Supplemental exposure to polystyrene nanoplastics synergistically amplifies calcium oxalate crystal–induced injury to renal tubular epithelium, accelerating the formation of calcium oxalate kidney stones

Abstract

Background

With the escalating issue of polystyrene microplastic pollution, microplastic particles have been detected in human urine. While calcium oxalate (CaOx) crystals are well-established mediators of renal stone formation, the role of microplastics, particularly polystyrene nanoplastics (PS-NPs), in promoting CaOx kidney stone formation remains unclear. This study aims to explore whether PS-NPs interact with CaOx crystals to enhance renal tubular epithelial cell injury and facilitate the formation of kidney stones.

Methods

Clinical CaOx kidney stone samples were analyzed using pyrolysis—gas chromatography-mass spectrometry (Py-GC/MS), which detected microplastic components. In vitro, human renal proximal tubular epithelial cells (HK-2) were exposed to calcium oxalate monohydrate crystals (1.5 mM), or cells were pretreated with PS-NPs (100 nm, 0.1 mg/mL) for 24 h, followed by the addition of 1.5 mM CaOx for co-treatment. Comprehensive mechanistic assessments, including whole-transcriptome RNA sequencing, crystal adhesion assays, macrophage chemotaxis and polarization analysis, ferroptosis biomarker quantification, lipid peroxidation measurement, and mitochondrial ferrous ion accumulation, were conducted. In vivo, a rat model of CaOx nephrolithiasis was induced by ethylene glycol (EG) with concurrent exposure to PS-NPs (4 mg/Kg·Day) via drinking water.

Results

Clinical analysis confirmed the presence of PS-NPs and other microplastics in human CaOx kidney stones. In vitro, exposure to PS-NPs significantly altered the morphology of CaOx crystals, promoting aggregation and enhancing adhesion to renal tubular epithelial cells. Combined exposure to PS-NPs and CaOx crystals exacerbated HK-2 cell injury through upregulation of VCAM1, CXCL8-driven macrophage chemotaxis and M1 polarization, and ferroptosis induced by xCT/GPX4 suppression. Transcriptomic analysis revealed LRP6 downregulation as a central regulator in these pathological processes. Overexpression of low-density lipoprotein receptor-related protein 6 (LRP6) alleviated cell damage and attenuated inflammatory responses. In vivo, PS-NPs co-exposure exacerbated renal CaOx deposition, ferroptosis in renal tubular epithelial cells, and inflammatory responses in the rat model.

Conclusion

Our study identifies PS-NPs as novel lithogenic cofactors that promote CaOx nucleation, enhance crystal adhesion to renal tubular epithelial cells, and amplify inflammation and ferroptosis through LRP6 downregulation. This suggests that microplastic pollution may be an emerging environmental risk factor for kidney stone pathogenesis.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12989-026-00661-0.

Introduction

Nephrolithiasis is a common urological disorder characterized by high incidence and frequent recurrence [1]. Elevated urinary oxalate and the presence of nascent calcium oxalate (CaOx) crystals have been shown to injure renal tubular epithelial cells, thereby promoting stone initiation and progression [2, 3]. The formation of urinary CaOx crystals is closely associated with supersaturation of oxalate and calcium ions, as well as injury to the tubular epithelium [4, 5].

In humans, CaOx crystals typically arise via heterogeneous nucleation, for which minute urinary impurities can provide nucleation sites. The potential health effects of microplastics (including nanoplastics) have attracted considerable attention in recent years [6–9]. In animal models, nanoplastics have been detected in multiple organs—including the kidney, spleen, lung, intestine, testes, and brain—indicating systemic distribution and tissue accumulation [10–13]. Within the kidney, accumulation of polystyrene nanoplastics exacerbates oxidative stress in renal tubular epithelial cells and triggers endoplasmic reticulum stress and mitochondrial dysfunction [14, 15]. Notably, oxidative stress and mitochondrial dysfunction are recognized contributors to the pathogenesis of nephrolithiasis [2, 16, 17].

Accordingly, we sought to determine whether urinary nanoplastics in patients with nephrolithiasis act synergistically with CaOx crystals to aggravate tubular epithelial injury and to promote CaOx crystallization and aggregation. In this study, we first examined nine clinically obtained calcium oxalate (CaOx) kidney stone specimens using pyrolysis–gas chromatography–mass spectrometry (Py–GC/MS) and confirmed the presence of multiple microplastic constituents; notably, polystyrene (PS)—a polymer that has garnered considerable attention in recent years—was detected in eight of the nine samples. Because CaOx crystallization proceeds through an initial nucleation step and micrometer-scale plastic particles are unlikely to directly participate in nucleation, we selected PS nanoparticles with a diameter of 100 nm for subsequent experiments [18]. We then synthesized CaOx crystals under laboratory conditions in the presence or absence of polystyrene nanoplastics (PS-NPs) to assess their impact on crystal formation. To elucidate mechanisms at the molecular level, we established a disease model by co-administering PS-NPs during CaOx crystal exposure and performed transcriptome sequencing. Guided by these data, we investigated how PS-NPs promote the initiation and progression of nephrolithiasis, focusing on pathways related to crystal adhesion, inflammatory responses, and oxidative stress.

Methods and materials

Chemicals

Calcium oxalate crystals were purchased from Sigma-Aldrich (USA), and ethylene glycol, sodium oxalate, calcium chloride, sodium chloride, and 1 M Tris–HCl (pH 7.4) were purchased from Solarbio (China). phorbol 12-myristate 13-acetate (PMA; Sigma P8139) were purchased from MedChemExpress (China). PS-NPs (100 nm, 2.5% w/v in ultrapure water, 20 mL) were commercially obtained from Jiangsu Zhichuan Technology (Jiangsu, China).

The plastic components in human CaOx stone samples were detected by Py-GC/MS

To investigate whether microplastic components are present within human kidney stones, we collected specimens from patients with calcium oxalate (CaOx) nephrolithiasis. Because our aim was to assess potential involvement of microplastics in stone formation rather than superficial adsorption onto the stone surface, we implemented a stringent decontamination procedure prior to analysis. Specifically, stones were boiled in PBS, then transferred into deionized water and washed twice on an orbital shaker (120 rpm, 2 min each) to remove surface-adherent microplastics as far as possible [19].

For comprehensive detection of microplastics contained within the stones, samples were pulverized to a fine powder using a ball mill. Sequential solvent extraction was then performed with chloroform, hexafluoroisopropanol, and xylene, followed by identification of microplastic constituents using pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS). Before analyzing the samples, a blank test was run to ensure no microplastic contamination in the instrument. Throughout all procedures, only glassware was used to avoid plastic contact and minimize the risk of contamination.

Synthesis and characterization of calcium oxalate crystals

Following established experimental protocols from previous studies [20, 21], we synthesized calcium oxalate crystals under controlled laboratory conditions using sodium oxalate and calcium chloride. To investigate the influence of PS-NPs on calcium oxalate crystallization, the synthesis process was conducted both with and without supplementation of PS-NPs, with the resultant experimental groups designated as the NC group and PS-NPs group respectively. The synthesized crystals were subsequently analyzed for zeta potential via Dynamic Light Scattering (DLS) and examined for morphological characteristics using Scanning Electron Microscopy (SEM).

Cell culture and treatment

The human proximal tubular epithelial cell line HK-2 was obtained from the China Center for Type Culture Collection (Beijing). Cell line authentication was performed via short tandem repeat (STR) profiling, and regular mycoplasma contamination testing was conducted. HK-2 cells were cultured in a Dulbecco's modified Eagle's (DMEM)/F12 medium (Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) and 1% penicillin (C0222, Beyotime Biotech Inc, China) at 37 °C and 5% CO₂ in a cell culture incubator (Binder, Tuttlingen, Germany). Upon reaching 70–80% confluence, cells were divided into three groups: the Control group (NC), which received DMEM/F12 base medium; the CaOx group, which was treated with 1.5 mM CaOx dissolved in the base medium; and the PS + CaOx group, in which cells were pretreated with PS-NPs for 24 h before exposure to 1.5 mM CaOx solution, with co-intervention lasting for 24 h. After 24-h interventions, conditioned media (CM) were collected, centrifuged at 300 g for 10 min, and immediately analyzed or stored at -80 °C.

‌The Tohoku Hospital Pediatrics-1 (THP-1) cell line was purchased from the China Center for Type Culture Collection and cultured in THP-1–specific complete medium (RPMI-1640 + 10%FBS + 0.05 mMβ-mercaptoethanol + 1% penicillin, CM-0233, Procell Life Science & Technology, China). At the beginning of the experiment, PMA (100 ng/mL) was used to induce M0 macrophages, followed by the addition of CM as needed for the experiment.

For the PS-NP intervention used in the cell experiments, commercially sourced PS-NPs were added to serum-free basal medium to a final concentration of 0.1 mg/mL. Serum-free conditions were used to minimize protein corona formation and thereby reduce potential alterations in zeta potential and particle morphology.

With respect to dosing, and in light of prior studies [12, 22, 23], we consider the commonly used concentration of 1 mg/mL to be excessively high and not representative of realistic environmental exposure. Based on reported human plastic-particle burdens—i.e., an estimated weekly intake of ~ 5 g for a 70-kg adult—and proportional distribution across body fluid volumes, the corresponding blood concentration is approximated at ~ 0.143 mg/mL. Accordingly, integrating these considerations with prior literature, we selected 0.1 mg/mL as the working concentration for our experiments.

Cell viability was detected and damage effect was calculated

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, CK04). After interventions, culture supernatants in 96-well plates were aspirated, and CCK-8 working solution (1:100 dilution) was added, followed by a 2 h incubation at 37 °C. Absorbance was measured at 450 nm using a microplate reader.

To determine whether CaOx and PS-NPs induced simple additive or synergistic cytotoxic effects, the Bliss independence model was applied. Based on previous studies [24, 25], cells were treated with 1.5 mM CaOx for 24 h or 0.1 mg/mL PS-NPs for 48 h. Theoretical cell viability was calculated by multiplying the viability values from individual PS-NP and CaOx treatments. Synergistic cytotoxicity was confirmed when the observed viability was significantly lower than the theoretical viability.

Transcriptome sequencing

To investigate the role of NPs in CaOx stone pathogenesis, comparative transcriptome sequencing was performed between CaOx-treated and CaOx + PS-NPs co-exposure groups. Following interventions, total RNA was extracted using the TRIzol™ reagent (Invitrogen) according to the manufacturer's protocol. Subsequent bioinformatic analyses were conducted using the Generover Cloud Platform.

Animals and treatment

Since drinking water is one of the main sources of NPs, combined with previous studies [26, 27], Male Sprague–Dawley (SD) rats (3–4 weeks old) were obtained from the Institute of Biomedical Research, Wuhan University. Animals were housed in the animal facility of Renmin Hospital of Wuhan University under standard conditions (12-h light/dark cycle, 22 ± 1 °C, 55 ± 5% humidity) with ad libitum access to food and water. Based on existing research [28], the total human intake of plastic particles can reach up to 5 g per week, which corresponds to approximately 10.2 mg/kg*day for a 70-kg adult. Because PS constitutes only one fraction of the ingested plastics, we accordingly reduced the dose of PS particles administered to the experimental animals.

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University (Approval No. 20230310F).

To model nanoplastic co-exposure during stone formation, animals were divided into:

(1) Normal control: Standard drinking water.

(2) Stone model: Water containing 0.75% ethylene glycol (EG).

(3) NPs co-exposure: Water with 0.75% EG + 4 mg/Kg*Day PS-NPs (100 nm PS).

All groups received standard chow. After 8-week interventions, rats were euthanized for serum collection and bilateral nephrectomy.

To investigate the role of low-density lipoprotein receptor-related protein 6 (LRP6) in lithogenesis, a subset of animals received daily intraperitoneal injections of salinomycin (6 mg/kg body weight), a LRP6 inhibitor that blocks Wnt-induced LRP6 phosphorylation and promotes its degradation. Pharmacological intervention continued throughout the experimental period.

Lentiviral transfection

When HK-2 cells reached 30–40% confluence, the medium was aspirated and replaced with lentiviral particles. Transduction proceeded for 12–16 h. After viral removal, cells were maintained in DMEM/F12 supplemented with 5 ng/mL puromycin for 48 h, followed by selection in 1.5 ng/mL puromycin for 5 additional days. Lentiviral constructs included: KD-CXCL8, KD-VCAM1, OE-LRP6 (All viruses from GeneChem, Shanghai). Knockdown/overexpression efficiency was validated via qPCR and WB to functional assays.

Total RNA extraction and quantitative PCR experiments

Total RNA was extracted from HK-2 cells and macrophages using TRIzol™ reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 1 μg total RNA using the Hiscript III Reverse Transcriptase kit (Yeasen Biotechnology, Shanghai, China). Quantitative real-time PCR (qPCR) was performed using SYBR qPCR Master Mix (Yeasen) with gene-specific primers (Supplementary Table S1). All primers were synthesized by Sangon Biotech (Shanghai, China). Relative gene expression was calculated using the ΔΔCt method with GAPDH as the endogenous control.

Crystal-cell adhesion assay

When HK-2 cells reached 90% confluence, they were treated for 24 h. Post-intervention, culture media were discarded and cells were washed three times with PBS on an orbital shaker (120 rpm, 2 min/wash, room temperature). Crystal adhesion was examined using phase-contrast microscopy, with quantitative analysis performed via ImageJ software.

Macrophage chemotaxis experiments

THP-1 monocytes were seeded in the upper chambers of Transwell inserts (Corning) at 1 × 10⁵ cells/mL and differentiated into M0 macrophages using 100 ng/mL PMA for 48 h. After removing non-adherent cells with PBS washes, conditioned media (CM) from differentially treated HK-2 cells were added to the lower chambers. Following 24-h co-culture, non-migrated cells in upper chambers were removed with cotton swabs. Migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde (30 min), stained with 0.1% crystal violet (30 min), and imaged using phase-contrast microscopy.

Macrophage polarization experiments

THP-1 monocytes were seeded into 12-well plates containing glass coverslips. After incubation with PMA under light-protected conditions for 24 h to induce differentiation into adherent M0 macrophages, the medium was replaced with fresh PMA-free medium for an additional 48 h to stabilize phenotypes. Conditioned media (CM) from experimental groups were then added for 24 h culture. Following treatment, qPCR and immunofluorescence assays were performed to detect macrophage polarization marker expression.

For animal experiments, SD rat kidney sections were stained with CD68 for macrophage labeling and CD86 for M1 polarization marker validation. Prepared sections were observed under a fluorescence microscope.

Reactive oxygen species (ROS)

The ROS Species Assay Kit was purchased from Solarbio (China) and utilizes the fluorescent probe DCFH-DA for the detection of reactive oxygen species. After cellular treatment, the probe working solution was prepared at a 1:5000 dilution according to the manufacturer's protocol. Following a 30-min incubation, samples were analyzed using flow cytometry.

Lipid peroxidation levels were measured

The BODIPY 581/591 C11 Kit (Thermo Fisher Scientific) was used to detect cellular lipid peroxidation levels. Following cellular interventions, cells were incubated with diluted probe according to the manufacturer’s instructions. Oxidized BODIPY (O-BODIPY) was observed at excitation/emission wavelengths of 488/510 nm, while reduced BODIPY (R-BODIPY) was detected at 581/591 nm using inverted fluorescence microscopy.

Determination of mitochondrial Fe²⁺

The Mito-FerroGreen mitochondrial ferrous ion probe (Dojindo) was employed to detect mitochondrial Fe²⁺ levels, correlating with ferroptosis severity. Post-intervention, probe working solution was prepared and incubated per manufacturer’s protocol. After incubation, cellular mitochondrial Fe²⁺ was visualized at 488/510 nm excitation/emission wavelengths using inverted fluorescence microscopy.

Mitochondrial membrane potential detection

The mitochondrial membrane potential was assessed using a JC-1 kit (C2006, Beyotime, China). In cells with high ΔΨm, JC-1 forms red-fluorescent J-aggregates (excitation/emission: 514/590 nm). In depolarized mitochondria, JC-1 remains as green-fluorescent monomers (excitation/emission: 510/527 nm). Post-intervention, cells were incubated with 2 mL JC-1 working solution (37 °C, 20 min), washed twice with staining buffer, and immediately imaged using an inverted fluorescence microscope (Olympus IX71, 20 × objective).

Antibodies

The following antibodies were used for WB, immunofluorescence, and immunohistochemical assays: Anti-LRP6(Abcam, ab134146), Anti-VCAM1(Proteintech, 11,444–1-AP), Anti-CXCR2(Proteintech, 20,634–1-AP), Anti-xCT (Proteintech, 26,864–1-AP), Anti-glutathione peroxidase 4 (GXP4) (Proteintech, 30,388–1-AP), Anti-ferritin light chain (FTL) (Proteintech, 10,727–1-AP), Anti-GAPDH (Proteintech, 10,494–1-AP), Anti-CD86(Abcam, ab317266), Anti-CD68(Abcam, ab303565).

Western blotting (WB)

Protein extracts from cells or tissues were obtained using RIPA lysis buffer. Equal protein quantities were mixed with 4 × Laemmli loading buffer, denatured at 95 °C for 5 min, and resolved on 4–12% Bis–Tris SDS–polyacrylamide gels. Electrophoresis was initiated at 80 V, then increased to 120 V after the bromophenol blue dye entered the separating gel, continuing until the dye front reached the gel bottom. Proteins were transferred to PVDF membranes via wet transfer (100 V, 60 min) with ice-cooling to prevent overheating. Membranes were blocked with 5% skim milk for 1 h at room temperature, probed with primary antibodies overnight at 4 °C, and washed three times with TBST. HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. After final washes, ECL substrate was applied and autoradiographic film was exposed in the darkroom. Band densities were quantified using ImageJ software.

Immunofluorescence

After PBS washing (twice), cells on coverslips were fixed with 4% paraformaldehyde (20 min), permeabilized with 0.1% Triton X-100 (room temperature), and blocked with 3% BSA (30 min, RT). Specimens were incubated with primary antibodies (12 h), washed with PBS, then stained with fluorescent secondary antibodies. Imaging was performed using a fluorescence microscope.

Rat kidney tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned. Sections were baked at 60 °C (30 min) for dewaxing, rehydrated through xylene/ethanol gradients, and subjected to microwave antigen retrieval in citrate buffer (pH 6.0; 10–15 min). After cooling and PBS rinsing, sections were blocked with 5% BSA (30 min, RT), incubated with primary antibodies (4 °C overnight), rewarmed, and washed. Fluorescent secondary antibodies were applied under light-protected conditions (1 h, RT). Following DAPI nuclear counterstain (5 min), sections were mounted with antifade medium and stored protected from light at 4 °C. Imaging was conducted within 48 h.

Immunohistochemistry (IHC)

Rat kidney tissue sections were deparaffinized. Distilled water washed the sections three times. Antigen retrieval and blocking treatments were performed. The sections were incubated with primary antibodies at 4 °C for 12 h, washed, then incubated with corresponding secondary antibodies. Color development, counterstaining, dehydration, and sealing followed. Observations and records were made under a microscope (BX53, Olympus, Japan).

Von Kossa staining

The Von Kossa staining method was employed to assess renal crystal deposition in rat kidney tissues. Paraffin-embedded kidney samples were sectioned, deparaffinized, and washed. Sections were immersed in silver nitrate solution, exposed to ultraviolet light for 10 min, washed, counterstained, dehydrated, and mounted. Observations were recorded using a microscope (BX53, Olympus, Japan).

Cell biochemistry assay

Glutathione (GSH) levels in cell samples were quantified using a commercial assay kit (A006-2-1, Nanjing Jiancheng) following the manufacturer’s protocol. Absorbance values at 405 nm were measured using a microplate reader (PerkinElmer, Waltham, MA, USA).

Statistical analyses

Data are presented as mean ± standard deviation (SD) from ≥ 3 independent experiments. Statistical analyses were conducted using GraphPad Prism 10.1.2 (GraphPad Software, La Jolla, CA). Two-group comparisons employed Student's t-test; multi-group comparisons used one-way ANOVA with Tukey's post hoc test. Statistical significance was defined as P < 0.05.

Results

A variety of plastic components exist in human kidney stone samples

Py–GC/MS analysis of nine human calcium oxalate kidney stone specimens revealed multiple polymer species, including PS, polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), polycarbonate (PC), and nylon. PS was present in 8 of 9 specimens; PE and PC were detected in 7 of 9 and 6 of 9 specimens, respectively (Fig. 1A, B).

Fig. 1.

Detection of microplastics in human calcium oxalate kidney stone samples, effect of PS-NPs on calcium oxalate crystal dynamics and transcriptome sequencing results. A, B Py–GC/MS analysis detected of plastic pollution in human calcium oxalate kidney stone clinical samples revealed specific peaks for PS, as well as distinct peaks for PE and PC. C Laboratory-synthesized CaOx crystals with added PS-NPs showed alterations in crystal morphology, resulting in increased size and the formation of sharp edges. D Dynamic light scattering (DLS) analysis revealed that the Zeta potential of CaOx crystals synthesized with PS-NPs was reduced, indicating an increased aggregation tendency. E Transcriptome sequencing results showed that additional exposure to PS-NPs led to significant changes in gene expression in CaOx kidney stone model HK-2 cells, with 531 genes upregulated and 256 genes downregulated. F–H Bioinformatics analysis of transcriptome data revealed enrichment of genes in pathways such as Wnt signaling, cytokine signaling, and other biological processes

PS-NPs can affect the morphology and aggregation of CaOx crystals synthesized in the laboratory

Under controlled laboratory conditions, CaOx crystals were synthesized with the supplemental addition of PS-NPs. Analytical results demonstrated that PS-NPs significantly altered CaOx crystal growth patterns, transforming their morphology from regular rhombic, brick-like structures to sharp-edged, blade-like configurations. Substantial adsorption of PS-NPs occurred on crystal surfaces and within interstructural crevices (Fig. 1C, Figure S1A). Dynamic light scattering (DLS) measurements revealed that PS-NPs reduced the absolute zeta potential of CaOx crystals (from − 9.23 to − 6.31 mV) (Fig. 1D), indicating weakened inter-crystal electrostatic repulsion and a consequent increased propensity for aggregation. Furthermore, we performed basic characterization of the PS-NPs under experimental conditions, including primary and hydrodynamic size, zeta potential, and polydispersity (Table S2). The weighted average of both the primary and hydrodynamic sizes of the PS-NPs remained stable around 113 nm, showing no significant changes with solvent variation, and the polydispersity index (PDI) was low (ranging from 0.018 to 0.0186). The zeta potential was also generally stable, with values around − 27.6 mV in the original solution and deionized water-diluted suspensions. In DMEM/F12 culture medium, the absolute value slightly decreased to − 19.2 mV.

Transcriptome sequencing results showed that the co-exposure of PS-NPs and CaOx crystals synergistically damaged RTECs and participated in stone formation through multiple pathways

Cell viability was assessed by CCK-8 at the concentrations described above to determine whether PS-NPs exert additive or synergistic effects on CaOx-induced tubular injury. Treatment with 1.5 mM CaOx reduced HK-2 viability by approximately 14.3%, whereas 0.1 mg/mL PS-NPs alone had no significant impact (mean inhibition ~ 1.2%, not statistically significant). Notably, the combined treatment yielded an average inhibition of 20.3%, exceeding the Bliss independence model prediction of 17.16% (Figure S1B), indicating that PS-NPs synergistically enhance CaOx cytotoxicity rather than merely add to it.

Subsequent transcriptomic sequencing compared HK-2 cells treated with CaOx monotherapy (1.5 mM) and CaOx + PS-NPs co-exposure (1.5 mM + 0.1 mg/mL).

Co-exposure induced 531 upregulated and 256 downregulated genes versus CaOx alone (Fig. 1E). KEGG enrichment analysis revealed significant pathway alterations in Cytokines, Wnt, and Hippo signaling, alongside critical biological processes including immune regulation and cytokine-receptor interactions (Fig. 1F–H). These findings guided focused investigations into crystal-cell adhesion and macrophage-epithelial crosstalk.

Exposure of PS-NPs aggravated the adhesion of calcium oxalate crystals to RTECs

Based on transcriptomic sequencing, several cell adhesion-related molecules were screened. PS-NPs exposure significantly upregulated vascular cell adhesion molecule 1 (VCAM1) expression (Fig. 2A). qPCR confirmed increased VCAM1 transcription (Fig. 2B). Western blotting further demonstrated elevated VCAM1 protein levels (Fig. 2C, D). In HK-2 cells co-treated with CaOx crystals and PS-NPs, crystal adhesion was markedly enhanced (Fig. 2E, F).

Fig. 2.

Additional exposure to PS-NPs increased CaOx crystal-cell adhesion. A Transcriptome sequencing results showed significant changes in multiple cell adhesion-related molecules, with a notable upregulation of VCAM1. B qPCR validation confirmed the transcriptome sequencing results, showing that additional PS-NPs exposure significantly elevated VCAM1 expression at the transcriptional level compared to the CaOx-only treatment group. C, D WB analysis further confirmed that PS-NPs exposure increased VCAM1 protein expression. E, F After additional PS-NPs exposure, the adhesion of CaOx crystals to HK-2 cells significantly increased. G, H Lentiviral-mediated VCAM1 knockdown reduced the PS-NPs-induced upregulation of VCAM1. I-J Lentiviral knockdown of VCAM1 led to a reduction in CaOx crystal adhesion to HK-2 cells following PS-NPs + CaOx intervention

To verify VCAM1's role in this adhesion, lentiviral VCAM1 knockdown was performed in HK-2 cells (Figure S1C). Post-knockdown, VCAM1 protein expression decreased significantly in PS-NPs + CaOx co-treated cells (Fig. 2G, H). Crystal adhesion assays confirmed that VCAM1 knockdown substantially reduced CaOx crystal adhesion on co-treated HK-2 cells (Fig. 2I, J).

Exposure to PS-NPs promotes CaOx-induced macrophage chemotaxis and inflammatory polarization

To investigate inflammatory signaling modulation by PS-NPs during CaOx stone pathogenesis, CM experiments were conducted (Fig. 3A, B). Compared to CaOx monotherapy, PS-NPs co-exposure significantly increased macrophage chemotaxis (Fig. 3C, D). Elevated expression of M1 marker CD86 confirmed enhanced pro-inflammatory polarization (Fig. 3E; Figure S1D). qPCR revealed significantly upregulated transcription of M1 markers (CD80, IL-6, TNF-α) in macrophages treated with PS-NPs + CaOx-CM (Fig. 3F).

Fig. 3.

Additional exposure to PS-NPs promotes macrophage chemotaxis and M1 polarization via the CXCL8-CXCR2 axis. A, B Schematic diagram of conditioned media extraction, chemotaxis, and polarization experiments. C, D Compared to the CaOx-only intervention group, conditioned media (CM) extracted after PS-NPs exposure significantly enhanced macrophage chemotaxis. E Immunofluorescence detection of CD68 and CD86 showed a significant increase in M1 polarization of macrophages cultured with PS-NPs + CaOx-CM. F qPCR of three common M1 polarization markers confirmed that additional PS-NPs exposure increased M1 polarization levels in macrophages treated with CM. G qPCR analysis confirmed that CXCL8 expression in HK-2 cells was significantly elevated at the transcriptional level in the PS-NPs + CaOx treatment group. H ELISA results showed that additional PS-NPs exposure significantly increased the secretion of CXCL8 in the CaOx kidney stone model. I, J Western blot analysis revealed a significant increase in CXCR2 protein expression in macrophages treated with PS-NPs + CaOx-CM. K CXCL8 knockdown significantly reduced CXCL8 secretion in macrophages treated with PS-NPs + CaOx-CM. L, M CXCL8 knockdown in HK-2 cells significantly reduced the chemotactic effect of CM from PS-NPs + CaOx-treated cells on macrophages. N CXCL8 knockdown in HK-2 cells significantly reduced the effect of PS-NPs + CaOx-CM on macrophage M1 polarization. O, P After CXCL8 knockdown, CXCR2 protein expression in macrophages treated with CM was significantly decreased

Transcriptomic screening identified upregulated CXCL8 expression, validated by qPCR (Fig. 3G). ELISA confirmed increased CXCL8 secretion in HK-2 supernatants following PS-NPs co-exposure (Fig. 3H). After CXCL8 binds to macrophage CXCR2 receptors, western blotting demonstrated significantly elevated CXCR2 protein in PS-NPs + CaOx-CM-treated macrophages versus CaOx alone (Fig. 3I, J).

For mechanistic validation, lentiviral CXCL8 knockdown was performed in HK-2 cells. Post-knockdown ELISA confirmed reduced CXCL8 secretion in co-exposure groups (Fig. 3K). Concomitantly, CXCL8 knockdown significantly attenuated PS-NPs-enhanced macrophage chemotaxis (Fig. 3L, M) and M1 polarization (Fig. 3N; Figure S1E). Furthermore, CXCR2 protein expression decreased in macrophages treated with CM from CXCL8-knockdown HK-2 cells (Fig. 3O, P).

PS-NPs exposure aggravated renal tubular epithelial cell ferroptosis in CaOx kidney stone cell model by decreasing xCT expression

Based on our previous findings demonstrating the involvement of ferroptosis in CaOx kidney stone formation [24, 29–31], we sought to investigate whether additional exposure to PS-NPs would further damage RTECs through ferroptosis-related pathways. Analysis of transcriptome sequencing data revealed that additional PS-NPs exposure significantly downregulated the expression of xCT, a key ferroptosis-related gene. Therefore, we first validated the transcriptome sequencing results using qPCR, confirming that xCT was indeed downregulated at the transcriptional level (Fig. 4A). Subsequently, we performed WB to detect ferroptosis-related proteins. We found that with PS-NPs exposure, the protein expression levels of xCT and GPX4 significantly decreased, while FTL protein expression significantly increased (Fig. 4B, C).

Fig. 4.

Additional exposure to PS-NPs exacerbates ferroptosis in the CaOx kidney stone model cells. A qPCR analysis confirmed the transcriptome sequencing results, showing that xCT expression at the transcriptional level was significantly decreased following additional PS-NPs exposure. B, C WB analysis revealed that ferroptosis-related proteins xCT and GPX4 were significantly reduced, while FTL expression significantly increased, confirming the exacerbation of ferroptosis. D GSH levels in HK-2 cells were significantly reduced following PS-NPs exposure. E–H After additional PS-NPs exposure, mitochondrial Fe2 + accumulation (E) significantly increased, lipid peroxidation levels (F) were significantly elevated, mitochondrial membrane potential (G) was reduced, and ROS levels (H) increased in the CaOx kidney stone model cells. I, J After ferroptosis inhibition with Fer-1, ferroptosis-related protein expression (xCT, GPX4) significantly increased, while FTL expression decreased, suggesting that the ferroptosis levels induced by PS-NPs exposure were significantly reduced. K–M After Fer-1 treatment, mitochondrial Fe2 + accumulation (K) was reduced, lipid peroxidation levels (L) decreased, and mitochondrial membrane potential (M) was restored in cells exposed to PS-NPs

Given that ferroptosis is characterized by iron accumulation, elevated cellular oxidative stress, and mitochondrial dysfunction, we conducted further related experiments. The results showed that with additional PS-NPs exposure, glutathione (GSH) levels significantly decreased in HK-2 cells (Fig. 4D), mitochondrial Fe²⁺ accumulated (Fig. 4E, Figure S1F), lipid peroxidation levels significantly increased (Fig. 4F, Figure S1G), mitochondrial membrane potential significantly decreased (Fig. 4G), and cellular ROS levels significantly increased (Fig. 4H).

Since our previous research has confirmed that xCT regulates ferroptosis during CaOx kidney stone formation [31, 32], we used the ferroptosis inhibitor Ferrostatin-1 (Fer-1, 8 μM) to directly inhibit ferroptosis and verify its role in CaOx kidney stone formation under additional PS-NPs exposure. The results demonstrated that after Fer-1 intervention, the protein expression levels of xCT and GPX4 significantly recovered, while FTL protein expression significantly decreased (Fig. 4I, J). Mitochondrial Fe²⁺ accumulation was reduced (Fig. 4K, Figure S1H), lipid peroxidation levels decreased (Fig. 4L, Figure S1I), mitochondrial membrane potential significantly recovered (Fig. 4M).

The upregulation of LRP6 expression could mitigate the damage to RTECs induced by the combined exposure to PS-NPs and CaOx crystals.​

Following the investigation of various pathological processes induced by additional PS-NPs exposure, we sought to identify a potential therapeutic target. Therefore, we re-analyzed the transcriptome sequencing data and identified a significant downregulation of low-density lipoprotein receptor-related protein 6 (LRP6), a key upstream gene in the Wnt signaling pathway. Considering the established roles of LRP6 in previous studies [33–35], we hypothesized that it might play a crucial regulatory role in this overall pathological process. To test this hypothesis, we first validated LRP6 expression using qPCR and WB. The results confirmed that PS-NPs exposure further reduced LRP6 expression at both the transcriptional level (Fig. 5A) and the protein level (Fig. 5B, C).

Fig. 5.

LRP6 is a potential therapeutic target in the process of CaOx kidney stone formation promoted by additional exposure to PS-NPs. A–C qPCR (A) and Western blot (WB) (B, C) analysis confirmed that LRP6 expression at both the transcriptional and protein levels was reduced in CaOx model cells exposed to PS-NPs. D, E In LRP6-overexpressing HK-2 cells, PS-NPs + CaOx intervention significantly decreased CaOx crystal adhesion. F, G WB analysis showed that LRP6 overexpression reduced VCAM1 expression after PS-NPs + CaOx intervention. H–J CM extracted from LRP6-overexpressing HK-2 cells significantly reduced macrophage chemotaxis (H, I) and M1 polarization levels (J). K The secretion of CXCL8 from PS-NPs + CaOx-treated HK-2 cells was also significantly reduced with LRP6 overexpression. L WB analysis revealed that CM extracted from LRP6-overexpressing PS-NPs + CaOx-treated HK-2 cells significantly reduced CXCR2 expression in macrophages. M–O After LRP6 overexpression in HK-2 cells, lipid peroxidation levels (M), mitochondrial Fe2 + accumulation (N), and intracellular ROS levels (O) were all significantly reduced after PS-NPs + CaOx co-treatment

Subsequently, we overexpressed LRP6 in HK-2 cells using a lentiviral vector (Figure S1J). Upon LRP6 overexpression, the adhesion of CaOx crystals, caused by the combined intervention of PS-NPs and CaOx, was significantly reduced (Fig. 5D, E). Western blot analysis confirmed significantly downregulated VCAM1 expression in co-exposed (PS-NPs + CaOx) HK-2 cells upon LRP6 overexpression (Fig. 5F, G). Furthermore, conditioned media (CM) from these HK-2 cells were harvested and used to treat macrophages. This intervention resulted in a significant decrease in macrophage chemotaxis (Fig. 5H–I) and M1 polarization levels (Fig. 5J, Figure S1K). Concurrently, CXCL8 levels in the supernatant of HK-2 cell cultures were significantly reduced upon LRP6 overexpression (Fig. 5K). Western blot analysis also indicated a reduction in CXCR2 protein expression in macrophages treated with this CM (Fig. 5L).

Additionally, the accumulation of mitochondrial Fe2 + (Fig. 5M, Figure S1L) and lipid peroxidation levels (Fig. 5N, Figure S1M) in HK-2 cells were markedly decreased. ROS levels were also reduced following LRP6 overexpression (Fig. 5O).

The addition of PS-NPs in drinking water promotes tubular calcium deposition, kidney injury and macrophage inflammatory activation in CaOx kidney stone model rats

To investigate whether PS-NPs exposure influences CaOx kidney stone formation at the in vivo level, we established a rat model. Calcium salt staining revealed that PS-NPs exposure significantly promoted renal calcium deposition (Fig. 6A), while fluorescence microscopy confirmed substantial renal accumulation of PS-NPs (Fig. 6B).

Fig. 6.

Additional exposure to PS-NPs in drinking water promotes CaOx kidney stone formation in SD rats. A Silver nitrate staining confirmed that PS-NPs exposure exacerbated calcium salt deposition in the kidneys of CaOx kidney stone model rats. B Fluorescently labeled PS-NPs were detected in the kidneys of treated rats, showing significant PS deposition. C–F Serum creatinine (C), blood urea nitrogen (D), renal injury marker NGAL (E), and renal ROS levels (F) were all significantly elevated in CaOx model SD rats following additional exposure to PS-NPs. G Immunofluorescence analysis revealed that VCAM1 protein expression in the kidneys of rats was significantly increased after additional exposure to PS-NPs. H Co-immunofluorescence staining for CD68 and CD86 indicated that PS-NPs exposure increased macrophage infiltration into the kidneys, accompanied by increased M1 polarization of macrophages. I Immunofluorescence analysis showed that xCT expression in the kidneys of CaOx kidney stone model rats was significantly reduced with additional PS-NPs exposure

Biochemical analysis of blood samples from experimental groups showed that PS-NPs exposure led to significantly elevated serum creatinine (CRE) (Fig. 6C) and blood urea nitrogen (BUN) levels (Fig. 6D). Immunohistochemical analysis confirmed significantly increased expression of the renal injury marker neutrophil gelatinase-associated lipocalin (NGAL) (Fig. 6E) and ROS (Fig. 6F) in rats exposed to PS-NPs. The immunofluorescence intensity of VCAM1 protein also increased in response to PS-NPs exposure (Fig. 6G). Concurrently, the number of M1-polarized macrophages infiltrating kidney tissue significantly increased (Fig. 6H). Immunofluorescence detection also indicated that additional PS-NPs exposure in the CaOx kidney stone model rats led to reduced protein expression of xCT (Fig. 6I).

Inhibition of LRP6 expression exacerbates PS-NPs exposure-induced renal injury and stone formation in a rat model of CaOx kidney stones

To validate the role of LRP6 in exacerbating CaOx kidney stone disease in rats exposed to PS-NPs, we intervened in the model rats with Salinomycin to inhibit LRP6 expression in the kidneys (Figure S1N). Subsequent analysis revealed that Salinomycin-treated PS-NPs + Stone model rats showed increased renal calcium salt deposition (Fig. 7A) and significantly elevated ROS levels (Fig. 7B). Additionally, CRE (Fig. 7C) and BUN (Fig. 7D) levels were further elevated, with a concomitant increase in NGAL expression (Fig. 7E). Immunofluorescence analysis showed that in Salinomycin-treated PS-NPs + Stone model rats, VCAM1 protein expression in the kidneys was further upregulated (Fig. 7F). Moreover, these rats exhibited increased macrophage M1 polarization levels (Fig. 7G). Immunofluorescence staining also indicated that Salinomycin intervention further reduced the protein expression levels of xCT (Fig. 7H).

Fig. 7.

Role of LRP6 in exacerbating CaOx kidney stone disease in PS-NPs exposed rats. A Silver nitrate staining shows increased renal calcium salt deposition in Salinomycin-treated PS-NPs + Stone model rats. B ROS levels were significantly elevated in the kidneys of Salinomycin-treated PS-NPs + Stone model rats. C, D Serum creatinine (CRE) (C) and blood urea nitrogen (BUN) (D) levels were further elevated in Salinomycin-treated PS-NPs + Stone model rats, indicating worsened renal function. E Neutrophil gelatinase-associated lipocalin (NGAL) expression was significantly increased in the kidneys of Salinomycin-treated PS-NPs + Stone model rats, indicating renal injury. F Immunofluorescence analysis revealed that VCAM1 protein expression in the kidneys was further upregulated in Salinomycin-treated PS-NPs + Stone model rats. G These rats showed increased macrophage M1 polarization levels, indicating an inflammatory response. H Immunofluorescence staining showed a significant reduction in xCT protein expression in Salinomycin-treated PS-NPs + Stone model rats, indicating inhibition of ferroptosis

Preliminary investigation of the effects of other plastics on CaOx kidney stone formation

Building on the polymer profiles identified in human CaOx stone specimens, we performed preliminary phenotypic assays with two additional common plastics—PE and PC. We found that supplementing with PE-NPs or PC-NPs likewise increased crystal–cell adhesion, macrophage chemotaxis, and ROS levels in HK-2 cells. However, because the present work focuses on the pro-pathogenic effects of PS-NPs (Fig. S2A–C), we do not pursue these observations further here.

Discussion

Accumulating evidence indicates that plastic pollution impacts human health through diverse pathways [36, 37]. Exposure routes for plastic components include dermal contact, inhalation, and dietary intake. As a major excretory organ, the kidney has garnered significant research attention [38, 39]. Meanwhile, emerging evidence has preliminarily suggested that long-term consumption of bottled water may exert adverse effects on human health, including a potential contribution to kidney stone formation [40]. Critically, this study provides the first evidence demonstrating that microenvironmental PS-NPs significantly promote the development and progression of CaOx kidney stone disease.​​

CaOx kidney stones represent a highly prevalent renal disorder, whose pathogenesis is closely linked to urinary solute supersaturation and renal tubular epithelial injury. Our investigation yielded a novel observation:​​ PS-derived plastics were detected within human CaOx kidney stone specimens. This finding prompted the pivotal question: Does PS merely adsorb onto existing stones, or does it actively participate in lithogenesis? Consequently, we investigated both fundamental etiological pathways of kidney stone formation.

For solutes to form crystals in supersaturated solutions, they must overcome the nucleation energy barrier [41]. Our in vitro experiments revealed that adding PS-NPs profoundly altered the morphology and zeta potential of synthesized CaOx crystals. We posit that within supersaturated urine, nanoplastics may serve as heterogeneous nucleation sites, substantially lowering the nucleation energy barrier and thereby promoting CaOx crystal formation. Furthermore, the reduction in absolute zeta potential enhances crystal aggregation—an effect that could precipitate the formation of obstructive crystal plugs in renal tubules. Notably, PS-NPs exposure yielded sharp-edged CaOx crystals with increased dimensions, presumably exacerbating their damaging potential to the renal tubular epithelium. Moreover, rigorous characterization of PS-NPs under the actual experimental conditions—particularly when suspended in culture medium—is essential, as proteins and other medium components can promote premature nanoparticle agglomeration [42]. In this study, the absolute magnitude of the zeta potential for PS-NPs suspended in DMEM/F12 basal medium was indeed lower than that measured in deionized water.

Tubular retention of CaOx crystals critically underlies kidney stone formation [43]. Retention occurs either when individual crystals grow beyond clearable dimensions or through aggregation into obstructive clusters. Retained crystals subsequently adhere tightly to and persistently interact with RTECs [44]. Significantly, our study demonstrates that PS-NPs exposure enhanced CaOx adhesion in both animal and cell models, with VCAM1 playing a pivotal role. VCAM1—a key cell adhesion molecule previously linked to infectious stones [45]—exhibited increased expression in our models, consistent with its established adhesion function. Moreover, PS-NPs-induced alterations in CaOx crystal zeta potential represent an additional mechanistic contributor to enhanced adhesion beyond upregulated adhesion proteins. These findings suggest future therapeutic strategies could target both antioxidant pathways and surface charge modulation to disrupt crystal adhesion.

Enhanced inflammatory responses during lithogenesis promote macrophage chemotaxis and M1 polarization [46, 47]. Critically, we observed that combined PS-NPs + CaOx exposure provoked significantly greater CXCL8 secretion from RTECs than CaOx alone. As CXCL8 is a potent chemoattractant that drives macrophage recruitment and M1 polarization [48, 49], its overproduction exacerbates local inflammation—a finding directly supported by our results showing that inhibiting tubular CXCL8 expression/secretio markedly reduced macrophage chemotaxis and M1 polarization.

Ferroptosis—an iron-dependent form of regulated cell death characterized by lethal lipid peroxidation [50]—is modulated by the xCT-GPX4 axis. This pathway suppresses ferroptosis by eliminating cytotoxic lipid peroxides [51, 52]. Our prior research definitively established ferroptosis as a key driver in CaOx nephrolithiasis [24, 29–31, 53]. Here, transcriptome sequencing revealed transcriptional downregulation of xCT, confirmed by qPCR. Consequently, attenuated xCT expression at both transcriptional and protein levels (with concomitant GPX4 reduction) intensified ferroptosis, amplifying renal tubular epithelial cell injury.

The Wnt/β-catenin signaling pathway is a crucial intracellular cascade that regulates multiple physiological and pathological processes, including cell growth, tissue homeostasis, inflammation, and apoptosis [54, 55]. LRP6 serves as a membrane co-receptor for canonical Wnt/β-catenin signaling, and its stable phosphorylation status and protein expression levels are essential [56–58]. Given the established broad regulatory roles of LRP6 across various physiological and pathological contexts, we hypothesized that its downregulation might play a critical role in mediating renal tubular epithelial cell injury, macrophage polarization, and chemotaxis induced by combined PS-NPs and CaOx crystal exposure. Our experimental results confirmed this hypothesis.

At the cellular level, lentiviral-mediated overexpression of LRP6 alleviated renal tubular epithelial cell injury, reduced crystal adhesion, attenuated ferroptosis, and diminished the inflammatory response, consequently decreasing macrophage chemotaxis and M1 polarization. At the animal level, intervention with the LRP6 inhibitor Salinomycin sodium reduced renal LRP6 expression in SD rats, exacerbating renal calcium salt deposition, increasing macrophage M1 polarization and infiltration, and elevating ferroptosis levels in RTECs. These findings collectively indicate the critical role of LRP6 in the pathogenesis and progression of PS-NPs-aggravated CaOx kidney stone disease.

Given that multiple plastic constituents were detected in human calcium oxalate stone specimens, we additionally conducted preliminary phenotypic assessments on two other microplastic types. Because this study primarily focuses on the role of PS in the pathophysiology of calcium oxalate nephrolithiasis, we did not comprehensively evaluate the effects of other plastics on stone formation. This limitation underscores the need for broader and more in-depth investigations into the relationships between environmental pollutants and urolithiasis in future work.

Several limitations inherent to this study should be acknowledged. First, the PS-NPs exposure concentration used in our experiments was derived by integrating reported levels of human plastic exposure from prior studies with concentrations commonly adopted in experimental models of other organ systems. While this strategy is theoretically reasonable and provides a pragmatic basis for dose selection, it must be recognized that environmental contaminants—including heavy metals and microplastics/nanoplastics—often display organ-specific biodistribution after entering the body. Regrettably, despite an extensive literature search, we were unable to identify quantitative data describing the burden or concentration of plastic particles within renal compartments, including the interstitium, renal vasculature, or tubular lumen. Moreover, kidney biopsy in patients with calcium oxalate nephrolithiasis is ethically unjustifiable given the benign nature of the disease. Accordingly, after careful consideration, we employed the calculated concentration to enable an initial, clinically relevant exploration of the impact of PS-NPs on calcium oxalate stone disease.

Second, salinomycin was used as a pharmacological inhibitor of LRP6 to interrogate its role in CaOx stone formation. Although salinomycin is widely recognized as an inhibitor of the Wnt/β-catenin pathway through suppression of LRP6, its action is not fully target-specific. As an ionophore, salinomycin can modulate membrane ion transport and perturb intracellular Na⁺, Ca²⁺, and H⁺ homeostasis, thereby influencing fundamental cellular physiology. In addition, salinomycin may elicit stress responses, including oxidative stress and endoplasmic reticulum stress, which could partially confound interpretation of injury-related mechanisms. Importantly, these potential off-target effects do not negate our overall conclusion regarding the pivotal involvement of LRP6 in the pathological process. Nevertheless, we anticipate that future studies employing more specific genetic approaches—such as renal tubular conditional LRP6 knockout models or retrograde lentiviral delivery via renal pelvic injection—will be required to more definitively validate the causal contribution of this target.

Finally, the Bliss independence model depends on highly precise and stable input data. Given the intrinsic variability of biological assays (e.g., CCK-8), we present single-concentration Bliss calculations to facilitate interpretation and provide a preliminary assessment of synergy. Future work should incorporate more stable multi-dose datasets to enable rigorous, concentration–response–based validation and computation.

In this study, we established both in vitro and in vivo CaOx kidney stone models with concurrent PS-NPs exposure. Through comprehensive investigation of crystal-cell adhesion, macrophage chemotaxis and polarization, and renal tubular epithelial injury, we demonstrated that widespread human PS exposure promotes CaOx kidney stone formation. We identified key regulatory genes (VCAM1, CXCL8-CXCR2, xCT) involved in these pathological processes and confirmed LRP6's pivotal regulatory role over these pathways. These results lead us to speculate: Could the increasing global burden of PS pollution be a significant contributing factor to the rising incidence and recurrence rates of kidney stones? Regardless, to our knowledge, this study is the first to demonstrate that PS-NPs—previously unrecognized lithogenic cofactors that are effective at least within certain concentration ranges—can interact with CaOx crystals to enhance their adhesion to renal tubular epithelial cells and, through modulation of LRP6 signaling, further amplify inflammation and ferroptosis. Collectively, our findings suggest that microplastic pollution may represent an emerging environmental risk factor for the development and progression of kidney stones, while also providing new mechanistic rationale and perspectives for targeting LRP6 as a potential therapeutic strategy.

Author Contribution

Xiaozhe Su, Yijun Yang and Heng Xiang contributed equally to this work and share the first authorship. Conceptualization: Caitao Dong, Heng Xiang, Yijun Yang Investigation: Xiaozhe Su, MingCheng Shi, Heng Xiang, Yufei Wang, Sixing Yang. Software: Xiaozhe Su, Qianlin Song, Caitao Dong, Qixuan Zhou, Qinhong Jiang, Bobo Chen, Yijun Yang. Analysis and Validation: Sixing Yang, Xiaozhe Su, Heng Xiang, Qixuan Zhou, Caitao Dong. Primary Draft Writing: Xiaozhe Su, Caitao Dong, Sixing Yang, Yunhan Wang, Qianlin Song, Yunhe Xiong.

Funding

This work was supported by the Natural Science Foundation of China, grant number No. 42107219and No. 82070723. The Nature Science Foundation of Hubei Province (Grant No. 2023AFB691). The Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University (Grant No. JCRCYR-2022-017). The Open Projects Funds for the Research Base of Regulatory Science for Medical Devices of Wuhan University, National Medical Products Administration (2023JDKF002).

Data availability

The original data can be made available upon reasonable request.

Conflicts of Interest

All authors declare that there are no conflicts of interest of any kind.

Ethical approval

The animal experiments in this study were approved by the Animal Ethics Committee of the Renmin Hospital of Wuhan University (Issue No. 20230310F). The collection and testing of human calcium oxalate kidney stone samples were reviewed by the Clinical Research Ethics Committee of Renmin Hospital of Wuhan University (Ethics No. WDRY2023-K183).