Isocitrate dehydrogenase 1 upregulation in urinary extracellular vesicles from proximal tubules of type 2 diabetic rats.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. Microalbuminuria is currently the most common non-invasive biomarker for the early diagnosis of DN. However, renal structural damage may have advanced when albuminuria is detected. In this study, we sought biomarkers for early DN diagnosis through proteomic analysis of urinary extracellular vesicles (uEVs) from type 2 diabetic model rats and normal controls. Isocitrate dehydrogenase 1 (IDH1) was significantly increased in uEVs from diabetic model rats at the early stage despite minimal differences in albuminuria between the groups. Calorie restriction significantly suppressed the increase in IDH1 in uEVs and 24-hour urinary albumin excretion, suggesting that the increase in IDH1 in uEVs was associated with the progression of DN. Additionally, we investigated the origin of IDH1-containing uEVs based on their surface sugar chains. Lectin affinity enrichment and immunohistochemical staining showed that IDH1-containing uEVs were derived from proximal tubules. These findings suggest that the increase in IDH1 in uEVs reflects pathophysiological alterations in the proximal tubules and that IDH1 in uEVs may serve as a potential biomarker of DN in the proximal tubules.

Rapid Isolation of Extracellular Vesicles Using a Hydrophilic Porous Silica Gel-Based Size-Exclusion Chromatography Column.

Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types and can be found in biological fluids, such as blood and urine. EVs play an important role in various physiological and pathological processes via cell-cell communication, highlighting their potential applications as diagnostic markers for diseases and therapeutic drug delivery carriers. Although various methods have been developed for the isolation of EVs from biological fluids, most of them exhibit major limitations, including low purity, long processing times, and high cost. In this study, we developed a size-exclusion chromatography (SEC) column device using hydrophilic porous silica gel (PSG). Owing to the resistance to pressure of the device, a rapid system for EV isolation was developed by connecting it to a flash liquid chromatography system furnished with a UV detector and a fraction collector. This system can be used for the real-time monitoring of eluted EVs by UV absorption without further analysis and separation of high-purity EVs from urine samples with high durability, reusability, and reproducibility. In addition, there were no significant differences between the PSG column- and conventional SEC column-isolated EVs in the proteome profiles and cellular uptake activities, suggesting the good quality of the EVs isolated by the PSG column. These findings suggest that the PSG column device offers an effective and rapid method for the isolation of intact EVs from biological fluids.