Changes in blood circulation in brain-dead organ donors can lead to severe ischemia-reperfusion injury, resulting in acute tubular necrosis and delayed organ function after kidney transplantation [21, 22]. During BD, an inflammatory storm occurs, which causes drastic inflammatory changes in the donor organ before transplantation [23, 24].
Studies support the role of apoptosis in acute kidney injury [25, 26]. Proximal tubular epithelial cells are susceptible to apoptosis, and damage to this region results in organ failure. However, preventing apoptosis alone cannot significantly improve renal function after transplantation; therefore, we aimed to explore the mechanism of pyroptosis in brain-dead donors. Here, our results suggested that pyroptosis was induced in kidney tissues after BD, and Z-YVAD-FMK treatment effectively improved the renal function and reduced renal injury thereafter.
We examined the expression of classical and non-classical pathway-related molecules in a brain-dead rat model. Caspase-1/11 belong to the proinflammatory caspase subfamily and play key roles in immune response-related signaling. Mice with caspase-1/11 gene knockout are more tolerant to Escherichia coli-induced septic shock than those lacking caspase-1 and IL-1β, suggesting that caspase-1/11 associated pathways act together in mice along with septic shock. Initially, caspase-1 and caspase-11 were thought to be associated with independent pathways; however, later, they were discovered as part of a complex regulatory network with mutual correlation and interaction [27, 28]. Here, we found that both caspase-1 and caspase-11 were increased in BD rats and associated with BD-induced kidney injury.
Cao et al. [29] confirmed that NLRP3 inflammasome activation mediated blood-brain barrier dysfunction in cerebral ischemia, and inhibition of the same reduced blood-brain barrier injury after ischemia [29]. In the BD model, expression of NLRP3 in the kidney was significantly increased, suggesting that it was one of the main receptors associated with inflammasome formation and initiation of the canonical pyroptotic pathway. NLRP3 promotes the activation of caspase-1, induces release of IL-1β and IL-18, and leads to renal injury, confirming that certain stimulating factors induced by BD activate NLRP3 in rats and promote occurrence of canonical pyroptosis thereafter.
GSDMD, a substrate of both caspase-1 and caspase-11/4/5, is primarily expressed in immune cells and shows unique structural characteristics of a perforating protein [30]. Here, a significant increase in GSDMD was detected in the kidneys of BD rats, demonstrating that GSDMD cleavage was necessary and sufficient for inflammatory caspase activation-induced pyroptosis. Both mRNA and protein expression of GSDMD in the BD + Z-YVAD-FMK group were significantly decreased, indicating that caspase-1 expression was inhibited by Z-YVAD-FMK, and the expression of GSDMD correspondingly decreased.
Pyroptosis is involved in the cryopreservation and auto-transplantation of mouse ovarian tissues, and its inhibition can improve ovarian graft function [31]. In our study, Z-YVAD-FMK effectively protected renal function in BD rats. In the H/R model, we verified that caspase-1, caspase-11, and GSDMD were significantly upregulated; whereas, addition of Z-YVAD-FMK abrogated this effect. NRK-52E cell viability decreased significantly in the H/R environment and Z-YVAD-FMK treatment increased the cell viability significantly.
Previous studies on pyroptosis [32] have mainly focused on the role of caspase-1 in the canonical pathway; here, we focused on whether caspase-11-mediated pyroptosis could be involved in BD-related organ injury. The level of caspase-11 in brain-dead kidney tissues was significantly increased, as determined by IHC and mRNA and protein expression. However, caspase-11-mediated atypical pyroptotic pathway was not affected by the caspase-1 inhibitor. Caspase-11 was knocked down by an siRNA, and the results revealed that H/R activated both canonical and non-canonical pyroptosis. Z-YVAD-FMK inhibited the expression of IL-1β and IL-18, thereby indicating the increased protective effect of Z-YVAD-FMK on cell viability after H/R. However, caspase-11 knockdown did not exhibit a protective effect on cell viability after H/R. Therefore, we concluded that canonical pyroptosis was the major pathway that affected H/R injury in NRK-52E cells.
This study has a few limitations. First, the maintenance time(6 h) in BD rats was limited. Second, we did not explore the role of pyroptosis in kidney injury beyond 6 h. Thus, further studies are required to understand the potential mechanisms of action.
In summary, our study shows that pyroptosis may occur in brain-dead donors, promote inflammation, and induce kidney injury, and could be considered as a therapeutic target for BD-induced kidney injury.