Periodic Reporting for period 3 - PAPA (Pathophysiology of Primary Aldosteronism)
Reporting period: 2020-01-01 to 2021-06-30
Arterial hypertension is classified as the leading preventable cause of premature death worldwide and it affects 20 to 40% of Western societies in an age dependent manner, amounting to more than 1.1 billion hypertensive individuals in the year 2017. Primary aldosteronism (PA) is the most common curable form of hypertension with a prevalence of 6-12% in the general population with hypertension. It is caused by the excessive, autonomous production of aldosterone from the adrenal glands resulting in refractory hypertension. If remaining undiagnosed, PA is a fatal disease leading to decreased quality of life, disabling cardiovascular morbidity and premature death by chronic heart failure, myocardial infarction, stroke and renal insufficiency. Pathological aldosterone secretion is caused by unilateral aldosterone-producing adenomas (APA) or idiopathic bilateral adrenal hyperplasia (BAH). We and others have shown, that primary aldosteronism is a major health threat affecting 2-3% of the total population. The recommended treatment option for patients with an APA is laparoscopic adrenalectomy, while patients with BAH are treated with mineralocorticoid receptor antagonists. Of high importance is the early detection of PA, because this has a significant impact on clinical outcome and survival due to severe cardiovascular adverse effects. Inherited forms of PA are extremely rare, with a worse clinical phenotype usually presenting with early onset hypertension, that is difficult to control by conventional medical treatment, and severe cardiovascular endo-organ damage in childhood or adolescence. These familial forms of PA (FH I-IV) arise from four different germline mutations. Currently, effective and targeted pharmacological treatment options are lacking and affected family members are often treated with bilateral adrenalectomy posing them at life-long risk of adrenal insufficiency. Hence, the ERC Advanced Grant PAPA is about to elucidate the humoral and molecular mechanisms underlying aldosterone excess and hypertension in PA to develop a pathophysiology-based classification for targeted treatments.
It is the first hypothesis of this proposal that the pathophysiology of PA is a process based on two ‘hits’: agonistic angiotensin II type 1 receptor (AT1R) autoantibodies and somatic mutations. Whereas agonistic autoantibodies induce proliferation and grossly changes adrenal cortex architecture towards diffuse or nodular hyperplasia, somatic mutations result in adenoma formation. It is the second hypothesis, that together, both factors induce not only aldosterone but also marked glucocorticoid excess. In the first 2.5 years we have analysed prevalence and binding characteristics of AT1R autoantibodies as a pathophysiologic basis of PA. These data demonstrate that patients with PA displayed different levels of AT1R activation with different responses to inhibition by losartan. Patients with BAH displayed higher losartan-independent affinity-isolated agonistic AT1R-Ab levels compared with patients with APA (P<0.01) and with normotensive individuals (P<0.0001). Taken together these data support our first hypothesis that agonistic AT1R antibodies may have a functional role in a subgroup of patients with PA. We are currently determining the effects of AT1R antibodies and genetic factors on cellular adrenal cortex models in vitro. In the next years we will extend these studies to specific in vivo genetic rodent models of PA. We also have quantified aldosterone and glucocorticoid excess as disease effectors of PA using liquid chromatography–mass spectrometry demonstrating that mineralocorticoid and glucocorticoid signatures can be found in plasma and urine of PA patients, and that these signatures are predictive for underlying somatic driver mutations as well as for metabolic and cardiovascular phenotypes. This is strong evidence for our second hypothesis. In summary, we found compelling evidence in support of the two main hypotheses of our grant.
It is the first hypothesis of this proposal that the pathophysiology of PA is a process based on two ‘hits’: agonistic angiotensin II type 1 receptor (AT1R) autoantibodies and somatic mutations. Whereas agonistic autoantibodies induce proliferation and grossly changes adrenal cortex architecture towards diffuse or nodular hyperplasia, somatic mutations result in adenoma formation. It is the second hypothesis, that together, both factors induce not only aldosterone but also marked glucocorticoid excess. In the first 2.5 years we have analysed prevalence and binding characteristics of AT1R autoantibodies as a pathophysiologic basis of PA. These data demonstrate that patients with PA displayed different levels of AT1R activation with different responses to inhibition by losartan. Patients with BAH displayed higher losartan-independent affinity-isolated agonistic AT1R-Ab levels compared with patients with APA (P<0.01) and with normotensive individuals (P<0.0001). Taken together these data support our first hypothesis that agonistic AT1R antibodies may have a functional role in a subgroup of patients with PA. We are currently determining the effects of AT1R antibodies and genetic factors on cellular adrenal cortex models in vitro. In the next years we will extend these studies to specific in vivo genetic rodent models of PA. We also have quantified aldosterone and glucocorticoid excess as disease effectors of PA using liquid chromatography–mass spectrometry demonstrating that mineralocorticoid and glucocorticoid signatures can be found in plasma and urine of PA patients, and that these signatures are predictive for underlying somatic driver mutations as well as for metabolic and cardiovascular phenotypes. This is strong evidence for our second hypothesis. In summary, we found compelling evidence in support of the two main hypotheses of our grant.
During the first 2.5 years of the running ERC project PAPA we achieved several milestones and focused on four main areas: We gained deeper insights in the classification of PA treatment as we were the lead researchers in an international study to develop consensus criteria for outcomes after unilateral adrenalectomy for unilateral PA (Williams TA et al., 2017). Additionally we established a scoring system which allows preoperatively to identify patients with a high probability of clinical remission after surgery (Burello et al., 2019). Furthermore, we focused on the pathophysiologic mechanism of PA treatment outcome and here we were the lead scientists in international multi-center retrospective studies that identified large numbers of patients with persisting aldosteronism after surgery (Williams TA et al., 2017; Williams TA et al., 2018). In additional potential utility to patient management, we identified peripheral venous mass spectrometry-based steroid profiles at baseline associated with patients with incomplete biochemical cure after surgery. With a view to directly translating scientific findings to clinical applications, we have exploited machine learning analysis of presurgical clinical variables to develop prediction tools for the classification of different forms of PA (Yang Y. et al., 2019). Regarding the studies related to agonistic AT1R antibodies in PA we could show that AT1R-Abs may play a role in patients with BAH which could feasibly exacerbate the effects of additional pathophysiological factors such as aldosterone-producing cell clusters. Some AT1R-Abs functions via a mechanism diverse from classical AT1R activation and implicate a role for losartan-independent biased AT1R agonism. Overall, the present study (Williams TA et al. 2019) suggests a role for agonistic autoantibodies to the AT1R in a subgroup of patients with PA, comprising those patients with adrenal hyperplasia. Finally, studies related to glucocorticoid co-secretion in PA were performed. We saw that cortisol co-secretion in PA appears to have an additional impact on cardiac remodeling. Treatment of PA by either adrenalectomy or mineralocorticoid receptor antagonist improves hypertrophy, but this effect was most pronounced in patients with high total glucocorticoid excretion.
In summary, within the first 2.5 years we have made exceptional advances in the basic research to elucidate the humoral and molecular mechanisms underlying aldosterone and cortisol excess and hypertension as we were able to identify specific genotype-phenotype signatures of PA via mass spectrometry based liquid biopsies leading to optimized prediction of blood pressure outcomes after treatment (Williams et al., 2017, Yang et al., 2019)
In summary, within the first 2.5 years we have made exceptional advances in the basic research to elucidate the humoral and molecular mechanisms underlying aldosterone and cortisol excess and hypertension as we were able to identify specific genotype-phenotype signatures of PA via mass spectrometry based liquid biopsies leading to optimized prediction of blood pressure outcomes after treatment (Williams et al., 2017, Yang et al., 2019)
Successful studies on AT1R autoantibodies are ongoing and we will establish AT1R-AA levels in patients with PA during long-term follow-up, its association with endocrine phenotypes and its effects on adrenal remodelling in BAH. Several manuscripts are planned for publication in 2020 as well as ongoing studies on functional activity and potential mechanism of action of AT1R-AAs.
The investigation of the effects of somatic APA mutations on adrenocortical cell growth will be completed by the end of 2019. Transcriptional analysis of resected adrenal samples from patients with unilateral PA will also be used to identify key genes and intracellular signaling pathways mediating deregulated cell growth. The study should be completed and published in 2020.
Based on the successful implementation of the LC-MS measurement of steroid profiles in blood samples as well as the development of statistical models required to interpret the profile data, we expect being able to discover more unique steroid profile finger prints correlated to specific clinical features or subtypes of PA. We also plan to correlate such profiles to the presence of activating AT1R autoantibodies to contribute to the understanding of pathophysiological mechanisms in patients affected by such autoantibodies. Additional analysis of urine samples from our patient cohorts will allow us a deeper understanding of steroid pathways involved in pathophysiology of subtypes of PA in future times.
The investigation of the effects of somatic APA mutations on adrenocortical cell growth will be completed by the end of 2019. Transcriptional analysis of resected adrenal samples from patients with unilateral PA will also be used to identify key genes and intracellular signaling pathways mediating deregulated cell growth. The study should be completed and published in 2020.
Based on the successful implementation of the LC-MS measurement of steroid profiles in blood samples as well as the development of statistical models required to interpret the profile data, we expect being able to discover more unique steroid profile finger prints correlated to specific clinical features or subtypes of PA. We also plan to correlate such profiles to the presence of activating AT1R autoantibodies to contribute to the understanding of pathophysiological mechanisms in patients affected by such autoantibodies. Additional analysis of urine samples from our patient cohorts will allow us a deeper understanding of steroid pathways involved in pathophysiology of subtypes of PA in future times.