Association Of Urinary Exosome And Clinically Worsened Diabetic Kidney Disease

BackgroundDiabetic kidney disease (DKD) is one of the major microvascular complications of diabetes and the most common cause of end stage of renal diseases (ESRD). The clinical stages of diabetic nephropathy are classified as follows:stage1, normoalbuminuria (urinary albumin/creatinine ratio (ACR<30mg/g creatinine); stage2, microalbuminuria (ACR from30-300mg/g creatinine); stage3, macroalbuminuria (ACR≧300mg/g creatinine and/or persistent proteinuria with serum concentration of creatinine<1.2mg/dl); stage4, chronic renal failure (serum concentration of creatinine≧1.2mg/dl with proteinuria); stage5, under chronic dialysis therapy.Previously, DKD was thought to be a unidirectional process that started with microalbuminuria and lead to end-stage renal failure. However, recent studies have shown that a large proportion of patients diagnosed with DKD may reverse back to normoalbuminuria. Therefore, the more sensitive and reliable markers for early detection of DKD are needed.Urine is one of the most useful resources for such a study and its collection is simple and non-invasive. In addition to soluble plasma proteins, urinary microvesicles, such as exosomes and microparticles, have recently been the targets of urine proteomic analysis. Urinary exosomes are membrane vesicles secreted by tubular cells with a diameter of40-100nm, while microparticles are membrane-shed vesicles with a size range between100and1000nm. Both the exosomes and microparticles are characterized by the same lipid bilayer, but the majority of microvesicles isolated from urine are thought to be exosomes. A previous report has shown that aquaporin-2was only identified in urinary exosomes and not in exosomes from other sources, thereby revealing a link between these exosomes and their urogenital tract origin. These exosomes may carry novel biomarkers for renal dysfunction and structural injury.Previous reports have indicated that a high level of plasma dipeptidyl peptidase-4(DPP4), also known as CD26, is positively correlated with Diabetes Mellitus (DM). DPP4degrades active glucagon-like peptide-1(GLP-1), an incretin released from L cells in the intestine after meal intake that enhances insulin secretion in a glucose-dependent manner. DPP4inhibitor showed blood glucose-lowering effects in both animal models of diabetes and patients with type2diabetes. Currently, the DPP4inhibitors such as sitagliptin, vidagliptin have been widely used as an adjunct to diet and exercise to improve glycemic control in adults with type2diabetes. DPP4is a membrane-associated peptidase and is widely expressed in all tissues. In the kidney, where the enzyme is exceptionally concentrated, it is located primarily in the cortex and found in the brush-border and microvillus fractions. On the basis of these findings, we hypothesized that DPP4might be a protein component of urinary exosome, and its activity might represent the concentration of exosome, secreted by tubular epithelial cells in the urine. To test this hypothesis, we have developed an ELISA method for DPP4on urinary exosome using a specific monoclonal antibody, AD-1.After it is immobilized on the plate, the antibody can specifically capture and purify the intact exosome from urine samples, and the DPP4activity in the exosome can be determined. With this assay, urinary exosome-bound DPP4activity in patients with type2diabetes mellitus was tested and compared with age-and sex-matched normal individuals. We hypothesized that the variable hydration state of individuals providing urine specimens may lead to some differences in water/salt content of urine, so we pooled all24-hour urine samples from an individual, then used a portion of that for our analysis. The applicability of exosome-bound DPP4as an early biomarker for determining the status of DKD was evaluated.ObjectivesThe present study was designed to identify the changes in exosome-DPP4levels in human urine and serum, and to determine whether there were correlations with the severity of DKD.Methods127patients with type2diabetes mellitus (T2DM) were divided into three groups according to the urinary albumin/creatinine ratio (UACR):microalbuminuria group (n=50); macroalbuminuria group (n=34) and normoalbuminuria group (n=43).34age-and sex-matched non-diabetic healthy subjects were selected as controls. Exosome-bound DPP4and free urinary DPP4were separated by a filtra-centrifugation method. The total exosome was captured by a specific monoclonal antibody, AD-1. DPP4activity was determined by measuring the cleavage of chromogenic free4-nitroaniline from Gly-Pro-p-nitroanilide at405nm with an ELISA plate reader. DPP4protein levels were determined by ELISA and Western blot. Simultaneous detections of glycated hemoglobin (liquid chromatography), blood cholesterol (chemical modification), creatinine (picric acid method), blood urea nitrogen (rate method) were used. Determination of urinary albumin was finished by Quickread101analyzer of Finland,and the results using the UACR report. Multivariate stepwise regression method was for statistical analysis.Results1. The exosomes exsisted in urine and serum,and its structure was lipid bilayer membrane.2. Comparison of the urinary and serum immuno-precipitates in the blot revealed that DPP4protein from human urine was consistent with its serum origin. Results showed that the exosome-bound type was the major form of DPP4in urine.3. The urinary exosome-DPP4of T2DM group was significantly higher compared to the controls. The urinary exosome-DPP4was positively correlated with UACR in T2DM patients. These findings suggested that the urinary level of exosome-bound DPP4was associated with the severity of DKD.ConclusionsThese findings indicate that urinary exosome-bound DPP4is a specific marker for DKD. The measurement of urinary exosome-bound DPP4may be a useful method for the screening and diagnosis of DKD in T2DM patients. BackgroundNot only type1diabetes is considerd as an autoimmune disease, the incidence of type2diabetes is closely related to immune system. Type2diabetes is on the genetic basis by a number of factors contributed to the patients with immune abnormalities.The patients with positive islet cell antibodies (ICA) have more severe P-cell damage than that with ICA negative.Diabetic kidney disease(DKD) is the most common chronic complication, but its pathogenesis is unknown.The early diagnosis and treatment of clinical work have become an important issue. T lymphocytes especially the helper T lymphocytes (Th) play an important role in the regulation of inflammatory responses. Interferon-gamma (IFN-y) direct toxic effects may be mediated by beta cells,and it stimulates macrophages and lymphocytes to destroy pancreatic β cells.Interleukin-4(IL-4) produced by T helper2cell (Th2) mediates humoral immunity, and regulates the production of antibody.The ratio of IFN-γ/IL-4is on behalf of T helper1cell (Th1) and Th2levels.The previous study has suggested that the activation of Th subsets and the imbalance of Th1/Th2involved in atherosclerosis, bronchial asthma and other inflammatory-related diseases in the pathological process, however,,it is not fully understood in the pathogenesis of type2diabetes.ObjectiveTo investigate the imbalance of urinary exosomal Th1/Th2and its correlation with diabetic kidney disease.MethodThirty healthy volunteers and120patients with type2diabetes mellitus(T2DM) were included. The healthy volunteers served as control. Urinary exosome-IFN-y and exosome-IL-4levels were determined by Enzyme-linked immunosorbent assay(ELISA).Multiple stepwise linear regression was used to analyze the relationship of exosome-Th1/Th2with glycated hemoglobin (HbA1c), cholesterol (CH), urinary albumin/creatinine ratio (UACR), creatinine (CR) and urea nitrogen (BUN).ResultsCorrelation analysis showed that urinary exosome-Th1/Th2was positively correlated with UACR (P=0.015) and BUN(P=0.001). Multiple stepwise linear regression analysis showed that BUN was the independent determinant for exosome-Th1/Th2(P=0.006).ConclusionsUrinary exosome-Th1/Th2correlates with early diagnosis evaluation of diabetic kidney disease. BackgroundChronic kidney disease has become an important global public health problem. It is reported that about1in every10people was suffered from kidney disease, and each year more than100,000people have died from cardiovascular disease associated with chronic kidney disease according to the announcement of the International Society of Nephrology on February20,2007. Chronic renal insufficiency caused by chronic kidney disease is a kind of common and frequently-occurring disease. The clinical symptom of chronic renal insufficiency may be hidden, the patients can have no symptom or obvious symptoms.The compensatory function of the kidney is extremely powerful, even when the kidney function has already lost more than50%,the patients may still have not any symptoms.Diabetic kidney disease, hypertension kidney disease, drug-induced nephropathy,1gA nephropathy and lupus nephritis are common chronic kidney diseases.If not treated,it will develop into chronic renal failure (CRF) and uremia. In addition to blood purification, peritoneal dialysis and kidney transplantation, there is no ideal treatment for the renal failure and uremia patients.ALP is widely distributed in the material exchange active cell membrane of human tissues, such as intestinal epithelium, liver and bile capillary membrane, and the arterioles and capillaries artery department of endothelium, et al.When cells were subjected to injury, degeneration and necrosis, intracellular enzymes may go into the urine probably due to cells damaged or organelle function changes inducing the obstacles of the synthesis or the decreased tubular cells reabsorption. Leucine aminopeptidase (LAP) is a kind of protease widespread in human tissues especially in intrahepatic. Unlike other liver enzymes, LAP can be detected not only in blood samples, but also in urine. LAP increased generally in acute nephritis, tubular injury, renal failure and toxic kidney damage.Urinary LAP activity changes in response to the injury of the proximal tubule. Glutamyl endopeptidase mainly distributed in kidney, brain, prostate and liver tissue,highest in the kidney.In the study, urinary exosome binding enzymes and free enzymes were detected in patients with chronic kidney disease. We look forward to finding specific markers associated with chronic kidney disease, so we can reach early prevention of end-stage kidney failure.ObjectiveTo explore the urinary exosome binding alkaline phosphatase (EXO-ALP), gamma-glutamylacyl transpeptidase (EXO-γ-GT) and leucine aminopeptidase (EXO-LAP) levels and free type of the above three enzymes in patients with stage Ⅳ chronic kidney disease.MethodsTen patients with stage Ⅳ type2diabetic kidney disease, ten cases of stage Ⅳ lupus nephritis, and ten cases of stage Ⅳ analgesic nephropathy were included. The14healthy volunteers served as control. Specific monoclonal antibody (AD-1) purified urinary exosome. The alkaline phosphatase was as the marker to identify the specificity of the antibody against exosome. Alkaline phosphatase isoenzyme separated by agarose gel method. The levels of EXO-ALP, EXO-γ-GT, EXO-LAP and free types of ALP, γ-GT and LAP in urine were measured by immunochromatography method.Results1AD-1antibody reacted with the free type of ALP and EXO-ALP respectively,and only the EXO-ALP can bind with the antibody. 2The reactants that EXO-ALP reacted with AD-1was done with agarose gel electrophoresis.The result showed that after the EXO-ALP binding with the antibody, the electrophoretic migration rate decreased.3Immunochromatographic result showed that urinary EXO-ALP, EXO-y-GT, EXO-LAP are higher than the free type of ALP, LAP and y-GT in normal people. Urinary EXO-ALP, EXO-y-GT, EXO-LAP and urinary free y-GT levels decreased,and the free type of ALP, LAP levels increased inversely in the patients with stage IV chronic kidney disease.ConclusionThe urinary exosome-bound enzymes in stage IV chronic kidney disease decreased, and the mechanism needs further study.