International Journal o f
Molecular Sciences
Review
Extracellular Vesicles Tune the Immune System in Renal
Disease: A Focus on Systemic Lupus Erythematosus,
Antiphospholipid Syndrome, Thrombotic Microangiopathy
and ANCA-Vasculitis
Martina Mazzariol, Giovanni Camussi and Maria Felice Brizzi *






Citation: Mazzariol, M.; Camussi, G.;
Brizzi, M.F. Extracellular Vesicles
Tune the Immune System in Renal
Disease: A Focus on Systemic Lupus
Erythematosus, Antiphospholipid
Syndrome, Thrombotic
Microangiopathy and ANCAVasculitis.
Int. J. Mol. Sci. 2021, 22,

4194. https://doi.org/10.3390/
      ijms22084194
      Academic Editor: Leonora Balaj
      Received: 29 March 2021
      Accepted: 16 April 2021
      Published: 18 April 2021
      Publisher’s Note: MDPI stays neutral
      with regard to jurisdictional claims in
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      Copyright: © 2021 by the authors.
      Licensee MDPI, Basel, Switzerland.
      This article is an open access article
      distributed under the terms and
      conditions of the Creative Commons
      Attribution (CC BY) license (https://
      creativecommons.org/licenses/by/
      4.0/).
      Department of Medical Sciences, University of Turin, Corso Dogliotti 14, 10126 Turin, Italy;
      marti.mazzariol@gmail.com (M.M.); giovanni.camussi@unito.it (G.C.)

• Correspondence: mariafelice.brizzi@unito.it; Tel.: +39-01-1670-6653
  Abstract: Extracellular vesicles (EV) are microparticles released in biological fluids by different cell
  types, both in physiological and pathological conditions. Owing to their ability to carry and transfer
  biomolecules, EV are mediators of cell-to-cell communication and are involved in the pathogenesis
  of several diseases. The ability of EV to modulate the immune system, the coagulation cascade, the
  angiogenetic process, and to drive endothelial dysfunction plays a crucial role in the pathophysiology
  of both autoimmune and renal diseases. Recent studies have demonstrated the involvement of
  EV in the control of renal homeostasis by acting as intercellular signaling molecules, mediators
  of inflammation and tissue regeneration. Moreover, circulating EV and urinary EV secreted by
  renal cells have been investigated as potential early biomarkers of renal injury. In the present
  review, we discuss the recent findings on the involvement of EV in autoimmunity and in renal
  intercellular communication. We focused on EV-mediated interaction between the immune system
  and the kidney in autoimmune diseases displaying common renal damage, such as antiphospholipid
  syndrome, systemic lupus erythematosus, thrombotic microangiopathy, and vasculitis. Although
  further studies are needed to extend our knowledge on EV in renal pathology, a deeper investigation
  of the impact of EV in kidney autoimmune diseases may also provide insight into renal biological
  processes. Furthermore, EV may represent promising biomarkers of renal diseases with potential
  future applications as diagnostic and therapeutic tools.
  Keywords: renal disease; autoimmune diseases; HUS; TTP; APS; antiphospholipid syndrome; vasculitis;
  systemic lupus erythematosus; lupus nephritis; pathogenesis; microparticles; extracellular
  vesicles; microvesicles; exosomes

4194. Introduction
      Extracellular vesicles (EV) are extracellular structures bounded by a phospholipid
      bilayer and released by different cell types in biological fluids (blood, urine, synovial
      fluids) through various mechanisms. First described as “platelet dust” by Peter Wolf
      in 1967, in recent years EV have gained interest as a commonly recognized important
      player in cell-to-cell communication, both in physiological and pathological conditions.
      This depends on their ability to transfer their cargo, consisting of proteins, lipids, or
      nucleic acids. Interestingly, EV have a role in different biological processes including cell
      proliferation and differentiation, inflammation, immune signaling, angiogenesis, and stress
      responses [1]. In this review, we will summarize the most recent data on EV biological
      features and their pathogenic role in immune-associated renal disease, and their potential
      use as disease biomarkers.
      Int. J. Mol. Sci. 2021, 22, 4194. https://doi.org/10.3390/ijms22084194 https://www.mdpi.com/journal/ijms
      Int. J. Mol. Sci. 2021, 22, 4194 2 of 20
      1.1. EV Nomenclature
      The most recently updated guidelines of the International Society for Extracellular
      Vesicles (ISEV) recommend using the term “extracellular vesicles” as “the generic term for
      particles naturally released from the cell that are delimited by a lipid bilayer and cannot
      replicate” [2]. EV were historically classified into three main subtypes according to their
      size, biogenesis and biological features: exosomes (the smallest EV generated from the
      invagination of the endosomal membrane) [1], microvesicles (or EV which are directly
      released from the plasma membrane) [3] and apoptotic bodies (the largest EV released by
      apoptotic cells) [4]. However, since a consensus has not yet emerged on specific markers
      defining each subtype, the ISEV guidelines recommend defining EV according to [2]:
       physical characteristics such as size (small EV and medium/large EV, with ranges
      defined, for instance, respectively, <100 nm or <200 nm (small), or >200 nm (large
      and/or medium)) or density (low, middle, high);
       biochemical composition (CD63+/CD81+􀀀 EV, Annexin A5-stained EV, etc.);
       descriptions of conditions or cell of origin (podocyte EV, hypoxic EV, large oncosomes,
      apoptotic bodies) [2].
      1.2. EV Isolation and Detection
      EV can be detected in almost all human fluids (blood samples, cerebrospinal fluid,
      synovial fluid, urine, bile, saliva, bronchoalveolar fluid) using different techniques depending
      on the EV population. In the pre-analytical phase, body sample collection, handling,
      and storage may impact EV concentration, composition, and function. Thus, to follow a
      preanalytical protocol tailored to the specific body fluid, the cell of origin of EV is crucial [5].
      After collection and isolation, EV can be analyzed through various methods. The most
      commonly exploited and recommended are the following:
       flow cytometry which detects EV passing through a laser beam. Modern flow cytometers
      may have many lasers and fluorescence detectors, which allow to label them with
      multiple conjugated antibodies using the same sample. Although widely used, the
      analysis using flow cytometry has limitations in detecting the smallest EV whose number
      and surface expression may be underestimated [3]. To circumvent this limitation,
      an alternative bead-based technique has been developed using specifically activated
      beads that capture EV with a cocktail of different exosome marker epitopes allowing
      subsequent simultaneous detection of multiple antigens [6];
       nanoparticle tracking analysis (NTA) which visualizes EV in the liquid phase by
      light scattering using a light microscope. A video is taken and the NTA software
      tracks the Brownian motion of individual vesicles and calculates their size and total
      concentration. NTA with fluorescent mode detects labeled vesicles and provides
      quantitative and qualitative analysis. NTA can detect vesicles smaller than those
      distinguished by conventional flow cytometry [3].
      1.3. EV and Cell-to-Cell Communication
      EV are shed into the extracellular environment under physiological and pathological
      conditions, and their release is increased by cellular stress conditions as inflammation,
      hypoxia, oxidative stress or infections [7]. Once released into the circulation, EV have a
      half-life ranging from a few minutes to a few hours, during which they can be taken up
      from cells by different mechanisms such as endocytosis or fusion with the membrane of the
      recipient cell [3]. Circulating EV mainly originate from platelets, erythrocytes, leukocytes,
      and endothelial cells; while urinary EV mainly derive from podocytes, tubular cells, and
      epithelial cells [8]. EV may transfer to the target cell receptors, allowing cell signaling
      in cells that are originally devoid of receptors or by enhancing their number. Moreover,
      EV may release or transfer proteins as cytokines and growth factors translating in the
      modulation of target cells and their extracellular environment. Furthermore, EV may
      transfer messenger RNAs (mRNAs) and microRNAs (miRNAs) to target cells, modulating
      their function or changing their phenotype [3].
      Int. J. Mol. Sci. 2021, 22, 4194 3 of 20
4195. EV in Physiological and Pathological Settings
      Based on their crucial role in cell-to-cell communication, EV have been involved in
      several processes such as coagulation and thrombosis, endothelial dysfunction, angiogenesis
      and immune modulation. Since all of these processes impact renal diseases, a brief
      discussion will be reported (Figure 1).
      Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 21
      may transfer messenger RNAs (mRNAs) and microRNAs (miRNAs) to target cells,
      modulating their function or changing their phenotype [3].
4196. EV in Physiological and Pathological Settings
      Based on their crucial role in cell-to-cell communication, EV have been involved in
      several processes such as coagulation and thrombosis, endothelial dysfunction,
      angiogenesis and immune modulation. Since all of these processes impact renal diseases,
      a brief discussion will be reported (Figure 1).
      Figure 1. EV in physiological and pathological processes. (A) EV and coagulation: EV enriched in phosphatidylserine (PS)
      and tissue factor (TF) can promote the activation of the coagulation cascade as well as platelet aggregation. (B) EV and
      endothelial activation: EV can modulate vasodilatation and vasoconstriction by activating endothelial cells, releasing
      cytokines, reducing nitric oxide (NO) production and increasing reactive oxygen species (ROS) production. (C) EV and
      angiogenesis: EV can modulate angiogenesis by increasing ROS production, downregulating the VEGF pathway,
      modulating the migration and proliferation of endothelial cells and matrix degradation. This figure has been created using
      Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License;
      https://smart.servier.com.
      2.1. EV and Coagulation
      Several autoimmune and renal diseases are associated with increased thrombotic risk
      and thrombotic events. EV are involved in the coagulation cascade as carriers of
      phosphatidylserine (PS) and tissue factor (TF) and are able to promote the activation of
      the coagulation cascade. PS can act as a binding site for the coagulation factors II, Va and
      Xa, enhancing the association between factor Xa and Va and the subsequent conversion
      of prothrombin to thrombin [9]. TF expressed on the EV surface can activate the extrinsic
      coagulation pathway by binding factor VIIa [10]. Interestingly, platelet-derived EV have
      Figure 1. EV in physiological and pathological processes. (A) EV and coagulation: EV enriched in
      phosphatidylserine (PS) and tissue factor (TF) can promote the activation of the coagulation cascade
      as well as platelet aggregation. (B) EV and endothelial activation: EV can modulate vasodilatation
      and vasoconstriction by activating endothelial cells, releasing cytokines, reducing nitric oxide (NO)
      production and increasing reactive oxygen species (ROS) production. (C) EV and angiogenesis:
      EV can modulate angiogenesis by increasing ROS production, downregulating the VEGF pathway,
      modulating the migration and proliferation of endothelial cells and matrix degradation. This figure
      has been created using Servier Medical Art templates, which are licensed under a Creative Commons
      Attribution 3.0 Unported License; https://smart.servier.com (accessed on 28 March 2021).
      2.1. EV and Coagulation
      Several autoimmune and renal diseases are associated with increased thrombotic
      risk and thrombotic events. EV are involved in the coagulation cascade as carriers of
      phosphatidylserine (PS) and tissue factor (TF) and are able to promote the activation of
      the coagulation cascade. PS can act as a binding site for the coagulation factors II, Va and
      Xa, enhancing the association between factor Xa and Va and the subsequent conversion
      of prothrombin to thrombin [9]. TF expressed on the EV surface can activate the extrinsic
      coagulation pathway by binding factor VIIa [10]. Interestingly, platelet-derived EV have
      higher pro-coagulant activity compared with activated platelets, possibly due to their
      enrichment in phosphatidylserine, glycoprotein IIb/IIIa, factor Xa, and P-selectin [11].
      Moreover, EV-mediated thrombotic events can be also promoted by their expression of the
      multimers of vonWillebrand factor and stabilization of platelet aggregation (Figure 1A) [9].
      2.2. EV and Endothelial Dysfunction
      Endothelial dysfunction is frequently associated with the pathogenesis of autoimmune
      diseases: EV contribute to endothelial regulation in physiological and pathological
      conditions. EV can reduce endothelial and macrophage nitric oxide production, controlling
      vasodilation. EV can also carry thromboxane A2 acting as vasoconstrictor and platelet
      Int. J. Mol. Sci. 2021, 22, 4194 4 of 20
      aggregation factor [1]. In addition, EV may increase the production of reactive oxygen
      species (ROS). Finally, as occurs in systemic vasculitis, activated neutrophils release EV expressing
      the myeloperoxidase (MPO) which by activating the myeloperoxidase-hydrogen
      peroxide-chloride system, leads to endothelial damage (Figure 1B) [12].
      2.3. EV and Angiogenesis
      Angiogenesis, defined as the growth of new blood vessels from pre-existing ones, is
      frequently involved in the pathogenesis of autoimmune diseases. EV may be involved
      in several angiogenesis-associated events [13] as matrix degradation, endothelial progenitor
      recruitment, and differentiation, migration and proliferation of endothelial cells.
      Endothelial- and macrophage-derived EV contain pro-angiogenic enzymes (MMP-2, MMP-
      9, MT1-MMP) which promote extracellular matrix remodeling and favor endothelial cell
      invasion [14]. On the contrary, lymphocyte-derived EV can inhibit angiogenesis by generating
      ROS and down-regulating the VEGF pathway (Figure 1C) [15].
      2.4. EV and Immune System Modulation
      EV may affect the immune response by activating innate and adaptive immunity
      and by participating in the formation of the immune complexes (IC), by acting as autoantigens.
      Immune cells release immunocompetent EV, which can modulate the immune
      response by regulating antigen presentation, NK/T cell activation, T cell polarization and
      immunosuppression (Figure 2) [16].
      In the immune system, natural killer (NK) cells have a role in innate immunity, whereas
      B and T cells are an essential part of adaptive immunity.
       Antigen presentation B cells can recognize foreign antigens, while T cells require
      antigen-presenting cells (APCs) for antigen recognition. Major histocompatibility
      complex class I (MHC I) and class II (MHC-II) present antigens to CD8+ and CD4+
      T cells, thereby activating the immune response. APCs- and B cells-derived EV
      express the MHC-I, MHC-II and the T-cell costimulatory molecules, thus may take
      part in the antigen presentation process and in the CD8+ and CD4+ T cell activation
      (Figure 2A) [17].
       Source of self-antigens and IC formation EV participate in the formation of IC. Indeed,
      EV can express both self-antigens and MHC complexes and may activate autoreactive
      T-cells in autoimmune disease. As an example, the synovial fluid of patients with
      rheumatoid arthritis contains IC composed of platelet-derived EV and autoantibodies
      against citrullinated peptides [18]. Similarly, in systemic lupus erythematosus (SLE),
      EV carry nuclear molecules, which represent a potential source of autoantigens and
      participate in IC formation. Furthermore, EV-associated ICs may affect the recognition
      and clearance of EV by phagocytes, leading to the accumulation of cell debris and
      triggering the autoimmune response (Figure 2B,C) [19].
       Role of adjuvants in innate immune response Leukocyte-derived EV activate the
      endothelium by upregulating adhesion molecules and releasing cytokines. This
      leads to leukocyte recruitment via platelet-derived EV, which promotes monocyte
      adhesion to the endothelium [20]. Dendritic cell-derived EV increase the NK cytotoxic
      activity and stimulate the release of proinflammatory cytokines by epithelial cells
      (Figure 2D) [21].
       Role in complement activation When the complement system undergoes activation, the
      membrane attack complex may be set down on blood cells and complement-coated EV
      may be released. C3-positive EV reflect the activation of the alternative pathway of the
      complement, while C1q-positive EV reflect the activation of the classic pathway [22].
      Moreover, EV may express complement regulators on their surface (complement
      receptor type 1, membrane cofactor protein, decay-accelerating factor also denoted as
      CD59), thereby inhibiting the membrane attack complex (Figure 2E) [23].
      Int. J. Mol. Sci. 2021, 22, 4194 5 of 20
      Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 21
      Figure 2. EV and immune system modulation: (A) Self-antigen presentation: antigen-presenting cells
      (APC) can vehicle self-antigens and release extracellular vesicles (EV) which present autoantigens
      to autoreactive T cells. (B) Self-antigen transfer: EV carrying self-antigens transfer them to APC
      which present them to T cells, triggering an autoimmune response. (C) Immune complex formation
      (IC): EV can participate in IC formation by carrying self-antigens that are bound by circulating
      autoantibodies. (D) Activation of innate immunity: EV can stimulate cytokine release and the upregulation
      of adhesion molecules on endothelial cells, which favor circulating immune cell recruitment.
      (E) Interaction with the complement system: the complement cascade can be activated on circulating
      leukocytes which can then release EV exposing complement molecules (e.g., C3, C1q). EV can also
      convey complement inhibitors and modulate the complement system. This Figure has been created
      using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0
      Unported License; https://smart.servier.com (accessed on 28 March 2021).
      Int. J. Mol. Sci. 2021, 22, 4194 6 of 20
4197. EV and Renal Intercellular Communication
      Recent studies have shown the relevance of EV in renal intercellular communication.
      Although their role has not been yet fully understood, their potential application as diagnostic,
      prognostic, or therapeutic biomarkers for various kidney diseases is currently being
      explored [8].
      In physiological conditions, the glomerular filtration apparatus prevents circulating
      EV from reaching the lumen of renal nephrons, thus circulating EV may stimulate kidney
      cells facing the vascular compartment and the immune cells [24]. However, EV are released
      from renal cells into the renal tubule and can be detected in urine. The release of renal
      EV may vary during renal disease, thus urinary EV have been proposed as potential
      biomarkers of kidney injury. Urinary EV are largely composed of EV released from renal
      cells as confirmed by proteomic analysis. Urinary EV carry specific proteins, mRNAs
      and miRNAs, which reflect their cellular origin. Indeed, EV from glomerular podocytes
      contain podocin and podocalyxin [25], EVs from proximal tubular cells express cubilin,
      megalin, aminopeptidase [26] and aquaporin-1; EVs from the Henle’s loop contain type 2
      Na-K-2Cl cotransporter, CD9 and Tamm–Horsfall protein [27]; EVs from collecting ducts
      carry AQP-2 and mucin-1 [28]. The majority of urinary EV are released from the first part of
      the renal nephron, with a limited contribution from the lower urinary collecting system [29].
      Interestingly, EV may have a role in cell-to-cell communication between the proximal and
      distal renal tubules. EV released from glomerular podocytes, via the urine flux, reach and
      can be taken up by the epithelial cells of the distal tubule and the collecting duct [30]. A
      recent study has investigated a potential hormonal mechanism that regulates EV uptake. It
      has been demonstrated that in vitro stimulation with desmopressin selectively stimulates
      the EV uptake in tubular cells, while a vasopressin antagonist reduced in vivo the uptake
      of EV injected within the renal tissue [31]. Moreover, EV may take part in the regulation of
      renal inflammation. Indeed, it was reported that EV from proximal tubular cells cultured
      in the presence of a dopamine receptor antagonist are able to reduce ROS production in
      distal tubular cells [32]. Interestingly, EV can modulate renal tissue repair and fibrosis
      after renal damage [33–35]. In vitro studies have demonstrated that hypoxic conditions
      increase the release of tubular cell-derived EV, which induced fibroblast activation and
      proliferation by the transfer of TGF mRNA [36]. EV may also control the ion transport, as
      EV from proximal tubule cells can decrease ENaC activity in the distal tubule and collective
      ducts [37]. Moreover, EV can also mediate the transfer of aquaporin 2 from cells of the
      upper collective duct to those of the lower collective duct, increasing water transport in
      the recipient cells [38]. Urinary EV may also exert protection against bacterial infection
      of the urinary tract. As a matter of fact, urinary EV carry antimicrobial peptides and can
      inhibit the growth of the most common urinary pathogen Escherichia coli by inducing their
      lysis [39]. Mesenchymal-to-epithelial transition is a crucial process in kidney development
      and regeneration. It has been reported that this process is mediated by tubular epithelial
      cell-derived EV which carry a specific subset of miRNAs, driving the differentiation of
      mesenchymal stem cells into epithelial cells [40]. Interestingly, mesenchymal stem cells
      and endothelial progenitor cells release EV that induce nephron regeneration and repair by
      promoting tubular proliferation and by inhibiting apoptosis [41,42].
4198. EV in Renal Disease
      4.1. Antiphospholipid Syndrome
      Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized
      by recurrent arterial or venous thrombosis and/or obstetric complications in the presence
      of antiphospholipid antibodies (aPL). APS is the most common cause of acquired
      thrombophilia and is associated with decreased survival. A severe form of APS, termed
      catastrophic antiphospholipid syndrome, occurs in <1% of patients with aPL and is associated
      with high mortality [43]. Despite the clinical relevance of this syndrome, its
      pathogenesis is not yet fully understood and recent studies have focused on EV as potential
      damage mediators.
      Int. J. Mol. Sci. 2021, 22, 4194 7 of 20
      Various studies have reported an increased level of EV in APS patients. Štok et al. [6]
      have compared APS patients, patients with idiopathic thrombosis and healthy controls and
      found that circulating EV were increased in patients with a history of thrombotic events
      (APS patients and patients with idiopathic thrombosis) compared to healthy subjects,
      suggesting a chronic cell activation even in the absence of an acute thrombotic event.
      This study demonstrated the presence of platelet, endothelial cell, lymphocyte, antigenpresenting
      cell-derived EV in patients with a history of thrombosis as well as in healthy
      controls. These EV express molecules involved in platelet/endothelial function, immune
      regulation extracellular matrix regulation and cell-to-cell adhesion. The analysis of EV
      surface proteins demonstrated an increased expression of CD8, CD44, CD133/1 and CD62P
      in the aPL patients. The increased expression of CD133/1 and CD62P on the EV surface in
      APS patients could reflect the increased endothelial and platelet activation, respectively, and
      their possible contribution to the thrombotic events [6]. Endothelial- and platelet-derived
      EV were increased in aPL patients, suggesting a chronic activation of endothelial cells and
      platelets. Interestingly, a correlation between the level of endothelial-derived EV and the
      level of anti-2GPI has been demonstrated which closely correlates with thrombosis [44].
      Breen et al. [45] have shown an increased level of endothelial- and platelet-derived EV
      in aPL patients compared with healthy subjects, while no difference between obstetric APS
      or asymptomatic aPL patients was detected. Moreover, plasma from patients with APS
      and from patients with SLE aPL+ or SLE aPL- increased the release of EV from cultured
      endothelial cells compared to the plasma of healthy subjects. Of note, only plasma from
      APS patients caused the release of EV with significant procoagulant activity [46]. aPL
      induces tissue factor (TF) synthesis in endothelial cells in vitro, and it has been reported
      that TF+ EV are elevated in aPL+ patients [44]. In particular, TF expression on endothelialderived
      EV is increased in APS patients compared to healthy subjects [47]. Moreover,
      Willemze et al. [48] have demonstrated that EV from APS patients display a higher TF
      activity compared to asymptomatic aPL+ patients.
      More recently, Mobarrez et al. [19] have compared anti-2GPI-positive SLE patients,
      aPL-negative SLE patients and healthy controls. They found that SLE patients are depleted
      of 2GPI-positive EV when compared to healthy subjects, and their level is particularly
      low in anti-2GPI-positive patients. They also found that 2GPI preferentially binds to the
      phosphatidylserine (PS)-positive EV, thus suggesting that anti-2GPI antibodies may bind
      to the 2GPI-PS complexes on EV resulting in the loss of EV 2GPI expression. 2GPI
      promotes the clearance of PS+ EV, thus the increased number of PS-negative EV may act as
      a possible source of autoantigens and thus trigger the autoimmune response [19].
      Regarding obstetric APS, pregnant women with APS had increased PS+ EV, endoglin+
      EV and endothelium-derived EV compared to healthy controls in the first and second
      trimester of pregnancy. Conversely, in the third trimester, higher levels of TF+ EV and
      platelet-derived EV can be detected. According to the authors, this finding could reflect the
      activation of both endothelial cells and platelets during pregnancy. In particular, high-risk
      APS patients (triple aPL positivity plus vascular thrombosis and/or severe pregnancy
      complications/placental insufficiency) have higher endoglin+ EV, TF+ EV and platelet
      derived-EV in all three trimesters, sustaining a major vascular activation. Interestingly,
      endoglin is expressed by vascular endothelium and by syncytiotrophoblasts and altered
      levels of soluble endoglin are linked to vascular disorders as pre-eclampsia [49].
      In conclusion, patients with aPL have an elevated level of circulating EV, which may
      reflect a state of systemic vascular activation. EV have been shown to act as procoagulant
      and proinflammatory mediators, thus their level may correlate with the thrombotic risk.
      However, at present, a clear relationship between elevated EV levels and thrombotic events
      is still missing (Table 1).
      Int. J. Mol. Sci. 2021, 22, 4194 8 of 20
      Table 1. Extracellular vesicles in antiphospholipid syndrome (APS).
      Study EV Biomarkers Cellular Origin of EV Study Findings Reference
      Štok, U.; et al.
      CD8, CD44, CD133/1,
      CD62P
      Platelets, endothelial
      cells, lymphocytes,
      antigen-presenting cells
      EV increased in patients with thrombotic events
      EV reflect endothelial and platelet chronic
      activation
      [6]
      Chaturvedi, S.; et al.,
      Breen, K.A.; et al.
      CD41, CD61, CD51,
      CD105
      Endothelial cells,
      platelets
      EV increased in aPL+ patients
      EV reflect endothelial and platelet chronic
      activation
      [44,45]
      Chaturvedi, S.; et al.,
      Willemze, R.; et al. Tissue factor (TF) Endothelial cells TF + EV increased in APS
      TF activity increased in EV from aPL+ patients [44,48]
      Mobarrez, F.; et al. 2GPI+ EV 2GPI+ reduced in SLE aPL+
      Anti-2GPI may bind to 2GPI expressed by EV [19]
      Campello, E.; et al.
      Phosphatidylserine (PS),
      Endoglin,
      Tissue factor (TF)
      Endothelial cells,
      platelets
      PS+ EV, endoglin+ EV and endothelium-derived
      EV increased in 1st and 2nd trimester of
      pregnancy;
      TF+ EV and platelet-derived EV increased in 3rd
      trimester of pregnancy
      Correlation with thrombosis and systemic platelet
      and endothelial activation in obstetric APS
      [49]
      4.2. Systemic Lupus Erythematosus
      Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized
      by the production of autoantibodies against nuclear antigens and the deposition of immune
      complex (IC) leading to systemic inflammation and tissue damage. Lupus nephritis (LN)
      is one of the most severe organ manifestations and a primary cause of morbidity and
      mortality [50].
      Studies on circulating EV in SLE patients revealed divergent results regarding their
      circulating level and characterization. EV were found increased in SLE [51,52], or decreased
      compared to healthy controls [53]. Those different results reflect the lack of standardized
      methods for EV evaluation and characterization.
      In SLE, circulating EV expose chromatin on their surface and may represent a source
      of nuclear antigens which can bound both the IgG and the complement, resulting in the
      formation of IC, which correlate with the disease activity and the vascular damage [52,54].
      Circulating EV can bind both IgG and IgM to form immune complexes (EV-ICs) and
      EV-IgG+ were positively correlated with the disease activity. Platelet-derived EV (PEV),
      mainly PEV-IgG+, stimulated monocytes in vitro changing their phenotype and promoting
      their inflammatory response [52]. Several studies have supported the role of the deoxyribonuclease
      DNASE1L3 as a genetic determinant of susceptibility [55,56]. Sisirak et al. [57]
      have found that DNASE1L3 can digest chromatin in apoptotic cell-derived EV and, in the
      absence of DNASE1L3, EV-associated DNA may gather in an extracellular environment
      and promote autoantibody production by autoreactive B cells. DNASE1L3 is produced by
      dendritic cells and macrophages, and its circulating level was inversely correlated with
      anti-dsDNA level [57].
      Platelet activation plays a key role in the pathogenesis of SLE. They promote T and
      B cell activation, NETosis, type I IFN production, and dendritic cell activation resulting
      in systemic organ damage. Platelet-derived EV are the most prevalent circulating EV in
      healthy subjects, while conflicting results have been reported regarding platelet-derived
      EV in SLE. Burbano et al. [52] have found an increased number of circulating EV (mainly
      platelet-derived EV) in patients with SLE compared to healthy controls. However, plateletderived
      EV level was unrelated to disease activity measured with SLEDAI score. Lopez
      et al. [58] have observed an increased level of platelet-, monocyte- and T lymphocytederived
      EV. Interestingly, the authors found that EV level is influenced by the disease
      activity and is related to the activation status of blood parental cells. They also reported
      that glucocorticoid therapy may influence EV production and T cell activation, as EV
      from patients treated with glucocorticoids induced the upregulation of CD25 and the
      Int. J. Mol. Sci. 2021, 22, 4194 9 of 20
      accumulation of IL-10 in T cells [58]. Moreover, circulating platelet-derived EV correlated
      with endothelial-independent vasodilatation in SLE [59].
      Patients with SLE have increased cardiovascular risk due to platelet activation and
      systemic endothelial activation. EV may be involved in this process, as EV and EV-ICs
      from SLE patients can activate endothelial cells by increasing the expression of adhesion
      molecules (CD54, CD102), the production of chemokine (CCL2, CCL5, IL-6) and the
      adherence of monocytes. EV may also mediate endothelial injury by increasing endothelial
      permeability through the alteration of cytoskeletal proteins leading to adhesion and
      migration of monocyte to the inflamed organs [60].
      Polymorphonuclear leukocytes (PMNs) are greatly involved in SLE pathogenesis, as
      PMNs showed generalized hyperactivity, with enhanced apoptosis and increased production
      of neutrophil extracellular traps (NETs) [61]. Moreover, increased oxidative stress
      has been observed in SLE, which may contribute to immune dysregulation [62]. Winberg
      et al. [63] have found that EV from SLE patients induced ROS production in the patient’s
      own PMNs re-suspended in autologous serum, particularly in patients with low circulating
      C3 level, which reflects disease activity. The ROS production partly depends on EV properties,
      serum components (including autoantibodies) and PMN hyper-responsiveness [63].
      In vitro studies have shown the proinflammatory effect of EV on blood-derived plasmacytoid
      dendritic cells and myeloid dendritic cells by increasing the expression of costimulatory
      molecules (CD40, CD80, CD83, CD86) and the release of proinflammatory cytokines
      (IL-6, TNF, IFN). Moreover, EV enhanced the formation of neutrophil extracellular traps
      (NETs) which may represent a source of nuclear autoantigens thus contributing to the
      pathogenesis of SLE and renal inflammation [64]. EV from patients with active LN contain
      a higher level of acetylated chromatin compared to patients with remissive LN, without
      LN, or healthy controls. Rother et al. [65] have found that the degree of EV acetylated
      chromatin determines their strength to stimulate neutrophils to form NETs.
      According to proteomic and flow cytometry analysis, circulating EV in SLE patients
      have an increased content of IgG and galectin-3 binding protein (G3BP), a glycoprotein
      that may contribute to the pathogenesis of SLE. G3BP is induced by type I IFN and exhibits
      a high binding capacity toward components of the glomerular basement membrane
      (GBM) [66], including collagen IV, fibronectin and galectin-3. Interestingly, patients suffering
      from lupus nephritis show a glomerular G3BP/IgG co-localization pattern specifically
      in the GBM, suggesting the presence of G3BP in the IC delivered either by EV from the
      circulation or locally formed [67]. In SLE patients, in vitro stimulation of peripheral blood
      mononuclear cells (PBMCs) with TLR-9 agonist increases the release of EV expressing both
      G3BP and dsDNA. This was particularly relevant in patients with active LN, compared to
      healthy donors [68].
      Urinary EV may reflect structural damage and renal dysfunction, and they have been
      investigated as potential LN diagnostic and prognostic biomarkers. Urinary podocytederived
      EV reflecting the glomerular podocyte damage are increased in LN and SLE
      patients compared to healthy controls. Interestingly, urinary EV level is correlated to the
      SLE disease activity index (SLEDAI) score, anti-dsDNA antibodies titer, proteinuria and
      histopathological lesions [69]. A recent study demonstrated that urinary EV expressing
      the high-mobility group box 1 molecule (HMGB1) are higher in SLE patients with active
      LN than in those without renal involvement, and correlate with proteinuria. HMGB1 is
      involved in the pathogenesis of several autoimmune diseases and it may be an important
      mediator in LN. Indeed, its expression is increased in glomerular endothelium and
      mesangium, and its blood and urinary level is increased in LN [70].
      Several studies have identified EV-derived miRNA as markers of renal damage which
      can also discriminate active LN [71]; miR-21, miR-150, and miR-29c were correlated to
      renal fibrosis and could predict the progression to the end-stage renal disease [72,73]. A
      unique circulating miRNA expression profile was detected in class IV LN [74] and urinary
      EV-derived miRNA have been proposed as peculiar biomarkers of class IV LN [75].
      Int. J. Mol. Sci. 2021, 22, 4194 10 of 20
      Recently, Garcia-Vives et al. investigated the miRNA expression profile of urinary EV
      in proliferative LN as a new potential prognostic biomarker. Patients with clinical responses
      to therapy are characterized by an increased level of miR-31, miR-107, and miR-135b-5p
      in urine and in renal tissue (mostly localized in epithelial tubular cells), compared to nonresponder
      patients. In vitro stimulation of tubular epithelial cells with proinflammatory
      cytokines increases the release of these miRNA, which can be taken up by endothelial cells
      and mesangial cells in responder patients [76].
      Recent studies have evaluated the role of mitochondria in autoimmune diseases.
      Activated cells can release EV containing mitochondria or free mitochondria which may
      stimulate immunity. Mobarretz et al. [77] have demonstrated the presence of circulating
      particles (approximately 3 m), and among them, a population of large EV carrying
      mitochondrial molecules (mitoEV) were found that both increased and associated with
      the disease activity in SLE patients. Furthermore, patients suffering from active LN have
      higher levels of mitoEV and IgG-coated mitoEV, suggesting that they may contribute to
      the formation of IC and thus be involved in renal damage [77].
      In conclusion, EV may play a crucial role in the pathogenesis of SLE, and particularly
      in LN-associated renal injury. For these reasons, EV have been proposed as potential
      biomarkers of disease in SLE patients as well as early biomarkers of renal damage in LN
      (Table 2).
      Table 2. Extracellular vesicles in systemic lupus erythematosus.
      Study EV Concentration Cellular Origin of EV EV Pathological Significance Reference
      Burbano, C.; et al.
      Increased in SLE
      compared to healthy
      controls
      platelet
      Formation of immune complexes,
      source of nuclear antigens,
      correlation with disease activity
      [52]
      López, P.; et al.
      Increased in SLE
      compared to healthy
      controls
      platelet, monocyte,
      T lymphocyte
      EV level correlated with:
      disease activity,
      glucocorticoid therapy,
      endothelial vasodilatation
      [58]
      Atehortúa, L.; et al. Endothelial cell activation,
      endothelial injury, [60]
      Winberg, L.-K.; et al.,
      Dieker, J.J.; et al.,
      Rother, N.; et al.
      In vitro stimulation of
      polymorphonuclear leukocytes with EV
      from SLE patients increased ROS
      production
      EV promote neutrophil activation and
      NETs production
      [63–65]
      Nielsen, C.T.; et al.,
      Rasmussen, N.S.; et al.
      IgG/galectin-3 binding protein (G3BP)+
      EV are involved in the pathogenesis of
      lupus nephritis
      [67,68]
      Lu, J.; et al.,
      Vanegas-García, A.; et al.
      Urinary podocyte-derived
      EV increased in SLE Urinary EV
      Urinary podocyte-derived EV level
      correlated with systemic disease activity
      and renal injury
      Urinary EV high-mobility group box 1
      molecule (HMGB1)+ were found to be
      higher in lupus nephritis
      [69,70]
      Felip, M.L.; et al.,
      Solé, C.; et al.,
      Navarro-Quiroz, E.; et al.,
      Li, Y.; et al.,
      Garcia-Vives, E.; et al.
      EV derived miRNA
      miR-21, miR-150, and miR-29c, miR-31,
      miR-107, and miR-135b-5p correlated
      with renal injury in lupus nephritis
      [72–76]
      Mobarrez, F.; et al.
      EV containing
      mitochondrial molecules
      (mitoEV)
      mitoEV were associated with disease
      activity, immune complex formation and
      renal damage
      [77]
      Int. J. Mol. Sci. 2021, 22, 4194 11 of 20
      4.3. Thrombotic Microangiopathies
      Thrombotic microangiopathies include different diseases characterized by microangiopathic
      hemolytic anemia and thrombocytopenia also potentially involving the kidney.
      Thrombotic microangiopathies include Shiga toxin-producing Escherichia coli (STEC-HUS)
      and thrombotic thrombocytopenic purpura (TTP).
      Haemolytic uremic syndrome (HUS) is characterized by nonimmune microangiopathic
      haemolytic anemia, thrombocytopenia and acute kidney injury. Typical HUS is
      subordinate to Shiga toxin-producing Escherichia coli (STEC-HUS) infection, which first
      colonizes the intestine and produces the toxin which enters the bloodstream and causes
      renal injury.
      In STEC-HUS, EV mainly derive from platelets, monocytes, neutrophils and red blood
      cells. Ståhl et al. have found EV expressing TF and phosphatidylserine potentially involved
      in the formation of microthrombi. Moreover, in the acute phase of the disease, circulating
      EV derived from platelets, monocytes, and neutrophils show deposition of C3 and C9
      on their surface. Interestingly, EV also express phosphatidylserine, which activates the
      coagulation factor V and X, thus enhancing and promoting thrombosis [78]. Those findings
      may reflect the systemic complement activation and the role of EV in the inflammatory and
      thrombogenic events in HUS [10].
      In vitro experiments showed that whole blood incubated with Shiga-toxin and/or
      STEC-lipopolysaccharide increased the release of TF-positive EV, C3- and C9-positive EV
      derived from platelets, monocytes and red blood cells [10,22]. It has been speculated that
      activated complement factors carried by EV can be transferred to recipient cells, driving
      cell damage [79].
      Since STEC are non-invasive bacteria, a small amount of Shiga toxin is present in the
      circulation; however, EV may transfer the toxin to the kidneys via peritubular capillaries.
      EV containing Shiga toxin were found within the kidney into renal cells, and in vivo
      experiments showed that EV enriched in Shiga toxin can reach renal cells through the
      glomerular and tubular basement membranes. In vitro studies also demonstrated that EV
      undergo endocytosis in glomerular endothelial cells, leading to cell damage [80].
      Shiga toxin interaction with circulating cells is mediated by two different receptors:
      the globotriaosylceramide (Gb3) toxin receptor and TLR4. Shiga toxin can be taken up
      by cells after binding to Gb3 or by cellular uptake of EV carrying the toxin derived from
      different host cells. EV expressing Shiga toxin are taken up by both Gb3-positive and
      Gb3-negative recipient cells. However, only Gb3-positive host cells are susceptible to
      toxin-induced cellular damage, reduced cellular metabolism and protein synthesis [81].
      Of note, renal endothelial cells express both Gb3 and TLR4. In vitro experiments using
      soluble Shiga toxin have shown that TLR4 acts as a Gb3 coreceptor, thus facilitating renal
      cell injury [79].
      TTP is a rare microangiopathic hemolytic anemia in which mutations of vWF protease
      (ADAMTS13) or autoantibodies against ADAMTS13 lead to the deposition of von
      Willebrand factor (vWF) multimers within capillaries. Systemic endothelial cell injury and
      platelet aggregation activate systemic microthrombosis mainly involving the brain and
      the kidney.
      A comparison of circulating EV in TTP patients and healthy subjects has shown
      that platelet-derived EV are the most represented EV subtype in both groups. Moreover,
      in TTP patients the platelet-derived EV level is significantly higher, as are EV express
      markers of platelet activation such as CD62p. Interestingly, platelet-derived EV have a
      procoagulant activity greater than platelets due to phospholipid expression on their surface
      which triggers the coagulation cascade [82].
      Other studies have found higher levels of platelet-derived and endothelial-derived
      EV in TTP. Endothelial-derived EV have shown procoagulant and proadhesive roles as
      they express CD62E (E-selectin), VWF, intercellular adhesion molecule 1 (ICAM-1), platelet
      endothelial cell adhesion molecules (PECAM-1; CD31) and endoglin (CD105) [83]. Tati
      et al. [84] have shown that circulating endothelial-derived EV are coated with C3 and
      Int. J. Mol. Sci. 2021, 22, 4194 12 of 20
      C9 and complement activated on platelets and glomerular endothelium. Complement
      activation may represent an ancillary phenomenon to platelet activation and endothelial
      injury, and thus may drive the microangiopathic process [84].
      Unfortunately, only a few studies have investigated the role of EV in TTP, and future
      research is needed to better understand the pathogenesis of TTP (Table 3).
      Table 3. Extracellular vesicles in thrombotic microangiopathies.
      Disease Study Cellular Origin of EV EV Biomarkers EV Pathological Significance Reference
      STEC-HUS Ståhl, A.-L.; et al.,
      Arvidsson, I.; et al.
      platelets, monocytes,
      neutrophils
      Tissue factor,
      phosphatidylserine
      (PS), C3, C9
      Promotion of thrombosis
      EV reflect complement
      activation
      [22,78]
      STEC-HUS
      Varrone, E.; et al.,
      Ståhl, A.-L.; et al.,
      Johansson, K.; et al.
      EV carrying Shiga
      toxin
      Delivery system of Shiga toxin
      to the kidney
      involvement in renal cell injury
      [79–81]
      TTP Tahmasbi, L.; et al.,
      Jimenez, J.J.; et al.
      platelets,
      endothelial
      cells
      CD62E (E-selectin),
      VWF, intercellular
      adhesion molecule 1
      (ICAM-1), platelet
      endothelial cell
      adhesion molecule
      (PECAM-1; CD31)
      and endoglin
      (CD105)
      Pro-coagulant and pro-adhesive
      roles [82,83]
      TTP Tati, R.; et al. Endothelial cells C3, C9 EV reflect complement
      activation [84]
      4.4. ANCA-Associated Vasculitis
      Systemic vasculitis consists of different syndromes characterized by blood vessel
      inflammation and multiple organ involvement. Small vessel vasculitis is often associated
      with anti-neutrophil cytoplasmic antibodies (ANCA), predominantly IgG autoantibodies
      directed against neutrophil cytoplasmatic constituents such as proteinase 3 (PR3, named
      cANCA) and myeloperoxidase (MPO, named pANCA). ANCA-associated vasculitis (AAV)
      comprises microscopic polyangiitis (MPA), granulomatosis with polyangiitis (Wegener’s)
      (GPA), and eosinophilic granulomatosis with polyangiitis (Churg-Strauss) (EGPA). AAV
      can affect small vessels in different organs resulting in pauci-immune glomerulonephritis,
      vasculitis involving the respiratory tract, and is associated with an increased risk of systemic
      thrombosis [85]. Despite new treatment options that have recently improved AAV
      prognosis, early diagnostic and prognostic biomarkers are still an unmet need.
      Patients with AAV have an increased risk of thromboembolic events due to the hypercoagulable
      state [86]. ANCA can induce neutrophil activation, neutrophil degranulation
      and release of neutrophil extracellular traps (NETs), which are involved in the development
      of vasculitis lesions [87]. Moreover, complement activation via the alternative pathway
      is crucial for the development of the disease [88]. In vitro experiments have found that
      both pANCA IgG and cANCA IgG can stimulate C5a-primed neutrophils to produce TFexpressing
      EV and TF-expressing NETs which, in turn, promote thrombin generation and
      the activation of the coagulation cascade [89]. Notably, Mendoza et al. [90] have measured
      the TF activity of EV and found higher TF activity in patients with AAV and associated
      venous thromboembolism, compared to patients without thrombotic events [90].
      Recent studies have demonstrated a potential role of platelet- and neutrophil-derived
      EV in the pathogenesis of vasculitis. Polyclonal ANCAs isolated from patients and chimeric
      PR3–ANCA induces the release of EV from primed neutrophils in vitro. Moreover, these EV
      are enriched in PR3 and MPO, exhibit thrombin-generating capacity, can bind the endothelium
      and induce its activation, induce ROS production and the release of proinflammatory
      cytokines (IL-6, IL-8) [91].
      An increased level of circulating platelet-, neutrophil- and endothelial-derived EV in
      patients with vasculitis has also been found [92]. In particular, a high level of neutrophil-EV
      Int. J. Mol. Sci. 2021, 22, 4194 13 of 20
      during the acute phase of the disease which decreased after steroid treatment has been
      reported, reflecting the key role of neutrophil activation. Of note, endothelial-EV level
      correlated with the Birmingham Activity Vasculitis Score (BVAS) and acute phase reactants,
      suggesting their potential application as disease biomarkers [92].
      The kinin system contributes to the inflammatory response and the development of
      vasculitis. The kinins regulate local blood pressure, promote inflammation, and capillary
      leakage. The kinins achieve their effect through B2 and B1 receptors. The B2 receptor is
      constitutively expressed and involved in inflammation and hyperalgesia, while the B1
      is upregulated during chronic inflammation (such as vasculitis), and controls neutrophil
      migration. Kahn et al. [93] have found increased circulating leukocyte-derived EV bearing
      the B1-kinin receptor during vasculitis. In particular, neutrophil-derived EV bearing the B1
      receptor were found docking to glomerular endothelial cells in kidney biopsies of patients
      with AAV. In vitro experiments showed that neutrophil-derived EV transfer functional B1-
      receptors to wild-type human embryonic kidney cells, which suggests a similar mechanism
      in vivo and could potentially promote kinin-associated inflammation. The observation
      that the main inhibitor of the kinin system, named the C1-inhibitor, is also involved in the
      inhibition of the release of EV enriched in B1-receptor has suggested a novel therapeutic
      target in vasculitis [93].
      Prikryl et al. [94] recently performed proteomic profiling of urinary EV isolated from
      patients with AAV and renal involvement and in healthy controls. The study showed
      different levels of proteins potentially involved in AAV pathogenesis. As an example, they
      found a significantly decreased level of Golgi mannosidases, such as MAN1A1, both in
      urinary EV and in the whole urine in active AAV. Interestingly, MAN1A1 is involved in
      protein glycosylation, which is considered to be involved in autoimmune disease and T cell
      activation, supporting its role in the pathogenesis of AAV. Moreover, they showed different
      levels of proteins related to neutrophil activation and degranulation, platelet regulation
      and podocyte-associated proteins, to name a few [94].
      Several studies have also shown that EV may contain enzymes linked to lipid metabo
      lism. Indeed, a comparison between EV in active GPA and healthy controls showed an
      increased content of leukotriene (LT)B4 and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in
      EV from GPA patients. Moreover, neutrophils primed with GM-CSF, and stimulated
      with EV recovered from GPA patients, generate ROS and release dsDNA. Interestingly,
      in vitro-primed neutrophils were stimulated by LTB4 and 5-oxo-ETE, thus EV carrying
      lipid enzymes may contribute to AAV pathogenesis [95]. In a recent study, Surmiak
      et al. [96] stimulated human umbilical endothelial cells (HUVEC) with EV from anti-
      PR3-activated neutrophils and analyzed their miRNA and mRNA content. They found a
      miRNA/mRNA profile consistent with the release of proinflammatory cytokines, which
      may be involved in endothelial injury in vasculitis. The most increased cytokines were
      IL-8, IL-33, Dickkopf-related protein 1 (DKK-1), soluble interleukin (IL)-1 like receptor-1
      (ST2) and angiopoietin-2. Interestingly this cytokine profile is similar to the circulating
      cytokine profile in GPA patients [96].
      Recent studies have investigated the role of EV in mediating endothelial injury in MPA.
      EV can be taken up by glomerular endothelial cells in vitro and can increase the release of
      soluble cellular adhesion molecules (sICAM-1 and sVCAM-1) leading to the injury of the
      glomerular endothelial barrier. A sequencing analysis of EV miRNA cargo in MPA patients
      revealed a different miRNA profile in MPA patients. In particular, a correlation between
      miR-185-3p, miR-125a-3p and clinical parameters, such as BVAS and 24-h urine proteins,
      was reported. Thus, the EV-miRNA content has been proposed as a biomarker of renal
      involvement in MPA [97].
      Circulating EV expressing MPO are elevated in AAV patients compared with healthy
      subjects. Interestingly, MPO+ EV expressing inflammatory biomarkers such as PTX3 and
      HMGB1 are associated with disease activity. Of interest, PTX3 is released by neutrophils
      during the inflammatory process, while HMGB1 may be associated with renal injury in
      AAV [98]. HMGB1 enhanced neutrophil activation and migration towards glomerular
      Int. J. Mol. Sci. 2021, 22, 4194 14 of 20
      endothelial cells in the presence of ANCA, leading to glomerular cell injury and the release
      of TF-positive EV and endothelin-1, which is involved in the fibrogenesis [99].
      The comparison of circulating EV expressing MPO in AAV patients and healthy
      controls reveals an increased expression of the complement components C3a and C5a on
      EV from AAV patients. Moreover, among AAV patients, C3a and C5a expression is higher
      in patients with active renal involvement compared to non-renal disease. Interestingly,
      the level of C3a and C5a expression on EV correlated with disease activity evaluated by
      BVAS [100].
      Platelet-derived EV were also found to be increased in AAV patients, particularly
      in MPO-positive patients with active disease in whom EV also expressed higher levels
      of chemokines, adhesion molecules, growth and apoptotic factors. Moreover, EV level
      correlated with the disease activity and the renal involvement, with serum creatinine and
      glomerular histologic lesions [101].
      Taken together, these results may provide insight into the role of EV in the pathogenesis
      of renal injury in AAV (Table 4).
      Table 4. Extracellular vesicles in ANCA-associated vasculitis.
      Study EV Biomarkers EV Cellular Origin Study Findings Reference
      Daniel, L.; et al. proteinase 3 (PR3),
      myeloperoxidase (MPO)
      EV released from primed
      neutrophils in vitro
      EV can induce endothelial activation,
      ROS production, cytokines release [91]
      Brogan, P.A.; et al.
      Platelets,
      neutrophils, endothelial
      cells
      EV level increased in vasculitis
      Decrease of neutrophil-derived EV
      after treatment
      Endothelial-derived EV correlated
      with disease activity
      [92]
      Kahn, R.; et al. B1 kinin receptor Leukocytes
      EV level increased in vasculitis
      Neutrophil-derived B1+ EV found
      on glomerular endothelial cells and
      renal injury
      [93]
      Prikryl, P.; et al. Urinary EV
      Proteomic EV profiling showed
      different regulation of proteins
      potentially involved in vasculitis
      pathogenesis
      [94]
      Surmiak, M.; et al.
      leukotriene (LT)B4,
      5-oxo-eicosatetraenoic
      acid (5-oxo-ETE)
      EV enriched in LTB4 and 5-oxo-ETE
      in granulomatosis with polyangiitis [95]
      Wang, Y.; et al.
      Sequencing analysis of EV miRNA
      cargo in microscopic polyangiitis
      identified a correlation between
      miR-185-3p, miR-125a-3p and both
      the clinical activity score and
      proteinuria
      [97]
      Manojlovic, M.; et al.
      myeloperoxidase (MPO),
      PTX3, high mobility group
      box 1 (HMGB1)
      PTX3+ and HMGB1+ EV correlated
      with disease activity
      HMGB1 potentially associated with
      renal injury
      [98]
      Antovic, A.; et al. myeloperoxidase (MPO),
      C3a, C5a
      MPO C3a+ and C5a+ EV increased
      in vasculitis, particularly in patients
      with renal involvement
      C3a and C5a expressed on EV
      correlated with disease activity
      [100]
      Miao, D.; et al.
      chemokines, adhesion
      molecules, growth and
      apoptotic factors
      Platelets
      Increased EV in vasculitis
      EV correlate with disease activity
      and renal injury
      [101]
      Int. J. Mol. Sci. 2021, 22, 4194 15 of 20
4199. Conclusions
      Recent studies have demonstrated the relevance of EV in physiological and pathological
      processes [7]. Due to their role in immune system modulation, EV are considered
      crucial actors in the pathogenesis of autoimmune diseases [1].
      Although current treatment options have improved their prognosis, autoimmune
      conditions remain rare diseases with high mortality and morbidity, particularly when
      the kidney is involved. Currently, kidney biopsy is the most important diagnostic and
      prognostic tool; however, recent investigations have focused on the identification and
      development of non-invasive biomarkers for early diagnosis and for the assessment of the
      disease activity.
      Recently, the impact of EV in autoimmune disease with renal involvement has been
      investigated [3]. EV numbers and features seem to correlate with pathological processes.
      In addition, EV have been reported to enhance the autoimmune process and promote
      renal injury. In APS, the EV circulating level has been shown to reflect endothelial and
      platelet chronic activation and may correlate with thrombotic events [6,44,49]. Moreover,
      EV can actively contribute to SLE pathogenesis as an autoantigen source, part of the
      immune complexes, endothelial and leukocytes activation promoters [52,63]. Of interest,
      the galectin-3 binding protein (G3BP)+ EV and urinary high-mobility group box 1 molecule
      (HMGB1)+ EV seem to be involved in lupus nephritis pathogenesis [67,68,70]. Likewise,
      EV contribute to renal injury in STEC-HUS [79–81]. In ANCA-associated vasculitis, EV are
      involved in endothelial activation and correlate with disease activity [91,94]. Of interest,
      HMGB1+ EV and B1-receptor + EV can be directly involved in renal cell injury and
      have been proposed as a novel therapeutic target [93,98]. Moreover, miRNA carried
      by circulating EV can be investigated as novel diagnostic biomarkers for both lupus
      nephritis [72,74,76] and ANCA-associated vasculitis [97].
      Encouraging discoveries on EV as diagnostic tools have been provided; however, being
      a recent research field, contradictory results reflecting the lack of standardized evaluation
      methods make their application in the clinical practice still challenging. Future studies
      should focus on defining standardized methods of EV collection and cargo evaluation. Of
      note, interesting observations have been reported in the research field, which need further
      investigation to be transferred in clinical settings.
      However, the rareness of these diseases made it difficult to investigate the potential
      clinical impact of EV as diagnostic and prognostic tools in large clinical studies. For this
      reason, multicenter studies are needed to collect relevant data.
      Despite these limitations, future studies on the role of EV in renal pathology should
      be pursued to better identify new targets in autoimmune diseases. Overall, the future
      challenge is to develop tools exploiting EV as diagnostic biomarkers, therapeutic targets or
      drug vectors for novel treatment options in autoimmune renal diseases.
      Author Contributions: M.M. contributed to data curation and writing the original draft; G.C. contributed
      to editing; M.F.B. contributed to writing, visualization, founding and editing. All authors
      have read and agreed to the published version of the manuscript.
      Funding: This work has been supported by grants obtained by MFB from Ministero dell’Istruzione,
      Università e Ricerca (MIUR) ex 60%.
      Institutional Review Board Statement: Not applicable.
      Informed Consent Statement: Not applicable.
      Data Availability Statement: Not applicable.
      Conflicts of Interest: G.C. is a component of the Scientific Advisory Board of UNICYTE. The other
      authors have no conflict of interest.
      Int. J. Mol. Sci. 2021, 22, 4194 16 of 20
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