The discovery of biomarkers for type 2 diabetic nephropathy by serum proteome analysis

Abstract

Diabetic nephropathy (DN) is a serious kidney complication of diabetes, and constitutes the leading cause of end-stage renal disease. The earliest clinical evidence of DN is microalbuminuria, a term which refers to the appearance of small but abnormal amounts of albumin in the urine. However, screening methods for DN, such as biomarker assays, are yet to be developed for type 2 DN. In the present study, in an attempt to identify the biomarkers for initial diagnoses of type 2 DN, the protein profiles of human sera collected from 30 microalbuminuric type 2 diabetic patients were compared with those collected from 30 normoalbuminuric type 2 diabetic patients, via 2-DE. As a result, a total of 18 spots were determined to have different protein levels in the microalbuminuric patients. Twelve spots had lower protein levels of approximately 50%, and the other six had higher levels of approximately 100-300% as compared to the spots of normoalbuminuric patients. These spots were identified with ESI-Q-TOF (ESI-quadrupole-TOF) MS. Among the identified proteins, vitamin D-binding protein (DBP) and pigment epithelium-derived factor (PEDF) were verified by Western blotting. The results of this study indicate that the DBP may be employed as diagnostic and monitoring biomarkers of type 2 DN, contingent on further study into the matter.

Proteomic analysis of glomeruli from streptozotocin-induced diabetic rats

“…The gel pieces were vortexed for 3 min in each solution. The gel pieces were incubated in digestion buffer containing 50 mM ammonium bicarbonate, 30% DMF solution, and 12.5 ng/µL trypsin at 37°C for 12 14 h. The peptides were recovered by two extractions with a solution containing 50% ACN and 5% TFA. The resultant peptide extracts were pooled, lyophilized in a vacuum centrifuge, and stored at 4°C for subsequent nano-LC-ESI-MS/MS experiments.…”

“…The samples were subsequently centrifuged at 19,326 × g for 15 min at 4°C to remove the cell debris pellet; the supernatants were transferred to new tubes for further analysis. Next, TCA/acetone precipitation of sample proteins was performed to remove salts as described previously with some modifications [12,13]. Four times the sample volume of TCA/acetone solution (10% TCA, 10% acetone, and 20 mM DTT) was added to the sample tubes.…”

Ultraviolet and tandem mass spectrometry for simultaneous quantification of 21 pivotal metabolites in plasma from patients with diabetic nephropathy

“…Thus, DN enormously influences the public health. Kim et al applied proteomics to DN [2,3]. They found out several protein biomarkers of DN, which can be linked to clinical applications [4].…”

Micro‐high‐performance liquid chromatography platform for the depletion of high‐abundance proteins and subsequent on‐line concentration/capturing of medium and low‐abundance proteins from serum. Application to profiling of protein expression in healthy and osteoarthritis sera by 2‐D gel electrophoresis

“…Protein spots with ratios of more than 1.5 were considered to be less expressed in OA sera and spots with ratios of less than 0.667 were considered to be more expressed in OA sera [21]. For inter-class differential analysis of OA treated (n = 3) vs. OA untreated (n = 3) Student's t-tests were performed for each match at a = 0.05 and spots having a significantly different %vol [22] were reported.…”