Investigating the physiology of ischaemia reperfusion injury of the mouse kidney and the efficacy of regenerative medicine therapies after acute kidney injury

Acute kidney injury (AKI) and chronic kidney disease (CKD) are a global cause of morbidity and mortality. AKI, commonly caused by poor perfusion of the kidneys, can cause CKD or hasten the progression to end stage renal failure. The current strategy for treatment of AKI caused by poor perfusion is to use supportive therapies to improve kidney perfusion and, if necessary, to provide renal replacement therapy (dialysis) until the kidneys start to function again. Cell-based regenerative medicine therapies have been proposed as a novel method of reducing or ameliorating AKI. Pre-clinical rodent studies have demonstrated efficacy of mesenchymal stromal cells (MSCs) and their derived extra-cellular vesicles (ECVs). Evidence suggests that the therapeutic effects of these therapies are mediated, at least in part, by their immunomodulatory effects. However, despite the reported beneficial effects in animals, this has not translated into clinical practice, leaving questions about the most appropriate timing, route of administration and dose required to ameliorate or reduce AKI after an ischaemic injury. The work in this thesis aimed to address these questions by first optimising the murine model of bilateral renal ischaemia reperfusion injury (r-IRI) to cause survivable but severe AKI. Once optimised, the efficacy of human umbilical cord mesenchymal stromal cells (hUC-MSCs) and their derived extra-cellular vesicles (hUC-ECVs) was assessed. Optimisation of the bilateral r-IRI model was undertaken in male BALB/c mice. Body temperature was maintained at 370C and clamping times of 25, 27.5 and 30 minutes assessed. Mice undergoing a clamping duration of 27.5 minutes were found to have a severe but generally survivable injury. Furthermore, the duration of anaesthesia before vascular clamp application was found to be associated with the severity of AKI and was standardised. hUC-MSCs and hUC-ECVs were then extensively investigated for efficacy in the standardised AKI model in BALB/c mice. Animals receiving hUC-MSCs by intracardiac injection immediately after surgery were found to have a significantly higher mortality rate compared to those receiving hUC-MSCs by intravenous injection at the same time point. Efficacy of intravenously delivered hUC-MSCs compared to a control group receiving phosphate buffered saline was not observed despite using two different doses (2.5x105 and 5x105 cells per animal). Administering a dose immediately after surgery and at 24 hours post-operatively was also not efficacious. Intravenous administration of 2x108 hUC-ECVs initially appeared to be efficacious but when the experiments was repeated with an increased sample size this could not be replicated, despite changing the dose (1x107, 1x108, 1x109) and administering a dose immediately after surgery and 24 hours post-operatively. Finally, administration of both hUC-MSCs and hUC-ECVs in an alternative mouse strain, C57JBL/6 mice, did not demonstrate efficacy. Most studies where efficacy of MSCs has been reported have used C57JBL/6 mice, which are known to have a pro-inflammatory (M1/Th1) immune response. BALB/c mice on the other hand have an anti-inflammatory (M2/Th2) response. To understand whether this difference in immune response alters the time-course or physiological function of the kidney after r-IRI, novel imaging methods were used to assess glomerular and tubular function after unilateral r-IRI in Albino BL6 and BALB/c mice. Glomerular function was assessed using multi-spectral optoacoustic tomography (MSOT) to monitor the clearance of IRDye800, a near infrared dye that is filtered through the glomeruli. Tubular function was assessed using single photon emission tomography (SPECT) to determine the uptake of 99mTc-DMSA. Glomerular function was found to substantially deteriorate immediately after severe r-IRI and rapidly recover in both strains. Albino BL6 mice then showed a later deterioration in glomerular function which progressed over three weeks following injury. Conversely, tubular function gradually deteriorated over 8-12 days after r-IRI before plateauing at a similar level of function in both strains. BALB/c mice appear to reach this plateau sooner than Albino BL6 mice, suggesting that there may be differences in the time-course of the response to kidney injury between the strains. Overall, efficacy of hUC-MSCs and hUC-ECVs has not been demonstrated within this thesis and highlights the need for robust, reproducible reporting of results. However, novel imaging therapies have been successfully used to demonstrate previously undescribed longitudinal changes in glomerular and tubular function and potential differences between mouse strains which may be used to deepen the understanding of the role of the immune system in r-IRI and could help to identify novel therapeutic targets to reduce the progression of AKI to CKD