P3561Detection and functional characterization of angiotensin receptor type 1 autoantibodies: establishment and clinical translation

Abstract

Abstract Circulating AT1R autoantibodies (AT1R-aabs) directed against the ECL2 of the AT1R with agonist-like activity are supposed to play a pathophysiological role in diseases associated with vascular and renal damage, such as preeclampsia and severehypertension (HT), but they are also thought to be involved in heart failure and primary hyperaldosteronism (PHA). Methods High-throughput screening assays aiming at a reliable detection of AT1R-aabs in sera from patients with HT and PHA were established. The agonist-like activity of AT1R-aabs was assessed by changes in intracellular calcium-levels using Fura2-QBT dye; the AlphaLISA Assay was used to assess induction of ERK1/2-phosphorylation in stably transfected AT1R-HEK-cells or in adrenocortical NCI-H295R cells. Results IgG isolated from sera of n=60 patients with PHA and n=164 with HT were screened for their capacity to increase [Ca2+]i or to activate ERK1/2. Sixteen out of 60 PHA-patients increased [Ca2+]i compared to none of the HT-patients, whereas in both disease-entities we detected AT1R-aabs inducing ERK1/2-activation with a similar prevalence (PHA: 41%, HT: 42%), indicating the existence of differentially acting AT1R-aabs. PHA-patients positive for ERK1/2-activating AT1R-aabs have significantly lower serum potassium- (3,8±0,1 vs. 4,1±0,1 mmol/l, p<0,05) and renin-levels (2,7±0,5 vs. 4,5±0,7 ng/l, p<0,05) together with an increased aldosterone concentration (341±37 vs. 236±20 ng/l, p<0,01) concordant with the disease phenotype. Similarly, higher BP values are observed in AT1R-aab positive HT-patients (syst/diast: 148/85 vs. 167/93 mmHg, p<0,0001) accompanied byhigher aldosteroneserum-levels (93±7 vs. 74±3 ng/l, p<0,05). In addition, ERK1/2-activation induced by either angiotensin II or by IgG isolated from patients with PHA or HT could be differentially blocked by the use of various signaling inhibitors. In order to elucidate if stimulating AT1R-aabs could be involved in an over-secretion of aldosterone due to sustained receptor-activation, we investigatedtheir effects on NCI-H295R-cells. At the transcriptional level, AT1R-aabs were able to induce a time-dependent upregulation of the key steroidogenic enzymes involved in aldosterone biosynthesis CYP21A1-, HSD3B2-, CYP11B1-, and in particular CYP11B2-mRNA (2fold over basal), with the maximum level achieved after 8 to 12 hours. Concordant withan agonist-stimulated internalization of AT1R,AT1R-mRNA was downregulated by AT1R-aabs (up to 25% of basal) providing direct evidence of a chronic receptor-stimulation by AT1R-aabs. Conclusion Functional assays based on AT1R-activation (Ca2+ measurements & ERK1/2-phosphorylation) are able to detect AT1R-aabs in 41% or 42% of patients with HT or PHA, respectively. Moreover, our data provide evidence that AT1R-aabs stabilize a specific AT1R-conformation distinct from that induced by angiotensin II thereby triggering a different intracellular signaling pattern resulting in chronic aldosterone production. Acknowledgement/Funding BMBF grant