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Regulated Cell Death at the Crossroad Between Ischemia-reperfusion Injury and Innate Immunity in Kidney Transplantation

Cucchiari, David MD1; Rovira, Jordi PhD2,3; Diekmann, Fritz MD, PhD1,2,3

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doi: 10.1097/TP.0000000000003128
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The ischemia-reperfusion injury (IRI) represents one of the most important factors that affect short- and long-term outcomes of kidney transplantation from deceased donors. The tissue damage induced by the IRI has its own clinical correlate in delayed graft function (DGF) and thanks to the liberation of damage-associated molecular patterns (DAMPs) can also predispose to the development of graft rejection.

To understand how IRI affects the graft is an important issue, because inhibiting the pathways involved in tissue damage could prevent the development of DGF and donor-specific alloimmunity. More than 20 years ago, Land et al described that the inhibition of oxidative damage by means of superoxide dismutase upon graft reperfusion could reduce the incidence of rejection in kidney transplant recipients.1

The tissue damage induced by the IRI mostly affects renal tubular epithelial cells (RTECs), given their high metabolic demands. However, it has become clear that cell death after IRI is not only a mere consequence of passive necrosis but also a regulated process of apoptosis. Interfering with programmed cell-death has led to an improvement in kidney function in many experimental models.2,3 However, clinical applicability has been limited by the redundancy of the apoptosis pathways that inevitably lead to RTECs death upon IRI.

In this issue of Transplantation, Jain et al analyzed the different pathways of injury that characterize DGF in a mouse kidney transplant model.4 Their results show that, in kidneys preserved in static cold storage (CI group), apoptosis is mediated by the intrinsic pathway via the activation of Caspase 9; this model showed only mild brush border at histological examination. On the other side, kidneys exposed to static cold storage and later transplanted (CI+Txp group) had minimal activation of Caspase 9 but significantly higher expression of the extrinsic pathway mediated by Caspase 8. This was also associated with important features of acute tubular necrosis and higher expression of tumor necrosis factor receptor. Mice of this group also displayed higher levels of tumor necrosis factor alpha (TNFα), the physiological ligand of tumor necrosis factor receptor.

The difference noted between the CI and the CI+Txp has been explained by Jain et al as a kinetic and mechanistic issue and seems reasonable. During cold storage, organized cell death relies on the intrinsic pathway, taking into account mitochondrial stress upon ischemia. However, when kidneys are exposed to the reperfusion injury, the extrinsic pathway of apoptosis prevails; the dissemination through the restored blood flow of the extrinsic mediators of death, such as TNFα, seems responsible for the activation of Caspase 8. The other notable finding of Jain et al was the up-regulation of necroptosis markers in the CI+Txp in comparison with the other group. Necroptosis is a programmed form of cell death that instead of apoptosis triggers tissue inflammation; its value has already been highlighted in animal models of kidney transplantation.5 It is driven by the activation of receptor interacting protein (RIP) 1 that interacts with RIP3 through the homotypic RIP homology interaction motif to form an autophosphorylated complex called the necrosome. This phosphorylates on turn the pseudokinase mixed-lineage kinase domain-like protein that, upon oligomerization, triggers programmed necrosis by lysis of the plasma membrane.6

The activation of RIP1 depends on external stimuli such as TNFα and engagement of toll-like receptors (TLRs), especially TLR3 and TLR4.6 TLRs are considered pattern recognition receptors that detect both pathogen-associated molecular patterns and DAMPs. They are expressed by dendritic cells, macrophages, B cells, natural killer cells, endothelial cells, and many other subtypes and activate the innate inflammatory cascade in response to tissue damage or pathogens.7

Taken together, all these findings indicate that cell death after IRI in kidney transplantation is not a chaotic event simply because of acute tubular necrosis following oxidative stress and impaired metabolic demands of the RTECs. It seems instead a highly regulated process in which the different pathways involved (extrinsic apoptosis and necroptosis) are activated by external stimuli. These are induced by the innate immune activation and the inflammation triggered by the IRI thanks to the DAMPs/pattern recognition receptors interplay.

Therefore, at least theoretically, blocking extrinsic apoptosis and necroptosis at the same time may improve graft injury. However, Sung et al recently demonstrated that RIPK3-/- caspase-8-/- double knockout mice exposed to IRI develop tissue injury by up-regulation of the intrinsic pathway.8 The logical conclusion is that after IRI, RTECs are committed to death, and if they cannot activate either extrinsic apoptosis or necroptosis, they can turn to the intrinsic pathway to accomplish it.

It is reasonable to assume that, given the redundancy of the system, a multimodal treatment is needed to inhibit all the possible pathways that lead to cell death after IRI.

However, given the importance of apoptosis and programmed necrosis in human biology, it is unlikely that this kind of approach would be tolerated in humans. Ex vivo machine perfusion of the kidney graft could constitute a possible way to inhibit specific pathways in the graft without damaging the host. In this setting, RNA interference (RNAi) through small interfering RNA and short hairpin RNA holds the potential to silence potential harmful genes before reperfusion. Several animal models have demonstrated the utility of RNAi in solid organ transplantation preventing IRI. RNAi has been tested in kidney transplantation targeting Caspase 3 and 8, Rel B, C3, Fas, CD40, or SHARP-2 with good results, reducing apoptosis, improving histology, and preserving renal function.9

So far, several lines of research have attempted to attenuate IRI with actions directed at the donor, the recipient, or the preservation solution. As the current experience suggests,4,8 the cell death response after KT is redundant, so it is likely that multitargeted treatment may represent a reasonable option. When moving from preclinical to clinical models, a possible way to administer safely this kind of therapy without harming the recipient is the ex vivo administration of small interfering RNA through perfusion machine.


The authors acknowledge the support from Redes Tematicas De Investigacion Cooperativa En Salud, REDINREN (RD16/0009/0023) and were cofunded by both ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (2017-SGR-1331).


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