondrial biogenesis and facilitation of OXPHOS in the kidneys by CS might be the potential mechanisms through which this traditional Chinese medicine protects against S-AKI. Detailed biochemical characterizations and subsequent in vitro experiments further confirmed that the reprogramming of mitochondrial energy metabolism and macrophage polarization by CS substantially contributed to its nephroprotective activity in S-AKI. Importantly, the exact mechanism through which CS stimulates mitochondrial biogenesis must be further explored. Nonetheless, our study highlights the potential value of CS in preventing and treating S-AKI, and provides novel insights to understand the mechanisms of its pleiotropic pharmacological activities.
