Application of CRYL1 protein in preparation of product for evaluating postoperative recurrence risk of calcium oxalate kidney stone combined with renal nipple calcium spot

Abstract

The invention belongs to the field of biomedicine, and particularly relates to application of CRYL1 protein in preparation of a product for evaluation, diagnosis or auxiliary diagnosis of postoperative recurrence risk of calcium oxalate kidney stone combined with renal nipple calcium spot, and the sequence of the CRYL1 protein is shown as SEQ ID NO.1. The invention discovers that the expression of the CRYL1 protein in renal tubules and collecting tube cells of renal papillary calcium spot tissues is reduced for the first time, and tests prove that the urine CRYL1 protein is a predictive factor and a predictive marker for postoperative recurrence of a patient with CaOx kidney stone combined with renal papillary calcium spot. The CRYL1 protein detection kit is further researched and developed to be used for detecting or evaluating the postoperative recurrence risk of patients suffering from CaOx kidney stone combined with renal nipple calcium spots, personalized follow-up visit and prevention schemes of the patients are promoted, and the CRYL1 protein detection kit has huge clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of CRYL1 protein in the preparation of a product for evaluating the postoperative recurrence risk of calcium oxalate nephrolithiasis complicated with renal papillary calcification plaque. Background Art

[0002] Calcium oxalate nephrolithiasis is a common disease in the urinary system, affecting approximately 8% of the population in China. [1, 2] The recurrence rate after surgery is high (about 30% - 50%) within 5 years. [3] The levels of urinary calcium, urinary oxalate, and urinary citrate are affected by dietary factors, making it difficult to predict the postoperative recurrence risk of patients with calcium oxalate nephrolithiasis. [4, 5] Other reported predictive indicators are not yet ideal (AUC is lower than 0.8). [6, 7] Currently, there is no effective predictive marker for the postoperative recurrence risk of calcium oxalate nephrolithiasis. Renal papillary calcification plaque originates from the deposition of calcium salts in the renal interstitium of the renal papilla. After breaking through the renal papillary mucosa, it becomes an ideal adhesion point for calcium oxalate crystals, thus inducing calcium oxalate nephrolithiasis. [8, 9] Renal papillary calcification plaque can be seen in almost 100% of patients with idiopathic calcium oxalate stones and 43% of patients with non - calcium oxalate stones. [10, 11] It is considered the initial lesion for the formation of calcium oxalate stones and is closely related to the postoperative recurrence of calcium oxalate nephrolithiasis.

[12] Therefore, based on urinary molecular markers related to renal papillary calcification plaque, it is expected to develop a predictive marker for the postoperative recurrence risk of patients with calcium oxalate stones complicated with renal papillary calcification plaque.

[0003] References: 1. Tan, S., D. Yuan, H. Su, et al., Prevalence of urolithiasis in China: a systematic review and meta - analysis . BJU Int, 2024. 133(1): 34 - 43. 2. Wang, B., X. Zheng, J. Xiong, et al., Characteristics of urinary stone composition among patients with urolithiasis: a retrospective study in China . BMJ Open, 2024. 14(11): e079431. 3. Kachkoul, R., G.B. Touimi, G. El Mouhri, et al., Urolithiasis: History, epidemiology, aetiologic factors and management . Malays J Pathol,2023. 45(3): 333-352. 4. Traxer, O., M. Corrales, and A. Sierra, Metabolic evaluation: is there really a future? Curr Opin Urol, 2022. 32(4): 373-378. 5. Coe, F.L., A. Evan, and E. Worcester, Pathophysiology-based treatment of idiopathic calcium kidney stones . Clin J Am Soc Nephrol, 2011. 6(8): 2083-92. 6. Elshal, A.M., H. Shamshoun, A. Awadalla, et al., Hormonal and molecular characterization of calcium oxalate stone formers predicting occurrence and recurrence . Urolithiasis, 2023. 51(1): 76. 7. Kavouras, S.A., H.G. Suh, M. Vallet, et al., Urine osmolality predicts calcium-oxalate crystallization risk in patients with recurrent urolithiasis . Urolithiasis, 2021. 49(5): 399-405. 8. Evan, A.P., E.M. Worcester, F.L. Coe, et al., Mechanisms of humankidney stone formation . Urolithiasis, 2015. 43 Suppl 1: 19-32. 9. Zhu, Z., F. Huang, M. Gao, et al., Osteogenic-LikeMicroenvironment of Renal Interstitium Induced by Osteomodulin Contributes toRandall's Plaque Formation . Adv Sci (Weinh), 2024. 11(40): e2405875. 10. Randall, A., THE ORIGIN AND GROWTH OF RENAL CALCULI . Ann Surg,1937. 105(6): 1009-1027. 11. Matlaga, B.R., J.C. Williams, S.C. Kim, et al., Endoscopicevidence of calculus attachment to Randall's plaque . Journal of Urology,2006. 175(5): 1720-1724. 12. Tamborino, F., R. Cicchetti, M. Mascitti, et al., Pathophysiologyand Main Molecular Mechanisms of Urinary Stone Formation and Recurrence . IntJ Mol Sci, 2024. 25(5): 3075. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of CRYL1 protein in the preparation of a product for evaluating the postoperative recurrence risk of calcium oxalate kidney stones combined with renal papillary calcification plaques.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions: Use of CRYL1 protein in the preparation of a product for evaluating, diagnosing or assisting in the diagnosis of the recurrence risk of calcium oxalate kidney stones combined with renal papillary calcification after surgery. The sequence of CRYL1 protein is shown as SEQ ID NO.1.

[0006] CRYL1 protein (Crystallin Lambda 1) is an important component of the lens as a crystallin protein and plays a key role in maintaining the transparency and refractive function of the lens. In addition, CRYL1 has dehydrogenase activity, catalyzing the dehydrogenation of L-gulonate to dehydro-L-gulonate and participating in NADPH-dependent metabolic reactions (https: / / www.ncbi.nlm.nih.gov / gene / 51084). CRYL1 protein is highly expressed in the renal tubules and collecting ducts of normal kidneys and may be related to inhibiting renal tubular injury and renal interstitial calcium salt deposition.

[0007] The amino acid sequence of CRYL1 protein is as follows: massaagcvv ivgsgvigrs wamlfasggf qvklydieqq qirnalenir kemklleqagslkgslsvee qlslisgcpn iqeavegamh iqvnppyyip lvelvphpet apttvdrtha lmkkigqcpmrvqkevagfv lnrlqyaiis eawrlveegi vspsdldlvm seglgmryaf igpletmhln aegmlsycdrysegikhvlq tfgpipefsr ataekvnqdm cmkvpddpeh laarrqwrde clmrlaklks qvqpq (SEQID NO.1).

[0008] Through a large number of experiments, the present invention discovers that the expression of CRYL1 protein is decreased in the renal tubules and collecting duct cells of renal papillary calcification tissues. The CRYL1 protein in urine can be used as a predictor and predictive marker for the recurrence of calcium oxalate kidney stones combined with renal papillary calcification after surgery, and has great application prospects in clinical practice.

[0009] Use of a substance for detecting CRYL1 protein in the preparation of a product for evaluating, diagnosing or assisting in the diagnosis of the recurrence risk of calcium oxalate kidney stones combined with renal papillary calcification after surgery. The sequence of CRYL1 protein is shown as SEQ ID NO.1.

[0010] In one preferred embodiment, the substance for detecting CRYL1 protein includes substances for detecting the content of CRYL1 protein by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immunological polymerase chain reaction or biotin-avidin technology.

[0011] In one preferred embodiment, the substance for detecting CRYL1 protein is a substance for binding to CRYL1 protein.

[0012] In one preferred embodiment, the substance for binding to CRYL1 protein is an antibody, polypeptide, protein or nucleic acid molecule.

[0013] The antibody may be a monoclonal antibody, polyclonal antibody, genetically engineered antibody, and may also be antibody variable region Fv, single-chain antibody ScFv, antigen-binding fragment Fab or Fab’, F(ab’)2, Fab’-SH and other antibody fragments, as well as antibody derivatives and the like.

[0014] In one preferred embodiment, the antibody is a CRYL1 antibody.

[0015] Based on the same inventive concept, the present invention also claims the use of a kit, the kit includes a substance for detecting CRYL1 protein, and the kit has at least one of the following uses: 1) For detecting the recurrence after the operation of calcium oxalate kidney stones combined with renal papillary calcification; 2) For diagnosing or assisting in diagnosing the recurrence after the operation of calcium oxalate kidney stones combined with renal papillary calcification; 3) For evaluating the recurrence risk of calcium oxalate kidney stones combined with renal papillary calcification after the operation.

[0016] In one preferred embodiment, the substance for detecting CRYL1 protein is a CRYL1 antibody.

[0017] In one preferred embodiment, the CRYL1 antibody is a CRYL1 rabbit antibody.

[0018] In one preferred embodiment, the detection sample of the kit is urine.

[0019] In one preferred embodiment, the kit further includes CRYL1 protein as a standard.

[0020] In one preferred embodiment, the kit further includes an HRP conjugate, a biotinylated detection antibody diluent, an HRP conjugate diluent, a washing buffer, a substrate and a stop solution.

[0021] The present invention for the first time discovers that the expression of CRYL1 protein is reduced in the renal tubules and collecting duct cells of the renal papillary calcification tissue, and through experiments, it is proved that the CRYL1 protein is a predictor and predictive marker for the postoperative recurrence of patients with CaOx kidney stones combined with renal papillary calcification. Furthermore, a detection kit for CRYL1 protein is developed to detect or evaluate the risk of postoperative recurrence of patients with CaOx kidney stones combined with renal papillary calcification, promoting personalized follow-up and prevention programs for such patients, and having great application prospects in clinical practice. Description of the Drawings

[0022] Figure 1 It is a result graph of single-cell sequencing; wherein, Figure 1 A is a diagram of the appearance of normal renal papilla tissue (NRP) and renal papillary calcification (RP) under endoscopy; Figure 1 B is a flowchart of the operation of single-cell sequencing; Figure 1 C is a UMAP dimensionality reduction clustering graph obtained by single-cell sequencing of RP tissue and NRP tissue; Figure 1 D is a result graph of single-cell sequencing; *P<0.05; **P<0.01; ***P<0.001.

[0023] Figure 2 is a result graph of the biological identification of renal papillary calcification (RP) tissue; wherein, Figure 2 A is a calcium salt staining (Von-Kossa) and immunohistochemical staining graph of renal papillary calcification (RP) tissue; Figure 2 B is a result graph of semi-quantitative analysis of immunohistochemical staining of RP and normal renal papilla tissue; Figure 2 C is a result graph of ELISA detection of urine from healthy physical examination population and patients with CaOx kidney stones combined with RP plaques.

[0024] Figure 3 It is a standard curve graph of detection well plate 1.

[0025] Figure 4 It is a standard curve graph of detection well plate 2.

[0026] Figure 5 It is a standard curve graph of detection well plate 3.

[0027] Figure 6 It is a fitting curve graph of urine CRYL1 protein and the postoperative recurrence rate of patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcification.

[0028] Figure 7 It is a ROC curve graph of urine CRYL1 protein predicting the postoperative recurrence of patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcification.

[0029] Figure 8ROC curve of the recurrence rate of calcium oxalate (CaOx) kidney stones combined with renal papillary calcification in patients detected by urinary CRYL1 protein at 5 years after surgery. Specific implementation manners

[0030] The present invention is not limited to the following specific implementation manners. Those of ordinary skill in the art can implement the present invention in other various specific implementation manners according to the content disclosed in the present invention, or any implementation manners that adopt the design structure and idea of the present invention and make simple changes or modifications fall within the protection scope of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0031] In the present invention, the inclusion criteria for patient material collection are as follows: (1) 18 years old ≤ age ≤ 75 years old; (2) mild hydronephrosis or no hydronephrosis; (3) no chronic kidney disease complicated with proteinuria; (4) the renal papilla at the material collection site is more than 3 cm away from the tumor margin and the renal papilla HE staining confirms that it is not invaded by the tumor. Renal papilla tissue samples are obtained from the excised specimens of radical nephrectomy in patients with renal cancer or ureteral cancer. Example 1

[0032] Single-cell sequencing The specific steps are as follows: (1)Tissue dissociation and single-cell suspension preparation: Renal papilla tissue samples were stored in GEXSCOPE tissue preservation solution (Singleron) and transported to Singleron Laboratory (Singleron Biotechnologies Co., Ltd., Nanjing, China) with ice packs. The renal papilla tissue samples were washed three times with Hank's balanced salt solution (HBSS, Gibco, catalog number: 14025-076), and then cut into small pieces of 1-2 mm. Subsequently, the tissue fragments were placed into a 15-ml centrifuge tube (Falcon, catalog number: 352095), 2 ml of GEXSCOPE tissue dissociation solution (Singleron) was added, and digestion was carried out with continuous stirring at 37°C for 15 minutes. The cells were filtered through a 40-μm sterile filter (Falcon, catalog number: 352340) and centrifuged at 300 g for 5 minutes (the centrifuge was manufactured by Eppendorf, model: 5810R). After removing the supernatant, the pellet was resuspended in 1 ml of PBS (Hyclone, catalog number: SA30256.01). To remove red blood cells, 2 ml of RBC lysis buffer (Roche, catalog number: 11814389001) was added to the cell suspension. The cells were centrifuged at 500×g for 5 minutes in a microcentrifuge at 15-25°C and resuspended in PBS (Hyclone, catalog number: SA30256.01). After sampling, trypan blue staining (Bio-RAD, catalog number: #1450013) was used, and the cells were counted under a microscope (Nikon, ECLIPSE Ts2). After ensuring that the concentration was 1×10^5 cells / ml and the cell viability exceeded 80%, subsequent sample processing could be carried out.

[0033] (2)Details and preliminary results of single-cell RNA sequencing: The cell suspension was adjusted to a concentration of 1×10^5 cells / ml in PBS. Subsequently, the single-cell suspension was loaded onto a microfluidic device, and according to the Singleron GEXSCOPE protocol, a scRNA-seq library was constructed using the GEXSCOPE single-cell RNA library kit (Singleron Biotechnologies). This process included cell lysis, mRNA capture, labeling of cells (barcoding) and mRNA (UMI), reverse transcription of mRNA into cDNA and amplification, and finally fragmentation of cDNA. Each library was diluted to 4 nM and mixed for sequencing. The mixed sample was sequenced on an Illumina HiSeq X with 150 bp paired-end reads. The raw reads were processed by fastQC and fastp to remove low-quality reads. The Poly-A tails and adapter sequences were removed by cutadapt. After quality control, the reads were further processed. The data was mapped to the reference genome GRCh38 (Ensembl release 92 annotation) using the STAR software. Gene counts and unique molecular identifier (UMI) counts were obtained by the featureCounts software. Based on these gene counts and UMI counts, an expression matrix file for subsequent analysis was generated.

[0034] The results of single-cell sequencing are as Figure 1 shown. The results showed a decreased expression of CRYL1 protein in the renal tubules and collecting duct cells of the renal papillary calcification tissue. Figure 1 A shows the appearance of endoscopic normal renal papillary tissue (NRP) and renal papillary calcification (RP), clearly showing the kidney stone adhering to RP (red arrow). Figure 1 B, Figure 1 C shows the UMAP dimensionality reduction clustering map obtained from RP tissue and NRP tissue from patients who underwent radical nephrectomy due to renal tumors after approval by the Ethics Committee of Xiangya Hospital, Central South University (approval number: 201705768) for single-cell sequencing. Figure 1 The sequencing results in D showed significantly lower expression of CRYL1 protein in proximal tubule epithelial cells, ascending thin limb cells, thick ascending limb cells, unassigned tubule cells, principal cells of the collecting duct, and intercalated cells of the collecting duct.

[0035] Von-Kossa staining Calcium salt staining was performed on renal papilla tissue, and the tissues were grouped into RP and NRP tissues. The specific implementation method was as follows: RP tissue and NRP tissue were obtained from patients who underwent total nephrectomy due to renal tumors. The collected tissues needed to be immediately fixed in a fixative containing 10% formalin. The fixation time was usually 24 to 48 hours to ensure good tissue morphology.

[0036] (1) Slide preparation: First, paraffin embedding was performed. The steps of paraffin embedding were as follows: Dehydration: The fixed tissue specimens were dehydrated in a gradient ethanol aqueous solution (the volume-mass of ethanol was 50%, 70%, 80%, 90%, and 100% respectively) to gradually remove the water in the tissues. Usually, each concentration of ethanol aqueous solution was soaked for 1 hour.

[0037] Clearing: The dehydrated tissues needed to be cleared with xylene or other clearing agents to remove alcohol.

[0038] Impregnation: The cleared tissues were immersed in molten paraffin. The temperature was usually set at 60°C, and the soaking time was about 1 hour to ensure complete penetration of paraffin.

[0039] Embedding: After the tissues were completely immersed in paraffin, they were taken out and placed in a mold, and cooled to room temperature to form paraffin blocks.

[0040] Then sectioning was performed. The steps of sectioning were as follows: Microtome setting: The paraffin block was fixed on the tray of the microtome, and the paraffin-embedded tissues were cut into thin slices using a microtome. Usually, the section thickness was 4 - 5 microns.

[0041] Sectioning process: Adjust the microtome to make the tissue surface flat. The cut sections should be carefully collected in water to prevent the sections from wrinkling or being damaged.

[0042] Steps for attaching sections to slides: The sections were taken out of the water and placed on a glass slide with adhesiveness. Then the sections were slowly heated to make them adhere to the slide.

[0043] Then, the paraffin sections were successively dewaxed and hydrated and then stained with Von-Kossa stain. The sections were placed on a baking oven at 65°C for baking for 2 hours to fully melt the paraffin and avoid subsequent decolorization during staining. The steps of dewaxing were as follows: After baking, the slides were immediately placed in xylene for dewaxing for 15 minutes while still hot, and this was repeated 3 times after changing xylene. The steps of hydration were as follows: The dewaxed slides were placed in ethanol aqueous solutions with gradually decreasing concentration (at room temperature) for hydration. The volume concentrations of the ethanol aqueous solutions were successively: absolute ethanol (extracting xylene) - absolute ethanol (changing cylinders) - 95% ethanol - 85% ethanol - 75% ethanol, each time for 5 minutes.

[0044] (2) Staining: Rinse the slide with hydrated section tissue three times for 3 minutes each with double-distilled water. After drying the distilled water around the specimen with filter paper, add Von-Kossa silver solution dropwise with a pipette and irradiate it under an ultraviolet lamp for 30 minutes. Then discard the staining solution, rinse it twice for 1 minute each with double-distilled water, and perform HE staining after adding hypo solution.

[0045] Perform immunohistochemical staining on the CRYL1 protein. The specific implementation method is as follows: (1) Slide preparation: Perform dewaxing - hydration treatment on paraffin sections in sequence and then perform tissue immunofluorescence staining. Place the slide on a 65°C baking machine for baking for 2 hours to fully melt the paraffin and avoid subsequent staining detachment. Dewaxing: Immediately after the baking is completed, place the slide in xylene while it is still hot for dewaxing for 15 minutes, and repeat 3 times after changing the xylene. Hydration: Place the dewaxed slide in ethanol aqueous solutions with gradually decreasing concentration (at room temperature) for hydration. The volume concentrations of the ethanol aqueous solutions are as follows: absolute ethanol (extracting xylene) - absolute ethanol (changing the cylinder) - 95% ethanol - 85% ethanol - 75% ethanol, each time for 5 minutes.

[0046] (2) Antigen retrieval: Take an appropriate amount of citrate antigen retrieval solution (PH = 6.0) and place it in a beaker. Heat it to boiling with a microwave oven, then put the hydrated slide into it, and continue heating for 10 - 15 minutes and then cool it naturally. Rinse it twice for 3 minutes each with double-distilled water, and then immerse it in PBS for washing three times for 3 minutes each.

[0047] (3) Blocking: After sucking away the PBS with filter paper, add 10% goat serum (animal serum of the same species origin as the secondary antibody) dropwise and block it at room temperature for 1 hour.

[0048] (4) Incubate with primary antibody: After sucking away the blocking solution, directly add an appropriate amount of diluted (1:150 to 1:300) primary antibody incubation solution (Abcam, ab231257) and incubate it overnight at 4°C.

[0049] (4) Incubate with primary antibody: After sucking away the blocking solution, directly add an appropriate amount of primary antibody (1:200 to 1:400) incubation solution and incubate it overnight at 4°C.

[0050] (5) Incubate with secondary antibody: After sucking away the primary antibody incubation solution, add pre-cooled PBS and rinse it three times for 3 minutes each, then add a horseradish peroxidase-conjugated secondary antibody dilution (1:300, Abcam) and incubate it at room temperature for 1 hour.

[0051] (6) DAB color development: After sucking away the secondary antibody incubation solution, rinse it three times for 3 minutes each with pre-cooled PBS. After removing the PBS, add DAB color development solution and observe the color development situation dynamically under the microscope. After the color development is completed, rinse it with tap water for 10 minutes to terminate the color development.

[0052] (7)Hematoxylin counterstaining: Counterstain with hematoxylin solution for 1 - 2 min, differentiate with 0.1% hydrochloric acid for 3 - 5 s, and then rinse with tap water for 10 min to terminate the staining.

[0053] (8)Mounting and observation: After dehydration - clearing - mounting, observe under a light microscope and take pictures.

[0054] The results are as Figure 2 shown in Figure A. Calcium salt staining (Von - Kossa) showed calcium salt deposition (brown - black) in the renal papillary calcification (RP) tissue, and immunohistochemical staining showed a significant decrease in CRYL1 protein in the RP tissue, indicating that CRYL1 protein was significantly decreased in both the renal papillary calcification tissue and urine of patients with calcium oxalate (CaOx) kidney stones.

[0055] Semi - quantitative analysis was performed on the immunohistochemical staining of RP (n = 12) and normal renal papillary tissue (NRP; n = 12). The results are as Figure 2 shown in Figure B, and the results showed that the staining depth was negatively correlated with the gray value.

[0056] Urine was collected from healthy physical examination subjects and patients with CaOx kidney stones complicated with RP plaques. The process of urine collection was as follows: After the patient emptied the bladder, timing started and this urine was not collected. All urine within the next 24 hours needed to be collected into a container. After 24 hours, the patient emptied the bladder again and this urine needed to be collected. The time and urine volume of each urination needed to be recorded during this period. If any urine was missed, it needed to be noted on the record sheet. The collected urine was stored in a container, sealed and refrigerated (2 - 8°C). After 24 hours, the urine was sent to the laboratory as soon as possible and then relevant urine analyses were performed.

[0057] The concentration of CRYL1 protein in urine was detected by ELISA, and the ratio of CRYL1 protein / urinary creatinine (Urinary CRYL1 protein / creatinine) was calculated for calibration.

[0058] The ELISA kit system is as follows: Table 1 ELISA kit system

[0059] In the table, unless otherwise specified, the percentage is generally the mass percentage.

[0060] The process of ELISA detection is as follows: Collect urine: Start timing after the patient empties the bladder. Do not collect this urine. All urine within the next 24 hours should be collected into a container. After 24 hours, the patient empties the bladder again, and this urine needs to be collected. The collected urine is stored in a container, which should be sealed and refrigerated (2 - 8°C). As soon as possible after the 24-hour period ends, send the urine to the laboratory and centrifuge the sample at 1000×g for 20 minutes at 2 - 8°C. Collect the supernatant for measurement.

[0061] Note: Collect samples in pyrogen- / endotoxin-free tubes.

[0062] Samples should be measured within 7 days when stored at 2 - 8°C. Otherwise, they must be aliquoted and stored at -20°C (≤1 month) or -80°C (≤3 months). Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely and mix well (do not vortex) before analysis.

[0063] If there are a large number of particulates in the sample, centrifuge or filter the sample before analysis.

[0064] Prepare samples: The sample concentration should be within the standard curve range. Since conditions may vary, the optimal dilution for each application should be determined.

[0065] All prepared samples should be used within 2 hours after dilution. Experiments are not recommended after 2 hours.

[0066] Prepare 1X wash buffer: 1. Dilute 30 mL of wash buffer concentrate (25X) with 720 mL of deionized water or distilled water. Label it as 1X wash buffer.

[0067] 2. Store the concentrate and 1X wash buffer at 2 - 8°C. Use the diluted buffer within 3 months.

[0068] Note: If crystals form in the concentrate, heat it in a 40°C water bath and mix gently until the crystals completely dissolve and the performance is not affected.

[0069] Prepare 1X biotinylated detection antibody solution: Note: The working solution should be prepared before use.

[0070] 1. Mix NHS-biotin and antibody at a ratio of 1:10 to prepare biotin-conjugated CRYL1 antibody (100X).

[0071] 2. Calculate the amount of biotinylated CRYL1 antibody required (100 μL / well) before the experiment. When preparing, prepare a slightly larger amount of solution than the calculated amount.

[0072] 3. Centrifuge the concentrated biotinylated detection antibody at 800×g for 1 minute at 2 - 8°C.

[0073] 4. Dilute the concentrated biotinylated detection antibody (100X) to 1X working solution with biotinylated detection antibody diluent.

[0074] Prepare 1X HRP conjugate solution: Note: Working solutions should be prepared prior to use.

[0075] 1. Calculate the required volume (100 μL / well) before the experiment. When preparing, a slightly larger volume of solution than the calculated amount should be prepared.

[0076] 2. Centrifuge the concentrated HRP conjugate at 800 × g for 1 minute at 2 - 8°C.

[0077] 3. Dilute the concentrated HRP conjugate (100X) to 1X working solution with HRP conjugate diluent.

[0078] Prepare diluted standards: Note: Use glass or plastic tubes to dilute the standards.

[0079] 1. Centrifuge the standards at 10,000 × g for 1 minute at 2 - 8°C to ensure the contents are at the bottom of the vial.

[0080] 2. Add 10 mL of sample diluent, let stand for 10 minutes and invert gently several times. After complete dissolution, mix thoroughly with a pipette. This resuspension produces a 0.1 mg / mL stock solution. Add 0.4 μL of the stock solution to 1000 μL of diluent to obtain a 400 pg / ml working solution.

[0081] 3. Take 7 tubes and add 500 μL of standards and sample diluent to each tube. Pipette 500 μL of the 400 pg / mL working solution into the first tube and mix to produce a 200 pg / mL working solution. Follow this step by pipetting the solution from the previous tube into the next tube. The last tube is considered blank and do not pipette the solution from the previous tube into it. The recommended dilution series is as follows: 400, 200, 100, 50, 25, 12.5, 6.25, 0 pg / mL.

[0082] Allow all components to reach room temperature before use. Mix all liquid reagents before use.

[0083] Determine the number of 8 - well strips required for the assay. Insert the strips into the frame for use. Repackage any unused strips and frames and store dry at - 20°C for future use. The silica gel packet in the bag keeps the plate dry.

[0084] After preparing the reagents, perform the assay. The assay procedure is as follows: 1. Bind antigen: Note: The solution should be added to the bottom of the ELISA plate wells, avoiding contact with the inner walls as much as possible and generating foam.

[0085] a. For the standard curve, add 100 µL of the standard to the appropriate wells. For the samples, add 100 µL of the pretreated sample to the wells.

[0086] b. Cover the plate with a plate seal and incubate at 37 °C for 90 minutes.

[0087] c. Aspirate the solution completely, do not wash.

[0088] 2. Add the biotinylated detection antibody: a. Add 100 µL of the biotinylated detection antibody working solution to each well.

[0089] b. Cover the plate with a plate seal and incubate at 37 °C for 60 minutes.

[0090] c. Aspirate the solution completely and wash the wells 3 times with 350 µL of 1X wash buffer. Pour out the solution from each well, add 350 µL of wash buffer to each well. Incubate for 1 minute and aspirate or pour out the solution from each well, and pat dry on a clean absorbent paper. Proceed to the next step immediately, ensuring that the wells do not dry out.

[0091] 3. Add the HRP conjugate: a. Add 100 µL of the HRP conjugate working solution to each well.

[0092] b. Cover the plate with a plate seal and incubate at 37 °C for 30 minutes.

[0093] c. Aspirate the solution completely and repeat the washing process 5 times as described in step 2.

[0094] 4. Add the substrate: a. Add 90 µL of the substrate reagent to each well.

[0095] b. Cover the plate with a plate seal and incubate at 37 °C for approximately 15 minutes. Protect the plate from light.

[0096] Note: The reaction time can be shortened or extended according to the actual color change, but not exceeding 30 minutes.

[0097] 5. Add the stop solution: a. Add 50 µL of the stop solution to each well. This step should be carried out in the same order as the substrate solution. Tap the sides of the plate gently to mix.

[0098] b. The solution in the wells will change from blue to yellow.

[0099] 6. Read the plate and generate the standard curve: 1. Preheat the microplate reader for about 15 minutes before OD measurement.

[0100] 2. Read the absorbance at 450 nm. Read the plate within 10 minutes after adding the stop solution.

[0101] 3. Use curve fitting software to generate a standard curve. The four-parameter algorithm provides the best standard curve fit. In the best case, the background absorbance can be subtracted from all data points (including standards, unknown samples, and controls) before plotting.

[0102] 4. Read the concentrations of the unknown samples and controls from the standard curve. Multiply the value obtained for the sample by the appropriate factor to correct for sample dilution.

[0103] Note: If the OD of the sample exceeds the upper limit of the standard curve, it should be retested using an appropriate dilution.

[0104] The data of the obtained standard curve are shown in the following table.

[0105] Table 2 Data of the standard curve

[0106] The standard curves fitted from well plates 1 - 3 are respectively as Figures 3 - 5 shown.

[0107] As can be seen from Table 2, as the concentration of CRYL1 protein decreases, the OD value also gradually decreases. The OD value of the standard curve decreases from about 2.4 at high concentration to 0.07 at low concentration. According to these data, there is a negative correlation between the CRYL1 protein concentration and the OD value, that is, the higher the CRYL1 protein concentration, the greater the optical density (OD value). This is in line with the standard results of ELISA testing, indicating that this experimental method has a good response at different concentrations. The OD values of the three well plates at each concentration are relatively consistent, indicating that this experiment has good repeatability.

[0108] ELISA was used to detect the CRYL1 protein concentration in urine and calculate the CRYL1 protein / urine creatinine ratio, and the results are as Figure 2 shown in C. Statistical analysis showed that the CRYL1 protein / urine creatinine ratio of CaOx stone formers with renal papillary stones (n = 25) was significantly higher than that of the healthy control group (n = 25), and this difference was statistically significant (* p <0.001).

[0109] To evaluate the precision between different batch tests, 10 repeated tests were performed on three human urine samples with low, medium, and high levels of CRYL1 protein.

[0110] Table 3 Repeated Tests of Human Urine Samples with Low, Medium, and High Levels of CRYL1 Protein

[0111] To evaluate the precision between intra-batch tests, repeated tests were performed on three human urine samples with low, medium, and high levels of CRYL1 protein, 20 times for each repetition, to determine the accuracy of the measurement.

[0112] Table 4 Twenty Repeated Tests of Human Urine Samples with Low, Medium, and High Levels of CRYL1 Protein

[0113] To evaluate the stability of the test, three urine samples with high concentrations of CRYL1 protein were selected, diluted (1:2; 1:4; 1:8), and then tested.

[0114] Table 5 Results of the Dilution Test

[0115] As can be seen from Table 5, at different dilution multiples, the floating range and average value indicate that the experiment for measuring CRYL1 protein has good stability and consistency. The results at different dilution multiples of 1:2, 1:4, and 1:8 all show that this test method has good adaptability to CRYL1 protein samples within different concentration ranges.

[0116] Three hundred and sixteen patients with calcium oxalate (CaOx) kidney stones combined with renal papillary calcification were tested. The inclusion criteria for patient sampling were: (1) 18 years old ≤ age ≤ 75 years old; (2) mild hydronephrosis or no hydronephrosis; (3) no chronic kidney disease complicated with proteinuria; (4) the renal papilla at the sampling site was more than 3 cm away from the tumor margin and the renal papilla was confirmed by HE staining not to be invaded by the tumor. Renal papilla tissue samples were obtained from the excised specimens of radical nephrectomy in patients with renal cancer or ureteral cancer. The data obtained from the test are shown in Table 5.

[0117] Table 6 Baseline Characteristics of a Cohort of 316 Patients with Calcium Oxalate (CaOx) Kidney Stones Combined with Renal Papillary Calcification

[0118] Recurrence definition: For patients with CaOx kidney stones without residue after lithotripsy and stone extraction, recurrence of kidney stones was detected by X-ray, B-ultrasound or CT during the 5-year follow-up after the operation. The samples for detection were from the urine samples collected before the operation (frozen). Chi-square test (https: / / libguides.library.kent.edu / spss / chisquare) was used for between-group analysis of binary data, and t-test (https: / / libguides.library.kent.edu / SPSS / IndependentTTest) was used for between-group analysis of continuous data.

[0119] By analyzing a cohort of 316 patients with CaOx kidney stones complicated with renal papillary calcification, it was found that there was no significant difference in traditional urinary calcium, urinary oxalate, and urinary citrate between the recurrence group and the non-recurrence group, while the ratio of urinary CRYL1 protein / urinary creatinine in the recurrence group was significantly decreased (Table 6).

[0120] And the process of fitting curve analysis for the data is as follows: (1) Data preparation: Ensure that the data has been preprocessed (such as removing outliers, standardization, etc.). Determine the independent variable (X) and the dependent variable (Y).

[0121] (2) Select a model: Select a suitable mathematical model according to the characteristics of the data, assuming that the data conforms to a linear relationship.

[0122] (3) Fit the model: Use the least squares method to fit the model parameters a and b.

[0123] (4) Evaluate the model: Evaluate the fitting effect of the model through statistical indicators (such as R², RMSE, etc.). R² (coefficient of determination): Measure the proportion of the model explaining the variation of the data. RMSE (root mean square error): Measure the deviation between the predicted value and the actual value.

[0124] (5) Prediction and visualization: Use the fitted model for prediction and draw the fitting curve. The results after fitting are as Figure 6 shown. The results show that the concentration of urinary CRYL1 protein in patients is negatively correlated with the recurrence rate of patients 5 years after the operation, and urinary CRYL1 protein is a predictor of postoperative recurrence in patients with CaOx kidney stones complicated with renal papillary calcification.

[0125] The concentration of urinary CRYL1 protein was detected in 316 patients with recurrent calcium oxalate (CaOx) kidney stones complicated with renal papillary calcification. The recurrence of patients was predicted by the concentration of urinary CRYL1 protein, and the ROC curve was made with sensitivity and specificity data. The process is as follows: (1) Train the model: Train a binary classification model (logistic regression) using the training data.

[0126] (2) Obtain the predicted probability: Use the model to predict the test set to obtain the probability that each sample belongs to the positive class.

[0127] (3) Set the threshold: Select multiple thresholds from 0 to 1 and calculate the TPR and FPR at each threshold.

[0128] (4) Plot the ROC curve: Use the FPR as the horizontal axis and the TPR as the vertical axis to plot the curve.

[0129] The results obtained are as Figure 7 shown. The ROC curve shows that the area under the curve for predicting the 5-year recurrence rate of patients using the urine CRYL1 protein concentration is 0.87.

[0130] Randomly select 200 patients with recurrent calcium oxalate (CaOx) kidney stones combined with renal papillary calcification for urine CRYL1 protein concentration detection. Take patients with urine CRYL1 protein content less than 86.7 pg / mg as recurrent patients. Compare the predicted results with the actual results to obtain data on sensitivity, specificity, and accuracy. Use the sensitivity and specificity data to plot the ROC curve. The process is as follows: (1) Train the model: Use the training data to train a binary classification model (logistic regression).

[0131] (2) Obtain the predicted probability: Use the model to predict the test set to obtain the probability that each sample belongs to the positive class.

[0132] (3) Set the threshold: Select multiple thresholds from 0 to 1 and calculate the TPR and FPR at each threshold.

[0133] (4) Plot the ROC curve: Use the FPR as the horizontal axis and the TPR as the vertical axis to plot the curve.

[0134] The results obtained are as Figure 8 shown. The ROC curve shows that the area under the curve for predicting the 5-year recurrence rate of patients using the urine CRYL1 protein concentration is 0.85, and the accuracy is 85.4%.

[0135] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Use of CRYL1 protein in the preparation of a product for evaluating, diagnosing or assisting in the diagnosis of the risk of postoperative recurrence of calcium oxalate kidney stones combined with renal papillary calcium plaques, the sequence of the CRYL1 protein being shown in SEQ ID NO.

1.

2. Use of a substance for detecting CRYL1 protein in the preparation of a product for evaluating, diagnosing or assisting in the diagnosis of the risk of postoperative recurrence of calcium oxalate kidney stones combined with renal papillary calcification, the sequence of the CRYL1 protein being shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The substance for detecting CRYL1 protein includes a substance for detecting the content of CRYL1 protein by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunocoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance, immuno-PCR or biotin-avidin technology.

4. The use according to claim 2, characterized in that: The substance for detecting CRYL1 protein is a substance for binding to CRYL1 protein.

5. The use according to claim 4, characterized in that: The substance binding to CRYL1 protein is an antibody, a polypeptide, a protein or a nucleic acid molecule.

6. The use according to claim 5, characterized in that: The antibody is a CRYL1 antibody.

7. An application of a kit, characterized in that: The kit includes a substance for detecting CRYL1 protein, and the kit has at least one of the following applications: 1) Used to detect the recurrence of calcium oxalate kidney stones combined with renal papillary calcium plaques after surgery; 2) Diagnosis or auxiliary diagnosis of calcium oxalate kidney stones combined with postoperative renal papillary calcium plaque recurrence; 3) To evaluate the risk of postoperative recurrence of calcium oxalate kidney stones combined with renal papillary calcium plaques.

8. The use according to claim 7, characterized in that: The substance for detecting CRYL1 protein is CRYL1 antibody.

9. The use according to claim 7, characterized in that: The substance for detecting CRYL1 protein is CRYL1 rabbit antibody.

10. The use according to claim 7, characterized in that: The test sample of the kit is urine; the kit also includes CRYL1 antibody, CRYL1 recombinant protein, HRP conjugate, biotinylated detection antibody diluent, HRP conjugate diluent, washing buffer, substrate and stop solution.

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