The worldwide shortage of standard brain dead donors has revived the use of kidneys from DCD. Therefore, with the required framework, DCD kidney transplant programs were reintroduced at the turn of the century. Experimental approach clearly defined that ischemic period is a crucial time which affects organ outcome . Since 1994, we developed several pre-clinical pig models mimicking clinical situations. The aim of this study was to decipher the deleterious effects induced in organ from DCD conditions to improve and adapt graft preservation. A previous study described the effect of both WI and CS in ex-vivo porcine model without a separated analysis of each condition . In addition in this report, the renal function was measured over a period of 3 hours. In our study, three types of IRI and their respective roles were compared: WI accompanied by no-reflow condition (shock, cardiac or aortic surgery or asystolic time) and CS involved in transplantation process, potentially preceded by WI in DCD conditions. In these conditions, the inflammatory and immune response which are key processes involved in repair as well as in chronic kidney fibrosis were studied at 3 hr, 3 days and 7 days for early stage after reperfusion and also at 3 months .
During the first week of reperfusion, renal function in groups exposed to CS was more affected than in WI group. Our auto-transplant setting is an emblematic situation of tolerance, in which IRI per se can break this tolerance and destines the graft to a degraded chronic outcome. The initiation of immune response appears in the early hours in an identical fashion in all conditions; however it is maintained and will expand in proportion to the level of injury, supporting the concept of immunity is a solid discriminatory tool between damage intensities. Interestingly, we correlated the number of adaptive immune cells recruited with plasma creatinin levels at the end of the first week of reperfusion underlining a direct relationship between the intensity of inflammatory process and pejorative graft outcome.
Oxidative stress and apoptosis are two major components of IRI, both closely associated to the inflammatory process . We demonstrated that CS combined or not with WI induced an overexpression of NADPH oxidase enzyme subunits during the first week of reperfusion in contrast to WI alone. This complex may participate in the superoxide anion production by inflammatory cells infiltrating the graft, or directly from the injured tissue. This pro-oxidative milieu was accentuated by a decreased expression of the major antioxidant enzymes: SODs already reported in a rodent model . These results indicate that severe ischemic conditions induce an early pro-oxidative microenvironment and that D3 is a critical time point to evaluate the red/ox balance. Apoptosis is also known to be an important response to ischemic injury . Our results indicate an early activation of the apoptotic process few hours after ischemia. The high level of cleaved caspase 3 staining at H3 and the return to basal values at D7 in WI group suggests an early transitory apoptotic process. However, the high level of injury in WI + CS group at H3 was associated with a high staining level but limited in CS group. On the other hand, at D7 this expression was similar in both CS groups, highlighting the need for early evaluation of such markers, since along with oxidative stress, this pathway is only discriminant in the early days of reperfusion. In these conditions, apoptosis markers could be primarily detected in tubular cells as previously described in human cadaveric kidney allograft . Taken together, these results support that ischemia condition and duration  could influence the level and the localisation of the apoptotic process.
Our study investigates the chronological development of early immunity in various preclinical ischemic conditions. Endothelial activation, a prerequisite in the initial recruitment of leukocytes to the site of injury, is a key of innate immunity response initiation commonly assessed by vascular endothelium markers expression such as selectin adhesion molecules. In our conditions, P-selectin mRNA expression was increased early after reperfusion (H3) in CS and WI + CS groups then reached control values at D7. However, WI did not affect this expression. Early immunity could be partially mediated by Damage Associated Molecular Pattern molecules (DAMPs) which bind the TLRs [16, 28, 29]. These DAMPs are released by cells that have been damaged by IR and thus closely associated to oxidative stress, apoptosis and necrosis [30, 31]. TLRs constitute an integral part of the innate immune response and play a pivotal role in IRI, though the role of each receptor is still unclear . The stimulated-TLRs pathway induces the production of pro-inflammatory cytokines such as IL-1β, TNFα, IL-6, IL-8 as well as expression of cell adhesion molecules like VCAM-1 on endothelial cells . In a recent study using deficient TLR4 mice, Pulskens et al. show that TLR4 initiates an exaggerated pro-inflammatory response upon renal IRI and subsequently controls the induction of an innate immune response . In our study, TLR2 expression followed the level of renal injury, particularly after D3. TLR4 overexpression was found in CS and WI + CS, but was not significantly different between both groups. Thus, TLR4 expression appears due to cold ischemic injury while TLR2 is a more discriminant marker of ischemic stress level. MCP-1 was significantly overexpressed in WI + CS from D3 supporting that the degree of injury seems to modulate the MCP-1 response. MCP-1 recruits monocytes and dendritic cells to the sites of tissue injury and inflammation. In addition, DAMPs are known to stimulate IL-1β secretion from the NLRP3 (NOD-like receptors Protein 3) inflammasome inducing pro-inflammatory cytokines production and immune cells invasion . As IL-1β, IL-6 could be produced by macrophages to stimulate immune response in damaged tissue leading to expand inflammation. Interestingly, we observed an IL1Rn mRNA (gene encoded to Interleukin-1 receptor antagonist) increased expression at D3 which could be appearing in response to IL-1β production. We reported an IL-10 mRNA over-expression by high intensity of IRI up to one week after reperfusion. As previously described, IL-10 could be a protecting agent against renal IRI decreasing TNFα expression and reactive oxygen species production . Our observed IL-10 levels could be linked to autologous immune response induced by CS as our results showed no differences between CS and WI + CS. In our results, this alarming environment, stimulated in the WI + CS condition was characterized by an increase of TLR2 and TLR4 mRNA supporting a DAMPs pathway, stimulating an upregulation from D3 of MCP1 and macrophages invasion.
The originality of this study was the graft follow-up during the first three months after transplantation. In accordance to the findings of the first week of reperfusion, we observed a deleterious effect of WI when is combined with CS. Indeed, the difference at D3 in term of renal function in each group was also observed at 3 months. As previously described in our DCD mimicking model , associating WI + CS, we observed an inflammatory response characterized by endothelial activation remaining until three months after reperfusion with an increase of MCP-1, and VCAM-1 expressions. These results were supported by lymphocytes and monocytes recruitment already described in this model seven days after reperfusion and maintained 3 months later. As these parameters did not reach statistical differences compared to control in WI or CS groups, these results highlight the direct relationship between the intensity of IR and chronic outcome. Above a certain degree of injury, specific pathological processes are rendered irreversible and will unavoidably lead to chronic failure.
Accordingly, we observed a level of tubular atrophy and fibrosis directly dependent on the severity of ischemia three months before. We underlined that WI alone also promotes fibrosis development although the inflammatory response was not present suggesting a stable process. As expected, CS group exhibited a collagen expression level between WI and WI + CS. Moreover, the development of renal fibrosis is still ongoing in WI + CS group, as indicated by profibrotic pathways activation analysis. While in WI or CS group they appear inhibited or reduced, we showed a trend towards increased for TGFβ and a significant rise of its first downstream mediator pSmad3 in WI + CS, indicate a maintain production of collagen and a progression of renal fibrosis accentuated by MMP-2 decrease expression supporting a less extracellular matrix degradation. In addition, these three parameters are not affecting three months after reperfusion by WI or CS conditions, conversely to BMP-7. In fact, CS group exhibited a high expression of BMP-7 in favour to a reduction of TGFβ pathway activation and an inhibition of collagen synthesis . Plasminogen pathway is also involved in extracellular matrix homeostasis and subsequent fibrosis development . We observed an upper expression of PAI-1 in ischemic groups which attained significant difference in WI + CS group which is associated to a down-expression of tPA compared to WI or CS groups. In these conditions, as BMP-7, the tPA expression is increased related to control value indicating a high extracellular matrix degradation which could explain, in part, the minor intensity of red Sirius staining in these groups.