Recent reports showed early progressive renal function decline precedes the onset of microalbuminuria, indicating the need for new factors predicting diabetic nephropathy (DN) that work alone or as a complement to urinary albumin and eGFR. To identify urinary proteins associated with DN, we carried out a 2D-DIGE-based proteomic analysis using the urine obtained from healthy control subjects (H; n=8), type 2 diabetic patients without nephropathy (T2DM; n=16), and stage II DN patients (DN2; n=16). From the total 3034 protein spots detected, a total of 34 proteins were identified as the candidate protein differentially excreted in DN2 or T2DM. Thirty proteins out of 34 candidates were then validated by multiple reaction monitoring assay using an independent sample set (H: n=14, T2DM: n=13, DN2: n=14, stages III to IV DN: n=17), where 26 proteins were validated to be differentially excreted in DN. Among them, the levels of deoxyribonuclease-1 and lysosomal alpha-glucosidase were significantly correlated with both ACR and eGFR, and were also significantly lower in patients with an eGFR < 60 compared with patients with an eGFR ≥ 60. We further analyzed gene expressions of DNase I and lysosomal alpha glucosidase in kidneys of DN patients using Nephroseq database (https://www.nephroseq.org), and found that DNase I gene was significantly downregulated in DN tubulointerstitum and glomeruli, and that lysosomal alpha glucosidase gene tend to decreased in DN tubulointerstitum. Finally, the urinary DNase I levels were found to be higher in patients who progressed to a more severe DN stage or had early renal decline than in patients who did not. These findings suggest a possible role for the identified proteins in the progression of DN, although further validation studies are required.
Y. Kaburagi: None. E. Ito: None. H. Unoki-Kubota: None.
Japan Society for the Promotion of Science