Application of urine exosome cathepsin D in diagnosis and treatment of type 2 diabetic nephropathy

Abstract

The invention relates to application of urine exosome cathepsin D (cathepsin D, CTSD) in preparation of reagents for diagnosis, differential diagnosis, disease monitoring, mechanism research and the like of type 2 diabetic nephropathy. At present, clinical diagnosis of diabetic nephropathy mainly depends on continuous proteinuria, estimation of reduction of glomerular filtration rate and elimination of kidney injury caused by other diseases. However, in clinical practice, part of patients show a transient proteinuria or eGFR decrease and the invasiveness of kidney needle biopsy, resulting in that such patients cannot be diagnosed in time. Urine is generated through kidney filtration and heavy absorption, is the second major common body fluid only second to blood in clinical diagnosis, and is also an important sample source for finding kidney injury and urinary system tumor markers. Through screening and verification, compared with a healthy control group and a type 2 diabetes kidney-injury-free group, CTSD expression in urine exosomes of patients with the type 2 diabetic nephropathy is increased, and the kit can be used for diagnosis, identification, monitoring and prognosis evaluation of the type 2 diabetic nephropathy.

Description

Technical Field

[0001] This invention relates to the application of cathepsin D (CTSD) in urinary exosomes in the preparation of reagents for the diagnosis, differential diagnosis, disease monitoring, and mechanism research of type 2 diabetic nephropathy. Background Technology

[0002] Diabetic kidney disease (DKD) is the most common microvascular complication in diabetic patients and a leading cause of chronic kidney disease and end-stage renal disease worldwide. The main pathological features of DKD are diffuse thickening of the glomerular basement membrane, mesangial expansion, and tuberous sclerosis. Currently, clinical diagnosis of DKD relies primarily on persistent proteinuria and an estimated decrease in glomerular filtration rate (eGFR), after ruling out kidney damage caused by other diseases. However, in clinical practice, some patients exhibit transient proteinuria or a decrease in eGFR, and the invasiveness of kidney biopsy can lead to delayed diagnosis in these cases. Over the past few decades, precise control of blood glucose, blood pressure, and blood lipids, the development and application of novel hypoglycemic drugs with nephroprotective effects, and vigorous promotion of healthy lifestyles have improved the prognosis of DKD patients and prolonged the progression to end-stage renal disease. However, the morbidity, mortality, and incidence of cardiovascular events in DKD patients continue to rise. Therefore, early and timely detection of kidney damage in diabetic patients and the implementation of effective clinical interventions are crucial for improving patient prognosis and quality of life.

[0003] Urine, produced by reabsorption through kidney filtration, is the second most commonly used body fluid in clinical diagnosis after blood, and is an important sample source for identifying biomarkers of kidney damage and urinary system tumors. Compared to blood samples, urine has advantages such as being non-invasive, repeatable, easy to collect and store, and having high patient compliance. Furthermore, the proteins and nucleic acids it contains are relatively stable and not easily degraded. Urine typically contains epithelial cells, blood cells, bacteria, viruses, and extracellular vesicles. Urinary exosomes are lipid bilayered, elliptical membrane-like vesicles with a diameter between 30 and 140 nm, secreted by the epithelial cells of the kidneys and urinary system. They contain nucleic acids, proteins, and lipids and can reflect the state of the secretory cells and the physiological and pathological state of the body. Since the discovery of urinary exosomes, numerous studies have shown that they can serve as a source of biomarkers for kidney disease, cancer, metabolic diseases, autoimmune diseases, urinary tract infections, and related human genetic diseases.

[0004] This invention investigated the changes in CTSD protein in urinary exosomes of patients with diabetic nephropathy. Validation showed that CTSD expression was significantly elevated in urinary exosomes of patients with type 2 diabetic nephropathy compared to healthy controls and those without nephropathy. In the screening and validation cohorts, correlation analysis showed that CTSD was associated with serum creatinine and eGFR levels. Receiver operating characteristic (ROC) curve analysis showed that it had good ability to distinguish between diabetic kidney disease (DKD) and could be used for the diagnosis, differentiation, monitoring, and prognostic assessment of type 2 diabetic nephropathy. Summary of the Invention

[0005] The purpose of this invention is to provide an application of urinary exosome protein CTSD in the preparation of formulations for the diagnosis, differential diagnosis, disease monitoring and mechanism research of type 2 diabetic nephropathy.

[0006] Preferably, the amino acid sequences of CTSD in the urinary exosomes are as shown in SEQ ID NO.1. (SEQ ID NO.1: 1 mqpssllpla lcllaapasa lvriplhkft sirrtmsevg gsvedliakg pvskysqavp) 61 avtegpipev lknymdaqyy geigigtppq cftvvfdtgs snlwvpsihc klldiacwih 121 hkynsdksst yvkngtsfdi hygsgslsgy lsqdtvsvpc qsassasalg gvkverqvfg 181 eatkqpgitf iaakfdgilg mayprisvnn vlpvfdnlmq qklvdqnifs fylsrdpdaq 241 pggelmlggt dskyykgsls ylnvtrkayw qvhldqveva sgltlckegc eaivdtgtsl 301 mvgpvdevre lqkaigavpl iqgeymipce kvstlpaitl klggkgykls pedytlkvsq 361 agktlclsgf mgmdipppsg plwilgdvfi gryytvfdrd nnrvgfaeaa rl).

[0007] Preferably, the formulation is a kit for detecting the expression level of CTSD in urinary exosomes of patients with type 2 diabetic nephropathy.

[0008] Preferably, the kit includes an immunization method for antigen-antibody reaction and a kit thereof, such as one or more aptamer antibodies or antibody fragments capable of specifically binding to CTSD.

[0009] Preferably, the kit further includes any one or a combination of several of the following components: solid support, diluent, control, standard, quality control, detection antibody, second antibody, second antibody diluent, luminescent reagent, washing solution, colorimetric solution, and stop solution.

[0010] Preferably, the standard includes CTSD standard and humanized labeled antibody standard; more preferably, the quality control includes CTSD quality control and humanized labeled antibody quality control; more preferably, the solid-phase carrier includes microparticles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, microplates or affinity membranes, and other carriers with equivalent functions.

[0011] Preferably, the solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, or a similar carrier.

[0012] The inventors first collected urine samples from healthy individuals, a group with type 2 diabetes without nephropathy, and a group with type 2 diabetic nephropathy. Urinary exosomes were extracted, and LC-MS / MS combined with bioinformatics analysis was used to construct a protein profile of urinary exosomes in patients with type 2 diabetic nephropathy and to screen for target proteins. The expression of the target proteins in urinary exosomes was further verified. The verification results showed that CTSD was highly expressed in the urine of patients with diabetic nephropathy compared to healthy individuals and the group with type 2 diabetes without nephropathy. ROC analysis results showed that urinary exosome CTSD has good sensitivity and specificity in the diagnosis and monitoring of type 2 diabetic nephropathy.

[0013] The following describes a preferred embodiment in detail with reference to the accompanying drawings: Attached Figure Description

[0014] Figure 1 Expression of CTSD in urine was determined by Western blotting and enzyme-linked immunosorbent assay (ELISA) in patients with type 2 diabetic nephropathy, type 2 diabetic nephropathy without nephropathy, and normal controls. (A: Western blotting results of the three groups; B: Comparison of band gray values ​​among the groups in the Western blotting results; C: Difference in CTSD protein concentration among the three groups in the ELISA; D: Difference in CTSD protein concentration at different UACR levels; E: Difference in CTSD protein concentration at different eGFR levels. ns: nosignificance; *: P <0.05;** P <0.01; ***: P <0.001).

[0015] Figure 2 Evaluation of the diagnostic efficacy of urinary exosome CTSD protein in type 2 diabetic nephropathy.

[0016] Figure 3 Identification of CTSD protein expression in kidney tissue of a mouse model of kidney injury from urinary exosomes. (A and B: Analysis of CTSD protein expression levels in glomeruli).

[0017] Figure 4 Expression and identification of CTSD protein in urine and urinary exosomes in a mouse model of kidney injury. (A: Western blot results of urinary exosomes from each group of mice; B: Gray value analysis of protein from each group of mice; C: Western blot results of urinary exosomes from each group of mice; D: Difference analysis of gray value of protein from each group of mice; *: P <0.05; ** P <0.01). Detailed Implementation

[0018] Example 1 Selection of research subjects and specimen collection As a screening cohort, referring to the diagnostic criteria of the Chinese Diabetes Society guidelines, 24 patients with a confirmed diagnosis of type 2 diabetic nephropathy were selected as the experimental group, and 24 patients with a confirmed diagnosis of type 2 diabetic nephropathy without nephropathy were selected as the disease control group. Simultaneously, 24 healthy subjects were collected as normal controls. Urine samples were collected from the above subjects, and urinary exosomes were extracted. LC-MS / MS technology was used to collect urinary exosome proteomics information. Differentially expressed proteins were compared among the experimental group, disease control group, and normal control group. Functional analysis of differentially expressed urinary exosome proteins was performed using GO and KEGG bioinformatics analysis to screen for differentially expressed proteins with CTSD.

[0019] Example 2 Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to verify the expression of CTSD in urinary exosomes. As a validation cohort, referring to the diagnostic criteria of the Chinese Diabetes Society guidelines, 25 patients with a confirmed diagnosis of diabetic nephropathy were selected as the experimental group, and 25 patients with a confirmed diagnosis of type 2 diabetes without nephropathy were selected as the disease control group. Simultaneously, 25 healthy subjects were collected as normal controls. Urine samples were collected from the above subjects, and urinary exosomes were extracted. Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to verify the expression of CTSD in urinary exosomes. Western blotting results showed that compared with the type 2 diabetes without nephropathy group, the expression level of CTSD protein in urinary exosomes was significantly increased in the type 2 diabetic nephropathy group, and the difference was statistically significant. Figure 1A, B). ELISA results showed that, compared with the type 2 diabetic group without nephropathy and healthy individuals, the concentration of CTSD protein in urinary exosomal tissues tended to increase in the type 2 diabetic nephropathy group, and the difference was statistically significant. Figure 1 C). Based on the UACR results and eGFR levels of patients in the type 2 diabetic nephropathy group, they were further subdivided into the same subgroup. The results showed that the concentration of urinary exosome CTSD protein differed significantly between the microalbuminuria group and the type 2 diabetic nephropathy-free group. Figure 1 D). Between the eGFR ≥ 90 mL / min / 1.73 m² and the type 2 diabetes mellitus without nephropathy groups, the urinary exosome CTSD protein level also showed an increasing trend, and the difference was statistically significant. Figure 1 E). The above results indicate that urinary exosome CTSD protein levels are associated with type 2 diabetic kidney injury.

[0020] Example 3 Evaluation of the diagnostic efficacy of CTSD in urinary exosomes ROC curves were constructed based on the CTSD data of the validation group, such as... Figure 2 As shown, the area under the ROC curve for urinary exosome CTSD in diagnosing type 2 diabetic nephropathy is 0.836, indicating that urinary exosome CTSD has good auxiliary diagnostic value for diabetic nephropathy. Figure 2 ).

[0021] Example 4 Establish a mouse model of kidney injury to validate CTSD A type 2 diabetic mouse model was established in C57BL6 / J mice using a high-fat diet combined with intraperitoneal injection of streptozotocin, inducing kidney injury. The expression levels of CTSD protein in mouse urine, urinary exosomes, and various organ tissues were investigated using Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. The results showed that CTSD protein expression was significantly increased in the glomeruli of mice with kidney injury, with statistically significant differences. Figure 3 A and B). In mice with kidney injury, the levels of CTSD protein in urine and urinary exosomes were significantly elevated, and the differences in gray values ​​between groups were statistically significant. Figure 4 A, B, C, and D).

[0022] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. Application of urinary exosome cathepsin D (CTSD) markers in the preparation of formulations for the diagnosis, differential diagnosis, disease monitoring and mechanism research of type 2 diabetic nephropathy.

2. The application according to claim 1, characterized in that, The CTSD amino acid sequence of the urinary exosomes is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The formulation is used to detect the expression level of CTSD in urinary exosomes in patients with type 2 diabetic nephropathy.

4. The application according to claim 3, characterized in that, in, The preparation includes an antigen-antibody detection immunoassay reagent; preferably, it includes a urine antigen-antibody detection immunoassay reagent.

5. The application according to claim 4, characterized in that, The reagent kit prepared by the reagent includes an immunoassay method for antigen-antibody reaction and a kit thereof, which can specifically bind to one or more of aptamer antibodies or antibody fragments.

6. The application according to claim 5, characterized in that, The kit also includes any one or a combination of several of the following components: solid support, diluent, control, standard, quality control, detection antibody, secondary antibody, secondary antibody diluent, luminescent reagent, washing solution, colorimetric solution, and stop solution.

7. The application according to claim 6, characterized in that, The standards include CTSD standards and humanized labeled antibody standards; the quality control materials include CTSD quality control materials and humanized labeled antibody quality control materials; the solid-phase carriers include microparticles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, microplates or affinity membranes, and other carriers with equivalent functions.

8. The application according to claim 6, characterized in that, The solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, or a similar carrier.

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