Abstract
Background
Despite growing evidence of ochratoxin A (OTA)-induced kidney toxicity, the underlying mechanisms remain elusive. Emerging evidence suggests that small extracellular vesicles (sEVs) act as mediators of intercellular communication to recipient cells during various physiological and pathological conditions. Given the distinctive properties of sEVs, it is hypothesized that OTA-induced sEVs might mediate the OTA-induced kidney pathogenesis.
Methods
To explore the involvement of sEVs in OTA-induced kidney toxicity, sEVs were isolated and characterized from OTA-exposed rat kidney epithelial cells (NRK52E). Later, these sEVs were used to treat NRK52E cells and Wistar rats to assess the impact of OTA-induced sEVs on kidney toxicity. Label-free proteomics was also performed on OTA-induced sEVs, and key proteins were identified and validated. The biodistribution of sEVs in rats was also assessed using live imaging. The role of validated protein/s in kidney toxicity was further confirmed via a gene silencing and overexpression study.
Results
OTA exposure increased sEV secretion into conditioned media of NRK52E cells and into the urine of Wistar rats. Interestingly, we found that OTA-induced sEVs cause similar kidney toxicity in vitro and in vivo systems as OTA exposure, and blocking of sEV secretion markedly alleviated OTA-mediated kidney toxicity. Proteomics analysis identified annexin A2 and fibrinogen-ɣ as common proteins detected in sEVs derived from OTA-exposed NRK52E cells or rat urine. However, immunoblotting validated that annexin A2 was the only sEV-associated protein, expressed significantly in both NRK52E and rat urine following OTA exposure. Notably, silencing of annexin A2 attenuated the ability of OTA-induced sEVs to cause kidney toxicity, whereas overexpression exacerbates it.
Conclusions
Our findings identify the annexin A2-enriched sEVs as key mediators of OTA-induced kidney toxicity. Annexin A2, along with other kidney injury markers, offers a promising non-invasive translational biomarker for early detection and monitoring of OTA-induced kidney toxicity.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12964-026-03023-7.
Keywords: Extracellular vesicles, sEVs, Ochratoxin A, Kidney fibrosis, Annexin A2
Background
Kidney diseases form a major public health concern globally and are the leading cause of morbidity and mortality [1]. Among them, chronic kidney disease (CKD) is one of the most prevalent forms of kidney disease, occurring due to different factors, including diabetes mellitus, hypertension, glomerular diseases, and exposure to environmental risk factors [2]. In recent years, a distinct type of chronic kidney disease has emerged, known as chronic kidney disease with unknown etiology (CKDu), characterized by chronic interstitial nephropathy (CIN) or fibrosis and sometimes leads to end-stage-renal disease. CKDu patients have no risk factors such as diabetes and hypertension. It has emerged as a significant public health concern, particularly among agricultural communities in Central America, Sri Lanka, India, and other developing countries [3, 4]. Growing evidence highlights a complex interplay of additional environmental, occupational, lifestyle, and social determinants heightening CKDu risk.
Key environmental risk factors include chronic exposure to nephrotoxic substances like heavy metals (e.g., arsenic, cadmium), pesticides, and mycotoxins. Mycotoxins are fungal secondary metabolites, considered a significant global food safety threat. Among different mycotoxins, ochratoxin A (OTA) is commonly found in various food commodities such as cereals, juices, dried fruits, wine, etc. The kidney is the main target organ of OTA-caused toxicity, as the organic anion transporter (OAT)−1/3, located predominantly on the basolateral surface of proximal tubule-epithelial cells (PTECs), facilitates its uptake and accumulation inside it [5]. Repeated accumulation of OTA exerts tubulointerstitial injury by inhibiting protein synthesis and promotes mitochondrial dysfunction, oxidative stress, lipid peroxidation, cell cycle arrest, and DNA damage [6]. Persistent tubular injury subsequently triggers the activation of pro-fibrotic signaling cascades, specifically TGF-β1 signaling, thereby resulting in the excessive accumulation of extracellular matrix (ECM) proteins, such as fibronectin, collagen-1, and α-SMA, and leading to kidney fibrosis [7]. In addition, various epidemiological studies have further suggested a potential association between higher OTA levels in blood and increased incidences of tubulointerstitial fibrosis, a key feature of CKDu [6, 8, 9]. However, many research gaps still persist that prevent a thorough comprehension of the exact molecular mechanism underlying OTA-induced kidney fibrosis.
During any pathophysiological condition, kidney tubular epithelial cells play a central role in intercellular communication, maintaining cell–cell communication by secreting soluble substances and small extracellular vesicles (sEVs) [10]. sEVs are the nanoscale vesicles originating from inward budding of the late endosomal membrane, playing pivotal roles as mediators in this intercellular exchange by carrying proteins, nucleic acids, lipids, etc. that can influence the function of recipient cells [11]. Earlier evidence suggests that sEVs are secreted by diseased cells containing distinct cargo that drives intercellular communication, ultimately leading to disease initiation and progression caused by environmental toxicants, including heavy metals, pesticides, and mycotoxins [12, 13].
Therefore, we hypothesized that signals induced by OTA exposure may be transmitted via sEVs through the transfer of specific cargo to recipient cells, thereby contributing to OTA-mediated kidney toxicity. In this study, normal rat kidney cells were exposed to OTA, after which sEVs were isolated and injected into Wistar rats. Proteomic analysis was conducted on sEVs derived from both OTA-treated NRK52E cells as well as the urine of OTA-exposed rats. The proteomic findings were subsequently validated by immunoblotting, and a knockdown as well as overexpression study were performed to validate the role of specific sEV-associated proteins in OTA-mediated kidney toxicity.
Methods
Chemicals and reagents
All chemicals and reagents used in the study are enlisted in Supplementary Table 1.
Cell culture and treatment
A Normal Rat Kidney Epithelial (NRK52E) cell line (Passage number 12) derived from rat proximal tubule epithelium, was continuously cultured using Dulbecco’s Modified Eagle Medium (DMEM) high glucose supplemented with 10% fetal bovine serum, sodium bicarbonate (13.5 g/L), and antibiotic–antimycotic solution (50 U/ml penicillin, 50 μg/ml streptomycin, 0.25 μg/ml amphotericin B) in a humidified incubator at 37 °C with 5% CO2. For long-term OTA treatment of NRK52E cells, OTA was dissolved in dimethyl sulfoxide (DMSO) and mixed with 10% FBS-containing DMEM and continuously applied to the adhered cells for two and four months. At 48 h intervals, the culture media were replaced with fresh OTA-containing media. Cells from the control group received vehicle (DMSO) treatment. To check the effect of long-term OTA-induced EVs on the expression of kidney injury markers, NRK52E cells were treated for 24 h with EVs (30 µg/ml) derived from four-month OTA-exposed NRK52E cells. For the EV inhibition study, OTA-treated or untreated NRK52E cells were exposed to 15 nM of dimethyl amiloride (DMA), a pharmacological inhibitor of EV release. The resulting conditioned media (with or without DMA) were used to treat four-month OTA-treated or untreated NRK52E cells.
Further, to validate the effect of OTA on human kidney derived proximal tubular epithelial cells and also to enhance the translational relevance of our findings, Human Kidney-2 (HK-2) cells were cultured in Defined Keratinocyte Serum-Free Medium (SFM) supplemented with (bovine pituitary extract (BPE; 0.05 mg/ml) and human recombinant epidermal growth factor (EGF; 5 ng/ml) in a humidified incubator at 37 °C with 5% CO2. To validate the effect of long-term OTA-induced EVs on the expression of kidney injury markers, HK-2 cells were treated for 24 h with EVs (30 µg/ml) derived from four-month OTA-exposed NRK52E cells.
Cell viability assay
To assess the dose-dependent effects of OTA on viability, NRK52E and HK-2 cells were grown overnight in a 96-well plate at a density of 3.0 × 103/well. Adhered cells were treated with different concentrations of OTA (0.1 to 10 µM) for 48 h. Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) and sulforhodamine B (SRB) assay following the manufacturer’s protocol. Absorbance was recorded at 570/630 nm for the MTT assay and 565 nm for the SRB assay by using a Cytation 5 multimode plate reader and imaging system (Bio-Tek, Winooski, VT, USA).
Isolation of EVs using a differential ultracentrifugation (dUC) method
EVs were isolated either from conditioned media or from rat urine using the differential ultracentrifugation technique. For EVs-isolation from conditioned media, NRK52E cells were grown in a culture dish (150 mm). At 70–80% confluency, the cultured medium was replaced with DMEM containing exosome-depleted FBS. After 48 h, conditioned media (CM) were collected and subjected to sequential centrifugation at 3000 × g for 10 min and 10,000 × g for 30 min to remove cells and larger vesicles, respectively. To eliminate particles larger than 200 nm, the CM was filtered through a 0.2 µm syringe filter and then concentrated using 100 kDa ultrafiltration tubes. The concentrated CM was then ultracentrifuged at 100,000 × g for 90 min at 4 °C using a Type 70.1 Ti Fixed-Angle Titanium Rotor (k-factor 36, Beckman Coulter Life Sciences, USA). The supernatant was carefully removed without disturbing the pellet. The EV-containing pellet was resuspended in cold PBS and subjected to a second ultracentrifugation under the same conditions to further purify the vesicles.
For urinary EV isolation, urine samples were collected and initially centrifuged at 5000 rpm for 10 min at 4 °C to remove cellular debris. The resulting supernatant was then centrifuged at 17,000 × g for 10 min at 4 °C to eliminate larger vesicles and contaminants. The supernatant (SN1) was collected for further processing. The pellet was resuspended in 250 µl of dithiothreitol (DTT; 200 mg/ml) and incubated at 37 °C for 10 min, with intermittent mixing on a vortex mixer every 2 min to dissociate any EVs entrapped in the Tamm-Horsfall protein (THP) mesh. Following incubation, the mixture was centrifuged again at 17,000 × g for 10 min at 4 °C, and the resulting supernatant (SN2) was collected and combined with SN1. The pooled supernatant (SN1 + SN2) was filtered through a 0.2 µm syringe filter to remove any remaining large vesicles and debris. The filtered sample was then ultracentrifuged at 200,000 × g for 1 h at 4 °C using a Type 70.1 Ti Fixed-Angle Titanium Rotor (k-factor 36, Beckman Coulter Life Sciences, USA). The EV pellet was washed once with cold PBS and subjected to a second ultracentrifugation at 200,000 × g for 1 h at 4 °C using the same rotor. After the final centrifugation, the supernatant was carefully discarded, and the EV pellet was resuspended in appropriate buffers based on further applications. To characterize these isolated EVs, nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting were performed as described below.
Nanoparticle tracking analysis
Nanoparticle Tracking Analysis (NTA) was conducted to determine the hydrodynamic size (nm) and concentration (particles per ml) of EVs using the NanoSight NS300 (Malvern, Worcestershire, UK). NTA was performed as illustrated in the earlier study [14]. Briefly, 1 ml of EV-containing PBS was injected into the NanoSight instrument, and their Brownian movement was recorded for 60 s. All instrument settings were kept consistent across experiments. To make the representative bar diagram, the average values for size and concentration of EVs (derived from conditioned media and urine) were used.
Immunogold transmission electron microscopy
The EVs were fixed using 2% paraformaldehyde (PFA) for 30 min at 4 °C and loaded onto a Formvar/carbon-coated transmission electron microscopy (TEM) grid (TED PELLA, INC., California, USA). After 20 min of incubation at room temperature (RT), negative staining was performed using uranyl acetate for visualization using TEM operated at 80 kV.
For immunogold-TEM, the fixed EVs were loaded onto a grid following 20 min of incubation. After washing using PBS, the grid was treated with 0.05 M glycine for 10 min to quench free aldehyde groups and then blocked with PBS containing 1% bovine serum albumin (BSA) for 30 min at RT. EVs were further incubated with anti-cluster of differentiation-9 (CD9) antibody for 3 h, and then with goat anti-rabbit IgG conjugated to gold particles (Invitrogen Life Technologies, Grand Island, NY) for 1 h at RT. The grid was subsequently washed twice with PBS to remove any unbound antibodies, followed by staining with uranyl acetate and allowed to air dry. As a negative control, the same steps were followed except that the incubation with the primary antibody was omitted. Finally, EVs on the grid were visualized using a TEM operated at 80 kV.
To study the biodistribution of exogenously injected EVs into rat kidneys, kidney tissues were first fixed with 2.5% glutaraldehyde and postfixed with 3% osmium tetroxide (OsO4) for 2 h. The samples were then dehydrated in a graded series of ethanol, embedded in Epon resin and sliced into 100 nm pieces. Final images were observed with a TEM at 80 kV. For all TEM-associated observations, we used a FEI Tecnai G2 Spirit system (Czech Republic).
EV protein quantification assay
The concentration of EV-associated protein was quantified with the Bicinchoninic Acid (BCA) assay using the Pierce BCA Protein Assay Kit, following the manufacturer’s instructions. Briefly, 1 mg/ml BSA was used as a standard. A mixture of distilled water and standard (5 standard-concentrations, including blank) or unknown samples was pipetted into a microplate in triplicate. BCA working reagent was prepared by mixing Reagent A and Reagent B (50:1) and adding to each well for 15 min at RT. Absorbance was recorded at 562 nm using Cytation 5 multimode plate reader and imaging system (BioTek, USA). The yield of total EV-associated proteins (µg) was calculated by multiplying the protein concentration (µg/µl) and the total volume (µl) of EV-resuspended PBS. The desired amount of EV proteins was used for further downstream processes.
Immunoblotting
OTA-treated and untreated NRK52E cells were lysed using RIPA buffer containing 0.02 M HEPES, 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.2 mM PMSF, 1 × protease inhibitor cocktail (PIC), and 0.2 M sodium orthovanadate. For tissue lysate preparation, 25 mg of kidney cortex tissue was homogenized in RIPA buffer using a tissue homogenizer. The homogenates were centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant was collected. Protein concentrations were determined using the BCA assay as described above.
For immunoblotting, EV, cell, or tissue lysates were mixed with Laemmli buffer and boiled at 95 °C for 5 min. Samples were loaded onto SDS-PAGE gels and electrophoresed. After overnight protein transfer, ponceau staining was performed to confirm the equal loading of EV protein. Membranes were blocked with 3% BSA in TBST for 1 h at RT. After washing, membranes were incubated overnight at 4 °C with the following primary antibodies: β-2-microglobulin (B2M; 1:2000), clusterin (1:1000), calbindin (1:2000), cystatin C (1:2000), fibronectin (1:1000), alix (1:2000), tumor susceptibility gene 101 (TSG-101; 1:2000), CD9 (1:1000), CD63 (1:1000), flotillin-1 (1:1000), calnexin (1:2000), GM130 (1:2000), annexin A2 (1:2000), fibrinogen-γ (1:1000), proliferating cell nuclear antigen (PCNA; 1:2000), and β-actin (1:10,000). The next day, membranes were incubated with HRP-conjugated secondary antibodies (anti-rabbit or anti-mouse) for 1 h at RT. After washing, protein bands were visualized using enhanced chemiluminescence (ECL) reagents and imaged using the ImageQuant LAS 500 system (GE Healthcare). Densitometric analysis of the bands was performed using ImageJ software. Band intensities were normalized to the corresponding loading control.
Uptake assay
To determine whether EVs were internalized by NRK52E cells after 24 h of treatment, EVs were pre-labelled with PKH26 fluorescent dye following the manufacturer's protocol. Briefly, EVs were resuspended in 1 ml of diluent C (tube 1), while in a separate tube (tube 2), 4 µl of PKH26 dye was mixed with 1 ml of diluent C. The contents of tube 1 and tube 2 were then combined and incubated at RT for 10 min.
As a control, a third tube (tube 3) was prepared, where the same concentration of PKH26 dye was resuspended in diluent C, to rule out any potential contamination by free dye. The labelling reaction was terminated by adding an equal volume of 1% BSA to tubes 1 and 3. The samples were then ultracentrifuged at 100,000 × g for 60 min at 4 °C to pellet the labelled EVs. The pellets were resuspended in PBS and subjected to another round of ultracentrifugation at 100,000 × g for 60 min at 4 °C to remove any remaining free dye. No pellet was observed in tube 3, confirming the removal of free dye.
The final pellet containing labelled EVs was resuspended in PBS and used to treat NRK52E cells for 24 h. After treatment, the cells were washed twice with PBS, fixed with 4% paraformaldehyde (PFA), and mounted with Fluoromount-G containing DAPI (Thermo Fisher Scientific, USA). Confocal images were captured using a Zeiss microscope (Germany) to visualize the internalized PKH26-labelled EVs.
Quantitative proteomics analysis of EVs using LC–MS/MS
Total proteins were isolated from EVs derived from conditioned media of untreated or chronic OTA-exposed NRK52E cells or rat-derived urine, using lysis buffer (5 mM DTT, 1 mM PMSF, 1% protease inhibitor cocktail, 0.1% PMSF, 1% NP-40, 0.15% sodium deoxycholate). To remove the contaminating salts, protein samples were passed through detergent-removal spin columns and quantified. An equal number of proteins from each group was further digested into polypeptides using an enzyme solution of Trypsin/Lys-C (1:30) overnight at 37 °C. The next day, proteolysis was stopped by adding 10% formic acid (final concentration 0.1%), and peptides were passed through C-18 spin columns (Waters Corporation, Milford, MA, USA) as instructed by the manufacturer. Subsequently, all samples were dried through a vacuum evaporator and reconstituted in 0.1% formic acid. Prepared samples were then analyzed using a nanoflow liquid chromatography system coupled with a Q-Exactive Orbitrap mass spectrometer equipped with a nano-electrospray ion source (Thermo Fisher Scientific, MA, USA). The peptide mixtures were loaded into a C18 reversed-phase column and separated with a 260 min gradient at a flow rate of 300 nl/min. Eluted peptides were analyzed using data-dependent MS/MS acquisition mode. The resulting raw data were processed and searched against the UniProt Rattus norvegicus database on the Proteome Discoverer 2.4 proteomics platform (Thermo Fisher Scientific, MA, USA), with the false discovery rate (FDR) threshold set at 1.0%. Tryptic searches allowed two missed cleavages, with cysteine carbamidomethylation as a fixed modification and N-terminal acetylation and methionine oxidation as variable modifications. Search settings included a 10 ppm precursor tolerance and 0.02 Da fragment error. Protein identification required one high-confidence peptide with a ≤ 1% FDR (Percolator). Label-free quantification (LFQ) was performed in Proteome Discoverer, applying a ≤ 1% FDR ion score threshold.
Gene Ontology (GO) enrichment analysis of the differentially expressed proteins was performed using the DAVID Functional Annotation Bioinformatics website. The LFQ intensities from PD were analyzed using Perseus (version 2.0.6.0, Max Planck Institute of Biochemistry, Germany). The protein list was filtered, and LFQ intensities were log2 transformed. Two-sample t-tests were used to compare the datasets, with imputation of missing values, followed by Benjamini–Hochberg multiple-testing correction [15, 16]. Proteins were identified as differentially expressed if the adjusted p-value between experimental groups was ≤ 0.05 and the fold change was at least 2 (s0 = 1).
In vivo study
Animals and ethics statement
Male Wistar rats weighing between 80–100 g were sourced from the in-house breeding facility of the CSIR-Indian Institute of Toxicology Research (IITR), Lucknow, India. The animals were housed under a maintained environment, with a temperature range of 22–25 °C and a 12 h light/dark cycle. They had unrestricted access to standard laboratory chow and water ad libitum. The study was carried out as per the guidelines established by the Institutional Animal Ethics Committee (IAEC) of CSIR-IITR (reference no. IITR/IAEC/07/23–58/24) and approved under the authorization of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, Government of India.
Animal treatment
After a seven-day acclimatization period, all rats were randomly allocated into 3 groups using a simple randomization method (based on the side of a coin), each comprising four animals. (i) V-control: Animals received corn oil through oral gavage (2 ml/kg b.wt.) along with PBS containing sEVs derived from control NRK52E cells via tail vein injection, seven days a week, serving as the vehicle control. (ii) OTA: Rats received OTA at a dosage of 210 μg/kg b.wt. via oral gavage (seven days/week). The dose for OTA was selected as per a study conducted by the National Toxicology Program, and subsequent studies showed that exposure to OTA at a dose of 210 μg/kg b.wt. led to kidney tubular cell neoplasms, epithelial degeneration, karyomegaly, and hyperplasia following 270 and 456 days of treatment [14, 17, 18]. (iii) sEVs: Rats received EVs via tail vein injection. For the EV treatment, previous studies have utilized a dose of 100 µg per animal of EVs derived from cells for intravenous injection in rats [19, 20]. 100 µg of EV proteins contain ~ 1 × 1011 to 5 × 1011 EV particles, a range consistent with circulating EV concentrations reported in pathological states [21, 22]. Therefore, in our study, we also administered 100 µg of EVs derived from chronically OTA-exposed or control NRK52E cells via tail vein injection to each Wistar rat. Animals with a body weight of less than 100 g were not considered for the study. Further, to validate the role of EVs in OTA-induced kidney toxicity in rats, we performed an EV inhibition study. Animals received daily intraperitoneal injections of DMA, a widely used pharmacological inhibitor for the secretion of EVs, at a dose of 10 mg/kg [23]. It inhibits Na+/H+ and Na+/Ca2+ exchange, affecting intracellular pH and calcium homeostasis, which are important regulators of multivesicular body trafficking and fusion with the plasma membrane. Ultimately leading to reduced release of EVs generated through both ESCRT-dependent and ESCRT-independent pathways in vitro and in vivo [24]. For this purpose, Wistar rats were grouped into four groups (n = 3). (i) V-control: Animals received corn oil through oral gavage (2 ml/kg b.wt.) along with PBS via intraperitoneal (I.P.) injection. (ii) DMA only: Animals received DMA via I.P. injection. (iii) OTA: Animals received OTA via oral gavage. (iv) OTA + DMA: Animals received OTA via oral gavage along with I.P. injections of DMA, which were initiated after 15 days post-OTA exposure, as at this time point OTA starts to cause its toxic effects on the kidney [25]. After a treatment period of eight weeks, urine was collected using metabolic cages, and subsequently, the animals were euthanized, blood was collected through cardiac puncture, and serum was separated. Vital organs (kidney, lung, heart, spleen, and liver) were harvested, and organ weight was measured subsequently. Throughout the study, the body weight of each animal was measured at seven-day intervals, while food and water consumption were monitored daily.
Folic acid is a well-known kidney toxicant at a specific dose. To make a renal fibrosis animal model, Wistar rats were allocated randomly into two groups (n = 3); the average weight was considered to be 300 g. (i) V-control: known as the vehicle control, received double-distilled water via intraperitoneal injection, and (ii) FA: received one-time folic acid (Sigma, GmbH) dissolved in distilled water (250 mg/kg) via intraperitoneal route [26]. In the current study, we followed the same experimental approach, including the dose and time points. After two weeks, urine was collected using metabolic cages for 24 h. Urine volume was measured over a 24 h urine collection period. Blood collection was performed through cardiac puncture. Body weight was measured at day 0, 7, and 14, after administration of folic acid or vehicle.
Analysis of organ weight ratio (KW/BW) and biochemical parameters
Upon sacrifice, the harvested organs were weighed to assess the effects of OTA treatment. For serum and urine biochemical analysis, samples were processed and analyzed using the AU480 fully automated clinical chemistry analyzer (Beckman Coulter). The biochemical parameters assessed included serum creatinine, urea, AST, ALT and urinary albumin (Siemens Healthcare, India).
Assessing the severity of kidney injury by estimation of glomerular filtration rate
For glomerular filtration rate (GFR) measurement, fluorescein isothiocyanate (FITC)-labelled sinistrin (2 µl/g b.wt.) was injected into rats via the lateral tail vein. A MediBeacon® transdermal GFR measurement system was attached to the dorsal side of the rat, specifically designed to monitor fluorescence transcutaneously [27]. Following injection, the device recorded fluorescence decay over a 30 min period, capturing the clearance kinetics of FITC-sinistrin from circulation. The half-life of fluorescence decay was used to calculate the GFR.
Analysis of kidney injury biomarkers by Luminex-based multiplex assay
To assess the effect of OTA and its derived EVs on kidney toxicity, we evaluated the expression of classical kidney injury biomarkers in OTA-exposed cells as well as in rats injected with OTA-induced sEVs, using the ProcartaPlex™ Rat Kidney Toxicity Panel 2, 5-plex. Cells from each group (48 h, 2 and 4-months) were washed twice with PBS, and then 300 µl of assay diluent buffer containing PIC (1x) was added. Next, the cells were scraped and collected in a centrifuge tube and then sonicated on ice at 38% amplitude (10 s on, 10 s off for three cycles). Subsequently, samples were centrifuged at 16,000 × g at 4 °C for 10 min, and the supernatant was stored for further processing.
For urinary kidney injury marker detection, collected urine samples were centrifuged at 5000 rpm for 10 min to remove debris. For kidney injury marker analysis, 50 µl of the capture bead mix was added to each well of a 96-well black plate, which was then placed on a hand-held magnetic plate washer. After 5 min, the bead mix was removed, and 150 µl of wash buffer was added to each well. Following the removal of the wash buffer, 50 µl of sample (cell lysate or urine) and standards were added to the appropriate wells and incubated for 2 h. The standard reconstitution process was carried out according to the manufacturer's protocol.
Histopathological analysis
Kidney tissues were harvested post-necropsy and immediately fixed in 10% neutral buffered formalin for proper preservation. Following fixation, tissues were embedded in paraffin and sectioned at a thickness of 5 µm using a fully automatic microtome (CUT 6062, SLEE, GmbH). To visualize histological alterations, tissue sections were stained with haematoxylin and eosin (H&E), Masson’s trichrome stain (MTS), or periodic acid–Schiff (PAS) using an Autostainer XL (Leica, GmbH). The stained slides were examined for histopathological changes in accordance with the nomenclature and diagnostic criteria established by the Society of Toxicologic Pathology (STP). Representative images were captured using a BX53 upright microscope (Olympus, Japan).
Immunohistochemical analysis of injury marker expression in renal tissue sections
The expression of kidney injury markers in renal tissue sections was assessed by immunohistochemistry (IHC) as described previously by Dev et al. [14]. Briefly, paraffin-embedded tissue sections were deparaffinized at 60 °C and rehydrated through a graded ethanol series. Antigen retrieval was performed by incubating the sections in citrate buffer (pH 6.0) at 100 °C for 1 h to unmask cross-linked epitopes.
Following antigen retrieval, the sections were washed twice with PBS (15 min each) and then incubated in 3% hydrogen peroxide in methanol at –20 °C for 30 min to quench endogenous peroxidase activity. After washing three times with PBS (10 min each), sections were blocked with 3% BSA at RT for 1 h. Subsequently, sections were incubated overnight at 4 °C with the following primary antibodies: B2M (1:200), calbindin (1:100), cystatin C (1:200), PCNA (1:50), fibronectin (1:50), and α-smooth muscle actin (α-SMA; 1:100). After two PBS washes (10 min each), sections were incubated with HRP-conjugated secondary antibodies (1:400) for 2 h at RT. This was followed by incubation with avidin–biotin complex (ABC) reagent for 30 min. Visualization was achieved using 3,3'-diaminobenzidine (DAB) as the chromogen, and nuclei were counterstained with haematoxylin. Slides were mounted using DPX mounting medium. Images were acquired using an Olympus BX53 light microscope (Japan), and immunoreactivity was quantified using ImageJ software. Negative control sections were processed identically but without incubation with the primary antibodies.
In vivo imaging of fluorescently labelled EVs for biodistribution study
To assess the biodistribution of exogenously injected EVs, they were labelled with PKH26 dye, as described above. The fur on the dorsal side (back) of the Wistar rats was carefully shaved and randomly divided into three groups (n = 3, weight 100–120 g). (i) V-control: rats received unlabelled EVs resuspended in PBS through tail-vein injection, serving as the vehicle control group; (ii) Dye only: rats received intravenous injection of PKH26 dye (2 × 10–6 M, 100 μl in PBS); and (iii) Labelled sEVs: rats received intravenous injection of PKH26-labelled EVs (100 µg, resuspended in 100 μl PBS). Images were acquired using an in vivo imaging system (IVIS Spectrum System, PerkinElmer Inc., MA, USA) at 5 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 24 h post-injection. Fluorescent detection was performed with excitation: 570 nm, and emission: 620 nm. Rats were subjected to 1.5–2% isoflurane throughout the imaging process. Quantitative analysis of total radiant efficacy, defined as the number of photons (p) that left a square centimeter of tissue per second and radiated into a solid angle of one steradian (sr), expressed as (p/s)/(μW/cm2), was conducted using Living Image Software coupled with the IVIS Spectrum System (PerkinElmer Inc., MA, USA).
To assess the exact source of fluorescence, ex vivo imaging was performed. At 24 h post-injection, all animals were euthanized, and the vital organs (liver, spleen, kidneys, heart, and lungs) were harvested and imaged with the same imaging system. After ex vivo imaging, kidneys were further snap frozen using liquid nitrogen and coated using cryomatrix and sectioned at −20 °C using a cryostat (Thermo, USA). Subsequently, sections were fixed using 4% PFA solution at 4 °C for 30 min. After washing, sections were further incubated with 0.1% Sudan Black B (SBB, dissolved in 70% ethanol) for 15 min at RT for quenching auto-fluorescence of tissues, as described in the previous study [28]. After washing, sections were then mounted with DAPI-containing mounting medium (Fluoromount-G, Thermo Fisher Scientific, USA), and photographs were captured using a Cytation 5 multimode plate reader and imaging system (BioTek, USA).
siRNA and plasmid transfection in NRK52E cells
Small interfering RNA (siRNA) targeting annexin A2 in NRK52E cells was purchased from Horizon Discovery (ON-TARGETplus Rat Anxa2 siRNA, catalogue no. L-092562–02–0005). For the overexpression study, an annexin A2 expression plasmid construct (Anxa2-GFP, Addgene plasmid # 107,196) was received as a gift from Volker Gerke & Ursula Rescher [29]. For transfection, cells were seeded in 6-well plates at approximately 50–60% confluence and transfected with either siRNA (25 nM) or Anxa2-GFP plasmid (1 µg) using Dharmafect reagent prepared in Opti-MEM according to the manufacturer’s instructions. siRNA or plasmid transfection reagent complexes were incubated for 20 min before being added to antibiotic-free medium, and cells were cultured for 48 h at 37 °C. A non-targeting scrambled siRNA served as a negative control. In overexpression experiments, cells transfected with an empty plasmid were used as a vector control. Knockdown or overexpression efficiency was assessed 48 h post-transfection by quantifying annexin A2 protein expression by immunoblotting.
Statistical analysis
All the experiments were conducted three times independently to ensure reproducibility and reliability of the findings. Statistical analyses were carried out using GraphPad Prism 8.0.2 (GraphPad Software, La Jolla, CA, USA). Data normality was assessed using the Shapiro–Wilk test, while homogeneity of variance was evaluated using the Brown-Forsythe test. To analyze differences between two groups, an unpaired t-test was employed, whereas for comparisons involving more than two groups, one-way or two-way ANOVA was performed. Followed by Dunnett's or Tukey’s multiple comparison tests were applied for post-hoc analysis, where appropriate. A threshold of p < 0.05 was considered statistically significant. Results were represented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 were considered to be significant. Non-significant results (p > 0.05) were annotated as ‘ns’.
Results
Low-dose OTA exposure caused toxicity to kidney proximal tubular epithelial cells in a time-dependent manner
We first examined the effect of OTA on NRK52E cells at different time points. For that, NRK52E cells were treated with different concentrations of OTA (100 nM-10 µM), and cell viability was assessed through MTT and SRB assays. The MTT assay showed that the relative cell viabilities were found to be increased by around 20–25% at 250 and 500 nM concentrations of OTA treatment compared to the control group (Fig. 1A). Similar to the MTT assay, the SRB assay also showed that the total protein mass was increased by 12–15% at 250 and 500 nM concentrations of OTA compared to control cells (Fig. 1B). Based on these results, we selected 250 and 500 nM concentrations of OTA for acute (48 h), sub-chronic (two months), and chronic (four months) exposure in NRK52E cells. As shown in Fig. 1C-G, the expression of kidney injury markers was increased in a time-dependent manner. Specifically, the higher expression of kidney toxicity markers was observed in two and four months of OTA-exposed NRK52E cells, compared to the 48 h exposure group. Consistent with the Luminex assay findings, immunoblotting results also confirmed the nephrotoxic effect of OTA. Clusterin, a kidney injury marker, showed a significant increase at four months, whereas cystatin C expression was elevated at 48 h as well as two and four months of OTA exposure (Fig. 1H and Supplementary Fig. 1A-B). Further, to confirm the effect of OTA-mediated cytotoxicity in human kidney proximal tubule-derived epithelial cells, the HK-2 cell line was treated with different concentrations of OTA. Notably, only 500 nM OTA concentration increased the survivability of HK-2 cells (Supplementary Fig. 1 C). Additionally, Luminex-based multiplex analysis showed an increased expression of kidney injury markers in HK-2 cells upon OTA-500 nM exposure (Supplementary Fig. 1D-H). Thus, the low-dose OTA exhibited a similar toxic response in both rat and human-derived kidney epithelial cells.
Fig. 1.
Low-dose exposure to OTA shows the alterations in the functional properties of NRK52E cells. A Graphical representation of the percent of cell viability measured using the MTT assay. B The SRB assay represents the percentage of total protein mass. Data were analysed by one-way ANOVA. C-G After exposure to OTA, the expression of kidney injury markers increased in NRK52E cells. Bar diagram showing that the concentration (pg/ml) of NGAL C, clusterin D, TIMP1 E, albumin F, and cystatin C G increased in a time-dependent manner. H Immunoblot analysis of kidney toxicity markers (clusterin and cystatin C) of treated and control NRK52E cells at different time points. The data were normalized to β-actin expression. The experiments were performed at least three times independently. Control = vehicle-treated NRK52E cells, LD = 250 nM OTA-treated NRK52E cells, and HD = 500 nM OTA-treated NRK52E cells
Chronic low-dose OTA exposure increased sEV secretion in NRK52E cells
Based on the above findings, our further aim was to check whether OTA exposure affects EV secretion from NRK52E cells in culture medium. In this regard, we isolated and characterized EVs from conditioned culture media of OTA-exposed NRK52E cells and characterized them using NTA, TEM, and immunoblotting. The NTA result showed that the hydrodynamic radius of EV particles ranged between 30–200 nm in all groups (Fig. 2A). Interestingly, 500 nM concentration of OTA (higher dose or HD) significantly enhanced the secretion of EVs in the conditioned culture media, while 250 nM OTA concentration (lower dose or LD) did not show any significant effect on EV secretion in the culture media, both for 48 h, 2 and 4 months (Fig. 2B). Subsequently, TEM analysis showed that the isolated EVs were small in size (30–200 nm) and spherical in shape (Fig. 2C). These tiny vesicles were termed small extracellular vesicles (sEVs). Moreover, these vesicles were enriched with sEV-markers such as CD63, CD9 and flotillin-1. Notably, the expression of these markers significantly increased solely at two and four-month intervals and remained unchanged at 48 h (Fig. 2D). In contrast, the negative sEV markers, calnexin (a marker for endoplasmic reticulum) and GM130 (a marker for Golgi apparatus), were not detected in isolated vesicle preparation (Fig. 2E). Furthermore, immuno-TEM analysis further confirmed that CD9 expression was increased on the surface of sEVs derived from four-month OTA-induced NRK52E cells (Fig. 2F). Taken together, significant alterations in the concentration of sEVs were evident after four months of sustained OTA exposure in NRK52E cells. Thus, four months of chronic OTA-exposed NRK52E cells-derived sEVs were used for further experiments.
Fig. 2.
Low-dose 2 and 4-month exposure to OTA induces sEV secretion in the conditioned media of NRK52E cells. A Chromatograms showing the size (x-axis) and concentration (y-axis) of exosomes isolated from 48 h (upper panel), 2-months (middle panel) and 4-months (lower panel) of OTA-exposed NRK52E cells. B Bar diagram representing the concentration of isolated EVs. C Characterization of transmission electron microscopy (TEM) representing the spherical shape and size (nm) of EVs isolated from OTA-treated cells (upper panel for 48 h, middle panel for 2 months, and lower panel for 4 months), original magnification X67000, 80 kV, scale bar 200 nm. D-E Representative images of immunoblotting for sEVs positive markers, CD63, CD9, flotillin-1 expression D and sEVs negative markers, calnexin and GM130 E in EVs isolated from both 48 h, 2 and 4-month OTA-exposed and control NRK52E cells. EVs were isolated from equal volumes of conditioned media, and equal amount of EV proteins was used for immunoblotting. F Immunogold-TEM images further validate the expression of CD9 protein on the surface of four-month OTA-induced sEVs. The upper lane shows X150000 magnification with a 100 nm scale bar, and the lower lane shows a zoomed-in view. The red arrow represents the CD9 expression on the surface of sEVs. 48 h = 48 h OTA-treated NRK52E cells, 2 M = two-month OTA-treated NRK52E cells, 4 M = four-month OTA-treated NRK52E cells, Control = vehicle-treated NRK52E cells, LD = 250 nM OTA-treated NRK52E cells, and HD = 500 nM OTA-treated NRK52E cells, WCL = whole cell lysate of NRK52E cells
Exposure of OTA-induced sEVs caused kidney toxicity in NRK52E cells
Low-dose chronic exposure to OTA increased the secretion of sEVs in the conditioned media of NRK52E cells. Furthermore, to confirm whether this increase correlates with OTA-induced toxicity, NRK52E cells were treated with sEVs derived from control, LD, or HD cells. As shown in Fig. 3A, after 24 h of treatment, sEVs were internalized by NRK52E cells. Subsequently, to assess their impact on toxicity, immunoblotting was performed in sEVs-treated NRK52E cells. A dose-dependent increase in the expression of clusterin, B2M, cystatin C, and fibronectin was observed in two and four-month OTA-exposed NRK52E cells compared to the control group, respectively (Fig. 3B-F). However, HD-sEVs markedly increased the expression of kidney injury markers in recipient cells, compared to control and LD-sEVs (Fig. 3B-F). Further, to confirm the toxic effects of OTA-induced sEVs, sEVs derived from four-month OTA-exposed NRK52E cells were used to treat HK-2 cells to assess whether these vesicles could also influence human-derived kidney epithelial cells. Interestingly, HD-sEVs increased the expression of kidney injury markers in HK-2 cells compared to control-sEVs-treated cells (Supplementary Fig. 2A-G). These findings suggested that the OTA-induced sEVs contributed to OTA-induced toxicity in kidney epithelial cells.
Fig. 3.
OTA-induced sEV treatment increased the expression of kidney injury markers in normal kidney cells. A Fluorescent staining confirms the intracellular uptake of NRK52E cell-derived sEVs after 24 h of treatment. B Immunoblotting analysis of the expression of kidney injury markers after 24 h of sEV treatment. C-F Bar diagram representing the densitometry analysis of the expression of clusterin C, B2M D, cystatin C E, and fibronectin F. The data were normalized by the expression of β-actin. C-sEVs = control cells-derived sEV-treated NRK52E cells,LD-sEVs = LD-derived sEV-treated NRK52E cells, and HD-sEVs = HD-derived sEV-treated NRK52E cells
Exogenous administration of OTA-induced sEVs mimicked the renal toxic effects of OTA in Wistar rats
After confirming the effect of OTA-induced sEVs on kidney proximal tubular epithelial cells in vitro, we next investigated whether exogenous administration of these vesicles could induce kidney toxicity in vivo, similar to direct OTA exposure. Therefore, we administered HD-derived sEVs (as they caused maximum toxicity in NRK52E cells compared to control or LD-derived sEVs) via tail vein injection into Wistar rats. Over the two-month treatment period, neither the sEVs-treated nor the OTA-exposed rats exhibited significant changes in body weight, food intake, or water intake compared to vehicle-treated rats (Supplementary Fig. 3A-C). However, analysis of organ and body weight ratios revealed that the kidney-to-body weight ratio (KW: BW) was significantly reduced in both sEVs and OTA-exposed groups (Fig. 4A), while the weights of other organs (liver, lungs, spleen, and heart) remained unchanged (Supplementary Fig. 4A-D). Interestingly, kidney function was impaired in both groups, evidenced by a significant drop in GFR (Fig. 4B). Moreover, serum levels of urea and creatinine, as well as urinary albumin, were notably elevated in both sEVs and the OTA group (Fig. 4C-E), while serum ALT and AST were not significantly altered (Supplementary Fig. 4E and F). Urinary kidney injury markers such as NGAL, albumin, cystatin C, clusterin, and TIMP1 were also markedly elevated in both treatment groups (Fig. 4F-J). A heightened level of histological abnormalities (including prominent tubular epithelial cell damage, glomerular shrinkage, loss of the brush border epithelial layer, and hypercellularity) was observed, as evidenced by H & E staining of kidney sections of both OTA and sEV-exposed rats, compared to vehicle control (Fig. 4K, upper row). In contrast, sEVs-treated rats did not show any abnormalities in other organs (lungs, liver, heart, and spleen), while the OTA-exposed rats showed multi-organ toxicity, including enlarged sinusoidal spaces in the liver, inflammatory cell infiltration and fiber disorganization in the heart, brown pigmentation indicating splenic melanosis, and thickened interalveolar septa and alveolar shrinkage in the lungs (Supplementary Fig. 4G). MTS further exhibited significant collagen deposition in the glomeruli, interstitium, and tubular membranes in both sEVs and OTA-treated rats, compared to vehicle-exposed rats (Fig. 4K, middle row). Moreover, PAS staining indicated substantial glycogen deposition in the glomeruli, interstitium, and tubules in both groups, while vehicle-exposed rat kidneys exhibited no such deposits (Fig. 4K, lower row). Further, IHC was performed to check the expression of kidney injury markers in kidney sections. The increased expression of kidney injury markers such as B2M, cystatin C, calbindin, PCNA, fibronectin, and α-SMA was observed in the kidney cortex of the sEV-exposed rats, similar to the OTA-exposed group (Fig. 4L and Supplementary Fig. 5 A). Notably, PCNA and fibronectin were also markedly expressed in the outer stripe of the outer medulla (OSOM) of the kidneys of both groups (data not shown). Further, immunoblotting analysis confirmed a significant upregulation of these kidney injury markers in both treatment groups, compared with vehicle control rats (Fig. 4M and Supplementary Fig. 5B).
Fig. 4.
Intravenous injection of OTA-induced sEVs caused histopathological changes and increased kidney injury markers in kidney tissue of rats. A The bar diagram depicting the kidney and body weight ratio reflects the condition of kidney. B Absolute GFR (µl/min) was also measured to check the filtration capacity of kidney. C-E The bar diagram represents the concentration (mg/dl) of serum creatinine (SCr) C, serum urea (SUrea) D, and urinary albumin (UAlb) E of vehicle control, OTA, and sEVs-exposed rats. F-J The representative bar graphs show the concentration (pg/ml) of urinary kidney injury markers, neutrophil gelatinase-associated lipocalin (NGAL) F, albumin G, cystatin C H, clusterin I, and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) J. All the data were analysed by One-way ANOVA. K Representative images showing the histopathological analysis of v-control, OTA and sEVs-exposed rats. H&E staining (upper row) representing that OTA (210 μg/kg b.wt.) and sEVs (100 μg) exposure-caused glomerular shrinkage (star symbol), hypercellularity in tubules (thick arrow symbol), proximal epithelial cell degeneration (asterisk symbol); MTS (middle row) showing the collagen deposition in tubulointerstitial space (blue color); PAS (lower row) representing the loss of brush border (hash symbol) glycogen deposition in the tubules and tubulointerstitial space (arrowhead symbol); magnification 20x, scale bar 50 µm. (L) Representative photographs of immunohistochemistry show the expression of B2M, cystatin C, calbindin, PCNA, fibronectin, and α-SMA; magnification 20x, scale bar 100 µm. (M) Immunoblotting of the expression of corresponding kidney injury markers. Numbers (1–4) indicate each animal in a given group. V-control = control sEV-injected and vehicle-treated rats, OTA = OTA-exposed rats, sEVs = OTA-induced sEV-injected rats, and N-control = negative control
In vivo live imaging confirms the higher renal accumulation of exogenously injected sEVs
We found that exogenous exposure of OTA-induced sEVs to Wistar rats mainly affected the kidneys, while the liver, lungs, spleen, and heart showed no significant changes. So, we further investigated the biodistribution of sEVs in rats by administering sEVs labelled with PKH26 through the lateral tail vein, and their biodistribution and accumulation were monitored over time. We observed an intense fluorescence signal that came from the lumbar region (lower back) of labelled-sEV-injected rats in a time-dependent manner. In dye-injected rats, fluorescence was detected slightly above the lumbar area, and the signal gradually disappeared 2 h after injection (Fig. 5A-B). Further, to confirm the appropriate organ where the signal was localized, ex vivo imaging was performed after 24 h of injection by collecting five vital organs (lungs, liver, kidneys, spleen, and heart). An intense fluorescence signal (dark blue colour) was detected in the kidneys, whereas minimal signal (red colour) was detected only in the liver, with no detectable signal from the heart, lungs, or spleen. In contrast, a minimal basal level fluorescence (green, yellow or red colours) was detected in kidney and liver of rats injected with dye alone or vehicle, while no signal was detected in the remaining organs (Fig. 5C-D). Fluorescence imaging of kidney cryo-sections also confirmed that most of the PKH26-fluorescence signal was present in the kidney of rats injected with labelled-sEVs, compared to the dye only group (Fig. 5E). Hereby, we speculated that the fluorescence signal observed in the kidney was primarily associated with the accumulation of PKH26-labelled sEVs rather than free dye alone. Additionally, TEM analysis of kidney cortex sections was performed to confirm whether accumulated particles in the cortex were sEVs or not. Analysis revealed a higher accumulation of spherical vesicles, resembling the size of sEVs (< 200 nm), in the extracellular space of tubular epithelial cells in the kidney cortex, consistent with the fluorescence imaging results (Fig. 5F).
Fig. 5.
Biodistribution of NRK52E cellderived sEVs in Wistar rat. A The in vivo (dorsal view) optical images represent the systemic distribution of labelled sEVs, dye only, and V-control groups. B Bar diagram representing the total radiant efficiency ([p/s]/[µW/cm2]) in both groups in a time-dependent manner. C Ex vivo imaging representing the accumulation status of labelled sEVs 24 h after injection. D The total radiant efficiency of kidney and liver of labelled-sEVs, dye only, and V-control group. E Photomicrograph representing the fluorescence imaging of a kidney section. Blue colour represents the nucleus, red colour dots representing the accumulated sEVs (indicated using a white arrow), magnification 40x, scale bar 50 µm. The control group received tail vein injection of PKH26 dye dissolved in PBS. F TEM images of the kidney cortex show accumulation of sEVs in both the V-control and treatment groups. The scale bar for the low-magnification (15000X) image is 1 µm, while the scale bar for the higher-magnification (452000X) image is 500 nm. Red arrow represents exosomes. For all analyses, three biological (n = 3) replicates were used. V-control = unlabelled sEVs injected rats, dye only = PKH26-injected rats, labelled sEVs = PKH26-labelled sEVs injected rats, control = PBS injected rats, sEVs = rats injected with sEVs of OTA-exposed NRK52E cells, TEC = tubular epithelial cells, and ES = extracellular space
Blockade of sEV secretion alleviated OTA-induced kidney toxicity
We found the toxic effects of OTA-induced sEVs on the kidney in both in vitro and in vivo conditions. To further validate its role in OTA-mediated kidney toxicity, we first reduced the sEV release into the conditioned media of OTA-exposed NRK52E cells using DMA treatment (Supplementary Fig. 6 A). Consistently, conditioned media of OTA-exposed cells significantly increased the expression of kidney toxicity markers in NRK52E cells. In contrast, this effect was attenuated when cells were treated with conditioned media derived from four-month OTA and DMA-exposed cells (Fig. 6A-B). Furthermore, these findings were validated in Wistar rats by administering DMA 15 days post-OTA exposure, as illustrated in Supplementary Fig. 6B. After 8 weeks of treatment, the concentration of urinary sEVs was significantly decreased after DMA administration in OTA-exposed rats, compared to the OTA-only group (Supplementary Fig. 6 C). Moreover, inhibition of sEV secretion significantly attenuated kidney weight reduction in OTA-exposed rats (Fig. 6C). In contrast, no significant alterations were found in liver, lungs, heart, and spleen weight in DMA-exposed rats, compared with OTA-exposed rats (Supplementary Fig. 6D-G). In addition, classical kidney injury markers, including serum urea and creatinine as well as urinary albumin level and the protein-based urinary kidney injury markers, including NGAL, clusterin, cystatin C, TIMP-1, and albumin level were significantly restored following DMA treatment (Fig. 6D-K). Furthermore, histopathological analysis revealed a marked reduction in structural alterations and collagen deposition in the kidney cortex after blockade of sEV secretion in OTA-exposed rats (Fig. 6L upper and lower panel, respectively). Moreover, kidney injury markers like B2M, cystatin C, and calbindin levels were significantly reduced in DMA-exposed rats (Fig. 6M). Importantly, the DMA-only control group did not exhibit any significant changes in kidney toxicity markers or histopathological alterations compared with the vehicle control group, indicating that DMA at the used dose does not independently affect kidney toxicity.
Fig. 6.
Blockade of EV secretion alleviates OTA-induced kidney toxicity. A-B Immunoblotting of the kidney injury markers in control or OTA-exposed NRK52E cells after DMA-mediated inhibition of EV secretion. C Bar diagram representing the kidney and body weight ratio across experimental groups. D-F The level of the classical kidney injury markers serum creatinine (SCr) D, serum urea (SUrea) E, and urinary albumin (UAlb) F, measured in V-control, OTA, OTA + DMA, and DMA-treated rats. G-K Bar diagram representing the concentration of the protein-based urinary kidney injury markers NGAL G, clusterin H, cystatin C (I), TIMP-1 J, and albumin K. L Representative histopathological images showing structural alterations in kidney tissue. H & E staining (left column) illustrating glomerular shrinkage (star symbol), hypercellularity in tubules (thick arrow symbol), proximal epithelial cell degeneration (asterisk symbol); MTS (right column) showing the collagen deposition in the tubulointerstitial space (blue colour). (M) Immunohistochemistry images showing the expression of kidney injury markers in kidney cortex region; magnification 20x, scale bar 100 µm. Each group contained three animals (n = 3). CTL = NRK52E cells treated with conditioned media from control NRK52E cells, CTL + DMA = NRK52E cells treated with conditioned media from DMA-exposed NRK52E cells, HD = NRK52E cells treated with conditioned media from four months OTA-exposed NRK52E cells, HD + DMA = NRK52E cells treated with conditioned media from four months OTA-exposed NRK52E cells treated with DMA, V-control = vehicle-treated rats, OTA = OTA-exposed rats, OTA + DMA = both DMA and OTA-exposed rats, and DMA = only DMA-exposed rats
OTA-exposed NRK52E cells-derived sEVs contain a higher number of kidney toxicity-associated proteins
Chronic exposure to OTA resulted in higher secretion of sEVs from NRK52E cells in the culture media. Notably, these sEVs were found to cause kidney toxicity when introduced to NRK52E cells as well as Wistar rats. To investigate the underlying mechanisms, we further looked for the most significantly affected sEV-associated proteins following OTA exposure. As recent studies have shown, EV-associated proteins play a key role in maintaining the normal as well as pathophysiological conditions of the kidney [30]. Therefore, the proteomic study was conducted on sEVs derived from 4-month OTA-exposed NRK52E cells. We found that a total of 541 proteins were identified in sEVs derived from control (control-sEVs), LD (LD-sEVs), and HD (HD-sEVs). Specifically, 122 proteins were identified in the control group, 178 proteins in the LD group, and 241 proteins in the HD group (Fig. 7A). Notably, 98 proteins were common across all three groups. Volcano plot analysis was performed to determine which proteins were significantly upregulated as well as downregulated in control-sEVs versus LD-sEVs or HD-sEVs groups. Following analysis of the results, it was found that LD-sEVs contained 101 significantly upregulated proteins, while no significantly downregulated proteins were detected. Whereas in the HD-sEVs group, 113 significantly upregulated and 1 downregulated protein were observed (Fig. 7B). Tables 1 and 2 list the significantly deregulated (upregulated as well as downregulated) proteins of control-sEVs versus LD-derived sEVs and control-sEVs versus HD-derived sEVs, respectively. Further, gene ontology study demonstrated that these proteins are involved in cell adhesion, negative regulation of apoptosis, response to hypoxia, protein folding, etc., which are the key biological processes involved in the initiation and progression of kidney toxicity (Fig. 7C).
Fig. 7.
Unlabelled proteomics analysis of OTA-exposed NRK52E cell-derived sEVs. A Venn diagram representing the distribution of sEV proteins between control, LD, and HD. (B) The volcano plot shows significantly upregulated and downregulated proteins in the LD vs the control group, as well as in the HD vs the control group. X-axis depicting the difference (Log2 fold change) and Y-axis depicting the –logP (significance). Statistical significance was calculated using two-sample t-tests followed by Benjamini–Hochberg correction. (C) Gene ontology (GO) enrichment analysis reveals that several biological processes were enriched, as indicated. C-sEVs = sEVs of vehicle-exposed NRK52E cells, LD-sEVs = sEVs of OTA-250 nM-exposed NRK52E cells, and HD-sEVs = sEVs of OTA-500 nM-exposed NRK52E cells
Table 1.
List of significantly upregulated proteins identified in the 250 nM OTA (LD)-induced sEVs vs control (C) sEVs group by LC/MS–MS analysis
| Accession | Description | Gene Symbol | Fold Change (Log2FC LDvsC) |
-Log (P- Value) |
|---|---|---|---|---|
| P62632 | Elongation factor 1-alpha 2 | Eef1a2 | 10.5 | 5.96E + 00 |
| Q63041 | Alpha-1-macroglobulin | A1m | 9.7 | 8.78E + 00 |
| P08650 | Complement C5 | C5 | 9.3 | 6.10E + 00 |
| P01346 | Insulin-like growth factor II | Igf2 | 8.9 | 5.63E + 00 |
| P16296 | Coagulation factor IX | F9 | 8.5 | 8.25E + 00 |
| P38652 | Phosphoglucomutase-1 | Pgm1 | 8.1 | 5.65E + 00 |
| Q5U300 |
Ubiquitin-like modifier-activating enzyme 1 |
Uba1 | 7.9 | 6.94E + 00 |
| P50399 | Rab GDP dissociation inhibitor beta | Gdi2 | 7.9 | 8.49E + 00 |
| O08651 |
D-3-phosphoglycerate dehydrogenase |
Phgdh | 7.8 | 7.42E + 00 |
| P11598 | Protein disulfide-isomerase A3 | Pdia3 | 7.6 | 5.15E + 00 |
| P55314 |
Complement component C8 beta chain |
C8b | 7.6 | 3.67E + 00 |
| P35444 | Cartilage oligomeric matrix protein | Comp | 7.6 | 4.49E + 00 |
| P62836 | Ras-related protein Rap-1A | Rap1a | 7.5 | 3.38E + 00 |
| Q3MHS2 | Zinc finger protein 830 | Znf830 | 7.4 | 7.41E + 00 |
| Q5XIM9 | T-complex protein 1 subunit beta | Cct2 | 7.4 | 4.92E + 00 |
| Q62894 | Extracellular matrix protein 1 | Ecm1 | 7.3 | 4.08E + 00 |
| P15800 | Laminin subunit beta-2 | Lamb2 | 7.1 | 3.82E + 00 |
| P04642 | L-lactate dehydrogenase A chain | Ldha | 7.0 | 3.09E + 00 |
| P06761 |
Endoplasmic reticulum chaperone BiP |
Hspa5 | 6.9 | 5.57E + 00 |
| P50503 | Hsc70-interacting protein | St13 | 6.8 | 5.43E + 00 |
| Q9WVC0 | Septin-7 | Septin7 | 6.8 | 6.35E + 00 |
| Q66HG4 | Galactose mutarotase | Galm | 6.8 | 6.78E + 00 |
| P61983 | 14–3-3 protein gamma | Ywhag | 6.8 | 4.78E + 00 |
| P10760 | Adenosylhomocysteinase | Ahcy | 6.7 | 2.14E + 00 |
| P05539 | Collagen alpha-1(II) chain | Col2a1 | 6.7 | 3.33E + 00 |
| P29534 | Vascular cell adhesion protein 1 | Vcam1 | 6.6 | 4.79E + 00 |
| P58751 | Reelin | Reln | 6.6 | 4.19E + 00 |
| P04762 | Catalase | Cat | 6.5 | 3.92E + 00 |
| Q66HD0 | Endoplasmin | Hsp90b1 | 6.5 | 3.91E + 00 |
| P62260 | 14–3-3 protein epsilon | Ywhae | 6.4 | 3.04E + 00 |
| O70199 | UDP-glucose 6-dehydrogenase | Ugdh | 6.4 | 4.29E + 00 |
| B3GNI6 | Septin-11 | Septin11 | 6.3 | 6.13E + 00 |
| P09811 | Glycogen phosphorylase, liver form | Pygl | 6.2 | 3.57E + 00 |
| P25113 | Phosphoglycerate mutase 1 | Pgam1 | 6.2 | 5.76E + 00 |
| P05197 | Elongation factor 2 | Eef2 | 6.2 | 4.32E + 00 |
| Q7TPB1 | T-complex protein 1 subunit delta | Cct4 | 6.2 | 6.49E + 00 |
| D3ZHA0 | Filamin-C | Flnc | 6.0 | 5.51E + 00 |
| P62804 | Histone H4 | H4c16;H4c2;Hist1h4m | 5.9 | 4.09E + 00 |
| Q9QZA2 |
Programmed cell death 6-interacting protein |
Pdcd6ip | 5.9 | 6.54E + 00 |
| P11442 | Clathrin heavy chain 1 | Cltc | 5.8 | 3.82E + 00 |
| Q6NYB7 | Ras-related protein Rab-1A | Rab1A | 5.7 | 2.63E + 00 |
| Q6RUV5 |
Ras-related C3 botulinum toxin substrate 1 |
Rac1 | 5.7 | 3.13E + 00 |
| Q8R2H2 | Integrin beta-3 | Itgb3 | 5.7 | 6.14E + 00 |
| Q07936 | Annexin A2 | Anxa2 | 5.6 | 2.84E + 00 |
| P25304 | Agrin | Agrn | 5.6 | 4.02E + 00 |
| Q9JLT0 | Myosin-10 | Myh10 | 5.6 | 4.46E + 00 |
| P13383 | Nucleolin | Ncl | 5.5 | 2.95E + 00 |
| P85968 | 6-phosphogluconate dehydrogenase, decarboxylating | Pgd | 5.5 | 3.03E + 00 |
| Q63610 | Tropomyosin alpha-3 chain | Tpm3 | 5.4 | 6.79E + 00 |
| P27653 |
C-1-tetrahydrofolate synthase, cytoplasmic |
Mthfd1 | 5.4 | 4.87E + 00 |
| P46462 |
Transitional endoplasmic reticulum ATPase |
Vcp | 5.2 | 3.78E + 00 |
| Q920P0 | L-xylulose reductase | Dcxr | 5.2 | 5.90E + 00 |
| P61589 | Transforming protein RhoA | Rhoa | 5.2 | 3.49E + 00 |
| Q63321 | Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 | Plod1 | 5.1 | 4.08E + 00 |
| Q08163 | Adenylyl cyclase-associated protein 1 | Cap1 | 5.0 | 5.24E + 00 |
| P46418 | Glutathione S-transferase alpha-5 | Gsta5 | 5.0 | 3.34E + 00 |
| Q4V7C7 | Actin-related protein 3 | Actr3 | 5.0 | 2.83E + 00 |
| P16638 | ATP-citrate synthase | Acly | 5.0 | 3.21E + 00 |
| P34058 | Heat shock protein HSP 90-beta | Hsp90ab1 | 4.9 | 4.76E + 00 |
| Q63347 | 26S proteasome regulatory subunit 7 | Psmc2 | 4.8 | 3.10E + 00 |
| O08618 |
Phosphoribosyl pyrophosphate synthase-associated protein 2 |
Prpsap2 | 4.7 | 1.92E + 00 |
| P12711 | Alcohol dehydrogenase class-3 | Adh5 | 4.6 | 2.82E + 00 |
| P68255 | 14–3-3 protein theta | Ywhaq | 4.6 | 2.80E + 00 |
| Q8CFN2 |
Cell division control protein 42 homolog |
Cdc42 | 4.5 | 2.17E + 00 |
| P47942 |
Dihydropyrimidinase-related protein 2 |
Dpysl2 | 4.5 | 3.43E + 00 |
| O35568 |
EGF-containing fibulin-like extracellular matrix protein 1 |
Efemp1 | 4.4 | 3.48E + 00 |
| P97603 | Neogenin (Fragment) | Neo1 | 4.4 | 4.07E + 00 |
| Q9Z1P2 | Alpha-actinin-1 | Actn1 | 4.4 | 3.40E + 00 |
| P05371 | Clusterin | Clu | 4.2 | 1.93E + 00 |
| P48500 | Triosephosphate isomerase | Tpi1 | 4.2 | 1.52E + 00 |
| P02454 | Collagen alpha-1(I) chain | Col1a1 | 4.0 | 4.97E + 00 |
| Q66HR2 |
Microtubule-associated protein RP/EB family member 1 |
Mapre1 | 3.9 | 2.63E + 00 |
| P85972 | Vinculin | Vcl | 3.8 | 4.68E + 00 |
| G3V928 |
Prolow-density lipoprotein receptor-related protein 1 |
Lrp1 | 3.8 | 2.36E + 00 |
| P82995 | Heat shock protein HSP 90-alpha | Hsp90aa1 | 3.6 | 6.12E + 00 |
| Q9WVC1 | Slit homolog 2 protein (Fragment) | Slit2 | 3.6 | 1.69E + 00 |
| P13084 | Nucleophosmin | Npm1 | 3.6 | 1.17E + 00 |
| O08628 |
Procollagen C-endopeptidase enhancer 1 |
Pcolce | 3.5 | 3.53E + 00 |
| Q9JLJ3 | 4-trimethylaminobutyraldehyde dehydrogenase | Aldh9a1 | 3.3 | 4.14E + 00 |
| P35952 | Low-density lipoprotein receptor | Ldlr | 3.2 | 2.00E + 00 |
| P62828 | GTP-binding nuclear protein Ran | Ran | 3.2 | 5.50E + 00 |
| P0DMW0 | Heat shock 70 kDa protein 1A | Hspa1a | 3.1 | 4.29E + 00 |
| Q5XIA3 |
tRNA wybutosine-synthesizing protein 4 |
Lcmt2 | 2.9 | 3.39E + 00 |
| Q91ZN1 | Coronin-1A | Coro1a | 2.8 | 3.21E + 00 |
| P63018 | Heat shock cognate 71 kDa protein | Hspa8 | 2.7 | 2.78E + 00 |
| P16617 | Phosphoglycerate kinase 1 | Pgk1 | 2.7 | 2.39E + 00 |
| P11980 | Pyruvate kinase PKM | Pkm | 2.7 | 6.16E + 00 |
| P63102 | 14–3-3 protein zeta/delta | Ywhaz | 2.6 | 3.09E + 00 |
| B0BNI5 | Olfactomedin-like protein 3 | Olfml3 | 2.6 | 3.36E + 00 |
| P04797 | Glyceraldehyde-3-phosphate dehydrogenase | Gapdh | 2.5 | 3.02E + 00 |
| Q64119 | Myosin light polypeptide 6 | Myl6 | 2.3 | 3.38E + 00 |
| O35303 | Dynamin-1-like protein | Dnm1l | 2.3 | 1.35E + 00 |
| O55096 | Dipeptidyl peptidase 3 | Dpp3 | 2.2 | 1.23E + 00 |
| P70490 | Lactadherin | Mfge8 | 2.1 | 1.77E + 00 |
| P0DP31 | Calmodulin-3 | Calm3 | 2.1 | 1.16E + 00 |
| P60711 | Actin, cytoplasmic 1 | Actb | 2.1 | 3.48E + 00 |
| P49744 | Thrombospondin-4 | Thbs4 | 2.1 | 3.13E + 00 |
| P02680 | Fibrinogen gamma chain | Fgg | 1.9 | 3.18E + 00 |
| Q7TMA5 | Apolipoprotein B-100 | Apob | 1.8 | 2.61E + 00 |
| P04764 | Alpha-enolase | Eno1 | 1.6 | 1.76E + 00 |
| P0C6B8 | Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 | Svep1 | 1.4 | 1.09E + 00 |
No downregulated proteins were identified. Log2FC LDvsC represents the fold change in protein abundance in low-dose OTA-exposed sEVs relative to control sEVs. Significant deregulated proteins were identified if the adjusted p-value between experimental groups was ≤ 0.05 and the Log2FC was at least 1
Table 2.
List of significantly upregulated and downregulated proteins identified in the 500 nM OTA (HD) -induced sEVs vs control (C) sEVs group by LC/MS–MS analysis
| Accession | Description | Gene Symbol |
Fold Change (Log2FC HDvsC) |
-Log (P- Value) |
|---|---|---|---|---|
| P01346 | Insulin-like growth factor II | Igf2 | 11.0 | 6.88E + 00 |
| P62632 | Elongation factor 1-alpha 2 | Eef1a2 | 10.6 | 7.46E + 00 |
| Q63041 | Alpha-1-macroglobulin | A1m | 10.5 | 8.98E + 00 |
| Q5U300 | Ubiquitin-like modifier-activating enzyme 1 | Uba1 | 9.9 | 8.16E + 00 |
| P08650 | Complement C5 | C5 | 9.8 | 7.06E + 00 |
| P38652 | Phosphoglucomutase-1 | Pgm1 | 9.0 | 6.29E + 00 |
| P50399 | Rab GDP dissociation inhibitor beta | Gdi2 | 8.9 | 7.88E + 00 |
| P06761 | Endoplasmic reticulum chaperone BiP | Hspa5 | 8.7 | 7.93E + 00 |
| P16296 | Coagulation factor IX | F9 | 8.6 | 8.10E + 00 |
| Q62894 | Extracellular matrix protein 1 | Ecm1 | 8.4 | 4.62E + 00 |
| P15800 | Laminin subunit beta-2 | Lamb2 | 8.4 | 4.51E + 00 |
| Q3MHS2 | Zinc finger protein 830 | Znf830 | 8.3 | 8.02E + 00 |
| O08651 | D-3-phosphoglycerate dehydrogenase | Phgdh | 8.0 | 7.57E + 00 |
| Q66HD0 | Endoplasmin | Hsp90b1 | 8.0 | 5.94E + 00 |
| Q6RUV5 | Ras-related C3 botulinum toxin substrate 1 | Rac1 | 8.0 | 4.16E + 00 |
| P85968 |
6-phosphogluconate dehydrogenase, decarboxylating |
Pgd | 7.9 | 8.11E + 00 |
| B3GNI6 | Septin-11 | Septin11 | 7.8 | 7.31E + 00 |
| Q9WVC0 | Septin-7 | Septin7 | 7.7 | 6.85E + 00 |
| P58751 | Reelin | Reln | 7.7 | 4.79E + 00 |
| P25113 | Phosphoglycerate mutase 1 | Pgam1 | 7.6 | 6.51E + 00 |
| P05539 | Collagen alpha-1(II) chain | Col2a1 | 7.6 | 3.83E + 00 |
| P62804 | Histone H4 |
H4c16;H4c2; Hist1h4m |
7.6 | 7.46E + 00 |
| P35444 | Cartilage oligomeric matrix protein | Comp | 7.6 | 4.45E + 00 |
| P05197 | Elongation factor 2 | Eef2 | 7.5 | 4.86E + 00 |
| P61983 | 14–3-3 protein gamma | Ywhag | 7.5 | 5.20E + 00 |
| Q6NYB7 | Ras-related protein Rab-1A | Rab1A | 7.5 | 3.57E + 00 |
| Q07936 | Annexin A2 | Anxa2 | 7.4 | 4.24E + 00 |
| P25304 | Agrin | Agrn | 7.3 | 5.22E + 00 |
| Q63321 | Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 | Plod1 | 7.3 | 6.00E + 00 |
| P62836 | Ras-related protein Rap-1A | Rap1a | 7.3 | 3.26E + 00 |
| P10760 | Adenosylhomocysteinase | Ahcy | 7.2 | 2.32E + 00 |
| P04642 | L-lactate dehydrogenase A chain | Ldha | 7.2 | 3.20E + 00 |
| Q9WVC1 | Slit homolog 2 protein (Fragment) | Slit2 | 7.2 | 3.61E + 00 |
| Q7TPB1 | T-complex protein 1 subunit delta | Cct4 | 7.1 | 6.20E + 00 |
| O70199 | UDP-glucose 6-dehydrogenase | Ugdh | 7.0 | 4.72E + 00 |
| Q9QZA2 | Programmed cell death 6-interacting protein | Pdcd6ip | 7.0 | 7.71E + 00 |
| Q66HG4 | Galactose mutarotase | Galm | 7.0 | 6.98E + 00 |
| P27653 | C-1-tetrahydrofolate synthase, cytoplasmic | Mthfd1 | 7.0 | 6.01E + 00 |
| P12711 | Alcohol dehydrogenase class-3 | Adh5 | 7.0 | 6.10E + 00 |
| Q8R2H2 | Integrin beta-3 | Itgb3 | 6.9 | 7.14E + 00 |
| P11598 | Protein disulfide-isomerase A3 | Pdia3 | 6.8 | 4.64E + 00 |
| P46462 | Transitional endoplasmic reticulum ATPase | Vcp | 6.8 | 5.04E + 00 |
| P04762 | Catalase | Cat | 6.7 | 3.81E + 00 |
| P16638 | ATP-citrate synthase | Acly | 6.7 | 6.03E + 00 |
| P35952 | Low-density lipoprotein receptor | Ldlr | 6.7 | 4.88E + 00 |
| P62260 | 14–3-3 protein epsilon | Ywhae | 6.7 | 3.07E + 00 |
| P11442 | Clathrin heavy chain 1 | Cltc | 6.5 | 4.76E + 00 |
| P29534 | Vascular cell adhesion protein 1 | Vcam1 | 6.3 | 4.47E + 00 |
| Q4V7C7 | Actin-related protein 3 | Actr3 | 6.2 | 3.51E + 00 |
| P55314 | Complement component C8 beta chain | C8b | 6.1 | 3.02E + 00 |
| P97603 | Neogenin (Fragment) | Neo1 | 6.1 | 5.46E + 00 |
| P50503 | Hsc70-interacting protein | St13 | 6.1 | 4.85E + 00 |
| Q5XIM9 | T-complex protein 1 subunit beta | Cct2 | 6.1 | 2.55E + 00 |
| P48500 | Triosephosphate isomerase | Tpi1 | 6.1 | 4.57E + 00 |
| D3ZHA0 | Filamin-C | Flnc | 6.1 | 6.25E + 00 |
| P13383 | Nucleolin | Ncl | 5.9 | 2.85E + 00 |
| Q63610 | Tropomyosin alpha-3 chain | Tpm3 | 5.9 | 6.73E + 00 |
| O08618 | Phosphoribosyl pyrophosphate synthase-associated protein 2 | Prpsap2 | 5.8 | 2.48E + 00 |
| Q920P0 | L-xylulose reductase | Dcxr | 5.8 | 5.90E + 00 |
| Q9Z1P2 | Alpha-actinin-1 | Actn1 | 5.7 | 4.73E + 00 |
| P02454 | Collagen alpha-1(I) chain | Col1a1 | 5.6 | 7.52E + 00 |
| P09811 | Glycogen phosphorylase, liver form | Pygl | 5.5 | 3.00E + 00 |
| Q08163 | Adenylyl cyclase-associated protein 1 | Cap1 | 5.5 | 5.27E + 00 |
| P61589 | Transforming protein RhoA | Rhoa | 5.4 | 3.13E + 00 |
| P38650 | Cytoplasmic dynein 1 heavy chain 1 | Dync1h1 | 5.3 | 2.88E + 00 |
| B0BNI5 | Olfactomedin-like protein 3 | Olfml3 | 5.3 | 7.13E + 00 |
| P46418 | Glutathione S-transferase alpha-5 | Gsta5 | 5.3 | 2.91E + 00 |
| P05371 | Clusterin | Clu | 5.2 | 2.44E + 00 |
| P13084 | Nucleophosmin | Npm1 | 5.2 | 2.16E + 00 |
| O35568 |
EGF-containing fibulin-like extracellular matrix protein 1 |
Efemp1 | 5.2 | 3.99E + 00 |
| P47942 | Dihydropyrimidinase-related protein 2 | Dpysl2 | 5.2 | 3.44E + 00 |
| P34058 | Heat shock protein HSP 90-beta | Hsp90ab1 | 5.1 | 4.97E + 00 |
| Q6P9T8 | Tubulin beta-4B chain | Tubb4b | 5.0 | 3.51E + 00 |
| P0C6B8 |
Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 |
Svep1 | 4.9 | 4.05E + 00 |
| O08628 | Procollagen C-endopeptidase enhancer 1 | Pcolce | 4.9 | 4.81E + 00 |
| Q8CFN2 | Cell division control protein 42 homolog | Cdc42 | 4.8 | 2.30E + 00 |
| P68255 | 14–3-3 protein theta | Ywhaq | 4.6 | 2.93E + 00 |
| Q63347 | 26S proteasome regulatory subunit 7 | Psmc2 | 4.6 | 2.74E + 00 |
| P0DMW0 | Heat shock 70 kDa protein 1A | Hspa1a | 4.4 | 6.50E + 00 |
| P63018 | Heat shock cognate 71 kDa protein | Hspa8 | 4.4 | 5.00E + 00 |
| Q9JLJ3 | 4-trimethylaminobutyraldehyde dehydrogenase | Aldh9a1 | 4.3 | 5.15E + 00 |
| P04797 | Glyceraldehyde-3-phosphate dehydrogenase | Gapdh | 4.2 | 6.40E + 00 |
| P11980 | Pyruvate kinase PKM | Pkm | 4.2 | 6.25E + 00 |
| O55096 | Dipeptidyl peptidase 3 | Dpp3 | 4.2 | 2.68E + 00 |
| P82995 | Heat shock protein HSP 90-alpha | Hsp90aa1 | 4.1 | 7.18E + 00 |
| P62828 | GTP-binding nuclear protein Ran | Ran | 4.0 | 6.71E + 00 |
| Q66HR2 | Microtubule-associated protein RP/EB family member 1 | Mapre1 | 4.0 | 2.66E + 00 |
| P85972 | Vinculin | Vcl | 4.0 | 4.60E + 00 |
| P16617 | Phosphoglycerate kinase 1 | Pgk1 | 4.0 | 4.37E + 00 |
| Q64119 | Myosin light polypeptide 6 | Myl6 | 3.7 | 5.64E + 00 |
| P49744 | Thrombospondin-4 | Thbs4 | 3.7 | 5.83E + 00 |
| G3V928 | Prolow-density lipoprotein receptor-related protein 1 | Lrp1 | 3.4 | 1.92E + 00 |
| Q5XIA3 | tRNA wybutosine-synthesizing protein 4 | Lcmt2 | 3.4 | 4.95E + 00 |
| P62982 | Ubiquitin-40S ribosomal protein S27a | Rps27a | 3.3 | 4.16E + 00 |
| Q7TMA5 | Apolipoprotein B-100 | Apob | 3.3 | 4.90E + 00 |
| Q9QWJ9 | Neuropilin-1 | Nrp1 | 3.3 | 1.75E + 00 |
| P04937 | Fibronectin | Fn1 | 3.2 | 6.05E + 00 |
| Q9EQT5 | Tubulointerstitial nephritis antigen-like | Tinagl1 | 3.2 | 5.41E + 00 |
| P60711 | Actin, cytoplasmic 1 | Actb | 3.2 | 7.39E + 00 |
| P04764 | Alpha-enolase | Eno1 | 3.1 | 7.09E + 00 |
| P63102 | 14–3-3 protein zeta/delta | Ywhaz | 3.1 | 4.02E + 00 |
| P70490 | Lactadherin | Mfge8 | 3.1 | 3.68E + 00 |
| Q9JLT0 | Myosin-10 | Myh10 | 3.1 | 2.68E + 00 |
| P69897 | Tubulin beta-5 chain | Tubb5 | 3.0 | 6.27E + 00 |
| Q91ZN1 | Coronin-1A | Coro1a | 2.9 | 3.16E + 00 |
| Q62812 | Myosin-9 | Myh9 | 2.9 | 6.11E + 00 |
| P02680 | Fibrinogen gamma chain | Fgg | 2.9 | 5.37E + 00 |
| Q5XIF6 | Tubulin alpha-4A chain | Tuba4a | 2.6 | 5.17E + 00 |
| Q8CHN8 | Mannan-binding lectin serine protease 1 | Masp1 | 2.5 | 4.40E + 00 |
| P63259 | Actin, cytoplasmic 2 | Actg1 | 2.3 | 3.46E + 00 |
| O35303 | Dynamin-1-like protein | Dnm1l | 2.1 | 1.13E + 00 |
| P08721 | Osteopontin | Spp1 | 2.0 | 1.46E + 00 |
| Downregulated proteins | ||||
| P15865 | Histone H1.4 | H1-4 | −2.3 | 2.35E + 00 |
Log2FC HDvsC represents the fold change in protein abundance in high-dose OTA-induced sEVs relative to control sEVs. Significantly deregulated proteins were identified if the adjusted p-value between experimental groups was ≤ 0.05 and the Log2FC was at least 1
OTA-exposure increased urinary sEV level and enrichment of kidney toxicity-associated proteins in rat-derived urinary sEVs
Chronic exposure to OTA altered the protein profile of normal rat kidney cells-derived sEVs. To further identify the sEV-associated protein cargos involved in OTA-induced kidney toxicity in a more complex in vivo system, a separate proteomic study was conducted in urinary vesicles. Following isolation, urinary vesicles were characterized using NTA, TEM, and immunoblotting. As shown in Fig. 8A-C, NTA resulted in increased urinary vesicle concentration, but the size was not significantly affected after OTA exposure in rats. TEM results confirmed that the isolated urinary vesicles were spherical in shape and smaller than 200 nm in diameter (Fig. 8D). Consistent with these observations, immunoblotting analysis showed an increased expression of sEV-associated proteins such as CD63, CD9, TSG-101, and alix in urinary vesicles derived from OTA-exposed rats, while calnexin and GM130 were not detected (Fig. 8E). After successful isolation of urinary sEVs derived from vehicle control (VC-sEVs) and OTA-exposed rats (OTA-sEVs), vesicles were lysed to collect proteins and further processed for proteomics analysis. As shown in the Venn diagram, a total of 312 proteins were identified in urinary sEVs; among them, 129 proteins were from the vehicle control and 183 proteins from the treatment group. (Fig. 8F). As shown in Fig. 8G, out of 147 proteins, 70 proteins were upregulated, and 20 proteins were downregulated in OTA-sEVs. Table 3 lists the significantly upregulated and downregulated proteins. Moreover, the gene ontology study revealed that the proteins detected in sEVs following OTA treatment were largely associated with various kidney toxicity-related biological pathways, such as acute-phase response, positive regulation of cell proliferation, response to hypoxia, fibrinolysis, complement system, etc. (Fig. 8H).
Fig. 8.
Unlabelled proteomics analysis of urinary sEVs derived from OTA-exposed rats. A Chromatogram representing the size (nm) (x-axis) and concentration (particles/ml) (y-axis) of EVs isolated from urine derived from both VC and OTA-induced rats. B and C Bar diagram representing the concentration (B) and size (C) of EVs. D Pictorial representation depicting the TEM analysis of EVs isolated from both v-control and OTA-exposed rats; original magnification 150000X, 80 kV, and scale bar 100 nm. E Immunoblotting of sEVs-associated markers, as well as a negative marker, calnexin. EVs were isolated from an equal volume of urine, and an equal volume of EV suspension was used for SDS-PAGE in both groups. F Venn diagram representing the total number as well as the common sEV-protein in vehicle and OTA-exposed animals. (G) The volcano plot shows significantly upregulated and downregulated proteins in the treatment vs. the vehicle control group. X-axis depicting the difference (Log2 fold change) and Y-axis depicting the –logP (significance). Statistical significance was calculated using two-sample t-tests followed by Benjamini–Hochberg correction. H Gene ontology (GO) enrichment analysis reveals that several biological processes were involved. VC = vehicle-exposed rats, OTA = OTA-exposed rats, VC-sEVS = urinary sEVs of vehicle-exposed rats, and OTA-sEVs = urinary sEVs of OTA-exposed rats, WCL = whole cell lysate of NRK52E cells
Table 3.
All significantly upregulated and downregulated proteins in urinary sEVs derived from OTA-exposed rats (T) vs vehicle-control (VC) sEVs
| Accession | Description | Gene Symbol |
Fold Change (Log2FC TvsVC) | -Log (P-value) |
|---|---|---|---|---|
| P14841 | Cystatin-C | Cst3 | 11.6 | 3.70E + 00 |
| P81828 | Urinary protein 2 | 10.9 | 3.09E + 00 | |
| P20059 | Hemopexin | Hpx | 9.9 | 3.20E + 00 |
| P06866 | Haptoglobin | Hp | 9.8 | 2.37E + 00 |
| P42854 | Regenerating islet-derived protein 3-gamma | Reg3g | 9.6 | 3.40E + 00 |
| P24090 | Alpha-2-HS-glycoprotein | Ahsg | 9.1 | 2.55E + 00 |
| A0A0G2JZV7 | Uncharacterized protein | 8.8 | 3.32E + 00 | |
| Q7TMB9 | Ab1-021 | Serpina3l | 8.6 | 3.22E + 00 |
| Q5PQU1 | Kininogen 1 | Kng2 | 8.3 | 4.61E + 00 |
| Q6IN22 | Cathepsin B | Ctsb | 8.2 | 3.73E + 00 |
| P27274 | CD59 glycoprotein | Cd59 | 8.0 | 3.31E + 00 |
| M0RBP7 | Uncharacterized protein | 7.9 | 3.05E + 00 | |
| M0R4Z4 | Uncharacterized protein | 7.9 | 2.44E + 00 | |
| D3ZEP5 | Uncharacterized protein | 7.5 | 3.83E + 00 | |
| Q07936 | Annexin A2 | Anxa2 | 7.5 | 2.76E + 00 |
| A0A0G2JZ73 | Alpha-1-antiproteinase | 6.9 | 3.49E + 00 | |
| Q64602 | Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial | Aadat | 6.7 | 3.15E + 00 |
| F7EPE0 | Prosaposin | 6.7 | 2.48E + 00 | |
| Q5VLR6 | BWK3 | 6.7 | 1.95E + 00 | |
| P81827 | Urinary protein 1 | 6.6 | 5.56E + 00 | |
| Q62930 | Complement component C9 | C9 | 6.5 | 2.88E + 00 |
| P10959 | Carboxylesterase 1C | Ces1c | 6.5 | 2.28E + 00 |
| Q01177 | Plasminogen | Plg | 6.4 | 1.24E + 00 |
| P02680 | Fibrinogen gamma chain | Fgg | 6.4 | 2.96E + 00 |
| M0RBK4 | Uncharacterized protein | 6.4 | 3.15E + 00 | |
| E0A3N4 | Serpina3n-like protein | 6.3 | 1.95E + 00 | |
| A0A0G2K4C9 | Prostatic spermine-binding protein | Sbp | 6.1 | 2.05E + 00 |
| E9MW47 | Immune CD300 receptor | Cd300le | 5.8 | 1.69E + 00 |
| A0A0G2JSH5 | Serum albumin | Alb | 5.8 | 5.92E + 00 |
| P12346 | Serotransferrin | Tf | 5.7 | 4.34E + 00 |
| G3V9M0 | Cystatin | Cyssl1 | 5.5 | 3.15E + 00 |
| A0A0G2K7I1 | Uncharacterized protein | 5.4 | 5.18E + 00 | |
| A0A0G2K6T8 | Uncharacterized protein | 5.4 | 2.91E + 00 | |
| A0A0G2K926 | Murinoglobulin-1 | LOC297568 | 5.1 | 4.22E + 00 |
| P20767 | Ig lambda-2 chain C region | 5.1 | 3.84E + 00 | |
| P04276 | Vitamin D-binding protein | Gc | 5.1 | 2.48E + 00 |
| P36953 | Afamin | Afm | 4.8 | 1.29E + 00 |
| A0A0G2K4K2 | Uncharacterized protein | 4.7 | 1.83E + 00 | |
| F1LTN6 | Uncharacterized protein | 4.7 | 3.76E + 00 | |
| M0RD98 | Uncharacterized protein | 4.5 | 3.07E + 00 | |
| P83121 | Urinary protein 3 | 4.5 | 4.05E + 00 | |
| Q9JJI4 | Alpha-2u globulin | Mup4l1 | 4.3 | 3.06E + 00 |
| G3V8G8 | Kallikrein 6 (Predicted) | Klk1c8 | 4.3 | 3.61E + 00 |
| Q4VBH1 | Ighg protein | Ighg | 4.1 | 1.25E + 00 |
| P02770 | Serum albumin | Alb | 4.0 | 3.41E + 00 |
| D3ZQR5 | Uncharacterized protein | 4.0 | 4.84E + 00 | |
| Q63430 | Cold shock domain-containing protein C2 | Csdc2 | 3.9 | 1.95E + 00 |
| G3V8H1 | Kallikrein 1-related peptidase B3 | Klk1 | 3.8 | 1.71E + 00 |
| P01015 | Angiotensinogen | Agt | 3.6 | 1.99E + 00 |
| F1LPG1 | Uncharacterized protein | 3.4 | 3.48E + 00 | |
| Q5I0L0 | Alpha-amylase | Amy1a | 3.3 | 4.20E + 00 |
| M0RBF1 | Complement C3 | C3 | 3.2 | 3.46E + 00 |
| A0A0G2JXF0 | Uncharacterized protein | 3.1 | 2.13E + 00 | |
| P21704 | Deoxyribonuclease-1 | Dnase1 | 3.0 | 2.72E + 00 |
| Q63530 | Phosphotriesterase-related protein | Pter | 3.0 | 1.67E + 00 |
| P15399 | Probasin | Pbsn | 3.0 | 3.47E + 00 |
| P05545 | Serine protease inhibitor A3K | Serpina3k | 2.9 | 1.75E + 00 |
| H6X320 | Pentaxin | Apcs | 2.9 | 2.52E + 00 |
| A0A0G2JSP1 | Uromodulin | 2.7 | 3.45E + 00 | |
| A0A0G2JUY4 | Uncharacterized protein | 2.7 | 2.54E + 00 | |
| P02780 | Secretoglobin family 2 A member 2 | Scgb2a2 | 2.6 | 4.63E + 00 |
| A0A0G2JV65 | 14–3-3 protein zeta/delta | 2.5 | 1.81E + 00 | |
| Q5M7V3 | LOC367586 protein | LOC367586 | 2.4 | 2.70E + 00 |
| A0A1W2Q6M4 | Uncharacterized LOC103691699 (Fragment) | 2.3 | 3.86E + 00 | |
| Q63015 | Common salivary protein 1 | Csap1 | 2.1 | 1.80E + 00 |
| Q6IE67 | Proteasome subunit alpha type | LOC100361067 | 2.0 | 1.13E + 00 |
| Downregulated proteins | ||||
| D3ZUQ1 | Lipase | Lipo1 | −8.4 | 2.61E + 00 |
| P07314 | Glutathione hydrolase 1 proenzyme | Ggt1 | −7.1 | 2.10E + 00 |
| Q64319 | Neutral and basic amino acid transport protein rBAT | Slc3a1 | −7.0 | 3.16E + 00 |
| A0A0H2UHX5 | Neprilysin | Mme | −7.0 | 2.34E + 00 |
| P10111 | Peptidyl-prolyl cis–trans isomerase A | Ppia | −5.9 | 3.01E + 00 |
| Q9QZT0 | CUB and zona pellucida-like domain-containing protein 1 | Cuzd1 | −5.5 | 1.64E + 00 |
| D3Z9U8 | S100 calcium-binding protein A7-like 2 | S100a7l2 | −5.4 | 2.10E + 00 |
| F1M9X2 | Pancreatic secretory granule membrane major glycoprotein GP2 | Gp2 | −4.6 | 1.07E + 00 |
| Q05175 | Brain acid soluble protein 1 | Basp1 | −4.2 | 1.86E + 00 |
| A0A0G2K1A2 | Myeloperoxidase | −4.2 | 2.93E + 00 | |
| P31977 | Ezrin | Ezr | −4.1 | 1.87E + 00 |
| Q4QQV8 | Charged multivesicular body protein 5 | Chmp5 | −3.1 | 2.85E + 00 |
| Q91XN5 | Prominin 1 | Prom1 | −2.9 | 2.18E + 00 |
| P15684 | Aminopeptidase N | Anpep | −2.7 | 1.54E + 00 |
| P63259 | Actin, cytoplasmic 2 | Actg1 | −2.4 | 1.69E + 00 |
| F1MAE5 | Uncharacterized protein | −2.3 | 2.59E + 00 | |
| Q45QL6 | Guanine nucleotide binding protein beta 2 (Fragment) | Gnb2 | −2.1 | 1.22E + 00 |
| P50123 | Glutamyl aminopeptidase | Enpep | −1.9 | 1.59E + 00 |
| D3ZS19 | Alpha-2-macroglobulin-like 1 | −1.6 | 1.32E + 00 | |
| P0DP31 | Calmodulin-3 | Calm3 | −1.1 | 1.78E + 00 |
Log2FC TvsVC represents the fold change in protein abundance in high-dose OTA-induced sEVs relative to vehicle control-derived sEVs Significantly deregulated proteins were identified if the adjusted p-value between experimental groups was ≤ 0.05 and the Log2FC was at least 1
Annexin A2 was enriched in sEVs derived from NRK52E cells and rat urine following OTA exposure
Our proteomics analysis identified several deregulated sEV-associated proteins, predominantly upregulated after OTA exposure in both in vitro and in vivo conditions. Among the significantly upregulated proteins, annexin A2 and fibrinogen-ɣ were the only two proteins consistently detected in sEVs derived from OTA-exposed NRK52E cells and OTA-exposed rats. These findings suggested their potential involvement in OTA-mediated kidney toxicity. To validate the proteomic study results, we performed immunoblotting to analyze the expression pattern of annexin A2 and fibrinogen-ɣ. As shown in Fig. 9A-C & Supplementary Fig. 7 A, annexin A2 levels were significantly increased in OTA-exposed cells, sEVs, and conditioned media in a concentration-dependent manner. However, fibrinogen-ɣ was undetected in sEVs, ruling out its potential involvement in OTA-mediated toxicity under these conditions. To further assess whether OTA influences annexin A2 expression in treated cells over very short exposure, we analyzed NRK52E cells exposed to OTA for 48 h. Furthermore, we also assessed annexin A2 expression in 48 h OTA-exposed HK-2 cells. Notably, annexin A2 expression was upregulated in both cell lines compared to control cells (Supplementary Fig. 7B and C). Moreover, the expression of annexin A2 was higher in OTA-induced rat urinary sEVs, while fibrinogen-γ expression remained unchanged (Fig. 9D and Supplementary Fig. 7D). Similarly, its expression was also increased in OTA-induced rat urine (Fig. 9E and Supplementary Fig. 7D). Tissue-level analysis of annexin A2 expression in OTA-exposed rat kidney tissues resembled the trend observed in cells and sEVs (Fig. 9F and Supplementary Fig. 7D). Additionally, immunohistochemical analysis showed the expression of annexin A2 being increased in OTA-exposed rat kidney tissue, specific to the glomerulus and intratubular space (Fig. 9G and Supplementary Fig. 7E). Further, to validate the expression of annexin A2 in other renal fibrosis models, we have established a folic acid-induced renal fibrosis model. Body weight was increased at day 7 and restored at day 14, compared to vehicle control (Supplementary Fig. 8 A). Subsequently, urine volume was substantially elevated at day 7 (and partially returned to normal by day 14), indicating that at least some aspects of renal function remained (at least transiently) abnormal (Supplementary Fig. 8B). An acute change in renal function, as determined by an increase in serum creatinine (SCr) and urea (SUrea) as well as urinary albumin was detected after FA injection (Supplementary Fig. 8C-E). EVs were isolated from collected urine at day 14, and the expression of annexin A2 was checked using immunoblotting. As shown in Fig. 9H and Supplementary Fig. 8 F, expression of annexin A2 was significantly increased in the FA-injected group, compared to the vehicle control, indicating the association between sEVs-associated annexin A2 upregulation and kidney toxicity.
Fig. 9.
Chronic exposure to OTA induced the expression and loading of annexin A2. A-C Immunoblot analysis of the expression of annexin A2 in OTA-exposed normal kidney cells (A), from derived sEVs (B) and from conditioned media (C). (D-F) Immunoblotting of annexin A2 in urinary sEVs (D), urine (E), and kidney tissue of OTA-exposed rats (F). (G) Immunohistochemical analysis representing the expression of annexin A2 in the OTA-exposed kidney section. Brown colour represents the expression of annexin A2. (H) The expression of annexin A2 in folic acid-exposed urinary sEVs. For sEVs-associated annexin A2, TSG-101 expression was used for normalization and annexin A2 expression in cells, tissue or urinary annexin A2, β-actin expression was used for normalization. Numbers (1–3) indicate each animal in a given group. Ctrl = vehicle-exposed NRK52E cells, LD = Low Dose (OTA-250 nM), HD = High Dose (OTA-500 nM), CM = conditioned Media, CM-sEVs = conditioned media-derived sEVs, V-ctrl or VC = vehicle-exposed rats, OTA = OTA-exposed rats, V-ctrl (sEVs) = urinary sEVs of vehicle-exposed rats, FA (sEVs) = urinary sEVs of folic acid-exposed rats, and N-ctrl = negative control
Annexin A2-enriched sEVs contributed to OTA-induced toxicity in NRK52E cells
Annexin A2 was the common protein significantly expressed in sEVs derived from OTA-exposed NRK52E cells and rat urine. But does annexin A2 really play a crucial role in OTA-mediated kidney toxicity? To answer this question, first, we silenced annexin A2 in OTA-exposed NRK52E cells. Silencing of annexin A2 did not cause any significant changes in the morphology and viability of NRK52E cells (Supplementary Fig. 9A-B). Interestingly, expression of injury markers was significantly decreased after annexin A2 silencing in both control and OTA-exposed NRK52E cells (Fig. 10A-C). In contrast, the expression of kidney injury markers was increased after annexin A2 overexpression in NRK52E cells (Fig. 10D-F).
Fig. 10.
Annexin A2 contributed to OTA-induced toxicity in NRK52E cells. A Representative immunoblot images of kidney injury markers in NRK52E cells after knockdown. B-C The bar graph represents the level of kidney injury markers in control (B) and OTA-exposed NRK52E cells (C) after annexin A2 knockdown. β-actin expression was used for normalization. (D) The expression of kidney injury markers in annexin A2 overexpressing NRK52E cells. (E–F) The bar graph showing the level of kidney injury markers in control (E) and OTA-exposed NRK52E cells (F) after annexin A2 overexpression. β-actin expression was used for normalization. CTL = vehicle-treated NRK52E cells, CTL + Scr = scrambled-treated NRK52E cells, HD + Scr = scrambled-treated four-month OTA-exposed NRK52E cells, HD + siAnx = annexin A2 siRNA-treated four-month OTA-exposed NRK52E cells. CTL + vector = control NRK52E cells transfected with empty plasmid, CTL + OE = control NRK52E cells transfected with Anxa2-GFP encoding plasmid, HD + vector = four-month OTA-exposed NRK52E cells transfected with empty vector, HD + OE = four-month OTA-exposed NRK52E cells transfected with Anxa2-GFP encoding plasmid
Further, to check the role of sEV-encapsulated annexin A2 in OTA-mediated kidney toxicity, we isolated sEVs derived from annexin A2-silenced OTA-exposed NRK52E cells and subsequently treated NRK52E cells. The level of annexin A2 in knockdown NRK52E cells-derived sEVs was also decreased (Fig. 11A). Furthermore, silencing of annexin A2 alleviated the ability of OTA-induced sEVs to induce injury markers in NRK52E cells. In contrast, OTA-induced sEVs derived from scrambled-treated cells retained the capacity to elicit toxicity in NRK52E cells (Fig. 11B-C). To further confirm the role of sEV-associated annexin A2 in OTA-induced kidney toxicity, we overexpressed annexin A2 in NRK52E cells. The level of annexin A2 was increased in sEVs derived from overexpressing cells (Fig. 11D-E). Notably, treatment of these sEVs markedly enhanced the expression of kidney injury markers in NRK52E cells (Fig. 11F-G). Collectively, as illustrated in Fig. 12, OTA treatment caused toxicity in tubular epithelial cells or kidney fibrosis-like alterations via increased secretion of sEVs as well as the sEVs-mediated shuttling of proteins, specifically annexin A2.
Fig. 11.
sEV-associated annexin A2 plays a major role in OTA-induced toxicity in NRK52E cells. A Expression of annexin A2 in silenced NRK52E cells-derived sEVs. An equal amount of sEV particles was lysed and loaded onto an SDS-PAGE gel. Ponceau staining was used for the loading control. B-C Immunoblotting of kidney injury markers in NRK52E cells treated with sEVs derived from knockdown cells. β-actin expression was used for normalization. D-E The expression of annexin A2 in Anxa2-GFP overexpression plasmid-transfected cells. TSG-101 expression was used for normalization. F Representative immunoblotting depicting the expression of kidney injury markers in NRK52E cells treated with sEVs of overexpressing cells. β-actin or α-tubulin expression was used for normalization. C-sEVs = NRK52E cells treated with sEVs derived from vehicle-treated NRK52E cells, Scr-sEVs = NRK52E cells treated with sEVs of scrambled-treated chronic OTA-exposed NRK52E cells, KD-sEVs = NRK52E cells treated with sEVs of annexin A2 siRNA-treated chronic OTA-exposed NRK52E cells, Vector = empty plasmid-transfected NRK52E cells, OE = Anxa2-GFP encoding plasmid-transfected NRK52E cells, Vec-sEVs = sEVs of empty plasmid-transfected NRK52E cells, OE-sEVs = sEVs of Anxa2-GFP encoding plasmid-transfected NRK52E cells
Fig. 12.
Schematic representation of the potential role of annexin A2-encapsulated sEVs in OTA-induced kidney toxicity in both in vivo and in vitro
Discussion
OTA is a naturally occurring food contaminant, globally recognized for its ability to cause kidney toxicity [11]. It causes glomeruli shrinkage, tubular degeneration, extracellular matrix production (ECM), and ultimately leads to fibrosis in the rat kidney [14]. Although the nephrotoxic effects of OTA are well-established, its mode of action remains elusive. To this end, Hou et al. showed that OTA decreased the viability of human kidney proximal tubule cells (HK-2) in a dose-dependent manner, with significant cytotoxic effects observed at a concentration of 1 µg/ml [31]. In agreement with these findings, the current study also showed that low-dose exposure of OTA (250 and 500 nM) did not cause any cytotoxicity to NRK52E cells. However, above these concentrations (≥ 1 µM), viability was significantly compromised. This phenomenon might have occurred due to the activation of the survival response of the cells against 250 nM and 500 nM concentrations of OTA, which ultimately leads to enhanced proliferation, while higher concentrations of OTA overwhelmed these protective responses, leading to reduced cell viability and increased toxicity. Moreover, these OTA concentrations were found to increase the expression of kidney injury markers like albumin, NGAL, clusterin, cystatin C, and TIMP-1 in NRK52E cells in a dose- and time-dependent manner without affecting viability. In addition, 500 nM OTA concentration increased survivability as well as the expression of kidney injury markers in HK-2 cells, also supporting the findings found in NRK52E cells. Further, OTA-exposed NRK52E cells-derived sEVs also increased kidney injury markers in HK-2 cells, indicating that there is likely something inside sEVs which may be responsible for toxicity. In this case, upregulation of these kidney toxicity markers indicates an early stress response that occurred prior to overt cytotoxicity or cell death. In agreement, Pyo et al. demonstrated that a 200 nM concentration of OTA activates various signaling cascades like apoptosis, epithelial-mesenchymal transition (EMT), and injury in HK-2 cells without affecting its viability [32]. However, it is critical to find the mechanism by which this toxicity was propagated after OTA exposure.
In recent years, EVs have gained attention for their potential to deliver various stress-responsive signals to neighbouring cells and propagate various toxicities in response to environmental pollutants, acting as a carrier for stress signals, inflammatory mediators, and even toxic cargo [33, 34]. In this context, Ngalame et al. revealed that arsenic-treated prostate epithelial cells recruit prostate stem cells into a cancer-like stem cell phenotype and secrete more exosomes compared to untreated cells [35]. In the case of OTA, two earlier studies have also shown that OTA exposure induces sEVs production, and those sEVs would be responsible for mediating the kidney toxicity [14, 36]. However, the mechanistic aspects by which OTA-induced sEVs contribute to toxicity have not yet been explored. Accordingly, our study explored the finding that 500 nM OTA concentration increased the secretion of sEVs from NRK52E cells. Notably, 250 nM OTA concentration increased the expression of the sEV-associated markers without enhancing sEV release from NRK52E cells, indicating a qualitative alteration in sEVs following two-month OTA exposure. In contrast, both qualitative and quantitative changes were observed in sEVs of four-month OTA-exposed NRK52E cells. Moreover, these sEVs increased the expression of kidney injury markers as found in chronic OTA-exposed NRK52E cells. In contrast, DMA-mediated inhibition of sEV release markedly alleviated this toxicity. In support of this notion, Liu et al. showed that exosomes derived from TGF-β1-treated tubular epithelial cells increase kidney interstitial fibrosis by activating fibroblasts, whereas inhibition of exosome secretion by DMA significantly attenuates fibrosis by activating fibroblast apoptosis [37]. Collectively, these findings suggest a potential role of sEVs derived from four-month OTA-induced NRK52E cells in mediating OTA-induced kidney toxicity. However, as HD-sEVs did not fully recapitulate the toxic effects of direct OTA exposure, the involvement of additional factors, including non-vesicular secreted components (like growth factors, cytokines, and other secreted proteins), may also contribute to the observed toxic manifestation in NRK52E cells.
Next, to check the physiological relevance of in vitro findings, the functions of OTA-induced sEVs in the in vivo system were explored. After exogenous injection of OTA-induced sEVs into Wistar rats, hallmarks of tubulointerstitial fibrosis, such as high collagen and glycogen deposition, increased expression of fibronectin, α-SMA, and other injury markers, were seen in kidney tissues. Similar to our findings, a prior study reported that exosomes derived from high glucose-exposed macrophages increased the expression of α-SMA, collagen-IV, fibronectin, and inflammatory cytokines in C57BL/6 mice [38]. In addition, Liu et al. showed that blockade of exosome secretion by DMA attenuates kidney fibrosis in Unilateral Ureteral Obstruction (UUO) and unilateral ischemia–reperfusion injury (UIRI) models [39]. In line with this finding, DMA-mediated reduction of sEV secretion in OTA-exposed rats markedly reduced kidney toxicity. However, DMA inhibits sEV secretion indirectly by inhibiting Na +/H + and Na +/Ca2 + exchange rather than through a specific blockade of the sEV secretion pathway; therefore, the possibility of off-target effects cannot be completely excluded. Future studies employing more specific approaches for sEV secretion, such as silencing of Rab27a/b, will be valuable to further strengthen the contribution of sEV-mediated OTA-induced kidney toxicity [40].
EVs secreted from proximal tubular epithelial cells have a strong kidney tropism in their tissue distribution. Liu et al. showed that EVs derived from TGFβ1-treated HK2 cells were deposited in both the kidney and liver following tail vein injection [10]. Interestingly, a similar organotropism is observed with OTA-induced sEVs. Following intravenous administration of these sEVs to Wistar rats, significant kidney-specific toxicity was observed, whereas no pathological or biochemical alterations were detected in other vital organs such as the liver, lungs, spleen, or heart. Furthermore, live imaging confirmed preferential accumulation of PKH26-labelled sEVs in the kidney, further supporting their kidney tropism and highlighting their role in targeted kidney toxicity. Although we assessed the biodistribution of OTA-induced sEVs, control sEVs were not included in the in vivo live imaging experiments. Therefore, further studies are needed to determine whether OTA-induced sEVs exhibit enhanced kidney tropism compared with control sEVs and to better define the organ-specific distribution of these vesicles.
It is well-known that cargo/s of EVs play a crucial role in mediating their toxic manifestations. Previous reports indicated that environmental factors such as heavy metals, pesticides, and toxic chemicals alter the EV cargo molecules, including mRNAs, proteins, and lipids, that further contribute to inflammatory diseases and disorders [13, 41]. Therefore, to identify the differentially expressed proteins in OTA-induced sEVs, proteomics was performed. Notably, we are the first to report that OTA significantly increased the number of proteins in sEVs derived from conditioned media of OTA-exposed NRK52E cells and urine of OTA-treated rats; most of the sEV-associated upregulated proteins (such as insulin-like growth factor 2, fibronectin, collagen 1, clusterin, cystatin C, septin-7/11, annexin A2, etc.) were found to be involved in the promotion or progression of kidney toxicity. In support of our findings, a study by Wu et al. also found similar results where high glucose-treated glomerular endothelial cells (GEC) release more exosomes that are highly enriched with TGFβ1 mRNA, which is involved in the impairment of podocytes [42]. Among various sEV-associated differentially expressed proteins, annexin A2 was the only common deregulated protein, which was found to be markedly upregulated in sEVs derived from OTA-exposed NRK52E cells and rat urine. The consistent upregulation of annexin A2 in both in vitro and in vivo scenarios suggest its probable association with the promotion and progression of OTA-caused kidney toxicity. Supportingly, Lin et al. showed that in the UUO model, annexin A2 mediates tissue plasminogen activator (tPA)-induced macrophage NF-κB activation, leading to sustained interstitial inflammation and fibrosis [43]. Building on this finding, to confirm the association between annexin A2 upregulation and OTA-induced kidney toxicity, we silenced annexin A2 from OTA-exposed NRK52E cells. Interestingly, the absence of annexin A2 significantly attenuated OTA-mediated toxicity in NRK52E cells. Notably, reduced expression of kidney injury markers was also observed in control NRK52E cells after annexin A2 silencing, without any significant changes in cellular morphology and viability, suggesting that annexin A2 may have a role in maintaining the basal level of stress markers (B2M, clusterin, and fibronectin), even in the absence of OTA exposure. Furthermore, increased expression of kidney injury markers after overexpression of annexin A2 confirms its contributory role in OTA-induced kidney toxicity. In addition, sEVs lacking annexin A2 exhibited a markedly reduced ability to induce kidney toxicity, whereas overexpression of annexin A2 in sEVs significantly induced the expression of kidney toxicity markers, confirming the role of sEV-encapsulated annexin A2 in kidney toxicity. Notably, the silencing of annexin A2 partially attenuated the OTA-induced kidney toxicity, suggesting that it is not the sole mediator of toxicity. The proteomic analysis revealed multiple differentially expressed proteins in OTA-induced sEVs, indicating that additional proteins may also participate in this pathogenesis. In addition, extracellular OTA may co-isolate during HD-sEV isolation, which may further contribute to the observed effects. Therefore, OTA-induced kidney toxicity is likely a multifactorial process mediated by a combinatorial effect of multiple sEV cargo proteins and sEV-associated OTA rather than a single effector molecule, warranting further studies.
To further delineate whether annexin A2 was specific to OTA-caused kidney toxicity or could be upregulated in kidney toxicity caused by other chemicals or injury, the level of annexin A2 was validated in the folic acid-induced kidney toxicity model. Interestingly, levels of annexin A2 were also high in urinary sEVs isolated from folic acid-treated rats. Consistent with our findings, a study by Rattanasinganchan et al. also found that folic acid-exposed rat urinary EVs have higher expression levels of different annexins, such as annexin-A11, A4, and A2 [26]. Based on these findings, it is speculated that annexin A2 could be a robust and early-response sEV-associated protein that may serve as a promising biomarker for the early detection of kidney problems across different pathological contexts. The upregulation of annexin A2 may be a key contributor in promoting inflammatory and fibrotic responses observed in OTA-induced kidney toxicity.
Conclusions
In summary, our findings demonstrate that OTA exposure upregulates the expression of annexin A2 inside sEVs, which potentially contributes to kidney toxicity, as evidenced in both in vitro and in vivo models. However, the exact mechanisms by which OTA-induced annexin A2 enrichment into sEVs and how sEV-associated annexin A2 subsequently mediates kidney toxicity remain to be elucidated. Further studies are warranted to determine whether annexin A2 functions independently or in concert with other sEV-associated cargo, and to identify the specific signaling pathways involved in OTA-induced, sEV-mediated kidney injury. Our findings suggest that annexin A2 could serve as a potential early biomarker for kidney disease. Nonetheless, validation using human samples is essential to establish its clinical relevance and utility for early diagnosis and monitoring of kidney injury. Furthermore, investigations using primary renal epithelial cultures are necessary to better reflect the in vivo conditions and strengthen the translational relevance of these findings, as the NRK52E cell line is transformed and may not fully recapitulate the physiological characteristics of normal renal epithelial cells. A limitation of this study is the use of chronically OTA-exposed NRK52E-derived sEVs in HK-2 recipient cells rather than chronically OTA-exposed HK-2-derived sEVs. Future studies establishing a chronic OTA-treated HK-2 model and investigating the role of OTA-induced HK-2 cells-derived sEVs in kidney toxicity will provide a greater translational relevance. In addition, this study has a relatively small sample size in the in vivo experiments, which may constrain the statistical strength of the findings.
Supplementary Information
Acknowledgements
Sourin Adhikary (S.A.) is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi for the award of a Senior Research Fellowship. Technical support of Mr. Jay Shankar for TEM, Mrs. Nidhi Arjaria for confocal microscopy. Ms. Saria Anjum, Mrs. Deepshikha, and Mr. Sagar for LC-MS/MS analysis is also acknowledged. I also thankful to Ms. Tazeem Fatima for helping to perform in vivo imaging. We are grateful to former Professor Susan M. Fischer, MD Anderson Cancer Center, Texas, USA, for critically reading the manuscript and providing editorial assistance. This research work is part of the PhD thesis of SA. The manuscript's CSIR-IITR communication number is IITR/SECC-PME/MSS/2025/033.
Abbreviations
- Anxa2
Annexin A2
- ALT
Alanine transaminase
- AST
Aspartate transaminase
- b.wt
Body weight
- CD9
Cluster of differentiation 9
- CIN
Chronic interstitial nephropathy
- CKDu
Chronic kidney disease with unknown etiology
- DMA
Dimethyl amiloride
- DMSO
Dimethyl sulfoxide
- DPX
Dibutylphthalate Polystyrene Xylene
- EVs
Extracellular vesicles
- EDTA
Ethylenediaminetetraacetic acid
- EGTA
Ethylene glycol tetraacetic acid
- FA
Folic acid
- FITC
Fluorescein isothiocyanate
- GFR
Glomerular filtration rate
- H&E
Haematoxylin and eosin
- IHC
Immunohistochemistry
- MTS
Masson’s trichrome stain
- MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- NGAL
Neutrophil gelatinase-associated lipocalin
- OTA
Ochratoxin A
- PAS
Periodic acid–Schiff
- PMSF
Phenylmethylsulfonyl Fluoride
- RIPA
Radioimmunoprecipitation assay
- sEVs
Small extracellular vesicles
Authors’ contributions
S.A. contributed to conceptualization, experimentation, data acquisition, data analysis and interpretation, manuscript writing and editing. I.D. quantified and characterized sEVs isolated from OTA-exposed NRK52E cells. A.R. performed proteomics analysis. P.R.J. contributed to histopathological staining of five vital organs. S.S. assessed the GFR of OTA-exposed or sEVs-injected rats. A.A. contributed to histopathological examination of five vital organs. K.M.A. helped in conceptualization, manuscript editing, and facilitated the study by providing necessary experimental tools and materials.
Funding
This work was financially supported by Anusandhan National Research Foundation (ANRF), ref. no. CRG/2022/003866.
Data availability
Raw files of two independent proteomics study (project) have been submitted to PRoteomics IDEntifications (PRIDE) Database (Perez-Riverol Y et al. 2025). The allocated accession no. for the first project, entitled “Proteomics analysis of culture media-derived extracellular vesicles isolated from OTA-exposed NRK52E cells” is PXD064968 (Token: u5d6UicXKpio) and for the second project, “Proteomics analysis of urinary EV-proteins derived from OTA-induced rat” is PXD064936 (Token: SMzOB4I0SY5v).
Declarations
Ethics approval and consent to participate
Animals were sourced from the in-house breeding facility of the CSIR-Indian Institute of Toxicology Research (IITR), Lucknow, India. The study was carried out as per the guidelines established by the Institutional Animal Ethics Committee (IAEC) of CSIR-IITR (reference number IITR/IAEC/07/23–58/24) and approved under the authorization of the Committee for Control and Supervision of Experiments on Animals (CCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, Government of India.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Raw files of two independent proteomics study (project) have been submitted to PRoteomics IDEntifications (PRIDE) Database (Perez-Riverol Y et al. 2025). The allocated accession no. for the first project, entitled “Proteomics analysis of culture media-derived extracellular vesicles isolated from OTA-exposed NRK52E cells” is PXD064968 (Token: u5d6UicXKpio) and for the second project, “Proteomics analysis of urinary EV-proteins derived from OTA-induced rat” is PXD064936 (Token: SMzOB4I0SY5v).