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Diabetic nephropathy is a devastating disease that affects a growing number of diabetic patients. A complete cure is very hard to achieve once the disease has been diagnosed, therefore the diagnosis of early stages in diabetic nephropathy has become a hot area. Numbers of molecules have been proposed to be potential biomarkers for this purpose. However, some problems still remain, such as discovering effective biomarkers to diagnose the disease before obvious clinical evidence appears. Thus, the main purpose of this study was to find plasma biomarkers for early diagnosis of type 2 diabetic nephropathy stage 1 and stage 2, as well as separating them from diabetes. 182 subjects (Chinese) were recruited for this study, including 50 healthy controls, 33 type 2 diabetic patients and 99 type 2 diabetic nephropathy patients (33 of these were stage 3). Important clinical indicators including proteinuria, serum creatinine, and urea nitrogen were measured and the glomerular filtration rate was estimated to assess kidney function; fasting blood glucose, postprandial blood glucose and glycated hemoglobin were measured to assess the blood glucose control. Key metabolites and genes in plasma samples were identified and determined using -omic and quantitative techniques. The potential biomarkers were then combined and carefully screened to determine the most informative ones for early diagnosis of type 2 diabetic nephropathy. An integrated biomarker system (IBS) incorporating 6 clinical indicators, 40 metabolites and 5 genes was established. Correlation analysis results revealed that most of the potential biomarkers significantly correlated with the 6 clinical indicators. Discriminant analysis results showed that the developed IBS gave the highest total predictive accuracy (98.9%). Significant test and receiver operating characteristic analysis results indicated that inosine had the highest sensitivity (0.889), specificity (1.000), positive predictive rate (1.000) and negative predictive rate (0.900) amongst the 48 potential biomarkers when separating patients with diabetes from patients with diabetic nephropathy stage 3. Finally, inosine with a cutoff of 0.086 mg L(-1) was combined with estimated GFR to differentiate between diabetic nephropathy stages 1 and 2 from diabetes. The results demonstrate that IBS combined with a proper statistical analysis technique is a powerful tool for biomarker screening.

It was reported that Hcy was related to the development of kidney disease, but it remains unknown whether Hcy is an independent biomarker for diabetic nephropathy. Analytical method for simultaneous determination of aminothiols among the Hcy metabolic cycle is desirable to discover other potential biomarkers. A high-performance liquid chromatography-electrospray tandem mass spectrometric (HPLC-ESI-MS/MS) method was established for simultaneous quantitation of Cysteine (Cys), total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), cystathionine (Cysta), methionine (Met), glutathione (GSH) and cysteinylglycine (Cys-gly) in plasma with N-(2-mercaptopropionyl)-glycine (MPG) as internal standard. The method had simple pretreatment without derivatization and the chromatograms show better separation of the eight aminothiols and the analytic time was 20 min. The results demonstrated that it provided an excellent linearity for all analytes over their respective concentration ranges and illustrated excellent precision and plasma recovery as well. Then, the method was applied in the case-control study of patients with diabetes mellitus (DM) and diabetic nephropathy (DN). In conclusion, it is an effective method to quantitate the concentrations of aminothiols in the human plasma. SAH and SAM were suggested as better potential biomarkers of DM and DN.