Calcium oxalate kidney stone biomarkers and uses thereof

Abstract

This application discloses a biomarker for calcium oxalate kidney stones and its application, relating to the field of pharmaceutical technology. Specifically, it proposes a biomarker for calcium oxalate kidney stones, namely isolithocholic acid. It reveals that isolithocholic acid directly targets and activates the PARP1 protein, inducing parthanatos in renal tubular epithelial cells, leading to cell damage and a microenvironment conducive to crystal adhesion, ultimately driving kidney stone formation. Isolithocholic acid is specifically elevated in feces, serum, kidney, and urine in a calcium oxalate kidney stone model, and its spatial localization is associated with areas of renal tubular damage, indicating its high relevance as a diagnostic or risk warning biomarker.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to calcium oxalate kidney stone biomarkers and their applications. Background Technology

[0002] Kidney stones are a prevalent urinary tract disease worldwide, with approximately 80% being calcium oxalate stones. This disease not only causes severe pain, urinary tract infections, and kidney damage, but also has a high recurrence rate, imposing a heavy health and economic burden on patients and society. The formation mechanism of calcium oxalate kidney stones is complex, and traditional research has mainly focused on the physicochemical processes of urine supersaturation (such as hyperoxaluria and hypercalciuria) and crystal nucleation, growth, and aggregation.

[0003] Currently, the clinical diagnosis of calcium oxalate kidney stones mainly relies on imaging examinations such as ultrasound, CT scans, and stone composition analysis. These methods often only detect stones after they have formed, lacking effective early risk prediction or warning biomarkers. Although some studies have attempted to use metabolomics to find characteristic molecules in urine or serum, the biomarkers found are often not specific enough or have unclear causal relationships with disease mechanisms, making clinical translation difficult. Summary of the Invention

[0004] In order to comprehensively address the above problems, this invention aims to provide a biomarker for calcium oxalate kidney stones and its application.

[0005] The first aspect of this invention provides a biomarker for calcium oxalate kidney stones.

[0006] A biomarker for calcium oxalate kidney stones, wherein the biomarker is isolithocholic acid.

[0007] Preferably, the biomarker is derived from fecal, serum, kidney tissue, or urine samples.

[0008] Preferably, the isolithocholic acid activates PARP1 to induce Parthanatos in renal tubular epithelial cells and promotes the formation of calcium oxalate kidney stones.

[0009] A second aspect of the present invention provides the use of a reagent for detecting isolithocholic acid in the preparation of products for detecting calcium oxalate kidney stones.

[0010] Preferably, the product includes a reagent kit, a probe, and a chip.

[0011] A third aspect of this invention provides the use of a PARP1 inhibitor in the preparation of a medicament for treating isolithocholic acid-induced calcium oxalate kidney stones.

[0012] Preferably, the PARP1 inhibitor is AZD5305.

[0013] Compared with the prior art, the advantages of this invention are as follows: (1) It was revealed that isolithocholic acid directly targets and activates PARP1 protein, induces Parthanatos in renal tubular epithelial cells, thereby creating a microenvironment for cell damage and crystal adhesion, which ultimately drives the formation of kidney stones.

[0014] (2) Isolithocholic acid was specifically elevated in feces, serum, kidney and urine in the calcium oxalate kidney stone model, and its spatial location was associated with the area of ​​renal tubular injury, indicating its high relevance as a diagnostic or risk warning marker. Attached Figure Description

[0015] Figure 1 This diagram illustrates the CaOx and Con rat models constructed and validated using the ethylene glycol and ammonium chloride drinking water method of this application. In the diagram, A is a schematic diagram of the construction of the CaOx and Con rat models; B is a schematic diagram of the body weight of the CaOx and Con rat models; and C is a schematic diagram of the urinary oxalate, calcium ion, and magnesium ion content of the CaOx and Con rat models.

[0016] Figure 2 This diagram illustrates the degree of kidney damage and CaOx crystal deposition in the CaOx and Con groups of rats constructed using the ethylene glycol and ammonium chloride drinking water method described in this application, based on histological staining.

[0017] Figure 3 This is a schematic diagram of TUNEL staining of the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0018] Figure 4 This is a schematic diagram of DHE staining of the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0019] Figure 5 This diagram illustrates the quantitative analysis of TUNEL counts and DHE staining in rats from the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0020] Figure 6 This is a schematic diagram of IHC analysis of kidney stone formation markers (CD44, MCP-1, and OPN) in rats of the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method in this application.

[0021] Figure 7 This is a schematic diagram of the renal function assessment of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0022] Figure 8 This application presents a schematic diagram of the detection of serum inflammatory factors in rats in the CaOx and Con groups, constructed using the ethylene glycol and ammonium chloride drinking water method.

[0023] Figure 9 This diagram illustrates the targeted bile acid metabolomics analysis of feces from rats in the CaOx and Con groups, constructed using the ethylene glycol and ammonium chloride drinking water method. A shows the OPLS-DA score of differential bile acids in the feces of rats in the CaOx and Con groups; B shows the volcano plot of differential bile acids in the feces of rats in the CaOx and Con groups; and C shows the VIP score of differential bile acids in the feces of rats in the CaOx and Con groups.

[0024] Figure 10 This is a schematic diagram of the targeted bile acid metabolomics analysis of the serum of rats in the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application. In the diagram, D is a schematic diagram of the OPLS-DA score of the differential bile acids in the serum of rats in the CaOx and Con groups; E is a volcano diagram of the differential bile acids in the serum of rats in the CaOx and Con groups; and F is a VIP score diagram of the differential bile acids in the serum of rats in the CaOx and Con groups.

[0025] Figure 11 This is a schematic diagram of the targeted bile acid metabolomics analysis of the kidneys of CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application. In the diagram, G is a schematic diagram of the OPLS-DA score of the differential bile acids in the kidneys of CaOx and Con groups; H is a volcano diagram of the differential bile acids in the kidneys of CaOx and Con groups; and I is a VIP score diagram of the differential bile acids in the kidneys of CaOx and Con groups.

[0026] Figure 12 This diagram illustrates the targeted bile acid metabolomics analysis of urine from rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application. J represents the OPLS-DA score of differential bile acids in the urine of rats in the CaOx and Con groups; K represents the volcano plot of differential bile acids in the urine of rats in the CaOx and Con groups; and L represents the VIP score of differential bile acids in the urine of rats in the CaOx and Con groups.

[0027] Figure 13 This is a Venn diagram showing the intersection of differential bile acids in the feces, serum, kidneys, and urine of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0028] Figure 14 This diagram illustrates the levels of isolithocholic acid in the feces, serum, kidneys, and urine of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0029] Figure 15 Volcano diagrams of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0030] Figure 16 The image shows a heatmap of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0031] Figure 17 This is a diagram showing the KEGG pathway analysis of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0032] Figure 18 This is a GO enrichment analysis diagram of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application.

[0033] Figure 19 This is a subcellular localization map of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0034] Figure 20 This is a GSEA analysis diagram of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application.

[0035] Figure 21 Volcano diagrams of differentially expressed proteins in the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0036] Figure 22 Principal component analysis diagram of differentially expressed proteins in the kidneys of rats in the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application.

[0037] Figure 23 The image shows a heatmap of differentially expressed proteins in the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0038] Figure 24 This is a GO enrichment analysis diagram of differentially expressed proteins in the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0039] Figure 25 This is a diagram showing the KEGG pathway analysis of differentially expressed proteins in the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0040] Figure 26 This is a subcellular localization map of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0041] Figure 27This is a GSEA analysis diagram of differentially expressed proteins in the serum of rats in the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application.

[0042] Figure 28 This is a schematic diagram of potential targets for isolithocholic acid in the PharmMapper database.

[0043] Figure 29 Venn diagram showing the intersection of isolithocholic acid targets in CTD, differentially expressed proteins in serum and kidney, and kidney stone-related genes in the CaOx group.

[0044] Figure 30 This diagram illustrates the protein expression of PARP1 in the kidneys and serum of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0045] Figure 31 This is a schematic diagram showing the IHC analysis of PARP1 protein expression in the kidneys of rats in the CaOx and Con groups constructed using the ethylene glycol and ammonium chloride drinking water method of this application.

[0046] Figure 32 This diagram illustrates the protein expression of PARP1 in the kidneys of rats in the CaOx and Con groups, constructed using the ethylene glycol and ammonium chloride drinking water method described in this application.

[0047] Figure 33 This is a schematic diagram showing the localization of isolithocholic acid and renal tubular damage in rats in the CaOx and Con groups constructed by the ethylene glycol and ammonium chloride drinking water method of this application.

[0048] Figure 34 A schematic diagram of PARP1 protein expression in the kidneys of healthy control patients and patients with calcium oxalate kidney stones, based on IHD analysis.

[0049] Figure 35 This is a schematic diagram of the CaOx and Con rat models constructed by injecting glyoxylic acid according to this application.

[0050] Figure 36 This diagram illustrates the protein expression of PARP1 in the CaOx and Con groups of rats constructed by injecting glyoxylic acid according to this application.

[0051] Figure 37 This application presents schematic diagrams of IHC, H&E, VK, and TUNEL staining of PARP1 protein in CaOx and Con rats constructed by glyoxylic acid injection.

[0052] Figure 38 This application presents schematic diagrams of blood urea nitrogen (BUN) and serum creatinine (Scr) levels in rats in the CaOx and Con groups constructed by injecting glyoxylic acid.

[0053] Figure 39 This is a schematic diagram of the isolithocholic acid treatment and PAPRA1 inhibitor treatment models in the CaOx and Con groups of rats constructed by injecting glyoxylic acid according to this application.

[0054] Figure 40 The diagram shows the blood urea nitrogen (BUN) and serum creatinine (Scr) levels in rats in the CaOx group, Con group, isolithocholic acid group, and PAPRA1 inhibitor group constructed by injecting glyoxylic acid according to this application.

[0055] Figure 41 This application presents schematic diagrams of staining for microCT, H&E, VK, Masson, TUNEL, and PARP1 proteins in the kidneys of rats in the CaOx group, Con group, isolithocholic acid group, and PAPRA1 inhibitor group constructed by injecting glyoxylic acid.

[0056] Figure 42 The diagrams show the SPR and MST results of isolithocholic acid and PARP1 protein in this application. A is a schematic diagram of the SPR analysis of isolithocholic acid and PARP1 protein; B is a schematic diagram of the equilibrium dissociation constant of the SPR analysis between isolithocholic acid and PARP1 protein; C is a schematic diagram of the MST analysis between isolithocholic acid and PARP1 protein; and D is a schematic diagram of the equilibrium dissociation constant of the MST analysis between isolithocholic acid and PARP1 protein in this application.

[0057] Figure 43 This diagram illustrates the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) between isolithocholic acid and PARP1 protein in this application. E represents the RMSD diagram, and F represents the RMSF diagram.

[0058] Figure 44 This diagram illustrates the radius of gyration, solvent-accessible surface area, and hydrogen bond contact between isolithocholic acid and PARP1 protein in this application; where G represents Rg, H represents SASA, and I represents hydrogen bond contact.

[0059] Figure 45 This is a schematic diagram illustrating the molecular docking of isolithocholic acid with human PARP1 protein and rat PARP1 protein according to this application.

[0060] Figure 46 This is a schematic diagram of the CETSA analysis between isolithocholic acid and PAPR1 protein in this application.

[0061] Figure 47This diagram illustrates the verification of isolithocholic acid in the healthy control group (HC group) and patients with calcium oxalate kidney stones (CaOx group) of this application. In this diagram, A is a sample selection diagram of the healthy control group (HC group) and patients with calcium oxalate kidney stones (CaOx group); B is a diagnostic diagram of the isolithocholic acid level in the samples of the healthy control group (HC group) and patients with calcium oxalate kidney stones. Detailed Implementation

[0062] The following is in conjunction with embodiments of the present invention. Figures 1-47 It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0063] The raw material information involved in this invention is as follows: Male SD rats, 6-8 weeks old, were sourced from Vital River Laboratory Animal Company. They were housed in an SPF animal facility with room temperature controlled at 20-24°C and humidity at 50%-70%. The environment was alternated between light and dark for 12 hours. Standard feed and drinking water were provided regularly. Experiments were conducted after one week of acclimatization.

[0064] All solvents used in this invention are commercially available analytical grade reagents, and the specific raw material information is shown in Table 1.

[0065] Table 1 Raw Material Information

[0066] This invention provides a biomarker for calcium oxalate kidney stones, specifically isolithocholic acid.

[0067] Isolithocholic acid is a derivative derived from lithocholic acid. The structural formula of isolithocholic acid is:

[0068] I. Construction and Characterization of a Rat Model of Calcium Oxalate Kidney Stones Twelve male SD rats were randomly divided into a control group (Con group) and a model group (CaOx group), with six rats in each group. Figure 1 As shown in A, the model group was allowed to drink water containing 1% ethylene glycol and 0.5% ammonium chloride for 4 weeks to induce the formation of calcium oxalate kidney stones; the control group was allowed to drink ordinary water for 4 weeks.

[0069] Physiological indicators: based on Figure 1 As shown in B, the model group rats experienced slow weight gain, according to Figure 1 As shown in C, mass spectrometry analysis revealed a significant increase in the concentrations of oxalate and calcium ions in the urine of rats in the model group. Urine biochemical analysis showed an increase in calcium ion concentration and a decrease in magnesium ion concentration in the model group.

[0070] Pathological analysis: The overall morphology of the tissue was observed using H&E staining. Paraffin sections were dewaxed, hydrated, stained with hematoxylin for the nuclei, stained with eosin for the cytoplasm, and then dehydrated and mounted. Next, Von Kossa staining was used to detect calcium salt deposition. The dewaxed sections were developed under light with silver nitrate solution and fixed with sodium thiosulfate; areas of calcium salt deposition appeared brownish-black. Finally, alizarin red staining was used to specifically label calcium oxalate crystals; staining at pH 4.2 resulted in an orange-red color for calcium oxalate. Figure 2 As shown, kidney tissue sections from rats in the model group and control group were stained with H&E, Von Kossa (VK, used to stain calcium salts) and Alizarin Red (AR). The staining revealed that the renal tubules of rats in the model group were significantly dilated, and a large number of brown-black (VK) or blue-green (AR) calcium oxalate crystals were deposited in the lumen, accompanied by atrophy and degeneration of renal tubular epithelial cells and interstitial inflammatory cell infiltration.

[0071] Cellular damage and oxidative stress: TUNEL (TdT-mediated dUTP nick end labeling) staining was performed on paraffin sections. The sections were dewaxed and hydrated, and incubated with proteinase K solution (20 μg / ml) at 37°C for 30 minutes to allow tissue penetration and expose DNA ends. A solution containing 0.1% Triton X-100 and 0.1% sodium citrate was added to permeabilize the sections. After standing at room temperature for 8 minutes, the sections were washed three times with PBS buffer for 5 minutes each time. The blocking solution was removed, and freshly prepared TUNEL staining solution was added. The TUNEL apoptosis detection kit was purchased from Wuhan Saiwei Biotechnology Co., Ltd. The sections were incubated at 37°C in the dark for 60 minutes. After incubation, the sections were washed three times with PBS buffer for 5 minutes each time. The nuclei were stained with DAPI nuclear dye and incubated at room temperature for 5-10 minutes. The sections were washed again with PBS buffer to remove excess nuclear dye and mounted with an antifluorescein quencher. Figure 3 As shown, TUNEL staining revealed a significant increase in kidney cell apoptosis / death in the model group.

[0072] DHE staining: OCT-embedded frozen kidney sections were thawed at room temperature, and then 5 μm of DHE working solution was added to cover the tissue. The tissue was incubated at 37°C in the dark for 30 minutes. Afterward, the tissue was gently washed three times with PBS to remove excess probe. The nuclei were then counterstained with DAPI staining solution for 2-5 minutes. After washing with PBS, the tissue was immediately mounted with an anti-quenching mounting medium. Observation was performed under a fluorescence microscope at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Areas positive for reactive oxygen species showed red fluorescence, while DAPI-labeled nuclei appeared blue. Figure 4 and Figure 5 As shown, DHE staining revealed a significant increase in reactive oxygen species levels in the kidney tissue.

[0073] Markers of kidney stone formation: Immunohistochemistry was used to detect the expression levels of kidney stone formation-related proteins MCP-1, CD44, and OPN in rat kidney tissue. The specific steps are as follows: 1. Dewaxing and hydration; 2. Antigen retrieval: Place the slides in Tris / EDTA antigen retrieval solution (pH 9.0), microwave on high for 7 minutes, and let stand for 30 minutes for antigen retrieval. Afterward, remove the slides, allow them to cool to room temperature, and rinse three times with PBS; 3. Blocking: Add 5% BSA as blocking solution and incubate at room temperature for 15 minutes to block non-specific binding sites; 4. Primary antibody incubation and washing: Add 20µL of primary antibody to cover the tissue surface, and then incubate overnight at 4°C. The primary antibody was purchased from Proteintech, USA, and included MCP-1 (1:500), CD44 (1:500), and OPN (1:200). PBS was used as a negative control. After 12 hours of incubation, rinse the slides three times with PBS for 5 minutes each time; 5. 6. Secondary antibody incubation and washing: Add biotin-labeled goat anti-rabbit IgG secondary antibody (primary antibody purchased from Proteintech, USA). Incubate at 37°C for 30 minutes, then wash three times with PBS for 5 minutes each time. 7. DAB staining: Stain with DAB solution, controlling the reaction time under a microscope. Stop the reaction immediately with tap water when brownish-yellow granules appear. 8. Blueing: Stain with hematoxylin for 1 minute, then wash with tap water to blue-re-stain the cell nuclei. 9. Dehydration and mounting: Photograph and examine under an upright microscope (OLYMPMS, Japan). Figure 6 As shown, immunohistochemistry revealed that the expression of osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1), and CD44 in the kidneys of the model group was significantly upregulated.

[0074] Kidney function and inflammation: such as Figure 7 As shown, serum creatinine and blood urea nitrogen levels were higher in the model group than in the control group, indicating impaired renal function in the model group; Figure 8 As shown, ELISA detection revealed a significant increase in serum levels of inflammatory factors IL-1β and IL-6.

[0075] The above test results indicate that a rat model of calcium oxalate kidney stones with typical pathological evidence has been successfully constructed.

[0076] II. Identification of Key Differential Metabolites Using Targeted Bile Acid Metabolomics After the administration was completed, feces, serum, kidney tissue, and urine were collected from rats in the control and model groups: (1) Fecal samples: Weigh 50 mg ± 2.5 mg of fecal samples from rats in the model group and control group. Add 500 μL of pre-cooled 70% methanol at 4°C to each fecal sample and shake to mix. Centrifuge at 12000 r / min for 10 min. Take 300 μL of the supernatant and let it stand in a -20°C refrigerator for 30 min. Repeat the above centrifugation steps. Take 200 μL of the supernatant and process it through a protein precipitation plate for later use. (2) Serum / urine samples: Weigh 50 μL of serum and urine samples from rats in the model group and control group. Add 250 μL of extraction solution containing 20% ​​acetonitrile methanol to each serum and urine sample. Remaining steps The same steps as for fecal samples were taken; (3) Kidney samples: Weigh 20 mg ± 2.5 mg of kidney samples from rats in the model group and control group, add a steel ball to each sample, add 10 μL of internal standard solution and 200 μL of 20% methanol acetonitrile for homogenization, shake and mix, and then let stand in a -20℃ refrigerator for 10 min; then, centrifuge at 4℃ and 12000 r / min for 10 min. After centrifugation, take out the supernatant and put it into a centrifuge concentrator for concentration treatment, so that the supernatant is completely dried in the centrifuge concentrator. After concentration, use 100 μL of 50% methanol aqueous solution to redissolve the sample and prepare it for analysis. Metabolomics analysis: Ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS) were used to analyze the above samples, and the results were analyzed according to... Figures 9-12 As shown, analysis of variance (ANOVA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used. Based on the statistical results of VIP value, fold change (FC), and ANOVA, the screening criteria for differentially expressed metabolites were determined: VIP > 1, P < 0.05, FC > 2 or FC < 0.5, and significant separation of bile acids between the model group and the control group in all four sample types.

[0077] Specifically, targeted bile acid metabolomics analysis was performed on each fecal sample. The PCA plot highlighted the differences in fecal bile acid profiles between the control group and the calcium oxalate group. Figure 9 As shown in A, OPLS-DA analysis revealed significant separation, indicating differences in bile acid profiles. Figure 9 In the B group, 12 significantly differentially regulated bile acids were identified, of which 3 were downregulated and 9 were upregulated in the model group. Figure 9 C in the table shows the differentially expressed bile acids, ordered in descending order of VIP scores.

[0078] Targeted bile acid metabolomics analysis was performed on each serum sample. The PCA plot highlighted the differences in serum metabolite profiles between the control and calcium oxalate groups. Figure 10 As can be seen from D in the data, OPLS-DA analysis showed significant separation, indicating differences in bile acid profiles. Figure 10 In the study, 19 significantly differentially regulated bile acids were identified, of which 17 were downregulated and 2 were upregulated in the model group. Figure 10 As shown in F, differentially expressed bile acids are displayed in descending order of VIP scores.

[0079] Targeted bile acid metabolomics analysis was performed on various kidney and urine samples. PCA plots validated the stability and reproducibility of the method and highlighted the differences in kidney and urine bile acid profiles between the control and calcium oxalate groups. Figure 11 G and Figure 12 As can be seen from J in the data, OPLS-DA analysis showed significant separation, indicating differences in the bile acid profiles of rat kidneys and urine. Figure 11 H in the kidney identified five significantly differentially expressed bile acids, based on Figure 12 K in the urine identified five significantly different bile acids. Figure 11 I and Figure 12 L in the figure represents the differentially expressed bile acids, ordered in descending order of VIP scores.

[0080] Differential metabolite screening: Using variable importance projection (VIP) > 1 and p < 0.05 as criteria, 12, 19, 5, and 5 differentially expressed bile acids were screened from feces, serum, kidney, and urine, respectively. Venn diagrams of the four groups of differentially expressed bile acids were plotted, and... Figure 13 As shown, isolithocholic acid is the only common differentially expressed bile acid that was significantly upregulated in all four samples.

[0081] Content verification: Quantitative analysis was performed on the above samples respectively, based on... Figure 14 It was found that, compared with the control group, the levels of isolithocholic acid in the feces, serum, kidneys and urine of rats in the model group were significantly increased.

[0082] The above results indicate that isolithocholic acid is a key bile acid molecule that is systematically elevated in the occurrence of kidney stones associated with high oxalate levels.

[0083] III. Proteomics and Network Pharmacology for Target Detection Proteomics analysis: Quantitative proteomics analysis using TMT labeling was performed on rat serum samples, based on... Figure 15It can be seen that in serum, volcano plots were created using thresholds of P<0.05 and |log2FC|>1 to visualize the distribution of differentially expressed proteins. The volcano plots showed that there were a large number of differentially expressed proteins in the model group and the control group, showing that 102 proteins were upregulated and 142 proteins were downregulated in the calcium oxalate group. Figure 16 A heatmap showing differentially expressed proteins. According to... Figure 17 KEGG pathway analysis showed significant enrichment in apoptosis, ferroptosis, and cell adhesion molecules, indicating that pathways related to cell death and oxidative stress were activated in the kidneys of the model group. Figure 18 GO analysis indicated that these differentially expressed proteins are mainly involved in functions such as tight junctions and cell-matrix adhesion. Subcellular localization of differentially expressed proteins in serum showed... Figure 19 Furthermore, according to Figure 20 GSEA analysis showed significant enrichment in necroptosis, motor proteins, and cell cycle components, which are closely related to the development of kidney stones.

[0084] Quantitative proteomics analysis of rat kidney samples using TMT labeling was performed, based on... Figure 21 The volcano plot shows that 909 proteins were upregulated and 457 proteins were downregulated in the calcium oxalate group. Figure 22 The PCA plots highlighted the differences in renal proteins between the control and calcium oxalate groups. Heatmaps of differentially expressed proteins showed... Figure 23 , Figure 24 GO analysis revealed that these differentially expressed proteins are primarily involved in inflammatory responses, extracellular matrix processes, calcium ion binding, cellular responses to hydrogen peroxide, and responses to nutrients. According to... Figure 25 KEGG pathway analysis revealed significant enrichment in pathways such as oxidative phosphorylation, ferroptosis, cell adhesion molecules, tryptophan metabolism, and necrosis / apoptosis. Subcellular localization of differentially expressed proteins in serum showed… Figure 26 According to Figure 27 GSEA analysis showed significant enrichment in cholesterol metabolism, apoptosis, and lysosomes, suggesting a potential role of bile acids in calcium oxalate kidney stones.

[0085] according to Figure 28 As shown, 292 potential targets of isolithocholic acid were obtained from the PharmMapper database, and 106 known calcium oxalate kidney stone-related genes were obtained from the CTD database. Intersections were then taken with differentially expressed proteins in rat serum and kidney. Figure 29 The intersection analysis results show that PARP1 is the only key target that appears in all three datasets. According to... Figure 30 Proteomics data showed that PARP1 protein levels were significantly elevated in the serum and kidneys of rats in the model group. Furthermore, according to... Figures 31-32 Immunohistochemical analysis and Western blot analysis were used to identify the expression of PARP1 protein in rat kidneys. The expression of PARP1 protein in the kidneys of rats in the CaOx group was increased.

[0086] Spatial localization verification: 10 μm thick frozen kidney tissue sections were prepared at -20℃ using a Leica CM1950 cryostat and mounted on indium tin oxide conductive glass slides, then vacuum dried for 30 minutes. Matrix spraying was performed at 60℃ using a nebulizer. The matrix was 15 mg / mL 2,5-dihydroxybenzoic acid (dissolved in a mixture of 90% acetonitrile and 10% water). Spraying parameters were set as follows: flow rate 0.1 mL / min, pressure 6 psi, 20 cycles, with a 5-second drying interval between each spray. Data were acquired using a MALDI-timsTOF mass spectrometry system (Bruker timsTOF flex) in positive ion mode. The mass-to-charge ratio detection range was m / z 50-1300 Da, the spatial resolution was 50 μm, and 400 laser shots were acquired per pixel. The mass spectrum signal intensity was normalized using the root mean square method. Metabolite structure identification was completed through secondary mass spectrometry analysis. Figure 33 It can be seen that the specific accumulation area of ​​isolithocholic acid in the kidneys of the model group highly overlaps with the area of ​​renal tubular damage shown by pathological staining, which directly proves the enrichment of this molecule in the lesion site.

[0087] Clinical sample validation: Kidney tissue samples were collected from patients with calcium oxalate kidney stones (CaOx) and healthy controls (HC) for immunohistochemical analysis. Figure 34 The results showed that the expression level of PARP1 protein in the kidney tissue of patients with calcium oxalate kidney stones was significantly higher than that in healthy controls, indicating that this target is also relevant in human diseases.

[0088] IV. Functional and Mechanism Verification according to Figure 35 As shown, PARP1 was knocked down in rat kidneys using AAV carrying shRNA targeting the PARP1 coding sequence, and the rats were fed a diet containing 1% isolithocholic acid. Figure 36 Western blot and Figure 37 Immunofluorescence staining confirmed successful PARP1 knockdown in kidney tissue. Figure 38 As shown, AAV-shPARP1-mediated PARP1 knockdown blocked the increase in BUN and Scr induced by isolithocholic acid treatment in CaOx rats. Figure 37H&E and VK staining results showed that PARP1 knockdown inhibited the aggravation of isolithocholic acid-induced CaOx deposition and renal tubular damage, and TUNEL staining suggested that PARP1 knockdown could block isolithocholic acid-mediated increase in apoptosis.

[0089] Rat experiment: For 14 consecutive days, 80 male SD rats were injected intraperitoneally with 80 mg / kg glyoxylic acid to establish a CaOx kidney stone model group (Gly model group). The 80 rats in the Gly model group were randomly divided into an isolicholic acid group and an AZD5305 group. The isolicholic acid group was fed with 1% isolicholic acid powder by weight, while the AZD5305 group was injected intraperitoneally with 1 mg / kg / d of the PARP1 inhibitor AZD5305, which was purchased from MedChemExpress, USA.

[0090] according to Figure 39 As shown, isolithocholic acid exacerbates weight loss in Gly rats, while the PARP1 inhibitor AZD5305 partially reverses this change. Meanwhile, according to... Figure 40 It is known that the PARP1 inhibitor AZD5305 can block the increase in BUN and Scr and the upregulation of inflammatory factors IL-1β and IL-6 induced by isolithocholic acid in the Gly model group.

[0091] Micro-CT scans were performed on each kidney sample. The fixed kidney specimens were placed in the micro-CT scanning chamber, parameters were adjusted, and the slow, precise mode was selected for scanning. In the SkyScan software system, the following scanning parameters were set: power 8W, voltage 80kV, current 120µA, and resolution 18µm 2000 x 1336. Under these conditions, the scans clearly showed the overall contour of the kidney and examined for the presence of high-density stone-like substances in the renal cortex and medulla. Figure 41 Micro-CT scans revealed significant renal calcium salt crystal deposition in Gly rats, which was further aggravated by allochlear acid, while the PARP1 inhibitor AZD5305 reduced crystal deposition. H&E and VK staining showed that the PARP1 inhibitor AZD5305 alleviated the aggravation of allochlear acid-induced CaOx deposition and renal tubular damage. Masson staining showed that the PARP1 inhibitor AZD5305 blocked allochlear acid-mediated renal fibrosis; according to... Figure 41TUNEL staining showed that the PARP1 inhibitor AZD5305 could inhibit isolicholic acid-mediated apoptosis. Furthermore, IHC analysis showed that AZD5305 could block isolicholic acid-induced enhancement of PARP1-positive staining. PAS staining indicated that AZD5305 could block isolicholic acid-mediated abnormal accumulation of cytoplasmic glycogen in renal tubular epithelial cells. Therefore, isolicholic acid promotes kidney stone formation mechanistically by targeting PARP1.

[0092] V. Binding affinity of isolithocholic acid to PARP1 protein Binding affinity was determined using surface plasmon resonance (SPR) technology on the Biacore T200 system: recombinant human PARP1 protein was immobilized onto the sensor chip using a standard amino-coupled method, with a capture level of approximately 12,000 RU; 200 μM isoallo LCA solution was injected at a flow rate of 30 μL / min for 90 seconds, followed by 180 seconds of dissociation monitoring. The binding constant and affinity values ​​were calculated by the system software. Figure 42 A and Figure 42 As shown in B, SPR analysis revealed that isolithocholic acid can bind directly to the PARP1 protein, with an equilibrium dissociation constant (KdK_dKd) of 4.31 μM.

[0093] Quantitative interaction characterization was performed using micro-thermophoresis on a Monolith NT.115 instrument. PARP1 protein was fluorescently labeled with RED-tris-NTA and incubated with serially diluted isoalloLCA in MST buffer. The MST buffer was 10X TNT Buffer (100 mmol / L Tris-HCl; 1.5 mol / L NaCl; 0.5% Tween 20; pH 7.8-8.2), purchased from Sangon Biotech (Shanghai) Co., Ltd. Detection was performed at 25℃ with 100% excitation intensity and 40% MST power. The dissociation constant was obtained by fitting using MO Affinity Analysis software. Figure 42 C and Figure 43 The MST results for D in the model show that its KdK_dKd is 65.7 μM.

[0094] Further cell thermal migration assays were employed. HK-2 and NRK-52E cells were treated with DMSO or 200 μM isoalloLCA for 24 hours, respectively. After gradient temperature heat treatment, the supernatant was collected by centrifugation, and changes in PARP1 thermostability were analyzed by Western blotting. Finally, computational simulations were used to obtain the high-resolution three-dimensional structure of PARP1 from the RCSB protein database. Molecular docking was performed using AutoDock Vina, and 100 ns molecular dynamics simulations were conducted using AMBER22 software. After hydration, ion addition, energy minimization, and equilibrium, 10,000 frames were sampled at 300 K and 1 bar. Trajectory analysis was performed using CPPTRAJ, and binding free energy was calculated using the MMPBSA.py tool. This multi-technology integration strategy comprehensively elucidated the specific interaction characteristics between isoalloLCA and PARP1 from three levels: in vitro binding, cell interaction, and computational simulation.

[0095] To evaluate the stability and conformational behavior of the protein-ligand (PARP1–isolithocholic acid) complex during simulation, root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and hydrogen bonds (Hbond) were analyzed. Figure 43 The root mean square deviation (RMSD) of the Cα atom in E indicates that the complex tends to stabilize within the first 2 ns of the simulation, and no significant fluctuations are observed in the 100 ns trajectory. According to... Figure 43 The root mean square fluctuation (RMSF) of F in the model shows that residues before position 200 have lower flexibility, while residues after position 200 have higher flexibility. According to... Figure 44 The radius of gyration (Rg) of G remained stable in the range of 48.6–48.9 Å, suggesting that no protein unfolding event occurred during the simulation. According to Figure 44 H- Figure 44 The surface area (SASA) and hydrogen bond analysis of protein I further confirmed that no significant conformational changes occurred during the simulation. Binding free energy calculations showed that the binding free energy of this complex was -42.36 kcal / mol, suggesting that the two form a stable complex with strong binding affinity.

[0096] In addition, according to Figure 45 Molecular docking results showed that the hydrogen bond energy between isolithocholic acid and human PARP1 protein was -9.4 kcal / mol, and the hydrogen bond energy between isolithocholic acid and rat PARP1 protein was -8.8 kcal / mol.

[0097] Cellular thermal migration assay (CETSA) results validated the thermodynamic stability of isolithocholic acid bound to PARP1: CETSA was used to assess the effect of isolithocholic acid on the thermostability of potential target proteins. The specific steps were as follows: Human renal tubular epithelial cells (HK-2) and rat renal tubular epithelial cells (NRK-52E) were treated for 24 hours with complete culture medium containing 200 μM isoalloLCA or an equal volume of DMSO (solvent control). After treatment, cells were collected, washed with PBS, and resuspended in appropriate lysis buffer. The cell suspension was divided into several aliquots and placed in a series of precisely temperature-controlled heating modules for heat shock treatment at a temperature gradient (37°C to 60°C), with each sample heated for 3 minutes. Immediately after heat shock, the samples were placed on ice to terminate the reaction, and then centrifuged at 12,000 rpm for 10 minutes at 4°C to separate the heat-denatured precipitated proteins from the soluble proteins. Carefully collect the supernatant (containing soluble protein whose thermal stability is not affected), add an appropriate amount of loading buffer, and boil to denature. Finally, use Western blot to detect the change in the content of the target protein under different temperature treatments, thereby analyzing whether isoalloLCA treatment changes the thermal melting curve (Tm) of the protein, i.e., its thermal stability. Figure 46 It was found that in HK-2 and NRK-52E cells, isolithocholic acid can enhance the stability of PARP1 in the 54–67℃ range. In summary, these results quantitatively confirm the interaction between isolithocholic acid and PARP1, and reveal changes in its binding affinity and the thermal stability of the protein after binding.

[0098] VI. Application of Biomarker Detection according to Figure 47 As shown in Figure A, morning stool, midstream urine, and fasting venous blood samples were collected from 30 patients at risk or suspected of having kidney stones (CaOx group) and 30 healthy controls (HC group). Isolithocholic acid content was determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS) as the standard method. The test results are shown below. Figure 47 As shown in B in the diagram.

[0099] according to Figure 47 As shown in Figure B, the level of isolithocholic acid in biological samples is high. Compared with the HC group, the concentration of isolithocholic acid in feces, serum, and urine was higher in the CaOx group. Receiver operating characteristic curve analysis showed that the areas under the curve (AUC) for isolithocholic acid in feces, serum, and urine were 0.772, 0.774, and 0.732, respectively, indicating that isolithocholic acid has moderate diagnostic ability in distinguishing between patients with calcium oxalate kidney stones and healthy individuals. All values ​​in the figure are expressed as mean, and the error bars represent the standard deviation. AUC: Area under the curve; Cl: Confidence interval.

[0100] In summary, this invention reveals for the first time that isolithocholic acid is a key pathogenic molecule and biomarker for calcium oxalate kidney stones, and elucidates its novel pathogenic pathway mediated by PARP1 in Parthanatos. This lays a solid scientific foundation for the early non-invasive diagnosis and risk warning of calcium oxalate kidney stones, as well as for the development of novel therapeutic strategies targeting PARP1 or regulating bile acid metabolism.

[0101] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker for calcium oxalate kidney stones, characterized in that, The biomarker is isolithocholic acid.

2. The calcium oxalate kidney stone biomarker according to claim 1, characterized in that, The biomarkers are derived from fecal, serum, kidney tissue, or urine samples.

3. The calcium oxalate kidney stone biomarker according to claim 1, characterized in that, The isolithocholic acid activates PARP1, inducing parthanatos in renal tubular epithelial cells and promoting the formation of calcium oxalate kidney stones.

4. Application of reagents for detecting isolithocholic acid in the preparation of products for detecting calcium oxalate kidney stones.

5. The application according to claim 4, characterized in that: The products include reagent kits, probes, and chips.

6. Application of PARP1 inhibitors in the preparation of drugs for the treatment of isolithocholic acid-induced calcium oxalate kidney stones.

7. The application according to claim 6, characterized in that: The PARP1 inhibitor is AZD5305.

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