MO289: IL33 Exerts Toxicity in the Heart as Secreted by Renal Inflammation

Abstract

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a major cardiovascular risk factor, regardless of the severity or the origin of the AKI. Among this risk, AKI imparts an array of signals and long-term physiologic alterations that can precipitate heart insufficiency and hypertrophy. However, few direct molecular mechanisms have been identified that show how AKI leads to cardiac disease and maladaptation. The aims of this project are to assess the role of IL33 in remote cardiac damage after AKI. METHOD AKI was induced in mice by ischemia–reperfusion injury of both right and left kidneys by 30min of reversible artery occlusion. To examine the role of renal inflammation more specifically, we used the unilateral ureteral obstruction (UUO) model, which does not cause acute injury. For UUO, an incision was made in the lower left quadrant of the abdomen and the left ureter was isolated and ligated permanently. Mice were treated to manipulate select signaling pathways with either Adenovirus-associated virus 9 (AAV9) or recombinant proteins. We generated AAV9 carrying the IL33 gene or an empty vector (EV) under a CMV promoter and injected 1012 viral particles intrathoracically in 6 to 9 days old pups. Recombinant IL33 (rIL33) or vehicle were injected intraperitoneally in IL33 KO adult mice after AKI (at D0, D1 and D2). Echocardiography was performed either at baseline or after 28 days of surgery. Hearts were fixed in 4% paraformaldehyde overnight and paraffin-embedded for fibrosis (picrosirius staining) and cardiomyocyte area measurement (WGA staining and ImageJ software). IL33 citrine reporter mice (IL33cit) in the heterozygous state were used to show IL33 expression. RESULTS Our data showed that UUO and AKI were strong inducers of cardiac dysfunction after 28 days in adult mice, measured with echocardiography (mean ejectional fraction (EF): 58, 46, 47% in Sham, AKI and UUO group respectively, P < .05). Cardiac pathology was also present such as increased fibrosis in hearts from WT mice after AKI compared with Sham (2.3%, 4.5%, 8.3% of fibrosis area in Sham, AKI and UUO respectively, P < 0.05) and an increased mean cardiomyocyte area indicative of hypertrophy (333, 597, 1246 µm2 in Sham, AKI and UUO respectively, P < .05). However, cardiac function and architecture were preserved either after 28 days of AKI or UUO in IL33 KO mice. In parallel, WT mice injected with AAV9-CMV-IL33 showed impaired cardiac function compared with mice injected with a control AAV9-empty vector after 8 weeks (EF 58% versus 42% in AAV-EV and AAV-IL33 respectively, P < .05). After AKI, AAV9-IL33 mice had even more impaired cardiac function compared to AAV-empty control mice (EF 42% versus 35% in AAV-EV and AAV-IL33 respectively). In IL33 KO mice, the injection of 1 µg of rIL33 in the early days of AKI restored cardiac dysfunction and fibrosis after 28 days of AKI (EF 59 versus 43% in Sham and AKI respectively, P < .05), yet vehicle injected mice were still protected. IL33 expression viewed with the Citrine reporter mice showed that it was locally downregulated in the heart after AKI, which was strictly localized in pericytes in the heart (PDGFRß+ cells). ELISA for protein levels of IL33 confirmed the reduction of this cytokine in hearts from WT AKI and UUO mice after 28 days compared with Sham (29.6, 4.2 and 2.7 fg of IL33/µg of protein in Sham, AKI and UUO hearts respectively, P < .05). CONCLUSION IL33 seems to exert toxicity in the heart as secreted by renal inflammation, which is against some previous reports in the literature where it was suggested to be a protective factor to the heart (whether released from the kidney or from within the heart).