Mesenchymal Stem Cells in the Treatment of Acute Kidney Injury (AKI), Chronic Kidney Disease (CKD) and the AKI-to-CKD Transition

Abstract

Acute kidney injury (AKI) and chronic kidney disease (CKD) are global health burdens that result in high economic costs to healthcare systems. AKI is a known risk factor for progression to CKD. The global absolute CKD prevalence increased by 28.2% from 2007–2017 among females and 25.4% among males. Current best clinical practice only slows the progress of renal fibrosis, the final pathological consequence of renal injury, in CKD. Kidney transplantation and dialysis are the only options for the management of kidney failure, which results in a significant burden on the health system. Hence innovative strategies are urgently needed to both prevent and treat CKD. Many studies have demonstrated that mesenchymal stem cells (MSCs) exert a therapeutic role through regenerating/repairing damaged tissues primarily through cargo secreted in extracellular vesicles. In recent years, the therapeutic effect of stem cells in the treatment of acute and chronic kidney disease have been primarily assessed in preclinical studies. This review outlines the safety and efficacy of MSCs in AKI, CKD and the AKI-to-CKD transition based on recent animal studies and clinical trials. It elucidates the antifibrotic mechanisms of MSCs and provides novel insights into MSC therapy in AKI, CKD and the transition from AKI to CKD.

INTRODUCTION

Acute kidney injury (AKI) refers to an abrupt decline in renal function that takes place over hours to days. It is associated with prolonged hospital stays and a significant increase in morbidity and mortality.^[1] In the long term there is a significant risk for the progression to chronic kidney disease (CKD) and renal failure via a maladaptive repair mechanism.^[2] This has been termed the AKI-to-CKD transition.^[3] The incidence of AKI is reported to be between 2%–7% of hospitalized patients and 5%–10% of ICU patients.^[4] The global prevalence of CKD has consistently been estimated to be between 11% and 13% and is associated with an increased risk of cardiovascular morbidity, premature mortality and a decreased quality of life.^[5] An alarming increase in the prevalence of CKD is expected due to increasing rates of diabetes mellitus, hypertension and obesity as well as an ageing population.^[6]

The progressive nature of CKD and the ensuing renal failure places a substantial burden on global healthcare resources. Kidney replacement therapy (KRT), including dialysis and kidney transplantation remain the only current treatment options.^[7] This sequalae is extremely burdensome for patients and gives rise to a low health-related quality of life. With worldwide use of KRT expected to more than double to 5.4 million people by 2030,^[8] novel therapies to prevent and treat CKD are in high demand. Mesenchymal stem cells (MSCs) derived therapies are receiving increasing attention as they can address multifaceted pathophysiology and have many advantages over other forms of cell-based therapy. Their therapeutic effect is credited to a paracrine mechanism based on immunomodulatory, anti-apoptotic, angiogenic, anti-fibrotic and chemoattractant activity.^[4] Furthermore, they are readily available, easy to isolate and expand.^[9]

PATHOPHYSIOLOGY OF AKI AND THE AKI-TO-CKD TRANSITION

The Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guidelines offer the most recent guidelines for the diagnosis and staging of AKI.^[10] It is a combination of the older Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease (RIFLE) and Acute Kidney Injury Network (AKIN) classification systems and defines AKI as an increased serum creatinine by ≥ 0.3 mg/dL (≥ 26.5 µmol/L) within 48 h or increase in serum creatinine to ≥ 1.5 times baseline, which is known or presumed to have occurred within the prior seven days, or urine volume < 0.5 mL/kg/h for 6 h. AKI is also traditionally classified according to anatomical categories that indicate the site and mechanism of injury; pre-renal, intrinsic and post-renal. With the major causes of AKI attributed to hypotension, heart failure and infection.^[11] Other risk factors for developing AKI include; ageing, liver failure, CKD, sepsis, shock and exposure to nephrotoxic agents such as cisplatin.^[12] The incidence of AKI in hospitalised patients is reported to be between 2%–7%, with an incidence of 5%–10% in intensive care units (ICU), where mortality may exceed 50%.^[4] In patients with type 2 diabetes, sodium-glucose co-transporter-2 (SGLT2) inhibitors provide protection against AKI as well as other major adverse kidney events, such as transplantation, dialysis and kidney related mortality.^[13]

Irrespective of the primary cause of AKI, an intrarenal inflammatory cascade is activated that may result in additional renal damage and irreversible renal fibrosis.^[14] Damage to the tubular epithelial cells (TECs) occurs via a process of “mitochondrial dysfunction, oxidative stress, metabolic changes, cell cycle arrest, dedifferentiation, induction of a senescence-related phenotype, secretion of inflammatory mediators, renin-angiotensin system (RAS) activation and epigenetic changes”.^[6] The inflammatory mediators involved include inflammatory cytokines interleukin (IL)-6, IL-8, tumor necrosis factor α (TNFα) and monocyte chemoattractant protein 1 (MCP1) as a result of endothelial dysfunction and tubular injury.^[14] This inflammatory environment is further amplified by reactive oxygen species (ROS) mediated mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NFκB).^[15] The mediators of inflammation can cause further tubular injury, either directly or through the recruitment of inflammatory cells, including macrophages.^[6] This inflammatory cascade often results in widespread apoptosis of the TECs.^[15]

After an insult to the TECs there is a recovery phase, where protective and regenerative mechanisms aim to restore the properties and functions of the TECs. Failure of these mechanisms has been termed maladaptive repair, which promotes kidney fibrosis and the progression to CKD.^[6] The key pathophysiological pathways that occur during maladaptive repair include capillary rarefaction, mitochondrial injury and metabolic dysregulation, epigenetic alterations, persistent inflammation, profibrogenic signal production and myofibroblast expansion. Due to the importance of TECs in regeneration after injury, it is thought that maladaptive repair in TECs plays a central role in the transition from AKI to CKD.^[16] The sequelae posed by this transition negatively impacts the health burden faced by patients and adds to the demands placed on already constrained KRT services.

PATHOPHYSIOLOGY OF CKD

CKD is defined as abnormalities of kidney structure or function, present for > 3 months, which has implications for other aspects of health and in particular cardiovascular risk factor modification, irrespective of the cause of CKD.^[17] Treatment of CKD is based on the underlying cause of kidney disease, the presence or absence of systemic disease and dominance within the glomerular or tubulointerstital compartments of the kidney or presumed pathologic-anatomic findings, according to glomerular filtration rate and levels of albuminuria.^[18] Staging is aimed to identify those who are at the greatest risk of progression and cardiovascular complications.

The aetiology of CKD varies globally, with diabetes, hypertension glomerulonephritis, genetic disease and CKD following AKI being the principal causes, particularly in high- and middle-income countries. Other risk factors include age (> 65 years), obesity, smoking, family history of kidney disease and in Australia, Aboriginal and Torres Strait Islander heritage.^[19] There are also many single and polygenic causes of CKD that contribute to its inherited susceptibility and progression.^[20] Regardless of the underlying aetiology of CKD, there is parenchymal kidney cell loss, chronic inflammation, fibrosis and reduced regeneration that contributes to its progression, as summarised in Figure 1. This in turn leads to irreversible nephron loss, kidney failure and premature death.^[6] Renal fibrosis is the final common pathway leading to kidney failure and is characterised by glomerulosclerosis, tubular atrophy and interstitial and vascular fibrosis.^[20] This final common pathway presents an opportunity for therapeutic intervention.

Figure 1.: The pathway of renal fibrosis. Renal fibrosis can be considered an extension of normal wound healing, with several key steps involved; ①. Kidney injury results in an inflammatory response and infiltration of a variety of inflammatory cells. ②. A release of fibrosis related factors such as growth factors, cytokines, and chemokines. ③. Imbalance in the synthesis and degradation of ECM and excessive accumulation of ECM in the renal interstitium. ④. Mesenchymal transition of innate renal cells and reduction of the number of intrinsic cells. ⑤. renal microvascular injury and capillary rarefaction. TGFβ1, transforming growth factor β1; SMAD3, small mothers against decapentaplegic 3; PDGF, platelet derived growth factor; MMP, matrix metalloproteinases; PAI1, plasminogen activator inhibitor 1; NFB, nuclear factor kappa B; PKC, protein kinase C; ERK, extracellular regulated protein kinases; PI3K, phosphatidylinositol-3 kinase; SOX9, SRY-Box transcription factor 9; JNK, c-Jun N-terminal kinase; ECM extracellular matrix.

Myofibroblasts are considered a critical cell type in the mediation of fibrosis. Resident mesenchymal stem cells are thought to be the key progenitor cell of myofibroblasts, with epithelial cells that have undergone a process of epithelial-mesenchymal transition (EMT) also contributing to the cell pool.^[21] Transforming growth factor β1 (TGFβ1) and the downstream activation of small mothers against decapentaplegic 3 (SMAD3) are understood be the key regulators driving fibrosis. Platelet derived growth factor (PDGF) is also considered to play a key role in the signalling for proliferation and recruitment of fibroblasts. There is reactivation of key developmental pathways that drive fibrosis and progression to CKD. These include the Notch pathway, Wnt/β-catenin pathway, SRY-Box transcription factor 9 (SOX9) transcription, protein kinase C (PKC)/extracellular regulated protein kinases (ERK), phosphatidylinositol-3 kinase (PI3K)/Akt and Sonic Hedgehog.^[6,22] Activation of c-Jun N-terminal kinase (JNK) signalling is a common feature in human kidney injury, and JNK pathway mediates kidney injury through interacting with the TGFβ/SMAD pathway.^[23] Specifically, it is JNK1 which plays a specific role in ischaemia-reperfusion injury (IRI) induced cell death in the proximal tubule, leading to acute renal failure.^[24] Myofibroblasts in turn produce excessive ECM proteins, mainly collagen 1 (COL-1), leading to tubulointerstitial fibrosis. Myofibroblasts are considered a critical cell type in the mediation of fibrosis. Alpha smooth muscle actin (α SMA) acts as a biomarker that indicates the level of myofibroblast activity.

MSCs can be derived from many different sources, with intrinsic properties that allow them to address several pathophysiological pathways [Figure 2]. The characteristics of MSCs include their plastic-adherence when maintained in standard culture conditions, expression of CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules, and capability to differentiate to osteoblasts, adipocytes and chondroblasts in vitro.^[25] MSCs are easy to obtain and culture, are capable of multilineage differentiation, have the potential for autologous or allogenic use, have immunoregulatory features and presently few ethical considerations regarding their use.^[26] As shown in Figure 2, MSCs derive from a variety of origins, including bone marrow (BM), adipose tissue (AD), Wharton's jelly (WJ), umbilical cord (UC), dental pulp, foetal tissues such as amniotic fluid and chorionic villi.^[24]

Figure 2.: Source and therapeutic effects of MSCs. MSCs can be derived from various sources including bone marrow, placenta, umbilical cord and Wharton's jelly, dental pulp, adipose tissue, peripheral blood as well as those that reside in the tissues. MSCs characteristically express CD105, CD73 and CD90. The therapeutic effects of MSCs are attributed to their immunomodulatory properties, the stimulation of angiogenesis and the inhibition of fibrosis, apoptosis and oxidation. MSC, mesenchymal stem cell; HGF, hepatocyte growth factor; bFGF, basic fibroblast growth factor; PGE2, prostaglandin E2; GSH, glutathione; VEGF, vascular endothelial growth factor.

The heterogenous origins of MSCs is reflected in their proliferative and differentiation potential, as well as paracrine mechanisms. For example, UC MSCs have higher rates of cell proliferation than AD MSCs and BM MSCs. BM MSCs and AD MSCs have a greater tendency to osteoblast differentiation, whereas it has been shown UC MSCs lack differentiation towards adipocytes.^[27] The origin of the MSCs also impacts the paracrine mechanisms by which they act, this can be seen in AD MSCs that secrete more pro-angiogenic molecules when compared to other MSC populations.^[28] This review will highlight the therapeutic paracrine mechanisms according to stem cell origin for AKI and CKD in animal models. Currently, bone marrow as a source of MSCs remains the most valued due to better documentation and wide use in both preclinical and clinical research.^[29]

It is thought that MSCs possess intrinsic properties resulting in immunomodulation, pro-angiogenesis, anti-inflammation, anti-apoptosis, and anti-oxidation, which are greatly beneficial for limiting acute injury and reversing chronic progression of fibrosis.^[16] Accumulating evidence has emphasized that MSCs are active in the release of several cytokines and growth factors, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), TGFβ, and insulin-like growth factor type 1 (IGF-1).^[30] These biological molecules can promote anti-apoptotic, anti-oxidative and pro-angiogenic effects, resulting in cell and tissue recovery. They also play critical roles in the modulation of renal blood flow, capillary permeability, endothelial cell survival and immunological responses.^[16]

The therapeutic effect of MSCs has been attributed mainly to their paracrine actions. Extracellular vesicles, which include apoptotic bodies, micro-vesicles and exosomes have gained recent attention as they are the fundamental paracrine effector of MSCs.^[31] It has been shown that the factors contained within the extracellular vesicles secretrome are responsible for the anti-apotpic, prevention of excessive fibrosis, the stimulation of angiogenesis and the immunomodulatory effects of MSCs.^[27]

MSCS IN AKI: KEY PATHWAYS, CYTOKINES AND TRANSCRIPTION FACTORS

There is a rapidly evolving body of evidence focusing on the use of MSCs in AKI. They are a promising therapeutic that targets multiple pathophysiological pathways to improve AKI and offers an attractive alternative to the current treatment paradigm. As such many animal and human trials have been established to assess the safety, efficacy and feasibility of MSC treatment. Although there is increasing amounts of research in the area, more data is still required to define the best route of administration of MSCs, the amount of cells needed per administration as well as the total number of injections, to understand the interaction between MSCs and other cells in the tissue, to identify any potentially adverse effects of MSCs and further establish profiles of MSCs to mitigate the heterogeneity of the cells. This review summarises the results of animal studies assessing the role of MSCs in AKI over the past 5 years [Table 1], as well as looking at the relevant clinical trials registered with http://clinicaltrials.gov at the National Institutes of Health (NIH).^[32]

Table 1 - Summary of animal models assessing the use of MSCs in AKI over the past 5 years

Author	AKI	Method of delivery	Type of MSC	Animal Model	Findings
Ashour et al. (2016)[33]	Cisplatin induced AKI	Tail vein	BM MSC, AD MSC, hAMSC	Rat	BM MSCs had the lowest serum creatine by day 30. All MSCs types were able to reduce oxidative stress markers and improve injury and regenerative scores.
Begum et al. (2019)[34]	Cisplatin induced AKI	Tail vein	AD MSC	Balb/c mouse	AD MSCs exert pro-proliferative, anti-inflammatory and anti-fibrotic effects.
Changizi-Ashtiyani et al. (2020)[35]	I/R induced AKI (45 minutes)	Tail vein	AD MSC	Wistar rat	AD MSCs administered at the time of I/R completely or partially protected the kidneys from I/R induced injuries.
Collett (2017)[36]	I/R induced AKI (40 minutes)	Suprarenal abdominal aorta	AD MSC	Sprague-Dawley rat	AD MSCs improve recovery from I/R injury by decreasing inflammation and preserving peritubular capillaries.
Condor et al. (2016)[37]	Cecal ligation and puncture model of sepsis.	Intraperitoneal	WJ MSC	Rat	Treatment with WJ MSCs improved the glomerular filtration rate, improved tubular function, decreased expression of NFκB and of cytokines, increased expression of eNOS and of Klotho, attenuated renal apoptosis, and improved survival.
Elhusseini et al. (2016)[38]	Cisplatin induced AKI	Tail vein	AD MSC	Sprague-Dawley rat	AD MSCs have both protective and regenerative abilities with consequent limitation of the development of renal fibrosis after the cisplatin induced acute tubular necrosis, largely through an anti-oxidative activity
Fahmy et al. (2017)[39]	I/R induced AKI (60 minutes)	Tail vein	UC MSC	Wistar rat	UC MSCs significantly improved creatinine, urea and uric acid levels in the serum. There was also a positive antioxidant efficacy of UC MSCs against the I/R induced oxidative stress. On histopathology UC MSCS restored healthy renal glomeruli and tubules injured by the I/R.
Hafazeh et al. (2019)[40]	I/R induced AKI (45 minutes)	Tail vein	AD MSC	Wistar rat	Treatment with AD MSC reduced tissue damage and oxidative stress while increasing antioxidant activity. In addition, it improved kidney function after 45 min ischemia and 24 h reperfusion.
Havakhah et al. (2018)[41]	I/R induced AKI (40 minutes)	Intra-parenchymal or intra-aortic.	BM MSC	Rat	Intra-parenchymal or intra-aortic administration of BM MSCs resulted in significantly better renal function and lower renal injury scores than untreated animals.
Ibrahim et al. (2018)[42]	Cisplatin induced AKI	Intraperitoneal	BM MSC	Rat	Injection of mesenchymal stem cells in rats with cisplatin-induced AKI improved the histopathological effects of cisplatin in renal tissues and kidney function tests were significantly improved.
Lee et al. (2018)[43]	I/R induced AKI (60 minutes)	Renal artery	BM MSC	Cynomolgus monkey	Histological findings and qRT-PCR analysis of Ngal mRNA in renal biopsy tissue showed that BM MSC promoted the recovery of tubular damage caused by AKI.
Lee et al. (2017)[44]	Cisplatin and gentamicin induced AKI	Renal corticomedullary junction	UC MSC	Dog	The group treated with AKI plus UC MSC had decreased blood urea nitrogen and creatinine levels, and showed an extended life-span and improved histological manifestations.
Lim et al. (2016)[45]	Cisplatin induced AKI	Intravenous	BM MSC	Dog	There was less fibrotic change and increased proliferation of renal tubular epithelial cells in the BM MSC treated dogs. Expressions of TNFα and TGFB were also lower.
Monteiro et al. (2018)[46]	Celiotomy and renal artery and vein clipped for 24 h.	Tail vein or intrarenal.	AD MSC	Wistar Rat	AD MSCs positively contributed to the replacement of necrotic tissue by renal tubular cells, vascularization of the renal parenchyma, and restoration of the organ function.
Moustafa et al. (2016)[47]	Cisplatin induced AKI	Either intravenous, intra-arterial or kidney sub capsular injection	BM MSC	Sprague Dawley Rat	Changing the route of MSCs injection does not have a major influence on the outcome. Future evaluation should focus on differences between the routes of administration considering the long term safety.
Osman et al. (2020)[48]	I/R induced AKI (90 minutes)	Injection directly into renal cortex	AD MSC, BM MSC	Dog	Stem cells protected against ischemic reperfusion injury in a canine model. AD MSCs provided better protection than BM MSCs.
Paglione et al. (2020)[49]	I/R induced AKI (45 minutes)	Parenchyma infusion.	OMSC	Rat	OMSCs induced an accelerated renal exocrine functional recovery, demonstrated by biochemical parameters and confirmed by histology showing that histopathological alterations associated with ischemic injury were less severe in cell-treated kidneys as compared with control groups (P < 0.05).
Perico et al. (2017)[50]	Cisplatin induced AKI	Intravenous	UC MSC	Mouse	UC MSC when transplanted into mice with acute kidney injury, stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3.
Rodrigues et al. (2017)[51]	I/R induced AKI (45 minutes)	Intravenous	UC MSC	Rat	Minimised renal fibrosis, decreased β-galactosidase expression and increased the expression of Klotho. Conclusions: Our data demonstrate that MSCs attenuate the inflammatory and oxidative stress responses occurring in AKI, as well as reducing the expression of senescence-related proteins and microRNAs.
Rosselli et al. (2016)[52]	Warm renal ischemia	Intravenous	BM MSC, AD MSC	Cat	This study did not support the use of allogeneic MSCs in AKI in the regimen described here. Type of renal injury, MSC dose, allogenicity, duration, and route or timing of administration could influence the efficacy MSCs.
Sakr (2017)[53]	Cisplatin induced	Tail vein	AD MSC	Wistar rat	AD MSCs almost restore the renal histological architecture. Increased tubular cell proliferation with marked reduction of the interstitial inflammation and fibrosis were also detected. However, some renal glomeruli and tubules showed degenerative changes
Selim et al. (2018)[54]	Cisplatin induced	Tail vein	BM MSC, AD MSC	Rat	MSC therapy has a favourable impact on lessening kidney derangement after AKI through monitoring the inflammatory response and restoring oxidant/anti oxidant homeostasis.
Selim et al. (2019)[55]	Cisplatin induced	Tail vein	BM MSC, AD MSC	Wistar rat	MSCs could ameliorated kidney function renal functions manifested by decreased urea, creatinine, and cys C levels; downregulation of p38; and upregulation of Bcl-2 and VEGF.
Sheashaa et al. (2016)[56]	I/R induced AKI (45 minutes)	Tail vein	AD MSC	Sprague Dawley rat	The use of AD MSCs was associated with significantly lowered injury scores at days 1 and 3; however, no significant effect was observed on day 7.
Tang et al. (2018)[57]	I/R induced AKI (30 minutes)	Tail vein	BM MSC	Mouse	The present study indicates that BM MSCs alleviate AKI via suppressing C5a/C5aR-NFκB pathway activation.
Xiu et al. (2018)[58]	(LPS)-induced acute kidney injury (septic-AKI)	Femoral vein	BM MSC	Rat	BM MSCs transplantation significantly reversed the already upregulated concentration of BUN and SCr, dramatically attenuated the event of the tissue injury, and prominently reduced mortality after AKI. These were paralleled by down-regulation of the level of TLR4 and NFκB.
Xu et al. (2020)[59]	Cisplatin induced AKI	Femoral vein	UC MSC	Sprague Dawley rat	UC MSCs protect against cisplatin induced AKI by suppressing HMGB1 expression and preventing cell apoptosis.
Zhang et al. (2017)[60]	I/R induced AKI (40 minutes)	Tail vein	AD MSC	Sprague Dawley rat	AD MSC treatment significantly decreased the number of apoptotic cells, the level of total urinary protein and serum creatinine, the expression of pro-inflammatory cytokines (IL-6, TNFα, IL-1 β, IFNγ, and TGFβ), and the inflammation-associated proteins (HGF and SDF1), but increased the expression of the anti-inflammatory cytokine, IL-10, and the anti-apoptotic regulator, Bcl2.
Zilberman-Itskovich et al. (2019)[61]	I/R induced AKI (60 minutes)	Tail vein	hA MSC	Rat	MSCs significantly decreased intra-renal levels of IL-1β and TNFα. Extensive activation of the complement system was ameliorated in the MSC treated groups. Renal functions improved in a U shaped dodse dependent manner.

BM: bone marrow, AD: adipose tissue, WJ: Wharton's jelly, UC: umbilical cord, hA: human amniotic, MSC: mesenchymal stem cell, AKI: acute kidney injury, VEGF: vascular endothelial growth factor, NFκB: nuclear factor kappa B, IL: interleukin, TNFα: tumor necrosis factor α, TGFβ: transforming growth factor β, HGF: hepatocyte growth factor, SDF1: stromal cell derived factor 1, TLR4: Toll-like receptor 4, HMGB1: high mobility group box 1, IFNγ: interferon gamma, Please check if this full name is correct.

There are many transcription factors, cytokines and signalling pathways that have been implicated in the pathogenesis of AKI. The animal studies included in this review have helped to elucidate some of these factors and how their upregulation or downregulation may attenuate AKI.

NFκB

NFκB is a transcription factor that mediates the immune response and is thought to play a key role in the pathogenesis of AKI. Condor et al. found that intraperitoneal injections of WJ MSCs decreased the expression of NFκB in rats with a sepsis induced model of AKI.^[37] Tang et al. found that in a mouse model, BM MSCs alleviated AKI through suppression of the C5a/c5ar NFκB pathway activation.^[57] Xiu et al. found that BM MSCs attenuated tissue injury and reduced mortality in rats with cisplatin induced AKI, with these observations paralleled by the down regulation of Toll-like receptor 4 (TLR4) and NFκB.^[58]

Klotho

Klotho is a multifunctional protein that plays a role in anti-oxidation, anti-apoptosis and anti-fibrosis. It is receiving increasing attention as a potential early biomarker in AKI.^[62] Klotho deficiency increases the expression of Wnt/β-catenin, activating the tubulointerstitial renal fibrotic response by arresting cells at the G2/M phase of the cell cycle, inducing the production of TGFβ and connective tissue growth factor.^[63] In rat models, Condor et al. found that WJ MSCs administered intraperitoneally, increased the expression of klotho after sepsis induced AKI.^[37] Similarly, Rodrigues et al. found that intravenous administration of UC MSCs also increased the expression of klotho in rats after ischemic AKI.^[51]

TNFα

TNFα is a pleiotropic cytokine that has many pro-inflammatory properties and is implicated in kidney disease. Lim et al. found that the administration of BM MSCs in dogs subjected to a cisplatin induced model of AKI resulted in reduced expression of TNFα.^[45] Zilberman-Itskovich et al. found a reduced expression of IL-1β and TNFα after the delivery of human amniotic (hA) MSCs in a rat model of ischemia induced AKI.^[61] Similarly, Zhang et al. found adipose tissue derived (AD) MSCs decreased the expression TNFα in an ischemia induced model of AKI in rats.^[60]

TGFβ

TGFβ is a multifunctional cytokine that promotes the transformation of fibroblasts into myofibroblasts, increasing ECM deposition and reducing ECM degradation, as well as inducing the EMT of tubular cells.^[64] Lim et al. found that the administration of BM MSCs reduced expression of TGFβ in dogs with cisplatin induced AKI.^[45] Zhang et al. found AD MSCs decreased the expression TGFβ in rats with an ischemic AKI.^[60]

SIRT 3

SIRT 3 is a mitochondrial sirtuin that regulates antioxidant activity. Perico et al. found that in mice with AKI, UC MSCs stimulate renal tubular cell growth and enhance mitochondrial function via SIRT 3.^[50] This was further confirmed by the finding that in SIRT 3 deficient mice with AKI, UC MSCs failed to induce renal protection.

MiRNAs

MiRNAs are non-coding RNAs that have been implicated as drivers of fibrotic signalling. Members of the miR-29 family have been found to suppress the protein phosphatase PPM1D, thus increasing apoptosis. miR-34a can induce a senescent profile in mesangial cells through reducing antioxidant activity.^[51] One study found that microRNAs (miR-29a and miR-34a) were overexpressed after IRI induced AKI and subsequently downregulated with treatment of UC MSCs.^[51]

VEGF

VEGF is a signalling protein involved in angiogenesis. Vascular damage is an early and important mediator of AKI and leads to renal deterioration and progressive loss of kidney functions.^[55] VEGF promotes renal repair following AKI by directly mediating mitogenic and anti-apoptotic effects on TECs, as well as stabilising microvascular density, diminishing capillary rarefaction and improving renal perfusion.^[3] Selim et al. found a downregulation of VEGF mRNA in rats with AKI and an upregulation of VEGF gene expression in the AKI group treated with AD MSCs.^[55]

B-Cell lymphoma 2

B-Cell lymphoma 2 (bcl 2) is a key regulator of apoptosis. Selim et al. found it to be downregulated in rats with AKI and upregulated after treatment with MSCs.^[55] Similarly, Zhang et al. found a downregulation of bcl 2 in rats after ischemic AKI, that was subsequently upregulated after delivery of AD MSCs.^[60]

Additionally, recent reports have demonstrated that hypoxic mesenchymal stem cells ameliorate acute kidney ischemia-reperfusion injury via enhancing renal tubular autophagy,^[65] and exosomal-miR-1184 derived from MSCs alleviates cisplatin-associated AKI in in vitro model using renal HK2 cells.^[66]

CLINICAL TRIALS OF MSC IN AKI

On the back of promising results in animal models there have been several notable clinical trials registered with clinicaltrials.gov.au at the NIH. NCT00733876 was a 15 participant, open label, phase 1 clinical trial assessing the safety of administering allogenic MSCs in patients at high risk of developing AKI after undergoing on-pump cardiac surgery. This was completed in

October 2013, reaching the primary endpoint of the absence of MSC specific adverse or serious adverse events. NCT01602328 evaluated the efficacy of allogenic MSCs in a phase 2, randomized, double-blind placebo-controlled trial in 156 participants. The intervention assessed a single administration of MSCs vs placebo for cardiac surgery patients (valve and/or CABG) with laboratory evidence of AKI within 48 h of removal from cardiopulmonary bypass. The treatment was found to be safe but MSCs did not decrease the time to recovery of kidney function (completed August 2014). NCT01275612 was a trial that assessed MSCs in cisplatin induced AKI in patients with solid organ cancers. This phase 1 study has since been withdrawn. NCT03015623 is a multi-centre, randomized, sham-controlled, double-blind, ascending-dose study of extracorporeal MSCs (SBI-101 Therapy) in subjects with AKI receiving continuous KRT. 24 participants are involved with an estimated completion date of December 2021; however the Overall Status shows active, not recruiting.

MSCS IN AKI-TO-CKD TRANSITION

In recent years it has been recognised that severe or repeated AKI can lead to CKD, contradicting the traditional paradigm that AKI and CKD are two separate entities. Zhao et al. found AKI patients who survive the acute phase will bear a 13-fold higher risk of developing CKD in their lifetime and for those patients with AKI at the RIFLE failure stage, the risk of progression to CKD is up to 41 times higher.^[16] RAS blockade remains the mainstay of treatment in delaying CKD progression in proteinuric kidney disease. However, no current therapy can prevent AKI or the AKI-to-CKD transition.^[6] MSCs are receiving increasing attention to address a desperate need for a therapy that either prevents CKD progression or allows regeneration in already fibrotic kidneys.

During the adaptive repair process after an AKI, surviving TECs undergo continuous four-phase mitosis (G0, G1, S, G2 and M) or may remain in the G1 phase to prevent proliferation of damaged DNA. TEC's may also stay in the G2/M phase under stressful conditions and actively produce profibrogenic signals such as TGFβ, c-jun NH2-terminal kinase (JNK), and epidermal growth factor receptor (EGFR), highlighting a pathophysiological link between AKI and CKD.^[16] Other growth factors that have been implicated in the course of the AKI-to-CKD transition via the regulation of inflammation include TGFβ, bone morphogenic protein 7 (BMP7), VEGF, and HGF. It is thought that targeting abnormal activation of these signals may prevent the AKI-to-CKD transition.^[3]

Andrade et al. suggest that the way in which MSCs could play a role in kidney regeneration is through a paracrine effect that allows TECs to avoid cellular senescence.^[63] Their group demonstrated in an experimental IRI model, that UC MSCs improved kidney function, downregulated the upregulated expression of senescence markers (β-gal, p21Waf1/Cip1, and p16INK4a) and miRs (miR-29a and miR-34a). These findings were consistent with the view that the therapeutic benefit of MSCs could be attributed to their paracrine functions. Compared with pharmacologic interventions, which target only one single aspect of the highly complex pathophysiological process during the AKI-to-CKD transition, MSCs may have the advantage of presenting multiple regenerative effects for organ protection.^[16]

A study by Zhu et al. found that administering adipose-derived MSCs in an IRI mouse model of AKI upregulated the expression of SOX9, promoted tubular regeneration, improved tubular cell cycle arrest in G2/M phase, attenuated AKI and mitigated subsequent renal fibrosis.^[67] SOX9 is a transcription factor that plays a major role in the development of multiple tissues and organs including the kidneys. SOX9 was also found to be a key player in the repair process of damaged TECs in research completed by Kumar et al.^[68] The principal findings of Zhu's study were that AD MSCs reduced renal pathological damage, promoted effective proliferation of TECs, improved cell cycle arrest of TECs, reduced renal ischemia and hypoxia and decreased the infiltration of inflammatory cells.^[67] These findings are in keeping with MSCs demonstrating their therapeutic effect through paracrine activity rather than differentiating into renal parenchymal cells.

MSCS IN CKD: FIBROSIS RELATED FACTORS AND THERAPEUTIC TARGETS

There is a great need to find therapeutic solutions that address the rising financial and health burdens posed by CKD. As fibrosis represents the final common pathway of CKD regardless of underlying aetiology, it follows that understanding and targeting the pathophysiology of fibrosis is necessary to evaluate and maximise the therapeutic potential of MSCs. MSCs are being considered for therapy in renal disease for their regenerative and paracrine properties. The secretion of paracrine factors are now recognised as the primary mechanism by which MSCs promote a regenerative environment conducive to healing.^[69] The use of MSCs in animal CKD models over the past 5 years [Table 2] and relevant clinical trials are discussed in this review.

Table 2 - Summary of animal models assessing the use of MSCs in CKD over the past 5 years

Author	CKD	Type of MSC	Animal Model	Findings
Akan et al. (2021)[70]	5/6 nephrectomy induced CKD model	hA MSC	Rat	Reduced TGFβ and COL-1 expression and increased protein and gene expression of BMP7.
Caldas et al. (2017)[71]	5/6 nephrectomy induced CKD model	BM MSC	Rat	Reduced TGFβ expression. Increased VEGF expression.
Cetinkaya et al. (2019)[72]	AA induced CKD	hA MSC	Rat	Reduced expression of COL-1. Reduced serum creatinine and urea levels.
Quimby et al. (2016)[73]	Ultrasonic evidence of CKD and biochemical evaluations performed 2 weeks apart	AD MSC	Cat	Although there was no adverse effects, significant improvement in renal function was not observed immediately after administration. Long-term follow-up is necessary to determine whether MSC administration affects disease progression in cats with CKD
Ramirez-Bajo et al. (2020)[74]	CsA nephrotoxicity	BM MSC	Mouse	BM MSCs induce an improvement in renal outcomes in an animal model of CsA nephrotoxicity, particularly if the inflammatory microenvironment is already established
Rota et al. (2018)[75]	ADR nephropathy	BM MSC, UC MSC, KP MSC	Rat	All types of MSC limited podocyte loss and glomerular endothelial cell injury and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis.
Thomson et al. (2019)[76]	International Renal Interest Society stage III CKD	AD MSC	Cat	Intra-arterial infusion of MSC into the renal artery in cats with CKD was feasible and safe within a 3-month postoperative period. Efficacy and long-term safety have yet to be established.
Vidane et al. (2017)[77]	Naturally occurring CKD	AD MSC	Cat	Significant improvement of renal function (decrease in serum creatinine and urine protein concentrations and increase in urine specific gravity). The kidney architecture and morphology did not change following the treatment.
Xing et al. (2019)[78]	UUO model	BM MSC	Mouse	Interstitial fibrosis was significantly attenuated in the MSC group. As well as inhibiting the loss of peritubular capillaries and increasing parenchymal cell proliferation. Proliferation of myofibroblasts were attenuated by MSC infusion.

BM: bone marrow, AD: adipose tissue, UC: umbilical cord, VEGF: vascular endothelial growth factor, MSC: mesenchymal stem cell, TGFβ: transforming growth factor β, CKD: chronic kidney disease, AA: aristolochic acid, CsA: chronic cyclosporine, ADR: Adriamycin, UUO: unilateral ureteral obstruction, hA: human amniotic, KP: kidney perivascular, BMP7: bone morphogenic protein 7.

It has been observed that in patients with CKD and increased collagen 1 (COL-1) levels, elevated tissue TGFβ1 expression and activated SMAD 3 were associated with glomerular and interstitial ECM accumulation.^[79] Another member of the TGFβ superfamily, BMP7 and its downstream SMAD1/5/8 signalling are downregulated in CKD.^[6]

A trial conducted by Akan et al. found that in a 5/6 nephrectomy induced CKD rat model, the administration of human amnion derived MSCs reduced TGFβ and COL-1 expression and increased protein and gene expression of BMP7.^[70] They concluded that increased TGFβ together with decreased BMP7 expression may cause fibrosis by EMT resulting in progressive kidney fibrosis. Caldas et al. found the administration of bone marrow MSCs in a 5/6 nephrectomy induced CKD rat model also reduced TGFβ as well as increasing VEGF expression.^[71] Cetinkaya et al. found that administration hA MSCs reduced the expression of COL-1 in an aristolochic acid (AA) induced CKD rat model.^[72]

Several clinical trials with stem cell therapy have been registered with http://clinicaltrials.gov assessing MSCs in various forms of CKD. NCT02166489 was a phase 1 clinical trial with 12 month follow up in 6 patients with autosomal dominant polycystic kidney disease (ADPKD), completed in January 2016. Using BM MSCs, they demonstrated safety and tolerability of intravenous transplanted autologous BM MSCs after 12 months of follow up.^[80] NCT02195323 assessed the 18 month safety and potential efficacy of autologous MSCs as a therapy for CKD. This study was completed in 2016. Safety and tolerability were demonstrated in 7 CKD patients, with a single dose infusion of autologous BM MSCs. NCT01840540 was a phase I study, involving 6 patients assessing autologous AD MSCs in vascular occlusive disease of the kidney. This was completed August 2017, with no results posted. Following on from this work NCT02266394 was conducted, which was a phase 1a dose-escalating clinical trial looking at autologous AD MSCs, in atherosclerotic renovascular disease (ARVD). The trial compared the administration of AD MSCs to standard medical therapy alone, with patients matched for age, kidney function and blood pressure. A total of 21 patients were treated with different dose levels (1.0, 2.5 and 5.0 × 10⁵ cells/kg) with 7 patients in each dosing group. It was found that in the treated cohort, mean renal blood flow in the stenotic kidney significantly increased in the entire cohort of stem cell infusion compared to the baseline (164 to 190 mL/min). Hypoxia, renal vein inflammatory cytokines, and angiogenic biomarkers significantly decreased following stem cell infusion. Mean systolic blood pressure in entire cohort of stem cell treatment significantly fell (144 to 136 mmHg) compared to the baseline and the mean GFR modestly but significantly increased from 53 to 56 mL/min.^[81]

DISCUSSION

The fibrosis related factors, growth factors, developmental pathways and inflammatory responses that have been implicated in AKI, CKD and the AKI-to-CKD transition have been reviewed, and the sequalae and paracrine activity of MSCs in therapeutic response have been described. The evidence supporting the use of MSCs has been highlighted in this review through the promising results of many animal studies that have been conducted over the past 5 years. These results provide a solid foundation from which to conduct further animal studies as well as robust clinical trials in the future.

The animal studies presented in Table 1 demonstrate the ability of MSCs to mitigate some of the key factors involved in the pathophysiology of AKI; including a reduction in NFκB, inflammatory cytokines (IL-6, TNFα, TGFβ, IL-1β, interferon gamma [IFNγ]), senescence related proteins and microRNAs and markers of oxidative stress. MSCs increased the expression of klotho, anti-inflammatory cytokine IL-10, eNOS, bcl-2 and VEGF as well as demonstrating improved histology on biopsy.

Attenuating AKI and preventing CKD is paramount in reducing the morbidity, mortality and economic burden posed by the sequelae of the AKI-to-CKD transition. MSCs were shown to upregulate the expression of SOX9, promote tubular regeneration and proliferation of TECs, improve TEC cell cycle arrest in G2/M phase, reduce renal ischemia and hypoxia and decrease the infiltration of inflammatory cells. These findings were in keeping with MSCs demonstrating their therapeutic effect through paracrine activity.^[16,63,67]

Fibrosis represents the final common pathway of CKD, regardless of the underlying aetiology and presents a suitable target for therapeutic intervention. In animal models as presented in Table 2, it has been demonstrated that MSCs are able to reduce TGFβ1 and COL-1 expression,^[70,72] whilst limiting podocyte loss and glomerular endothelial cell injury.^[75] MSCs were able to increase protein and gene expression of BMP7 and VEGF, as well as ameliorated kidney function in animal models of CKD.^[72,74,77] With limited evidence supporting the use of MSCs in the reversal of late-stage fibrosis, future studies may benefit from focussing on the prevention of CKD or delaying progression in its early stages.

A limitation of this literature review stems from the heterogenous nature of the current MSC treatment paradigm. This includes a lack of standardised collection, isolation, culture and storage procedures; varied potency, dosing, timing and route of delivery; allogenic, autogenic and xenogeneic MSCs being utilised as well as varied origins of MSCs. This heterogeneity can make it difficult to compare results of both animal and clinical trials. All these factors must be addressed in order to facilitate the future clinical translation of MSC therapy. Another limitation that needs to be considered in the context of MSC therapy is the availability of the grafted cells at the site of injury. Future studies should compare the efficacy of targeted administration of MSCs directly into the renal parenchyma with intravenous administration. This may help elucidate the so called pulmonary first pass effect, where MSCs may initially get trapped in the lungs as they travel through the systemic circulation.

The data considered in this review across animal and clinical trials found MSCs to be a safe treatment. This was consistent with a recent systematic review and meta-analysis into the safety of MSC therapy,^[82] which concluded that MSCs appear safe. However, future studies need to allow for systematic monitoring and reporting of adverse events to establish a more appropriate safety profile for the use of MSCs.

In summary, MSCs are a promising therapeutic solution to address an unmet need in AKI, CKD and the AKI-to-CKD transition. The animal studies included in this review have demonstrated the feasibility, efficacy and safety of MSC therapy in these conditions. Clinical trials are yet to replicate the same level of efficacy.