Sex Differences in Dynamics of Necroptosis in Ischemia-Reperfusion-Induced AKI

Background: Ischemia reperfusion induced injury (IRI) is an important cause of acute kidney injury (AKI). Interruption of the arterial blood supply to a donor kidney leads to renal hypoxic injury and cell death, but blood flow restoration also result in reperfusion injury and organ dysfunction. Necroptosis has been recently identified as an essential factor in the pathogenesis of IR-AKI. However, sex differences in the dynamics of necroptosis in kidney IRI have not been a focus of study until now. Methods: Male and female C57BL/6J mice (n=3-5 male or female mice/ group) were induced AKI via bilateral renal pedicle clamping for 18 minutes at 37 degrees Celsius. Plasma, urine and kidney tissue were collected at 0, 3, 6, 12, 24, 48, and 72 hours post reperfusion and plasma creatine, BUN and morphological damage were assessed by PAS staining and TUNEL staining. Necroptosis activation was equantified by the phosphorylation status of the necroptosis factors RIPk1, RIPk3, MLKL, and inflammatory factors (IL-6, IL-10 and TNF-α) post I/R injury. The effects of different sex hormone on the dynamic of necroptotic activation was assessed by ovariectomy to the female mice followed by IR-AKI. Kidney injury and function were evaluated and compared. Results: Activation of necroptosis started at 3 hours post reperfusion in male mice, and around 6 hours after reperfusion in the female mice. The male mice exhibited a stronger and longer activated necroptosis status than females based on the phosphorylation measurement by western blotting. This sexual dimorphism closely correlated with sex differences in kidney injury dynamics. The plasma creatinine was 0.35±0.04 and 0.32±0.06 mg/dL for male and females, respectively, at 3 hours of reperfusion. Which dramatically increased to 2.05±0.18 at 48 hours of reperfusion for the males and gradually increased to 0.94±0.13 mg/dL. Deficiency in sex hormone mitigated the sex difference in the dynamic of the necroptotic activation and consequently in kidney injury. Conclusion: Our study is the first to describe the sex differences in the dynamics of necroptosis and necroinflammation in a mouse model of IRI-AKI. Our findings provide a different time frame in monitoring kidney injury in males and females and suggest that although treatments targeting necroptosis may be effective even if given 3 hours after reperfusion, the treatment in male should start earlier than females.