Abstract

   This study aims to investigate the role of hypercalciuria and
   pyroptosis in the formation of calcium oxalate kidney stones.
   Bioinformatics analysis was performed to compare the correlation of
   pyroptosis scores and cell adhesion scores between Randall’s plaques
   and normal tissues from kidney stone patients. For the in vitro
   experiments, we investigated the effects of high concentrations of
   Ca^2+ on the pyroptosis and adhesion levels of renal tubular epithelial
   cells and examined the adhesion levels and crystal aggregation of the
   cells in high Ca^2+ concentrations environment by knockdown and
   overexpression of the key pyroptosis gene, GSDMD, and we verified the
   effects of Ca^2+ concentration on pyroptosis and adhesion levels,
   kidney injury, and crystal deposition by in vivo experiments.
   Bioinformatic results showed that the scores of pyroptosis and cell
   adhesion in Randall’s plaques of patients with kidney stones were
   significantly higher than those in normal tissues, and pyroptosis was
   highly positively correlated with cell adhesion. In vitro and in vivo
   experiments showed that high concentrations of Ca^2+ activated the
   NLRP3/Caspase-1/GSDMD pathway of pyroptosis through ROS and
   up-regulated the expression of adhesion-related proteins, and GSDMD
   could regulate the adhesion level of renal tubular epithelial cells by
   mediating the level of pyroptosis, thereby affecting the adhesion and
   deposition of calcium oxalate crystals. Our findings reveal that the
   Ca^2+-induced classical pyroptosis pathway may be a potential mechanism
   to promote calcium oxalate kidney stone formation, which provides new
   insights into the etiology of kidney stones.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-91460-8.

   Keywords: Pyroptosis, Cell adhesion, Hypercalciuria, Reactive oxygen
   species, Kidney stones

   Subject terms: Renal calculi, Kidney diseases, Mechanisms of disease

Introduction

   Kidney stones are a urologic disease with relatively high incidence and
   recurrence rates worldwide, with prevalence rates of 7–14%, 5–9%, and
   1–5% in North America, Europe, and Asia, respectively^[38]1. The
   majority of kidney stones are calcium oxalate (CaOx) stones^[39]2. The
   renal tubules are the main kidney components injured by stone
   obstruction and calcium oxalate^[40]3,[41]4. The main process of renal
   stone formation is urine supersaturation, crystal nucleation, and
   migration to the surface of renal tubular epithelial cells
   (RTECs)^[42]5, and the deposition of crystals in the kidneys leads to
   oxidative stress and upregulates the expression of inflammatory
   factors^[43]6, which promotes RTECs damage. The membrane structures of
   the injured RTECs such as microvilli and tight junctions will be
   disrupted, thereby increasing cell adhesion to crystals, and the
   adhesion is critical for the nucleation and growth of stones^[44]7.

   Randall’s plaque is considered the precalculus lesion to renal calculi,
   and the precalculus lesions were classified into Randall’s plaque and
   Randall’s plugs, termed Randall’s plaque type I and II^[45]8. Randall’s
   plaques play an important role and are a prerequisite for calcium
   oxalate stone formation and growth in idiopathic calcium oxalate stone
   formers, and approximately 75% of calcium oxalate stones are formed
   attached to sites of Randall’s plaques^[46]9,[47]10.

   Pyroptosis is a newly discovered type of programmed cell death and
   natural immune response of the body^[48]11,[49]12, which is widely
   involved in the development and regulation of many diseases, such as
   metabolic diseases^[50]13,[51]14, renal fibrosis^[52]15,[53]16, and the
   development and metastasis of many tumors^[54]17,[55]18. Kidney stones
   are also one of the metabolic diseases and its pathogenesis is still
   unclear. It has shown that molecules of the classical pyroptosis
   pathway are related to the formation of kidney stones^[56]19,[57]20.
   RTECs could undergo oxidative stress injury in the environment of CaOx
   crystals, leading to the activation of NLRP3 inflammasome and the
   production of inflammatory factors through pyroptosis^[58]21,[59]22,
   while inhibiting NLRP3 expression can reduce the pyroptosis level of
   RTECs and alleviate the damage caused by CaOx crystal deposition in
   kidney tissue^[60]23. In addition, vitexin could down-regulate the
   expression of pyroptosis-related proteins, and inhibit the apoptosis of
   RTECs and the expression of adhesion-related proteins in kidney stone
   model mice, indicating that inhibition of pyroptosis has a protective
   effect on kidney stones^[61]24.

   The most common metabolic abnormality in patients with kidney stones is
   hypercalciuria, which accounts for approximately 40–60% of kidney stone
   cases^[62]25,[63]26. Hypercalciuria can induce crystal formation in the
   renal tubule lumen, leading to supersaturation, precipitation, tubular
   obstruction, cellular damage, and a significant increase in the risk of
   kidney stones^[64]27. Most of the previous studies demonstrated the
   effects of oxalate and CaOx on pyroptosis. This study is the first to
   investigate the mechanism of pyroptosis triggered by elevated Ca^2+ on
   kidney stone formation. Moreover, the 24-hour urinary Ca^2+
   concentration of patients with recurrent stones was nearly 50% higher
   than that of the nonrecurrent group, which suggests that hypercalciuria
   is also a major predictor of kidney stone recurrence^[65]28. Therefore,
   exploring the potential relationship between Ca^2+-induced pyroptosis
   and crystal adhesion will not only provide new insights and targets for
   the pathogenesis of kidney stones, but also be of great significance
   for the prevention of clinical kidney stone recurrence.

Results

Identification of DEGs and enrichment analysis

   We screened a total of 525 differentially expressed genes (DEGs), which
   are shown in the volcano plot and heatmap (Fig. [66]1a, b). The DEGs
   identified in [67]GSE73680 contained 297 upregulated genes and 228
   downregulated genes based on |log2FC| > 0.5 and p < 0.05. Then, Gene
   Ontology (GO) enrichment analysis was performed on the upregulated and
   downregulated genes (Fig. [68]1c), and the enriched functions included
   nephron tubular epithelial cell differentiation, mesenchymal cell
   differentiation, metanephric collecting duct development, mesenchyme
   development, and cell differentiation involved in kidney development.
   The enrichment results were visualized with a cnetplot (Fig. [69]1d,
   e), where each node represents a gene, and each edge represents an
   interaction between two genes. Cnetplots are used to accurately
   determine key signaling pathways and their upstream and downstream
   genes. In the lower-left corner of the figure, the genes involved in
   the differentiation of renal epithelial cells are interconnected. The
   graph links genes and biological concepts (such as GO terms or Kyoto
   Encyclopedia of Genes and Genomes [KEGG] pathways) into a network for
   viewing important genes involved in multiple enriched pathways that
   possibly belong to multiple annotation categories. The results
   demonstrated the top three functions with the most significant p-value:
   regulation of nephron tubular epithelial cell differentiation,
   regulation of epithelial cell differentiation involved in kidney
   development and nephron tubular epithelial cell differentiation, as
   well as the core genes that regulate these functions: STAT1, PAX2,
   PAX8, PROM1 and CD24 (Fig. [70]1e). The results of KEGG pathway
   enrichment analysis showed that cell adhesion, lysosomal, and oxidative
   phosphorylation pathways that may be associated with cell death were
   active in the kidney stone group (Fig. [71]1f).

Fig. 1.

   [72]Fig. 1
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   (a) Volcano plot and (b) heatmap of differentially expressed genes
   between the Randall plaque tissues of CaOx stone formers and normal
   tissues of CaOx stone patients (group P vs. group N). (c-e) GO
   enrichment analysis. (f) Enrichment analysis of the KEGG pathway.

WGCNA of coexpression modules related to pyroptosis and cell adhesion

   The results of sample clustering showed that there were no outliers. To
   ensure that the interaction between genes conforms to a scale-free
   distribution to the greatest extent, we first determined the soft
   threshold of the data and generally chose the lowest soft power
   threshold with a scale-free topology Model Fit greater than or equal to
   0.8. Here, we chose 8 as the soft threshold for the subsequent analysis
   (Fig. [74]2a). A total of 21 modules were clustered by constructing the
   coexpression network (Fig. [75]2b). After determining the existence of
   21 coexpression modules in the gene expression data, we used the sample
   grouping information as a phenotype to analyze the correlation between
   each module and the N, C, and P groups. Red represents a positive
   correlation between the expression module and grouping, while blue
   represents a negative correlation (Fig. [76]2c). For example, the MEred
   module was positively correlated with group P, with a correlation of
   0.29, p < 0.05.

Fig. 2.

   [77]Fig. 2
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   WGCNA of coexpression modules related to pyroptosis and cell adhesion.
   (a) Determination of soft threshold. (b–c) Dendrogram of cluster
   modules. (group P: Randall’s plaque tissues from CaOx stone formers,
   group N: normal tissues from CaOx stone patients, and group C: healthy
   controls). (d) Ca^2+ pathway enrichment analysis. (e) Heatmap of
   correlation between coexpression modules and different samples.
   Coexpression network of genes related to (f) pyroptosis and (g) cell
   adhesion in the MEred module. (h) Coexpression network of
   pyroptosis-related genes in the MEblack module. (i)Coexpression network
   of GSDMD. (j) Interaction analysis of GSDMD.

   The Ca^2+ pathway plays an important role in kidney stones. We
   performed enrichment analysis on the screened modules, and the results
   showed that the turquoise module contained the largest number of Ca^2+
   pathway-related genes (Fig. [79]2d). Therefore, we screened the
   turquoise module as a key module associated with kidney stones. The
   results of single-sample enrichment analysis showed that the MEBlue,
   MEroyalblue, MEred, and MEblack modules were positively correlated with
   pyroptosis (Fig. [80]2e). Combined with the above analysis, the MEBlue,
   MEroyalblue, MEred, MEblack, and turquoise modules were selected as the
   key modules associated with pyroptosis and the Ca^2+ pathway in kidney
   stone development. We further looked at the hub core genes that
   appeared in the pyroptosis gene set in these four modules and mapped
   the network using Cytoscape (Fig. [81]2f–i), where the labeled
   pyroptosis-related genes are in the center of the network, including
   MUC20, MST1, SLA16A4, VDR, and HKDC1 in the red module, which are
   related to epithelial cell differentiation (Fig. [82]2f). The MEblack
   module was associated with immune regulation and included NLRP7, IL-32,
   and PRF1 (Fig. [83]2h). The MEred module was most closely related to
   renal tubular epithelial cells, and we examined the coexpression
   network of genes in this module that were regulated by genes related to
   cell adhesion, among which the hub genes were MUC1, EPCAM1, CCN3, and
   ATP1B1 (Fig. [84]2g). In the gene coexpression network of the turquoise
   module, although a small number of pyroptosis-related genes were
   involved in this module, the classical pyroptosis gene gasderminD
   (GSDMD) was included (Fig. [85]2i). Subsequently, we found that the
   genes interacting with GSDMD included NLRP3, cysteinyl aspartate
   specific proteinase-1 (Caspase-1), GSDMD, IL-1β, and IL-18 through
   STRING protein interaction analysis (Fig. [86]2j).

Functional enrichment analysis of key pyroptosis modules

   GO enrichment analysis was used to identify the biological functions of
   the above MEblue, MEroyalblue, MEred, and MEblack modules. The results
   showed that the MEblue module was related to the regulation of mRNA
   catabolic processes (Supplementary Fig. 1a), the MEroyalblue module was
   associated with RNA variable shearing (Supplementary Fig. 1b), the
   final MEred module was related to the regulation of renal tubular
   development and differentiation of renal tubular epithelial cells
   (Supplementary Fig. 1c), and the MEblack module was associated with the
   immunomodulation of lymphocyte-mediated immunity, activation of signal
   transduction by immune response, complement activation, B-cell-mediated
   immunity, immunoglobulin-mediated immune response, and humoral immune
   response (Supplementary Fig. 1d).

Differential expression analysis of modular genes and scoring for pyroptosis
and cell adhesion

   We compared the expression levels of the core pyroptosis- and cell
   adhesion-related genes in the modules of groups P and N. In the MEblue
   module, the expression levels of the pyroptosis-related genes ANXA2 and
   CHMP4A were upregulated in group P, while ASIC1 was downregulated in
   group P (Fig. [87]3a). The pyroptosis-related genes HKDC1, MST1,
   SLC16A4, and VDR and the cell adhesion-related gene MUC1 were highly
   expressed in group P in the MEred module (Fig. [88]3b, c). However, the
   pyroptosis-related genes in the MEblack module showed no significant
   difference between groups P and N (Fig. [89]3d). The MEred module,
   which was the most relevant to the function of epithelial cells, was
   used to identify the core pyroptosis-related genes SLC16A4, HKDC1, VDR,
   MST1, and MUC20 and the core cell adhesion-related genes MUC1, ATP1B1,
   CCN3, and EPCAM and to single-sample gene set enrichment analysis
   (GSEA) was used to score the samples of the P and N groups. The results
   showed that the pyroptosis and cell adhesion scores of group P were
   significantly higher than those of group N (Fig. [90]3e, f). Finally,
   we analyzed the correlation between the pyroptosis score and cell
   adhesion score, and the results showed that pyroptosis was highly and
   positively correlated with cell adhesion (Fig. [91]3g, R = 0.91).

Fig. 3.

   [92]Fig. 3
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   Expression levels of core pyroptosis-related genes in the (a) MEblue
   and (d) MEblack modules, expression levels of core (b) pyroptosis- and
   (c) cell adhesion-related genes in the MEred module, and scores of (e)
   pyroptosis and (f) cell adhesion in group P and N (group P: Randall’s
   plaque tissues from CaOx stone formers, and group N: normal tissues
   from CaOx stone patients). (g) Correlation between pyroptosis score and
   cell adhesion score.

High Ca^2+ promoted renal tubular epithelial cell injury

   The CCK-8 results showed a gradual decrease in cell viability with
   increasing concentrations of Ca^2+, and the lactate dehydrogenase (LDH)
   assay results showed that the level of LDH released from the cells and
   the degree of cell damage increased as the concentration of Ca^2+
   increased. Cell viability was significantly decreased when the
   concentration of Ca^2+ reached 10 mM (Fig. [94]4a), while LDH release
   was significantly increased when the concentration of Ca^2+ reached 20
   mM compared to the control treatment (Fig. [95]4b). According to the
   above experimental results, we used 10 mM Ca^2+ for subsequent
   experiments.

Fig. 4.

   [96]Fig. 4
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   In vitro: Ca^2+ promotes renal tubular epithelial cell injury in a
   concentration- and time-dependent manner. Cell viability (a) and LDH
   activity in the media (b). Pyroptosis- and adhesion-related protein
   expression in the HK-2 cells using ELISA (c) and Western Blot images
   and their graphical quantification (d). *p < 0.05; **p < 0.01;
   ****p < 0.0001.

   It has been shown that 5 mM Ca^2+ is the normal physiological level to
   which renal tubular epithelial cells may be exposed^[98]27. ELISA
   (Fig. [99]4c) and Western Blot (Fig. [100]4d) results also showed that
   the expression levels of the pyroptosis-related proteins NLRP3,
   Caspase-1/p20, GSDMD, GSDMD-N, IL-1β, IL-18, and the adhesion-related
   proteins osteopontin (OPN), CD44, monocyte chemotactic protein-1
   (MCP-1), and integrin beta 1 (ITGB1) were not significantly different
   in the HK-2 cells compared with the Control group after treatment with
   5 mM of Ca^2+ for 24 h, 48 h, and 72 h, so we used 5 mM as a control
   for subsequent experiments. In addition, 20 mM Ca^2+ was used to
   confirm the effect of a high Ca^2+ concentration on cell damage in a
   concentration-dependent manner.

High Ca^2+ promoted pyroptosis and adhesion changes in HK-2 cells

Pyroptosis-related microstructural changes in Ca^2+-treated HK-2 cells

   The transmission electron microscopy results showed that HK-2 cells in
   the control group had normal morphology and intact cell membranes.
   Compared with the 5 mM Ca^2+ group, the cells in the group treated with
   a high concentration of Ca^2+ showed obvious pyroptosis-related
   microstructural changes such as abnormal cell morphology, local damage
   to the cell membrane, cell-matrix overflow, free organelles in some
   areas and many vacuoles (Fig. [101]5a), and the above situation was
   more severe in the 20 mM Ca^2+ group than in the 10 mM group.

Fig. 5.

   [102]Fig. 5
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   In vitro: High Ca^2+ upregulates the expression of pyroptosis- and
   adhesion-related proteins in a concentration- and time-dependent
   manner. The microstructural changes of cells in the 5mM, 10 mM, and 20
   mM Ca^2+ groups: abnormal cell morphology, local damage to the cell
   membrane (red arrow), cell-matrix overflow, free organelles in some
   areas (green arrow) and many vacuoles (blue arrow) using a transmission
   electron microscopy (a). Pyroptosis- and adhesion-related protein
   expression in the HK-2 cells using Western Blot images and their
   graphical quantification (b) and ELISA (c). *p < 0.05; **p < 0.01;
   ***p < 0.001.

Ca^2+ treatment increased the expression levels of pyroptosis and
adhesion-related proteins

   The Western blot (Fig. [104]5b) and ELISA (Fig. [105]5c) results showed
   that the expression levels of pyroptosis-related proteins NLRP3,
   Caspase-1/p20, GSDMD, GSDMD-N, IL-1β, and IL-18 and adhesion-related
   proteins OPN, CD44, MCP-1, and ITGB1 were significantly increased in
   HK-2 cells treated with Ca^2+ (10 mM and 20 mM [n = 3]), and the effect
   of Ca^2+ on the expression levels of these proteins was concentration-
   and time-dependent.

GSDMD expression regulated pyroptosis in HK-2 cells

   We regulated the pyroptosis level of HK-2 cells by knocking down and
   overexpressing GSDMD, a key gene in the classical pyroptosis pathway.
   The qPCR results showed that the mRNA expression levels of the
   pyroptosis-related genes GSDMD, IL-1β, and IL-18 were significantly
   downregulated and upregulated with GSDMD knockdown and overexpression
   after 10 mM Ca^2+ treatment for 24 h, 48 h, and 72 h, respectively
   (Supplementary Fig. 2a). However, the mRNA expression levels of NLRP3
   and Caspase-1 were not affected by changes in the expression levels of
   GSDMD (Supplementary Fig. 2a). In addition, similar results were
   demonstrated by Western blot (Supplementary Fig. 2b) and ELISA
   (Supplementary Fig. 2c).

GSDMD expression regulated the adhesion of HK-2 cells

   We similarly regulated the level of pyroptosis by knocking down and
   overexpressing GSDMD to investigate the effect on HK-2 cell adhesion.
   The qPCR results showed that the mRNA expression levels of the
   adhesion-related genes OPN, CD44, MCP-1, and ITGB1 were significantly
   decreased in the sh-GSDMD group and increased in the GSDMD group
   (Fig. [106]6a), respectively. The Western blot (Fig. [107]6b) and ELISA
   (Fig. [108]6c) results also showed that the expression levels of these
   proteins were positively correlated with the expression levels of
   GSDMD. In addition, the results of scanning electron microscopy
   (Fig. [109]6d) also showed that the number of adherent crystals on the
   surface of the sh-GSDMD group was significantly lower than that on the
   surface of the sh-NC group when treated with COM and 10 mM Ca^2+, while
   the results were the opposite in the GSDMD group, and the number of
   adherent crystals on the cell surface was positively correlated with
   time.

Fig. 6.

   [110]Fig. 6
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   In vitro: GSDMD regulates the expression of adhesion-related proteins
   in HK-2 cells. Adhesion-related protein expression in the HK-2 cells
   using qPCR (a), Western Blot images and their graphical quantification
   (b), and ELISA (c). The number of adherent crystals (red arrow) on the
   cell surface of the four groups (sh-NC, sh-GSDMD, GSDMD-NC, and GSDMD)
   using a scanning electron microscopy (d). Significant differences in
   the GSDMD knockdown and overexpression groups compared to their
   negative controls. *p < 0.05; **p < 0.01; ***p < 0.001.

Ca^2+ treatment promoted the production of ROS in HK-2 cells to activate the
NLRP3 inflammasome

Ca^2+ treatment induced oxidative stress injury in HK-2 cells

   HK-2 cells were treated with 5 mM, 10 mM, and 20 mM Ca^2+ for 24 h. The
   results of the laser confocal microscopy showed that the level of
   reactive oxygen species (ROS) in the high Ca^2+ concentration treatment
   group was higher than that in the 5 mM Ca^2+ group (Fig. [112]7a, b).
   The results of flow cytometry also showed that the levels of ROS were
   significantly increased in the 10 mM and 20 mM Ca^2+ groups compared
   with the 5 mM Ca^2+ group (Fig. [113]7c).

Fig. 7.

   [114]Fig. 7
   [115]Open in a new tab

   In vitro: Interconnectivity between oxidative stress and NLRP3
   activation in HK-2 cells stimulated with Ca²⁺ (5 mM, 10 mM, and 20 mM
   [n = 6]) at 24 h, and pretreated or not with NAC (5.0 mM [n = 3]) at
   2 h. Immunofluorescence images (a) and their graphical quantification
   (b), flow cytometry (c), cell viability (d), Western Blot images and
   their graphical quantification for NLRP3 expression (e). **p < 0.01;
   ***p < 0.001; ****p < 0.0001.

ROS activated NLRP3 inflammasome

   The CCK-8 results showed that the cell viability of HK-2 cells
   decreased after treatment with different concentrations of the ROS
   inhibitor N-acetylcysteine (NAC) (1.0, 2.5, 5.0, 7.5, 10 mM) for 2 h.
   The cell viability significantly decreased in the group treated with
   7.5 mM NAC compared with that of the control group (Fig. [116]7d).
   Therefore, a concentration of 5 mM was used to pretreat the cells for
   2 h. Then, the cells were treated with 10 mM Ca^2+ for 24 h. The
   Western Blot results showed that compared with the 10 mM Ca^2+ group,
   the NAC + 10 mM Ca^2+ group exhibited significantly decreased
   expression levels of NLRP3 (Fig. [117]7e).

High Ca^2+ up-regulated the level of pyroptosis and adhesion to promote renal
tissue damage and calcium oxalate crystal deposition

   We established a mouse model of kidney stones and intervened in the
   urinary calcium concentration in model mice by using potassium citrate
   (PC). The results of the automatic biochemical analyzer showed that the
   urinary calcium concentration of the glyoxylic acid (GA) group was
   significantly higher than that of the Control group, while the urinary
   calcium concentration of GA + 20% PC group was significantly decreased
   (Fig. [118]8a). Subsequently, qPCR (Fig. [119]8b), Western Blot
   (Fig. [120]8c), and immunofluorescence (IF) (Fig. [121]9) results
   showed that the expression levels of pyroptosis-related proteins NLRP3,
   Caspase-1/p20, GSDMD/-N, IL-1β, and IL-18 in the kidney tissues of the
   GA group were significantly higher than those of the Control group, and
   compared with the GA group, the expression of related proteins in the
   kidney tissue of the GA + 20% PC group was significantly
   down-regulated. We next examined the effect of reducing the urinary
   calcium concentration on renal function and histologic changes in the
   mice. Deleterious tissue changes in the medulla (tubular necrosis and
   casts formation) by periodic acid-Schiff (PAS) staining were more
   evident in kidneys from the mice in the GA group than Control group and
   GA + 20% PC group (Fig. [122]8d). In addition, The results revealed
   that the mice in GA + 20% PC group have a decrease in blood urea
   nitrogen (BUN) and serum creatinine (Scr) as compared with the GA group
   (Fig. [123]8e).

Fig. 8.

   [124]Fig. 8
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   In vivo: Treatment with potassium citrate (PC). (a) 24-hour urinary
   calcium levels. Pyroptosis-related protein expression in the kidney
   tissues using qPCR (b) and Western Blot images and their graphical
   quantification (c). Periodic acid-Schiff (PAS) staining (d), serum
   creatinine, and blood urea nitrogen levels (e). Significant differences
   in the GA group compared to the Control and GA + 20% PC groups.
   *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 9.

   [126]Fig. 9
   [127]Open in a new tab

   In vivo: Treatment with potassium citrate (PC). Pyroptosis-related
   protein expression in the kidney tissues using immunofluorescence
   images and their graphical quantification. Significant differences in
   the GA group compared to the Control and GA + 20% PC groups.
   **p < 0.01; ****p < 0.0001.

   To confirm the results of Ca^2+ on the in vitro cell lines at the level
   of adhesion using an in vivo mouse model, we similarly examined the
   effect of decreasing urinary calcium concentration on the level of cell
   adhesion and the development of crystal deposition in mouse renal
   tissues. qPCR (Fig. [128]10a), Western Blot (Fig. [129]10b), and IF
   (Fig. [130]11) results showed that the expression levels of
   adhesion-related proteins OPN, CD44, MCP-1, and ITGB1 in the kidney
   tissues of the GA group were significantly up-regulated compared with
   the Control group, and the expression of these proteins in the kidney
   tissue of the GA + 20% PC group was significantly lower than that of
   the GA group. Moreover, the development of crystal deposition by Von
   Kossa staining was less obvious in kidneys from the mice in the
   GA + 20% PC group than GA group (Fig. [131]10c).

Fig. 10.

   [132]Fig. 10
   [133]Open in a new tab

   In vivo: Treatment with potassium citrate (PC). Adhesion-related
   protein expression in the kidney tissues using qPCR (a) and Western
   Blot images and their graphical quantification (b). Von Kossa staining
   (c). Significant differences in the GA group compared to the Control
   and GA + 20% PC groups. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 11.

   [134]Fig. 11
   [135]Open in a new tab

   In vivo: Treatment with potassium citrate (PC). Adhesion -related
   protein expression in the kidney tissues using immunofluorescence
   images and their graphical quantification. Significant differences in
   the GA group compared to the Control and GA + 20% PC groups.
   ****p < 0.0001.

   Together, these results from Figs. [136]8, [137]9, [138]10 and [139]11
   suggest that reducing urinary calcium concentration may lead to a
   decrease in glyoxylate-induced renal CaOx crystal deposition.

Discussion

   The exact pathogenesis of kidney stones is still unclear, and its high
   incidence and recurrence rates greatly affect human life and health,
   demonstrating the importance of actively studying the mechanism of
   kidney stones.

   Idiopathic hypercalciuria is an important risk factor for the
   occurrence and development of calcium-containing kidney stones^[140]26.
   Ca^2+ is the cationic component of crystals, and exposure to high
   concentrations of Ca^2+ can lead to oxidative stress damage in
   RTECs^[141]29. However, CaOx stone formation is associated with
   oxidative stress damage, increased crystal adhesion, and the
   inflammatory response that occurs during the pyroptosis of
   RTECs^[142]19,[143]20,[144]29. In our in vitro experiments, high Ca^2+
   caused oxidative stress damage to RTECs to generate ROS, which
   subsequently activated the initiation factor of pyroptosis, the NLRP3
   inflammasome. Relevant studies have shown that increased intracellular
   generation of ROS can cause DNA damage and cell apoptosis^[145]30,
   activate the NLRP3 inflammasome^[146]31, and result in inflammatory
   damage to renal tubular epithelial cells^[147]22,[148]32. Venegas et
   al.^[149]33 found that pyroptosis and the resulting inflammatory
   response were significantly reduced when the NLRP3 gene was knocked
   down or antagonized by an inhibitor, suggesting that NLRP3 is
   critically involved in pyroptosis. In our study, we identified the
   promoting role of Ca^2+ in oxidative stress injury-induced NLRP3
   inflammasome activation and pyroptosis in renal tubular epithelial
   cells. Considering these previous studies, Ca^2+ might be involved in
   the pathophysiological process of pyroptosis and the subsequent
   inflammatory response of RTECs.

   CaOx crystals can activate the NADPH oxidase complex to produce ROS
   through the renin-angiotensin system^[150]21, and increased
   intracellular generation of ROS can also upregulate the expression of
   adhesion-associated proteins through the p38 MAPK signaling pathway,
   which increases cell adhesion to crystals and promotes the attachment
   of crystals during stone formation^[151]34. Phosphorylated proteins,
   such as p38 and c-Jun, play an important role in the regulation of this
   process^[152]7,[153]35,[154]36. It has been shown that molecules
   closely associated with changes in the adhesion of RTECs, OPN, CD44,
   and MCP-1 are the most widely studied proteins that CaOx crystal
   attachment depends upon^[155]37–[156]39 and play an important role in
   the formation of calcium-containing kidney stones^[157]40. ITGB1 is
   widely expressed in epithelial cells and promotes cell adhesion and
   growth^[158]41. In our DEG analysis, the expression of OPN was
   significantly upregulated in Randall’s plaques in patients with kidney
   stones. We also demonstrated that high Ca^2+ increased the expression
   levels of the adhesion-associated proteins, which revealed that Ca^2+
   can increase the adhesion of RTECs.

   Bioinformatics analysis showed that the pyroptosis and cell adhesion
   scores of Randall’s plaque tissues were significantly higher than those
   of normal tissues, and there was a highly positive correlation between
   these two functions (R = 0.91). Relevant studies have found that CaOx
   crystals exacerbated pyroptosis in HK-2 cells, whereas overexpression
   of miR-141-3p downregulated pyroptosis-associated proteins to reduce
   pyroptosis levels, followed by a significant slowing of the rate of
   kidney stone formation^[159]23. Joshi et al.^[160]21 also found a
   significant decrease in the expression levels of renal tubular
   epithelial cell adhesion-associated proteins OPN, HA, and CD44, as well
   as a significant reduction in the rate of CaOx stone formation after
   reducing pyroptosis levels by knocking out the gene NLRP3 in mice. In
   this study, we regulated the pyroptosis level of renal tubular
   epithelial cells by knocking down and overexpressing GSDMD, the core
   pyroptosis-related gene. Our results showed that the expression levels
   of the adhesion-related proteins were significantly downregulated by
   GSDMD knockdown and increased by GSDMD overexpression. The results of
   crystal adhesion experiments similarly demonstrated that the adherent
   crystals on the cell surface of the renal tubular epithelial cell lines
   with GSDMD knockdown were significantly reduced and cell damage was
   significantly attenuated compared to those of the negative control
   group, whereas the opposite was true for the renal tubular epithelial
   cells with GSDMD overexpression.

   Citrate is readily absorbed in the intestine^[161]42 and decreases
   calcium absorption and urinary calcium excretion^[162]43,[163]44.
   Increased urinary citrate would also bind urinary calcium, removing the
   calcium from the pool available for binding with phosphate or oxalate.
   In addition, citrate metabolism to bicarbonate results in systemic
   alkalinization, which directly decreases bone resorption and increases
   renal tubule calcium reabsorption, thereby reducing the level of
   calcium in urine^[164]44,[165]45. The decrease in urinary calcium by
   itself would favorably reduce urinary supersaturation for
   calcium-containing kidney stones. In our in vivo validation experiment,
   we reduced the urinary calcium concentration in kidney stone model mice
   by potassium citrate, which resulted in the down-regulation of renal
   pyroptosis levels and their release of inflammatory factors,
   improvement of renal injury, and attenuation of renal adhesion levels
   and deposition of CaOx crystals in response to the reduction in urinary
   calcium concentration. These results revealed that Ca^2+ can increase
   the adhesion of renal tubular epithelial cells by inducing pyroptosis,
   which results in the aggregation and growth of crystals, and the
   supersaturation and continuous accumulation of crystals ultimately lead
   to the formation of kidney stones. However, the limitation of this
   study was not considered in humans, and further studies should be
   conducted in clinical CaOx kidney stone formers to support this
   evidence.

   In conclusion, we revealed the mechanism by which the Ca^2+ and cell
   adhesion interaction exerts its effect by the NLRP3/Caspase-1/GSDMD
   classical pyroptosis pathway (Fig. [166]12). Our study provides new
   mechanistic insight into the regulatory role of hypercalciuria in CaOx
   nephrocalcinosis disease and may aid in the development of novel
   therapeutic strategies targeting Ca^2+ and pyroptosis-related pathways.
   Treatments such as reducing urinary Ca²⁺ concentrations or inhibiting
   the NLRP3 inflammasome pathway may be effective in preventing kidney
   stones.

Fig. 12.

   [167]Fig. 12
   [168]Open in a new tab

   A schematic diagram for the underlying mechanism of the Ca^2+ in
   regulating calcium oxalate stone formation. High concentrations of
   Ca^2+ activates the NLRP3/Caspase-1/GSDMD pathway of pyroptosis through
   ROS and up-regulated the expression of adhesion-related proteins,
   thereby affecting the adhesion and deposition of crystals, and
   eventually leads to the formation of calcium oxalate kidney stones.

Materials and methods

Bioinformatics analysis

Data obtained and processing

   We downloaded the mRNA profiles of the [169]GSE73680 dataset from the
   public Gene Expression Omnibus (GEO) database managed by the National
   Center for Biotechnology Information- NCBI (Bethesda, EUA).
   [170]GSE73680 contained 29 CaOx stone patients and 6 healthy controls.
   We performed differential gene screening and functional enrichment
   analysis to compare the DEGs related to the function of renal tubular
   epithelial cells in Randall’s plaques and normal tissue from calcium
   stone patients, analyzed the coexpressed gene modules related to
   pyroptosis and cell adhesion by weighted gene coexpression network
   analysis (WGCNA), and calculated the pyroptosis score and cell adhesion
   score by single-sample GSEA using the GSVA package in R. Group P:
   Randall’s plaque tissues from calcium stone patients (n = 29), Group N:
   normal tissues from calcium stone patients (n = 27), and Group C:
   normal tissues from healthy controls (n = 6).

Screening DEGs and biological function analysis

   The [171]GSE73680 expression matrices were log2 transformed and
   normalized by the R function “normalizeBetweenArrays”. Based on the
   cutoff value of p-value < 0.05 and the absolute log2 |fold change
   (log2FC)| > 0.5, [172]GSE73680 successfully adopted the LIMMA package
   to select DEGs. Then, GO and KEGG analysis were used to rank gene
   functions and pathways by applying the “clusterProfiler” R
   package^[173]46.

GSEA

   GSEA was performed to identify the most significantly enriched pathways
   between group P and group N using the clusterProfiler package in R.

Weight gene co-expression network analysis (WGCNA)

   WGCNA is an analysis method to analyze the gene expression patterns of
   multiple samples, which can cluster genes with similar expression
   patterns and analyze the association between modules and specific
   traits or phenotypes. In this study, to identify the genes associated
   with different traits, WGCNA was performed on the [174]GSE73680
   dataset, and kidney stones, pyroptosis, cell adhesion, and Ca^2+
   pathways were analyzed as traits by WGCNA.

   The specific steps of the analysis were as follows: (1) Sample
   clustering to check the overall correlation of all samples in the
   dataset. We first clustered the samples and removed outlier samples to
   ensure the accuracy of the analysis. (2) Constructing sample clusters
   and clinical traits. (3) Soft threshold determination. A rational soft
   power threshold was recommended for the adjacency matrix through the
   “pickSoftThreshold” function built into the “WGCNA” package from 1 to
   20. (4) Construction of coexpression networks. Genes with similar
   expression profiles could be attributed to various gene modules by
   employing the dynamic tree-cutting function. We set the module’s
   correlation coefficient threshold and merged homologous modules in a
   dynamic cluster tree. (5) Correlation analysis of modules and clinical
   characteristics. The following procedure evaluates the expression-trait
   correlation plotted in various random color modules. The highest
   correlated module eigengene (ME) was identified as the key gene group
   with gene significance (GS) p < 0.05. (6) Screening of Ca^2+ pathway
   modules. (7) Correlation analysis between modules and pyroptosis. We
   used the GSVA R package and pyroptosis gene set to perform a
   single-sample enrichment analysis of each sample to evaluate the
   correlation between pyroptosis and the modules.

Cell culture

   The normal human proximal tubular epithelial cell line HK-2 was kindly
   provided by Procell Life Science & Technology Co., Ltd. (Wuhan, China)
   and maintained in Dulbecco’s modified Eagle’s medium and F12 medium
   (DMEM/F12) supplemented with 10% fetal bovine serum (ExCell, Shanghai,
   China) and 1% penicillin/streptomycin in a humidified atmosphere of 5%
   CO2.

   All experiments were repeated multiple times at different times and the
   sampling is sufficient to ensure statistically significant results.

Cell viability assay

   Five 96-well plates were used to culture HK-2 cells at different times.
   Cells were incubated with 100 µL of culture medium containing different
   concentrations (1, 5, 10, 20, 30, 40 mM [n = 5]) of CaCl[2] (Yuanye,
   Shanghai, China) for 24 h, 48 h, and 72 h to release Ca^2+ ions and
   induce hypercalciuria, and different concentrations (1.0, 2.5, 5.0,
   7.5, 10 mM [n = 5]) of NAC (Proteintech, USA) for 2 h. Subsequently, 10
   µL CCK-8 reagent (Proteintech, USA) was added to each well and
   incubated in a constant-temperature incubator at 37 °C for 2 h. The
   optical density (OD) was read at 450 nm on a microplate reader and the
   results were expressed as a line chart. We used a concentration of
   Ca^2+ that significantly reduced the viability of HK-2 cells but not
   less than 50% for subsequent experiments.

LDH release

   Media were aliquoted into selected wells of the 96-well plate. A LDH
   assay kit (Nanjing Jiancheng, China) was used to determine the percent
   LDH release. The substrate and chromophoric reagent supplied with the
   kit were added to the blanks (acclimatization media), positive control,
   and all samples. The plate was incubated for 15 min at 37 ℃. The stop
   solution, supplied with a kit, was added to all of the wells, and the
   plate was incubated for 3 min at room temperature. A microplate reader
   was used to read the OD at 440 nm and the results were expressed as a
   line chart. We used a concentration of Ca^2+ that did not cause damage
   to HK-2 cells for subsequent experiments.

Reagents and transfection

   To knock down and overexpress of GSDMD, lentivirus GSDMD and a negative
   control were synthesized by Generalbiol (Anhui, China) and purchased.
   Lentivirus was diluted with 500 µL of Opti-MEM medium at 10^8
   transduction units (TU)/mL containing polybrene (5 mg/mL) and incubated
   with cells for 12 h. The medium was then replaced with DMEM/F12 and
   cultured for 24 h. Then, subsequent screening with puromycin yielded
   stably transfected cell lines. Sh-NC (HK-2 cells transfected with blank
   silencing lentivirus, n = 3), Sh-GSDMD (HK-2 cells with silenced GSDMD
   expression, n = 3), GSDMD-NC (HK-2 cells transfected with blank
   overexpressing lentivirus, n = 3), GSDMD (HK-2 cells with overexpressed
   GSDMD expression, n = 3). These groups were concomitantly stimulated
   with calcium oxalate monohydrate (COM-100 µg/mL) and Ca²⁺ (10 mM) for
   24 h, 48 h, and 72 h.

Animal procedures

   All experimental procedures were in accordance with the regulations on
   the management of experimental animals of Kunming Medical University
   and approved by the Animal Ethics Committee. C57BL/6 N male mice (6–8
   weeks old) were used in the experiments. To establish the CaOx
   nephrocalcinosis model, each C57BL/6 N male mouse received either
   intraperitoneal saline or GA (75 mg/kg, 200 µL) every day for two
   weeks^[175]47. In the Ca^2+ intervention groups, PC was used to reduce
   urinary calcium concentration in mice^[176]44, and each mouse was
   gavaged with 20% PC (2 g/kg, 200 µL) solution daily for two weeks while
   the other groups were gavaged with an equal amount of saline. So the
   mice were divided into three groups: Control group (gavaged with saline
   and received intraperitoneal saline, n = 6), GA group (gavaged with
   saline and received intraperitoneal GA, n = 6), and GA + 20% PC group
   (gavaged with 20% PC and received intraperitoneal GA, n = 6). After two
   weeks, 24-h urine was collected from each group of mice using metabolic
   cages, and blood was collected by enucleation of eyeballs, and then the
   mice were euthanized by a physical method (cervical dislocation), and
   the kidneys were removed by laparotomy for further assessment. Urine
   and blood were analyzed by an automatic biochemical analyzer and kidney
   tissue sections were stained with periodic acid-Schiff (PAS) and Von
   Kossa to assess tubular injury and crystal formation/adhesion, and then
   quantified as the percent area of crystal deposition per kidney section
   with Image-Pro-Plus.

Observation of cellular microstructural changes

   HK-2 cells were harvested with Ca^2+ (5 mM, 10 mM, and 20 mM [n = 3])
   for 24 h, 48 h, and 72 h. Cell clusters were fixed by the addition of
   2.5% glutaraldehyde and wrapped using 1% agarose, followed by
   dehydration, embedding, sectioning, double staining with uranium-lead,
   and drying overnight, and images were acquired and analyzed by using a
   transmission electron microscopy (FEI Tecnai G2 Spirit, USA). The
   results were expressed as original images.

RNA extraction and qPCR

   Total RNA was extracted from the control and experimental groups using
   a total RNA extraction kit (TIANGEN, China) and then reverse
   transcribed using a reverse transcription kit (Vazyme, China). qPCRs
   were conducted on an Applied Biosystems 7500 system with ChamQ
   Universal SYBR qPCR Master Mix (Vazyme, China). All amplifications were
   followed by a dissociation curve analysis of the amplified products.
   Relative RNA expression of the gene was calculated using the 2^(−ΔΔCt)
   method and normalized to GAPDH expression^[177]47. The results were
   expressed as a histogram. Specific primers were designed using NCBI and
   the sequence identity was confirmed with BLAST. The primers used in our
   research are presented in Supplementary Table 1.

Western blot

   Protein extracts were separated by SDS-PAGE, transferred to PVDF
   membranes, and incubated overnight at 4 °C with primary antibodies.
   Then, the membrane strips were incubated with the appropriate
   horseradish peroxidase-conjugated secondary antibodies. The protein
   bands were visualized using an ECL kit (Proteintech, USA), and ImageJ
   software was used to calculate the relative density of proteins. The
   results were expressed as a histogram.

ELISA

   The cells were treated with Ca^2+ (5 mM, 10 mM, and 20 mM [n = 3]) for
   24 h, 48 h, and 72 h, the culture medium was collected and centrifuged
   to obtain the supernatant, and the levels of the pyroptosis-related
   inflammatory factors IL-1β and IL-18, and the adhesion-related MCP-1 in
   the culture supernatant were measured by using an ELISA kit
   (Proteintech, USA). The results were expressed as a histogram.

Histology and immunofluorescence

   Kidneys from mice were fixed for 72 h with 4% buffered polyformaldehyde
   before embedding in paraffin. Serial sections at 5 μm thick were
   obtained for histologic analysis. Hematoxylin and eosin (H&E) staining
   involved standard procedures.

   For IF, sections were incubated with the primary antibodies. To enhance
   antigen exposure, the sections were treated with 10 mM sodium
   citrate/pH 6.0 at 98 °C for 15 min for antigen retrieval. The sections
   were incubated with 3% BSA blocking solution, and then were incubated
   with the primary antibody at 4 ℃ overnight. After rinsing with PBS, the
   sections were incubated for 50 min with secondary antibody, washed, and
   then incubated with DAPI staining solution for 10 min. After rinsing
   again, the tissue autofluorescence was quenched using an
   autofluorescence quencher, and the sections were sealed using an
   anti-fluorescence quenching sealer, and finally the sections were
   observed using a fluorescence microscope (Nikon ECLIPSE C1, Japan) and
   images were captured (Nikon DS-U3, Japan). The results were expressed
   as original images.

Antibodies

   The antibodies used in this study are listed in Supplementary Table 2.

COM crystals preparation and crystal adhesion analysis

   Calcium oxalate monohydrate (COM) crystals were prepared as described
   previously^[178]48. Cells were seeded in round coverslips and incubated
   in a culture medium containing 100 µg/mL COM^[179]37 and 10 mM Ca^2+
   for 24 h, 48 h, and 72 h. Cells were washed 3 times with PBS, fixed
   with 2.5% glutaraldehyde at 4 °C in the dark. Then, they were
   subsequently washed, dehydrated, dried at the critical point, and
   metal-coated to observe crystal adhesion on the cell surface under
   scanning electron microscopy (Hitachi SU8100, Japan).

ROS assay and effects on the NLRP3 inflammasome

   HK-2 cells were cultured in a laser confocal dish and 6-well plates,
   and exposed to Ca^2+ (5 mM, 10 mM, and 20 mM, [n = 6]) for 24 h. Then,
   the cells were treated with 2,7-dichlorofluorescein diacetate
   (DCFH-DA)^[180]49 for 30 min, and the intracellular ROS levels were
   observed by laser scanning confocal microscopy (LSCM). The cells in the
   6-well plates were collected and resuspended in PBS, and intracellular
   ROS production was determined using flow cytometry with excitation and
   emission at 488 and 525 nm, respectively.

   The cells were pretreated with the ROS inhibitor NAC (5 mM, [n = 3])
   for 2 h. Subsequently, each group of cells was incubated with 10 mM
   Ca^2+ for 24 h. Western blot was used to detect the expression level of
   the NLRP3 inflammasome proteins in the cells. ImageJ software was used
   to calculate the relative density of ROS and proteins.

Data analysis

   GraphPad Prism 9.4 and SPSS 26.0 software were used for statistical
   analysis, and quantitative data were normally distributed and analyzed
   by a two-tailed t-test or one-way ANOVA. The results are expressed as
   the mean ± SEM. p < 0.05 for the difference was defined as
   statistically significant.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [181]Supplementary Material 1^ (90.2MB, docx)

Acknowledgements