Use of serum soluble CD146 as a biomarker for acute kidney injury

Abstract

The present application relates to the field of biomedicine and discloses the use of serum soluble CD146 as a biomarker for acute kidney injury. The present invention studies the role of sCD146 in AKI in mice and human samples and finds that sCD146 may become a valuable biomarker for AKI and can effectively predict the severity and prognosis of the disease.

Description

Technical Field

[0001] The present application relates to the field of biomedical hospital, and more specifically, to the use of serum soluble CD146 as a biomarker for acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a common clinical critical illness that threatens human health, with an estimated 1.7 million deaths each year. Although some patients can reverse renal function through active treatment after AKI occurs, 12.6%-18.2% of patients still develop chronic kidney disease (CKD), which brings high medical expenses and economic burdens due to dialysis, kidney transplantation and drug treatment. However, the process and mechanism that mediates the transformation of AKI to CKD are still not fully understood. At the same time, there is a clear lack of sensitive and specific biomarkers that can reliably assess the progression and prediction of AKI.

[0003] The cell adhesion molecule CD146 is associated with a variety of biological processes, including tissue regeneration, tumor progression, inflammation, and fibrosis. Although CD146 mainly functions as a membrane receptor, under certain pathological conditions, it can be proteolytically cleaved and its soluble form (sCD146) can be released into the circulation. Circulating sCD146 has become a potential biomarker for some diseases, such as nervous system infection, diabetic retinopathy, and diabetic nephropathy. However, its value in AKI has not been studied. Summary of the invention

[0004] The purpose of the invention of this application is to explore the role of serum soluble CD146 in AKI and to provide a use of serum soluble CD146 as a biomarker for acute kidney injury.

[0005] In a first aspect, the present application provides the use of serum soluble CD146 as a biomarker for acute kidney injury.

[0006] In a second aspect, the present application provides the use of serum soluble CD146 in preparing a screening or prognosis kit for acute kidney injury.

[0007] In a third aspect, the present application provides the use of a reagent for detecting the content of serum soluble CD146 in the preparation of a screening or prognosis kit for acute kidney injury.

[0008] In summary, this application has the following beneficial effects: The present invention has conducted research on the role of sCD146 in AKI in mouse and human samples, and found that sCD146 may become a valuable biomarker for AKI and can effectively predict the severity and prognosis of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Results of sCD146 determination in the serum of mice in different groups: sCD146 in IRI mice was significantly higher than that in the control group (IRI-Sham); sCD146 in UUO mice was significantly higher than that in the control group (UUO-Sham). *P<0.05, *** P<0.001, showing statistical significance.

[0010] Figure 2 :The results of serum sCD146 determination in human AKI patients and healthy controls: The sCD146 in AKI patients was significantly higher than that in the healthy controls. **** P<0.0001, showing statistical significance.

[0011] Figure 3 : Serum sCD146 in human AKI patients is correlated with commonly used clinical indicators: it is correlated with renal function sCr, BUN, eGFR, CysC, etc.

[0012] Figure 4 :ROC analysis of sCD146 in diagnosing AKI; Figure 5 : Serum sCD146 in human AKI patients and whether AKI progresses to CKD: The serum sCD146 level in patients who progress to CKD is higher than that in patients who recover from AKI. * P<0.05, there is statistical difference. DETAILED DESCRIPTION

[0013] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0014] It is worth noting that the materials and methods used in the examples are all existing methods unless otherwise specified.

[0015] 1. Materials and Methods 1. Experimental reagents

[0016] 2. Experimental instruments

[0017] 3. Experimental animals 3.1 General Six-week-old male wild-type mice were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. The mice were housed in the animal laboratory of Shanghai Sixth People's Hospital (SPF grade). The mice were fed a natural diet and had a standard 12-hour light / 12-hour dark cycle. All operations involved in this experiment were approved by the Animal Ethics Committee of Shanghai Sixth People's Hospital. Mice were fed until 8 weeks of age for acute kidney injury (AKI) modeling.

[0018] 3.2 Preoperative preparation of mice The weight of mice was measured, and male mice aged 8 weeks and weighing >20g were selected for modeling, with at least 6 mice in each group. The mouse abdominal hair was shaved with a mouse depilatory device, and the right lower abdomen of the mouse was disinfected with 75% disinfectant alcohol. A 1ml syringe was used to absorb the Avertin anesthetic, which was slowly inserted into the right lower abdomen of the mouse. After a breakthrough feeling and blood withdrawal, the anesthetic was injected. The injection dose was 0.2mL / 10g mouse body weight. After the injection, the mouse was observed for anesthesia. After it stopped moving, the pain reflex disappeared, and the breathing and heartbeat were stable, it was placed on the operating table, the mouse limbs were fixed, and the mouse abdomen was disinfected twice with 75% disinfectant alcohol. The abdominal surgical area was exposed by laying a hole towel.

[0019] Surgical instruments such as scissors and surgical forceps are sterilized by high temperature and high pressure before the operation. The experimenters wear surgical caps, masks, disposable sterile surgical gowns, and sterile surgical gloves. Items that are not suitable for high temperature and high pressure sterilization, such as constant temperature pads required during the operation, are surface disinfected with disinfectant and disinfected with ultraviolet light before the operation to ensure sterility during the operation as much as possible.

[0020] 3.3 Establishment of bilateral ischemia-reperfusion injury (IRI) model of AKI in mice The mouse abdomen was cut open layer by layer along the midline white line, and the fascia and fat tissue were bluntly separated with forceps to expose the bilateral kidneys. The fat and fascia tissue around the bilateral renal pedicles were separated, and a non-invasive microaneurysm clip (Fine Science Tools, Cambridge, UK) was placed on the renal pedicle for 30 minutes to induce renal ischemia. At this time, the color of the kidney should change from bright red to purple-black. The abdominal opening was covered with gauze soaked in 0.9% saline preheated at 37°C, and the mouse was placed on a 37°C constant temperature pad throughout the process to ensure the body temperature of the mouse. After 30 minutes, the non-invasive microaneurysm clip was removed and the kidney was observed for several minutes. The reperfusion was successful when the kidney returned from purple-black to dark red. After washing the abdominal cavity with 0.9% saline, 5-0 surgical sutures were used to suture layer by layer. After suturing, the mouse was placed on a 37°C constant temperature pad until it recovered. In the control sham operation group, the abdomen was opened after anesthesia, and the abdominal cavity was sutured layer by layer after exposing the kidney for half an hour. The IRI model was established for 14 days.

[0021] 3.4 Establishment of the unilateral ureteral obstruction (UUO) model of AKI in mice After the mice were anesthetized to a satisfactory degree, a midline incision was made on the mouse abdomen to expose the left ureter, and the surrounding fat fascia tissue was bluntly separated. Two points 2 to 3 mm apart in the middle and upper part of the ureter were selected and ligated separately using 4-0 surgical sutures. After the ligation was completed, surgical scissors were used to cut between the ligation sites. After the abdominal cavity was cleaned with 0.9% saline, 5-0 surgical sutures were used to suture layer by layer. After suturing, the mice were placed on a 37°C constant temperature pad until they recovered. After anesthesia, the sham operation group underwent laparotomy, and the abdominal cavity was sutured layer by layer after the left ureteral tissue was separated. The UUO model was established for 7 days.

[0022] 3.5 Anti-infection in mice after surgery After the mice were modeled, 80,000 units of sodium penicillin (dissolved in 0.2 ml of 0.9% saline) were intraperitoneally injected once a day for 3 days. The abdominal suture was observed for bleeding and exudation, and the vitality of the mice was restored.

[0023] 3.6 Collection of mouse serum samples After the AKI model of mice was established for a sufficient period of time, the mice were anesthetized with Avertin, and the abdominal cavity of the mice was cut open layer by layer after routine surgical disinfection. Blood was collected from the abdominal aorta of the mice with a 1ml syringe. The blood samples were left to stand at room temperature for 1 hour and then centrifuged at 4°C and 3000 rpm for 15 minutes. The upper serum was collected and stored in a -80°C refrigerator for subsequent related experimental tests. After the blood was collected, the mouse chest was opened upwards and the mouse was euthanized by cervical dislocation.

[0024] 4. Clinical samples 4.1 General The clinical sample collection was approved by the Ethics Committee of Shanghai Sixth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Informed consent was obtained from all patients before the samples were collected. A total of 32 patients diagnosed with AKI in a single center of our hospital from March 2023 to July 2024 were selected, with a follow-up time of at least 3 months, and 20 healthy volunteers in a single center of our hospital in July 2024 were included in the control group. Basic information and blood samples of patients and healthy volunteers were collected.

[0025] 4.2 Collection of serum samples from subjects and determination of biochemical indices Professional laboratory doctors collected blood from patients through venous blood sampling, obtained serum from patients through centrifugation, and used Roche's fully automatic biochemical analyzer to measure biochemical indicators. Excess serum was stored at -80°C for subsequent experiments.

[0026] 4.3 Patient follow-up and chronic kidney disease (CKD) criteria AKI patients were followed up for at least 3 months, with routine follow-up visits to monitor clinical indicators (at least 2 times) and to record changes in biochemical indicators. CKD was determined based on the patient's estimated glomerular filtration rate (eGFR) standard, and CKD stage 1 (eGFR>90 ml / min / 1.73m 2 ) is difficult to judge clinically. Only eGFR < 90 ml / min / 1.73m 2 That is, patients with overt CKD of at least CKD stage 2 were included in the analysis.

[0027] 5. Serum soluble CD146 (sCD146) determination 5.1 Determination of mouse serum sCD146 5.1.1 Preparation of working fluid The kit was equilibrated at room temperature for 30 minutes, the concentrated wash solution was diluted 1:20 with distilled water, the anti-CD146 peroxide was diluted 1:20 with the sample diluent, and the human lyophilized plasma of known concentration was resuspended with 0.5 mL of distilled water. The standard was diluted by the gradient dilution method as follows:

[0028] 5.1.2 Preparation of samples to be tested The sample to be tested was slowly thawed in a 4°C refrigerator. After it was completely thawed, 15 μL was taken and diluted with sample diluent at a ratio of 1:20, and mixed by gently pipetting.

[0029] 5.1.3 ELISA reaction Add 200 μL of gradient standards and diluted samples to the 96-well plate coated with CD146 and incubate at room temperature for 30 minutes. Discard the sample supernatant and wash with diluted washing solution, 300 μL per well, wash 5 times, and tap the plate each time to ensure that the liquid is removed as much as possible. Then add 200 μL of diluted anti-CD146 peroxide to each well and incubate at room temperature for 30 minutes. Discard the sample supernatant and wash 5 times as described above. After protecting from light, add 200 μL of TMB colorimetric solution to each well, incubate at room temperature for 5 minutes, and then add 100 μL of stop solution to each well.

[0030] 5.1.4 Microplate reader readings After placing the 96-well plate in the microplate reader, measure the absorbance at a wavelength of 450nm. Take the logarithm of the absorbance value and concentration of the gradient standard and draw a standard curve, requiring R square>0.99. Calculate the concentration of the sample to be tested according to its absorbance value and multiply it by the dilution factor of 20 to obtain the final mouse serum sCD146 value.

[0031] 5.2 Human serum sCD146 assay 5.2.1 Reagent preparation Prepare 1X Wash Buffer PT by diluting Wash Buffer PT 10X with deionized water. Mix 5 mL WashBuffer PT 10X with 45 mL deionized water to make 50 mL 1X Wash Buffer PT. Prepare 3 mL of antibody complex by mixing 300 µL 10X capture antibody and 300 µL 10X detection antibody with 2.4 mL Antibody Diluent.

[0032] 5.2.2 Preparation of standard products Add sample diluent NS as indicated on the standard bottle and mix thoroughly to obtain a 19200 pg / mL standard. Perform a gradient dilution as indicated in the following subscripts:

[0033] 5.2.3 Preparation of samples to be tested The sample to be tested was thawed slowly in a 4°C refrigerator, 2 μL was taken and diluted with sample diluent at a ratio of 1:20, and then diluted at a ratio of 1:25 (the final dilution factor was 1:500), and mixed by gently pipetting.

[0034] 5.2.4 ELISA reaction Add 50 μL of each sample and standard to a pre-coated 96-well plate, add 50 μL of antibody complex to each well, seal the plate, and incubate on a shaker at 400 rpm at room temperature for 1 hour. Discard the supernatant and wash the plate 3 times with 1X Wash Buffer PT, adding 350 μL to each well each time, leaving the liquid for at least 30 seconds, and remove as much liquid as possible after each wash. After washing, add 100 μL of TMB developer to each well, incubate on a shaker at 400 rpm at room temperature in the dark, add 100 μL of stop solution to each well, and mix thoroughly.

[0035] 5.2.5 Microplate reader readings After placing the 96-well plate in the microplate reader, the absorbance is measured at a wavelength of 450nm, and a standard straight line is drawn according to the absorbance and concentration of the standard, requiring R square>0.99. The concentration of the sample to be tested is calculated according to its absorbance value, multiplied by a 500-fold dilution factor, to obtain the value of human serum sCD146.

[0036] 6. Statistical analysis GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, US) and SPSS 22.0 software (IBM, Armonk, NY, US) were used for statistical analysis and graphics. Quantitative data were expressed as mean ± SD. All data were tested for normality and homogeneity of variance using the Shapiro-Wilk test and the Levene test. Normally distributed data with homogeneous means were analyzed using one-way ANOVA and two-sided unpaired t-tests; data with unequal variances were analyzed using the Welch's t-test. P < 0.05 was considered statistically significant.

[0037] 2. Experimental Results 1. The serum sCD146 level in mice with AKI was significantly higher than that in the sham operation group pass Figure 1 It can be seen that in two AKI models of mice, bilateral IRI and UUO models, the serum sCD146 of mice was significantly increased compared with the control sham operation group.

[0038] 2. The serum sCD146 level in AKI patients was significantly higher than that in the healthy volunteers control group pass Figure 2 It can be seen that the sCD146 measurement of 32 human AKI patients and 20 healthy volunteer controls showed that sCD146 was significantly increased in the AKI patient group.

[0039] 3. Correlation between serum sCD146 and clinically commonly used renal function indicators in human AKI patients The basic information and laboratory test results of the 32 AKI patients and 20 healthy controls included in the study are shown in the following table. There were no differences in gender, age, or liver function between the AKI patient group and the healthy control group, but the hemoglobin level and renal function of the AKI group decreased.

[0040] General conditions and laboratory test results of human AKI patients and healthy controls (P<0.05 was considered statistically significant):

[0041] Combining the above table with Figure 3 It can be seen that the serum CD146 level of AKI patients was highly positively correlated with the patients' peak serum creatinine (sCr) level (r=0.697), blood urea nitrogen (BUN) level (r=0.777) and cystatin C (Cys C) level (r=0.600), and moderately negatively correlated with eGFR (r=0.569), indicating the reliability of sCD146 as a clinical indicator.

[0042] 4. ROC analysis of sCD146 in the diagnosis of AKI The serum creatinine, urea nitrogen and serum sCD146 levels of healthy controls and AKI patients were compared, and the ROC curve was used to calculate the area under the ROC curve (AUC) to evaluate the predictive value of the indicators.

[0043] For reference Figure 4 , serum creatinine (the main criterion for AKI diagnosis): AUC=0.9438, p<0.0001; Urea nitrogen: AUC = 0.8516, p < 0.0001; Serum sCD146: AUC=0.8219, p=0.0001; The results showed that sCD146 was sensitive and specific in diagnosing AKI.

[0044] 5. Serum sCD146 in human AKI patients may predict the risk of progression of AKI to CKD The relationship between AKI progression to CKD and serum CD146 levels in patients was analyzed to explore the potential prognostic role of serum sCD146 in AKI. Figure 5 , 22 of the 32 patients eventually developed CKD during a follow-up period of at least 3 months. The serum sCD146 levels of these patients who developed CKD were significantly higher than those of AKI recovery patients before renal biopsy, indicating that serum sCD146 may indicate the prognosis of AKI.

[0045] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Use of serum soluble CD146 as a biomarker for acute kidney injury.

2. Use of serum soluble CD146 in the preparation of an acute kidney injury screening or prognosis kit.

3. Use of a reagent for detecting serum soluble CD146 content in the preparation of an acute kidney injury screening or prognosis kit.

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