Abstract Body (Do not enter title and authors here): Background: Diabetes mellitus (DM) elevates the risk of acute kidney injury (AKI); however, the underlying mechanisms are not fully elucidated. Necroptosis, a form of regulated cell death mediated through activation of RIPK1/RIPK3/MLKL signaling, contributes to the development of AKI. We previously reported that suppression of autophagy after renal ischemia/reperfusion (I/R) underlies the aggravation of AKI in type 2 DM.
Hypothesis: Impaired autophagy amplifies necroptosis signaling in the diabetic kidney following I/R.
Methods: AKI was induced by unilateral nephrectomy and 30-min renal artery occlusion/24-hour reperfusion in the contralateral kidney in OLETF, obese type 2 DM rats, and its non-diabetic control LETO. To inhibit autophagy in LETO, chloroquine (CQ, 10 mg/kg/day) was subcutaneously administered from 7 days before I/R. Rapamycin was injected to OLETF 30-min prior to ischemia to restore autophagic activity in the kidney. Renal tissues sampled before and after I/R were analyzed.
Results: The serum creatinine (sCr) levels after I/R were higher in OLETF than in LETO (3.8±0.3 vs. 1.9±0.4 mg/dl, P<0.05). CQ treatment elevated sCr levels in LETO while rapamycin decreased the levels in OLETF. Immunoblot analyses showed that renal protein levels of RIPK1, RIPK3, and MLKL were similar in LETO and OLETF at baseline, but RIPK1 and RIPK3 were increased after I/R in both rats. Notably, the upregulation of RIPK3 after I/R were more pronounced in OLETF than in LETO. Suppression of autophagy by CQ enhanced the increase in RIPK3 levels by I/R in LETO, while restoration of autophagy by rapamycin reduced the RIPK3 levels in OLETF. Immunostaining for RIPK3 revealed that tubular cells were sites of RIPK3 upregulation observed in OLETF and CQ-treated LETO. To determine whether changes in RIPK3 protein levels were regulated at the transcription level, we measured RIPK3 mRNA level. qPCR analysis of renal tissues demonstrated that RIPK3 mRNA levels were increased by I/R in LETO and OLETF, but the upregulation was more pronounced in OLETF than LETO. The upregulation of RIPK3 mRNA after I/R was enhanced by CQ treatment in LETO and was suppressed by rapamycin in OLETF, suggesting that the impairment in autophagy promoted transcription of RIPK3.
Conclusion: Compromised autophagy augments RIPK3-mediated necroptosis signaling via the upregulation of RIPK3 transcription in the diabetic kidney following I/R.