Does the route matter? A preclinical review of mesenchymal stromal cell delivery to the kidney

Mesenchymal stromal/stem cell (MSC) therapy has been thoroughly tested in preclinical animal models and holds great promise for the treatment of kidney diseases. It is becoming increasingly evident that the efficacy of MSC therapy is dependent on several factors including dosage, the tissue source of MSCs, the route of delivery and timing of administration. In a time where MSC therapy is moving from preclinical research to clinically therapeutic use, the importance of choice of delivery method, modality, and administration route increases. In this review, we provide an overview of the different MSC delivery routes used in preclinical kidney disease models, highlight the recent advances in the field, and summarize studies comparing delivery routes of MSCs to the kidney.


INTRODUCTION
The pathophysiology of kidney diseases is complex and includes multiple biological processes including inflammation, fibrosis, and a maladaptive immune response.Currently, there are no effective treatment strategies that can halt or reverse the progression of fibrosis [1].Cellular therapies in the form of mesenchymal stromal/stem cells (MSCs) have shown great promise in several preclinical models of kidney diseases and are currently applied in several clinical trials treating different renal disorders, such as chronic kidney disease (CKD), acute kidney injury (AKI), diabetic nephropathy (DN), and lupus nephritis [2].MSCs possess anti-inflammatory, immunomodulatory, and regenerative effects.Whereas regenerative effects are thought to be of less importance in renal disease, MSC's immunomodulatory effects by interaction with cells of both the innate and the adaptive immune system are believed to be the major actors of MSC's therapeutic effects [3].MSCs initiate immunomodulation by suppressing both T-and B-cell function and proliferation as well as prompting polarization of macrophages into anti-inflammatory phenotypes [4].
Secreted factors including extracellular vesicles (EVs) are believed to be responsible for a substantial part of the therapeutic effects, as conditioned media from MSCs have displayed therapeutic effects in several preclinical models of AKI and CKD [5].However, the molecular mechanisms of how MSCs mediate their therapeutic effect in renal disease models are not fully understood.
Although the secretome of MSCs has been extensively studied [6], the secreted factors responsible for mediating kidney protection are still debated.Prostaglandin E2 (PGE2) has been extensively studied in relation to its renoprotective effects [7,8].PGE2 is secreted by MSCs in response to inflammatory stimuli and has been shown to stimulate macrophages to transition from a proinflammatory to an anti-inflammatory phenotype [9].In line with this, MSC-secreted PGE2 can reduce renal ischemia/reperfusion injury by increasing activation of T regulatory cells [10].Similarly, the angiogenic factor, vascular endothelial growth factor (VEGF), plays a central role in mediating MSCs' renoprotective effects.VEGF is secreted by MSCs, and it has been demonstrated that VEGF knockdown in MSCs reduce the therapeutic effect of MSCs in an AKI rat model [11].Although VEGF and PGE2 are often mentioned in relation to MSC therapy in kidney diseases, the renoprotective effects of MSCs are likely mediated by a combination of different secreted factors.
So far, clinical trials have repeatedly proven the safety of MSC therapy but have failed to build on the positive results obtained in preclinical models of kidney diseases [12].The clinical use of MSCs is in its beginning stage, but it is evident that factors like histocompatibility, and MSC donor variability have a significant impact on the efficacy of MSC therapy.Other variables like MSC dosage, timing of administration, the tissue source of MSCs, cryostorage, and the administration route, can all affect clinical outcome.In this review, we will provide an overview of the different types of MSC delivery routes used in preclinical kidney disease research and compare local vs systemic administration.

MSC ADMINISTRATION ROUTES
In publications from 2004 to 2022, we found in total 469 preclinical studies investigating the therapeutic effects of MSCs in kidney disease.In these studies, administration of MSCs can be categorized as systemic delivery or local delivery to the kidney (Fig. 1).Most studies used systemic delivery (total of 74%), whereas only 25% of the studies used local delivery (Fig. 2).

Systemic administration
In studies applying systemic MSC delivery, intravenous (IV), intraperitoneal (IP), and intra-arterial delivery make up the vast majority (Fig. 2).Due to the convenience and easy accessibility, the tail vein is by far the most used site of injection for systemic delivery in preclinical models, while alternative routes such as the carotid artery or aorta have been used more sparingly.In addition, some studies have delivered MSCs via IP injections.In this review, IP injection is classified as systemic administration, although, it is not clear whether the MSCs enter the vascular system after injection into the peritoneum.However, MSCs injected IP have been reported to reach the kidney parenchyma [13].
Vascular injection of MSCs has been shown to lead to thrombosis in both preclinical animal models as well as in humans [14], however, in general vascular administration of MSCs is considered safe with only minor adverse events [15].T€ ogel et al. [16] compared arterial and venous MSC injection in an AKI mouse model and found that recruitment of MSCs to the injured kidney was superior after arterial delivery.This might be due to the pulmonary first-pass effect of IV administered MSCs which results in a greater extent of entrapment of MSCs in the capillary bed of the lungs than intra-arterial administered MSCs.Pulmonary entrapment of MSCs is dependent on cell size and the ability to adhere to the vascular tissue Fig. 1.Common delivery methods of MSC therapeutics to kidney.Examples of methodological papers: intravenous [55], intra-renal [34], subcapsular injection [34,56] and renal artery injection [57].[17].Vascular cell adhesion molecule (VCAM-1) is important for adhesion, as blockage of its counter ligand significantly improves the number of MSCs reaching the arterial system [17].Moreover, a greater number of MSCs escape lung tissue entrapment when given as a second bolus injection compared to a single injection, presumably due to receptor saturation from the first injection [17].
It has been shown that MSCs entrapped in the lungs are phagocytosed by macrophages, which can change the secretion profile of the macrophages to promote more anti-inflammatory properties [4].Moreover, endocrine activity of lung entrapped MSCs can mediate effects in the kidney.Thus, despite systemic entrapment MSCs still possess the ability to mitigate kidney injury and carry out systemic immunomodulatory effects [18,19].Although the majority of MSCs end up in the lungs, reports have shown that MSCs can reach sites of injury after systemic delivery.After introduction of MSCs into the bloodstream, engraftment at the site of injury is dependent on surface receptors such as the CXCR4 chemokine receptor [20] and CD44 [21] expressed on MSCs.CD44 knockdown or CD44 blocking antibodies prevented renal entrapment of MSCs along with recovery of renal function in an AKI mice model [21].Although tracking studies of MSCs after systemic delivery have showed poor engraftment of MSCs in the kidney, it has been demonstrated that renal injury results in an increased numbers of MSCs reaching the kidney tissue, indicating that vascular injected MSCs have preference for damaged tissue [22].This is however still debated as a recent study combining bioluminescence and magnetic resonance imaging found that kidney injury did not correlate with enhanced engraftment [23].Importantly, the sensitivity of the detection method could influence the conclusion, as made evident in a study by Schubert et al. [24] comparing in vivo bioluminescence tracking with PCR-based detection on IV-injected luciferase transgenic MSCs in a cisplatin-induced AKI mouse model.Here, in vivo bioluminescence imaging only detected luciferase active cells in the lungs and none in the kidney.However, the more sensitive PCR analysis revealed that luciferase-expressing MSCs were indeed present in the kidney and the number of cells positively correlated with the grade of kidney injury [24].This perhaps points to the shortcomings of some detection methods used for tracking MSCs and shapes the conclusion made upon them.More advancement in the field of cell tracking will be essential in order to gain more insight into the engraftment in kidney tissue and fate of systemically administered MSCs.
Methods to improve entrapment of MSCs in the injured kidney might include transient or stable expression of genes or activation of specific surface markers and chemokine receptors improving the ability of MSCs to detect damaged tissue.CXCR4, important for engraftment in injured tissue, has been overexpressed in bone marrow-derived MSCs.
Here, CXCR4 overexpression led to increased engraftment in injured kidneys, resulting in enhanced restoration of kidney function after AKI compared to na€ ıve MSCs [25].Similarly, another study has investigated the role of hyaluronic acid, a ligand of CD44, related to the engraftment of MSCs into an AKI mouse model.The addition of hyaluronic acid dose-dependently promoted engraftment to the injured kidney [21].Alternatively, methods using ultrasound have been explored to improve MSC entrapment in the injured kidney.Ultrasound-targeted microbubble destruction in which small gas bubbles facilitate vascular permeability [26] has been found to improve MSC engraftment and recovery from kidney injury [27].

Local administration
To circumvent entrapment of MSCs in the lungs local MSC administration directly to the kidney has been evaluated in several preclinical models of kidney disease.Local MSC delivery to the kidney include injection directly into renal parenchyma (intra-renal), subcapsular injection, injection into the renal artery as well as transplantation of an MSC sheet onto the kidney (Fig. 1).Moreover, local delivery of MSCs can also be accomplished during ex vivo machine perfusion prior to kidney transplantation [28].Intra-renal (38%) and renal arterial (34%) injection are the most frequently used for local delivery with injection under the capsule (16%) being the third most prevalent (Fig. 2).
The type of local delivery method results in a different distribution of MSCs in the kidney.Ex vivo kidney perfusion and renal artery injection utilize the vascular system.Here, the majority of cells end up in the glomeruli and less in the renal parenchymal tissue [29][30][31].MSCs transplanted by subcapsular injection are often delivered together with a cell scaffold, but the literature is conflicting as to whether the MSCs infiltrate the kidney parenchyma or not [32,33].In a recent study, Huang et al. [34] compared subcapsular administration of MSCs packed in a collagen matrix with intra-renal injection in a mouse model of AKI.Here, they showed that subcapsular administration produced better MSC survival and therapeutic effect over direct intra-renal injection.They speculated that the improved efficacy could be explained by a larger area of kidney tissue covered by subcapsularinjected MSCs spanning the entire kidney, compared with the direct intra-renal injection only reaching the proximate area of the site of injection [34].Interestingly, transplantation of an MSC sheet onto the kidney has emerged as a novel local route for targeted delivery [35].Cell sheets are made by harvesting MSCs without disrupting extracellular matrix and cell-cell contacts.MSCs connected in the cell sheet do not infiltrate the renal parenchyma but mediate their therapeutic function through paracrine signaling.However, formation of blood vessels that connect the kidney tissue with the transplanted MSC sheet has been observed in a study with a VEGF overexpressing MSC sheet.This suggests that a vascular connection can be formed between the MSC sheet and the kidney tissue under pro-angiogenic conditions [35].
Local delivery of MSCs have various disadvantages compared to systemic delivery.Local administration is often invasive, more complicated, and requires additional training of the medical practitioner.

SYSTEMIC VS LOCAL DELIVERY
The majority of the studies outlined in Fig. 2, whether using systemic or local delivery of MSCs, report a renoprotective effect compared to untreated controls.However, most of these studies only investigated one type of MSC administration.Studies that directly compare different administration routes of MSCs will be more informative in identifying which delivery routes are most effective for certain renal disorders.In Table 1, we have summarized the outcomes of studies that directly compared local and systemic MSC delivery routes in preclinical models of kidney disease.We found 12 studies investigating AKI, two studies investigating DN, and no studies investigating other kidney diseases, including CKD.The majority of studies using AKI models report no statistical difference between local and systemic delivery regarding effects of treatment, survival, and engraftment of cells [36][37][38][39][40][41][42].Five studies report better outcomes of local delivery compared to systemic administration [13,30,31,35,43].In one study, systemic MSC delivery outperformed local administration via the renal artery at the highest investigated dose of 1 9 10 6 MSCs when analyzing serum creatinine [30].A recent performed meta-analysis comparing arterial, IV or intra-renal delivery of MSCs in AKI rat models found that the improvement of renal function was significantly better than that of the placebo group.Moreover, it was shown that the therapeutic effect of systemic administration of MSCs by IV was better than that of the local delivery by intra-renal injection [44].The comparison included 50 studies of which most only investigated one delivery method compared to the placebo group.
We performed an additional analysis, including only studies that directly compared local vs systemic MSC administration in preclinical AKI models.The statistical comparison was based on serum creatinine measured 24-48 h after MSC intervention.The comparison found that both systemic (mean difference À 0.73 mg/dL; 95% CI, À1.06, À0.39; p < 0.0001) and local (mean difference À 1.03 mg/dL; 95% CI, À1.48, À0.58; p < 0.00001) MSC administration significantly decreased serum creatinine (Fig. 3A,  B) compared with untreated controls, consistent with the meta-analysis performed by Wanyan et al. [44].However, the relative change in serum creatinine decrease was not significantly different between local and systemic MSC administration (p = 0.11; Fig. 3C).Taken together, this comparison analysis indicates that in an AKI model the MSC route of delivery is not significantly impacting the therapeutic effect of MSC on kidney function measured by serum creatinine.However, the number of studies included in this analysis is limited and more animal studies, especially evidence from direct comparisons, are needed to explore the best administration strategies of MSCs.For other renal diseases, studies comparing local and systemic delivery of MSC are scarce.In a rat model of streptozotocin-induced DN, systemic administration by single or multiple tail vein injections was compared with injection into the liver parenchyma and by local subcapsular injection.The authors found that all delivery methods except single tail vein injections effectively attenuated DNassociated rise in blood glucose [45].In another study, local delivery using a MSC cell sheets and systemic IV administration were compared in a rat model of spontaneous DN [46].Locally administration via the MSC sheets had better effects on proteinuria, albuminuria, and chronic inflammation than IV administration of MSCs but both administration routes had similar ameliorating effects on kidney function measured by serum creatinine.One study performed a comparison between local and systemic MSC delivery in a preclinical CKD model [47].Here, MSCs were delivered locally by a catheter system into the renal artery or by IV injection in a rat model where CKD was induced by an adenine diet.The authors concluded that their local administration was superior in engraftment and survival; however, kidney function was not investigated.
Additional studies directly comparing systemic and local delivery in specific kidney diseases are needed in the future.Moreover, in these studies, it might be beneficial to also evaluate other MSC parameters such as MSC dosage, timing of MSC administration and MSC tissue origin.The impact of MSC tissue origin on the efficacy of MSC  applications is an ongoing discussion.Thus, factors such as dosing, inherent secretome and immunomodulatory properties of different sources of MSCs must be taken into consideration [48].Due to a lack of comparative studies, it is not possible to reveal whether the source of MSCs determines the optimal delivery route.These considerations may not only apply to MSC-based therapies but also be useful for other cell therapies aiming to treat kidney disease, including renal stem cells.

EXTRACELLULAR VESICLES
Part of the therapeutic effect of MSCs is induced by the secretion of 30-1000 nm large EVs, which are carriers of proteins and microRNA [49].In recent years, cell-free treatment with MSC-derived EVs has been explored as an alternative for MSC therapy as they hold several advantages over MSCs including the ability to cross biological barriers [50].EVs have been reported to have improved or similar effects compared to MSCs in a rat model of AKI, where intra-renal arterial EV administration mitigates kidney damage, oxidative stress, and inflammation [51].
The small size of EVs results in completely different tissue distribution than MSCs.EVs seem to bypass the pulmonary first-pass effect, but instead accumulate in the liver and spleen [50].However, there have also been reports of EV accumulation in the damaged kidney after systemic administration [52].Preclinical testing of EVs in kidney disease have used both systemic and local delivery (Fig. 2), but despite the increasing use of EVs in preclinical models of kidney disease, no studies have compared delivery routes and its effect on therapeutic function.

CLINICAL STUDIES
We categorized a total of 50 clinical trials listed on clinicaltrials.govwith the general term 'mesenchymal stem cells OR mesenchymal stromal cells' and the disease term 'kidney diseases', according to delivery route.In total, 25 studies were designed for systemic injection by IV or intra-arterial infusion, three use local delivery by injection into the renal artery or directly into the renal parenchyma, one used intramuscular delivery, while the remaining trials did not specify the delivery route.Thus, in a clinical setting vascular administration is highly favored, which is most likely driven by the less-invasive nature and the ease of the procedure compared to local administration via the renal artery or injection into the renal parenchyma.However, local MSC administration via the renal artery has been proven feasible and safe in patients with renal vascular disease, in which 1, 2.5 or 5 9 10 5 MSCs/kg were administered into the renal artery by a catheter [53].
One has to keep in mind that the doses of MSCs used for preclinical testing are many-fold higher than the ones used for clinical trials and it might be unrealistic to reach the same number of cells in humans [54].Potentially, local delivery, resulting in a greater dose of MSCs at the diseased kidney, could be an option to bridge this gap.

CONCLUSION
The application of MSCs in preclinical and clinical kidney research is expanding, and studies of the therapeutic effects and the best administration route of MSCs are in high demand.Currently, MSCs are predominantly administered by IV injections in preclinical animal models and in clinical trials.Importantly, different administration routes can have an impact on the survival and homing rate of MSCs.Moreover, the timing of MSC injection, the number of injections as well as the number of cells per injection are important aspects which needs further investigations.Our statistical comparison of systemic and local delivery of MSC to the kidney in preclinical AKI models found that both delivery modalities improved kidney function.However, from the limited number of studies directly comparing systemic and local administration, it was not possible to draw a definitive conclusion on the optimal delivery route, particularly across different kidney diseases.Thus, there is a need for additional comparative studies investigating the impact of the administration modalities in kidney diseases and we encourage these types of studies.

Fig. 3 .
Fig. 3. Statistical comparison of the effect of MSC delivery route on kidney function.(A) Forest plots displaying the effect of systemically delivered MSCs on decreasing serum creatinine in comparison to control and (B) local delivery of MSC compared to control.Means are displayed in mg/dL serum creatinine.(C) Forest plot displaying the effect of systemic administration of MSC on decreasing serum creatinine compared to local delivery.Means are displayed as fold change compared to respective control animals.The inclusion criteria were: (1) Preclinical models of AKI, (2) systemic and local administration of MSC at the same dosage, (3) placebo control included (4) quantification of serum creatinine 24-48 h after intervention.Forest plots and statistics were generated using Review Manager (RevMan) version 5.4.1.The Cochrane Collaboration, 2020.95% CI, 95% confidence interval; IV, Inverse variance; SD, standard deviation.

Table 1 .
Comparison between local and systemic administration of mesenchymal stem cells in preclinical models of kidney disease Ó 2023 The Authors.APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and

Table 1 (
continued)OnceThe table only includes studies that directly compare the effect of local and systemic administration on the kidney.The corresponding column lists outcomes that were statistically improved by either local (L) or systemic (S) delivery when compared to each other.Arrows indicate whether outcomes were decreased (↓) or increased (↑) compared with the intervention group.In cases where systemic and local administration had an equal effect, the term 'No difference' (ND) is indicated.AD, adipose tissue-derived; AKI, acute kidney injury; BM, bone marrow-derived; BUN, blood urea nitrogen; DN, diabetic nephropathy; GDM, gestational diabetes mellitus; GSH,