Use of a compound in preventing recurrence of calcium oxalate kidney stones

Abstract

This invention belongs to the biomedical field and relates to the application of a compound in preventing the recurrence of calcium oxalate kidney stones. The application of the compound, or its pharmaceutically acceptable salts, esters, solvates, crystal forms, or prodrugs, in the preparation of reagents for preventing the recurrence of calcium oxalate kidney stones is described. The structure of the compound is as follows: [structure details missing]. The calcium oxalate kidney stones originate from Randall plaques. Extensive experimental evidence demonstrates that Benathin can enhance intercellular adhesion by repairing the adhesion protein network damaged by hydroxyapatite (HAP), thereby reversing epithelial damage, preventing epithelial cell detachment, and effectively protecting the renal papillary mucosal barrier, thus reducing the risk of stone formation and recurrence of calcium oxalate kidney stones.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of a compound in the preparation of a formulation for preventing the recurrence of calcium oxalate kidney stones. Background Technology

[0002] Calcium oxalate nephrolithiasis is a common urinary tract disease with a high recurrence rate (approximately 30%-50%) after surgery. Randall's plaque is considered the initial lesion for stone formation, but its molecular mechanism remains unclear. Randall's plaques, located at the apex of the renal papilla or in the surrounding interstitial tissue, are found in almost 100% of patients with calcium oxalate stones and 43% of those without, and are considered ideal sites for the growth and formation of calcium oxalate and calcium phosphate stones. Calcium oxalate nephrolithiasis originating from Randall's plaque refers to calcified plaques (i.e., Randall's plaques) in the renal papillary interstitium as the initial lesion. When the transitional epithelial cells of the renal papilla covering the plaque are damaged and slough off, the plaque core (mainly hydroxyapatite, HAP) is exposed to the urine. Calcium oxalate crystals then heterogeneously nucleate, aggregate, and grow on this basis, ultimately forming calcium oxalate nephroliths. However, traditional drugs (such as thiazides and citrates) only regulate urine composition and cannot block the core pathological process of Randall plaques.

[0003] Prior art CN 120214156 A discloses the application of CRYL1 protein in the preparation of products for assessing, diagnosing, or assisting in the diagnosis of the risk of recurrence after surgery for calcium oxalate kidney stones complicated with renal papillary calcification. The sequence of CRYL1 protein is shown in SEQ ID NO.1. This invention is the first to discover that the expression of CRYL1 protein is reduced in the renal tubular and collecting duct cells of renal papillary calcification tissue, and experimentally demonstrated that urinary CRYL1 protein is a predictor and predictive biomarker for postoperative recurrence in patients with CaOx kidney stones complicated with renal papillary calcification. However, inhibitors of CRYL1 protein can only serve as predictive biomarkers and cannot be used as agents for the treatment and prevention of recurrence of calcium oxalate kidney stones. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for a compound, specifically its application in preventing the recurrence of calcium oxalate kidney stones.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The use of a compound or a pharmaceutically acceptable salt, ester, solvate, crystal form, or prodrug thereof in the preparation of a reagent for preventing recurrence of calcium oxalate kidney stones, wherein the structure of the compound is as follows:

[0007] ;

[0008] The calcium oxalate kidney stones originated from Randall plaques.

[0009] The compound of this invention is named Benathin, also known as (2,3-dihydroxybenzoyl)arginine-threonine, with CAS number 143651-45-0. Its molecular formula is C2. 17 H 25 N5O7, molecular weight 411.41. It contains benzoyl groups. Benarthin contains arginine and an amide bond (Arg), forming a zwitterionic structure. Its molecular backbone includes β-alanine and an aromatic ring, stabilized by a network of hydrogen bonds. Existing research shows that Benarthin is a novel pyrrolidone carboxypeptidase (PG-peptidase) inhibitor isolated from the culture medium of *Streptomyces xanthophaeus* MJ244-SF1. However, no studies have explored the association between Benarthin and the prognosis of Randall plaque-associated calcium oxalate kidney stones. This invention, through extensive experimental verification, demonstrates that Benarthin can serve as a reagent for preventing the recurrence of Randall plaque-associated calcium oxalate kidney stones, showing great promise for clinical application.

[0010] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0011] In one preferred embodiment, the reagent is formulated into a pharmaceutically acceptable dosage form using the compound as the active ingredient and a pharmaceutically acceptable carrier or excipient.

[0012] In one preferred embodiment, the carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants commonly used in the pharmaceutical field.

[0013] In one preferred embodiment, the carrier is selected from one or more of starch, sucrose, cellulose derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, agar, calcium carbonate, sodium bicarbonate, quaternary ammonium compounds, hexadecyl alcohol, kaolin, soap clay, talc, calcium stearate, magnesium stearate, polyethylene glycol, flavorings, sweeteners, and flavoring agents.

[0014] In one preferred embodiment, the weight percentage of the active ingredient is 0.1% to 99.51%.

[0015] In one preferred embodiment, the dosage form of the drug is any one of granules, capsules, tablets, injections, infusions, or suppositories.

[0016] All of the above-mentioned dosage forms of drugs can be prepared using conventional methods in the pharmaceutical field. For example, the active ingredient can be mixed with one or more carriers and then formulated into the desired dosage form.

[0017] In one preferred embodiment, the compound can repair the damaged mucosal barrier in Randall plaques.

[0018] In one preferred embodiment, the compound can inhibit Randall plaque exposure.

[0019] In one preferred embodiment, the compound inhibits epithelial cell shedding from Randall plaques.

[0020] Based on the same inventive concept, this invention also claims a reagent for preventing the recurrence of calcium oxalate kidney stones, said reagent having a compound or its pharmaceutically acceptable salt, ester, solvate, crystal form or prodrug as the active ingredient, the structure of which is as follows:

[0021] ;

[0022] The calcium oxalate kidney stones originated from Randall plaques.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention, using optical microscopy and TUNEL apoptosis staining at the cellular level, confirms that the addition of the compound Benathin can completely reverse the toxic effects of HAP crystals on normal renal papillary transitional epithelial cells, protecting cell survival and maintaining normal morphology. Immunofluorescence staining confirms that Benathin can enhance intercellular adhesion by repairing the adhesion protein network damaged by HAP, thereby reversing epithelial damage and preventing epithelial cell detachment. In vivo experiments confirm that Benathin can effectively protect the renal papillary mucosal barrier by maintaining epithelial integrity, thereby reducing the risk of stone formation. Furthermore, at the effective dosage used in this invention, long-term oral administration of Benathin exhibits good biocompatibility.

[0025] This invention marks the first shift from treating existing diseases to preventing future ones, making it suitable for high-risk groups (such as patients with recurrent kidney stones) and reducing the need for repeated surgeries. Compared to the cumbersome administration procedures of existing technologies, Benathin simplifies the prevention process, is suitable for long-term use, and is particularly suitable for patients with chronic kidney stones, improving treatment sustainability. Attached Figure Description

[0026] Figure 1 Ureteroscopic images showing normal renal papillary tissue and the appearance of renal papillary calcification.

[0027] Figure 2This is a comparison image of Von-Kossa staining between normal renal papillae and Randall plaque renal papillae (RP).

[0028] Figure 3 Results of normal renal papillary transitional epithelial cells under different treatment conditions; among them, Figure 3 A shows optical micrographs of normal renal papillary transitional epithelial cells under different treatment conditions; Figure 3 B shows TUNEL staining of normal renal papillary transitional epithelial cells under different treatment conditions; Figure 3 C shows immunofluorescence staining of normal renal papillary transitional epithelial cells under different treatment conditions; Figure 3 D shows the Western blot (WB) images of normal renal papillary transitional epithelial cells under different treatment conditions.

[0029] Figure 4 Umod for different processing groups - / - Pathological features of the mouse model and the in vivo repair effect of Benathin on the renal papillary mucosal barrier; among which, Figure 4 A is Umod - / - Schematic diagram of the mouse model; Figure 4 B represents Umod from different processing groups. - / - Western blot diagram for identifying pathological features in a mouse model; Figure 4 C represents Umod for different processing groups. - / - Von-Kossa staining and immunofluorescence staining images of mice; Figure 4 D represents Umod for different treatment groups. - / - Immunofluorescence staining image of mice.

[0030] Figure 5 For long-term oral administration of Benathin to counteract Umod - / - Histological safety assessment of major organs in mice. Detailed Implementation

[0031] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0032] Example 1

[0033] First, the direct physical association between Randall plaque (RP) and calcium oxalate (CaOx) kidney stones was established. High-resolution images were recorded using the endoscope's built-in imaging system. The inclusion criteria for patients with calcium oxalate kidney stones originating from Randall plaque were:

[0034] 1. Clear stone composition: The patient must have calcium oxalate (CaOx) stones. This is usually confirmed by performing infrared spectroscopy or X-ray diffraction analysis on the stones collected after surgery.

[0035] 2. Direct endoscopic observation (key criterion): During ureteroscopy, the physician must visually observe the presence of characteristic Randall plaques (typically appearing as creamy or white calcified patches located subepithelially at the tip of the renal papilla). Furthermore, calcium oxalate stones must be observed growing directly onto these plaques.

[0036] The exclusion criteria are:

[0037] To ensure that the study focuses on idiopathic calcium oxalate stones caused by Randall plaques, the following patients are typically excluded: 1. Non-calcium oxalate stones: Stones whose composition is not CaO. x 1. Primary causes; 2. Stones with clear secondary causes: patients with primary hyperparathyroidism, distal renal tubular acidosis, or enterogenic hyperoxaluria (such as Crohn's disease or after intestinal bypass surgery); 3. Non-Randall plaque mechanisms: even with calcium oxalate stones, if Randall plaques are not observed endoscopically, or if the stone is found to be caused by a "tubular blockage" in the renal tubule, these patients may be excluded or classified into different groups.

[0038] This study involved in vivo ureteroscopic examination of renal papillary calcification (RP) in patients with calcium oxalate kidney stones originating from Randall plaques, and normal renal papillary tissue (NRP) in a control group undergoing ureteroscopy for non-calcified causes. The renal calyces were systematically examined, with a focus on the morphology, color, and presence of calcified plaques (RP) or attached stones in the renal papillae (NRP). High-resolution images were recorded using the endoscope's built-in imaging system.

[0039] The results are as follows Figure 1 As shown in the image, the appearance of normal renal papillary tissue (NRP) and renal papillary calcification plaques (RP) under endoscopy is clearly visible. Normal renal papillary (NRP) surfaces are smooth and free of deposits, while RP renal papillae show calcium salt deposits (black arrows) with CaOx stones (yellow crystals) adhering to their surfaces. CaOx kidney stones specifically adhere to the calcified plaques on the renal papillae, whereas the surface of normal renal papillae is smooth and free of deposits.

[0040] Other non-calculous nephrectomy control groups were patients who had to have their kidneys removed due to other diseases (most commonly kidney tumors). From their removed kidneys, renal papillary tissue that appeared completely normal in appearance and histology and was far from the lesion was selected as the normal control group.

[0041] To detect calcium salt deposition in renal papillary tissue and simultaneously observe tissue morphology, this embodiment employed the von Kossa staining method. Paraffin sections of renal papillary tissue were obtained from clinical studies approved by the Ethics Committee of Xiangya Hospital (Ethics Nos.: 201603035; 202103089). These samples were collected from renal papillary biopsy samples obtained from patients undergoing ureteroscopic surgery for calcium oxalate kidney stones originating from Randall plaques (RP group) and from renal papillary tissue from control groups who underwent nephrectomy for other non-calculous reasons (NRP group). Informed consent was obtained from all patients.

[0042] The preparation method for paraffin sections of renal papillary tissue is as follows: Fresh renal papillary tissue is immediately immersed in a 4% paraformaldehyde solution for fixation. After fixation, the tissue block is dehydrated sequentially through a series of ethanol solutions (75%, 85%, 95% to 100% anhydrous ethanol). Subsequently, the tissue block is cleared using xylene. Next, the cleared tissue block is transferred to molten paraffin for infiltration, allowing for complete penetration. After infiltration, the tissue block and paraffin are cooled and solidified together in an embedding mold to form a hard paraffin block. Finally, the paraffin block is cut into thin slices with a thickness of 3-5 micrometers using a microtome, and the slices are flatly attached to a glass slide and baked to complete the section preparation.

[0043] Paraffin sections of renal papilla tissue were prepared for analysis. The Von Kossa staining kit (purchased from Solaribo, China, Cat# G3282) was used, strictly following the manufacturer's standard protocol. In short, the sections were dewaxed to water, immersed in silver nitrate solution, and irradiated under strong light (such as UV lamp) for 30-60 minutes until the calcium salts turned dark brown. After treatment with sodium thiosulfate, counterstaining was performed. Counterstaining was performed using a hematoxylin-eosin (H&E) staining kit (Solaribo, Cat# G1120) to clearly visualize the background structures such as the cell nucleus and cytoplasm. Using this method, calcium salt deposits were specifically labeled as black or dark brown granules, while the cell nucleus appeared blue-purple, and the cytoplasm and extracellular matrix appeared as varying shades of pink.

[0044] The staining results are as follows Figure 2 As shown. The results indicated that, compared to normal tissue with an intact transitional epithelial cell layer (NRP), RP tissue exhibited extensive epithelial cell shedding and mucosal barrier loss above the calcium salt deposition areas. Figure 2 This pathological change exposes the hydroxyapatite (HAP) deposits that form the plaque core directly to the urine, providing ideal attachment sites for the heterogeneous nucleation of CaOx crystals, thus initiating the stone formation process. Therefore, repairing this damaged mucosal barrier has been identified as a key strategy for preventing this type of kidney stone.

[0045] Example 2

[0046] Cell culture and treatment, TUNEL staining, Western blotting (WB), and immunofluorescence staining.

[0047] (1) Cell culture and treatment.

[0048] The cells used in this invention were primary human renal papillary transitional epithelial cells. These cells were isolated from human renal pelvis tissue using collagenase digestion. Immunofluorescence was used to identify the cells, with the co-expression of E-cadherin and keratin-13 as a positive indicator. All cells used in subsequent experiments were selected from passages 3 to 6. The cell culture conditions were as follows: F-12K medium (Servicebio, China) containing 10% fetal bovine serum (FBS, Beyotime, China, Cat# C0258), 100 U / mL penicillin, and 100 μg / mL streptomycin (Servicebio, China, Cat# G4003) was used, and the cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0049] To verify the protective effect of the small molecule compound Benathin against hydroxyapatite (HAP)-induced epithelial cell damage, cultured renal papillary transitional epithelial cells were divided into four groups for treatment: ① Normal control group (NC group): Normally cultured renal papillary transitional epithelial cells without any additional treatment; ② HAP treatment group (HAP group): Cell cultured cells were treated with 100 μg / mL HAP; ③ Solvent control group (HAP+DMSO group): HAP was added along with 0.1% dimethyl sulfoxide (DMSO, Beyotime, China, ST038); ④ Benathin treatment group (HAP+Benarthin+DMSO group): HAP was added along with 0.1% dimethyl sulfoxide (DMSO, Beyotime, China, ST038), and then 10 μM of the small molecule compound Benathin (MCE, HY-117738) was added. Cells from each of the above experimental groups were treated under the same culture conditions for 3 consecutive days. Cells were then collected for subsequent cell morphology observation, apoptosis detection, and analysis of related protein expression.

[0050] (2) TUNEL staining

[0051] Apoptosis was quantitatively analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. The reagents used were a one-step TUNEL apoptosis detection kit (Beyotime, China, Cat#C1086). Cultured cells were washed once with PBS (phosphate-buffered saline, Beyotime, Cat#C0221A), then fixed with 4% paraformaldehyde solution (PFA, Beyotime, Cat#P0099) for 30 minutes at room temperature. After fixation, cells were washed with PBS, and then treated with PBS containing 0.3% Triton X-100 (Beyotime, China, P0096) for 5 minutes at room temperature to increase cell membrane permeability. 100 μL of TUNEL assay solution was added to the treated cells, and the cells were incubated in the dark at 37°C for 60 minutes. The cells were then thoroughly washed three times with PBS and mounted using mounting medium containing anti-fluorescence quencher (DAPI, Beyotime, Cat#P0131). Finally, apoptotic cells were observed using a fluorescence microscope (Leica TCSSP8 X, Germany), and images were acquired for analysis.

[0052] The results are as follows Figure 3 As shown in A and 3B, HAP and HAP+DMSO have significant cytotoxicity to normal renal papillary transitional epithelial cells and can induce apoptosis, with the apoptosis rate of HAP+solvent being even higher; while the addition of Benathin can completely reverse this toxic effect, protect cell survival and maintain normal morphology, and its apoptosis rate is much lower than that of the HAP group.

[0053] (3) Western blotting (WB)

[0054] Cells from each experimental group (NC group, HAP group, HAP+DMSO group, HAP+Benarthin group) were collected, and total protein was extracted using RIPA lysis buffer (Beyotime, China, Cat# P0013B) containing 1% PMSF (phenylmethylsulfonyl fluoride, Beyotime, Cat# ST506) and 1% phosphatase inhibitor mixture (NCM Biotech, China, Cat# P003). The protein concentration of each sample was then accurately determined using a BCA protein quantification kit (Beyotime, China, Cat# P0012). A suitable amount of total protein sample was loaded onto a 12% SDS-PAGE gel (GenScript, China, Cat# M01215C) for electrophoresis. After electrophoresis, the separated proteins were transferred to a 0.45 μm PVDF membrane (Millipore, Cat# IPVH00010) using a wet transfer method. PVDF membranes were blocked with TBST buffer (Beyotime, China, Cat# ST673) containing 5% skim milk powder (BD, Cat# 232100) at room temperature for 1-2 hours, and then incubated overnight with specific primary antibodies at 4°C. The primary antibodies used in this invention included: ITGB1 antibody (CST, #4706, 1:1000), E-cadherin antibody (CST, #3195, 1:1000), PXN antibody (Abcam, ab32084, 1:1000), and antibody against the internal reference protein GAPDH (CST, #5174, 1:2000). The next day, after washing with TBST buffer, the membranes were incubated with the corresponding horseradish peroxidase (HRP) conjugated secondary antibodies (anti-rabbit: CST, #7074, 1:3000; anti-mouse: CST, #7076, 1:3000) at room temperature for 1 hour. Finally, development was performed using enhanced chemiluminescence (ECL) reagent (NCM Biotech, China, Cat# P10100), and protein band images were captured and recorded using a chemiluminescence imaging system (ChemiDoc XRS, Bio-Rad, USA). The grayscale value of each protein band was analyzed and calculated using ImageJ software (NIH, USA). The relative expression levels of each target protein (ITGB1, E-cadherin, PXN) were calculated by comparing the grayscale values ​​with those of the internal control protein GAPDH.

[0055] The results are as follows Figure 3As shown in Figure D, HAP treatment reduced the protein expression levels of ITGB1, E-cadherin, and PXN by approximately 60%, 70%, and 55%, respectively. After Benathin intervention, the expression levels of these three key adhesion proteins were significantly restored, with PXN, E-cadherin, and ITGB1 returning to approximately 83.4%, 83.8%, and 94.5% of their normal levels, respectively (p<0.01), and their intracellular localization also returned to normal. Figure 3 D).

[0056] (4) Cell immunofluorescence staining

[0057] Cells were seeded onto coverslips at approximately 25% confluence and treated in osteogenic medium for 3 days as required for the experiment. Before staining, the cell layer was washed three times with pre-chilled PBS and then fixed with 4% PFA for 20 minutes at room temperature. Blocking was performed for 1 hour at room temperature using 10% normal goat serum (Beyotime, China, Cat# C0265). Cells were incubated overnight at 4°C with primary antibodies (ITGB1 antibody, CST, #4706, 1:200; E-cadherin antibody, CST, #3195, 1:200; or PXN antibody, Abcam, ab32084, 1:200). The next day, after washing with pre-cooled PBS, cells were incubated with fluorescent secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit secondary antibody, Invitrogen, A-11034, 1:500; or Alexa Fluor 647-conjugated anti-mouse secondary antibody, Invitrogen, A-21235, 1:500) at room temperature in the dark for 1 hour. Nuclear counterstaining and imaging: DAPI (Servicebio, China, Cat# G1012) was added and incubated for 5 minutes to counterstain the cell nuclei. Finally, slides were mounted with mounting medium containing an anti-fluorescence quencher (Beyotime, Cat# P0126), and images were acquired and analyzed using a fluorescence microscope (Leica TCS SP8 X, Germany).

[0058] The results are as follows Figure 3 As shown in C, treatment with HAP and HAP+ solvent severely disrupted the cell adhesion protein network, leading to abnormal aggregation of ITGB1, while the expression of E-cadherin and PXN was significantly downregulated. Figure 3 C). Benathin can significantly upregulate the expression of ITGB1, E-cadherin, and PXN.

[0059] These results indicate that Benathin can enhance intercellular adhesion, reverse epithelial damage, and prevent epithelial cell detachment by repairing the adhesion protein network damaged by HAP.

[0060] Example 3

[0061] Umod - / - Gene knockout mice were used as a Randall plaque-associated kidney stone model. The Umod gene in this mouse model is located on mouse chromosome 7 and was constructed by targeted deletion of exons 3 through 7. All experimental mice were purchased from Cyagen Biosciences Co., Ltd., China, and genotyping was performed by DNA extraction and PCR amplification from tail samples. The experiment consisted of two parts: model identification and drug intervention.

[0062] (1) Model validation: To verify Umod - / - A mouse model was successfully constructed that can simulate the early pathological changes of Randall plaques. Wild-type (WT) and Umod mice were collected at different time points. - / - Kidney tissue from mice (n=3 per group) was analyzed as follows:

[0063] Western blotting (WB): WT and Umod samples were collected from 4-week-old infants. - / - Mouse kidney tissue was homogenized with RIPA lysis buffer (same as in Example 2) and total protein was extracted. Subsequent steps, including BCA quantification, SDS-PAGE electrophoresis, membrane transfer, blocking, incubation with primary antibody (Umod antibody, Santa Cruz, sc-390326, 1:500; GAPDH antibody, CST, #5174, 1:2000) and secondary antibody, and ECL imaging, were all the same as in Example 2.

[0064] Von-Kossa staining and immunofluorescence staining: WT and Umod samples were collected from 6-month-old infants. - / - Mouse kidney tissue was fixed with 4% PFA, embedded in paraffin, and sectioned. The same procedure as in Example 1 was followed using a Solaribo (China, Cat# G3282) kit to detect calcium salt deposition in the renal papillary interstitium.

[0065] Immunofluorescence staining: The procedure was the same as in Example 2. The sections underwent antigen retrieval and blocking, and were stained with primary antibodies (Keratin-13 antibody, Abcam, ab92551, 1:200; E-cadherin antibody, CST, #3195, 1:200; Pxn antibody, Abcam, ab32084, 1:200) and corresponding secondary fluorescent antibodies (Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647). Cell nuclei were counterstained with DAPI. The expression and distribution of epithelial markers were observed using a confocal microscope.

[0066] (2) Benathin drug intervention experiment: 6-week-old Umod - / - Mice were randomly divided into three groups (n=6 in each group) for treatment: Model control group (NC group): Umod - / - Mice were fed standard drinking water. The solvent control group (HP-β-CD group) received drinking water with 2% hydroxypropyl-β-cyclodextrin (a solubilizer, Sigma-Aldrich, Cat# H107). The Benarthin treatment group received drinking water containing 2% hydroxypropyl-β-cyclodextrin with Benarthin added at a dose of 100 mg / kg / day. A normal control group (WT group) was also established, where normal mice were fed standard drinking water. After 3 months of continuous administration, the mice were sacrificed, and renal papilla tissue was collected and sectioned for subsequent analysis.

[0067] Mouse kidney tissue sections were stained using a double immunofluorescence staining method. The specific steps were as follows: First, the paraffin sections were dewaxed and hydrated. Specifically, the paraffin sections (glass slides) were sequentially immersed in: xylene (I) for 15 minutes; xylene (II) for 15 minutes; 100% (anhydrous) ethanol for 5 minutes; 95% ethanol for 3 minutes; 85% ethanol for 3 minutes; and 75% ethanol for 3 minutes. Ethanol solutions less than 100% were aqueous solutions. Finally, the sections were rinsed in tap water for 5 minutes to ensure complete hydration, ready for subsequent staining. Then, heat-mediated antigen retrieval was performed using antigen retrieval buffer (pH 6.0, Solaribo, Cat#G1202). The sections were placed in citrate antigen retrieval buffer (pH 6.0, Solaribo, Cat#G1202), heated to boiling using a microwave oven, maintained at a gentle boil for 15 minutes, and then allowed to cool naturally to room temperature. Subsequently, to eliminate background signals, sections were blocked for 1 hour at room temperature using 10% goat serum (Beyotime, China, Cat# C0265). Next, two primary antibodies were simultaneously incubated: rabbit-derived anti-Pxn antibody (Abcam, ab32084, 1:200) and mouse-derived anti-Keratin 13 antibody (Abcam, ab92551, 1:200) overnight at 4°C. Finally, images were acquired using a confocal microscope system (Leica TCS SP8 X, Germany), and image analysis was performed using ImageJ software (NIH, USA) and its Coloc 2 plugin. The expression levels of Pxn and Keratin-13 proteins were semi-quantitatively assessed by measuring the mean fluorescence intensity (MFI) of the defined regions of interest (ROIs). The co-localization of Pxn and Keratin-13 was analyzed by calculating the Pearson correlation coefficient (PCC) to assess the structural integrity of the epithelial barrier.

[0068] The results are as follows Figure 4 As shown. Western blot (WB) analysis clearly confirmed that, compared with wild-type (WT) mice, Umod... - / - The complete absence of Umod protein expression in mouse kidney tissue proves that the model was successfully constructed. Figure 4 A, 4B). Von-Kossa staining shows that Umod... - / - Significant calcium salt deposition was observed in the renal papillary interstitium of mice, successfully mimicking the early pathological changes of Randall plaques, while this phenomenon was not observed in WT mice. Figure 4 C). Immunofluorescence staining results showed that, compared to the well-structured epithelial layer in WT mice, Umod... - / - The expression of epithelial markers Keratin-13, E-cadherin, and Pxn in mice was disordered and downregulated, strongly demonstrating a congenital defect in their mucosal barrier function. Figure 4 C). The above results confirm that Umod - / - Mice are an ideal in vivo model for studying Randall plaque-related mucosal barrier damage and drug repair.

[0069] Based on the above model, this invention evaluated the in vivo efficacy of Benathin. Umod... - / - Mice were divided into a model control group (NC), a solvent control group (HP-β-CD), and a Benathin treatment group (100 mg / kg / day, orally administered). Immunofluorescence staining results showed that in the renal papilla tissue of mice in the model control and solvent control groups, the epithelial structure remained loose, with a broken distribution of the epithelial marker Keratin-13 (red) and weak expression of Pxn (green). In contrast, after oral treatment with Benathin, the mucosal barrier structure of the mouse renal papilla was significantly repaired. Microscopically, Pxn expression showed a continuous high-intensity signal, and Keratin-13 completely covered the entire epithelial layer, with significant co-localization of the two signals, forming a structurally intact and tightly connected epithelial barrier. Figure 4 D). These in vivo experimental results confirm that Benathin can effectively protect the renal papillary mucosal barrier by maintaining epithelial integrity, thereby reducing the risk of stone formation.

[0070] To evaluate the safety of Benathin as a long-term prophylactic drug, histopathological analysis of major organs was performed on mice that had been orally administered the drug for 3 months. Hematoxylin-eosin (H&E) staining was performed on the kidneys, heart, liver, spleen, and lungs using a hematoxylin-eosin (H&E) staining kit (Solaribo, Cat# G1120) to clearly visualize background structures such as cell nuclei and cytoplasm. The detailed steps are as follows: First, paraffin sections were dewaxed sequentially in xylene I and xylene II, followed by hydration in a gradient of 100%, 95%, 85%, and 75% ethanol baths and water. Next, the sections were immersed in hematoxylin staining solution to stain the cell nuclei. After washing with water, rapid differentiation was achieved with 1% hydrochloric acid ethanol, followed by rinsing with tap water to terminate differentiation. Subsequently, Scott's blue solution or a weakly alkaline solution such as dilute ammonia was used for blue reversion, resulting in a blue-purple color in the cell nuclei, followed by washing with water again. Next, the sections were placed in eosin staining solution to counterstain the cytoplasm and extracellular matrix, followed by a brief wash with water. After staining, the sections were dehydrated using a gradient of 75%, 85%, 95%, and 100% ethanol, and then cleared with xylene I and xylene II. Finally, neutral resin was added, and the sections were sealed with coverslips. Staining results: The cell nuclei appeared blue-purple, while the cytoplasm and extracellular matrix appeared in varying shades of pink or red.

[0071] The results showed that, compared with the model control group and the solvent control group, no obvious inflammatory infiltration, cell necrosis or other pathological damage or tissue structural abnormalities were observed in the major organs of the mice treated with Benathin. Figure 5 This result demonstrates that, at the effective dose used in this invention, long-term oral administration of Benathin exhibits good biocompatibility.

[0072] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a reagent for preventing the recurrence of calcium oxalate kidney stones, characterized in that, The structure of the compound is as follows: ； The calcium oxalate kidney stones originated from Randall plaques.

2. Use according to claim 1, characterized in that, The reagent uses the compound as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier or excipient.

3. Use according to claim 2, characterized in that, The carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants commonly used in the pharmaceutical field.

4. Use according to claim 2, characterized in that, The carrier is selected from one or more of the following: starch, sucrose, cellulose derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, agar, calcium carbonate, sodium bicarbonate, quaternary ammonium compounds, hexadecyl alcohol, kaolin, soap clay, talc, calcium stearate, magnesium stearate, polyethylene glycol, and flavoring agents.

5. Use according to claim 2, characterized in that, The weight percentage of the active ingredient is 0.1% to 99.51%.

6. Use according to claim 2, characterized in that, The dosage form is any one of granules, capsules, tablets, injections, or suppositories.

7. The use according to any one of claims 1 to 6, characterized in that, The compound prevents the recurrence of calcium oxalate kidney stones by repairing the damaged mucosal barrier in Randall plaques.

8. Use according to any one of claims 1 to 6, characterized in that, The compound prevents the recurrence of calcium oxalate kidney stones by inhibiting Randall plaque exposure.

9. Use according to any one of claims 1 to 6, characterized in that, The compound prevents the recurrence of calcium oxalate kidney stones by inhibiting the shedding of epithelial cells from Randall plaques.

Images