Application of urine TRPV5 as biomarker in chronic kidney disease diagnosis and risk assessment

Abstract

According to the application of the urine TRPV5 as the biomarker in chronic kidney disease diagnosis and risk assessment, it is found for the first time that the TRPV5 and the detectable fragment thereof exist in the urine, the content of the TRPV5 and the detectable fragment thereof in the urine is in positive correlation with the chronic kidney disease CKD, the CKD can be diagnosed by detecting the content of the TRPV5 in the urine, and the application of the TRPV5 and the detectable fragment thereof in the diagnosis and risk assessment of the chronic kidney disease in the diagnosis and risk assessment of the chronic kidney disease is achieved. The invention also provides a specific in-vitro diagnosis method and a kit. According to the in-vitro diagnosis method, multiple defects of the existing CKD diagnosis technology are overcome, a brand-new diagnosis tool which is noninvasive, sensitive and specific and can be used for staging and prognosis evaluation is provided, and the in-vitro diagnosis method has remarkable scientific and technological advancement and wide clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of clinical diagnostic technology, and in particular to the application of urinary TRPV5 as a biomarker in the diagnosis and risk assessment of chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is a global public health problem, characterized by its high incidence, low awareness, and high risk of progression to end-stage renal disease. Early diagnosis is crucial for slowing disease progression and improving patient outcomes.

[0003] Currently, the clinical diagnosis and staging of CKD mainly rely on serum creatinine (SCr), estimated glomerular filtration rate (eGFR), urine albumin / creatinine ratio (ACR), and renal biopsy. However, serum creatinine levels are affected by various non-renal factors such as age, sex, race, and muscle mass, resulting in insufficient sensitivity. eGFR, estimated by formula, is not sensitive enough in the early stages of mild renal function decline (CKD stages 1-2), often only showing significant changes when renal function loss exceeds 50%, thus failing to provide early warning.

[0004] The urine albumin / creatinine ratio (ACR) is a key indicator for diagnosing glomerular damage (such as diabetic nephropathy). However, it mainly reflects damage to the glomerular filtration barrier, and its sensitivity is low for CKD types that present with tubulointerstitial lesions as the initial or main manifestation.

[0005] Kidney biopsy is the gold standard for pathological diagnosis, clearly identifying the type and extent of damage. However, it is an invasive procedure with risks such as bleeding and infection, and cannot be used for routine screening, dynamic monitoring, or population surveys.

[0006] In summary, existing technologies have limitations such as insensitivity to early renal tubular specific damage, invasiveness, or susceptibility to interference. There is an urgent clinical need for non-invasive biomarkers that can specifically and sensitively reflect the health status of renal tubules.

[0007] Transient receptor potential vanilloid 5 (TRPV5) is a highly calcium-selective epithelial cell calcium channel, mainly distributed in the distal convoluted tubule and apical membrane of the connecting tubule in the kidney. It is responsible for calcium reabsorption and acts as a "gatekeeper protein" for renal calcium metabolism. TRPV5 plays a central role in maintaining calcium homeostasis in the body.

[0008] Currently, some literature has revealed the association between TRPV5 and kidney disease at the tissue level. For example, in the academic journal *Journal of Clinical Investigation* (2003, 112(12), 1906–1914), Joost G J Hoenderop et al. published a paper entitled "Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5," which pointed out that TRPV5 knockout mice exhibited significant renal calcium loss and decreased bone mineral density. Another article published in *Journal of the American Society of Nephrology* (2004; 15(3): 549-57.), entitled "Downregulation of Ca(2+) and Mg(2+) transport proteins in the kidney explains tacrolimus (FK506)-induced hypercalciuria and hypomagnesemia," found that the immunosuppressant tacrolimus downregulates TRPV5 expression in the renal tubules and induces hypercalciuria.

[0009] Mechanism of Action and Gaps in Existing Technologies: These studies consistently demonstrate that TRPV5 protein expression changes in kidney tissue under disease conditions. However, all these studies are limited to the analysis of kidney tissue itself, and their techniques (such as gene knockout, tissue Western blotting, and immunohistochemistry) are invasive and cannot be translated into clinical diagnostic tools. To date, no existing technology has been disclosed or suggested. TRPV5 protein or its hydrolyzed fragments are present in the supernatant of human urine.

[0010] The concentration of TRPV5 protein in urine is correlated with the diagnosis of CKD and the severity of proteinuria.

[0011] These proteins in urine can be detected noninvasively using commercial immunological assays (such as ELISA) and developed into a novel clinical diagnostic biomarker. Summary of the Invention

[0012] To address the shortcomings of the existing technologies, this invention provides an application of urinary TRPV5 as a biomarker in the diagnosis and risk assessment of chronic kidney disease (CKD), thereby overcoming the lack of in vitro diagnostic methods for early-stage CKD in the existing technologies and providing a novel, non-invasive solution for in vitro diagnosis and risk assessment of CKD.

[0013] In a first aspect, the present invention provides the use of TRPV5 protein or characteristic fragments thereof in the preparation of in vitro diagnostic or screening products for chronic kidney disease.

[0014] In particular, reagents for detecting TRPV5 protein or its characteristic fragments are used in the preparation of products for the in vitro auxiliary diagnosis of chronic kidney disease in subjects.

[0015] In existing technologies, research on TRPV5 focuses on its molecular physiological mechanisms in cellular calcium ion transport. Its known uses are limited to basic scientific research tools. For example, commercially available TRPV5 antibodies and ELISA kits (e.g., the TRPV5 ELISA kit from Kanglang Pharmaceutical Co., Ltd., catalog number KL131079Hu) explicitly state in their instructions that they are "For Research Use Only," not for in vitro diagnostics. Their original purpose was to detect TRPV5 protein expression levels in cell lysates and tissue homogenates to study its expression regulation mechanisms in various physiological and pathological models (such as osteoporosis, hypertension, and kidney calcium stones).

[0016] This invention, by integrating animal models and human clinical studies, reveals and validates for the first time an innovative finding: in CKD, especially in the early stages (CKD stages 1-2), the concentration of TRPV5 protein in urine shows a specific and significant upregulation. This phenomenon is highly consistent with the upregulation of transcription and protein expression in kidney tissue, indicating that it reflects an active pathophysiological response of the kidney to chronic injury.

[0017] This invention, based on large-scale clinical data (12,588 CKD patients), found that reduced 24-hour urinary calcium excretion occurs early in CKD, suggesting impaired renal tubular calcium reabsorption in the early stages of CKD. Through bioinformatics analysis, the key calcium reabsorption channel protein TRPV5 was identified as being upregulated in the kidneys of CKD patients. Furthermore, the protein was extracted from human morning urine, and Western blotting confirmed that its content in the urine of CKD patients was significantly higher than that in healthy controls, suggesting its great potential as a urinary biomarker.

[0018] In a CKD animal model, this invention demonstrated a significant upregulation of TRPV5 in renal tissue across three dimensions: transcription, protein translation, and tissue distribution. This molecular event directly led to changes in functional output: decreased urinary calcium excretion occurred simultaneously with a specific increase in urinary TRPV5 protein levels. Crucially, using a mouse model with conditional knockout of the Trpv5 gene in renal tubular epithelial cells, this invention proved that urinary TRPV5 protein originates from renal expression, confirming its biological basis as a biomarker of kidney-specific damage.

[0019] This invention also provides the application of TRPV5 protein or a characteristic fragment thereof in the preparation of products for predicting the risk of chronic kidney disease.

[0020] This invention also provides the application of TRPV5 protein or a characteristic fragment thereof in the preparation of prognostic monitoring products for chronic kidney disease.

[0021] Preferably, the application is characterized by a significant increase in TRPV5 protein expression, or a significant increase in the content of its characteristic fragments or hydrolyzed fragments, which indicates that the patient under test has chronic kidney disease or is at high risk of developing chronic kidney disease or has a poor prognosis.

[0022] Preferably, the product is a detection reagent or detection kit; the reagent is used to detect the expression level of TRPV5 protein or the content of its characteristic fragments; The reagent is used for ELISA, protein / peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay; preferably, the reagent is used to detect the content of the TRPV5 protein or its characteristic fragments or hydrolyzed fragments by antigen-antibody reaction.

[0023] Preferably, the test reagent or test kit is used to detect urine, and more preferably urine supernatant.

[0024] Preferably, the judgment principle is to determine a preset threshold based on healthy people. When the content of TRPV5 protein or its characteristic fragment or its hydrolyzed fragment in the sample to be tested is significantly higher than the preset threshold, the patient to be tested is in chronic kidney disease or at high risk of developing chronic kidney disease.

[0025] Secondly, the present invention provides a kit for in vitro diagnosis or screening of chronic kidney disease, containing a detection reagent for the content of TRPV5 protein characteristic fragments, wherein the kit is used to detect urine supernatant.

[0026] Preferably, the detection threshold of the kit is 297.6 pg / mL. A value higher than this threshold indicates that the patient whose urine sample is being tested has chronic kidney disease or is at risk of developing chronic kidney disease.

[0027] Preferably, when the kit detects CKD1 and / or CKD2, the detection threshold is 309.0 pg / mL. A value higher than this threshold indicates that the majority of the patient in the urine sample is in CKD1 and / or CKD2.

[0028] Existing research on TRPV5 is entirely confined to the scientific research field. Its detection methods (such as Western blotting and immunohistochemistry) rely on invasive kidney tissue biopsy, which has fatal drawbacks such as high operational risks, inability to repeat sampling, and poor patient compliance. Therefore, it is completely impossible to transform it into a large-scale, non-invasive clinical diagnostic application.

[0029] Furthermore, existing technologies have not revealed the presence of TRPV5 protein or its hydrolyzed fragments in urine and their diagnostic value. Although literature has demonstrated the correlation between changes in tissue TRPV5 and kidney disease, existing technologies have never disclosed or suggested the presence of TRPV5 protein or its hydrolyzed fragments in human urine supernatant, nor have any studies explored the association between its urine concentration and the clinical course of CKD. This makes it impossible for those skilled in the art to naturally conceive of developing it as a non-invasive urine biomarker. However, this invention discovers that TRPV5 is rapidly upregulated in the early stages of CKD, followed by a slight decrease, but its overall expression remains higher than in healthy individuals. Therefore, elevated expression in the kidneys and urine actually represents the occurrence and development of CKD.

[0030] The present invention has achieved at least the following beneficial effects: (1) This invention is the first to discover and confirm the presence of TRPV5 protein or its hydrolyzed fragments in urine and to associate it with the diagnosis of CKD. The prior art has never disclosed or suggested that TRPV5 protein can be detected in human urine supernatant, let alone established any connection between its concentration changes and the diagnosis and treatment of CKD.

[0031] (2) This invention reveals a novel pattern of "consistent upregulation" of urinary TRPV5 concentration in CKD, overturning the conventional understanding that "kidney damage leads to reduced protein loss." This invention, through animal models, confirms that this upregulation in urine is completely consistent with the upregulation of renal tissue at the levels of mRNA, protein, and spatial distribution, and is accompanied by functional changes in urinary calcium reabsorption. This elucidates a novel "active compensation / adaptive response" mechanism, rather than a simple leakage, providing a new perspective for understanding the pathophysiology of CKD.

[0032] (3) This invention breaks through the limitations of existing technologies that rely solely on invasive renal biopsy or serological indicators (such as eGFR) with insufficient sensitivity. It provides for the first time a method that can specifically reflect the health status of renal tubules by collecting urine non-invasively. In particular, it establishes a critical value system for urine TRPV5 concentration for clinical diagnosis. ROC analysis shows that the area under the curve (AUC) of urine TRPV5 in diagnosing CKD can reach 0.883 (95% CI: 0.830-0.936). At the optimal cut-off value, the diagnostic sensitivity is as high as 95%, the specificity is as high as 71%, and the accuracy is 78%, which fully demonstrates its excellent diagnostic ability. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a heatmap showing the correlation between urinary electrolyte excretion (urinary calcium) and renal function in Embodiment 1 of the present invention.

[0035] Figure 2 This is a volcano diagram of differentially expressed genes for ion transporters in the GSE66494 dataset of Embodiment 1 of the present invention.

[0036] Figure 3 This is a graph showing the results of urinary TRPV5 protein WB content in healthy individuals and CKD patients in Example 1 of this invention. Figure 3 A is the WB result image. Figure 3 B represents the statistical results.

[0037] Figure 4 This is a Western blot (WB) image showing the TRPV5 protein content in the kidneys of mice in the adenine-fed group and the control group, according to Example 2 of this invention. Figure 4 A is the WB result image. Figure 4 B represents the statistical results.

[0038] Figure 5 This is an immunofluorescence distribution map of TRPV5 protein in kidney slices of mice in the adenine-fed group and the control group in Example 2 of the present invention.

[0039] Figure 6 This is a graph showing the analysis results of urinary calcium / creatinine excretion and urinary TRPV5 content in CKD model group mice and Trpv5 conditional knockout group mice in Example 2 of this invention. Figure 6 Figure A shows the urinary calcium / creatinine excretion ratio in the CKD model group. Figure 6 Figure B shows the results of urinary TRPV5 content in the CKD model group. Figure 6 The graph with C represents the urinary calcium / creatinine excretion ratio in the Trpv5 conditional knockout group. Figure 6 The figure shows the results of urinary TRPV5 content in the Trpv5 conditional knockout group (D).

[0040] Figure 7 This is a graph showing the distribution of TRPV5 concentration in urine collected in Example 3 of the present invention.

[0041] Figure 8 This is a graph showing the distribution of TRPV5 concentration in urine from the verification collection of Example 3 of the present invention.

[0042] Figure 9 This is the ROC curve of urine TRPV5 for predicting CKD diagnosis in Example 3 of the present invention.

[0043] Figure 10 This is the ROC curve of urine TRPV5 / UCr for predicting CKD diagnosis in Example 3 of the present invention.

[0044] Figure 11 This is a comparison of the ROC curve of urine TRPV5 predicting CKD diagnosis and the ROC curve of urine TRPV5 / UCr predicting CKD diagnosis in Example 3 of the present invention.

[0045] Figure 12 This is a graph showing the correlation between urinary TRPV5 levels and eGFR, 24-hour urinary protein (Upro), and urinary calcium excretion in Example 3 of this invention. Figure 12 Figure A shows a schematic diagram illustrating the correlation between urinary TRPV5 levels and eGFR. Figure 12 Figure B shows the correlation between urinary TRPV5 levels and 24-hour urinary protein (Upro). Figure 12 C represents a schematic diagram illustrating the correlation between urinary TRPV5 levels and urinary calcium excretion.

[0046] Figure 13 This is a graph showing the results of urine TRPV5 content and the severity of urinary protein in Example 3 of the present invention. Figure 13 Figure A shows the box plot of the distribution of urinary TRPV5 content under different 24-hour urinary protein groups. Figure 13 Figure B shows the correlation curve results between 24-hour urinary protein levels and urinary TRPV5 content.

[0047] Figure 14 This is the ROC curve of urine TRPV5 content in Example 3 of the present invention for predicting the diagnosis of massive urinary protein (≥3.5g / d).

[0048] Figure 15 This is the ROC curve of urine TRPV5 content in Example 3 of the present invention for predicting the diagnosis of early CKD (stage 1-2). Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] Example 1: Discovery, Focusing and Preliminary Identification of Urinary TRPV5 Protein This embodiment provides a process for identifying the target molecule TRPV5 in human urine, based on clinical observations, through bioinformatics, and verifying its status as a potential biomarker.

[0051] 1. Clinical data review and analysis: A retrospective analysis was conducted on electronic medical record data from 12,588 inpatients and outpatients diagnosed with CKD at the First Medical Center of the General Hospital of the Chinese People's Liberation Army. Serum calcium, 24-hour urinary calcium levels, and renal function-related parameters (serum creatinine, blood urea nitrogen, eGFR, etc.) were analyzed. Patients were classified into CKD stages 1, 2, 3a, 3b, 4, and 5 based on their estimated glomerular filtration rate (eGFR) and the clinical guidelines' definition of CKD. The results are shown in Tables 1 and 2.

[0052] Table 1 Table 2 CKD: Chronic kidney disease; eGFR: estimated glomerular filtration rate; urinary CaV, urinary PV: represent the 24-hour urinary calcium and phosphorus excretion, respectively; urinary Ca / Cr, urinary P / Cr: represent the ratio of urinary calcium and urinary phosphorus to urinary creatinine, respectively; FECa, FEP: represent the fractional excretion of urinary calcium and urinary phosphorus, respectively.

[0053] As shown in Tables 1 and 2, the 24-hour urinary calcium excretion of patients with CKD stages 1-5 decreased with the increase of CKD stage. The urinary calcium excretion of CKD stages 1-5 were 2.70 (1.49, 4.41) mmol / 24h, 1.92 (0.99, 3.42) mmol / 24h, 1.36 (0.66, 2.59) mmol / 24h, 0.93 (0.51, 1.68) mmol / 24h, 0.80 (0.45, 1.41) mmol / 24h, and 0.70 (0.40, 1.20) mmol / 24h, respectively.

[0054] As can be seen, from CKD stage 1 to stage 5, the median urinary calcium excretion gradually decreased from approximately 2.70 mmol / 24h to approximately 0.70 mmol / 24h. Of particular note is that this significant decrease in excretion began as early as CKD stage 2 (a decrease of approximately 28.9% compared to stage 1), rather than the traditional view that it only occurs in end-stage renal disease.

[0055] 2. Bioinformatics analysis: The dataset GSE66494 was downloaded from the GEO database. Differential gene expression analysis was performed using the limma package in R. A significance threshold of log2(FC) > 1.0 and adj. P-value < 0.05 was used to screen for differentially expressed ion transporter genes in CKD kidney tissue. The results are as follows: Figure 1 and Figure 2 As shown.

[0056] Depend on Figure 1 As shown in Figure A, urinary calcium excretion is significantly positively correlated with eGFR, indicating that the better the renal function, the higher the level of urinary calcium excretion; conversely, decreased renal function leads to decreased urinary calcium excretion. At the same time, urinary calcium excretion is significantly negatively correlated with urinary protein, meaning that the higher the level of urinary protein, the lower the urinary calcium excretion. Figure 1 B showed a positive correlation between urinary calcium excretion and serum calcium levels, indicating that increased urinary calcium is accompanied by increased serum calcium. Furthermore, according to... Figure 2 Bioinformatics analysis showed that TRPV5 was significantly upregulated in the kidney tissue of CKD patients, with a fold change of 3.02 (log2FC = 1.591, corrected P = 0.0008).

[0057] 3. Preliminary verification of the efficacy of TRPV5 as a clinical biomarker for CKD. Three CKD patients and three healthy volunteers were selected, and midstream urine samples were collected upon waking in the morning. Within two hours of collection, urine samples were centrifuged at 4°C and 3000×g for 15 minutes. The supernatant was aliquoted and immediately stored at -80°C to avoid repeated freeze-thaw cycles.

[0058] Urine supernatant protein was extracted using a liquid protein extraction kit (Solepro; EX1150) and quantified using a BCA protein quantification kit. 20 μg of total protein was subjected to 10% SDS-PAGE gel electrophoresis. The primary antibody was rabbit anti-human TRPV5 polyclonal antibody (Abcam, ab137028), diluted 1:1000, and incubated overnight at 4°C. The secondary antibody was HRP-labeled goat anti-rabbit IgG (Beyotime, A0208), diluted 1:1000, and incubated at room temperature for 2 hours.

[0059] The results are as follows Figure 3 As shown.

[0060] Depend on Figure 3 As can be seen from the Western blot results, a clear TRPV5 protein band was visible in the 130-55 kDa region in CKD patients, and the signal intensity in the urine of CKD patients was on average 16.31 ± 7.34 times that of healthy controls.

[0061] Example 2: Animal model validation of the source and mechanism of urinary TRPV5 This embodiment verifies the causal relationship between renal TRPV5 expression and urinary TRPV5 levels in an animal model, clarifying its biological origin.

[0062] 1. Animal models: CKD model: Male C57BL / 6J mice aged 7-8 weeks were selected and fed adenine (0.25%) for 4 weeks to establish a CKD model. The control group was fed a control diet.

[0063] Gene knockout model: Cdh16cre+ / -&Trpv5fl / fl mice were constructed using the Cre-loxp system, and tubule-specific Trpv5 gene knockout was induced by injection of tamoxifen (120 mg / kg, once every other day for 3 consecutive times).

[0064] 2. Sample collection and testing: Urine collection: Urine was collected from mice in metabolic cages 4 weeks after adenine (0.25%) feeding and 2 weeks after tamoxifen knockout.

[0065] Kidney histology: Total RNA was extracted using the TRIzol method, reverse transcribed using the PrimeScript RT kit, and qPCR was performed using Gapdh as an internal control. The results were then followed by nucleic acid electrophoresis using the SYBR Green method. (The results are shown below.) Figure 4 As shown.

[0066] Immunofluorescence: After antigen retrieval, frozen sections were stained with primary antibody (Alomone; ACC-035), diluted 1:100, for LTL (proximal tubule marker) and DAPI staining. The results are as follows. Figure 5 As shown.

[0067] Other detection indicators and methods: Urinary calcium: Measured using an o-cresolphthalein complex ketone method calcium assay kit.

[0068] Urinary TRPV5: Measured using a mouse TRPV5 ELISA kit (Kanglang Biotechnology, KL-TRPV5-Mu).

[0069] The urinary calcium / creatinine excretion and TRPV5 content of mice in the CKD model group and the Trpv5 gene knockout group were statistically analyzed, and the results are as follows: Figure 6 As shown.

[0070] Depend on Figure 4 It was found that the expression level of TRPV5 mRNA in the kidneys of mice fed with adenine (0.25%) was 2.06 times that of the control group (P<0.001). Figure 5 The study showed significantly enhanced TRPV5 protein expression and distal-connecting tubule staining in the CKD model group, indicating that the renal tissue actively upregulated the transcription and synthesis of TRPV5 under CKD conditions.

[0071] Depend on Figure 6 It was found that the 24-hour urinary calcium / creatinine excretion of mice in the CKD model group (0.18 ± 0.01) was significantly lower than that in the control group (0.31 ± 0.07), while the urinary TRPV5 content (3831 ± 1681 pg / mL) was significantly higher than that in the control group (1324 ± 527 pg / mL). The urinary calcium / creatinine excretion of Trpv5 conditional knockout mice (0.34 ± 0.10) was significantly higher than that of the control group (2.08 ± 0.39), and their urinary TRPV5 content (1400 ± 460 ng / mL) was significantly lower than that of the control group (7750 ± 1880 ng / mL). This indicates that TRPV5 is a key molecule regulating urinary calcium excretion, and its loss of function leads to hypercalciuria. In the CKD model, the increase in urinary TRPV5 that accompanies the decrease in urinary calcium may reveal the compensatory upregulation of the renal tubular calcium reabsorption mechanism and / or leakage due to renal tubular cell damage in the disease state.

[0072] Example 3: Clinical efficacy verification of urinary TRPV5 in diagnosing CKD 1. Purpose In an independent human cross-sectional study, the diagnostic efficacy of urinary TRPV5 for CKD was finally confirmed using a standardized ELISA method.

[0073] 2. Materials and Methods 2.1 Research Subjects: Exploratory set: 44 urine samples (36 CKD patients and 8 healthy controls).

[0074] Validation set: 149 independent samples (105 CKD cases and 44 disease controls).

[0075] 2.2 Detection Method: ELISA Assay: 1. Sample Addition and Incubation: Add 100 μL of diluted urine supernatant sample and standard to a microplate coated with TRPV5 capture antibody. Reserve blank wells. 2. Incubation Conditions: Incubate at 37℃ for 90 minutes to allow for complete antigen-antibody binding. 3. Washing: Discard the liquid in the wells and wash thoroughly twice with PBS containing 0.05% Tween-20, soaking for 30 seconds each time, then pat dry. 4. Adding Enzyme-Label Secondary Antibody and Incubation: Add 100 μL of biotinylated human TRPV5 antibody and incubate at 37℃ in the dark for 60 minutes. 5. Washing: Repeat the above washing steps three times. 6. Enzyme Conjugation Working Solution: Add 100 μL of enzyme conjugation working solution to all wells except the blank wells, and develop color at 37℃ in the dark for 30 minutes. 7. Washing: Repeat the above washing steps five times. 8. Color Development and Termination: Add 100 μL of TMB substrate solution (including blank wells) and incubate at 37°C in the dark for 15-20 minutes. When a clear blue gradient appears in the high-concentration standard wells, add 100 μL of stop solution to terminate the reaction; the solution will immediately turn yellow. 9. Detection: Immediately measure the absorbance (OD value) of each well using a microplate reader at a dominant wavelength of 450 nm. 10. Concentration Calculation: Plot a standard curve based on the OD values ​​of a series of standard concentrations (0, 15.6, 31.25, 62.5, 125, 250, 500, 1000 pg / mL). Calculate the absolute concentration of TRPV5 (in pg / mL) in each sample automatically using the curve equation.

[0076] 2.3 Statistical Analysis: R (4.4.0) software was used. Intergroup comparisons were performed using parametric tests (T-tests) or nonparametric Mann-Whitney U tests. Diagnostic efficacy was assessed by plotting ROC curves. Correlation was analyzed using Spearman rank correlation analysis.

[0077] 3. Results Exploration set results as follows Figure 7 As shown, the urinary TRPV5 concentration in the CKD group was 221.9 pg / mL (IQR: 166.9-363.6), while that in the control group was 40.4 pg / mL (IQR: 34.9-43.0) (P<0.0001), indicating a significantly elevated level of TRPV5 in the urine of CKD patients, with a median concentration 5.5 times that of the healthy control group. This result further confirms the abnormal expression of TRPV5 in the pathological state of CKD from a clinical perspective, laying a solid foundation for its use as a novel biomarker.

[0078] Validation set: Concentration differences, such as Figure 8As shown: the urinary TRPV5 concentration in the CKD group was 382.1 (IQR: 279.1-529.4) pg / mL, while that in the control group was 162.4 (IQR: 88.6-246.6) pg / mL (P<0.0001), indicating...

[0079] Diagnostic efficacy such as Figure 9 , Figure 10 , Figure 11 As shown: by Figure 9 ROC curve analysis showed that urinary TRPV5 had an AUC of 0.883 (95% CI: 0.830-0.936) for predicting CKD, with an optimal cutoff of 297.6 pg / mL, exhibiting a sensitivity of 95% and a specificity of 71%. Figure 10 It can be seen that TRPV5 / UCr has an AUC of 0.838 (0.764-0.913) for predicting CKD, an optimal cutoff value of 40.307, a sensitivity of 63%, and a specificity of 90%. Figure 11 It can be seen that both urinary TRPV5 and its corrected ratio to creatinine (TRPV5 / UCr) demonstrate excellent efficacy in predicting CKD, with no statistically significant difference between the two. In summary, the raw urinary TRPV5 concentration (AUC = 0.883) is highly suitable as an early screening indicator for CKD due to its high sensitivity of up to 95%, minimizing missed diagnoses; while the creatinine-corrected TRPV5 / UCr ratio (AUC = 0.838), with its high specificity of 90%, is more valuable in the disease confirmation stage and can effectively reduce misdiagnosis.

[0080] The correlation analysis results between urinary TRPV5 levels and renal function and urinalysis indicators are as follows: Figure 12 As shown. Specifically, urinary TRPV5 levels were significantly negatively correlated with eGFR (r = -0.277, P<0.001), meaning that as renal function declined (eGFR decreased), urinary TRPV5 levels gradually increased. Figure 12 A). Furthermore, urinary TRPV5 levels were significantly positively correlated with 24-hour urinary protein quantification (Upro) (r = 0.690, P<0.001), suggesting that urinary TRPV5 levels increase with increasing proteinuria severity. Figure 12 B). Furthermore, a significant negative correlation was found between urinary TRPV5 levels and 24-hour urinary calcium excretion (UCa) (r = -0.424, P < 0.001), indicating that elevated urinary TRPV5 levels are accompanied by decreased urinary calcium excretion. Figure 12 C).

[0081] Depend on Figure 13It can be seen that urinary TRPV5 levels are clearly positively correlated with the severity of proteinuria. Specifically, in different grades of proteinuria ( Figure 13 A), there were significant differences in the distribution of TRPV5 (p<0.05), and it showed a monotonically increasing trend with the increase of urinary protein quantification. Figure 13 B). This indicates that urinary TRPV5 can serve as a quantitative characterization of the degree of kidney damage. Figure 14 It can be seen that the AUC value of urinary TRPV5 in predicting massive proteinuria (≥3.5g / d) is 0.863 (0.802-0.925).

[0082] Early CKD diagnostic efficacy results such as Figure 15 As shown: In early CKD (CKD stage 1-2, n=38), the urinary TRPV5 content is significantly increased. The AUC in predicting early CKD is 0.940 (0.874, 1.000). At this time, the optimal cutoff value based on the Youden index is 309.0 pg / mL, with a sensitivity of 95%, specificity of 80%, and accuracy of 88%.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of TRPV5 protein or its characteristic fragments in the preparation of in vitro diagnostic or screening products for chronic kidney disease.

2. Application of TRPV5 protein or its characteristic fragments in the preparation of products for predicting the risk of chronic kidney disease.

3. Application of TRPV5 protein or its characteristic fragments in the preparation of prognostic monitoring products for chronic kidney disease.

4. The application according to any one of claims 1-3, characterized in that, The application described is that a significant increase in TRPV5 protein expression, or a significant increase in the content of its characteristic fragments or hydrolyzed fragments, indicates that the patient under test has chronic kidney disease or is at high risk of developing chronic kidney disease or has a poor prognosis.

5. The application according to any one of claims 1-3, characterized in that, The products described are detection reagents and detection kits; the reagents are used to detect the expression level of TRPV5 protein or the content of its characteristic fragments. The reagent is used for ELISA, protein / peptide chip detection, immunoblotting, microbead immunoassay, or microfluidic immunoassay; preferably, the reagent is used to detect the content of the TRPV5 protein or its characteristic fragments or hydrolyzed fragments by antigen-antibody reaction.

6. The application according to claim 4, characterized in that, The test reagent or test kit is used to detect urine, preferably urine supernatant.

7. The application according to claim 5, characterized in that, The judgment principle is to determine a preset threshold based on healthy people. When the content of TRPV5 protein or its characteristic fragments or its hydrolyzed fragments in the sample to be tested is significantly higher than the preset threshold, the patient to be tested is in chronic kidney disease or at high risk of developing chronic kidney disease.

8. A kit for in vitro diagnosis or screening of chronic kidney disease, characterized in that, A reagent kit containing the content of TRPV5 protein characteristic fragments is used to detect urine supernatant.

9. The reagent kit according to claim 7, characterized in that, The detection threshold of the kit is 297.6 pg / mL. A value higher than this threshold indicates that the patient whose urine sample is being tested has chronic kidney disease or is at risk of developing chronic kidney disease.

10. The kit according to claim 7, characterized in that, When the kit detects CKD1 and / or CKD2, the detection threshold is 309.0 pg / mL. A value higher than this threshold indicates that the majority of the patient in the urine sample is in CKD1 and / or CKD2.

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