Application of urine flna as a marker in preparation of a reagent kit for diagnosis of diabetic nephropathy

Abstract

The application discloses application of urine FLNA as a marker in preparation of a diabetes nephropathy disease diagnosis kit. A reagent for quantitatively detecting FLNA protein in urine is applied in preparation of a diabetes nephropathy diagnosis reagent or an auxiliary diagnosis reagent. As a preferred embodiment of the application, the reagent for detecting urine FLNA is an ELISA reagent for quantitatively detecting FLNA content in urine. The patent identifies a series of proteins in urine in the progress of diabetes nephropathy through urine protein spectrum, and then constructs a method based on urine protein capable of being used for diabetes nephropathy diagnosis through ELISA technology. FLNA is used as a detection target for the diagnosis of diabetes nephropathy. The ROC curve for identifying diabetes nephropathy and diabetes non-nephropathy according to FLNA content in urine shows that the sensitivity and specificity are 91.07% and 70.21% respectively.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical diagnosis, and relates to application of FLNA from urine in preparation of a diabetes nephropathy disease diagnosis kit. BACKGROUND

[0002] According to the latest data in 2022, the incidence of diabetes in China is about 0.9%. According to the data of the seventh population census of China, there are 141178 million people in China. According to this calculation, there will be 1270 million new cases of diabetes every year. Diabetes has become an important public health problem in China. Diabetic nephropathy is one of the main microvascular complications of diabetes. According to statistics, about 25% to 35% of diabetic patients will have symptoms of kidney disease. Diabetic nephropathy has become the primary cause of CKD (chronic kidney disease) in hospitalized patients in China. Statistics show that diabetic nephropathy has become the main cause of end-stage renal disease, and about 30% to 50% of end-stage renal disease is caused by diabetic nephropathy. Therefore, it is urgent to pay great attention to the prevention and treatment of diabetic nephropathy!

[0003] Diabetes develops into diabetic nephropathy for a long period of time, which provides valuable time for the prevention and treatment of diabetic nephropathy. Studies have shown that if diabetic nephropathy patients can be confirmed in the early screening process and treated effectively, the risk of end-stage renal disease and death of diabetic nephropathy patients will be significantly reduced. Therefore, it is urgent to develop a convenient, fast and accurate method for early diagnosis of diabetic nephropathy. The appearance of urinary albumin is the earliest stage that can be detected in clinical practice at present, but once clinical proteinuria appears, the renal function will be progressively decreased until renal failure. Once proteinuria appears, the kidney damage is often irreversible. In addition, albuminuria lacks specificity for the diagnosis of DKD, and some DKD patients may show negative urinary protein in the early stage, so the method has a high rate of missed diagnosis. At present, it is still not known which patients will eventually develop diabetic nephropathy due to genetic and environmental factors. Changes in the vascular system and glomerulus, including podocytes, mesangial cells and changes in the filtration barrier, play an important role in the pathophysiology of diabetic kidney. In addition, the diabetic environment has a major impact on the renal tubular system. Therefore, these markers of cell damage in urine are expected to become markers for the diagnosis of diabetic nephropathy.

[0004] Filamin A (FLNA) is a high-molecular-weight cytoskeletal protein that forms a network by cross-linking actin and links it to the cell membrane. FLNA is involved in the formation of the cytoskeleton, anchoring various proteins in the cytoskeleton, and regulating cell adhesion and migration. It is involved in signal transduction, cell proliferation and differentiation, pseudopod formation, vesicle transport, tumor resistance, and genetic diseases by binding to interacting proteins. Studies have shown that the phosphorylation level of FLNA is related to the prognosis of various tumors, including colorectal cancer, hepatocellular carcinoma, pancreatic cancer, and oral squamous cell carcinoma, suggesting that it can be used as a candidate biomarker and potential therapeutic target for the prognosis of these tumors. In addition, a 2017 study showed that FLNA combined with FLNB and keratin 19 (KRT19) in serum can enhance the diagnosis of prostate cancer by prostate-specific antigen (PSA), suggesting that FLNA can be used as a circulating biomarker. However, there is no report on the relationship between FLNA and diabetic nephropathy. SUMMARY

[0005] The purpose of the present application is to provide a urine protein marker related to diabetic nephropathy.

[0006] Another purpose of the present application is to provide the use of the marker.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] The urine protein marker related to diabetic nephropathy is selected from the following proteins: FLNA.

[0009] The use of the urine protein marker of the present application in the preparation of a diagnostic reagent for diabetic nephropathy.

[0010] The use of a reagent for quantitatively detecting FLNA protein in urine in the preparation of a diagnostic reagent or an auxiliary diagnostic reagent for diabetic nephropathy.

[0011] As a preferred embodiment of the present application, the reagent for detecting urine FLNA is an ELISA reagent for quantitatively detecting the content of FLNA in urine.

[0012] Advantages:

[0013] The present application identifies a series of proteins in urine during the progression of diabetic nephropathy through urine proteomics, and then constructs a method for diagnosing diabetic nephropathy based on urine proteins through ELISA technology. FLNA is used as a detection target for the diagnosis of diabetic nephropathy. The ROC curve based on the content of FLNA in urine to distinguish diabetic nephropathy from non-renal diseases in diabetic patients shows that the sensitivity and specificity are 91.07% and 70.21%, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Urinary mass spectrometry of patients with diabetic non-renal disease and diabetic nephropathy

[0015] Figure 2 Preliminary verification of urinary mass spectrometry results

[0016] Figure 3 Expression localization of FLNA and expression changes in DN cases

[0017] Figure 4 Differences in the amount of urinary FLNA in patients with diabetic non-renal disease and diabetic nephropathy

[0018] Figure 5 An ROC curve showing that urinary FLNA content can identify diabetic nephropathy from diabetic non-renal disease is shown. DETAILED DESCRIPTION

[0019] Example 1 Urinary proteome analysis of patients with diabetic non-renal disease and diabetic nephropathy

[0020] (1) Collect 5 ml of morning urine from each of 3 patients with diabetes and from each of 3 patients with DN confirmed by kidney puncture. Centrifuge at 3000 rpm for 5 minutes and take the supernatant.

[0021] (2) Inclusion criteria for patients with diabetic non-renal disease: a) age >= 18 years; b) type 2 diabetes (WHO diagnostic criteria), negative for microalbuminuria, eGFR > 60, normal liver and kidney function, C) first treatment of diabetes, no retinopathy. Exclusion criteria: a) age >= 65 years; b) combined with other complications and kidney diseases. Inclusion criteria for patients with diabetic nephropathy: a) age >= 18 years; b) type 2 diabetes (T2DM) patients confirmed by biopsy as having DN. Exclusion criteria: a) age >= 65 years; b) non-diabetic nephropathy complications or acute kidney injury at the time of kidney biopsy.

[0022] (3) Perform proteome analysis on the urine, and analyze the function of the differential proteins in the urine by bioinformatics methods to find proteins with potential diagnostic markers. The volcano plot shows the proteome analysis results, with 413 proteins increased and 35 proteins decreased.

[0023] Example 2 Preliminary verification of FLNA expression

[0024] According to the above sample collection criteria, further collect 200 ul of morning urine from each of 20 patients with diabetic non-renal disease and from each of 20 patients with diabetic nephropathy confirmed by kidney puncture. Centrifuge at 3000 rpm for 5 minutes and take the supernatant. Use ELISA to verify the proteome results:

[0025] (1) The kit is taken out of the refrigerated environment and placed in a room temperature environment for 15-30 minutes before use.

[0026] (2) Establish a standard curve: 1 hole (no sample and enzyme standard reagent, the same as each hole), set up 6 standard holes. Add 50ul of corresponding standard to the first to sixth holes of the enzyme standard coating plate, and the seventh hole is used as a blank control. Repeat the operation in the eighty-ninth to ninety-third holes, and make duplicate holes.

[0027] (3) Add sample: add 40ul of sample diluent to each hole of the sample to be tested, then add 10ul of sample to be tested. Add sample to the bottom of the enzyme standard plate, try not to touch the hole wall, and mix gently. The sampling time should not exceed 5min

[0028] (4) Incubation: seal the plate with sealing film and incubate at 37°C for 30min.

[0029] (5) Wash the plate: carefully remove the sealing film, discard the liquid in each hole, shake dry, add enough washing solution to each hole, stand for 30s, then discard, pat dry, repeat 5 times, and dry on filter paper.

[0030] (6) Add enzyme: add 50ul of enzyme standard reagent to each hole, except for the blank hole, seal the plate with sealing film, and incubate at 37°C for 60min.

[0031] (7) Wash the plate

[0032] (8) Color development: add 50ul of color developing agent A to each hole, then add 50ul of color developing agent B, mix gently, and develop color at 37°C for 10min.

[0033] (9) Termination: add 50ul of termination solution to each hole to terminate the reaction. At this time, the blue color will change to yellow, and the determination should be made within 15min.

[0034] (10) Measurement: zero the blank hole, measure the absorbance (OD value) of each hole at 450nm wavelength in sequence, and calculate the data.

[0035] The results are shown in Figure 2 The amount of FLNA in the urine of patients with diabetic nephropathy was significantly higher than that of patients with diabetic non-nephropathy.

[0036] Example 3 Expression localization of FLNA

[0037] (1) Collect kidney cancer adjacent kidney tissue as normal control and diabetic nephropathy patients in examples 1 and 2;

[0038] (2) Make frozen sections respectively;

[0039] (3) Use tubular and podocyte specific markers SGLT2 and Synaptopodin to detect the expression of FLNA in the kidney tissue of patients with diabetic nephropathy

[0040] As Figure 3 shown, compared with normal control group, FLNA was expressed in renal tubules (SGLT2 specific marker) and podocytes (Synaptopodin specific marker) of diabetic nephropathy patients, and the expression in renal tubules and podocytes of diabetic nephropathy patients was increased.

[0041] Example 4 Urinary FLNA as a diagnostic marker for diabetic nephropathy

[0042] (1) Further according to the sample collection criteria in Examples 1 and 2, 27 cases of diabetic non-renal disease patients and 36 cases of diabetic nephropathy patients were collected, and 200ul of morning urine was centrifuged at 3000rpm for 5 minutes, and the supernatant was taken;

[0043] (2) The expression amount of urinary FLNA was detected by ELISA, and the steps were the same as in Example 2;

[0044] (3) The detection results in Examples 2 and 4 (47 cases of diabetic non-renal disease patients and 56 cases of diabetic nephropathy patients) were integrated, and ROC curve analysis was performed.

[0045] As Figure 4 shown, the detection results showed that compared with diabetic non-renal disease patients, the content of FLNA was significantly higher in diabetic nephropathy patients. The area under the curve of receiver operating characteristic curve (ROC curve) for FLNA protein to distinguish diabetic nephropathy and diabetic non-renal disease was 0.8682 (95% CI: 0.8008-0.9355), and the sensitivity and specificity were 91.07% and 70.21% respectively. Figure 5 )

Claims

1. The use of a reagent for detecting urine FLNA in the preparation of a kit for the auxiliary diagnosis of diabetic nephropathy.

2. Use according to claim 1, characterized in that The reagent for detecting urine FLNA is an ELISA reagent for quantitatively detecting the content of FLNA in urine.

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