Introduction
Diabetes mellitus (DM) is a metabolic disease. It is mainly caused by insulin resistance or impaired insulin production and has reached epidemic proportions worldwide [
1,
2]. At present, there are more than 537 million people with diabetes mellitus worldwide, and the number continues to rise [
3]. Type II diabetes mellitus accounts for more than 90% of these cases, and the number of people with type II diabetes mellitus is expected to reach 700 million by 2045 [
4], which is a serious threat to human life and health.
To date, diabetes mellitus remains incurable, and all patients with type I diabetes mellitus and advanced type II diabetes mellitus are treated with exogenous insulin [
3]. As a chronic disease, diabetes mellitus needs lifelong monitoring. The current clinical indicators are still limited in detection. Long-term and repeated invasive blood drawing tests have caused physical and mental pain and burden to patients [
5‐
7]. How to establish objective monitoring methods and minimize the trauma and mental burden of patients is one of the most challenging problems at present.
Urine is one of the most attractive samples for large-scale noninvasive clinical screening programs [
8]. Urine is a potential source of disease biomarkers that could replace plasma. Exosomes are tiny membrane vesicles that are secreted by most cells in the body and range in diameter from 30 to 150 nm. Exosomes are derived from the endocytosis pathway of plasma membrane invagination and are a new library of biomarkers for discovery [
9]. Exosomes are secreted by different cell types under normal and pathological conditions, and their contents and functions vary accordingly [
10,
11]. Studies have found that exosomes are associated with pregnancy [
12,
13], metabolism [
14], cardiovascular disease [
15], the central nervous system [
16] and cancer progression [
17]. In recent years, the continuous development of urine proteomics has provided room for the development of urine exosome protein biomarkers [
18]. Although large clinical studies are required to validate these exosome biomarkers, the potential of urine exosomes as a noninvasive alternative to current diagnostic tests warrants further investigation.
Coagulation is the process in which coagulation factors are activated in a certain order to produce thrombin, and then converts fibrinogen into fibrin. The coagulation process needs to be finely regulated, and its coherence is of great importance [
19]. Coagulation factors are mainly produced by the liver and circulate in the blood. Prothrombin, also known as factor II (F2), is the precursor of thrombin. Prothrombin is activated and hydrolyzed to thrombin by factor X and plays a key role in physiological and pathological coagulation processes [
20]. Recent studies found that coagulation factors are also involved in tissue repair and inflammatory expression, including nervous system inflammation [
20,
21].
As a chronic metabolic disease, diabetic patients often exhibit a hypercoagulable state. And hypercoagulability is an important factor for the occurrence and progression of diabetes. Studies have found that persistent elevation of blood glucose leads to prothrombin and fibrinogen glycosylation, which incompletely activates the coagulation cascade. At this point, the levels of many coagulation factors in the plasma increase, such as fibrinogen, factor VII, factor IX, factor XII, etc. [
21,
22]. After that, the blood coagulation function and fat metabolism of the human changed significantly, which in turn promoted the occurrence and development of diabetes mellitus. Therefore, the monitoring of coagulation function has important clinical significance. It is also very important to understand the changes and influences of the expression of coagulation factors in the urine exosomes of diabetic patients to study the occurrence and development of diabetes mellitus.
The purpose of this study was to verify the expression of coagulation-related proteins in urine exosomes by means of proteomics and to explore the role of coagulation factors in the pathogenesis of diabetes mellitus. In addition, we evaluated its diagnostic and monitoring efficacy for diabetes mellitus, thereby providing suitable, sensitive, and specific biomarkers for diabetes monitoring.
Materials and methods
Patients
Patients with diabetes mellitus admitted to Beijing Shijitan Hospital from September 2020 to April 2021 were included as the study subjects, and healthy people with age and sex matching were selected as the healthy control. Diabetic patients had fasting blood glucose (FBG) ≥ 7.0 mmol/l, glycated hemoglobin (HbA1c) ≥ 6.5% or OGTT 2-hour blood glucose ≥ 11.1 mmol/L. The discovery cohort consisted of 30 patients with diabetes mellitus and 30 healthy controls. The test verification cohort consisted of 24 patients with diabetes mellitus and 24 healthy controls. And the validation cohort included 36 patients with diabetes mellitus and 36 healthy controls.
Clinical data and routine test indexes of subjects were collected retrospectively. All subjects were free of hematuria, proteinuria and ketosis. Urinary diseases and tumors were also excluded, and patients with a history of drug use for 2 weeks were excluded before urine samples were collected. All subjects provided informed consent prior to inclusion in the study and specimen collection. All procedures were carried out in accordance with the ethical standards of the Declaration of Helsinki and approved by the Ethics Committee of Beijing Shijitan Hospital.
Thirty milliliters of clean morning urine was collected from the subjects, and the urine samples were centrifuged at 1500 g speed for 10 min and 10,000 g speed for 30 min to remove dead cells and debris. The exosomes were then collected using the size exclusion SEC method (qEV10/35 nm, IZON, Shanghai, China) [
23‐
25]. Samples were stored at -80℃ until use. The morphology of exosomes was detected by transmission electron microscope (TEM); the size and concentration of exosomes were identified by nanoparticle tracking analysis (NTA); and the exosome markers and common exosome negative protein were analyzed by western blotting analysis.
Mass spectrometry analysis of urine exosomes
Experiments were performed on a QExactive HF-X mass spectrometer (Thermo Fisher), and mass spectra were acquired in data independent acquisition (DIA) mode with a full scan range of m/z 350–1500 and resolution of 120,000 (m/z 200). Mass spectrometry results were queried in the SwissProt human database within UniProt (
www.uniprot. org) using the proteome discovery software suite (Thermo Fisher Scientific v2.1). At the protein level, each protein contained at least one unique peptide using false discovery rate (FDR) of 1% as a filter. Proteins with a fold change > 1.5 and p value < 0.05 were considered significantly different.
Western blotting
To further verify the proteomic analysis results of urine exosomes, we conducted western blotting experiments. After the exosomes were lysed, protein of equal mass was loaded onto a 12% Tris-HCl SDS-polyacrylamide gel. Transferred to PVDF membrane by Trans-Blot Turbo Transfer System (Bio-Rad, California, USA), and shaken for 2 h at room temperature after blocking with 5% skim milk in TBST. Then, the primary antibody (1:1000 dilution; Abcam, Cambridge, UK) was added and incubated overnight. Washed three times with TBST for 15 min each time, and added the horseradish peroxidase-labeled secondary antibody (diluted by 1: 2000; Bios, Beijing, China), and incubated at room temperature for 2 h. After that, they were washed three times with TBST and detected by enhanced chemiluminescence (ECL).
Enzyme linked immunosorbent assay
The validation cohort included 36 patients with diabetes mellitus and 36 healthy controls who were age- and sex -matched. Urine exosomes were lysed with RIPA, and then the protein concentration was measured. The total mass of fixed protein was 10 µg, and the loading amount was adjusted to 100 µl with sample buffer. Then, a sample of 10 µl was added into each well using the YuanJu Biotechnology Center (Shanghai, China) ELISA kit. The concentration of target protein in exosomes was determined three times for each sample according to the instructions. The unknown sample concentration was calculated according to the standard curve, and the unit of target protein expression in exosomes was defined as pg/ml.
Statistics
All experimental data were presented as the mean ± standard deviation (SD), and statistical analysis was performed by GraphPad Prism 8.0 (GraphPad, La Jolla, CA, USA) software. Student’s t-test was used for comparison of differences between groups. Pearson correlation was used for correlation analysis. The diagnostic performance of the target protein was evaluated using receiver operating characteristic curve (ROC) analysis. P < 0.05 was considered statistically significant.
Discussion
The coagulation is a continuous process, including the formation of prothrombin activator, thrombin formation, and fibrin production. Abnormalities at each stage can lead to disease. Patients with long-term diabetes mellitus greatly increase the risk of vascular diseases and microvascular complications, which seriously threaten the life and health of patients. It is of great significance to explore the expression changes of coagulation-related proteins in diabetic patients for the monitoring of diabetes mellitus and the control of diabetes complications.
In this study, a total of 8 coagulation-related proteins were identified in the urine exosomes of diabetic patients, and F2 protein with elevated expression was screened out. After that, ELISA, mass spectrometry, and western blotting were further used to verify the changes in F2 protein concentration in urine exosomes of diabetic patients, which were consistent with the protein changes observed in the discovery cohort. The F2 protein in urine exosomes can be used as a potential biomarker for monitoring diabetes mellitus and has clinical application value.
Coagulation factors are important proteins. The increase in F2 protein and thrombin production in the diabetes mellitus indicate that the blood of patients is in a state of hypercoagulation and that the activation of the coagulation system is enhanced. This is consistent with the results of previous studies [
22]. Prothrombin is encoded by the F2 gene, which is located on chromosome 11 [
26]. The gene contains 14 exons of 21 kb, and its structural integrity is essential for life and an important indicator for coagulation function monitoring. After vascular injury, prothrombin can be converted into active thrombin by prothrombinase. Thrombin is a macromolecular complex composed of factor Xa, factor Va, calcium ions, and phospholipids. Thrombin can convert fibrinogen to fibrin, activate platelets and increase endothelial permeability, thereby preventing blood loss at the site of injury and promoting vascular remodeling [
27‐
29]. The integrity of the prothrombin structure is essential for life, and mice lacking prothrombin die prematurely in the early embryonic stage [
30].
Diabetes mellitus is a hypercoagulable state [
31], especially in patients with uncontrolled diabetes mellitus. Glycemic control is one of the important elements to assess in patients with diabetes mellitus. In this study, the level of F2 expression in the urine exosomes of patients at a high level was significantly lower than that of patients at a general level. For diabetic patients with HbA1c ≥ 8%, blood glucose control was not ideal, and F2 expression was lower. This may be because when the blood glucose control of diabetic patients worsens, the increase in plasma glucose and insulin leads to a rapid and substantial increase in the circulation of thrombin production [
32]. In this study, the level of F2 expression in the urine exosomes of patients at a general level was significantly higher than that of the normal controls. Meanwhile, when we removed the data of the high level group and reanalyzed the area under the curve of F2 protein, as shown in Figure
S1, we found that the area under the curve of F2 protein is larger at this time. It is speculated that urine exosome protein F2 may have higher clinical value for the early diagnosis of diabetes mellitus. In conclusion, F2 can be used as a monitoring indicator of diabetes mellitus, especially for blood glucose control monitoring, which has very important clinical value.
Studies have shown that cholesterol plays a crucial role in determining cell membrane properties and function, and the accumulation of cholesterol in pancreatic β cells can lead to cell dysfunction [
33]. High levels of TG and LDL and low levels of HDL are associated with insulin resistance and are independent factors in insulin development [
34]. Elevated blood glucose occurs when the function of pancreatic β cells in the body to increase insulin release fails to compensate for the degree of insulin resistance. In vitro experiments have shown that HDL increases skeletal muscle glucose uptake and stimulates pancreatic β cells to synthesize and secrete insulin [
35,
36]. Hypertension can aggravate insulin resistance, thus exacerbating the occurrence and development of diabetes. In this study, we found a strong positive correlation between urine exosome F2 and TG concentration in blood through exploration. F2 and TG concentrations are increased in diabetic patients, which may be one of the reasons why diabetic patients are more likely to be complicated with vascular lesions. In addition, we found that the F2 protein concentration was not associated with blood HDL-C in the healthy controls but was strongly negatively associated with blood HDL-C in the diabetes mellitus group. This correlation gives us a guess about the F2 protein. With the disorder of glucose metabolism in the human body, the concentration of F2 protein increases, which will affect the expression of HDL-C. Low levels of HDL-C cause insulin resistance, which in turn contributes to the development and progression of diabetes mellitus. The rise in blood sugar and insulin disorder further lead to abnormal changes in blood lipids [
37]. Of course, at present, this is just our guess, and the specific reason needs to be further studied in the molecular mechanism.
This study is the first to report the expression of coagulation-related proteins in urine exosomes of diabetic patients. In ROC curve analysis, we found that urine exosome protein F2 has good diagnostic ability and could be a potential biomarker for monitoring diabetic changes. Early detection of individuals at risk of developing diabetes mellitus may benefit for the implementation of preventive treatment. Urine samples have unique advantages in disease screening. Urine exosomes can meet the need for novel markers of diabetes mellitus and can be used for noninvasive, rapid and simple outcome determination [
38].
The biomarker found in this study needs to be further evaluated and verified by many samples before being used in clinical practice. In the future, this study will be applied to the clinical monitoring of diabetes mellitus and could benefit a large population.
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