20 eight-week-old male C57BL/6 mice, weighing between 20 and 25 grams, were obtained from Jiangsu GemPharmatech Co., Ltd. The mice were kept in a standard SPF animal facility. The mice were randomly assigned to either a control group or a stone model group, with each group containing ten mice. The stone model group was given a daily intraperitoneal injection of glyoxylic acid solution at a dose of 80 mg/kg for a period of seven days. Daily intraperitoneal injections of sterile physiological saline, matching the volume and frequency, were administered to the control group. An overdose of 1% pentobarbital sodium (150 mg/kg) was used to anesthetize the mice, which were then euthanized by cervical dislocation 24 hours after the last injection. Bilateral kidneys were promptly excised. The left kidneys were quickly frozen in liquid nitrogen and kept at –80 °C for later transcriptome sequencing analysis. Meanwhile, the right kidneys were cut sagittally and preserved in 4% paraformaldehyde at 4 °C for a day, and then underwent routine dehydration and paraffin embedding for Von Kossa staining and immunofluorescence staining. A second cohort of mice was established using the same protocol, with these samples used for subsequent real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot validation experiments. The animal experiment was approved by the Animal Experimental Ethical Inspection Form of Southeast University (20250909003).

At the Affiliated Zhongda Hospital of Southeast University, clinical samples were collected over the period from January 2020 to October 2024. All participants provided informed consent for the collection of samples and involvement in the study.

Inclusion criteria for stone group: (i) Patients who underwent nephrectomy due to renal dysfunction caused by kidney stones; (ii) Postoperative stone composition analysis confirmed calcium oxalate as the primary component; (iii) Age 18–75 years.

Inclusion criteria for control group: (i) Patients who underwent nephrectomy for renal cell carcinoma; (ii) Paracancerous renal tissues located at least 2 cm from the tumor edge; (iii) Intraoperative frozen pathology confirmed normal renal tissue without tumor infiltration.

Exclusion criteria (both groups): (i) Systemic autoimmune diseases; (ii) Active infection; (iii) Other renal diseases (e.g., polycystic kidney disease, diabetic nephropathy); (iv) Preoperative immunosuppressive therapy or chemotherapy.

Three patients were enrolled in each group. Postoperative pathological examination confirmed the diagnosis in all cases. Approval for the study was granted by the ethics committee at Affiliated Zhongda Hospital of Southeast University (ZDYJLY(2017)-49), in accordance with the Helsinki Declaration.

Sections of kidney tissue embedded in paraffin were deparaffinized, hydrated, and then immersed in silver nitrate solution. Sections were exposed to ultraviolet light for 20 minutes, followed by washing with distilled water. Residual silver salts were removed by treatment with 5% sodium thiosulfate solution for 2 minutes to fix the staining. Sections were counterstained with nuclear fast red for 2 minutes to clearly visualize the nuclei, then proceed with the standard processing procedure. Images were captured after observing the staining results with a BX60 Olympus optical microscope (Tokyo, Japan). ImageJ software (version 1.8.0, NIH, USA) was used to perform quantitative analysis of the area proportion of black calcium salt deposits.

The TRIzol reagent (Beyotime, Shanghai, China) was employed to extract total RNA from renal tissues, which was then converted into cDNA using the cDNA Synthesis Kit (Servicebio, Wuhan, China). qRT-PCR was performed using the SYBR Green qPCR Master Mix in a 20 µL reaction volume [18]. The 2^{-Δ⁢Δ⁢Ct} method was employed to determine the relative expression levels of target genes, using $\beta$-actin as the reference gene. Each sample included three technical replicates, and each experimental group comprised five biological replicates. Primer sequences are detailed in Supplementary Table 1.

Stored at –80 °C, kidney tissues were homogenized in cold RIPA lysis buffer with added protease and phosphatase inhibitors. Protein concentration was assessed and standardized via the bicinchoninic acid (BCA) method. Equal amounts of protein (10 µg) were mixed with loading buffer and denatured by heating at 100 °C for 8 minutes. Samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and transferred to a polyvinylidenefluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk in tris buffered saline and tween (TBST) at room temperature for 2 hours. After washing with TBST, the membrane was incubated with diluted primary antibodies (Supplementary Table 2) at 4 °C overnight. Following further TBST washes, the membrane was incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 1 hour. Protein signals were detected using enhanced chemiluminescence (ECL) chemiluminescent substrate in a chemiluminescence imaging system [19]. The relative expression of target proteins was calculated by analyzing the grayscale intensity of target bands with ImageJ software (version 1.8.0, NIH, Maryland, USA), using $\beta$-actin as the internal reference protein.

Kidney tissue sections embedded in paraffin were sliced into 5 µm thick pieces, deparaffinized, and rehydrated. Antigen retrieval was conducted using a sodium citrate buffer at high temperature. For permeabilization, sections were treated with 0.3% Triton X-100 in PBS for 15 minutes at room temperature. This condition is essential for both cytoplasmic (C1QC) and extracellular matrix (COL3A1) protein detection. The sections were then blocked with 3% BSA for 1 hour at room temperature. After blocking, sections were incubated overnight at 4 °C with primary antibody. After PBS washes, they were treated with secondary antibody at room temperature for 1 hour, shielded from light. Nuclei were counterstained using 4^′,6-Diamidino-2-phenylindole (DAPI). A BX60 Olympus fluorescence microscope (Tokyo, Japan) with cellSens software (v4.3, Tokyo, Japan) was used to observed the stained sections, and captured the images.

Human samples (n = 3 per group) and mouse samples (n = 5 per group) were used for transcriptome sequencing. In brief, total RNA was extracted using TRIzol reagent in accordance with the manufacturer’s instructions, and library construction was performed on the extracted total RNA. The libraries were subsequently sequenced on the Illumina NovaSeq 6000 platform (Astrocyte, Hangzhou). Quality control of the raw read data was performed using fastp (v0.20.0, China). The processed read data were aligned to the reference genomes (mm10/GRCm38 for mice and hg38 for humans) using HISAT2 (v2.2.1, Maryland, USA). Gene expression levels were quantified using FeatureCounts (v2.0.1, Victoria, Australia) as ‘fraction of reads per million mapped reads’ (FPKM).

To understand the roles of differentially expressed genes (DEGs), gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted. GO classification encompasses Cellular Component (CC), Biological Process (BP), and Molecular Function (MF).

The R software (version 3.5.3, Auckland, New Zealand) was used in this study. The DESeq2 R package was conducted used to analysis the differential expression of sequencing data. DEGs were identified with the criteria of p 0.01 and $|$log2FC$|$ $\ge$2.5. The R packages ggplot2 and pheatmap were employed to generate volcano plots and heatmaps, and Venn diagrams were created using the VennDiagram R package. The clusterProfiler R package was used to conduct GO functional and KEGG pathway enrichment analyses, with a p-value of less than 0.05 deemed statistically significant.

Von Kossa staining was performed on mouse kidney sections to specifically label calcium salt deposits. As shown in Fig. 1A, staining results revealed prominent brownish-black positive deposition signals in the stone group, primarily concentrated in the renal tubular lumen and renal papillary interstitial regions. The deposition morphologies exhibited a granular or sheet-like distribution. In contrast, no such specific calcium deposits were observed in the normal group. Quantitative analysis using ImageJ software showed that the percentage of positive staining area was significantly higher in the stone group compared to controls (stone group: 12.5% $\pm$ 1.6% vs. control group: 0% $\pm$ 0%; p 0.001, n = 5) (Fig. 1B).

Fig. 1.

Von Kossa staining of mouse kidneys. (A) Representative images of kidneys from the normal group and the kidney stone group. (B) Quantitative bar charts showing the percentage of positive staining area in kidneys from the normal group and the kidney stone group. Individual data points are shown as dots. Scale bar: 2 mm, Scale bar: 100 µm. Statistical significance was determined by independent samples t-test: ***p 0.001.

Transcriptome sequencing analysis was conducted on both human (Fig. 2A) and mouse (Fig. 2B) samples. According to the screening criteria, we identified a total of 132 DEGs (91 up-regulated and 41 down-regulated) in human samples and 1123 DEGs (901 up-regulated and 222 down-regulated) in mouse samples (Supplementary Table 3). Finally, 8 DEGs were found to be shared between human and mouse samples, namely COL3A1 , TYROBP, MMP2, BASP1, COL5A1, ITGAX, C1QC, and S100A8, all of which were upregulated in both the mouse stone group and human patient stone group (Fig. 2C).

Fig. 2.

Screening of DEGs. (A) Volcano plot of DEGs between human kidney stone samples and normal kidney samples (n = 3 per group). (B) Volcano plot of DEGs between mouse kidney stone samples and the control kidney samples (n = 5 per group). (C) Venn diagram of DEGs between human and mouse. Red dots indicate upregulated genes, while blue dots represent downregulated genes. DEGs, differentially expressed genes.

To validate the expression differences of the eight candidate genes at the transcriptional level, qRT-PCR was performed on an independent set of mouse tissue samples (n = 5 per group). The results showed that the relative mRNA expression levels of COL3A1 and C1QC were significantly increased in the stone group. The expression trends of these genes were consistent with the transcriptome sequencing data, confirming their stable upregulation at the transcriptional level in the kidney stone model (Fig. 3). No statistically significant differences were observed in the expression of the other six genes between the normal and kidney stone groups.

Fig. 3.

qRT-PCR validation of the relative mRNA expression of candidate genes in the mouse kidney samples. ***Data are presented as mean $\pm$ SD (n = 5 per group). Individual data points are shown as dots. Statistical significance was determined by independent samples t-test: ***p 0.001; ns, not significant. Y-axis: Relative mRNA expression (fold change normalized to $\beta$-actin). qRT-PCR, real-time quantitative polymerase chain reaction; SD, standard deviation.

To further validate the gene expression at the translational level, Western blot analysis revealed a significant upregulation of COL3A1 protein expression in the stone group compared to the control group, as illustrated in Fig. 4A, aligning with the mRNA expression trends and indicating consistent differential expression of COL3A1 from transcription to translation. In contrast, the protein expression level of C1QC showed no statistically significant difference between the normal and kidney stone groups (Fig. 4B).

Fig. 4.

Protein Expression of COL3A1 and C1QC in normal and kidney stone groups (n = 5 per group). (A) Western blot analysis of COL3A1 protein expression in mouse kidneys. (B) Western blot analysis of C1QC protein expression in mouse kidneys. Individual data points are shown as dots. Statistical significance was determined by independent samples t-test: *p 0.05; ns, not significant. COL3A1, collagen type III alpha 1 chain; C1QC, complement C1q C chain.

Immunofluorescence results shown in Fig. 5 revealed that compared to the normal group, the fluorescence intensity of COL3A1 was significantly enhanced in the renal tissue of the stone group, with clear regional enrichment. This enrichment was primarily concentrated in the interstitial regions of the renal medulla where stone deposition occurred. This finding further supports the upregulation of COL3A1 expression in kidney tissue associated with stone formation.

Fig. 5.

Immunofluorescence staining of COL3A1 in mouse kidneys. High-resolution representative images showing COL3A1 (red) and DAPI (blue) staining in control and stone group. DAPI, 4^′,6-Diamidino-2-phenylindole. Scale bar: 2 mm (1 $\times$), 100 µm (10 $\times$), 25 µm (40 $\times$).

Functional enrichment analysis was conducted on the DEGs identified in mouse and clinical patient renal tissues. As shown in Fig. 6, a high degree of consistency in functional enrichment pattern was observed between the two datasets. GO analysis revealed significant enrichment at the BP level, with terms related to immune response-activating signaling pathways, leukocyte-mediated immunity, and activation of immune responses. At the CC level, DEGs were predominantly localized to structures such as the external side of the plasma membrane, collagen-containing extracellular matrix, and blood microparticles. In terms of MF, the DEGs were enriched in platelet-derived growth factor binding. KEGG pathway analysis indicated that in the animal model, DEGs were significantly enriched in pathways such as complement and coagulation cascades, IL-17 signaling, and TNF signaling. In human samples, significant enrichment was found in pathways related to graft-versus-host disease, antigen processing and presentation, and phagosome. The core functions of the cross-species shared DEGs in kidney stone formation primarily involve two key processes: structural remodeling of the renal extracellular matrix and activation of local immune responses.

Fig. 6.

GO and KEGG enrichment analyses of DEGs. (A) GO and KEGG enrichment analyses of DEGs in human. (B) GO and KEGG enrichment analyses of DEGs in mouse. GO, gene ontology; KEGG, kyoto encyclopedia of genes and genomes. The pathway marked in red is common to both humans and mouse.