Urinary Proteomic Research To Identify Novel Biomarkers For Early Diagnosis Of Type 2 Diabetic Nephropathy

Diabetic nephropathy (DN) as a common and severe chronic complication of type 2 diabetes mellitus has rapidly become an important public health problem. In the western world, DN is now the single most common cause of ESRD, account for approximately 44% of all ESRD cases. Therefore, early detection of the risk of DN before advanced renal damage has occurred is an obviously important goal, this goal is made difficult by the fact that much of the important diabetic renal structural injury can occur in absolute clinical silence. Although analysis of kidney tissue may provide clues to determine which patients may progress to DN, current clinical practice does not allow for routine kidney biopsies because the procedure is invasive.Urine can provides much information of kidney disease, and has been defined as a fluid biopsy of the kidney, so the research on urine has received much concern. At present, microalbuminuria is considered as the best noninvasive available marker for DN risk, but recent studies have proved it has inadequate specificity and sensitivity. Because many factors affect its accuracy, for example, when the inflammation and bleeding of urinary system happened, plasma albumin will leak out to the urine and lead to the increasing of microalbuminuria. Besides, when microalbuminuria occurs, DN has developed to the stage of III. Thus, only monitor microalbuminuria can't realize early diagnosis of DN, looking for a new diagnostic method is needed.Recently, with the development of proteomics technology, the urinary proteomes has been demonstrated to identify novel proteins, or protein patterns, that may serve as biomarkers for DN. In the present study, we used urinary proteomic approach of two-dimensional gel electrophoresis (2-DE) and two dimensional fluorescence difference gel electrophoresis (2D-DIGE) to identify differentially expressed proteins in urine samples, which were from type 2 diabetes patients with normoalbuminuria (DM group), microalbuminuria (DN1 group), macroalbuminuria (DN2 group) and healthy control group. Then, the differentially expressed proteins were identified by MALDI-TOF mass spectra and analyzed by bioinformatics. At last, we performed further studies on partly identified proteins to evaluate their early diagnostic value of DN. The research contents were divided into five parts as below.Part I : Analysis of 2-DE for the differentially expressed proteins in type 2 diabetic nephropathyObjective To establish the two-dimensional gel electrophoresis (2-DE) profiles and explore the differentially expressed proteins among type 2 diabetes patients with normoalbuminuria (DM), microalbuminuria (early DN), macroalbuminuria (overt DN) and healthy controls.Methods Urine samples were collected from type 2 diabetes patients with normoalbuminuria (UAER<30mg/24h, DM group), microalbuminuria (30300mg/24h, DN2 group) and control group (n=8 in each group). The urinary protein of all urine samples was isolated by cold acetone precipitation after dialysis. The rehydrated IPG strips containing protein samples were isoelectric focusing (IEF), after IEF, the equilibrated strips are transferred to the vertical SDS gel. After silver stained, the gel images were analyzed using the software PDQuest, then the differentially expressed protein spots were found.Results Using the proper method stated above, satisfactory 2-DE maps of urinary protein was obtained and the preliminary analysis results were reported. After analysis by the PDQuest software, 217±42, 178±36, 135±31 and 98±27 (mean±SD) protein spots were visualized on each gel of DN2, DN1, DM and control group, respectively. By quantitative and statistical analysis among the proteomic maps from four groups, a total of 16 protein spots were markedly differentially expressed in DN group (abundance ratio>2, p<0.01). Among these 16 protein spots, 9 were up-regulated and 7 were down-regulated in DN group.Conclusion Urinary proteomics method of 2-DE is feasible to be applied in the study of type 2 diabetes patients with nephropathy and without nephropathy. A few differentially expressed protein spots in type 2 diabetes patients with nephropathy can be found by 2-DE method.Part II: Analysis of 2D-DIGE for the differentially expressed proteins in type 2 diabetic nephropathyObjective To overcome the disadvantage of poor repeatability by ordinary 2-DE, we used another proteomic method, the two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), to explore the differentially expressed proteins among type 2 diabetes patients with normoalbuminuria(DM), microalbuminuria (early DN), macroalbuminuria (overt DN) and healthy controls.Methods Urine samples were collected from type 2 diabetes patients with normoalbuminuria (DM group), microalbuminuria (DN1 group), macroalbuminuria (DN2 group) and healthy control group (n=8 in each group, same with part I). The urine samples were mixed equally in each group, and then the urinary protein was isolated by cold acetone precipitation after dialysis. The prepared urine protein samples from four groups were labeled with Cy3 or Cy5 fluorescent dyes, and the internal standard (a pool of equal amounts from all samples) was labeled with Cy2. Then, isoelectric focusing (IEF) was performed in an Ettan IPGphor II apparatus with 24cm immobilized pH gradient (IPG) strips. After IEF, SDS-PAGE was run using an Ettan DALT twelve system. After scanning using Typhoon scanner, the gel images were analyzed statistically by DeCyder software to explore the differentially expressed protein spots in DN group. Results We obtained the satisfactory 2-D DIGE maps of urinary protein from DN2, DN1, DM and healthy control groups. The different color scanning images can be seen after scanned for Cy2, Cy3 and Cy5 using different wavelength of laser 488 nm, 532nm and 633nm. After the analysis by the DeCyder software, more than 400 protein spots were visualized on each gel scanning image. By quantitative comparison with internal standard, a total of 21 protein spots were markedly differentially expressed in DN1 and DN2 groups (abundance ratio>2, p<0.01). Among these 21 protein spots, 13 were up-regulated and 8 were down-regulated in DN group.Conclusion Urinary quantitative proteomics method of 2D-DIGE is feasible to be applied in the study of type 2 diabetes nephropathy. 2D-DIGE is more accurate and convenient than ordinary 2-DE method, and more differential protein spots can be obtained using 2D-DIGE method.Part III: Mass spectrometry identification and basic bioinformatics analysis of the differentially expressed proteinsObjective To identify the differential expressed proteins based on mass spectrometer and bioinformatics analysis from 16 differential expressed protein spots selected by 2-DE technique and 21 protein spots selected by 2D-DIGE technique.Methods A total of 37 differential expressed protein spots were cut out and subjected to enzymatic digestion with trypsin. Subsequent protein identification was carried out using matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS) on a reflective setting. The mass spectrometry data was then searched in comparison with the human subset of the Swiss-Prot/TrEMBL protein database using GPS explorer software with a MASCOT search engine.Results After mass spectrometry identification and bioinformatics analysis, 8 proteins were identified from 16 differential expressed protein spots selected by 2-DE technique. Among them, 6 proteins were significantly up-regulated in DN groups, which were epithelial-cadherin, serum albumin, zinc-alpha-2 glycoprotein, orosomucoid, retinol-binding protein and kininogen. Another 2 proteins were significantly down-regulated in DN groups, which were uromodulin and transthyretin. Besides, 10 proteins were identified from 21 differential expressed protein spots selected by 2D-DIGE technique, including 7 proteins significantly up-regulated and 3 proteins down-regulated in DN group. The up-regulated protein were epithelial-cadherin, serum albumin, zinc-alpha-2-glycoprotein, orosomucoid, retinol-binding protein,prostaglandin-H2D-isomerase and Ig kappa chain C region. The down-regulated protein were AMBP protein, uromodulin and haptoglobin. It is noteworthy that there were 6 proteins identified by both 2-DE and 2D-DIGE technique, which were epithelial-cadherin, serum albumin, zinc-alpha-2 glycoprotein, orosomucoid, retinol-binding protein and uromodulin.Conclusion The protein biomarkers associated with type 2 diabetic nephropathy could be identified by MALDI-TOF mass spectrometry and bioinformatics analysis. The research on differential proteins identification is helpful to the early diagnosis and pathogenic mechanism study of DN.Part IV: The study on the relationship between E-cadherin and type 2 diabetic nephropathyObjective To analyze the concentration changes of urinary E-cadherin in type 2 diabetic patients with nephropathy and without nephropathy, and evaluate its diagnostic value for diabetic nephropathy; To detect the expression of E-cadherin in renal tissue of type 2 diabetic nephropathy patients, and analyze its role in pathogenesis of diabetic nephropathy.Methods Western blot method was performed to verify the expression of E-cadherin in urine samples, which were from type 2 diabetes patients with normoalbuminuria (DM group), microalbuminuria (DN1 group), macroal- buminuria (DN2 group) and control group (n=6 in each group). Meanwhile, the concentration of urinary E-cadherin was measured by ELISA detection in urine samples from DM, DN1, DN2 and control group (n=40 in each group). From this data, the sensitivity and specificity of urinary sE-cadherin for diagnosis of DN were calculated by formula. Besides, we performed immunohistochemical staining to analyze the expression of E-cadherin in human renal biopsies from type 2 diabetic nephropathy patients (n=5) and normal renal tissue (n=4).Results The results of western blot demonstrated E-cadherin did not be detected in control group, but the soluble 80kDa fragment of E-cadherin (sE-cadherin) was detected in DM, DN1 and DN2 groups. Meanwhile, we found sE-cadherin was weakly expressed in DM group, but significantly expressed in DN1 and DN2 groups, especially in DN2 group. The ELISA data also demonstrated urinary sE-cadherin/Cr was markedly increased in DN1 and DN2 groups versus DM or control group (2751.5±164 and 5839.6±428 vs. 721.9±93 or 652.7±87 ug/g; p<0.001), and markedly elevated in DN2 group versus DN1 group (5839.6±428 vs. 2751.5±164 ug/g; p<0.01). But no significant difference of urinary sE-cadherin/Cr was found between DM group and control group (721.9±93 vs. 652.7±87 ug/g; p>0.05). The sensitivity and specificity of urinary sE-cadherin for diagnosis of DN were calculated as 78.8% (95%CI, 74-83%) and 80% (95%CI, 65-91%). Pearson correlation and stepwise regression analysis indicated urinary sE-cadherin/Cr was high positive correlation with UAER and serum creatinine (r=0.861, r=0.713; p<0.01), and serum creatinine was the only independent influential factor of urinary sE-cadherin/Cr (R~2=0.831, F=56.72; p<0.01). Besides, immunohistochemical stain showed E-cadherin was mainly expressed in membrane and cytoplasm of renal tubular epithelial cells, and its expression was markedly decreased in DN kidneys versus normal kidneys.Conclusion Urinary sE-cadherin has a potential clinical diagnostic value for DN, which is elevated in DN patients, and gradually increased with the development of nephropathy. Besides, E-cadherin may participate in the pathogenesis of DN, whose expression was markedly decreased in kidneys of DN.Part V: The study on the relationship between urinary orosomucoid and type 2 diabetic nephropathyObjective To detect the concentration changes of urinary orosomucoid in type 2 diabetes patients with normoalbuminuria, microalbuminuria, macroalbuminuria and healthy control group; To analyze the relationship between urinary orosomucoid and type 2 diabetic nephropathy, and evaluate its diagnostic value for diabetic nephropathy.Methods The urinary level of urinary orosomucoid was detected by immunoturbidimetry assay in urine samples, which were from 160 type 2 diabetes patients with normoalbuminuria (DM group), microalbuminuria (DN1 group), macroalbuminuria (DN2 group) and healthy control group (n=40 in each group, the same with part IV). Then, we calculated the urinary orosomucoid excretion rate (UOER), to analyze the changes of UOER in different stages of DN. Besides, Pearson correlation analysis and multivariate logistic regression analysis were performed to evaluate the influencing factor of UOER. Meanwhile, the sensitivity and specificity of UOER for diagnosis of DN were calculated.Results The data of immunoturbidimetry assay showed UOER was gradually increased in normo-, micro- and macroalbuminuria group versus control group (0.91±0.37, 1.87±0.72, 2.95±0.84 vs. 0.42±0.19 ug/min, P<0.05). The result indicated UOER increased in early stage of DN and gradually increased with the development of DN. Besides, Pearson correlation analysis indicated UOER was positively correlated with UAER, serum creatinine and CRP (r=0.781, 0.695, 0.401, respectively; P<0.05). And multivariate logistic regression analysis showed increased UOER was an independent risk factor for DN (OR=2.88, P<0.01). After the calculation by formula, the sensitivity and specificity of UOER for diagnosis of DN were 75.8% (95%CI, 71-82%) and 69% (95%CI, 58-83%).Conclusion UOER is increased in early stage of type 2 diabetic nephropathy, and gradually increased with the development of nephropathy. Besides, increased UOER is an independent risk factor of DN, and it also has potential diagnostic value for DN.