Specific renal parenchymal-derived urinary extracellular vesicles identify age-associated structural changes in living donor kidneys

Abstract

Non-invasive tests to identify age and early disease-associated pathology within the kidney are needed. Specific populations of urinary extracellular vesicles (EVs) could potentially be used for such a diagnostic test. Random urine samples were obtained from age- and sex-stratified living kidney donors before kidney donation. A biopsy of the donor kidney was obtained at the time of transplantation to identify nephron hypertrophy (larger glomerular volume, cortex per glomerulus and mean profile tubular area) and nephrosclerosis (% fibrosis, % glomerulosclerosis and arteriosclerosis). Renal parenchymal-derived EVs in cell-free urine were quantified by digital flow cytometry. The relationship between these EV populations and structural pathology on the kidney biopsy was assessed. Clinical characteristics of the kidney donors (n=138, age range: 20-70 years, 50% women) were within the normative range. Overall, urine from women contained more EVs than that from men. The number of exosomes, juxtaglomerular cells and podocyte marker-positive EVs decreased (p<0.05) with increasing age. There were fewer total EVs as well as EVs positive for mesangial cell, parietal cell, descending limb of Henle's loop (simple squamous epithelium), collecting tubule-intercalated cell and monocyte chemoattractant protein-1 markers (p<0.05) in persons with nephron hypertrophy. The number of EVs positive for intercellular adhesion molecule-1, juxtaglomerular cell, podocyte, parietal cell, proximal tubular epithelial cell, distal tubular epithelial cell and collecting duct cells were fewer (p<0.05) in persons with nephrosclerosis. EVs carrying markers of cells from the renal pelvis epithelium did not associate with any indices of nephron hypertrophy or nephrosclerosis. Therefore, specific populations of EVs derived from cells of the glomerulus and nephron associate with underlying kidney structural changes. Further validation of these findings in other cohorts is needed to determine their clinical utility.

Keywords: arteriosclerosis; exosomes; fibrosis; glomerulosclerosis; microparticles; microvesicles; nephron hypertrophy; urinary vesicles.

Fig. 1

Example of fluorescence dot (quadrant) plots showing fluorophore spectra separate of markers used to identify urinary EVs in this study. (a) Isotype controls (fluorescein (FITC)-conjugated immunoglobulin G (IgG) versus phycoerythrin (PE)-conjugated IgG); (b) annexin-V (phosphatidylserine) versus CD63 (exosome); (c) intercellular adhesion molecule-1 (ICAM-1) versus monocyte chemoattractant protein-1 (MCP-1); (d) tissue factor (TF) versus vascular cell adhesion molecule -1 (VCAM-1); (e) beta-adrenergic receptor-1 (β-1 AR) versus renin; (f) SM22 alpha versus annexin-V-PE; (g) nephrin versus podocin; (h) cytokeratin 8 (CK 8) versus caludin-1; (i) URAT-1 versus megalin; (j) aquaporin-1 versus SLC14A2; (k) uromodulin versus epidermal growth factor receptor (EGFR); (l) SLC12A3 versus prominin; (m) aquaporin-2 versus V-ATPase; (n) cytokeratin 20 versus cytokeratin 19.

Fig. 2

Example of biopsies morphometric observation indices of nephron hypertrophy and nephrosclerosis/glomerulosclerosis. (a) Two functional non-sclerotic glomeruli; (b) 2 globally sclerosed glomeruli; (c) 5 consecutive 0.2 mm² circles (green) in which full (red) or partial (cyan) tubules are labelled, and their area subsequently quantified after exclusion of all non-tubular structures (yellow); (d) area of cortical fibrosis and tubular atrophy outlined in blue dashed line; (e) the percent intimal thickening (arteriosclerosis) was determined by the area of intima (between yellow and blue boundaries) divided by the area of intima and lumen (within blue boundary). Examples of different degrees of intimal thickening are shown in E1–no thickening, E2–moderate thickening and E3–severe thickening.

Fig. 3

Shows the overall changes of specific populations of urinary EVs between sexes and with older age, nephron hypertrophy and nephrosclerosis. (−) indicates decrease and (+) indicates increase. Abbreviations/renal cell markers: annexin-V, microvesicles; aquaporin-1, simple squamous epithelium of descending limb of Henle's loop; aquaporin-2, principal cells of collecting tubule; β1-AR (beta-1 adrenergic receptor), juxtaglomerular cells; CD63, exosomes; CK8 (cytokeratin 8), parietal cells; claudin-1, parietal cells; GSG, globally sclerotic glomeruli; ICAM-1, intercellular adhesion molecule-1; megalin, simple cuboidal epithelium of proximal tubule; MCP-1, monocyte chemoattractant protein-1; nephrin, podocytes; NSG, non-sclerotic glomeruli; prominin-2, simple cuboidal epithelium of distal tubule; SM22 alpha, mesangial cells; TF, tissue factor; V-ATPase, intercalated cells of collecting tubule.