Roles for Exosome in Various Kidney Diseases and Disorders

Exosome is a nanoscale vesicle with a size range of 30–100 nm. It is secreted from cell to extracellular space by exocytosis after fusion of multivesicular body (MVB) (formed by endocytic vesicles) with plasma membrane. Exosome plays several important roles in cellular homeostasis and intercellular communications. During the last two decades, exosome has acquired a wide attention to explore its additional roles in various aspects of cell biology and function in several organ systems. For the kidney, several lines of evidence have demonstrated 1that exosome is involved in the renal physiology and pathogenic mechanisms of various kidney diseases/disorders. This article summarizes roles of the exosome as the potential source of biomarkers, pathogenic molecules, and therapeutic biologics that have been extensively investigated in many kidney diseases/disorders, including lupus nephritis (LN), other glomerular diseases, acute kidney injury (AKI), diabetic nephropathy (DN), as well as in the process of renal fibrosis and chronic kidney disease (CKD) progression, in addition to polycystic kidney disease (PKD), kidney transplantation, and renal cell carcinoma (RCC). Moreover, the most recent evidence has shown its emerging role in kidney stone disease (or nephrolithiasis), involving inflammasome activation and inflammatory cascade frequently found in kidney stone pathogenesis.


INTRODUCTION
Exosome is a nanoscale extracellular vesicle with a size range of approximately 30-100 nm and spherical shape surrounded by lipid bilayers (Huotari and Helenius, 2011;Raimondo et al., 2011). Initially, exosome had been thought to serve only as an exocytic vesicle to shed some intracellular and membrane components out of the cells (Pan et al., 1985;Johnstone et al., 1987). However, recent evidence has revealed its more significant roles as the messenger cargo for intercellular communications (Hessvik and Llorente, 2018). Exosome is originated from small intraluminal vesicle inside multivesicular body (MVB) (also known as late endosome) that is subsequently fused with cellular plasma membrane to expel such nano-sized vesicle to the extracellular space (Hessvik and Llorente, 2018). Therefore, the exosome contains greater proportion of membrane proteins and MVB-formation proteins as compared to the parental cells (Bobrie et al., 2011;Colombo et al., 2014;Park et al., 2018). The biogenesis of exosome also enables transportation of various receptors, proteins, genetic materials [e.g., DNA, messenger RNA (mRNA), and microRNA (miRNA)] and lipids to the target cells, which incorporate these information-rich molecules from exosome by three major pathways, including receptor-ligand interaction, direct fusion with plasma membrane, and endocytosis (Bobrie et al., 2011;Colombo et al., 2014;Park et al., 2018).
Exosome can be found in several biological fluids, e.g., blood, seminal fluid, cerebrospinal fluid, synovial fluid, breast milk, saliva, bile, ascitic fluid, amniotic fluid, pleural fluid, and urine (Lin et al., 2015). For the renal system, recent studies have investigated the compositions of urinary exosomes secreted from different segments of the nephron and their relevance to the renal physiology and pathophysiology of kidney diseases (Morrison et al., 2016;Pomatto et al., 2017;Stahl et al., 2019). In addition, roles for exosomes derived from serum/plasma/blood, renal tubular cells, renal tissues, glomerular endothelial cells, mesenchymal stem cells (MSCs), urinary stem cells, cancer cells, and macrophages related to kidney diseases have been also examined. This review summarizes the current knowledge of roles for exosome in pathogenic mechanisms, biomarker discovery and therapeutics of various kidney diseases/disorders, particularly lupus nephritis (LN), other glomerular diseases, acute kidney injury (AKI), diabetic nephropathy (DN), as well as in the process of renal fibrosis and chronic kidney disease (CKD) progression, in addition to polycystic kidney disease (PKD), kidney transplantation, and renal cell carcinoma (RCC) ( Table 1). Finally, the emerging role of exosome in kidney stone disease is also emphasized.

EXOSOME AND THE RENAL PHYSIOLOGY
Kidney is one of the vital organs responsible for homeostasis to maintain and regulate the normal physiology of human body.
Exosome has been proposed to play roles in cell-cell communications within nephron segments that affect the renal physiology (Stahl et al., 2019). Several proteomics studies have revealed that the majority of exosomes in the nephron are originated mostly from glomerular podocytes and tubular cells at proximal convoluted tubule, thick ascending limb of the Henle's loop, distal convoluted tubule and collecting duct (Pisitkun et al., 2004;Miranda et al., 2010;Vlassov et al., 2012;Dear et al., 2013). In addition, there are numerous membrane vesicles with the diameter of 30-50 nm and positive MVB markers found in the pellet derived from the normal urine of healthy individuals (Pisitkun et al., 2004). Moreover, these urinary exosomes are enriched with proteins and transporters from each part of the nephron, e.g., podocin and podocalyxin from glomerular podocytes, aquaporin (AQP)-1 from proximal convoluted tubule, type-2 Na + -K + -2Cl − cotransporter from thick ascending limb of the Henle's loop, Na + -Cl − cotransporter from distal convoluted tubule, and AQP-2 from collecting duct (Pisitkun et al., 2004).
The roles for exosome in the renal physiology have been investigated in both in vitro and in vivo settings. For water transport, exosomes secreted from vasopressin analogue-treated collecting duct cells expressed high level of AQP-2 and subsequently induced neighboring cells to up-regulate expression of AQP-2 to enhance water reabsorption (Street et al., 2011;Radin et al., 2012;Miyazawa et al., 2018). For intra-nephron communications, several studies have implicated that exosome is involved in proximal-distal signaling (Prunotto et al., 2013;Gildea et al., 2014;Merchant et al., 2017). For example, a proteomics study has demonstrated that exosomes derived from glomerular podocytes could pass through the upstream proximal renal tubule and transferred information to the downstream distal renal tubular epithelial cells (Prunotto et al., 2013). Similarly, another study has also shown that exosomes derived from proximal renal tubular cells treated with fenoldopam (a dopamine receptor agonist) could be transferred to distal renal tubule and collecting duct, and further modulated production of reactive oxygen species (ROS) in the cells lining these tubular segments (Gildea et al., 2014). In addition, exosomes derived from LLC-PK1 proximal renal tubular cells expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which could be delivered to distal convoluted tubule and collecting duct (Jella et al., 2016). GAPDH-expressing exosomes then induced conformational changes of epithelial sodium channel (ENaC) and reduced sodium ion reabsorption capability of the affected cells (Jella et al., 2016).
Apart from function involving the renal physiology, urinary exosomes also act as immune effectors to protect from bacterial infection in the urinary tract. The intact human urinary exosome carries several innate immune proteins, including lysozyme-C, dermcidin, mucin-1, calprotectin, and myeloperoxidase, all of which potently inhibit the growth of various pathogenic and non-pathogenic Escherichia coli strains (Hiemstra et al., 2014). Taken together, under the normal state of the kidney, urinary exosome acts as the shelter for bioactive molecules to effectively transmit the functional substances from the upstream renal tubular cells to the downstream tubular cells that finally influence functions or activities of the effector cells. Intravenous administration of extracellular vesicles (mainly exosomes) derived from rat renal tubular cells could improve ischemia-induced renal injury. (Dominguez et al., 2017) Therapeutics Renal tubular epithelial cells (human) Intravenous administration of exosomes derived from human renal tubular cells could improve ischemia-induced renal injury. (Dominguez et al., 2018) Therapeutics MSCs (human) Exosomes derived from bone marrow MSCs were rich with miR-199a-3p and could prevent ischemia/reperfusion-induced AKI by increasing expression of miR-199a-3p in renal cells. (Zhu et al., 2019a) Sepsisinduced AKI Biomarker Urine (human) Increased urinary exosomal NGAL and activating transcription factor 3 in sepsis-induced AKI patients. (Panich et al., 2017) Biomarker
-Increased production of IL-1b (a marker for inflammasome activation) in exosomes derived from the COM-exposed macrophages that activated several functions of inflammatory cells, including monocytes, macrophages, and T-cells.
-Knockdown of vimentin by siRNA could abolish the effects of exosomes derived from the COM-exposed macrophages on monocytic and T-cell migration and phagocytic activity of macrophages.  Pathogenic mechanism Macrophages (human) -Exosomes derived from COM-exposed macrophages had changes in levels of proteins involved in cytoskeletal and actin binding, calcium binding, stress response, transcription regulation, immune response, and ECM disassembly.
-COM-induced exosomes caused IL-8 overproduction from renal tubular cells and enhanced neutrophil migration.
-COM-induced exosomes could bind with COM crystals more tightly than the control exosomes and subsequently activated COM crystal invasion through the ECM.

ROLES OF EXOSOME IN VARIOUS KIDNEY DISEASES/DISORDERS Lupus Nephritis and Other Glomerular Diseases
To date, urinary exosome has been widely proposed as the promising source of non-invasive biomarkers for diagnostics and prognostics to predict severity and progression of various kidney diseases, including glomerular disorders (Hoorn et al., 2005). In a murine model of LN, it has been shown that miR-26a expression in urinary exosomes significantly increased along with progressive glomerular injury (Ichii et al., 2014). The data obtained from this study has implicated that miR-26a regulates podocyte differentiation and cytoskeletal integrity and its increase might serve as the marker for the injured podocytes in glomerulonephritis (Ichii et al., 2014). Transcriptomics study in humans has shown that miR-146a was increased in urinary exosomes derived from active LN patients and might serve as the diagnostic biomarker for discriminating active LN from healthy controls and systemic lupus erythematosus (SLE) patients without LN (Perez-Hernandez et al., 2015). In addition, early phase of renal fibrosis and chronicity in LN patients could be predicted by a decline in urinary exosomal miR-29c (Sole et al., 2015). Similarly, the anti-inflammatory miRNAs (i.e., let-7a and miR-21) were down-regulated in urinary exosomes derived from patients with LN and might serve as the non-invasive biomarkers to classify the clinical stage of LN (Tangtanatakul et al., 2019). Furthermore, urinary exosomal miR-3135b was increased in patients with active LN class IV as compared to those with inactive LN class IV and healthy controls, whereas miR-654-5p was increased only in LN class IV with cellular crescent (with very poor prognosis) (Li et al., 2018b). For other glomerular diseases, a previous study using a proteomics approach has shown that urinary exosomal aminopeptidase N, vasorin precursor, a-1-antitrypsin, and ceruloplasmin could differentiate immunoglobulin A (IgA) nephropathy from thin basement membrane nephropathy and healthy controls (Moon et al., 2011). In another study, level of fibroblast-specific protein 1 (FSP1) has been shown to increase in extracellular vesicles (including exosomes) derived from patients with active crescentic glomerulonephritis (Morikawa et al., 2019). Interestingly, its level was decreased after immunosuppressive therapy (Morikawa et al., 2019).
In addition to proteins, exosomal mRNAs and miRNAs also serve as the valuable markers for glomerular disorders. For example, level of urinary exosomal miR-193a was greater in children with primary focal segmental glomerulosclerosis as compared to those with minimal change nephrotic syndrome (NS) . Urinary exosomal excretion and chemokine (C-C motif) ligand-2 (CCL2) mRNA level were increased in IgA nephropathy and correlated with the disease activity (Feng et al., 2018). Additionally, 30 urinary exosomal miRNAs were markedly increased in children with idiopathic NS . Among these, miR-194-5p and miR-23b-3p correlated well with urinary protein level . These data strengthen that urinary exosomes are rich with the information or molecules that can be further developed to serve as the diagnostic and prognostic biomarkers for LN and other glomerular diseases.

Acute Kidney Injury
Similar to LN, exosome has gained a wide attention as a source of pathogenic molecules, biomarkers, and therapeutic compounds in AKI. For pathogenic mechanisms and biomarker discovery, studies in a rat model have revealed that levels of urinary exosomal AQP-1 and AQP-2 protein and mRNA were reduced in animals with ischemia/reperfusion-induced AKI (Sonoda et al., 2009;Asvapromtada et al., 2018). The decreased levels of aquaporins in renal tubules could affect water handling of the nephron and might reflect the progressive development of AKI (Nielsen et al., 2002). In addition, different sets of exosomal miRNAs could be used as the biomarkers to classify AKI progression in ischemia/reperfusion injury. For example, the increased levels of miR-16, miR-24, and miR-200c were detected in urinary exosomes at an early (or injury) phase, whereas the increases of miR-125 and miR-351 were found at the late (or fibrotic) stage of ischemia/reperfusion injury (Sonoda et al., 2019a).
A most recent transcriptomics study has revealed that exosomal miR-19b-3p derived from renal tubular epithelial cells was elevated in mice with lipopolysaccharide-induced AKI (Lv et al., 2019). This molecule has been proposed to play a critical pathogenic role in tubulointerstitial inflammation by recruiting macrophage infiltration (Lv et al., 2019). Moreover, studies of cisplatin-induced AKI have revealed the increased level of fetuin-A but decreased level of AQP-2 in urinary exosomes from the AKI animals (Zhou et al., 2006;Sonoda et al., 2019a;Sonoda et al., 2019b). In another study, exosomes derived from patients with sepsis-induced AKI carried high amounts of neutrophil gelatinase-associated lipocalin (NGAL) and activating transcription factor 3 (Panich et al., 2017). All of these data indicate that changes in levels of the mentioned urinary exosomal proteins may serve as the biomarkers for diagnosis of AKI and prediction of its severity.
For therapeutics, there is increasing evidence implicating the renoprotective role of exosomes, particularly those derived from MSCs, for attenuation and/or prevention of AKI (Aghajani Nargesi et al., 2017). Administration of extracellular vesicles derived from human Wharton's jelly MSCs could decrease renal injury and improve renal function after ischemia/ reperfusion-induced renal damage in rats (Zhang et al., 2014;Zhang et al., 2016). High expression of C-C motif chemokine receptor-2 (CCR2) on exosomes derived from MSCs could reduce the level of its ligand (CCL2) and suppress its effects to induce macrophage infiltration in mice with ischemia/ reperfusion-induced AKI . Exosomes derived from adipose MSCs could protect ischemia/reperfusion-induced AKI (Lin et al., 2016). Similarly, exosomes derived from bone marrow MSCs were rich with miR-199a-3p and could prevent ischemia/reperfusion-induced AKI by increasing expression of miR-199a-3p in renal cells (Zhu et al., 2019a). Additionally, intravenous administration of extracellular vesicles (mainly exosomes) derived from rat and human renal tubular cells could improve ischemia-induced renal injury (Dominguez et al., 2017;Dominguez et al., 2018).
In other models of AKI, exosomes derived from sera of animals with remote ischemic preconditioning and myoblasts under hypoxia and reoxygenation preconditioning could prevent sepsis-induced AKI in vivo and in vitro, respectively, by enhancing the expression of miR-21 (Pan et al., 2019). Also, transferring insulin growth factor-1 (IGF-1) receptor to cisplatin-treated renal tubular cells and co-incubation of these injured cells with soluble IGF-1 and exosomes derived from bone marrow MSCs significantly increased cell proliferation (Tomasoni et al., 2013). Moreover, exosomes derived from umbilical cord MSCs could prevent cisplatin-induced AKI via 14-3-3z-dependent pathway (Jia et al., 2018). Although all of these studies were carried out in animals, their results hold a promise for further validation in the clinical setting.

Chronic Kidney Disease
The roles of exosome have also been extensively investigated in CKD, particularly in DN and renal fibrosis models. A recent study has shown that type 1 DN was associated with increases in urinary excretion of exosomal plasmin, prostasin, and urokinase as well as the proteolytic activation of ENaC that might contribute to the dysfunction of Na + excretion and hypertension (Andersen et al., 2015). Interestingly, exosomes derived from high-glucose-treated glomerular endothelial cells played a crosstalk role to activate epithelial mesenchymal transition (EMT) and fibrotic changes of glomerular mesangial cells and podocytes in DN Wu et al., 2017). High-glucose also enhanced secretion of exosomes from macrophages, which then caused mesangial proliferation and activated inflammatory cascade in the renal tissue (Zhu et al., 2019b). Knockdown of TGF-b1 significantly reduced such effects of exosomes indicating that TGF-b1 serves as an important mediator for interactions or cross talks between macrophages and renal cells (Zhu et al., 2019b). In addition, advanced glycation end products (AGEs) activated TGF-b-Smad3 signaling pathway and induced secretion of a transcription factor Elf3 through exosomes from the murine podocytes (Sakurai et al., 2019). Moreover, urinary exosomal Elf3 was detectable only in DN patients, not in those with minimal change NS and healthy controls (Sakurai et al., 2019).
In renal fibrosis, a study using unilateral ureteric obstruction (UUO) model has indicated that transforming growth factor-b1 (TGF-b1) mRNA was transported by exosomes, which might activate fibroblast proliferation and development of renal fibrosis (Borges et al., 2013). Additionally, a recent proteomics study has demonstrated an increased level of secreting transglutaminase-2 (a fibrosis-activating enzyme) through exosomal pathway in UUO mice and suggested that its increase might serve as a prognostic biomarker to predict the progression of renal fibrosis (Furini et al., 2018).
In other studies of CKD, urinary exosomal miR-29c and miR-181a were decreased and correlated with the degree of chronicity or renal fibrosis in CKD patients (Lv et al., 2013;Khurana et al., 2017;Chun-Yan et al., 2018). Urinary exosomal miR-200b was decreased in CKD patients and the degree of decrease was greatest in urinary exosomes derived from cells other than those of proximal renal tubules (Yu et al., 2018). By contrast, urinary exosomal miR-21 was increased in CKD patients and inversely correlated with estimated glomerular filtration rate (eGFR) (Lange et al., 2019). Urinary exosomal ceruloplasmin was increased in animals with passive Heyman nephritis and also in CKD patients (Gudehithlu et al., 2019). Interestingly, its increase in animals was detected prior to the onset of proteinuria and thus might serve as an early biomarker to predict the renal histopathological change (Gudehithlu et al., 2019).
For therapeutics, the study in DN has shown that exosomes derived from bone marrow MSCs suppressed infiltration of dendritic cells into the kidney by regulating expression of intercellular adhesion molecule-1 (ICAM-1) (Nagaishi et al., 2016). These exosomes also inhibited renal fibrosis and reduced production of pro-inflammatory cytokines, i.e., tumor necrosis factor-a (TNF-a) and TGF-b1 (Nagaishi et al., 2016). In addition, exosomes derived from urinary stem cells carried growth factor, TGF-b1, angiogenin, and bone morphogenetic protein-7 that might play roles in vascular regeneration and cell survival in an early phase of DN (Jiang et al., 2016). In the UUO model, exosomal miR-let7c derived from MSCs could attenuate fibrotic process in TGF-b-treated renal tubular epithelial cells . Taken together, these findings indicate the potential roles of exosome for therapeutic intervention in CKD in both DN and UUO renal fibrosis models.

Polycystic Kidney Disease
In an early phase of proteomics studies of urinary exosomes, multiple gene products related to PKD were detected in the urine, implicating that they were excreted into the urine via exosomal secretion (Pisitkun et al., 2004;Hogan et al., 2009). Comparing to those derived from healthy individuals, urinary exosomes derived from autosomal dominant PKD (ADPKD) patients with PKD1 gene mutation had decreased levels of polycystin-1 and polycystin-2, but increase of transmembrane protein 2 (Hogan et al., 2015). A subsequent proteomics study has demonstrated that complement components C3 and C9 were increased in urinary extracellular vesicles (including exosomes) derived from ADPKD patients with or without progressive CKD, whereas urinary exosomal villin-1, periplakin, and envoplakin were increased only in ADPKD patients with progressive CKD (Salih et al., 2016). More recently, the study in both animal model and in PKD patients has shown the increase in urinary exosomal activator of G protein signaling 3 (Keri et al., 2018). Furthermore, a most recent study has reported the increased urinary exosomal CD133 that could effectively discriminate ADPKD patients from those with medullary sponge kidney (Bruschi et al., 2019). These findings underscore the important role of exosomes as the source of the non-invasive biomarkers for diagnostics and prognostics of PKD.

Kidney Transplantation
Currently, renal biopsy is the gold standard for the diagnosis of kidney transplant rejection. However, such diagnostic procedure is invasive and is frequently accompanied by hematuria and other biopsy-related complications. Recently, much attention has been given to urinary and blood-derived exosomes as alternative means for monitoring the graft rejection. Indeed, exosomes served as the source of potential non-invasive biomarkers to facilitate the diagnosis of cell-mediated and antibody-mediated allograft rejection. The presence of intragraft infiltration of T-cells is one of the hallmarks for the diagnosis of acute cellular rejection after kidney transplantation. A recent study has shown the higher level of CD3-positive urinary exosomes in patients with acute cellular rejection that could reflect T-cell infiltration in the renal allografts (Park et al., 2017). Similarly, a subsequent proteomics study has demonstrated the increased levels of urinary exosomal tetraspanin-1 and hemopexin in patients with T-cell-mediated rejection (Lim et al., 2018). For antibody-mediated rejection, the increased mRNA levels of gp130, CCL4, TNFa, SH2D1B, CAV1, and atypical chemokine receptor 1 were found in plasma exosomes derived from patients with allograft rejection .
Additionally, the information obtained from exosomes also leads to better understanding of the mechanisms and pathogenic events after kidney transplantation. For example, the increased levels of 50-kDa and 75-kDa subunits of g-ENaC were found in urinary exosomes derived from albuminuric kidney transplant recipients and was associated with hypertension (Hinrichs et al., 2018). Moreover, the decreased level of AQP-2 was detected in urinary extracellular vesicles (including exosomes) 1-day after kidney transplantation together with increased urinary volume and decreased urinary osmolality (Oshikawa-Hori et al., 2019). However, such decrease of AQP-2 in urinary extracellular vesicles and diuresis were recovered by day-6 posttransplantation, suggesting the temporary decrease in the renal expression of AQP2 and impairment of renal tubular water handling during the first few days after transplantation that might explain the acute diuresis phenomenon after kidney transplantation (Oshikawa-Hori et al., 2019).
For biomarker discovery, exosomes could be used for monitoring toxicities from immunosuppressive drugs. For example, the increased urinary exosomal Na + -K + -2Cl − cotransporter was observed in cyclosporine-A-treated kidney transplant recipients (Esteva-Font et al., 2014). Similarly, levels of Na + -Cl − cotranspoter and its phosphorylated form were greater in urinary exosomes derived from tacrolimus-treated kidney transplant recipients with hypertension (Rojas-Vega et al., 2015). Interestingly, the increases in both of these cotransporters were associated with high blood pressure, which is a common complication of kidney transplantation during immunosuppressive therapy.
Overall, these findings strengthen the important roles of exosome as the source of pathogenic molecules and noninvasive biomarkers in kidney transplantation. Exosome-based monitoring during the early post-transplantation phase may help the clinicians to maintain and perhaps prolong the allograft survival and function.

Renal Cell Carcinoma
Exosome also plays roles in pathogenic mechanisms of RCC. For evasion of immune surveillance, an in vitro study has indicated that exosomes from primary RCC cells of patients with clear cell RCC (ccRCC) and from RCC cell line had increased level of TGF-b1 that further mediated natural killer cell dysfunction (Xia et al., 2017). For cancer progression, exosomes derived from cancer stem cells (CSCs) of RCC patients promoted proliferation and EMT of ccRCC (Wang et al., 2019b). Moreover, the same set of data also showed that CD103-positive exosomes served as the biomarker for metastatic ccRCC (Wang et al., 2019b).
For therapeutics, the in vitro study on a RCC cell line showed that CD8 + T-cells stimulated by exosomes derived from RCC cells combined with granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin (IL)-12 exerted autologous anti-cancer effects (Xu et al., 2019). Taken together, exosomes play several roles in RCC, from the pathogenesis to diagnostics, prognostics and therapeutics.

Emerging Role of Exosome in Kidney Stone Disease
Kidney stone disease ("nephrolithiasis" or "urolithiasis") is a common but under-estimated disease of the world involving 1-20% of worldwide population, depending on geographical areas (Thongboonkerd, 2008;. Its incidence/prevalence has been increasing in both industrialized and developing countries (Ziemba and Matlaga, 2017;Alelign and Petros, 2018). Calcium oxalate (CaOx) is the most common constituent in kidney stone matrix accounting approximately 80% of all kidney stones analyzed (Schubert, 2006). The stone formation frequently starts with supersaturation of urinary calcium and oxalate ions to form CaOx crystals inside the renal tubules (Thongboonkerd et al., 2006). After crystallization, CaOx crystalline particles can increase their sizes by crystal growth and self-aggregation and then be retained inside renal tubules by crystal-cell adhesion (Tsujihata, 2008).
Between the two common hydrated forms of CaOx crystals, CaOx monohydrate (COM) is more prominent and more pathogenic than CaOx dihydrate (COD) in kidney stone disease (Semangoen et al., 2008a;Semangoen et al., 2008b;Thongboonkerd et al., 2008;Peerapen et al., 2018). By differences in adhesive capability, atomic lattice, binding kinetics and surface atomic pattern of these two hydrated forms, COM crystal adheres more tightly to renal epithelial cell surface and is then internalized into the cells and subsequently transferred to the renal interstitium (Chiangjong and Thongboonkerd, 2012;Kanlaya et al., 2013;Chaiyarit et al., 2016;Chiangjong and Thongboonkerd, 2016;. In addition to forming stone(s) in the renal interstitial locale (Randall's plaque's model) (Alelign and Petros, 2018;Wiener et al., 2018), the crystals deposited in the renal interstitium can then trigger several cascades of cellular response, e.g., increases in production of prostaglandin E, ROS, and inflammatory chemokines/cytokines (Umekawa et al., 2002;Khan, 2004;Khan, 2014;Mulay et al., 2014). In particular, the increased synthesis of chemokines [i.e., osteopontin, MIP-1 (macrophage inflammatory protein-1), RANTES (regulated upon activation normal T cell expressed and secreted), and MCP-1 (monocyte chemoattractant protein-1)] in the renal interstitium can activate and attract monocytes, macrophages, and other leukocytes to the crystal-deposited locales to serve as effector cells involving the inflammatory processes (Umekawa et al., 2002;Okada et al., 2010).
Although many efforts have been made to extensively investigate the roles for exosome in several other kidney diseases, the contributions of exosome in kidney stone disease remain under-investigated (Table 1). Indeed, the involvement of exosome-like vesicles in kidney stone disease had initially been discovered in rat models (Nagasawa et al., 1992;Khan et al., 2012). These studies have suggested that crystal deposition in renal papillae might start from nanoscale membrane vesicles derived from brush border of the injured renal cells that could induce nucleation, crystallization, and growth of the causative crystals at the periphery within a collagen framework (Khan et al., 2012). In humans, patients with kidney stones (stone formers) showed differential levels of urinary exosomal secretion when compared with the healthy volunteers without kidney stone (Jayachandran et al., 2015). Later, additional evidence has suggested that hyperoxaluria (an aggravating or etiologic factor for kidney stone formation) caused cytotoxicity in HK-2 cells and activated exosomal secretion (He et al., 2017). Nevertheless, the biological relevance of the increased exosomal secretion in kidney stone disease remains under-investigated.
Macrophage is an effector cell responsible for elimination of CaOx crystals deposited in the renal interstitium but, on the other hand, can aggravate or worsen tissue inflammation in kidney stone disease by autocrine, paracrine, and/or cytokine mechanisms (Kusmartsev et al., 2016). Recently, a combined expression and functional study has been conducted to examine proteome changes in exosomes derived from macrophages after exposure to COM crystals using a gel-based proteomics technology followed by several functional assays . The data have shown that exosomes derived from COM-exposed macrophages had changes in levels of proteins involved in immune regulation, i.e., T-cell activation and homeostasis, Fcg receptor-mediated phagocytosis, interferon-g (IFN-g) regulation, and cell migration . Functional assays revealed an increase in production of IL-1b (a marker for inflammasome activation) in exosomes derived from the COM-exposed macrophages. Additionally, these exosomes activated several functions of inflammatory cells, including monocytes, macrophages, and Tcells. Moreover, exosomes derived from the COM-exposed macrophages caused an increase in production of proinflammatory cytokine (IL-8) in monocytes. Interestingly, knockdown of expression of vimentin (one of the significantly increased proteins in exosomes derived from the COM-exposed macrophages identified by proteomics approach) by smallinterfering RNA (siRNA) could abolish the effects of exosomes derived from the COM-exposed macrophages on monocytic and T-cell migration and phagocytic activity of macrophages . The data obtained from this study indicate that macrophage-derived exosome is involved, at least in part, in immune process and inflammatory cascade frequently found in kidney stone pathogenesis.
In addition to the inflammasome activation, a most recent study using label-free, gel-free, quantitative proteomics approach has identified 26 proteins whose levels were significantly changed in exosomes derived from the COM-exposed macrophages as compared to the control exosomes derived from the untreated macrophages . These proteins with significantly altered levels were involved mainly in cytoskeletal and actin binding, calcium binding, stress response, transcription regulation, immune response, and extracellular matrix (ECM) disassembly. Functional assays have shown the IL-8 overproduction from renal tubular cells treated with these COM-induced exosomes that also enhanced neutrophil migration. In concordance, these COM-induced exosomes were more fragile, thereby were easily to release their intraluminal contents for inflammasome activation. Interestingly, these COM-induced exosomes could bind with COM crystals more tightly than the control exosomes and subsequently activated COM crystal invasion through the ECM , a process that relies on plasminogen-plasmin activity on the COM crystal surface (Chiangjong and Thongboonkerd, 2012;Chiangjong and Thongboonkerd, 2016). These findings strengthen the pathogenic roles of macrophage-derived exosome, particularly in the inflammatory cascade of kidney stone disease induced by COM crystals in the renal interstitium.

CONCLUSIONS AND FUTURE PERSPECTIVES
The aforementioned studies have demonstrated the important roles for exosome as the vesicular cargo for carrying and transferring molecular mediators for cell-cell communications and signal transduction. For example, exosomes derived from the urinary system are involved in intercellular communications under the physiologic and pathologic states. Their release during development and progression of kidney diseases may exert dual effects, i.e., aggravating the disease severity while promoting tissue repair. Exosome not only plays a role in the pathogenic mechanisms of kidney diseases but also serves as the valuable source of potential non-invasive biomarkers for diagnostics and prognostics (Hoorn et al., 2005). Comparing to the conventional urinary and circulating biomarkers, exosome carries and is enriched with specific sets of biomarker molecules, particularly receptors, proteins, genetic materials (e.g., DNA, mRNA, and miRNA) and lipids that are much less abundant in the urine and circulating blood. Therefore, exosomal markers provide the advantages for biomarker discovery in specific diseases that involve abnormalities of such molecules carried by exosome. Because these biomarker molecules are carried inside the exosomal cargo, they are more stable in biological fluids as compared to the other freely circulating molecules. In addition to serving as the important source of non-invasive biomarkers, exosome also provides the therapeutic potential as well (Tomasoni et al., 2013;Nagaishi et al., 2016;Aghajani Nargesi et al., 2017). Details of the roles for exosomes in various kidney diseases/disorders obtained from previous and recent studies are summarized in Table 1. Nevertheless, it should be noted that exosomes have not yet entered into the clinical practice, which of course needs clinical trials and validation in large cohorts.
In kidney stone disease, the emerging role of exosome has been recently reported to serve as the mediator to promote kidney stone generation at the initial phase of inflammatory cascade by activation of inflammasome . Although the mentioned recent studies have provided convincing evidence for the pathogenic role of macrophage-derived exosome in kidney stone disease, there are still many challenges that need further elucidations. For example, roles of exosomes derived from cells other than macrophages (e.g., renal cells aligning various segments of the nephron) should be also investigated. In addition, large-scale analyses of exosomes in kidney stone research should not be limited only to proteomics but can be done by other "omics" studies (e.g., transcriptomics, lipidomics, metabolomics, interactomics, etc.) as well. Moreover, it would be interesting to observe the effects of exosomes derived from specific cell types on the disease course using animal models or ex vivo setting. However, isolation or purification of exosomes from animal model or raw biological fluids is still challenging because some components, such as high-density lipoproteins, chylomicrons, and microvesicles, have their size ranges close to that of exosomes (Yuana et al., 2014). Recently, several efforts have been made to optimize the isolation/purification of exosomes (Gheinani et al., 2018;Ayala-Mar et al., 2019;Doyle and Wang, 2019;Hou et al., 2019;Huang et al., 2019;Wang et al., 2019a;Yang et al., 2019). Nevertheless, the outcome is still unsatisfactory. Therefore, it is essential to further optimize the isolation methods or develop new techniques to isolate/purify exosomes more efficiently. Finally, genetic manipulation of specific transcript or protein compositions of the exosome would yield better understanding of the mechanisms for exosomal involvement in kidney stone disease. Having done so, the roles for exosome in kidney stone disease will be much clearer.

AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and approved it for publication.

ACKNOWLEDGMENTS
The author is grateful to Dr. Nilubon Singhto for assistance in gathering some references. This work was supported by a Mahidol University research grant and the Thailand Research Fund (IRN60W0004).