(583) Loss of AGTR2 Confers Protection from Hepatic Steatosis and Mortality in Ketogenic CF Mice

Background: Cystic fibrosis liver disease (CFLD) affects ~27-35% of patients [Columbo, 2002], and is the third cause of death in CF patients after lung disease and transplantation complications [Kobelska-Dubiel, 2014]. While cirrhosis is the classic phenotype of CFLD, hepatic steatosis is most prevalent, occurring in up to 60% of patients. Steatosis on its own is not indicative of CFLD, but is associated with fibrosis and cirrhosis in CF patients, and may also be present independent of classic CFLD, with similarities to non-alcoholic fatty liver disease [Ayoub, 2018]. Steatosis occurs in the CF mouse, and CF mice exposed to high-fat, ketogenic diets fail to thrive, have high levels of morbidity, and exhibit severe hepatic steatosis compared to wild-type mice that thrive under the same conditions with no detectable steatosis. A study in rats demonstrated that infusions of angiotensin II, a component of the renin-angiotensin signaling pathway, increased triglyceride production, resulting in accumulation of free fatty acids in the liver, which is a hallmark of steatosis. Pharmacological inhibition of the angiotensin II type-2 receptor (AGTR2) suppresses this phenomenon, normalizing plasma TG and FFA levels [Ran, 2005]. Interestingly, a large CF genome-wide association study identified a genetic region containing AGTR2 as associating with CF lung disease severity, and our studies concluded that absence or antagonism of AGTR2 improves pulmonary function indices in CF mice carrying various CFTR mutations [Darrah, 2019]. The goal of this study was to determine whether loss of AGTR2 in CF mice would not only improve pulmonary function, but would correct the CF-specific steatosis phenotypes evident during ketogenic challenge.


Methods: F508del CF mice, C57BL/6 WT, and Agtr2/F508del double knockout mice were weighed and fasted on aspen bedding for 24hrs before given free-access to a high fat, ketogenic diet. Mice were monitored for 3 days. On the 4^th day hepatic steatosis was visually assessed for indicative white, punctiform appearance of the liver. Liver samples and blood were obtained for mass spec analyses, and liver was fixed for Oil Red staining.

Results: All mice lost 5-10% bodyweight post-24-hr fast. After access to high-fat food, CF mice failed to thrive over the course of the ketogenic challenge, progressively losing weight, while both WT and Agtr2/CF mice stabilized and gained weight by day 4. Macroscopically, CF mice developed severe steatosis, while the WT and Agtr2/CF mice displayed normal, healthy liver color and texture. Oil red staining in the CF liver revealed larger, more numerous oil droplets, indicating lipid invasion as compared to both WT and Agtr2/CF mice. Pending mass spec analysis on blood and liver will reveal any differences in lipid composition between the groups.

Conclusions: Not only does the loss of AGTR2 act as an effective modifier of CF pulmonary disease, it also seems to confer some protection against lipid invasion, steatosis, and failure to thrive in CF mice during metabolic challenge. CF mice that lack AGTR2 are nearly indistinguishable from their WT counterparts, suggesting that CFTR-associated steatosis can be reduced or potentially eliminated by the loss or blocking of AGTR2. This protection against steatosis could have a marked impact on the occurrence of fibrosis and cirrhosis and other liver diseases in the adult CF population. Taken together, these data suggest AGTR2 as a valuable CFTR-independent target for pharmacological therapy in CF.

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