Excretion Patterns of Urinary Extracellular Vesicles in Proteinuric and Nonproteinuric Diabetic Kidney Disease over the Course of a Day

Background: Over 540 million adults have diabetes, and 20% will develop diabetic kidney disease (DKD), the leading cause of end stage renal disease. Existing clinical markers for diabetic nephropathy lack early and specific diagnostic ability; however, urine can be used as a noninvasive biopsy. Urinary extracellular vesicles (uEVs) are explored as early, noninvasive biomarkers of kidney disease by identifying specific sites of organ damage. We wanted to understand the excretion patterns of uEV over the course of a day to optimize urinary biomarker detection in DKD. Methods: Ten patients (6M, 4F) with Stage 3 DKD (GFR 30-59 mL/min) collected every urine void separately over 24 hours. Five patients had microalbuminuria (albumin=20-200 mg/24 hours), four had subnephrotic proteinuria (protein>500 mg/24 hours), and one had nephrotic range proteinuria (protein>3500 mg/24 hours). uEVs were isolated using differential centrifugation with low ionic strength buffer to reduce uromodulin. Specific gravity and pH were assessed through a urine dipstick (Siemens Multistix 10SG). Protein and creatinine content were analyzed through a Bradford and Jaffe reaction assay. uEV size and concentration were determined by Nanoparticle Tracking Analysis (PMX-120, Zetaview). uEV excretion patterns were analyzed using GraphPad Prism with a Friedman Test and linear mix modeling for correlations. Results: 84 urine voids were processed into separate uEV enriched pellets. The average for participant age was 74 +/- 5.7 years, serum creatinine was 1.8 +/- 0.3 mg/dL, and eGFR was 37 +/- 9.0 mL/min/1.73 m2. Across all voids, the protein/creatinine ratio ranged from 2.653 to 6521 mg/g, and uEV concentration varied over the day by 100-fold from 3.6x107 to 2.2x109 particles/mL urine. uEV concentration positively correlated with urine creatinine concentration and specific gravity. There was a statistically significant difference between micro- and macroalbuminuric patients’ uEV concentration. Conclusion: Study of uEV excretion over the course of a day is highly variable, reflecting the dynamic nature of urine components. Our data shows a strong correlation of uEV counts with urinary creatinine concentration and diabetic kidney disease stages, thus advancing strategies for uEV biomarker development in DKD. Funding: Clinical Revenue Support