Characterization of diabetic nephropathy by urinary proteomic analysis: Identification of a processed ubiquitin form as a differentially excreted protein in diabetic nephropathy patients

Background: Identification of markers for prediction of the clinical course of diabetic nephropathy remains a major challenge in disease management. We established a proteomics approach for identification of diabetic nephropathy-related biomarkers in urine. Methods: We used SELDI-TOF mass spectrometry and SAX2 protein arrays to compare protein profiles from urine of 4 defined patient groups. Samples from patients with type 2 diabetes (DM; n = 45) without nephropathy and without microalbuminuria (DM-WNP), patients with DM with macro- or microalbuminuria (DM-NP; n = 38), patients with proteinuria due to nondiabetic renal disease (n = 34), and healthy controls (n = 45) were analyzed. Anionic exchange, reversed-phase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. Results: A protein with m/z 6188 (P < 0.0000004) was strongly released in the urine of healthy controls, patients with proteinuria due to nondiabetic disease, and DM-WNP in contrast to DM-NP patients. An m/z 14 766 protein (P < 0.00008) was selectively excreted in the urine of DM-NP patients, whereas the protein with m/z 11 774 (P < 0.000004) was significantly excreted by patients with proteinuria and DM-NP. The m/z 11 774 and m/z 14 766 mass peaks were identified as γ₂-microglobulin and UbA52, a ubiquitin ribosomal fusion protein, respectively. The protein with m/z 6188 was identified as a processed form of ubiquitin. Conclusion: The release of high amounts of UbA52 in urine of DM-NP patients could serve as a diagnostic marker, whereas the lack of the short form of ubiquitin raises interesting questions about the pathophysiology.