This study was designed to investigate the changes of urinary microvesicle-bound uromodulin and total urinary uromodulin levels in human urine and the correlations with the severity of diabetic kidney disease (DKD). 31 healthy subjects without diabetes and 100 patients with type 2 diabetes mellitus (T2DM) were included in this study. The patients with T2DM were divided into three groups based on the urinary albumin/creatinine ratio (UACR): normoalbuminuria group (DM,
Diabetic kidney disease (DKD) is a major complication of diabetes mellitus and the most frequent cause of end-stage renal disease (ESRD) [
It has been found that many cells can produce microvesicles, including hematopoietic cells, reticulocytes, B/T lymphocytes, dendritic cells, mast cells, platelets, intestinal epithelial cells, astrocytes, neurons, fetal cells, and tumor cells [
Urinary microvesicles, including microparticles and exosomes, have recently been the targets of urine proteomic analysis [
Urinary microvesicles are derived from the kidney and contain proteins secreted by the kidney tissue mostly. Based on this, several recent articles have reported new potential markers of kidney disease in urinary microvesicles [
We hypothesised that uromodulin might be a protein component of urinary microvesicles, and the microvesicle-bound uromodulin represents kidney underlying protein alterations. To confirm this hypothesis, we have developed an enzyme-linked immunosorbent assay (ELISA) method for urinary microvesicles-bound uromodulin using a specific monoclonal antibody, AD-1, developed by Deng et al. [
In this study, 31 age- and sex-matched Chinese Han nondiabetic healthy subjects and 100 patients with type 2 diabetes mellitus (T2DM) were recruited from the Second Hospital of Shandong University. The patients with T2DM were divided into three groups based on the urinary albumin/creatinine ratio (UACR): normoalbuminuria group (DM,
All patients with hypertension or proteinuria were treated with angiotensin converting enzyme inhibitors (ACEI) and/or angiotensin receptor blockers (ARB), patients with diabetes were treated with human insulin or repaglinide, and patients with hypercholesterolemia were treated with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors. No additional medicines were taken within 1 week of urine collection. Exclusion criteria were as follows: tumors, urinary tract disorders, pregnancy, known renal diseases other than DKD, decompensated heart failure, chronic inflammatory diseases, prostatic diseases (in males), hematologic diseases, liver diseases, and recent myocardial infarction or unstable angina within the past 6 months.
Urine samples were collected from all subjects with a 24 h period. Two 0.95 ml aliquots of the urine sample were collected and neutralized with 50
The following methods were used to assay the biochemical parameters in the serum samples: liquid chromatography for glycosylated hemoglobin (HbA1c), chemical modification method for triglyceride (TG), cholesterol (CH), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C), Jaffe-kinetic assay for creatinine (CR), and urease-GLDH method for blood urea nitrogen (BUN).
The microvesicle specific monoclonal antibody (AD-1) was initially obtained from the serum of liver cancer patients with anti-human membrane associated liver alkaline phosphatase [
Use the tris buffer solution (10 mmol/L Tris, 10 mmol/L NaCl, and 10 mmol/L NaN3, pH 8.5) to dilute the concentration of goat anti-mouse antibody to 10 ug/ml. Distribute the diluted antibody to the 96-well plate (Costar Co. Ltd., USA, 50
Total urinary uromodulin was determined by a sandwich ELISA kit (R&D System). The verification of the specificity of the ELISA method has been proven in our previous article [
Measurement data, which met normal distribution, were presented as mean ± SEM, and comparisons were tested statistically using one-way analysis of variance followed by the appropriate post hoc test for determining statistical significance among various groups. Urinary microvesicle-bound uromodulin and total urinary uromodulin, which did not meet normal distribution, were presented as median (
The anthropometric and biochemical characteristics of baseline are shown in Table
Baseline anthropometric and biochemical characteristics.
Parameter | Control group | DM group | DN1 group | DN2 group |
---|---|---|---|---|
Number ( | 31 | 46 | 32 | 22 |
Age (year) | 56.5 ± 7.85 | 55.02 ± 11.41 | 52.79 ± 15.12 | 51.56 ± 17.09 |
Gender (male/female) | 15/16 | 28/18 | 13/19 | 12/10 |
UACR (mg/g) | 5.75 ± 2.63 | 9.8 ± 4.38 | 91.6 ± 46.44 | 642.79 ± 332.86 |
HbA1c (%) | 4.62 ± 0.43 | 8.45 ± 2.72 | 8.98 ± 2.56 | 9.63 ± 2.3 |
CR (mmol/l) | 51.13 ± 13.17 | 61.78 ± 15.21 | 70.58 ± 21.19 | 95.03 ± 46.11 |
BUN (mmol/l) | 4.33 ± 0.84 | 4.21 ± 0.99 | 6.75 ± 2.97 | 6.03 ± 2.41 |
Systolic blood pressure (mmHg) | 139.5 ± 12.66 | 142.68 ± 20.82 | 138.7 ± 14.8 | 162.57 ± 19.97 |
Diastolic blood pressure (mmHg) | 79.25 ± 10.78 | 82.47 ± 9.87 | 81.5 ± 12.66 | 83.43 ± 9.62 |
CH (mmol/l) | 4.83 ± 0.92 | 4.9 ± 1.62 | 4.13 ± 1.3 | 5.58 ± 2.3 |
TG (mmol/l) | 2.03 ± 0.95 | 1.68 ± 0.94 | 1.78 ± 0.43 | 1.53 ± 0.76 |
LDL-C (mmol/l) | 1.78 ± 0.4 | 2.76 ± 0.74 | 2.21 ± 0.8 | 2.22 ± 0.53 |
HDL-C (mmol/l) | 1.24 ± 0.57 | 1.3 ± 0.3 | 1.08 ± 0.25 | 1.09 ± 0.3 |
DM = type 2 diabetes mellitus with normoalbuminuria; DN1 = type 2 diabetes mellitus with microalbuminuria; DN2 = type 2 diabetes mellitus with macroalbuminuria; UACR = urinary albumin/creatinine ratio; HbA1c = glycosylated hemoglobin; TG = triglyceride; CH = cholesterol; LDL-C = low density lipoprotein cholesterol; HDL-C = high density lipoprotein cholesterol; CR = creatinine; BUN = blood urea nitrogen.
The levels of urinary microvesicle-bound uromodulin in DN1 and DN2 groups were significantly higher than those in control group and DM group (
The changes of urinary microvesicle-bound uromodulin and total urinary uromodulin.
Group | | Urinary microvesicle-bound uromodulin (pg/ml) | Total urinary uromodulin (pg/ml) |
---|---|---|---|
Control | 31 | 0.251 (0.233, 0.277) | 0.469 (0.330, 1.309) |
DM | 46 | 0.262 (0.247, 0.569) | 1.083 (0.719, 1.349) |
DN1 | 31 | 0.298 (0.279, 0.311) | 1.320 (1.022, 1.457) |
DN2 | 19 | 0.337 (0.272, 0.352) | 1.047 (0.319, 1.453) |
Correlation analysis showed that the levels of urinary microvesicle-bound uromodulin were positively correlated with UACR, HbA1c, systolic blood pressure, and total urinary uromodulin (
The correlation analysis among urinary microvesicle-bound uromodulin and total urinary uromodulin and variables (
Variables | Urinary microvesicle-bound uromodulin |
---|---|
UACR | 0.002 |
HbA1c | 0.034 |
Systolic blood pressure | 0.049 |
Total urinary uromodulin | 0.000 |
The stepwise multiple linear regression analysis among urinary microvesicle-bound uromodulin and total urinary uromodulin and variables (
Variables | Urinary microvesicle-bound uromodulin |
---|---|
UACR | 0.000 |
HbA1c | 0.046 |
Systolic blood pressure | 0.200 |
Total urinary uromodulin | 0.064 |
The cellular and molecular mechanisms that lead to DKD are not completely identified. It is known that the renal functional changes are associated with cellular and extracellular derangements in both the glomerular and tubulointerstitial compartments [
The purification technology of the microvesicles is mainly supercentrifugation and filtration. Because the supercentrifugation needs expensive equipment and a very long period of time, we use the filter instead of the supercentrifugation. However, the filtration did not separate the microvesicles that the diameter less than 300 nm [
In our study, the excretion of total urinary uromodulin in the T2DM patients without albuminuria and with microalbuminuria was significantly increased compared with healthy subjects. It is consistent with previous studies that uromodulin expression in the early stages of DKD increased in the T2DM patients [
AD-1 staining is only present on the brush border of proximal tubular cells in the cortex (S1 and S2 segments) and in the outer strip of the outer medulla and in the medullary rays (S3 segments) [
The increased excretion of urinary microvesicle-bound uromodulin was correlated with the UACR, HbA1c, systolic blood pressure, and total urinary uromodulin. In a further multiple stepwise linear regression analysis, UACR and HbA1c were independent determinants for urinary microvesicle-bound uromodulin. This result suggested that the urinary microvesicle-bound uromodulin may be a specific marker of DKD.
Recent study has shown that some patients with diabetes have advanced renal pathological changes and progressive kidney function decline even if urinary albumin levels are in the normal range, indicating that albuminuria is not the perfect marker for the early detection of DKD [
This study has limitations such as small number of subjects, wide distributions for age, no GFR, different duration of diabetes, and effect of drugs. Further improvements are needed in our future study. However, all these findings indicate that urinary microvesicle-bound uromodulin is a specific marker for DKD and potentially may be used to predict the onset and/or monitor the progression of DKD.
All the authors declare that there are no competing interests.
Funding for this study was provided by the Research Grant from the National Science Foundation for Yong Scholars of China (81500554), the Science and Technology Development Plan Project of Shandong Province (2015GSF118084), and the Youth Found of The Second Hospital of Shandong University (Y2014010005).