AKI is characterized by an abrupt and frequently irreversible loss of excretory kidney function and represents a major health concern in hospitalized patients worldwide that is associated with substantial morbidity and mortality. While many surviving patients may recover renal function to pre-injury levels, AKI is a major risk factor for the development of CKD or end stage renal disease, adding to the clinical burden of this disorder [30, 31]. The pathophysiology of AKI is complex, and the majority of therapeutic measures are supportive modalities. In some cases, treatments intended to avoid ongoing damage following AKI have met with disappointing results [32], and effective therapeutic options for promoting kidney regeneration or repair following AKI remain scarce.

Over the past few decades, there has been much interest revolving around cell-based therapies for application in regenerative medicine and cell-based strategies have shown the potential to affect the course of AKI in pre-clinical models. This includes seminal studies by Lange et al., demonstrating that systemic administration of bone marrow-derived MSCs to post-ischemic rats significantly improved kidney excretory function at days 2 and 3 after cell therapy, increased tubular epithelial cell proliferation, decreased apoptosis and tissue damage [13]. Similar reno-protective effects of MSCs have been described in several other reports [6, 7, 11,12,13,14]. It has been suggested that the MSC-derived protective activity is due to the release of a variety of released soluble factors, including VEGF [33].

ASCs are a type of MSC that have several advantages relative to bone marrow-derived MSC, as they are abundant in the adult human body and relatively easy to obtain. Multiple studies, including our prior study, have demonstrated the efficacy of hASCs in preclinical models of AKI, in which they improve renal function, reduce tissue inflammation, enhance tubular repair, preserve vascular integrity, and lower mortality [14, 15].

The mechanism of MSC/ASC-mediated protection is not fully understood. Originally, considered as a source of adult progenitor cells to mediate tissue repair, there are discrepant reports that injected MSCs home to the site of tissue injury [11,12,13, 34]. In our own study, suprarenal injection of ASCs preserved kidney function in the absence of homing [15], suggesting that the therapeutic effect is mediated by factors liberated from ASC and/or modulation of distant cells or tissues, such as cells comprising the immune system. Indeed, numerous recent reports describe soluble factors in conditioned media or in media-derived exosomes that promote vascular repair or inhibit inflammation [9, 19]. These activities are important as one considers the potential application of ASCs to the point-of-care for patients in the ICU with AKI. We suggest that the therapeutic use of ASC secretome offers both logistical and potential safety advantages versus direct administration of cells.

The timing of treatment initiation in pre-clinical models should seek to represent real-world scenarios to assess the potential efficiency of a therapy for AKI. It is likely that significant tissue injury is already present by the time the diagnosis of AKI is made [35]. For this reason, treatments should focus on recovery following the establishment of AKI. In the current study, significant injury was established 24 h post-IRI. In rats, this time-point is associated with severe tubular damage via multiple forms of cell death, impaired vascular function, including vasoconstriction and vascular and tubular congestion, and a fall in GFR. Depending upon the severity of the initial injury in rat I/R models, recovery of serum creatinine occurs over the course of 5–8 days, while tubular morphology may not be complete for several weeks [4]. Other aspects of renal structure and function may not be fully restored; for example, a population of tubular cells may assume a novel phenotype associated with a failed repair response, and there is evidence of persistent inflammation and permanent vascular rarefaction [4, 5, 31]. All of these have been proposed to play an important role in the transition of AKI to subsequent CKD and fibrosis, and all of these may benefit from trophic, angiogenic and anti-inflammatory activity from ASC-derived secretome.

The current report contains several unique elements highlighting the potential utility of ASC-CS in the setting of AKI: 1) Unlike previous reports using intravenous administrations of cells, there was a rapid response of improved renal function noticeable within 1 day following a single-subcutaneous injection of ASC-CS. Given that many effects of ASC are ascribed to Secreted factors, it is likely cells, which effectively represent prodrugs relative to secretome, may have delayed onset of action versus secretome. In addition, the subcutaneous route of ASC-CS administration was selected based on the recent report by Bogatcheva et al., which demonstrated the utility of a similar approach to alleviate lung inflammation in a model of influenza in mice [23]. This approach may have pharmacokinetic and/or biodistribution properties favorable for the observed effects. Although a second injection was provided 48 h after the first injection, the temporal trajectory of recovery suggests that the recovery was predominantly due to the effects of the initial injection. 2) As described before, the model used for randomization was chosen to avoid any potential imbalance in the severity of AKI between the treatment groups and was geared to address recovery from AKI rather than initiation. 3) Because our sample size was sufficient to subdivide into groups based on injury severity, we were able to determine that ASC-CS was effective in improving recovery in the most severely injured subset of rats. As rats from the lower half of the creatinine range have less severe tissue damage, the more rapid recovery and lack of additional therapeutic benefit in this group of rats should not be surprising.

ASCs can release multiple growth factors, chemokines, cytokines, and extracellular vesicles. For example, trophic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), hepatocyte growth factors (HGF), and transforming growth factor β1 (TGF-β1) have been recognized as the main mediators of the pro-angiogenic, immunomodulating, and anti-apoptotic effects of ASCs [36]. The ASC-CS generated under conditions described here also contains large amounts of VEGF and IL-8. While the characterization of secretome is important to evaluate consistency for production, and may provide clues to mechanism, it is important to point out that the specific factor or factors that mediate the protective effects reported here are uncertain. Indeed, the beneficial effects are likely mediated via combined activity by multiple paracrine factors [37, 38].

The reduction of Th17 cells reported here is consistent with our previous report using an intra-arterial injection of ASCs at the time of reperfusion [15]. Th17 cells are the most abundant lymphocyte population induced in rat kidney following I/R [27], and inhibition of its primary secreted cytokine IL17 protects against early acute injury and CKD progression [25]. In the current report, we demonstrate that ASC-CS reduced in vitro activation of lymphocytes primed by injury to express the Th17 phenotype. To the best of our knowledge, this is the first demonstration that ASC-secretome may directly inhibit lymphocyte activation to the Th17 phenotype.

In our previous report, we also demonstrated that human ASCs injected intra-arterially protected against the loss of peritubular capillaries following AKI [15]. It is possible that ASC-CS also has a similar protective effect on vascular damage since Ang-2 levels, which have been shown to correlate with vascular rarefaction [28, 29], were attenuated by ASC-CS. Unfortunately, in the current study, there was a limited availability of appropriately fixed tissues that prevented us from directly measuring capillary density.

Despite our attempts to minimize bias by performing randomization based on a quantitative assessment of renal function prior to treatment initiation, the rat model contains several inherent weaknesses. Included among these whether surgery- induced AKI in young healthy rats recapitulates elements of recovery in critically ill human patients with AKI. In addition, the current study did not assess long term function and structure at later time points to determine if the treatments may mitigate the development of chronic kidney disease, fibrosis, and hypertension frequently reported following recovery from AKI [4, 5, 31].