Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers?

Abstract

Extracellular vesicles from the urine and circulation have gained significant interest as potential diagnostic biomarkers in renal diseases. Urinary extracellular vesicles contain proteins from all sections of the nephron, whereas most studied circulating extracellular vesicles are derived from platelets, immune cells, and the endothelium. In addition to their diagnostic role as markers of kidney and vascular damage, extracellular vesicles may have functional significance in renal health and disease by facilitating communication between cells and protecting against kidney injury and bacterial infection in the urinary tract. However, the current understanding of extracellular vesicles has derived mostly from studies with very small numbers of patients or in vitro data. Moreover, accurate assessment of these vesicles remains a challenge, in part because of a lack of consensus in the methodologies to measure extracellular vesicles and the inability of most techniques to capture the entire size range of these vesicles. However, newer techniques and standardized protocols to improve the detection of extracellular vesicles are in development. A clearer understanding of the composition and biology of extracellular vesicles will provide insights into their pathophysiologic, diagnostic, and therapeutic roles.

Keywords: biomarker; exosomes; extracellular vesicles; kidney disease; microparticles.

Figure 1.

Biogenesis of EVs. Exosomes are believed to be released from intracellular MVBs when they fuse with the cell membrane. Exosomes, here shown in yellow, ranging from 30 to 100 nm display the same membrane orientation as the cell of origin. In contrast, MPs, shown in blue, ranging from 100 to 1000 nm are thought to be the product of exocytic budding and consist of cytoplasmic components and phospholipids. Exosomes and MPs can be involved in various biologic processes. They can play a role in antigen presentation by transferring MHC molecules and antigen. They can directly activate cell surface receptors, transfer receptors, or deliver factors, such as transcription factors, protein, and various RNAs (including miRNA and mRNA). Internalization pathways have not been clearly delineated. Possibilities include (1) membrane fusion, (2) micropinocytosis, and (3) receptor-dependent endocytosis.

Figure 2.

Example images of IFCM. Comparison of MPs with other cells/debris. Column 1 shows a bright-field image (BF) of two red blood cells (RBCs) and a white blood cell (WBC) with debris. The size of these RBCs and WBC is about 6–8 µm. Column 2 shows scatter of the laser of these same cells seen in column 1. Column 3 indicates that the debris seen in BF is positive for Annexin V (AV) staining and therefore, could be apoptotic material. Column 4 shows the same WBC (big red circle) and a WBC-derived MP (small red dot). The size of the MP is <1 µm, although it is not measurable with this technique yet. Column 5 shows overlay of all four columns. SSC, side scatter.

Figure 3.

Imaging FCM detects more total and Annexin negative MPs compared to conventional FCM. Annexin-negative and -positive EVs detected by FCM and IFCM using the ImageStream X Machine (Amnis, Seattle, WA). Absolute number of MPs per microliter of platelet-free plasma from three different runs by FCM with and without imaging using split samples. Absolute numbers were calculated for both the Annexin V (AV) -positive (green) and -negative (gray) populations. FCM runs 1 (FC1) and FC2 used forward scatter (FSC) as a threshold, and FC3 (arrow) used side scatter (SSC), because recent publications indicated that larger angles of scatter provided better resolution for small particles. In all cases, the ISX detected more total MPs per microliter and AV-negative MPs per microliter than FCM. Although conventional FCM had higher AV positive counts in two runs, this could be explained by the difference in data acquisition by ISX and FCM. Reprinted from reference , with permission.

Figure 4.

Electron microscopy images of EVs. (A–C) Cryo-EM images from an EV prep showing EVs of three sizes (approximately 100, approximately 500, and approximately 1000 nm, respectively). (D) Distribution of the range of sizes of EVs detected in platelet-poor plasma by scanning EM. Reprinted from reference , with permission.

Figure 5.

Origin of EVs in renal diseases. Proteomic analysis has identified uEVs, including exosomes from glomerular, tubular, prostate, and bladder cells.^, Here, we show a schematic drawing of the glomerulus and kidney tubule. Several markers of podocyte damage have been identified in uEVs, including podocalyxin, podoplanin, and WT-1.^, A protective role of EVs derived from MSCs has been described in models of kidney damage. It is suggested that these EVs from MSCs are involved in transfer of mRNA, miRNA, and proteins and reprogramming of their phenotypes. Solute and water transporters have been identified on uEVs: sodium potassium chloride cotransporter (NaKCl) from the thick ascending limb, sodium chloride cotransporter (NCC) from the distal tubule, and AQP-2 deriving from the collecting duct. Figure adapted from previous publications.^,,