Novel delivery of cellular therapy to reduce ischaemia reperfusion injury in kidney transplantation

Abstract: Ex-vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimise organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC(R)) possess potent immunomodulatory properties which could prove beneficial in minimising subsequent ischaemia reperfusion injury. We investigated the potential reconditioning capability of MAPC cells in kidney NMP. Methods: Pairs (5) of human kidneys from the same donor were simultaneously perfused for 7 hours. The right or left kidney was randomly allocated to receive MAPC treatment. Serial samples of perfusate, urine and tissue biopsies were taken for comparison with the control paired kidney. Results: MAPC-treated kidneys demonstrated improved urine output (p<0.01), decreased expression of the kidney injury biomarker NGAL (p<0.01), improved microvascular perfusion on contrast enhanced ultrasound (cortex p<0.05, medulla p<0.01), downregulation of IL-1{beta} (p<0.05) and upregulation of IL-10 (p<0.05) and Indolamine-2, 3-dioxygenase (p<0.05). A mouse model of intraperitoneal chemotaxis demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (p<0.01). Immunofluorescence revealed pre-labelled MAPC cells home to the perivascular space in the kidneys during NMP. MAPC therapy was not associated with detrimental physiological or embolic events. Conclusion: We report the first successful delivery of cellular therapy to a kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant functional parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to clinical transplantation.


Introduction
Currently the UK kidney transplant waiting list stands at over 5000 patients and the average waiting time is 3 years 1 . The organ shortage has led to increased use of organs from donation after circulatory death (DCD) and extended criteria donors (ECD) to bridge the gap between supply and demand 2 . Concerns regarding inferior transplant outcomes from DCD & ECD organs can lead to under utilization of this valuable resource 3,4 . DCD & ECD organs are more susceptible to damage from ischaemia reperfusion injury (IRI) manifesting as delayed graft function (DGF) and this can diminish graft survival 5,6 . IRI is the result of hypoxia followed by restoration of blood flow resulting in microvascular dysfunction, inflammation, immune activation and tissue injury 7 .
IRI is an unavoidable consequence of solid organ transplantation and results in DGF in up to 50% of kidney transplants 8 . As the transplant community becomes increasing reliant on marginal donors, new therapeutic approaches to reduce IRI and optimise utilisation of ECD kidneys are leading to a greater focus on improving organ preservation.
Normothermic Machine Perfusion (NMP) is a method of organ preservation that facilitates restoration of cellular metabolism, reviving the organ ex vivo to resume normal physiological functions 9 . This method has many potential benefits over traditional, static cold storage (SCS), including the opportunity to undertake real-time objective assessments of organ quality prior to transplantation. Over the past 10 years, a number of NMP techniques and commercially available devices have been adopted into clinical practice for kidney, heart, liver and lung transplantation 10,11 . Nasralla et al recently reported the first international, multicentre, randomised controlled trial of 220 liver transplants investigating liver NMP for organ preservation. This study clearly demonstrated NMP had significant advantages compared with SCS. There was a 50% reduction in graft injury (measured by hepatocellular enzyme release), a 50% reduction in organ discard, and a safe increase in preservation times 12 . This promising study has confirmed NMP as a viable, realistic technology with wider implications for its translation into other solid organ types and organ reconditioning.
Kidney NMP was first described in 2008 13 . In this system, a paediatric cardiopulmonary bypass machine and membrane oxygenator provides an ex vivo kidney with oxygenated red blood cells suspended in crystalloid at 37 o C 14 . A national multicentre phase 3 randomised controlled trial is currently underway to investigate the effectiveness of this technology in reducing DGF rates 15 .
NMP provides a unique opportunity to deliver organ-directed, reconditioning therapies. By establishing an isolated ex vivo platform with a metabolically active organ, therapies targeting ischaemia reperfusion injury can be delivered to the kidney 16 . This could prove to be transformative, allowing delivery of stem cell or gene therapies directly to the kidney reducing off-target, systemic effects in recipients.
Multipotent Adult Progenitor Cells (MAPC) are adult, bone-marrow derived stromal cells first described in 2002 17 . MAPC cells represent an attractive 'off-the-shelf' cell therapy option as they lack MHC Class II, or co-stimulatory molecules (CD80, CD86 & CD40) and have low levels of MHC Class I expression meaning they are non-immunogenic 18 . MAPC cells release antiinflammatory, immunomodulatory and pro-tolerogenic cytokines thereby limiting infiltrating pathogenic immune cells 19 . MAPC cells have also demonstrated potent in vitro immunosuppressive effects on T-cell proliferation 20 . Maximal immunosuppressive effects were achieved in the most pro-inflammatory environments suggesting possible benefit in the highly inflammatory, marginal organ.
These in vitro findings have been validated by a number of animal models confirming the immunomodulatory capacity of MAPC cells in a transplant setting. In a rat allogeneic heterotopic . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint heart transplant model (Lewis-into ACI) allogeneic MAPC cells could successfully replace standard pharmacological immunosuppression to maintain long-term graft survival mediated by local heart-specific effects of T regulatory cells 21 .
There have also been a number of successful clinical trials harnessing the immunomodulatory potential of MAPC treatment for Graft versus Host Disease 22  Our study aims to investigate the possible benefit of delivering MAPC cell therapy directly to marginal human kidneys in a pre-clinical NMP model.
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint

Results
Kidneys included in pre-clinical series 5 pairs of kidneys (n=10) were included in the study ( Table 1). The donors included represent a heterogeneous cohort with ages ranging from 52 to 77 years, but all were from either DCD donors or had characteristics consistent with ECD status. Cold ischaemic times were significantly extended due to the delays inherent in a kidney being offered for research only purposes. All the pairs of kidneys were declined for transplant due to a suspicion of non-renal malignancy identified at the retrieval operation.

Renal Physiology
Serial measurements of physiological parameters were recorded during NMP. Perfusate samples were analysed in real-time to assess adequate oxygenation, metabolic requirements and biochemical parameters. These were compared between the pairs of kidneys, control vs MAPC treated, and demonstrated equivalent organ physiology associated with MAPC cell infusion. For clinically relevant markers, potassium, lactate, renal blood flow & renal resistance, the kidneys were well matched throughout the 7-hour perfusion timeline ( Figure 1 Panel A-D).
During NMP the ureter of the kidney was cannulated and attached to a urometer to facilitate hourly urine output measurement and sampling. In the kidneys treated with MAPC cells there was significantly higher urine output compared to control kidneys during NMP, p<0.01 (Figure 1,

Kidney Injury Biomarkers
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org /10.1101/19005546 doi: medRxiv preprint There are a number of validated biomarkers regularly used in kidney transplant or acute kidney injury research, including Kidney Injury Marker 1 (KIM-1) and Neutrophil Gelatinase-Associated Lipocalin (NGAL) 28 . With the advent of NMP technologies we can analyse these biomarkers during perfusion to evaluate reconditioning. In this study, we assessed both the levels of urinary

Ex vivo Ultrasound
To determine if the administration of a MAPC cell bolus was associated with any effects on the renal microvasculature we performed a number of imaging studies using ultrasound. MicroFlow Imaging (MFI) Doppler ultrasound was performed. MFI is an ultrasound technology that can provide high resolution detail on blood flow within small vessels. This technique was used to assess blood flow following MAPC treatment and revealed restored blood flow within the renal medulla 4 hours after MAPC cell infusion ( Figure 1, Panel G, indicated by white arrow). The same effect was not seen in control kidneys.
Alongside MFI, contrast enhanced ultrasound (CEUS) was performed after 60 minutes of NMP (before MAPC cell infusion) and 4 hours later. This was primarily to investigate if the cell bolus was associated with micro-emboli or occlusion of the renal microvasculature as the mean diameter of a MAPC cell is greater than a capillary. CEUS provides a quantifiable assessment of microvascular perfusion and, demonstrated a significant improvement in both cortical and . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  (19). Cells expressing IDO catabolize tryptophan (Trp) to suppress effector T cells and activate Foxp3-lineage regulatory CD4 T cells (Tregs) 29 . IDO activity was assessed by measuring Trp and the catabolite kynurenine (Kyn) in the perfusate using HPLC. Data revealed that MAPC treated kidneys had significantly higher IDO activity (elevated Kyn:Trp ratio) following 7 hours of perfusion when compared to IDO activity in paired control kidneys, p<0.05 ( Figure 2, Panel C).
MAPC perfusate secretome effect on HMEC-1 endothelial cell line model . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org /10.1101/19005546 doi: medRxiv preprint An in vitro endothelial cell line model was used to better understand the impact of the MAPC secretome on kidney vascular endothelium and microvascular integrity. As MAPC cells are a biologically reactive product, the secretome produced by the cells is dependent upon the microenvironment in which they are delivered. Indeed, this is one of the attractive mechanisms of action we hope to harness through MAPC cell delivery to a marginal kidney during NMP -the cells should react to this pro-inflammatory, ischaemic environment and produce the required growth factors and mediators for optimum reconditioning. To model this in vitro, samples of acellular perfusate taken after 7 hours of NMP were added to Human Microvascular Endothelial Cells (HMEC-1) cells in culture. ICAM-1 (activation status) and S1PR1 (microvascular barrier integrity) expression was analysed in the HMEC-1 cells in response to perfusate from pairs of kidneys (untreated vs MAPC treated) (Figure 2 Panel D-G). ICAM-1 protein expression was significantly increased in the control group, p<0.001. However, in the MAPC treated group this upregulation was not as marked, p<0.05. In contrast, S1PR1 gene and protein expression in HMEC-1 cells was downregulated by control perfusate, however, MAPC perfusate maintained S1PR1 gene expression at unstimulated levels, p<0.05; and increased protein expression when compared with control perfusate, p<0.001). This preservation of S1PR1 protein was also seen on immunofluorescence in the tissue of NMP perfused kidneys, p<0.05 ( Figure 2, Panel H-J).

MAPC secretome effect on mouse intraperitoneal chemotaxis assay
To evaluate if the MAPC secretome in the perfusate during NMP had an impact on leucocyte chemotaxis we used a small animal model. Samples of acellular perfusate were injected intraperitoneally to mice. There were 4 treatment groups, each containing 5 mice, as described in the methods section. Six hours after injection the intraperitoneal space was lavaged to harvest the immune cell infiltrate which was analysed using flow cytometry (supplementary figure 1, Panel . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint C-E). CD45+ was used as a leucocyte marker and Ly6G+ for neutrophils. Control perfusate led to a significant increase in peritoneal neutrophils compared to crystalloid injections alone. This increase was not seen in perfusate from MAPC treated kidneys, p<0.01 ( Figure 2, Panel J-L).

Determining the physical distribution of NMP administered MAPC cells
MAPC cells were pre-labelled with a red fluorescent dye in order to understand cell fate following intra-arterial delivery during kidney NMP. To achieve this, following 7 hours of perfusion, the kidneys were de-cannulated and anatomical regions of interest sampled. Samples were taken from the cortex, medulla, artery and collecting system of the kidney and visualised using fluorescent immunohistochemistry and confocal microscopy. Nuclear counter-staining was with DAPI (blue).
Additional staining for endothelial cell marker (CD31 -green) and a proximal tubular epithelial cell marker (Aquaporin-1) was performed to aid co-localisation of MAPC cells within the kidney's architecture. This revealed that the majority of the red labelled MAPC cells were to be found in the glomeruli and around the peritubular capillaries kidneys, ( author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint circulating live MAPC cells to be 21%. In contrast, the passenger leucocyte population had a mean live proportion of 44%.
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint Table 1 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint typical confocal microscopy image from kidneys treated with MAPC cells. The white letter (G) identifies the glomerulus. Panel C is a typical section taken from the kidney's medulla, vessels identified by the CD31 green stain are identified by the green (V). Panel D is a high magnification image taken of 3 MAPC cells that are resident in the interstitium next to a peritubular capillary. Panel E depicts the confocal images of a blood vessel from the renal cortex with a MAPC cell in the lumen and cells that have mobilised out of the vessel into the nearby tissue.
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint

Discussion
MAPC therapy has demonstrated significant promise in treating a number of clinical conditions associated with inflammation and ischaemia 23,24,25,30 . Clinical trials have demonstrated MAPC cells have a robust safety profile. Therefore, the translation of MAPC therapy to minimise ischaemia reperfusion injury in kidney transplantation represents a promising avenue for further exploration. Here we have described the first reported series of human kidneys successfully treated with a cellular therapy using normothermic machine perfusion.
Kidneys treated with MAPC therapy demonstrated an improvement in functional parameters and biomarkers of kidney injury. A higher volume of urine output and lower urinary NGAL during NMP has previously been correlated with improved clinical outcomes following transplantation 31,32 . Improving these key parameters with MAPC treatment is suggestive of less tissue damage and improved cellular metabolism. We have also demonstrated a significant improvement in microvascular perfusion during NMP using CEUS. The improved blood flow seen in MAPC treated kidneys could be mediated by PGE2 or IDO, both potent vasodilators, which are reported to be part of the MAPC cell secretome in pro-inflammatory environments 33 .
Cytokine profiling revealed that MAPC therapy during kidney NMP was associated with an antiinflammatory, pro-tolerogenic cytokine profile. This included decreased expression of IL-1β, a pro-inflammatory cytokine associated with endothelial activation. Our group have previously correlated lower levels of IL-1β during ex vivo lung perfusion with better clinical outcomes following lung transplantation 34 . There was also upregulation of pro-tolerogenic, antiinflammatory IL-10 and this was accompanied by increased IDO activity. MAPC cells modulating differential cytokine expression in perfusate may be key in their potential role of minimising IRI.
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint Tracking the MAPC cells after delivery in the NMP circuit has revealed cells could be found throughout the kidney. Previous studies demonstrated MAPC cells have a powerful ability to home to hypoxic, damaged areas of the endothelium where they migrate into the tissue, taking up residence within the perivascular space 35,36,37 . Indeed, in a pre-clinical study of acute myocardial infarction the cells actively migrated into the ischaemic, damaged myocardium following transarterial delivery 38 . This may be mirrored in our NMP model -MAPC cells are homing to hypoxic areas within a marginal kidney where they take temporary residence. In this environment, the cells release an anti-inflammatory secretome which is having beneficial effects, resulting in In 2018-19, 320 kidneys were not transplanted in the United Kingdom. The vast majority of these organs were rejected because of anticipated failure at reperfusion from excessive IRI. In addition, an unknown number of kidneys were not retrieved because of fears over primary non-function, despite adequate function in the donor prior to death. Previous attempts at discovering a pharmacological therapy to effectively minimise IRI have been unsuccessful (46) due to the high levels of redundancy inherent in IRI pathways. We have shown that MAPC therapy combined with EVNP interacts with multiple pathways and improves physiological function in a measurable and . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint reassuring way. Based on these results we believe up to 50% of discarded kidneys and an unknown number of un-retrieved kidneys could be transplantable with MAPC therapy during EVNP

Conclusion
This is the first reported series of cell therapy successfully delivered directly to human donor kidneys in an isolated ex vivo perfusion platform. Kidneys treated with MAPC cells during NMP demonstrate improvement in clinically relevant functional parameters and a reduction in injury and pro-inflammatory biomarkers. This may be mediated by changes to circulating cytokines or through secreted soluble anti-inflammatory mediators. NMP represents a novel cell therapy delivery system. This is a paradigm shift, providing an exciting opportunity to directly treat organs prior to transplantation to minimise ischaemia reperfusion injury. A future clinical trial evaluating this modality of delivery could result in the transplantation of otherwise discarded organs, thereby reducing the transplant waiting list and offering hope to patients with renal failure.
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint

Ethics
Ethical approval for the use of human donor kidneys declined for transplantation was granted by local Research Ethics Committee (16/NE/0230). Consent for recruitment of the organ into a research study was obtained from the donor's next of kin by specialist nurses in organ donation.
Currently 15% of kidneys retrieved from organ donors across the UK are deemed unsuitable for

Human Kidney Normothermic Machine Perfusion
On arrival at our centre kidneys were surgically prepared on ice. The renal artery was cannulated to facilitate connection to the NMP circuit. The ureter was also cannulated so urine output could be measured and sampled. Kidney pairs were perfused simultaneous for 7 hours with an oxygenated red-cell-based perfusate at a mean temperature of 36.5 o C and mean arterial pressure of 75mmHg, according to published protocols 46 . The volume of perfusate in the circuit was kept constant by matching the urine output with a crystalloid solution via continuous infusion. All physiological parameters accessible during NMP were recorded and analysed including: perfusate blood gas analysis, biochemical analysis, urine production, flow rate, and scored according to the validated quality assessment tool (7).

MAPC treatment
The MAPC cells used in this study were obtained in collaboration with Athersys Inc, Ohio, and were provided as pre-dosed cryovial aliquots of 25x10 6 cells. The MAPC cells were research grade . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org /10.1101/19005546 doi: medRxiv preprint Human MultiStem® cultures that were isolated from human bone marrow with consent from a healthy donor as previously described 47 . Prior to release, the MAPC cells were confirmed to express phenotypic markers 48 , be of >95% purity, and were fluorescently labelled with cytoplasmic dye CellTracker Red CMPTX (ThermoFisher).
For MAPC treatment of kidneys during NMP, following 60 minutes of perfusion, the right or left kidney was randomly allocated to a prescribed MAPC dose (50x10 6 cells). In the 60 minutes preceding infusion the MAPC aliquots were gently thawed in a water bath at 37 o C and then kept on ice until ready for delivery. Immediately prior to delivery cells were re-suspended in 10ml of perfusate. Control kidneys simply received a 10mL bolus of perfusate. The chosen MAPC dose was extrapolated from previous phase 2/3 clinical trials using MAPC cells for systemic infusion and calculated based on the average weight of a human kidney and volume of fluid in the perfusion circuit 27 .

Contrast Enhanced Ultrasound
Contrast Enhanced Ultrasound (CEUS) was performed during NMP. This technique utilises microbubbles of inert gas (sulphur hexafluoride in a phospholipid shell) to increase ultrasound signal return. Each bubble is approximately 2-3μm in diameter, allowing it to pass through the capillary bed but not the interstitium. CEUS has made it possible to assess the distribution of perfusion at a microcirculation level 49 . We have previously had success with this technique on porcine kidneys on a cold perfusion circuit and in NMP livers (16). This technique investigated whether MAPC cells cause microemboli within the microcirculation by comparing CEUS on kidneys pre and post MAPC infusion. One minute CEUS clips obtained were analysed using QLab 8.1 (Philips, Bothwell, WA, USA) CEUS quantification software. CEUS was performed using a Philips EPIQ7 Ultrasound machine. During NMP CEUS recordings were taken at 60 minutes . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint (before MAPC infusion) and 4 hours later. A sterile sheath covered the CEUS probe and it was placed directly onto the perfusing kidney. The probe was kept stationary in a longitudinal section during contrast infusion. Contrast media (0.5mL bolus of Sonovue) was delivered via a 3-way tap on the renal artery cannula. Cineloops were acquired of the renal parenchyma and 3 standardised 5mm regions-of-interest (ROI) were captured (the interlobar artery, cortex and medulla) to record perfusion gradients. To quantify perfusion during CEUS, the Area Under the Curve (AUC) was calculated for each ROIs. A ratio of the parenchymal ROI AUC (medulla or cortex) was calculated in reference to the vessel ROI. These ratios (rAUC) were used to quantify differences in tissue perfusion. The rAUC from the start and end of perfusion were compared.
MicroFlow Imaging Ultrasound was also performed using the Philips EPIQ7 machine. MFI Ultrasound is designed to detect blood flow within the small vessels at high resolution with minimal artefact. This is displayed using overlaid blue Doppler signal and can aid in subjective interpretation of perfusion. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

ELISA
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint manufacturer's instructions alongside technical support from an experienced MSD technician. The method of detection utilised an electrochemi-luminescent label conjugated to the detection antibody.

High Performance Liquid Chromatography
Culture supernatants and perfusate were acidified with sodium acetate, pH=4, to give a final concentration of 15mM. Protein was precipitated with 1:10 perchloric acid 60%; samples were centrifuged, filtered and, then, analysed by HPLC to quantify kynurenine and tryptophan as described 50 .

In vitro endothelial cell line perfusate stimulation model
An Immortalised endothelial cell line (HMEC-1 cells, ATCC) was used to investigate the impact of the MAPC perfusate secretome on endothelial function. HMEC-1 cells were cultured to confluence and were treated with acellular perfusate diluted in modified MCDB131 media. In chamber slides, cells were stimulated with 25µl of acellular perfusate to evaluate protein expression using immunohistochemistry. In 6-well plates, HMEC-1 cells were stimulated with 250 µl of acellular perfusate to evaluate gene expression using RT-qPCR. In both situations cells were stimulated for 4 hours. RNA extraction was performed on-column using Qiagen RNeasy Plus Mini-kit as per manufacturer's instructions. cDNA synthesis was carried out using the Tetro cDNA synthesis kit. RNA sequence quantification was carried out using TaqMan RT-qPCR on a StepOnePlus™ Real-Time PCR System. Per well 2μl of cDNA, 7μl RNase free water, 10μl SensiFAST™ Probe Hi-ROX Kit and 1μl of primer (ICAM1 TaqMan Hs00164932_m1, S1PR1TaqMan Hs00173499_m1) was added in a 96 well PCR plate. GAPDH was utilised as a housekeeping gene (TaqMan Hs02786624_g1).

Mouse Intraperitoneal Chemotaxis Assay
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Samples were fixed in formalin and stored as paraffin embedded blocks. These were cut into 4µM sections at a later date for fluorescence microscopy imaging using a Zeiss Axioimager.

Immunofluorescence co-localisation staining of tissue sections.
Cut sections of perfused kidneys were de-waxed for 10 minutes in Xylene. The sections were then rehydrated through graded ethanol (99%, 90%, and 70%). Sections were placed in a container into . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint a pressure cooker with 1.5L of Tris/EDTA and heated for 2mins. Blocking was performed with 10% Goat serum, 100ul per slide for 30 minutes at room temperature. Blocking buffer was removed and 100ul of primary antibody diluted in added ((S1PR1, ThermoFisher Scientific PA1-1040, 1:50; CD31, Abcam, ab182981, 1:2000; Aquaporin-1 Abcam ab168387, 1:100). Slides were incubated in the fridge at 4°C overnight. The primary antibody was washed three times in 0.05% Tween for 5 minutes. Then 100ul of the relevant secondary antibody (anti-rabbit Cy5 ThermoFisherer Scientific 1:100; anti-rabbit FITC, Abcam, ab97050 1:200, anti-rabbit Dylight550, Immunoreagents 1:100) was added and incubated for 1hour at room temperature. Following a further 3 washes, slides were incubated with 0.1% Sudan Black B in 70% EtOH to quench tissue auto-fluorescence. Coverslips were mounted with Vectashield Antifade Mounting medium with DAPI (Vectorlabs) and sealed. Since fluorescent staining fades over time it was ensured that sections were imaged and analysed taken within 2 weeks. Fluorescent imaging was performed using a Zeiss Axioimager or for high resolution Leica SP2 UV AOBS. Images were processed and analysed using Zen, LAS X or Fiji software.  CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Cell filter studies
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint incubated with 1:2000 DAPI for 15 minutes prior to analysis. Flow cytometry was performed using the BD Fortessa X20 and the results analysed using FlowJo™ software.

Statistical analysis
Continuous variables are reported as mean ± standard deviation where appropriate. When comparing between control and MAPC treated kidney pairs from the same donor a two-sided paired t-test was used. If another treatment group was included analysis was carried out using one- . . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint

Supplementary Material
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint
. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/19005546 doi: medRxiv preprint