The effects of adipose-derived regenerative cells in preclinical kidney injury models

Chronic kidney disease is a common health issue affecting approximately 10% of the UK population. Advanced kidney failure necessitates kidney transplantation or dialysis in affected patients. The optimal treatment for end-stage kidney failure is kidney transplantation. Although the number of deceased donor kidney transplants has increased, the number of patients waiting for kidney transplantation continues to rise. Therefore, to make more kidneys available for transplantation, it is important to improve kidney transplantation techniques by using effective perfusate solutions, developing methods for organ storage, and applying additional treatments to the transplant kidney to optimise outcomes. Adipose-derived regenerative cells (ADRCs) are a heterogeneous cell population derived from adipose tissue. These cells are composed of endothelial progenitor cells, pericytes, and adipose stromal cells. Previous studies have revealed the potential therapeutic effects of ADRCs. These effects might help kidney grafts recover from ischaemia-reperfusion injury in kidney transplantation. My project aims to examine the therapeutic effects of ADRCs on injured kidney models to assess their potential utility for optimising kidneys for transplantation. I used three approaches for investigation: 1) a systematic review to investigate the effect of mesenchymal stem cells (MSCs) on solid organ transplantation models; 2) testing kidney organoids as a complex structural model for examining the effects of ADRCs on cellular communication and organoid protection; and 3) proximal tubular cell culture to study the therapeutic effects of ADRCs. Since oxidative stress is a hallmark of injury in ischemia-reperfusion injury (IRI), hydrogen peroxide was used as an injury inducer to mimic kidney transplantation by increasing oxidative stress in both the kidney organoid model and the proximal tubular cell model. A systematic review was conducted using the search engine program Rayyan. Data were collected from 1991 to 2020 using specific terms. Two researchers assessed these data for validity. The results showed that MSCs have anti-apoptotic, anti-fibrotic, tubular injury protection, and anti-oxidative stress properties. These findings reveal the effects of MSCs on organ transplantation in the preclinical and clinical literature to date. In studies of ADRCs on injured kidney organoid models, embryonic kidneys at days 13.5–14.5 were collected and processed into cell pellets. These pellets were cultured for 14 days for organogenesis. Organoids were assigned to four groups for study: non-injury, injury, ADRC-treated non-injury, and ADRC-treated injury groups. Injured organoids were induced using 10 µM hydrogen peroxide for 60 minutes, and ADRCs at 10% of embryonic cells were used in the ADRC-treated groups. After assignment, organoids were cultured for 14 days before collection and downstream assays. The results showed organoid protection by maintenance of organoid size after injury, revealing the pro-survival properties of ADRCs. In proximal tubular cell culture experiments, NRK-52E cells were used and assigned to four groups (similar to the previous study). These cells were incubated with 500 µM hydrogen peroxide for 4 hours, and ADRCs were added at a 10% ratio of total NRK-52E cells. Laboratory assays including trypan blue exclusion test, western blot, real-time polymerase chain reaction (RT-PCR), flow cytometry, caspase-3 activity assay, and nanoparticle tracking analysis were performed after 24 hours of ADRC treatment. The results showed that ADRCs tended to decrease cell death markers and increase live cell markers in the ADRC-treated injured group and decreased NGAL gene expression. These findings reveal tubular protection from injury by ADRCs in injured proximal tubular cells. This thesis shows that ADRCs had positive effects in organ transplant models in systematic reviews, exhibited pro-survival effects on kidney organoids, and reduced kidney injury markers in NRK-52E cells. Therefore, ADRCs are a promising additional treatment to diminish IRI in transplant kidneys.