Abstract 227: Megalin Regulates Angiotensinogen and Contributes to Atherosclerosis

Abstract

Objective: Angiotensinogen (AGT) is the only substrate of the renin-angiotensin system to generate angiotensin peptides, including angiotensin II (AngII), a critical contributor to atherosclerosis. AGT interacts with megalin in proximal convoluted tubules of kidney. The purpose of this study was to determine effects of megalin on AGT, AngII, and atherosclerosis in mice. Methods and Results: Male C57BL/6 mice were injected subcutaneously with vehicle (PBS) or megalin second generation antisense oligonucleotides (ASO) for 5 weeks. Inhibition of megalin was confirmed by more than 80% reduction of megalin mRNA in kidney. Urine was collected using metabolic cages after 5 weeks of vehicle or ASO injections. At termination, blood was collected with EDTA and protease inhibitor cocktail to measure plasma concentrations of renin and AngII, respectively. Urine AGT and renin concentrations were profoundly increased, accompanied by reduction of renal, but not plasma, AngII production. To determine whether megalin inhibition influences atherosclerosis, male LDL receptor -/- mice were injected with vehicle, control ASO, or megalin ASO for 13 weeks. Western diet was started 1 week after the first injection and continued for 12 weeks. Atherosclerosis was quantified by en face analysis of the aortic intimal surface from the ascending aorta to 3 mm proximal to the left subclavian artery branch. Megalin ASO administration led to more than 70% reductions of megalin mRNA in kidney. Consistent with the effects in C57BL/6 mice, AGT concentrations were significantly higher in urine (Vehicle, control ASO and megalin ASO groups: 85 ± 10, 109 ± 71, and 4616 ± 637 ng/ml, respectively; P<0.001 by one way ANOVA with Holm-Sidak method). Plasma total cholesterol concentrations did not differ among groups. Megalin inhibition reduced atherosclerotic lesion area compared to the other two groups (Percent lesion area in Vehicle, control ASO and megalin ASO groups: 23 ± 2, 20 ± 2, 12 ± 1 %, respectively; P < 0.001 by one way ANOVA with Holm-Sidak method). Conclusions: Inhibition of megalin increased urine AGT and renin, reduced renal AngII concentrations, and diminished hypercholesterolemia-induced atherosclerosis in mice.