GASDERMIN-D MUTATION IS PROTECTIVE AGAINST RENAL ISCHEMIA REPERFUSION INJURY

Abstract

Introduction: Ischemia reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and delayed graft function following kidney transplantation. The desire to limit IRI and graft injury has driven further exploration of the underlying pathophysiology. We hypothesize that pyroptosis, a pro-inflammatory form of cell death, has an important role in IRI and AKI. This pathway converges to the downstream cleavage and of the Gasdermin-D protein, resulting in the release of the N-terminus and pore formation in the cell membrane and cell death. We examine the effects of Gasdermin-D mutation on inflammation in acute kidney injury. Materials and Methods: Male C57BL/6 mice were exposed to ethyl-N-nitrosourea mutagenesis, leading to a loss-of-function, single nucleotide polymorphism (isoleucine to asparagine mutation, I105N) in the Gasdermin-D gene. Age- and gender-matched littermate control wild-type, heterozygous and homozygous Gasdermin-D mice were subjected to bilateral renal IRI (36 degrees Celcius, 22 min) and sacrificed 24-hours post-reperfusion for assessment of renal function, histology and biomolecular phenotyping. Similar experiments were performed with chimeric mice to delineate if a mutation in the parenchyma (kidney) or hematopoietic (particularly immune) cells drive the key effects of pyroptosis in IRI. Chimers were created with by whole body irradiation of the recipient mouse, followed by infusion of syngeneic donor bone marrow. The impact of bilateral renal IRI was assessed 8-weeks post-engraftment. Results and Discussion: Homozygote (HOM) and heterozygote Gasdermin-DI105N mice were protected from renal IRI in a gene-related, dose-dependent manner when compared to wild-type (WT), with lower mean serum creatinine (15.7, 48.1 and 85.5µmol/L respectively, p<0.001), less histological tubular injury and cell death (1.8, 3.6 and 5.1 TUNEL+ve cells/hpf, p<0.01). Mutant mice also had reduced expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6, RANTES). This data supports the hypothesis that Gasdermin-D and the pyroptosis pathway has significant impact on the severity of renal injury and has an important role in renal IRI. Chimeric models indicated that susceptibility to IRI may be driven by the parenchymal (renal tubular) cells when matched for age, gender and recipient mutation status. There was no significant difference in serum creatinine in HOM Gasdermin-D mice receiving bone marrow from either HOM or WT donor (36.5 vs 56 µmol/L respectively, p=0.72), nor was there difference in the WT mice receiving bone marrow from HOM or WT donors (114.5 vs 128.5 µmol/L respectively, p=0.96). Conclusion: Mice with impaired pyroptosis, from loss-of-function mutation in the Gasdermin-D (I105N), were protected from renal IRI. This clearly demonstrates the importance of the pyroptosis in acute kidney injury and manipulation of this pathway is an attractive target to mitigate inflammation and cellular death following injury.