Extracellular Vesicles As Novel Players in Kidney Disease

“Urine can provide us day by day, month by month, and year by year with a serial story of the major events within the kidney.” Dr. Thomas Addis, the pioneer in renal physiology, came to this conclusion in 1948. The discovery of urinary extracellular vesicles (uEVs) as molecular mimics of kidney cells has taken his view to a new level (Figure 1). uEVs include nano-sized vesicles that bud outward from the cell plasma membrane of healthy or dying cells (microvesicles or apoptotic bodies, respectively) or are excreted via multivesicular bodies in a regulated process (exosomes). True to Dr. Addis’ view, uEVs contain disease- and site-specific markers from cells lining the kidney’s tubules and urinary tract,1 making them an invaluable addition to markers of kidney dysfunction such as serum creatinine and proteinuria, which are nonspecific and late to manifest. Evolving evidence demonstrates uEVs’ potential to predict disease earlier than conventional markers. Indeed, the fifirst uEV-based biomarker was recently Food and Drug Administration approved in the field of urology, where a uEV RNA signature serves as a noninvasive screening method for prostate cancer. Other than their role in diagnosis and prognosis, uEVs are increasingly studied as novel messengers in renal disease mechanisms and in the context of regenerative medicine. Recently, a position statement was published by the Urine Task Force of the International Society of Extracellular Vesicles to advance rigor and standardization of uEV analysis and accelerate the clinical application of uEVs.

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1 uEVs FOR DIAGNOSIS AND PROGNOSIS 

The use of uEVs for diagnosis and prognosis in liquid biopsies hinges on the fact that extracellular vesicles (EVs) retain the properties of the cell from which they are formed. uEV diagnostic studies can be broadly divided into two approaches: (1) approaches focused on single-EV analysis (for example, by Nanoparticle Tracking Analysis or flow cytometry) and (2) approaches focused on bulk EV analysis, such as proteomics or RNA profiling. The focus of single-EV approaches is generally enumeration of uEVs and targeted phenotyping, such as detecting EV’s cell of origin and specific cargo from glomerular and tubular cells. Early studies suggested that elevations in uEV levels may be indicative of underlying disease. For example, urinary podocyte EVs were increased in diabetic kidney disease in advance of albuminuria,2 and elevations in uEV subpopulations were reported in preeclampsia.3 Many early studies are notable for heterogeneity in methodology and subsequent challenges with external validation. Several use suboptimal instrumentations, lack appropriate controls or antibody validation, and fail to include orthogonal approaches to confirm vesicle isolation. A recent study showcases a modern approach for uEV assessment by flow cytometry, which will be essential for the transition from a research tool to a clinical test.4 

A comprehensive validation of vesicle isolation is used including multiple protein markers. The authors also established key controls: buffer, buffer plus reagents, uEVs without reagents, and a lysed vesicle preparation to reduce false positives as well as “molecules of equivalent soluble fluorochrome” beads to standardize fluorescent intensity units. These key steps will allow for interlaboratory comparison of results regardless of instrument or software. The second approach to uEV diagnostics is a bulk assessment of uEV content. A seminal study by Pisitkun et al.5 from 2004 established the feasibility of proteomic assessment of uEVs and identified fundamental challenges, such as the interference of Tamm–Horsfall protein. Similar approaches to EV cargo assessment have been described for EV-bound nucleic acids, lipids, and metabolites. A key assumption when inferring changes to the kidney from those seen in uEVs is that the molecular composition of uEVs mirrors those of the kidney. Recent work by Wu et al.6 appears to support this. Using a proteomic approach, the authors showed strong correlations between uEV proteins and those found in whole kidneys. Changes to protein expression in whole kidneys following a physiologic stimulation (high K1) were reflected in uEVs providing strong support for the use of uEVs as surrogate measures of kidney pathology.

uEVs FOR FUNCTION AND REGENERATION 

There is increasing interest in the functional and regenerative role of uEVs. Cells are able to communicate by releasing EVs, which modulate processes in recipient cells.7–9 Three types of such communication have been described in the kidney: (1) intranephron communication that may explain how glomerular (e.g., podocyte damage with proteinuria) and tubular damage (e.g., hypoxia) causes interstitial fibrosis,9 (2) intratubular communication (between tubular segments),7 and (3) circulation to kidney communication, which was demonstrated in patients with active vasculitis and shown to bear B1-kinin receptors.8 Understanding uEV-derived signaling pathways could ultimately lead to new therapeutic targets in renal disease. However, for functional studies, it is important to study pure EV preparations and contrast those fractions with isolated non-EV material (“EV corona” or non-EV eluate).1 

The researcher is starting to translate the in vitro findings to in vivo models, including the use of labeled or tagged EVs, and to validate findings in human cohorts. For example, Lv et al.10 used a transwell culture system to demonstrate that tubular epithelial cells stimulated by albumin produce EVs containing the inflammatory cytokines C-C Motif Chemokine Ligand 2 (CCL2). EVs from these albumin-treated tubular epithelial cells were injected into mice and induced tubular injury in an in vivo model. In addition, CCL2 messenger RNA in uEVs was found in patients with proteinuric IgA nephropathy, supporting the translational potential.10 The therapeutic role of stem cell-derived EVs has long been studied in acute kidney disease and CKD. Very recently, the regenerative role of uEVs from healthy donors was demonstrated in a glycerol-induced AKI model.11 uEVs improved renal recovery, stimulated tubular cell proliferation, and reduced expression of inflammatory and injury markers, restoring endogenous Klotho loss. The authors performed extra purification steps to obtain pure EVs, thorough EV characterization of EV (CD81, CD63), and non-EV–related proteins (calreticulin). The authors also included several key controls, including non-EV fractions and EVs from nonrenal sources.

FUTURE DIRECTIONS 

uEVs have been studied for .17 years since the hallmark publication by Pisitkun et al.5 in 2004. Why is interest in uEVs as novel players in kidney disease ever increasing? In fact, 2004 launched an era of increasing rigor in uEV studies, where the field progressively overcame key roadblocks to clinical application. Biology and cargo loading of EVs were established; new isolation methods were developed, leading to purer EV isolates.1 Quality markers were developed, normalization methods of uEVs were researched and validated, and 12 and minimal reporting requirements were put in place (reviewed in ref. 1). Meanwhile, uEV research has ever increased because of the promises that uEVs hold. Most importantly, uEVs are enriched “baskets” of information on molecular processes and pathways that can be traced back to one cell type. Thus, they are potentially more sensitive than secreted proteins or RNA in urine, and they may also be more specific. Indeed, several attempts have been made to study EVs specific to certain tubule segments. However, this remains challenging, as many protein markers recognize only intracellular epitopes, which may necessitate permeabilization of uEVs by detergents.12 A list of proteins that may be used for this purpose as outlined in the recently published uEV position paper.1 Although the isolation of uEVs is still very time-consuming and labor intensive, high-throughput uEV characterization methods are being developed and characterized12 with the potential to speed up the process of getting kidney biomarkers clinically applicable, such as markers of transplant rejection that could bypass the need for kidney biopsy.13 Many of these advancements have increased the complexity of information that can be retrieved from uEVs. Therefore, the current challenges have shifted from finding isolation and characterization methods sensitive enough to study these novel messengers to improving specificity by (further) optimizing normalization methods, quality control, and thorough reporting. Here, we extend recommendations from the uEV position paper1 with a selected collection of resources for uEV research (Table 1). Ultimately, addressing these challenges will lead to the fast and accurate methods with low variation necessary for clinical application. These next steps could make it possible for uEV-based approaches to replace kidney biopsies within the next decade.

for more information:ali.ma@wecistanche.com