The term Acute kidney injury (AKI) indicates a sudden worsening of renal function due to acute renal damage, with consequent accumulation of nitrogenous waste products and alteration of hydrosaline and acid-base homeostasis. In the past decades, several criteria have been proposed in order to uniform the definition of AKI: the recommendation statements of 2012 Kidney Disease Improving Global Outcomes (KDIGO) Clinical Practice Guideline define AKI as a rise in the serum creatinine (sCr) level by 0.3 mg/dl within 48 hours, or a 1.5-fold increase from baseline within prior 7 days; or oliguria (<0.5 ml/kg/h for 6 hours). In addition, AKI is staged for severity – from mild stage 1 to most severe stage 3 – according to serum creatinine values and urine output, usually classifying patients requiring renal replacement therapy (RRT) as stage 3 KDIGO. Several causes may induce AKI in patients with or without underlying chronic kidney disease (CKD); a potential classification is based on pathophysiological mechanisms of renal injury such as kidney hypoperfusion (pre-renal AKI), parenchymal kidney diseases (intra-renal or parenchymal AKI, which includes acute tubular necrosis, ATN) and obstruction of the urinary tract (post-renal AKI). Incomplete recovery of an AKI event due to persistence of renal pathophysiologic process can lead to AKI-CKD transition, especially in patient with some degree of pre-existent CKD ( Figure 1 ). As for AKI epidemiology, incidence and prevalence are not well defined due to different AKI definitions. Despite these limitations, a metanalysis of Susantitaphong et al. (1) observed that in-hospital AKI incidence was 22% – using 2012 KDIGO AKI definition – while it reached 57% in intensive care units (ICU) according to the multinational AKI-EPI study (2). Overall AKI incidence seems to be rising in the United States and it is associated with higher health care costs, greater long-term care, increased risk of CKD and hospital mortality (3, 4). This increase especially affects Afro-American population, due to genetic factors which also condition a reduced number of nephrons. A large metanalysis (5) and recent studies (6) confirmed that Black race is an independent risk factor for AKI.

Similar to CKD, other factors associated to the rise of AKI incidence are older age, increasing burden of comorbidities (e.g. hypertension, diabetes mellitus, CKD, heart failure, sepsis, cancer), improved clinician’s awareness (leading to inclusion of less severe forms), growing use of nephrotoxic drugs and increasing frequency of surgical and angiographic procedures (7, 8). Among these, older age is strongly associated with AKI incidence through multifactorial mechanisms. Renal senescence reduces nephron number and functional reserve, predisposing to relapsing AKI episodes and also to maladaptive repair, incomplete recovery and AKI-CKD transition. This process can be considered as a form of accelerated renal senescence and will be analysed in a specific Section (9, 10).

Different causes related to ischemia-reperfusion injury due to hypovolemia and/or hypoperfusion, administration of nephrotoxic drugs and the presence of sepsis/septic shock have been identified as hallmarks for AKI development. Recent studies have also provided new insights into complex AKI pathophysiology, with remarkable progress especially in the field of sepsis-associated AKI (s-AKI).

Sepsis is a dysregulated immune response to infection that causes multiple organ dysfunction: the immune response after a septic insult is characterized by unbalanced hyperinflammation and immune suppression (11). Sepsis-associated excessive inflammation is sustained by several cell types including leukocytes (neutrophils, macrophages, natural killer cells), endothelial cells (EC), cytokines, complement products, and the coagulation system (12). The widely used concept of “cytokine storm” refers to the release of proinflammatory cytokines such as TNF-alpha, IL-1b, IL-12, and IL-18 that may contribute to organ injury (12). However, a complex network of several mediators is embedded in sepsis-associated multiple organ dysfunction such as the release of extracellular traps by neutrophils (NETs) (13), the complement activation resulting in the release of the anaphylatoxins C3a and C5a, the immunothrombosis in the microvasculature mainly triggered by tissue factor that initiates blood coagulation by forming a complex with clotting factor (F) VIIa, thereby inciting blood coagulation by activating FX and FIX (14). These alterations have been also recently described in COVID-19-associated AKI (15).

Sepsis represents nowadays the most frequent cause of AKI in critically ill patients admitted to Intensive Care Units (ICU) with pathogenic mechanisms similar to those described above for immune dysfunction. Indeed, experimental and clinical studies aimed to evaluate kidney perfusion clearly demonstrated that during sepsis, AKI can develop in the presence of a normal or even increased renal blood flow: this finding suggested the presence of other key mechanisms of tissue injury such as microvascular derangement, endothelial dysfunction, inflammation, metabolic reprogramming and sub-lethal injury of tubular epithelial cells, overthrowing the simplistic old paradigm based on kidney hypoperfusion (16, 17). Recent works have further shed light on these etiopathogenetic mechanisms: for example, interaction of damage-associated molecular pattern (DAMPs) and pathogen- associated molecular pattern (PAMPs) molecules with Toll-like Receptors (TLRs) expressed on renal tubular epithelial cells (RTEC) and other renal resident cells can activate several pro-inflammatory pathways (18) and mitochondrial DNA (mDNA) damage due to oxidative stress also plays a key role in inducing RTEC dysfunction (19). Basically, PAMPs and DAMPs can be freely filtered by glomeruli, a process favored by sepsis-induced increased permeability, thus reaching the tubular lumen and inducing functional alterations of RTECs; moreover, microvascular damage of ECs located in the peritubular capillaries may enhance metabolic alterations of RTECs (20).

Innate and adaptive immunity activation during AKI have been increasingly recognized to play an important role in AKI. Cross-talk between RTECs and immune cells through a network of cytokines seems to be essential to induce T cell phenotype changes which affect AKI evolution (21). In this setting an important role of IL-15 has been delineated. RTECs express IL 15 and its receptors and intrarenal IL 15 levels inversely correlate with AKI severity in experimental models. Inhibition of RTECs apoptosis during pathological stress appears to explain IL 15 protective effects (22) Furthermore, IL 15 can reduce extracellular matrix (ECM) synthesis by myofibroblasts and monocyte chemoattractant protein (MCP-1) release by macrophages and also inhibits Transforming Growth Factor-β (TGF-β) 1-induced RTEC epithelial-mesenchymal transition (EMT): these multiple anti-fibrotic effects suggest a role in the prevention of AKI-CKD transition (23, 24).

Despite progress in understanding AKI pathophysiology, however, efforts to develop targeted therapies has not led to robust results yet (16) and treatment is still mainly supportive (e.g. hemodynamic stabilization, dose adjustment or discontinuation of nephrotoxic drugs, antibiotic therapy in s-AKI) (25–28). Also in s-AKI, neither pharmacological approaches nor extracorporeal blood purification therapies aimed at removing PAMPs and DAMPs have led to a significant improvement of in-hospital AKI incidence and mortality (16).

In this setting, stem-cell (SC) therapy and SC-derived extracellular vesicles (EVs) represents a new frontier in the treatment of acute and chronic kidney disorders, because of their anti-inflammatory, immunomodulatory and regenerative properties. Recent studies observed the beneficial therapeutic properties of Mesenchymal Stromal Cells (MSCs) in ischemic AKI, renal transplantation, lupus nephritis and diabetic nephropathy (29, 30). These actions are mainly paracrine and mostly mediated by transfer of EVs containing microRNAs, mRNAs, and proteins that reprogram cell functions via immunomodulatory and regenerative effects (31), as detailed in the following section.

In this review, we summarize the state-of-the-art knowledge on EVs derived from different types of stem cells (SCs) as therapy of AKI, focusing on impact of EVs on different pathophysiological mechanisms underlying toxic, ischemic and s-AKI (29–31) and AKI-CKD transition.

SCs are unspecialized cells with self-renewal capacity, which can potentially differentiate into any cell type of organism. Their therapeutic potential is revolutionizing regenerative medicine and is providing promising applications also in the field of Nephrology (32).

Among progenitor cells, MSCs represent the most studied type over the last decades: MSCs are adult multipotent stromal cells with a high proliferative potential, derived from non-hematopoietic precursors. They can differentiate into mesenchymal (osteocytes, adipocytes and chondroblasts) and non-mesenchymal lineages (33). Initially found in bone marrow (BM) (34), they were subsequently isolated from multiple fetal and adult tissues such as adipose tissue (AD-MSCs), umbilical cord blood (UC-MSCs), fetal membrane (FM-MSCs) and human placenta (hP-MSCs) (35–37). This makes them one of the most accessible SC type and an attractive candidate source to develop products for cell therapies (38). Due to the ease of preparation, MSCs remain the most common option among cellular therapies and have already proven to be safe and effective in reducing AKI in experimental models and clinical trials, displaying cytoprotective, regenerative and immunomodulatory properties (39, 40).

Other types of SCs have been recently investigated as a therapy for AKI, including inducible Pluripotent Stem Cells (iPSCs). These are derived from differentiated adult cells (e.g. keratinocytes, fibroblasts) which are induced into pluripotency by exposing them to specific reprogramming factors through viral vectors and directed towards renal lineages such as podocyte progenitors (41). Administration of iPSC has proved to be effective in a rat model of AKI, reducing oxidative stress and inflammation (42).

Spermatogonial stem cells (SCCs) have also been shown to be capable of differentiating into pluripotent stem cell lines, converting into embryonic-like SCs and differentiating into renal tubular-like cells. Also, this type of SC has shown promise in restoring kidney function after AKI (43).

Endothelial progenitor cells (EPCs), a BM-derived progenitor type able to circulate in the bloodstream, play a major role in vascular integrity by protecting ECs and promoting angiogenesis and recovery also in AKI models (44).

Of interest, the beneficial effects of all these SC types are predominantly mediated by paracrine/endocrine actions, via secretion of growth factors and especially EVs. The latter represent crucial components of cellular secretome and mediate intercellular communication through transfer of bioactive molecules between originator and recipient cells, especially mRNAs and microRNAs (miRNAs), modifying phenotype and function of target cells (45, 46). As their parental cells, EVs are not immunogenic and may successfully activate regenerative processes in injured cells and tissues (47, 48).

Recently, EVs released from renal cells themselves have been investigated as therapy for AKI: kidney-derived MSCs (49), glomerular and tubular renal progenitors (50), renal tubular epithelial cells (RTEC) (51) and even urinary EVs (uEVs) from healthy subjects have shown initial promising results in this setting (52). The efficacy of SC-derived EVs has been demonstrated in different settings including numerous studies showing an interesting cross-kingdom communication: EVs from different eukaryotic and prokaryotic kingdoms are involved in many processes including host-pathogen interactions and modulation of cellular functions (53). Of note, some studies showed that exogenous dietary RNAs of plant and animal origin are protected from food processing and gut microenvironment through encapsulation within EVs. EV-carried RNAs (in particular miRNAs) are able to exert biological activities between the host and gut microbiota influencing organ function in the recipient after ingestion (54). Our group has already demonstrated the horizontal transfer of mRNAs and miRNAs from SC-derived EVs of human origin in rat cells: indeed, in a rat model of anti-Thy1.1-induced mesangioproliferative glomerulonephritis, EVs released from human Endothelial Progenitor Cells (EPCs) horizontally transferred to rat mesangial cells distinct mRNAs coding for Factor H, CD55 and CD59, thus inhibiting complement-induced apoptosis and C5b-9/C3 mesangial cell deposition (55). Moreover, the same type of EVs protected the kidney from ischemia-reperfusion injury in rats by delivering their RNA content, the miRNA cargo of which was shown to contribute to reprogramming hypoxic renal endothelial and tubular epithelial cells to a regenerative program (56).

EV-based therapeutic approach has some advantages compared to cell-therapy. First, EVs exhibit a superior efficacy profile as they pass through the blood-tissue barriers and efficiently reach injured cells (57). Second, no adverse immune responses have been reported in patients undergoing allogeneic administration of SC-EVs and no evidence of oncogenic potential of SC-EVs has been reported. In fact, they can inhibit tumor growth by interfering with cell cycle and inducing apoptosis and/or necrosis of cancer cells (58, 59). Thus, EVs represent a feasible, cell-free therapeutic alternative and their role has recently been investigated in several renal diseases (45, 46), especially in AKI. This is the setting in which therapeutic properties of SC-EVs and the rationale for their employment has been better defined and represents the main focus of this Review.

A growing body of pre-clinical studies indicates a potential therapeutic role of EVs derived from MSCs and other progenitor cell types especially in IRI-induced AKI. EVs can shuttle miRNAs and other genetic material into injured renal cells – such as RTEC and ECs – and epigenetically re-programme them. This leads to activation of signaling pathways, which exerts multiple beneficial effects within three main areas (60, 61) ( Figure 2 ).

The combination of these effects can heal injured RTECs and ECs, thus promoting regression of AKI (66). Of note, pre-treatment with RNAase consistently abolished these effects, indicating that mRNA and miRNA transfer from SCs to injured renal cells is crucial in this healing process (67). Ferguson et al. identified 23 top-miRNAs which account for over 79% of total miRNAs in MSC-derived exosomes and seem to mediate the predominant effects, targeting 5481 genes (68). A comprehensive review of miRNA repertoire carried by SC-derived EVs for AKI treatment has been recently published and it is beyond the scope of this work (69).

As previously mentioned, while most studies were performed with EVs derived from BM-MSCs, UC-MSCs and AD-MSC, other EV sources have been recently employed, including kidney resident populations (50, 70) and uEVs (71). Potential mechanisms of actions and main mediators, with a focus on miRNAs, will be briefly analyzed. A summary of the main studies on EVs as therapy of AKI is outlined in Table 1 , in which they are categorised according to type of originating cell type (49–51, 56, 65–67, 72–96).

Recent studies have demonstrated that maladaptive repair after an AKI episode can predispose to evolution towards CKD and end-stage renal disease (ESRD) (127–129). Different factors appear to contribute to maladaptive repair during AKI, including oxidative stress, DNA damage, microvascular rarefaction, tubular loss, early fibrosis induced by endothelial-to-mesenchymal transition (EndMT) and pericyte-to-mesenchymal transition (PMT), lymphocyte infiltrates, inflammatory cytokine storm (128, 129). Pathophysiology of AKI-CKD transition has been the focus of research and complement system activation is increasingly recognised as having an essential role in this inflammatory scenario, which is closely related to renal senescence (130). These aspects and the potential of EVs as a tool to treat AKI and prevent AKI-CKD transition will be analysed in the following sections.

The use of SC-derived EVs as a therapeutic tool to deliver growth factors, proteins and genetic material to injured renal resident cells is promising in different AKI fields: however, several obstacles still limit their translation to clinic and have been recently reviewed (162, 163).

Biochemical composition is not defined and can vary depending on parental cell but also on surrounding milieu (e.g. inflammation, hypoxia). EVs released from the same cell-type may even have contradictory effects: for example, hypoxic RTECs have proved beneficial in alleviating tubular damage and fibrosis but injured RTECs can also release EVs which contribute to amplify inflammation (164). Moreover, focusing on SC-derived EVs, a different phenotype may depend on donor characteristics (autologous vs. heterologous, age, gender, presence of comorbidities or transient inflammatory states, etc.)

Furthermore, EV production or uptake mechanisms by kidney resident cells or infiltrating inflammatory cells are not completely defined and an intact glomerular filtration barrier could prevent EVs from reaching podocytes and tubular cells (165).

Finally, lack of good manufacturing practice standards and high-scale EV production hinder clinical application. At the moment, the clinical use of EVs is not classified as cell therapy and their mechanisms of action look like more to administration of a drug rather than a real cell therapy. Moreover, despite the development of new isolation procedures, cGMP production of SC-derived EVs for clinical application seems still to depend on an ultracentrifugation step to be performed within a cell factory.

Despite these limits, EV-based therapy has many strengths and is opening new therapeutic perspectives for a condition currently treated only with supportive therapy.

In general, EV lipid and surface protein composition (e.g.CD47) limits phagocytosis by circulating monocytes and prolongs blood half-life if compared to liposomes or other nanoparticles employed to carry drugs. EVs usually express integrins and adhesion molecules which allow to enhance their homing to inflamed or injured tissue: moreover, EVs protect RNA from degradation after their intravenous administration and at tissue level.

Technologies such as tangential flow filtration (TFF) appears to allow large-scale production of high-quality, reproducible EVs from AD-MSCs, paving the way for potential widespread clinical application in AKI (166). Other technological advances may potentiate EV qualitative therapeutic properties. For example, EV encapsulation could make their therapeutic content (e.g. miRNAs, mRNAs, proteins) more protected and stable. Nanomedicine techniques may help engineering EV features (size, shape, surface charge) in order to enable them to pass specific biological barriers, including glomeruli. Decoy exosomes have been proposed to antagonize inflammatory mediators (167). Another therapeutic approach is that of transfecting MSCs with specific miRNA mimics in order to enrich them with selected miRNAs. These enriched EVs proved to be more effective than naïve ones, potentially allowing the use of a lower amount of them (168). Combination therapy of pulsed focused ultrasound (pFUS) and EVs has proved more effective than either approach alone in reversing AKI-related inflammation through suppression of heat shock protein 70 – mediated NLPR3 inflammasome (169).

An interesting tool to increase therapeutic potential of MSCs is the adoption of three-dimensional (3D) culture of human placental MSC (hPMSCs), which proved to be more effective than two-dimension (2D) culture in preventing renal damage when injected in a mouse model of IRI-induced AKI (170). MSC 3D spheroid structures enable increased cell-cell interactions and enhance MSC trophic and immunomodulatory functions, with more reproducible clinical outcomes in many preclinical models (171) including cisplatin-induced AKI (172).

We have already discussed the potential of hypoxia and collagen matrix encapsulation to enhance EV protective effects, paving the way to new possibilities of therapeutic manipulation (106, 107, 116). Of note, the use of EVs avoid the possible adverse effects associated with whole cell therapies such as pulmonary embolism, vascular thrombosis, maldifferentiation and tumorigenesis (173).

A growing body of evidence based on pre-clinical studies suggests that EVs released from MSCs of different origin and from other SC types could be effective to treat toxic, ischemic and septic AKI through direct interference with multiple etiopathogenetic mechanisms of tubular and endothelial damage. A network of cytoprotective, regenerative and immunomodulatory EV properties is being defined. EV-based therapy could prevent renal fibrosis and AKI-CKD transition, also through inhibition of complement-mediated processes such as EndMT and PMT. Hypoxia-conditioned and engineered EVs with enhanced therapeutic properties are promising new tools. Even though some technological hurdles must still be overcome before widespread clinical application, EV-based therapies may become a cornerstone for the treatment of the most common forms of AKI in the near future.

MQ and VC designed and wrote the initial manuscript. GM and AC designed Figures (Created with BioRender.com) and Tables and arranged References. SB, AS, RF, EG, and VF reviewed the article focusing on experimental models. GC wrote the paragraph on AKI-CKD transition and all authors critically revised the whole article.

This study was partially funded by the Italian Ministry of Education, University and Research (MIUR) program “Departments of Excellence 2018-2022”, AGING Project – Department of Translational Medicine, University of Piemonte Orientale (UPO) and by local grants of the University of Piemonte Orientale (UPO, FAR) to M.Q. and V.C.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.