Investigation into the action of mesenchymal stromal cells in renal ischaemia reperfusion injury

Abstract

Renal ischaemia reperfusion injury (IRI) is a common contributor to acute kidney injury, arising from clinical conditions such as kidney transplantation, sepsis and cardiac bypass surgery. Any condition leading to a reduction in renal blood flow followed by reintroduction of perfusion leads to a characteristic pattern of tubular injury worsened by the cascade of inflammatory and immune events triggered by reperfusion. This can have significant consequences including death and need for dialysis and there is no known specific treatment. Mesenchymal stromal cells (MSC) are rare immunomodulatory cells which have been shown to skew the immune response towards an anti-inflammatory response, inhibit dendritic cell (DC) maturation and suppress T cell function. Early literature suggested that MSC may be renoprotective in IRI. The goal of this study was to examine the effect of MSC in an in vitro human tubular epithelial cell (TEC) model and in an in vivo murine IRI model, with particular emphasis on the impact of MSC on CD4+ T cells and DC.Firstly, a model mimicking IRI was developed using an immortalised proximal TEC line exposed to hydrogen peroxide. Human MSC were isolated from placenta and bone marrow and characterised to ensure they were consistent with literature descriptions of MSC. Cell viability of TEC in this model was assessed by annexin V and propidium iodide in a transwell model and cytotoxicity by lactate dehydrogenase (LDH) release in a co-culture model, at the time of hydrogen peroxide administration, with or without MSC. Exposure of TEC to MSC or prestimulated MSC did not alter cell viability or cytotoxicity at 24 hours. TEC exposed to MSC conditioned media were found to have reduced cytotoxicity by LDH release but no difference in cell viability.The next goal of this work was to assess the impact of exposure of TEC to MSC or MSC conditioned media in the presence of CD4+ cells and DC. CD4+ cells and DC were isolated from donated peripheral blood mononuclear cells using magnetic cell sorting. Addition of CD4+ cells and/or DC to TEC or TEC/MSC in the model of IRI did not lead to any reduction in cell viability or cytotoxicity. The protective effect of MSC conditioned media against cytotoxicity was confirmed but there was no additional apparent effect mediated by immune cells.The aim of the second half of this study was to study MSC in renal IRI in an in vivo murine model (C57 Bl/6 mice). A model of bilateral IRI using flank incisions and clamping of the renal hilum for 20 minutes followed by reperfusion was validated. A pilot study was undertaken administering green fluorescent protein (GFP+) murine MSC either intravenously (IV), intraperitoneally (IP) or via subcapsular (SC) injection. GFP+ MSC were located in the kidney after IP and SC injection but not after IV administration. Renal histology was unchanged by any MSC administration method compared to the control groups. There was a non-significant reduction in creatinine and urea in the SC group. A larger experiment compared MSC with vehicle administered SC. MSC were not protective against renal IRI as analysed by renal histology or serum renal function tests. A third experiment was undertaken using a model of unilateral nephrectomy and then contralateral ischaemia-reperfusion with administration of GFP+ MSC via intra-arterial injection into the carotid artery. MSC were found in the kidney and were generally located near the tubules – either within the tubular lumen or in the peritubular interstitium. However, there was no change in the severity of renal histopathology or renal function 24 hours post IRI.In summary, in these models, MSC conditioned media led to a reduction in cytotoxicity without change in overall cell viability although MSC were not protective against renal IRI. This is consistent with previous studies which have demonstrated mixed findings in murine studies with MSC, although more consistent renoprotection in rat MSC IRI studies. Analysis of the literature reveals very few human in vitro studies of MSC/TEC with which to compare this work. Recently, MSC have been found to secrete extracellular vesicles (EV) which can be characterised and quantified. These EV range in size and in protein content. Direct administration of MSC-derived EV have been consistently protective against damage from renal IRI. It is possible that the protective effect of MSC conditioned media seen in the in vitro work of the current study was due to EV. Results from the current work would suggest that further investigation into EV use and application may be of more benefit than continuing to study MSC as a cellular therapy.