Tungsten toxicity on kidney tubular epithelial cells induces renal inflammation and M1-macrophage polarization

Tungsten is widely used in medical, industrial, and military applications. The environmental exposure to tungsten has increased over the past several years, and few studies have addressed its potential toxicity. In this study, we evaluated the effects of chronic oral tungsten exposure (100 ppm) on renal inflammation in male mice. We found that 30- or 90-day tungsten exposure led to the accumulation of LAMP1-positive lysosomes in renal tubular epithelial cells. In addition, the kidneys of mice exposed to tungsten showed interstitial infiltration of leukocytes, myeloid cells, and macrophages together with increased levels of proinflammatory cytokines and p50/p65-NFkB subunits. In proximal tubule epithelial cells (HK-2) in vitro, tungsten induced a similar inflammatory status characterized by increased mRNA levels of CSF1, IL34, CXCL2, and CXCL10 and NFkB activation. Moreover, tungsten exposure reduced HK-2 cell viability and enhanced reactive oxygen species generation. Conditioned media from HK-2 cells treated with tungsten induced an M1-proinflammatory polarization of RAW macrophages as evidenced by increased levels of iNOS and interleukin-6 and decreased levels of the M2-antiinflammatory marker CD206. These effects were not observed when RAW cells were exposed to conditioned media from HK-2 cells treated with tungsten and supplemented with the antioxidant N-acetylcysteine (NAC). Similarly, direct tungsten exposure induced M1-proinflammatory polarization of RAW cells that was prevented by NAC co-treatment. Altogether, our data suggest that prolonged tungsten exposure leads to oxidative injury in the kidney ultimately leading to chronic renal inflammation characterized by a proinflammatory status in kidney tubular epithelial cells and immune cell infiltration.


Introduction
Exposure to pollutants including metals has been recognized as a risk factor for chronic kidney disease (CKD) development (Orr and Bridges 2017;Tsai et al. 2021).Tungsten (W, atomic number 74) is a naturally occurring element possessing exceptional industrial features such as having the highest melting point, high density, flexibility, tensile strength, and conductivity.Therefore, it is widely used in the manufacturing of electronics, implanted medical devices, tools, munitions, and other industrial and military applications (Bolt and Mann 2016;Lemus and Venezia 2015;Yu et al. 2023).Owing to its broad use, occupational and environmental exposure to tungsten is increasing, and it has been detected in soil, ground sources, and potable water (Du et al. 2022;Keith et al. 2007;Shi et al. 2022).In the clinical setting, it has been reported that patients exposed to treatments based on tungsten display high levels of tungsten in the blood and urine (Domingo 2002;Lalak and Moussa 2002;Tajima 2001Tajima , 2003)).Notably, it appears that tungsten can bioaccumulate as evidenced in a cohort of breast cancer patients with a tungsten-based shield during intraoperative radiotherapy in which tungsten was detectable in their urine following several months postsurgery (Bolt et al. 2015).Emerging evidence shows that tungsten may lead to toxic effects in organs such as the lungs, kidneys, bones, and intervertebral disks; however, more research is needed to fully understand the potential health risks of chronic tungsten exposure (Grant et al. 2022;Grant et al. 2021;Miller et al. 2021).
Once tungsten enters the body, it is excreted mainly through the kidneys and gut, but it also accumulates in the bone, spleen, colon, liver, brain, intervertebral disk, and the kidneys (Grant et al. 2021;Guandalini et al. 2011).The kidneys are vulnerable since they are not only a site of bioaccumulation but are also responsible for tungsten excretion.Kidneys are particularly susceptible since tungsten induces mitochondrial dysfunction and oxidative injury in renal mitochondria (Cheraghi et al. 2019).Only a few studies have evaluated the potential kidney toxic effects of chronic tungsten exposure.In the rat, daily gavage of tungsten for 90 days produced mild to severe basophilia in renal cortical tubules of male and female rats at doses of 125 and 200 mg/kg/day (McCain et al. 2015).Moreover, exposure of male rats to 500 ppm of tungsten for 28 days leads to increased reactive oxygen species (ROS) generation and lipid peroxidation (Sachdeva et al. 2022).We recently showed that chronic oral exposure to tungsten in male mice for 1 to 3 months induced fibrotic tissue accumulation in the kidneys accompanied by increased levels of myofibroblast markers, extracellular matrix, and matricellular proteins (Grant et al. 2022).In addition, we observed vacuole formation in renal tubular epithelial cells following tungsten exposure; these alterations were associated with a reduction in the glomerular filtration rate, indicating that tungsten nephrotoxicity may predispose to CKD (Grant et al. 2022).The notion that tungsten exposure contributes to CKD development is supported by one study in the population from San Luis Valley in Colorado, in which it was found that higher urinary tungsten concentrations were associated with decreased time to CKD, independently of diabetes or hypertension.Moreover, doubling tungsten concentrations in the urine were associated with a 27-31% higher risk of developing CKD within 5 years (Fox et al. 2021).
Some studies in male rodents have shown that tungsten can induce hepatic and pulmonary damage mainly by producing oxidative injury and increasing the levels of inflammatory cytokines and macrophage activation (Armstead and Li 2016;Mao et al. 2021;Roedel et al. 2012).Tungsten hepatotoxicity was characterized in mice in which intraperitoneal injection of 10 or 20 mg/kg of tungsten trioxide nanoparticles for 14 days induced liver injury and increased ROS accumulation and proinflammatory mediators including NFκB, IFNγ, TNF-α, and IL4.Of note, melatonin showed a protective effect against tungsten-induced alterations through reducing oxidative stress (Mao et al. 2021).In the lung, intratracheal instillation of 1 to 4 mg of a heavy metal mixture consisting of at least 92% tungsten and analysis after 24 h in rats revealed pulmonary inflammation and increased ROS generation (Roedel et al. 2012).To further explore the nephrotoxic effects of chronic tungsten exposure, in this study, we investigated the effect of orally administered tungsten on renal inflammatory changes in male mice and whether oxidative stress is the main mechanism leading to these alterations.

Animal model
We used male C57BL/6J mice purchased from The Jackson Laboratory (USA).The mice were housed Vol.: (0123456789) within the Maisonneuve-Rosemont Hospital Research Center animal facility and fed Harlan Teklad rodent diet (#2018 Envigo, Canada) and water ad libitum.All experiments were conducted according to the Canadian Council on Animal Care guidelines for the care and use of laboratory animals and under the supervision and approval of our local animal care committee, Comité de protection des animaux du Centre Intégré Universitaire de Santé et de Services Sociaux (CIUSSS) de l 'Est-de-l'île-de-Montréal (Approved Protocol No. 2018-1261), in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) Guidelines.At 5 weeks of age, twentyfour mice were divided into two treatment groups: (1) control (CTL) group receiving acidified water (12 mL 5 N hydrochloric acid (HCl) in 20 L water) or (2) sodium tungsten (NaW) group consisting of mice receiving 100 ppm of tungsten in their drinking water for 1 or 3 months as previously reported (Grant et al. 2022) (n = 6 per treatment group/time point).Sodium tungstate dihydrate (Na 2 WO 4 •2H 2 O; Sigma-Aldrich) was dissolved in acidified water (1.79 g Na 2 WO 4 •2H 2 O:1 g W) and was replaced every 2 or 3 days to limit conversion to polytungstates (Kelly et al. 2013).Following 1 or 3 months of treatment, the mice were euthanized under isoflurane anesthesia, and the kidneys were collected.A portion of the kidney was further processed for histological analysis, and the other portions were flash-frozen in liquid nitrogen for quantitative polymerase chain reaction (qPCR) and western blot analyses.

Immunofluorescence
The kidneys were fixed in 10% formaldehyde, dehydrated, and embedded in paraffin.The tissue was sectioned (5 μM) and was subjected to antigen retrieval in citrate solution at pH 6.The sections were blocked with anti-donkey serum 5% and labeled with anti-LAMP1 (1:200; Cell signaling), anti-CD45-FITC (1:200; BioLegend), anti-CD11b-FITC (1:200; Bio-Legend), and anti-F4/80-AF647 (1:100; BioLegend).For LAMP1, the slides were subsequently exposed to donkey anti-rabbit AF647-conjugated (1:400 Jackson ImmunoResearch Laboratories).Fluoroshield with DAPI (Millipore-Sigma) was used for nuclear staining and mounting.Slides were imaged using a Zeiss AxioObserver.Z1 inverted microscope coupled to an X-Cite 120LED Boost High-Power LED illumination system.Images for quantitative analysis were captured with a 20× objective, and the number of positive cells was determined as the average of positive cells in at least 8 fields per kidney section.

Cell culture
The human proximal tubule cell line HK-2 (derived from male normal kidney) and the RAW 264.7 murine macrophage cell line (derived from male mouse) were obtained from American Type Culture Collection (ATCC, USA) and maintained in a humidified atmosphere of 5% CO 2 at 37°C with Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) (Gibco/ Life Technologies, USA) or DMEM supplemented with 10% FBS, respectively.For tungsten treatment, HK-2 or RAW cells were seeded in 6-well plates and allowed to adhere for 24 h.After the 24-h adhesion time, the media was replaced with DMEM containing tungsten to a final concentration of 100 ppm for 48 to 216 h depending on the assay.Tungsten was dissolved in acidified water and then diluted in DMEM before supplementation.Vehicle was used in control wells.
For the experiments with N-acetylcysteine (NAC) addition, NAC was dissolved in water and added at a 1 mM concentration.For conditioned media experiments, HK-2 cells were treated for 72 h as described above, next, fresh DMEM media was added to allow conditioned media enrichment for an additional 48 h.The media was then collected and added to RAW cells seeded in 6-well plates for a 48-h incubation.

Quantitative real-time PCR
The total RNA was extracted from frozen tissue or fresh cells using TRIzol reagent (Invitrogen).The RNA obtained from the aqueous fraction was then purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol.Next, we synthesized cDNA using 1 μg of total RNA and the SuperScript VILO cDNA Synthesis Kit with DNase (Invitrogen).The transcript levels were analyzed by quantitative real-time PCR (SYBR green fluorescence detection) in an ABI 7500 Real-Time PCR System (Applied Biosystems) using the primer pairs listed in Supplementary Table 1 and SsoAdvanced Universal SYBR Green Supermix (BioRad).All samples were measured with technical triplicates and normalized against 18S and GAPDH as endogenous controls.The relative quantification of all gene expressions was performed using the comparative 2 −DDCT method (Livak and Schmittgen 2001).
Hydrogen peroxide detection assay HK-2 cells were seeded in 6-well plates on glass coverslips coated with Fibronectin (0.5 μg/mL).Cells were cultured in HK-2 medium supplemented with 100 ppm tungsten for nine days.Media was changed every 3 days.Detection of cellular hydrogen peroxide was performed using a Hydrogen Peroxide Assay Kit following the manufacturer's guidelines (ab138874, Abcam).Cells were imaged on a Zeiss LSM800 confocal microscope with Airyscan (Carl Zeiss, Oberkochen, Germany) using an excitation 490 nm and emission 525-nm filter set.
Cell viability assays RAW cells were seeded in triplicate on a 96-well plate and allowed to adhere for 6 h.Next, control, tungsten, or conditioned media were added.Cell confluency was monitored using the Incucyte Live Cell SX5 system (Sartorius) as specified in the manufacturer's instructions.The plate was photographed every 2 h for 96 h.The data was analyzed using the Incucyte software.
Alamar blue assay was used to determine the effects of tungsten on HK-2 cell viability.HK-2 cells (5 × 10 5 ) were seeded in 6-well plates and cultured in 2-mL medium supplemented with the indicated amounts of tungsten (0, 5, 15, 50, 100, and 200 ppm) for up to 9 days.Every 3 days including day 0, pellets were incubated with 2-mL medium containing 10% resazurin (alamarBlue reagent, Thermo Fisher Scientific, Waltham, MA, USA, Cat.#DAL1025) for 3 h.Supernatant (100 μL) was added to a black 96-well plate and quantified for reductions in resazurin by measuring changes in its fluorescence (Ex 540 nm/Em 590 nm) using a spectrophotometer.Results were normalized to day 0 within each treatment group.All experiments were performed in triplicate.Pellets were rinsed in PBS prior to the addition of fresh medium supplemented with the indicated concentrations of tungsten.

Statistical analysis
The results are presented as means ± standard error.For multiple group comparisons, the statistical significance was calculated by ANOVA followed by Dunnett's multiple-comparison post hoc test.To compare between two groups, a two-tailed Student's t-test was performed.We considered p values < 0.05 as statistically significant.Statistical analysis Vol.: (0123456789) and graphical representation were performed using GraphPad Prism v9.1.0. 5.

Chronic tungsten exposure induced lysosome accumulation in tubular epithelial cells
We previously showed that daily treatment with water containing sodium tungsten (100 ppm) over a period of 1 or 3 months induced markers of renal fibrosis and tubular cell vacuolization in mice (Grant et al. 2022).To further characterize vacuole/granule formation in tubular epithelial cells, we stained kidney sections from mice exposed to tungsten over a 1-or 3-month period against LAMP1, a marker of lysosome formation that has been reported to be involved in toxicant-induced proximal tubule nephropathy (Vervaet et al. 2020).In control mice, we observed a diffuse cytoplasmic localization of LAMP1 that switched to a granular localization in mice exposed to tungsten at 1 or 3 months of treatment (Fig. 1A and B).Moreover, we found an increased number of LAMP1 + tubules in the kidneys of mice exposed to tungsten (Fig. 1) Thus, chronic tungsten exposure in mice induced LAMP1-positive lysosome accumulation in renal tubular epithelial cells.Chronic tungsten exposure stimulated renal inflammation Since tungsten exposure induced a toxic effect on tubular epithelial cells (Fig. 1), we hypothesized that this toxic effect could trigger an inflammatory response in the kidney.Therefore, we next evaluated the effect of chronic tungsten ingestion on the renal recruitment of inflammatory cells.Following 1 month of daily tungsten exposure, we observed a significant increase in the infiltration of leukocytes (CD45 + ), myeloid cells (CD11b + ), and macrophages (F4/80 + ) to the kidney interstitial space as shown in the representative immunofluorescence images (Fig. 2A) and the quantification of positively stained cells by highpower field (Fig. 2B).A similar pattern of increased infiltration was found for CD45 + and CD11b + positive cells in mice receiving tungsten for 3 months, whereas macrophage accumulation was unchanged at this time point as compared to controls (Fig. 3A and B).
It has been described that injured tubular epithelial cells develop a proinflammatory status characterized by increased NFkB activity and proinflammatory cytokine production (Kirita et al. 2020).In mice exposed to tungsten in their drinking water for 3 months, we observed that the enhanced infiltration of inflammatory cells was associated with increased levels of the p65-and p50-NFkB subunits, without changes following a 1-month treatment (Fig. 4A and B).In addition, we detected increased renal mRNA levels of inflammatory cytokines known to enable communication between injured epithelial cells and leukocytes (Kirita et al. 2020), including Csf1, Il34, and Ccl7 at month 1 (Fig. 4C) and Il34, Ccl7, and Cxcl2 following 3-month exposure to tungsten (Fig. 4D).

Tungsten treatment induces proinflammatory changes and oxidative stress in kidney tubular epithelial cells
In order to acquire a thorough understanding of the effects of tungsten exposure on tubular epithelial cells, we carried out a systematic examination at several time periods, specifically at 3, 6, and 9 days.By analyzing the cellular response at these different time periods, we expected to gain insight into the temporal dynamics of tungsten-induced alterations and their possible consequences for cellular function and inflammatory processes.First, we confirmed if the inflammatory changes induced by tungsten in the kidney are triggered by an inflammatory status in tubular epithelial cells, using a proximal tubule cell line (HK-2 cells) exposed to tungsten 100 ppm in vitro.Following 72-h exposure of HK-2 cells to tungsten, we observed an increase in the phosphorylation of p65-NFkB and in the protein levels of p50-NFkB (Fig. 5A).Moreover, incubation of HK-2 cells with tungsten for 72 h promoted an increase in the mRNA levels of CSF1, IL34, CXCL2, and CXCL10 (Fig. 5B); similar proinflammatory cytokines that were upregulated in the kidney tissue of mice chronically exposed to tungsten (Fig. 4C  and D).This inflammatory state was accompanied by an oxidative environment induced by tungsten as evidenced by an increase in the reactive oxygen species (ROS) generation as detected by a fluorescent probe for hydrogen peroxide/H 2 O 2 following 9 days of tungsten exposure (Fig. 6A).Moreover, the antioxidant enzyme catalase was significantly diminished with 6 days of tungsten treatment at 100 and 200 ppm, without altering the levels of other antioxidant enzymes such as SOD1 or thioredoxin (Fig. 6B).In addition, 100 and 200 ppm of tungsten had reduced viability of HK-2 cells from day 3 to 9 following tungsten exposure, as compared to control cells (Fig. 6C).These data suggest that tungsten induces a toxic effect on tubular epithelial cells that leads to increased ROS generation, reduced viability, and triggers an inflammatory response.

Conditioned media from epithelial cells or direct tungsten exposure modulated macrophage polarization
To evaluate if the release of inflammatory mediators by epithelial cells exposed to tungsten would influence inflammatory cell phenotype, we treated RAW macrophages with conditioned media (CM) from HK-2 cells exposed to tungsten.Following 48 h of treatment with the conditioned media from tungsten-treated HK-2 cells, the RAW cells showed a reduction in the levels of the M2-antiinflammatory macrophage polarization marker CD206 and an increase in the levels of the M1-proinflammatory polarization markers iNOS  and IL-6 (Fig. 7A).Of note, these effects were partially prevented in RAW cells that were treated with CM from HK-2 cells under co-treatment with tungsten and N-acetylcysteine (NAC), as an antioxidant to prevent the ROS generation induced by tungsten (Fig. 7A).This data suggests that tungsten induces oxidative stress in HK-2 cells that leads to the production of proinflammatory mediators that modify macrophage phenotype.
Finally, to investigate if tungsten would have a direct effect on inflammatory cell activation once they are recruited to the kidney, we also evaluated the influence of direct tungsten treatment on RAW macrophage polarization and if this effect could be prevented by NAC co-treatment.Tungsten treatment of RAW cells for 48 h induced a reduction in the levels of the M2-marker CD206 with a concomitant upregulation of the M1-markers iNOS and IL-6 levels.These changes were prevented by NAC treatment (Fig. 7B).Despite the marked changes in macrophage activation, RAW cell proliferation was unaffected by either conditioned media from tungsten-treated HK-2 cells or direct tungsten treatment (Sup.Fig. 1).

Discussion
Chronic kidney disease is a health problem that affects around 850 million people worldwide (Jager et al. 2019).Environmental exposure to pollutants including metals has the potential of increasing the susceptibility to develop kidney disease and/or accelerating its progression (Orr and Bridges 2017;Tsai et al. 2021).In a recent study, we showed that chronic oral tungsten exposure for 90 days led to functional and structural renal damage in male mice as evidenced by a reduction in the glomerular filtration rate and by the presence of tubular vacuoles and fibrotic tissue, accompanied by increased expression of matricellular proteins and the myofibroblast marker αSMA (Grant et al. 2022).Here, we further expanded on the nephrotoxic effects of chronic tungsten exposure by showing that tungsten promotes the upregulation of inflammatory cytokines and infiltration of inflammatory cells to the kidney, mechanisms that might contribute to chronic renal injury and fibrosis.Fig. 7 RAW macrophage proinflammatory phenotype induced by tungsten and conditioned media from tungsten-treated HK-2 cells.A RAW cells were treated for 48 h with conditioned media collected from HK-2 cells treated with water (Ctl), sodium tungsten 100 ppm (NaW), or NaW 100 ppmplus n-acetylcysteine 1 mM (NaW + NAC).B RAW cells were treated for 48 h with water (Ctl), sodium tungsten 100 ppm (NaW), or NaW 100 ppm plus n-acetylcysteine 1 mM (NaW + NAC).
For (A) and (B), the cell lysates were analyzed by western blot to evaluate the levels of Mannose receptor (CD206), inducible nitric oxide synthase (iNOS), arginase-1 (Arg1), or interleukin 6 (IL-6).Representative blot images and their respective densitometric analysis are shown.GAPDH was used as the loading control.*p<0.05 and **p<0.01,ANOVA followed by Dunnett's multiple-comparison post hoc test Indeed, persistent inflammation is a well-known risk factor for CKD progression (Mihai et al. 2018;Yilmaz et al. 2007), and tungsten has been shown to induce inflammatory responses in other organs.Male mice exposed to tungsten (15ppm) for 4 weeks in the drinking water showed tungsten accumulation in lumbar intervertebral disk and increased levels of the inflammatory cytokines TNF-α and interleukin-1β (Grant et al. 2021).Female mice exposed to inhaled tungsten particles (1.7 mg/m 3 <1 mm) had increased lung levels of IL-1β and CXCL1 accompanied by infiltration of neutrophils and macrophages to the lungs (Miller et al. 2021).Our data shows that the kidney is also a target for inflammatory effects following chronic tungsten exposure as shown by increased infiltration of leukocytes, myeloid cells, and macrophages.Moreover, we found increased expression of inflammatory cytokines that have been shown to be released by injured epithelial cells to facilitate leukocyte chemotaxis (Kirita et al. 2020), such as Csf1, Il34, Ccl7, and Cxcl2.This inflammatory signature was also upregulated in human proximal tubule epithelial cells that were treated with tungsten, suggesting that prolonged tungsten exposure induces an inflammatory state in epithelial cells that leads to the release of inflammatory mediators and inflammatory cell recruitment in the kidney (Fig. 8).In agreement with our findings, it has been reported that in primary cultures of human renal proximal epithelial cells that were isolated from diabetic and non-diabetic individuals, tungsten stimulated the secretion of proinflammatory cytokines such as IL-6, IL-8, and MCP-1 (Bertinat et al. 2017).While the upregulation of inflammatory cytokines was observed at 1-and 3-month exposure, NFkB upregulation was only observed after 3 months of tungsten exposure.Increases in inflammatory cytokine expression after 1 month of tungsten treatment might be explained by activation of other inflammatory pathways or transcription factors known to be implicated in renal inflammation such as STAT1/ STAT6 (O'Brown et al. 2015) or the cGAS-STING pathways (Maekawa et al. 2019).
The current study evaluated the effect of chronic exposure to 100 ppm of tungsten in the drinking Fig. 8 Schematic representation of tungsten effects on renal inflammation.Chronic tungsten ingestion leads to oxidative injury in tubular epithelial cells which creates a proinflammatory status characterized by increased production of proinflammatory cytokines that mediate the attraction of leukocytes, myeloid cells, and macrophages to the kidney.In addition, tungsten has direct effects on macrophages by promoting its polarization towards a proinflammatory phenotype.These effects are attenuated by an antioxidant treatment with N-acetylcysteine.Schematic was created with BioRe nder.com Vol:.( 1234567890) water.A limitation of our study is that we did not evaluate whether lower concentrations of tungsten could also lead to kidney inflammation.The concentration of 100 mg/L (100 ppm) of tungsten used in our in vivo study was chosen based on our cell viability and antioxidant enzyme expression experiments showing 100 ppm having significant effects in our dose-response profiles.In addition, as pointed out, a similar concentration was used in our previous study based on circumstances for its effects on fibrosis (Grant et al. 2022).This concentration is less than the levels measured in a study performed in the population of Fallon, Nevada, a city with facilities for tungsten refining, where the exposure levels of tungsten reached as high as 934 ppm (mg/kg); consequently, children and adults were reported to have elevated levels of tungsten in their urine (median of 0.97mg/L) (Rubin et al. 2007).Other studies have also shown kidney alterations in rats exposed to tungsten.Pregnant Sprague Dawley rats were exposed to sodium tungsten in drinking water (125 to 2000 mg/L) from gestation day 6 through lactation day 20, and the pups were exposed to tungsten from postnatal day 12 to 3 months or 2 years in their drinking water.Tungsten accumulated in the kidney of the rats from month 3 to 18 of exposure, with evidence of suppurative inflammation in the renal tubules of the rats exposed to 1000 mg/L of tungsten.Similarly, only the kidney displayed histological lesions in male/female mice exposed to 1000 and 2000 mg/ mL for 3 months, while after a 2-year exposure, tungsten, induced a significant tubular injury in male mice exposed to 500, 1000, and 2000 mg/L of tungsten and in the female mice exposed to 1000 and 2000 mg/L (National Toxicology 2021).These observations are in agreement with our findings in male mice.Tungsten pellet implanted in the gastrocnemius muscle of the rat lead to increase urinary tungsten excretion in the period of study (1 to 12 months) and induced a transient increase in the urinary excretion of retinol binding protein as a marker of tubular injury at 9 months post-implant, without alterations on other kidney injury markers at any time point (Hoffman et al. 2021).
Immune cell infiltration to the kidney has an essential role in the regulation of injury and repair mechanisms (Lee et al. 2017;McWilliam et al. 2021).Depending on the microenvironment and stimulus received by macrophages, they can polarize to a "classically" M1-proinflammatory or an "alternative" M2-antiinflammatory phenotype that plays dual roles in kidney injury (Lee et al. 2011).Macrophages expressing M1 markers such as CD80, CD64, or iNOS are proinflammatory and produce cytokines including IL-6, TNF-α, and IL-1β, whereas macrophages that express M2 markers such as CD206 or arginase-1 are essentially antiinflammatory (Liu et al. 2014;Meng et al. 2015).Several intermediate and dynamic populations have also been described.During kidney damage, enhanced M1 macrophage accumulation amplifies the initial injury and leads to further damage and fibrosis (Jo et al. 2006;Ko et al. 2008).Here, we showed that tungsten exposure stimulated the infiltration of macrophages to the kidney but also modulated macrophage polarization to an M1-proinflammatory phenotype characterized by the upregulation of iNOS and IL-6 and the downregulation of CD206.In agreement with our findings, THP-1 macrophages exposed to nanoparticles composed of a tungsten carbide-cobalt mix displayed an increase in the proinflammatory cytokines IL-1β and IL-12 while stimulating the M1 phenotype as determined by higher levels of CD40 following nanoparticle exposure (Armstead and Li 2016).Similarly, intratracheally instillation of powder mixtures consisting mostly of tungsten (>90%) promoted lung inflammation and upregulation of genes associated with oxidative and metabolic stress responses in male rats.Moreover, this effect was linked to macrophage activation, ROS generation, and neutrophilia (Roedel et al. 2012).A study in male Fischer 334 rats exposed to 2 or 10 mg/m 3 of tungsten trioxide nanoparticles for 6 h/day for 4 weeks found only transient increases in neutrophil number and cytokine-induced neutrophil chemoattractant but no evidence of histological injury in the lung (Marui et al. 2022).In another study, 10 mg/kg of nano-tungsten carbide suspension was perfused into the lungs of SD rats by tracheal instillation, for 3 or 8 weeks, and leads to increased inflammatory cytokine levels and alveolar structural alterations (Zhou et al. 2023).
In addition to inflammation, tungsten might induce tissue injury by increasing ROS generation.Using an in vitro timeline-based approach, we were able to track and evaluate the changing impacts of tungsten on important cellular signaling pathways, gene expression patterns, oxidative stress levels, and cell viability.Such a Vol.: (0123456789) thorough examination of several time periods offers a more nuanced understanding of how tungsten exposure affects tubular epithelial cells over time, which helps in the discovery of potential time-dependent mechanisms behind its toxic effects.Specifically, we found that in HK-2 cells, tungsten treatment increased H 2 O 2 production and reduced the levels of the antioxidant enzyme catalase.Moreover, the addition of NAC, a potent antioxidant (Ezerina et al. 2018), prevented the effects of HK-2 cell-conditioned media on promoting M1 polarization of RAW macrophages.These observations suggest that the oxidative stress induced by tungsten in proximal tubular epithelial cells leads to a proinflammatory state that signals immune cell recruitment to the kidney and macrophage activation.This is in agreement with observations suggesting that kidney mitochondria are susceptible to tungsten-induced alterations leading to oxidative injury (Cheraghi et al. 2019) as evidenced in male rats in which exposure to 500 ppm of tungsten in the drinking water for 28 days induced renal (ROS) generation and lipid peroxidation (Sachdeva et al. 2022).The pro-oxidant effects of tungsten have also been evaluated in other organs.In male Wistar rats, sodium tungsten (100 ppm) administration in the drinking water for 3 months induced oxidative stress as determined by increased levels of oxidized glutathione and thiobarbituric acid reactive species in the liver and spleen, while these effects were prevented by NAC co-administration (Sachdeva and Flora 2014).Moreover, this study found reduced blood activity of antioxidant enzymes including δ-aminolevulinic acid dehydratase and catalase accompanied by increased blood ROS detection, effects prevented by NAC (Sachdeva and Flora 2014).In the liver of male mice, tungsten trioxide nanoparticles caused hepatic structural and functional alterations, an effect that was mediated by increased oxidative stress and prevented by melatonin pre-treatment as an antioxidant (Mao et al. 2021).Male mice exposed to tungsten particles by inhalation (9, 23, or 132 mg/m 3 ) 45 min/day, 5 days/week for 2 weeks, had no indication of lung inflammation; nevertheless, a dose-dependent increase in ROS generation was observed (Kovago et al. 2022).Similar to our observation of increased ROS in HK-2 cells following tungsten exposure, it has been documented that tungsten also induces increased ROS generation in HEK-293 (kidney) and HepG2 (liver) cell lines (Sachdeva and Maret 2021).
Altogether, our data demonstrate that chronic oral exposure to sodium tungsten (100 ppm) for 30 or 90 days induces epithelial cell ROS generation and renal inflammation characterized by immune cell infiltration and by the upregulation of inflammatory cytokines in male mice.Our study only addressed the effects of chronic tungsten administration in male mice.Therefore, future studies should determine whether tungsten exposure has similar effects in female animals.In addition, RAW macrophages were polarized towards an M1 proinflammatory phenotype when directly exposed to tungsten or when treated with conditioned media from proximal tubule cells treated with sodium tungsten, effects that were prevented by an antioxidant treatment.

Fig. 3
Fig. 3 Tungsten exposure for 3 months increases the infiltration of CD45 + and CD11b + cells.A Immunofluorescence images of kidneys from mice treated with sodium tungsten 100 ppm (NaW) in the drinking water for 3 months showing CD45 + , leukocytes (green); CD11b + , myeloid cells (green);

Fig. 4
Fig.4Chronic tungsten exposure promotes renal inflammation.Western blot analysis was performed to evaluate the kidney levels of the NFkB subunits p65 and p50 following sodium tungsten (NaW) exposure for (A) 1 month or (B) 3 months.Representative blot images with their respective densitometric analysis are shown.GAPDH was used as the loading con-

Fig. 5
Fig. 5 Tungsten induces an inflammatory state in human proximal tubule HK-2 cells.HK-2 cells were treated with sodium tungsten 100 ppm (NaW) or water (Ctl) for 72 h.A Western blot analysis to quantify the levels of the NFkB subunits p65 and p50 and p65 phosphorylation (Ser-536) following NaW exposure.Representative blot images and their respective den-

Fig. 6
Fig.6Tungsten promotes oxidative stress in human proximal tubule HK-2 cells.A HK-2 cells were treated with sodium tungsten 100 ppm (NaW) or without (0 ppm, Ctl) for 9 days and detected for intracellular hydrogen peroxide accumulation.B Western blot analyses to determine the levels of superoxide dismutase 1 (SOD1), thioredoxin, and catalase.β-actin or