Alginate oligosaccharide prevents renal ischemia-reperfusion injury in rats via MRC1-mediated pathway

Abstract

Acute kidney injury (AKI) is a clinical syndrome that is defined as a sudden decline in renal function and characterized by inflammation and tubular injury. Alginate oligosaccharide (AOSC), a natural product obtained from alginate by acidolysis and hydrolysis, shows activities of antioxidant, immunomodulation, and anti-inflammation. In this study, we investigated the potential of AOSC in the treatment of AKI. Renal ischemia-reperfusion (I/R) was induced in male rats by clipping both the renal artery and vein for 45 min followed by reperfusion for 24 h. The rats were treated with AOSC (100 mg/kg, i.g.) before surgery. At the end of the experiments, both kidneys were collected for protein, mRNA measurement, or histological analysis. We showed that AOSC pretreatment significantly improved glomerular and tubular function in the kidney of I/R rats. AOSC markedly inhibited I/R-induced activation of TLR4/MyD88/NF-κB/IL-1β inflammatory signaling and prevented apoptosis in the kidney. In HK2 cells subjected to hypoxia/reoxygenation (H/R) stimulation, AOSC (250–1000 μg/ml) dose-dependently prevented pro-inflammatory responses and cell apoptosis. Transcriptomic analysis revealed that I/R increased the expression levels of mannose receptor type C1 (MRC1) in the kidney, which was markedly inhibited by AOSC. Molecular docking showed that AOSC interacted with E725, N727, E733, T743, S745, and N747 of MRC1 through hydrogen bonds. MRC1 gene knockout significantly improved renal function and attenuated I/R-induced kidney inflammation and apoptosis in mice. In line with this, AOSC failed to prevent I/R-induced kidney injury in MRC1 gene knockout mice. UPLC analysis showed that the protection of AOSC in HK2 cells subjected to H/R was likely attributed to MRC1-mediated intracellular endocytosis. In conclusion, AOSC prevents I/R-induced AKI, which is at least partially mediated by MRC1.

Introduction

Acute kidney injury (AKI) is a common clinical syndrome characterized by a sudden loss of excretory kidney function, which is associated with considerable morbidity and mortality and a heavy cost burden. Patients with AKI have high risks of developing chronic kidney disease and end-stage renal disease. The main causes of AKI include ischemia-reperfusion (I/R) injury, sepsis, and exposure to nephrotoxins, whose pathological mechanisms are similar to those of microvascular derangement and tubular epithelial cell dysfunction [1]. Despite advances in preventive strategies and support measures, no specific therapeutic strategies for AKI are available [2].

I/R injury is commonly encountered in clinical settings such as during transplantation, sepsis-driven hypotension, major surgery, or decompensated cardiac failure [2–4], presenting with a reduced glomerular filtration rate (GFR) and impaired tubular function. I/R injury is characterized by acute tubular necrosis (ATN), including loss of brush border, flattening, focal loss, death of tubular epithelial cells, dilation of tubules, and infiltration of inflammatory cells [5, 6]. Inflammation and tubular epithelial cell death are widely accepted as typical features of I/R-induced AKI [3].

I/R-induced cell damage initiates an inflammatory cascade, which continues and exacerbates disease progression, resulting in the loss of tubular cells. In I/R, initial hypoxia followed by the production of reactive oxygen species (ROS) due to reperfusion initiates injury events, leading to apoptosis, necrosis, and severe inflammatory responses. These inflammatory responses include the infiltration and activation of inflammatory cells (e.g., macrophages, dendritic cells, and lymphocytes) and release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and chemokines [7, 8]. Among them, TNF-α and IL-1β are known to cause renal dysfunction and injury following I/R [9]. Tubular epithelial cells can also contribute to inflammation. In addition to generating pro-inflammatory and chemotactic cytokines (e.g., TNF-α, monocyte chemoattractant protein-1 [MCP-1], and IL-1β), tubular cells express Toll-like receptors (TLRs), complement and complement receptors, and class II major histocompatibility complex (MHC) molecules, thereby participating in the regulation of inflammation [5, 10]. TLR4 is a pattern recognition receptor (PRR) type of transmembrane receptor [11]. Tubular epithelial cells constitutively express TLR4, the expression of which increases during kidney I/R injury [12, 13]. Excessive activation of TLR4 triggers the production of various inflammatory factors, such as TNF-α, IL-1β, and IL-6, accompanied by neutrophil and macrophage infiltration [12, 14]. MyD88 is the most important transduction signal after TLR4 stimulation, which leads to the activation of the transcription factor NF-κB, followed by the production of pro-inflammatory cytokines and chemokines [12, 14]. As inflammation is a crucial factor in renal I/R injury, suppressing inflammatory responses may be beneficial in managing I/R injury and maintaining renal function [7, 8, 15]. Nevertheless, specific therapeutic options to target tubular inflammation and necrosis remain limited in clinical practice [16, 17].

The mannose receptor is a member of the C-type lectin family, containing C-type lectin domains that play an important function in ligand recognition. Mannose receptor C type 1 (MRC1) mediates sugar recognition through C-type carbohydrate recognition domains [18], thus playing a major role in diverse biological processes, including the regulation of circulating levels of glycoproteins and innate and adaptive immunity [18]. MRC1 is mainly expressed on macrophages, immature dendritic cells, endothelial cells [19], and kidney glomerular mesangial cells [20, 21]. Recent single-cell sequencing studies have shown that MRC1 is also expressed on proximal tubular cells [22] (https://www.proteinatlas.org/), suggesting that these cells are involved in MRC1-mediated immunomodulation.

Alginates are linear polysaccharides derived from brown seaweeds; these polymer compounds are composed of α-L-guluronic acid (G) and its C-5 epimer β-D-mannuronic acid (M). Alginate oligosaccharide (AOSC) is a natural product obtained from alginate by acidolysis and enzymatic hydrolysis. AOSC may have different structures and physiological functions, depending on the hydrolysis methods used. AOSC has recently drawn attention because of its physical and chemical properties (such as low molecular weight, viscosity, and excellent solubility in water) as well as its antioxidant, immunomodulatory, anti-diabetic, anti-inflammatory, and anti-tumor activities [23–25], which make it an ideal candidate for pharmaceutical purposes [23]. AOSC treatment has been found to decrease the expression of inflammatory markers such as IL-1β [26]. Alginate-derived Se-polymannuronate has been shown to suppress lipopolysaccharide (LPS)-induced apoptosis [27] and the production of IL-6, IL-1β, and TNF-α [28], as well as attenuate LPS-induced over-activation of MAPK and NF-κB inflammatory signaling [29, 30]. Recently, AOSC was shown to ameliorate gouty arthritis via activating Nrf2-dependent antioxidant signaling and suppressing ROS-mediated NLRP3 inflammasome activation [31]. However, whether AOSC exerts a protective effect against I/R-induced AKI remains unknown.

The current study aims to investigate whether AOSC prevented I/R-induced kidney injury and potential molecular mechanisms. The experiments were carried out using animal models of kidney I/R injury and cellular hypoxia/reoxygenation (H/R) model in the human proximal tubular epithelial cell line (HK2). We found that AOSC prevented I/R-induced kidney injury likely through an interaction with MRC1. Our observations suggested that alginate oligosaccharide may have therapeutic potential for I/R-induced AKI.

Materials and methods

Animals and protocols

Male Sprague-Dawley rats were purchased from Guangdong Medical Laboratory Animal Center. Mice with MRC1 gene knockout (MRC1 KO) on the C57BL/6 background were kindly provided by Dr. Jun Chen, Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University. For identification of the genotypes, DNA extracted from both wild-type (WT) and MRC1 KO mice was amplified by PCR and then used for agarose gel electrophoresis. All animals were housed in an animal facility with a 12-h light/12-h dark cycle at 24 °C, with water and chow ad libitum. All animal experiments were approved by the Experimental Animal Ethics Committee at Sun Yat-sen University (Guangzhou, China).

Protocol 1

Male Sprague-Dawley rats were divided into the following three groups: SHAM, I/R, and I/R + AOSC. I/R was induced in the bilateral kidneys by clipping both the renal artery and vein for 45 min followed by reperfusion for 24 h. Rats in the I/R + AOSC group were gavaged with AOSC (100 mg/kg, Alginate oligosaccharide, Shanghai Haitang Pharmaceutical Technology Co., LTD) before surgery. Throughout the ischemic period, rats were maintained at a body temperature of approximately 37 °C using a temperature-controlled heating system.

Protocol 2

Male WT C57BL/6 and MRC1 KO mice were divided into the following six groups: WT-SHAM, WT-I/R, WT-I/R + AOSC, KO-SHAM, KO-I/R, and KO-I/R + AOSC. I/R was induced in the bilateral kidneys by clipping both the renal artery and vein for 30 min followed by reperfusion for 24 h. Mice in the I/R + AOSC group were gavaged with AOSC (100 mg/kg) before surgery.

All the rats and mice were maintained in metabolic cages during experimentation. The daily water intake and urine output were monitored. At the end of the experiments, all animals were anesthetized, and both kidneys were collected and prepared for protein, mRNA measurement, or histological analysis.

Blood and urine chemistry

At the end of the experiments, blood samples were collected from the vena cava for biochemistry. The osmolality of the urine and serum was determined by freezing point depression (OM 806 odometer; Löser, Berlin, Germany). All plasma and urine electrolytes were measured by a standardized and certified program using an automatic biochemical analyzer (AU5800, BECKMAN COULTER, USA) in the hospital central laboratory (The 1st affiliated hospital, Sun Yat-sen University).

tGFR determination in rats

The rats were exposed to the skin above the right kidney by hair removal cream one day before measurement. After 24 h of I/R experiment, the rats were anesthetized with isoflurane, and GFR was measured. Rats were injected retro-orbitally with FITC-sinistrin (5 mg/100 g body wt; MediBeacon, Germany). A miniaturized imager device (MediBeacon) was used to detect fluorescence in the skin on the shaved back over 1 h. GFR was calculated on the basis of the kinetics of fluorescence decay.

Kidney RNA sequencing

Total RNA of kidney tissues was prepared using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instructions, and genomic DNA was removed using DNase I (TaKara, Japan). Then RNA quality was determined by 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only a high-quality RNA sample (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, >2 μg) was used to construct a sequencing library. The RNA purification, reverse transcription, library construction, and sequencing were performed at DIATRE Biotechnology, Shanghai, China, using Illumina Nova Seq (Illumina, San Diego, CA) according to the manufacturer’s instructions. FastQC initially processed raw sequence reads for quality control, and then adapter sequences and poor quality reads were removed. Quality filtered reads were then mapped to the reference genome using STAR, and only uniquely mapped reads were kept. Sam files were converted to Bam format using Samtools. Cufflinks software was used to compute gene expression levels and differences. DEGs were identified using R statistical package software DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2.html) (fold change ≥2 and P-value < 0.05) with a false discovery rate (FDR) cutoff <0.05. KEGG functional enrichment analysis was performed to identify which DEGs were significantly enriched in KEGG signaling pathways at a Bonferroni-corrected P-value < 0.05 compared with the whole-transcriptome background. KEGG enrichment analysis was performed by KOBAS 2.1.1 (http://kobas.cbi.pku.edu.cn/download.php). Besides, protein−protein interactions of genes were analyzed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) algorithm (http://www.string-db.org/).

Cell culture and treatment

Human proximal tubular cells (HK2 cells) were obtained from ATCC and grown in DMEM/F12 (Corning) containing 10% FBS (Quacell Biotechnology) and 1% penicillin/streptomycin (Corning) and maintained at 37°C in a 5% CO₂ atmosphere.

The HK2 cells were seeded on 6-well plates (Thermo Fisher Scientific) for 24 h and were serum-starved for 12 h. An in vitro hypoxia/reoxygenation (H/R) model was induced as described previously[32], with or without AOSC (250 μg/mL) or mannan (MRC1 inhibitor, 5 mg/mL, Sigma-Aldrich) pretreatments. The mineral oil overlay model could induce the nutrient/oxygen deprivation and metabolite accumulation, which was analogous to ischemia in vivo. HK2 cells were washed with PBS before the addition of PBS supplemented with 1.5 mmol/L CaCl₂ and 2.0 mmol/L MgCl₂. A layer of mineral oil (Sigma, USA) was deposited onto the surface to induce hypoxia for 2 h followed by oil removal and medium refreshment for reoxygenation in HK2 cells. All the cells were then collected for the protein or RNA analyses.

Cell viability assay

Cell viability was determined by CCK8 assay based on the principle of a purple formazan product formation by mitochondrial dehydrogenase of viable cells. Briefly, the HK2 cells were seeded in 96-well plates and cultured with different known concentrations of AOSC (ranging from 25 to 1000 μg/mL) for 24 h with or without the administration of hypoxia for 2 h and reoxygenation for 24 h, respectively. Subsequently, 5 mg/mL CCK8 solution was added to each well, and the cells were incubated at 37 °C for 4 h. After incubation, the supernatant was removed, and 150 μL of DMSO was used to dissolve formazan crystals. Optical density (OD) was measured at a wavelength of 474 nm (Multiskan MK3, Thermo Fischer Scientific Corp).

siRNA transfection

Transfection of HK2 cells with MRC1 siRNA or negative control siRNA (RIBOBIO, China) was performed using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s instructions. The knockdown efficiency of MRC1 was determined by immunoblot at 48 h post-transfection (Supplementary Data Fig. S1).

Histologic analysis and immunofluorescence

For histology, kidney tissues were fixed with 4% paraformaldehyde for paraffin embedding. Tissue slices (4 μm) of the kidney were prepared and stained via H&E staining, TUNEL staining, and immunohistochemistry (IHC). Paraffin-embedded kidney sections used for IHC studies were dewaxed, rehydrated, and incubated with MRC1 (Santa Cruz, sc-58986), KIM-1 (Cell Signaling Technology,14971), or NGAL (Cell Signaling Technology, 44058) primary antibodies overnight at 4 °C. The next day, sections were incubated with goat anti-rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti-mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific) secondary antibodies for 1 h at room temperature, treated with diaminobenzidine, and counterstained with hematoxylin. Images were captured by a digital scanning microscope (Leica DM2000). H&E staining was used to detect the changes in renal structure and morphology. TUNEL staining was used to detect cell death in the kidney.

Western blotting

HK2 cells or kidney samples were lysed in protein lysis buffer for 15 min on ice before protein was extracted. Western blotting was performed by electrophoresis and incubation with primary antibodies against MRC1 (Santa Cruz, sc-58986), KIM-1 (Cell Signaling Technology, 14971), NGAL (Cell Signaling Technology, 44058), TLR4 (Santa Cruz, sc-52962), MyD88 (Santa Cruz, sc-74532), TNF-α (Santa Cruz, sc-52746), IL-6 (Santa Cruz, sc-57315), p-STAT3 (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 9139), p-NFκB-p65 (Cell Signaling Technology, 3033), NFκB-p65 (Cell Signaling Technology, 8242), IL-1β (Cell Signaling Technology, 12242), Bcl-2 (Cell Signaling Technology, 15071), Bax (Cell Signaling Technology, 2772) and cleaved-caspase 3 (Cell Signaling Technology, 9664), followed by the addition of horseradish peroxidase-labeled secondary antibodies goat anti-rabbit IgG (H+L) (catalog 31460, Thermo Fisher Scientific) and goat anti-mouse IgG (H+L) (catalog 31430, Thermo Fisher Scientific). The blots were visualized in an ECL detection system (catalog FD8020, FDbio), and densitometric analysis was performed using AlphaEase software.

Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen). The amount and quality of RNA were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific). 1 μg total RNA was reverse-transcribed to cDNA, and then quantified by real-time PCR using SYBR green master mix (AG-BIO) on the StepOnePlus system (Applied Biosystems). The sequences of the primers used are listed in supplementary materials (Supplementary Data Tables S1 and S2).

Cellular thermal shift assay (CETSA)

CETSA experiments were performed according to a published protocol [33]. Briefly, the HK2 cells were seeded in 6-well plates with 250 μg/mL AOSC and cultured for 24 h. Control cells were incubated with the same volume of the vehicle containing media. The cells were resuspended in PBS (containing 1 mmol/L PMSF) post-incubation and distributed equally into seven PCR tubes, with 100 mL volume per PCR tube. The cells were then heated in a thermal gradient ranging from 42 to 82 °C for 3 min. Post freeze–thaw processing twice with liquid nitrogen, the supernatant was separated by centrifugation at 12,000 × g for 30 min and collected in fresh tubes. 20 µL of the supernatant was loaded onto an SDS-PAGE gel and processed further for immunoblot analysis.

Virtual screening and molecular docking

In this study, the crystal structure of the C-type lectin-like domain of MRC1 (PDB ID: 7JUE) and AOSC (PDB ID:5Z9T) was downloaded from the RCSB Protein Data Bank (PDB) (https://www.rcsb.org/). Docking was performed using AutoDock Vina using standard methods, with flexibility allowed in the ligand, but not the protein. All of the PDB molecules were converted to PDBQT format for docking using AutoDock Tools. A grid box with dimensions of 22 Å × 32 Å × 40 Å was used to encompass the substrate-binding region of the C-type lectin-like domain. The binding pose with the greatest binding energy was saved for further analysis in PyMOL.

Preparation and quantification of AOSC

Alginate lyases were utilized to depolymerize alginate, resulting in a mixture of AOSs and AOSC (with a specific degree of polymerization) that was prepared after purification. As described in our recent study [31], High-Performance Liquid Chromatography (HPLC) analysis indicated that the homogeneity of AOSC exceeded 95%, and the molecular masses of AOSC determined by mass spectrometry are 528 kDa [31]. The total AOSC amount in cell samples was quantified using HPLC. In this procedure, 200 µl of the cell sample was mixed with 800 µl of 1% methanoic acid and then vortexed vigorously for 30 s. The mixture was then centrifuged at 12,000 rpm for 10 min, and the resultant supernatant was filtered through a 0.22 µm syringe filter. The HPLC analysis detected the presence of AOSC in the samples by using parameters such as isocratic elution at a flow rate of 1 mL/min, a sample injection volume of 10 µl, and detection at a wavelength of 230 nm. For this study, the mobile phase was a 30/70 combination of acetonitrile and 1% methanoic acid.

Statistics

Data are presented as the means ± SEM. Data were analyzed by one-way ANOVA and Newman-Keuls tests for multiple comparisons. Statistical significance was accepted at the P < 0.05 level. Values represent means ± SEM of three independent sets of experiments in cell culture studies. All data were analyzed and plotted using GraphPad Prism 6.0 software.

Results

AOSC prevented I/R-induced filtration decline and tubular injury in rats

As shown in Table 1, compared with sham-operated controls (SHAM rats), rats with renal I/R had significantly increased plasma creatinine levels, which were markedly prevented by AOSC treatment (Table 1). Correspondingly, AOSC significantly inhibited the decrease in the GFR in rats with I/R (Fig. 1b). Further, rats with I/R showed increased urine output and fractional sodium excretion and decreased urine osmolality compared with SHAM rats, indicating impaired renal water and sodium handling (Table 1). These parameters were markedly improved by AOSC treatment (Table 1). Protein expression (Fig. 1c) and mRNA levels (Fig. 1d, e) of two tubular injury markers, KIM-1 and NGAL, were significantly upregulated in the kidney cortex and outer medulla of rats with I/R, which were at least partially normalized by AOSC treatment (Fig. 1c–e). AOSC treatment also prevented histological changes (e.g., loss of brush border and dilation of tubules) in the kidneys of rats with I/R (Fig. 1c). These data suggested that AOSC inhibited the decline in renal filtration and improved tubular functions in rats with renal I/R.

Table 1.

Physiological parameters in rats.

Group	SHAM	I/R	I/R + AOSC
UO (ml)	7.40 ± 1.26	11.49 ± 1.17*	6.77 ± 0.87#
PCr (μmol/L)	24.4 ± 0.4	146.2 ± 27.5*	28.7 ± 4.5#
Posm (mOsm/kg·H2O)	290.6 ± 7.9	280.5 ± 3.1	290.5 ± 3.9
Uosm (mOsm/kg·H2O)	2085 ± 280	834 ± 68*	1595 ± 44#
P-Na+ (mmol/L)	140.6 ± 5.0	138.2 ± 2.6	135.0 ± 4.5
P-K+ (mmol/L)	3.12 ± 0.13	5.39 ± 1.24*	2.93 ± 0.05#
FENa (%)	0.94 ± 0.10	5.56 ± 1.69*	1.07 ± 0.38#

SHAM sham-operated group, I/R renal ischemia-reperfusion group, I/R + AOSC I/R group with AOSC, UO urine output, P_Cr plasma creatinine, U_osm urine osmolality, P_osm plasma osmolality, P-Na⁺ plasma sodium, P-K⁺ plasma potassium, FE_Na fractional excretion of sodium.

*P < 0.05 compared with SHAM. ^#P < 0.05 compared with I/R, n = 8 in each group.

Fig. 1. AOSC prevented I/R-induced filtration decline and tubular injury in rats.

a Schematic diagram of an animal experiment. The red arrow indicates the time point of intragastric administration of AOSC (100 mg/kg), and the red and blue arrow indicates the time point of renal ischemia-reperfusion surgery. The rats were killed after reperfusion for 24 h. b The MediBeacon tGFR detection system monitors the changes in GFR in rats 24 h after surgery. c The top picture shows the representative H&E staining of kidney tissue sections of rats in each group, * indicates damaged renal tubules, and the middle and bottom pictures show the immunohistochemical staining of KIM-1 and NGAL, respectively. Magnification: ×400. d and e QPCR was used to detect the levels of KIM-1 and NGAL mRNA levels in rat kidney tissue. Data are shown as mean ± SEM (n = 8 in each group); *P < 0.05, compared with Sham group; ^#P < 0.05, compared with I/R group.

AOSC prevented I/R-induced renal inflammation and apoptosis in rats

Inflammation plays a crucial role in the pathophysiology of AKI resulting from I/R. Semiquantitative immunoblotting analysis demonstrated that protein abundances of TLR4, MyD88, phosphorylated NF-κB, and IL-1β in the kidney cortex and outer medulla were significantly increased in rats with renal I/R and significantly decreased after AOSC treatment (Fig. 2a, b). Immunohistochemistry showed marked increases in the interstitial labeling of F4/80 in the kidneys of rats with I/R, which were not seen after AOSC treatment (Fig. 2c, d), indicating that AOSC inhibited macrophage infiltration in the kidneys with I/R. Macrophages can be polarized into two main types: M1-type and M2-type [34, 35]. M1-type polarization is characterized by an increase in pro-inflammatory cytokines, such as IL-1β, TNF, and IL-6, whereas M2-type polarization is linked to immunosuppression and tissue repair [35]. In rats with I/R, the mRNA levels of the M1 markers IL-1β, TNF, and iNOS were markedly increased compared with those in SHAM rats, while AOSC significantly inhibited mRNA expression of these markers (Fig. 2e). In contrast, AOSC failed to suppress increases in the mRNA levels of the M2 markers IL-4, IL-10, and Arg1 in the kidneys of rats with I/R (Fig. 2f). These data suggested that AOSC could affect macrophage polarization, promoting a switch from pro-inflammatory M1- to anti-inflammatory M2-dominant macrophages, which may accelerate renal repair/regeneration processes, leading to favorable outcomes.

Fig. 2. AOSC prevented I/R-induced renal inflammation and apoptosis in rats.

a Semiquantitative immunoblots reacted with TLR4, MyD88, p-NFκB, NFκB, IL-1β. b Corresponding densitometric analyses of protein levels of TLR4, MyD88, p-NFκB, and IL-1β corrected by β-actin. c and d Immunohistochemistry of F4/80 in the kidney of SHAM, I/R, and I/R + AOSC rats. Magnification: ×400. e and f Analysis of mRNA levels by quantitative real-time PCR for TNF-α, IL-6, iNOS, IL-4, IL-10, and Arg1 under renal I/R in rats. Data are shown as mean ± SEM (n = 8 in each group); *P < 0.05, compared with SHAM group; ^#P < 0.05, compared with I/R group. g Semiquantitative immunoblots reacted with Bcl-2, Bax, and cleaved-caspase 3. h Corresponding densitometric analyses of protein levels of Bcl-2, Bax, and cleaved-caspase 3 corrected by β-actin. i Representative photomicrographs of TUNEL staining in the kidney of SHAM, I/R, and I/R + AOSC rats. Magnification: ×400 (upper) and ×800 (lower). j The percentage of cells with TUNEL staining was evaluated. Data are shown as mean ± SEM (n = 8 in each group); *P < 0.05, compared with SHAM group; ^#P < 0.05, compared with I/R group.

The proximal tubular epithelium can also undergo apoptosis or necrosis in response to I/R injury. Compared with SHAM rats, the Bcl-2/Bax ratio, a marker of apoptosis, was markedly decreased in the kidney cortex and outer medulla of rats with I/R; this change was clearly prevented by AOSC (Fig. 2g, h). Similarly, the abundance of cleaved-caspase 3, another marker of apoptosis, was dramatically upregulated after I/R but was markedly inhibited by AOSC (Fig. 2g, h). Consistent with these findings, the TUNEL study confirmed an increase in apoptosis in the kidneys of rats with I/R compared with SHAM rats, which was significantly attenuated by AOSC treatment (Fig. 2i, j).

AOSC prevented H/R-induced pro-inflammatory response and apoptosis in HK2 cells

We examined whether AOSC prevented H/R injury in HK2 cells. A cell survival assay showed that AOSC treatment increased HK2 cell viability in a dose-dependent manner under both normal and H/R conditions (Supplementary Data Fig. S2). In HK2 cells, the protein abundances of TLR4, phosphorylated NF-κB, and cleaved IL-1β gradually increased with time and peaked in the 24th hour after reoxygenation (Supplementary Data Fig. S3a, b), indicating that pro-inflammatory responses occurred after hypoxia and reoxygenation. AOSC prevented H/R-induced pro-inflammatory responses in a dose-dependent manner. AOSC, starting from the dose of 250 µg/mL, markedly downregulated the protein expression of TLR4, phosphorylated NF-κB, and cleaved IL-1β (Supplementary Data Fig. S3c, d). This dose was used in the subsequent analyses.

In HK2 cells with H/R, 250 µg/mL AOSC treatment remarkably decreased the protein expression of several pro-inflammatory markers (Fig. 3a, b) and prevented H/R-induced apoptosis, as indicated by the increased Bcl-2/Bax ratio and decreased cleaved-caspase 3 protein abundance (Fig. 3c, d). Flow cytometry (Fig. 3e) and TUNEL staining (Fig. 3f, g) demonstrated that AOSC attenuated the apoptosis of HK2 cells with H/R.

Fig. 3. AOSC prevented H/R-induced pro-inflammatory responses and apoptosis in HK2 cells.

a Semiquantitative immunoblots reacted with TLR4, MyD88, p-NFκB, NFκB, IL-1β. b Corresponding densitometric analyses of protein levels of TLR4, MyD88, p-NFκB, and IL-1β corrected by β-actin. c Semiquantitative immunoblots reacted with Bcl-2, Bax, and cleaved-caspase 3. d Corresponding densitometric analyses of protein levels of Bcl-2, Bax, and cleaved-caspase 3 corrected by β-actin. e Programmed cell death was detected by flow cytometry using Annexin V-FITC/PI apoptosis detection kit in HK2 cells pretreated with AOSC, followed by H/R. f Representative photomicrographs of TUNEL staining in the HK2 cells of CTL, H/R, and H/R + AOSC group. Magnification: ×400. g The percentage of cells with TUNEL staining was evaluated. H/R Hypoxia/Reoxygenation. Data are shown as mean ± SEM (n = 6 in each group); *P < 0.05, compared with CTL group; ^#P < 0.05, compared with H/R group.

Identification of MRC1 as a potential target of AOSC in the kidneys of I/R rats by transcriptomics and bioinformatics

To gain further insights into the molecular mechanisms by which AOSC protected against I/R-induced kidney injury, we subjected the kidneys of rats with I/R to RNA sequencing in the absence or presence of AOSC to identify the genes that were significantly involved. Notably, we identified 1068 genes that were significantly upregulated and 1259 genes that were dramatically downregulated in response to I/R compared with the SHAM group (Fig. 4a). Further, 246 genes were significantly upregulated and 264 genes were markedly downregulated in the IR + AOSC group compared with the I/R group (Fig. 4b). Subsequently, a predicted protein–protein interaction (PPI) network was constructed by inputting the differentially expressed genes (DEGs) into the STRING platform for topological analysis in Cytoscape (Fig. 4c). Gene impact values were calculated from pathway topology analysis. As depicted in Fig. 4c, members of the CXC motif chemokine gene family, vascular cell adhesion molecule 1, mannose receptor C type 1 (MRC1), and complement C3a receptor 1, were identified as core nodes (hub genes) based on their high network degree values. As shown in Fig. 4d and Supplementary Data Table S3, transcriptome sequencing data revealed that MRC1 expression was augmented in the I/R kidneys compared with the SHAM kidneys, while AOSC treatment decreased MRC1 expression. To further verify whether the protective role of AOSC was associated with MRC1, we examined the expression of the MRC1 protein and gene in rat kidneys. Renal MRC1 protein and mRNA expression levels were increased significantly after I/R, while AOSC treatment caused a marked reduction in MRC1 mRNA and protein expression in rats with I/R (Supplementary Data Fig. S4a–c). KEGG pathway enrichment analysis revealed that DEGs were predominantly involved in cellular processes such as the chemokine signaling pathway, TNF signaling pathway, NF-κB signaling pathway, and Toll-like receptor signaling pathway (Fig. S5a). Besides, GO enrichment of the transcriptomic analysis results illustrated that DEGs were enriched in the chemokine activity-associated pathways, chemokine-mediated signaling pathway, and immune response-associated pathways (Fig. S5b).

Fig. 4. Identification of MRC1 as a potential target of AOSC in the kidney of I/R rats by transcriptomics and bioinformatics.

a Differentially expressed genes between I/R and SHAM. b Differentially expressed genes between I/R and I/R + AOSC. Red represents up-regulation and green represents down-regulation. Intersections of differentially expressed genes were screened by Venn diagrams. c Interaction gene topology network diagram. d Cluster analysis diagram of differentially expressed genes. Red represents up-regulation, and blue represents down-regulation. In the green box, mrc1 gene is indicated.

Next, using CETSA, we determined whether AOSC functions by interacting with MRC1 on HK2 cells. The thermal stability of human MRC1 on AOSC-treated HK2 cells increased with increasing temperature (from 37 °C to 87 °C) in an AOSC concentration-dependent manner (0–1000 μM) (Fig. 5a, b, Supplementary Data Table S4), suggesting a direct interaction between AOSC and MRC1. To further explore the binding of AOSC to MRC1, molecular docking for MRC1 bound to AOSC was performed. The molecular structure of AOSC and the domains of MRC1 involved in binding are presented in Fig. 5c. MRC1 is a complex protein with 10 domains, of which the C-type lectin-like domains (CLTDs) have been proven to bind to mannose. Because mannose is similar to AOSC in structure, we speculated that the CLTDs would function in AOSC binding and internalization. Therefore, the crystal structure of one CLTD of human MRC1 was downloaded from the Protein Data Bank (PDB ID: 7JUE) and used for molecular docking. The binding score was identified using AutoDock tools, and the binding sites were visualized using PyMOL software. As shown in Fig. 5d, AOSC interacted with E725, N727, E733, T743, S745, and N747 of the CLTD of MRC1 through hydrogen bonds, providing a possible protein–sugar binding mode. Based on the results of CETSA and molecular docking, we assumed that AOSC acts by binding to the MRC1 receptor on the cell surface.

Fig. 5. The interaction between AOSC and MRC1.

a MRC1 temperature-dependent thermal transition analysis. b Dose-dependent thermal transition analysis of MRC1. c Molecular structure formula for AOSC and protein domains involved in MRC1. MRCI consists of 10 domains. CR, FNIII, and CTLD indicate cysteine-rich domain, fibronectin type II domain, and C-type lectin-like domain, respectively. The substrates of each domain are indicated above. d Molecular docking of AOSC onto one CTLD. AOSC and the residues involved in AOSC binding are shown in stick, and hydrogen bonds are indicated with orange dashed lines.

MRC1 gene knockout (KO) ameliorated I/R-induced kidney injury in mice

As MRC1 may be involved in I/R-induced kidney injury, MRC1 KO mice were subjected to renal I/R injury. As expected, in wild-type (WT) mice, AOSC markedly prevented the I/R-induced increase in plasma creatinine and improved tubular function, as indicated by the decreased urine output and fractional sodium excretion and increased urine osmolality in mice with renal I/R. Importantly, mice with I/R developed proteinuria compared with SHAM mice, which was also decreased by AOSC treatment (Table 2). Interestingly, in MRC1 KO mice with renal I/R, plasma creatinine levels increased significantly compared with KO-SHAM-control mice but were not as high as those in WT mice with I/R. In contrast to WT mice with I/R, AOSC failed to reduce plasma creatinine levels in KO mice with I/R (Table 2). Fractional sodium excretion was increased and urine osmolality was decreased in KO mice with I/R, but did not approach the corresponding levels in WT mice with I/R. AOSC did not affect sodium excretion or urine osmolality in KO mice with I/R. Proteinuria in KO mice with I/R was not as severe as that in WT mice with I/R, and AOSC was unable to ameliorate proteinuria in the former group of mice (Table 2). Renal histology showed tubular damage and increased ATN scores in WT mice with I/R, which were prevented by AOSC. MRC1 KO mice exhibited moderate tubular damage that was not affected by AOSC (Fig. 6a, b). Protein (Fig. 6c, e) and mRNA (Fig. 6d, f) expression levels of KIM-1 and NGAL were dramatically increased in WT mice with I/R but were inhibited by AOSC, while in KO mice with I/R, KIM-1 and NGAL expression levels were moderately upregulated and not affected by AOSC treatment (Fig. 6c–f). These data suggested that KO of MRC1 alone ameliorated I/R-induced kidney injury and that the protective effect of AOSC is probably associated with MRC1 expression.

Table 2.

Physiological parameters in mice.

	MRC1 WT			MRC1 KO
Group	SHAM	I/R	I/R + AOSC	SHAM	I/R	I/R + AOSC
UO (ml)	0.79 ± 0.07	1.49 ± 0.13*	0.96 ± 0.15*#	0.86 ± 0.12	1.13 ± 0.17	1.02 ± 0.17
PCr (μmol/L)	11.9 ± 0.9	132.1 ± 29.3*	53.4 ± 15.2*#	12.1 ± 0.5	81.5 ± 23.2& #	77.4 ± 23.6&
Posm (mOsm/kg·H2O)	309.8 ± 8.2	305.0 ± 2.8	306.8 ± 8.8	309.2 ± 6.0	301.7 ± 5.4	295.0 ± 6.9
Uosm (mOsm/kg·H2O)	3297 ± 192	764 ± 72*	1581 ± 273*#	3058 ± 316	1039 ± 43	1626 ± 384
U-Alb (μg/24 h)	2.4 ± 0.9	127.2 ± 10.2*	36.3 ± 8.9*#	2.0 ± 0.5	54.0 ± 11.8& #	43.8 ± 10.9&
P-Na+ (mmol/L)	154.1 ± 0.5	155.1 ± 0.8	156.4 ± 1.2	156.6 ± 1.2	155.1 ± 0.9	156.8 ± 0.9
P-K+ (mmol/L)	3.29 ± 0.10	5.27 ± 0.74*	3.92 ± 0.36#	3.65 ± 0.29	5.23 ± 0.69&	4.59 ± 0.25
FENa (%)	0.03 ± 0.006	0.28 ± 0.03*	0.09 ± 0.02*#	0.03 ± 0.002	0.12 ± 0.02& #	0.11 ± 0.03&

MRC1 WT MRC1 wild type, MRC1 KO MRC1 knockout, SHAM sham-operated group, I/R renal ischemia-reperfusion group, I/R + AOSC I/R group with AOSC, UO urine output, P_Cr plasma creatinine, P_osm plasma osmolality, U_osm urine osmolality, U-Alb urine albumin, P-Na⁺ plasma sodium, P-K⁺ plasma potassium, FE_Na fractional excretion of sodium.

*P < 0.05 compared with WT-SHAM. ^#P < 0.05 compared with WT-I/R. ^&P < 0.05 compared with KO-SHAM. n = 4–6 in each group.

Fig. 6. MRC1 gene knockout ameliorated I/R-induced kidney injury in mice.

a Representative H&E staining of kidney tissue sections of mice in each group, * indicates damaged renal tubules, Magnification: ×400. b Acute tubular necrosis (ATN) score of injured kidneys in WT (wild-type) or MRC1 KO mice. c Immunohistochemistry of Kim-1 in the kidney of WT or MRC1 KO mice, Magnification: ×400. d QPCR was used to detect the levels of KIM-1 mRNA levels in mice kidney tissue. e Immunohistochemistry of NGAL in the kidney of WT or MRC1 KO mice, Magnification: ×400. f QPCR was used to detect the levels of NGAL mRNA levels in mice kidney tissue. Data are shown as mean ± SEM (n = 6–10 in each group); *P < 0.05 compared with WT- SHAM; ^#P < 0.05 compared with WT-I/R; ^&P < 0.05 compared with KO-SHAM; ^$P < 0.05 compared with KO-I/R.

MRC1 gene KO prevented I/R-induced renal inflammation and apoptosis in mice

In WT mice with I/R, the MRC1 protein abundance in the kidney cortex and outer medulla was dramatically increased compared with that in WT-SHAM mice, which was markedly inhibited by AOSC. MRC1 KO was associated with moderate decreases in the protein abundances of TLR4, MyD88, phosphorylated NF-κB, and IL-1β in the kidney cortex and outer medulla of mice with I/R compared with WT mice with I/R, and AOSC failed to further decrease these inflammatory markers in KO mice with I/R (Fig. 7a, b). In MRC1 KO mice, mRNA levels of pro-inflammatory cytokines (TNF-α, IL-6, iNOS, MCP-1, and IL-1β) and anti-inflammatory cytokines (Arg1 and IL-10) were moderately upregulated compared with KO-SHAM mice, which were not affected by AOSC (Fig. 7c). MRC1 KO caused a mild increase in the Bcl-2/Bax ratio and a slight decrease in cleaved-caspase 3 protein abundance (Fig. 7d, e), as well as a decrease in apoptosis (Fig. 7f, g), in the kidneys of mice with I/R compared with WT-I/R mice. AOSC contributed little to anti-apoptotic effects in the kidneys of KO mice with I/R.

Fig. 7. MRC1 gene knockout prevented I/R-induced renal inflammation and apoptosis in mice.

a and b Representative immunoblots and corresponding densitometry analysis of MRC1, TLR4, MyD88, p-NFκB, NFκB, and IL-1β protein abundance in WT or MRC1 KO mice treated with AOSC. β-actin was used as a loading control. Data are shown as mean ± SEM (n = 6 in each group). c QPCR was used to detect the levels of TNF-α, IL-6, iNOS, MCP-1, IL-1β, IL-18, Arg1, IL-10, and MRC1 mRNA in the kidney of mice. Data are shown as mean ± SEM (n = 6–10 in each group); *P < 0.05 compared with WT-SHAM; ^#P < 0.05 compared with WT-I/R; ^&P < 0.05 compared with KO-SHAM; £P < 0.05 compared with WT-I/R + AOSC. d and e Representative immunoblots and corresponding densitometry analysis of Bcl-2, Bax, and cleaved-caspase 3 protein abundance in WT or MRC1 KO mice treated with AOSC. β-actin was used as a loading control. Data are shown as mean ± SEM (n = 6 in each group). f Representative photomicrographs of TUNEL staining in the kidney of WT or MRC1 KO mice treated with AOSC. g The percentage of cells with TUNEL staining was evaluated. Data are shown as mean ± SEM (n = 6–10 in each group); *P < 0.05 compared with WT- SHAM; ^#P < 0.05 compared with WT-I/R; P < 0.05 compared with KO-SHAM.

MRC1 inhibition or silencing prevented H/R-induced pro-inflammatory responses and apoptosis in HK2 cells

In vitro, the MRC1 inhibitor mannan significantly inhibited H/R-induced pro-inflammatory responses in HK2 cells, similar to the inhibitory effect of AOSC (Fig. 8a, b). MRC1 silencing by siRNA significantly attenuated the protein abundances of TLR4, MyD88, phosphorylated NF-κB, and IL-1β in HK2 cells with H/R, and AOSC failed to further inhibit these protein abundances (Fig. 8c, d). Likewise, MRC1 silencing suppressed apoptosis in HK2 cells in response to H/R, which was not affected by AOSC (Fig. 8e, f).

Fig. 8. MRC1 inhibition or silence prevented H/R-induced pro-inflammatory responses and apoptosis in HK2 cells.

a and b Representative immunoblots and corresponding densitometry analysis of MRC1, TLR4, MyD88, p-NFκB, NFκB, and IL-1β protein abundance in HK2 cells pretreated with mannan and/or AOSC followed by H/R for 24 h. β-actin was used as a loading control. Data are shown as mean ± SEM (n = 6 in each group); *P < 0.05, compared with CTL group; ^#P < 0.05, compared with H/R group. c and d Representative immunoblots and corresponding densitometry analysis of TLR4, MyD88, p-NFκB, and IL-1β protein abundance in HK2 cells transfected with MRC1 siRNA followed by H/R. e and f Representative immunoblots and corresponding densitometry analysis of Bcl-2, Bax, and cleaved-caspase 3 protein abundance in HK2 cells transfected with MRC1 siRNA followed by H/R. Data are shown as mean ± SEM (n = 6 in each group); *P < 0.05 when compared with scramble siRNA group; ^#P < 0.05 when compared with scramble siRNA-H/R group.

AOSC protected HK2 cell injury induced by H/R via binding to MRC1

To further examine whether the protective effect of AOSC against H/R-induced cell injury was mediated by MRC1, membrane and cytoplasmic proteins were extracted. H/R was found to markedly increase the protein abundance of MRC1 in both the membrane and cytoplasm. Membrane MRC1 expression was almost completely inhibited, while cytoplasmic MRC1 expression was markedly decreased after AOSC treatment (Fig. 9a, b). Subsequently, the cytoplasmic and membrane proteins were analyzed by ultra-performance liquid chromatography (UPLC) against a 1 mg/mL standard AOSC. AOSC existed in both membrane and cytoplasmic forms in HK2 cells, but AOSC distribution was greater in the cytoplasm than in the membrane (Fig. 9c). MRC1 can be internalized via clathrin-coated vesicles [36]. Chlorpromazine (CPZ, a clathrin inhibitor) treatment of HK2 cells is known to inhibit the internalization of MRC1 from the cell membrane into the cytoplasm [19]. Indeed, CPZ significantly reversed the AOSC-induced decrease in the protein abundances of TLR4, MyD88, NF-κB, and IL-β in HK2 cells with H/R (Fig. 9d, e). These data indicate that the distribution of AOSC from the membrane to the cytoplasm and its anti-inflammatory effects at least partially require the involvement of the MRC1 receptor. After silencing MRC1, MRC1 protein abundances in both the membrane and cytoplasm were markedly decreased compared with WT HK2 cells with H/R (Fig. 10a, b). UPLC revealed that both membrane and cytoplasmic AOSC decreased markedly when MRC1 was absent in HK2 cells with H/R (Fig. 10c). These results suggest that MRC1 is at least partially essential for AOSC distribution to the membrane and cytoplasm in an H/R model.

Fig. 9. AOSC protected HK2 cell injury induced by H/R, likely by interacting with MRC1.

a and b Representative immunoblots and corresponding densitometry analysis of MRC1 protein abundance in HK2 cells pretreated with AOSC followed by H/R for 24 h. β-actin was used as a loading control. Data are shown as mean ± SEM (n = 4 in each group). c Determination of AOSC in HK2 cell membrane and cytoplasmic protein by UPLC, and the chromatogram of 1 mg/ml AOSC standard was used for comparison. d and e Representative immunoblots and corresponding densitometry analysis of MRC1, TLR4, MyD88, p-NFκB, NFκB, and IL-1β protein abundance in HK2 cells treated with AOSC and/or CPZ followed by H/R for 24 h. β-actin was used as a loading control. Data are shown as mean ± SEM (n = 6 in each group); *P < 0.05 when compared with CTL group; ^#P < 0.05 when compared with H/R group.

Fig. 10. MRC1 mediates the entry of AOSC into the cytoplasm of HK2 cells during hypoxic reoxygenation injury.

a Representative immunoblots of MRC1 in HK2 cells transfected with MRC1 siRNA. b Corresponding densitometry analysis of MRC1 in HK2 cell membrane and cytoplasmic protein corrected by β-actin. Data are shown as mean ± SEM (n = 3 in each group); *P < 0.05 when compared with control group; ^#P < 0.05 when compared with H/R group. c Determination of AOSC in HK2 cell membrane and cytoplasmic protein by UPL, and the chromatogram of 1 mg/ml AOSC standard was used for comparison.

Discussion

This study demonstrated that AOSC prevented I/R-induced kidney injury and improved glomerular and tubular function, probably by inhibiting inflammation and apoptosis in the kidney. MRC1 was found to mediate the anti-inflammatory and anti-apoptotic effects of AOSC against I/R injury. AOSC treatment dramatically prevented the decrease in GFR in rats with renal I/R and thus decreased the plasma creatinine levels. Meanwhile, AOSC at least partially maintained tubular function, as indicated by the recovery of urine sodium excretion, urine output, and urine osmolality, together with improved tubular morphology and protein expression of tubular injury markers. These data support a protective effect of AOSC against I/R-induced kidney injury, which is probably attributable to the inhibition of inflammation and apoptosis.

Proximal tubular epithelial cells are sensitive to I/R-induced AKI and tend to undergo degeneration, apoptosis, necrosis, and shedding [37]. Damaged tubular cells synthesize and secrete cytokines, chemokines, and damage-associated molecular patterns, triggering the recruitment and activation of inflammatory immune cells. The chemokines and cytokines released by these immune cells and tubular cells serve as effectors for a positive feedback pathway, enhancing inflammation and cell injury [16, 38, 39]. TLR4 expression is higher in renal proximal and distal tubular epithelial cells than in other intrinsic renal cells [40, 41]. In I/R-induced kidney injury, epithelial cells in kidney tubules express high levels of TLR4 [13, 14]. TLR4 deficiency protects mice from I/R injury-mediated tubular damage [12]. Interestingly, we found that AOSC treatment markedly downregulated the protein expression of TLR4 and its downstream signaling component MyD88 in the kidneys of rats with I/R, which inhibited the nuclear translocation of NF-κB (marked by a decrease in NF-κB phosphorylation) and thus suppressed the transcription of pro-inflammatory cytokines such as IL-1β and TNF-α. AOSC also decreased macrophage infiltration and mRNA levels of the inflammatory cytokines TNF-α, IL-6, and iNOS in kidneys with I/R injury, but did not affect the mRNA expression of anti-inflammatory cytokines. Moreover, the AOSC-induced inhibition of inflammation was associated with significantly attenuated apoptosis in kidneys with I/R injury. H/R injury in tubular cells is a crucial pathophysiological process in I/R injury, involving apoptosis, inflammatory responses, and excessive ROS generation [42]. In HK2 cells, AOSC promoted cell viability and proliferation after H/R and inhibited pro-inflammatory responses and apoptosis. These findings suggest that AOSC administration in rats with renal I/R injury prevented the loss of renal function, probably via anti-inflammatory and anti-apoptotic effects, consistent with the findings of previous studies emphasizing the anti-inflammatory effect of AOSC [23–25].

Next, we investigated the mechanism by which AOSC protected I/R-induced kidney injury. Transcriptomic data demonstrated that the top 10 upregulated genes in the I/R kidneys were associated with chemotaxis and cytokine/chemokine signaling, and their expression was decreased by AOSC. The 11th gene, Mrc1, which encodes the MRC1 protein that binds to sugar, drew our attention. MRC1 plays a major role in diverse biological processes, including the regulation of circulating levels of glycoproteins and innate and adaptive immunity [18]. MRC1 was found to be expressed on proximal tubular cells [22], indicating the involvement of these cells in MRC1-mediated immunomodulation. MRC1 can bind to and internalize a variety of endogenous and exogenous ligands, most potently mannose, N-acetylglucosamine, and fucose residues [18, 19, 43]. We thus presumed that AOSC acts by binding to MRC1, as we found a close interaction between them. MRC1 was found to be stable after binding to AOSC in a concentration-dependent pattern in HK2 cells. Molecular docking theoretically showed that AOSC interacts with the E725, N727, E733, T743, S745, and N747 of MRC1 via hydrogen bonds. Interestingly, two solved CLTD–sugar complex structures (PDB ID: 7JUE and 7JUH) demonstrated that N727 and N747 are critical for carbohydrate binding, which is consistent with our molecular docking results. Structural studies have revealed that MRC1 adopts extended and flexible conformations under physiological or various pH conditions, which change the spatial contact between domains and may affect carbohydrate binding [44, 45]. The detailed AOSC–MRC1 binding mechanism was not examined in the present study, and whether MRC1 is the only receptor to which AOSC binds remains to be clarified, as other receptors in the MRC family share a similar pattern of ligand binding [46].

MRC1 is involved in a variety of pro- and anti-inflammatory responses, depending on the ligands and co-receptors [18, 47, 48]. MRC1 is expressed in the mesangial cells of the kidney [20, 21]. An early study showed that MRC1 deficiency was associated with substantial protection from glomerulonephritis [49]. Compared with WT mice, MRC1-deficient mice displayed well-preserved renal architecture, normal renal function, and minimal proteinuria in nephrotoxic nephritis [49]. Our data support the pro-inflammatory role of MRC1 in the I/R-affected kidney. The expression levels of MRC1 protein and mRNA were significantly upregulated in HK2 cells and kidneys after H/R or I/R injury, and this was associated with enhanced inflammation and apoptosis. MRC1 KO remarkably prevented histological injury and functional decline, in addition to suppressing inflammation, in kidneys subjected to I/R-induced injury. MRC1 inhibition/knockdown in HK2 cells also markedly attenuated H/R-induced pro-inflammatory responses and apoptosis. These data suggest that MRC1 at least partially mediated I/R-induced kidney injury.

Kidney tubular epithelial cells may function as professional immune cells, modulating both innate and adaptive immune responses [47]. For example, tubular epithelial cells in the kidney express class I and II MHC molecules, which are essential in mediating the interaction of tubular cells with immune cells. Class II MHC molecules and TLRs co-localize on the surfaces of tubular epithelial cells in lipid rafts and synergize in the activation of intracellular pathways [50]. Recent single-cell sequencing studies have shown that MRC1 is also expressed on proximal tubular cells [22], suggesting that these cells are involved in MRC1-mediated immunomodulation. MRC1 is considered a “non-canonical” PRR that can bind to endogenous molecules and pathogens, mediating physiological clearance and acting as a bridge between homeostasis and immunity [47]. Therefore, MRC1 and TLRs may collaborate with each other, both playing important roles in I/R-induced kidney injury.

Interestingly, AOSC dramatically downregulated MRC1 protein and mRNA expression and decreased inflammation and apoptosis in the I/R kidneys of WT mice; however, it failed to further rescue I/R injury in MRC1 KO mice, indicating that the protective effect of AOSC was probably mediated through MRC1. The intracellular region of MRC1 lacks known signaling domains; thus, it is possible that it assists other receptors in their signaling cascades [18, 47]. MRC1 probably induces the expression of target genes by assisting other receptors (such as TLRs) in their signaling cascade [47]. For example, pathogens induce interactions between MRC1 and TLR2 [47, 51, 52], probably due to a functional complex formed by MRC1 and TLR2 on the cell surface that facilitates signal transduction. Interestingly, MRC1 was shown to modulate the production of pro-inflammatory cytokines by interacting with TLR4 [47]. Recent studies have shown that chitin-oligosaccharides bind to the mannose receptor together with TLR4 in cultured cells, resulting in signaling toward a pro-inflammatory phenotype [18, 53]. Unlike chitin-oligosaccharides, AOSC inhibited the TLR4-MyD88–NF-κB signaling pathway in our H/R and I/R models, which is probably attributable to molecular structure differences between the two oligosaccharides. Nevertheless, it is plausible that AOSC modulates pro-inflammatory responses via binding to MRC1.

MRC1 is constantly recycled between the plasma membrane and the early endosomal compartment, even without ligand binding. In normal conditions, 10%–30% of the receptor is found at the cell surface and the remaining 70% is localized intracellularly [47]. To further examine how MRC1 mediates the protective effects of AOSC, MRC1 protein was extracted from both the plasma membrane and cytoplasm of HK2 cells with H/R. H/R significantly induced MRC1 protein expression in the plasma membrane and cytoplasm, which was remarkably decreased by AOSC treatment. The expression of MRC1 extracted from the plasma membrane showed a greater decrease than that extracted from the cytoplasm, presumably because MRC1 was internalized with AOSC and delivered into the cytoplasm (Fig. 11). Consistent with this finding, more AOSC was found intracellularly in HK2 cells with H/R, while in HK2 cells with MRC1 silencing, HPLC analysis detected little intracellular AOSC after H/R. These data suggest that AOSC ameliorated I/R-induced injury probably through AOSC–MRC1 internalization. This is supported by the finding that CPZ prevented the AOSC-induced inhibition of MRC1 and pro-inflammatory responses. CPZ is known to affect the redistribution and assembly of clathrin-coated pits [54], which are crucially involved in MRC1 recycling. MRC1 internalization and deliverance into the endosomal system occurs via clathrin-coated vesicles, which involves the polymerization of clathrin and adapter protein complexes [47]. Blocking MRC1 internalization by CPZ was found to prevent the protective effect of AOSC in HK2 cells after H/R. However, the mechanism by which AOSC prevented the activation of TLR4 intracellular signaling remains elusive, and further studies are warranted. It is also possible that the mechanism by which AOSC provides protection against I/R injury involves simple blocking or antagonism of MRC1; however, further investigation into this possibility is needed.

Fig. 11. Hypothesized mechanism diagram of alginate oligosaccharides alleviating renal I/R injury by inhibiting MRC1.

① I/R induces MRC1 protein expression (left) and activation of TLR4 signaling (right) in renal tubular epithelial cells. AOSC binds to MRC1 and the complex is internalized via endocytosis. ② The AOSC–MRC1 complex is disassembled in the epithelial cells. MRC1 trafficking back to the apical plasma membrane. ③ The released AOSC may inhibit TLR4-MyD88-NFκB signaling pathway, decrease production and secretion of inflammatory cytokines, such as IL-1β. ④ The released AOSC may also prevent cell apoptosis.

In conclusion, AOSC was shown to have a protective effect on renal I/R injury, including the normalization of glomerular and tubular function and inhibition of inflammation and apoptosis, promoting the recovery against I/R injury. The observed protective effect of AOSC is at least partially mediated by MRC1, likely through MRC1 internalization intracellularly, alleviating inflammation and cell death after I/R injury. Our data indicated that targeting the mannose receptor and its various carbohydrate-recognizing abilities might be potentially therapeutically interesting.

Author contributions

BEL: experimental design, animal surgery, collection and assembly of data, data analysis and interpretation, and manuscript writing. LSL: animal surgery, collection and assembly of data, data analysis and interpretation. XYW, MYH, and YL: conducting experiments, data analysis, and interpretation. XY, MHW, ML, QQZ, HLZ, and MCC: conducting experiments. ZDL, QQL, and XG: preparation and characterization of AOSC, Molecular docking with this study. WDW, QHL, WZL, and CLL: financial support, conception and design, manuscript revision, final approval of manuscript. All authors read and approved the final manuscript.

Competing interests

Dr. Zhen-de Liu is one of the inventors on patent applications related to this work filed by Haitang (Jiangsu) Biotechnology Co, Ltd (ZL 202110831844.0 and PCT/CN2021/107883). All other authors declare that they have no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-025-01545-3.