So thank you very much, everybody, for coming to the last symposium of the 2022 U.S. ENDO meeting. It's delightful to have such a full room for this wonderful symposium, so thank you for all coming along. And this is the session on advances and novel perspectives in mineralocorticoid receptor antagonism. And our first speaker today is Professor Gail Adler, Professor of Medicine at Harvard Medical School and Chief of the Cardiovascular Endocrine Section in the Division of Endocrinology and she also works in diabetes and hypertension at Women and Brigham's Hospital. And we are looking forward to your presentation today, Gail, on metabolic effects of steroidal and non-steroidal mineralocorticoid receptors. Thank you very much. So thank you very much. I'm delighted to be here and to present this work to you. I have no financial relationships to disclose, but I really want to say thank you to NIH for my research funding. So what I was going to do today, since I'm this first speaker, is give you an overview of steroidal and non-steroidal MR antagonists, then discuss the role of the mineralocorticoid receptor in the pathophysiologic consequences of obesity and diabetes, and finally, because I'm a translational researcher, discuss how findings in mice relate to findings in humans. So aldosterone was discovered in 1952 and a friend of mine who's in sabbatical in London sent me this picture about two weeks ago indicating that it was at the Middlesex Hospital Medical School in London that aldosterone was discovered by James Tate, Sylvia Simpson Tate, and Hilary Grundy. The FDA approved the use of the first mineralocorticoid receptor antagonist, spironolactone, in 1960, and it was used to treat hypertension in edemonous states such as cirrhosis, heart failure, and nephrotic syndrome. It had a pretty good affinity for the mineralocorticoid receptor, but it wasn't that selective. So there were concerns with having gynecomastia and menstrual irregularities, and then because it's blocking the actions of the mineralocorticoid receptor, hyperkalemia. So 40 years later, we have finarinone, another MR antagonist that is more selective for the mineralocorticoid receptor, and it's approved for hypertension treatment and then for heart failure with a reduced ejection fraction. And I just want to say what makes a steroid, and you probably can't see this, but do you see the four rings, one, two, three, four, that you see in aldosterone, spironolactone, and empleronone? And that's why they're called steroidal MR antagonists. So now we have these non-steroidal MR antagonists, and the esaxaranone was approved in Japan for the treatment of hypertension, and then finarinone has come out. And what I'd like you to see is that they have a very high affinity for the mineralocorticoid receptor, and they're very selective. But I wanted to say a little bit about what happened in the 20 years between 2000 and 2021. So during this time, it became evident that type 2 diabetes and obesity is associated with excess activity of the mineralocorticoid receptor and excess aldosterone. And a variety of potential mechanisms have been proposed for the increased aldo production and MR activation. It's been thought that you can have increased activity of your classical aldosecretogogues, Ang-2-potassium, and ACTH. Also, it's been shown that adipose tissue makes aldosterone secretogogues, such as leptin, which then stimulate the adrenal to make aldosterone. People have shown that adipose tissue itself can make aldosterone, and that's relatively small amounts, but that may be very important for adipose tissue around vasculature. And finally, people have reported that there's increase in the level expression of the mineralocorticoid receptor in diabetes, increased expression of RAC1, which can activate the mineralocorticoid receptor. And there are also potential changes in a variety of sort of MR modulating proteins. And all of this leads to increased MR activation. So consistent with this hypothesis, we and others have shown that you can get increased aldosterone production in people who are overweight and obese. And we've shown that increased aldosterone levels associate with the number of metabolic syndrome components. So if you have higher serum aldosterone, this is associated with an increased number of metabolic syndrome components. Also, there were multiple rodent models of obesity, the OB-OB, the DB-DB, the high fat fed mouse, the fructose fed mouse, the Western diet fed mouse. An MR blockade has been shown to decrease renal damage, including albuminuria and glomerular damage. In addition, there were multiple studies in humans showing that if you add an MR antagonist to ACE inhibition or ARB therapy, you decrease albuminuria. But the big problem was that there were no large scale clinical trials showing that MR blockade reduces the progression of diabetic kidney disease or reduces cardiovascular events in individuals with type two diabetes on ACE inhibitor or ARB therapy. So the finerenone trials did, I think did a fabulous job in addressing this problem. So we know that the prognosis of chronic kidney disease is worse if you have more albuminuria. So as you go this direction, your prognosis is worse. And actually as your EGFR declines, your prognosis is worse. So these red boxes are indicating poor prognosis from chronic kidney disease. So the FIGARO trial enrolled patients that I've highlighted here in blue. And they had stage two to four kidney disease with moderately increased albuminuria. Or if they had severe albuminuria, they had stage one to two kidney disease. And the outcome for this group was a cardiovascular outcome. It was time to cardiovascular death, nonfatal MI, nonfatal stroke or hospitalization for heart failure. The study that was done at the same time was a FIDELIO trial. And what they're doing is they're recruiting this group that I've highlighted in gray. And this is more severe kidney disease. And the outcome for them was time to renal failure, renal death, or a sustained greater than 40% decrease in EGFR. There was, everyone who was included in this trial already had to be on an ACE inhibitor or an ARB. Because they're worried about the effects of finerenone on potassium, CRMK had to be 4.8 or less when they were enrolled. And they had to have pretty good blood pressure control because their blood pressure was less than 160 millimeters of mercury. FIDELI, I don't know where the names come from. I'm really sorry. FIDELI was a pre-specified pooled analysis. They basically said, we're gonna take all of these people that we're studying, and we're gonna look at the cardiovascular outcomes and the kidney outcomes. And the only thing they did was that the kidney outcome was changed to have a sustained greater than 57% decrease in EGFR. So this is the result from the Figaro, which is the people who have less chronic kidney disease. It was a median follow-up of 3.4 years. And there's a 13% decrease in cardiovascular outcomes. FIDELIO is looking at the renal outcome. It was a median follow-up of 2.6 years. And you have an 18% decrease in the kidney outcomes. When you combine all this data together, you now have about 13,000 people who have been randomized to finarinone, 10 to 20 milligrams a day, or placebo. And what you can see is you've got a 14% decrease in your cardiovascular outcomes and a 23% decrease in your kidney outcomes. There is, okay, there we go. There is 12% of the participants in the finarinone group did develop hyperkalemia, but I think they only had to disempanel about one to 2%. What they tended to do was they would go back down on the dose of finarinone if they needed to. And it was hyperkalemia was 5.9% in the placebo. What's, I think, really interesting is about 7% of the participants were on SGLT2 inhibitors and 7% on the GLP-1 receptor agonist. And the cardiovascular and renal benefits of finarinone were not modified if people were on these agents. So that suggests that adding an MR antagonist on top of these agents is going to be beneficial. And just recently, there was a post hoc analysis of the finarinone trials and the Credence trials, which is an SGLT2 inhibitor. And when they took into account the different enrollment criteria, what they said is that there was about a 26% to 30% risk reduction with finarinone and SGLT2 inhibitors. So we can't say that one is better than the other. They both seem to be effective and we clearly need more trials to look at these outcomes. So, okay, so in 2021, FDA approves finarinone for treatment of CKD associated with type 2 diabetes. So let me, the structures of these, you can see are a little different. So let me talk about this a bit. So we know that when aldosterone, which is, I've highlighted here, interacts with the mineralocorticoid receptor, it recruits coactivators, binds to the hormonal response element, and triggers transcription. When the steroidal MR antagonist binds to the MR, you still have the coactivators binding, but you do get a marked reduction in transcription. What's different is that these non-steroidal MR antagonists are really bulky if you look at how big they are there. And when they bind to the mineralocorticoid receptor, the coactivators can't bind and you're really blocking transcription. So this is another chart that's sort of trying to show differences between your steroidal and non-steroidal MR antagonists. So the tissue distribution is a little bit different in that the steroidal ones tend to be, accumulate more in the kidney than in the heart, whereas in the non-steroidal MR antagonists, they're balanced equally in the kidney and the heart. The non-steroidal are less lipophilic and they don't cross the blood-brain barrier. So finarion has a very short half-life with no active metabolites. Spironolactone is not active, it's a prodrug, but its active metabolites last a long time and have a very long half-life. I talked about affinity before. I think it's just to say at this point, all of these drugs are effective for organ protection. We don't, and the steroidal MR antagonists are very effective for blood pressure protection. We don't have good trials yet for finarion and blood pressure protection. The major trials we have are in CKD and there's a minimal decrease in blood pressure, but if you look at RALS, where MR antagonists were very protective in reducing cardiovascular disease, they had a minimal effect on blood pressure too. So I think as we get more trials, we're gonna see, hopefully, that these non-steroidals are also very good at lowering blood pressure. So in all of these models of obesity, we had seen in the animals marked decreases in albuminuria and glomerular damage. And yes, this translates to humans. So the next thing, what I would like to talk about is there was so much data from these animal studies saying that being on an MR antagonist is going to improve glucose homeostasis. And they did GTTs in animals and they looked at HOMA and they did ITTs, insulin tolerance tests. Every thing looked great. And I said, this is wonderful. We're gonna go and we're gonna go into humans and we're gonna show that this is gonna improve diabetes. So the first thing we did was we took 84 healthy people on a high salt diet. We showed that ANG2 stimulated aldo is associated with an increased BMI. We did multiple variant regression analysis to predict the insulin sensitivity index. ANG2 stimulated aldo was a predictor of impaired insulin sensitivity. We go, okay, this is all good. Will MR blockade improve insulin resistance? So our first study was in 32 non-diabetic, insulin-resistant, normal-tensive obese men and women. We randomized to spironolactone or placebo for six weeks. The outcomes were HOMA-IR and an insulin sensitivity index calculated from the oral glucose tolerance test. We had nice decreases in blood pressure with spironolactone, no change with placebo, but no effect of insulin on insulin sensitivity. So we said, okay, we're not treating long enough. It's the wrong patient population. Let's try again. So this time, this is now a study that we did with Steve Grinspoon and Suman Srinivasa where we recruited 46 individuals with HIV, well controlled on ART. They had increased weight circumference and they had baseline abnormal glucose homeostasis. They either had to have a fasting blood sugar between 100 to 126, abnormalities on the oral glucose tolerance test or fasting hyperinsulinemia. We randomized to a plurinone, 50 milligrams or placebo. We went out six months. We said, we're gonna do the gold standard euglycemic hyperinsulinemic clamp to assess insulin sensitivity. And there's no effect of a plurinone and insulin sensitivity at all. So then we said, okay, now let's go back and try again. And this time, this is Matt Luther at Vanderbilt who's spearheading this. And we said, let's go to patients with primary aldosterone. We know they have elevated aldosterone. If we treat them, are we going to improve glucose homeostasis? So we had non-diabetic patients with primary aldo. They all had elevated aldo levels. The mean was 33. The renin was suppressed at 0.3. They had an elevated aldo to renin ratio. They didn't suppress with saline suppression. They were studied prior to treatment and then three to 12 months after PA treatment. So we were grabbing them sort of between the adrenal vein sampling and before they had their adrenalectomy, which is not easy to do, to convince people to come in and do a hyperglycemic clamp to assess insulin production and a euglycemic hyperinsulinemic clamp to assess insulin sensitivity. But we did it. And I think this is the results of the hyperglycemic clamp studies. So what you're doing is you're infusing glucose into an individual to maintain the blood sugar at 20 milligrams per DL. And then you're measuring the amount of insulin secreted. And we focus on the initial secretion and then a steady state. And you also look at C-peptide. And what you can see is that before we treated them, they had lower insulin levels. After treatment, there was a increase in insulin. So this looks like excess aldosterone leads to lower levels due to decreased insulin secretion. So then we went and we did the euglycemic hyperinsulinemic clamp studies where you give a constant infusion of insulin at a low dose and a higher dose. You're adjusting the rate of glucose to give a target glucose of 95 milligrams per DL. And this shows you the insulin levels pre-treatment and post-treatment. And what you can see is post-treatment, they have higher insulin levels even though we're infusing the same amount of insulin each time. And we did an insulin clearance and it shows that after treatment, the insulin clearance is reduced. So it looks like excess aldo levels lead to lower insulin levels due to increased insulin clearance and decreased insulin secretion. So the model we came up with, that Matt came up with, was say primary aldo has decreased insulin levels. The person compensates. So if they can compensate well, they have an increase in insulin sensitivity in normal glucose tolerance. If you can't compensate, you're on the left-hand side and then you have impaired glucose tolerance. So what we're saying is if you treat primary aldo, meaning you reduce aldo levels or you block the MR, you're gonna get an increase in insulin levels due to increases in secretion and decreases in clearance, potentially a compensatory decrease in insulin sensitivity. But in those with prediabetes or diabetes and primary aldo, glycemic control could improve. So the future questions is will non-steroidal MR antagonists have similar effects on insulin levels? And we tried to control very much for this, but remember increases in glucocorticoids and decreases in potassium can affect insulin secretion. So it is possible that some of the effects are that we've taken out the adrenal and that was a diseased adrenal and it could have made some glucocorticoids that we were not able to detect the differences. So we do need more studies on this. But Matt did one other thing is he wanted to look at eats and these are lipid signaling molecules that are formed from arachidonic acid by cytochrome P450s. They're anti-inflammatory, they're vasodilatory, they increase insulin secretion and sensitivity. And what he showed was that when you treat primary ALDU, you get an increase in total eats and that's driven by an increase in 14, 15 eats. So yes, we think changes in glucose metabolism translate to humans, but it's a little tricky and it's probably gonna depend on patient populations. And I think this is why with the finerenone trials, hemoglobin A1C stayed the same between studies. Meta-analysis have not shown consistent effects and there have been studies like in CKD where Spiro did improve insulin sensitivity, but maybe they had, that's a unique population. White adipose tissue is, so let me go back. Blocking the MR decreases inflammation in adipose tissue, promotes browning of white adipose tissue, activates brown adipose tissue, decreases liver fat. This is just showing that in the DBDB mouse, you have increases in pro-inflammatory factors, increases in MCP1, you give a plurinone and they go down. So the study that I talked about earlier where we took the people with the HIV and we put them on a plurinone versus placebo, you can see that at the end of six months, a plurinone reduces MCP1 and increases HDL as compared to placebo. We also showed that at baseline, aldo levels predicted liver fat and they predicted the presence of liver disease, but we saw no change in liver fat with a plurinone treatment. There was a decrease in intramyocellular lipids with a plurinone, but we need future studies to see if longer treatments could affect liver fat or accumulation. So potentially we have an effect there. And finally, I wanted to end with vascular data. So coronary flow reserve can be assessed in humans. It's adenosine stressed myocardial blood flow divided by rest. It's an integrated measure of the coronary micro and microvascular system and impaired CFR is a strong predictor of cardiovascular mortality. So we did a study. We said, we're gonna take patients with type two diabetes without known coronary artery disease. No one could be a smoker. We were going to take them off all their antihypertensives, put them on an alloprosol, an ACE inhibitor, add amlodipine if they needed it. If they weren't on a statin and their LDL was greater than a hundred, we added simvastatin. We optimized blood sugar control. We made everything pretty well controlled. I learned a tremendous amount from Rajesh Garg who was a diabetologist working with me. And then we randomized them to spironolactone, hydrochlorothiazide and K and placebo. And we did cardiac PET to assess coronary flow reserve at the baseline and at the end of treatment. And what you can see is treatment with spironolactone significantly increased coronary flow reserve as compared to hydrochlorothiazide, as compared to placebo and as compared to the combination. Greater improvements in coronary flow reserve were associated with spironolactone use, lower baseline coronary flow reserve, statin use, weight loss and being non-Caucasian. So what's the clinical relevance of this? Well, there is a recent paper showing that a 0.1 unit increase in CFR is associated with an 8% decrease in the hazard of cardiovascular events. So if we've increased not 0.1, but 0.4, we could have a profound effect on cardiovascular events. Andrea Haas is junior faculty with us. And she wanted to look at sex differences in coronary flow reserve. So this is taking that same data set. And she looked at coronary flow reserve in males and females and showed that it was significantly lower in the females, even after you adjust for age, race, BMI, hemoglobin A1C, statin use, and systolic blood pressure. And women have a 14% lower CFR as compared to men. And what's interesting is, remember, CFR is stress divided by rest myocardial blood flow. Stress divided by, and what's really different is that the women have this 31% increase in rest myocardial blood flow. And because the stress myocardial blood flow doesn't increase, your ratio ends up being suppressed. So you would think this increase in rest myocardial blood flow might be a good thing, but it doesn't appear to be. And what you can see here is that the increases in rest myocardial blood flow associate with more diastolic dysfunction. So we don't know why the women have greater alterations in coronary microvascular function. I wanna show you one thing that we did look at. At baseline, we had performed angiotensin infusions in all of our men and women on a liberal sodium diet. And what you can see is that the change in aldo with angiotensin II is increased in women, am I right? Okay, it's increased in women versus men. And this is important because women with diabetes have MIs earlier and they have a higher mortality as compared with men and death from coronary artery disease is three times higher in women versus men. So the model we have is as you go from lean and healthy to diabetic, to diabetic with cardiovascular disease, you're having more MR mediated injury and there may be sex differences in this. And I wanna say thank you, thank you, thank you to all of my collaborators, everybody who's done all this work. Sorry. Thank you. Thank you so much, Gail. I'm so sorry, we're running out of time. But we have time for a couple of quick questions and thank you for that wonderful introduction to the symposium. Very nice presentation, thanks a lot. I'm Adina Togu from University of Michigan. I was wondering what are your thoughts about the divergence in target organ protection and blood pressure effects with the Fidelio Fidneranone study? And the reason I ask, to me, those are a little bit surprising, at least based on the lessons we know from primary outdoor studies. So in primary outdoor, when we use an MR antagonist, be it spironolactone or pleurinone, we usually obtain blood pressure control way before we are able to reverse renin. And then from the retrospective studies from Brigham and Women, we know that the end organ protection effects are when we are able to reverse renin suppression, at least in the retrospective studies. Although I would say that in those studies it's hard to know if maybe the people who achieved a reversal of renin suppression maybe had a milder PA phenotype. So, especially so, second reason is, with Fidneranone having such a short half-life and it was dosed once a day, I'm just surprised that we didn't see much effect on blood pressure, yet we saw a humongous effect on renal protection. Thank you. So I would say, in our animal studies, where we give Elname to raise blood pressure and give angiotensin to, and we cause a lot of aldo-media damage, when we give a steroidal MR antagonist, we prevent the damage without affecting blood pressure. So we get, and we see similar protection with steroidal and non-steroidal MR antagonists. So I think you can get the protection without the blood pressure reduction. And then in humans, why doesn't it go together? And part of it is, I think if you take somebody with primary aldo who's not on any other medications and you start an MR antagonist, you get great blood pressure reduction. When you look at these trials where they have people on multiple medications and you're adding another medication, I think that's when you don't get as much blood pressure reduction. So thank you, Gail, for this really beautiful overview of what the older studies you have done over the last years. I just wonder, in your studies on insulin sensitivity, I know the numbers were small, but could you look into sex differences? And they were really small. Yeah, so there's no way to have an idea about that. I don't have an idea about the sex differences. I'm sorry on that. Hello, Bernal, Washington University. The question is changes in dexascanning fat percentage. There was any studies during this process that the antagonists will change the obesity? Yeah, we have lots of studies, not lots, but we have some studies that saying visceral adiposity tracks with an increase in aldo. But when we block aldo, we're not seeing decreases in fat. We tried it. We looked at pericardial fat with that big study in patients with diabetes. We didn't see any decrease. And so we're hoping to see that. We're not yet. I think there's a question online. We have 136 people online for this session, too, which is very exciting. Welcome to all our listeners today. So the question is related to the, just the previous question there. Do you see any, with Emma, blockade activating brown fat, is there a weight loss effect? I'm not sure which study, I didn't say which study, but I guess it's not just about percentage change in fat depots. I'm sort of trying to extrapolate to you. I don't think that, no substantial weight loss. I'll say in our patients with type two diabetes that we put on an MR antagonist, there were a couple who were on the MR antagonist who lost a lot of weight, but it wasn't universal. And there may be unique characteristics about some people who they might lose weight, but I don't think it's a general phenomenon. Right, okay. Okay, one very quick question, a quick answer. So how do you differentiate this group of patients from those with primary hyperaldo? Where do you draw the line? The CKD? No, no, patients who have diabetes, obesity, and higher aldosterone levels. So which is the chicken or the egg? Which is coming first? Well, I mean, I don't know that we have any studies in which we've taken people with obesity, CKD, and then applied our normal criteria for assigning primary aldo. We've sort of retrospectively gone back and just said in a group of... Actually, I can't answer the question. It's a good question. It's a really good question and I give up. Okay. Thank you so much, Gail. Thank you very much. We'll move on now. So I'll move to the next speaker, Dr. Jonathan Barret de Timal. He's Associate Researcher at the Maison of Rosemont Hospital in Montreal, and he will present No Steroidal MRIs in Cardio-Renal Medicine from Length to Bedside. Thank you. Good morning, everyone. I'm very happy to be here today. And I would like to thank the organizers for the opportunity to share today some of the most recent advances on non-steroidal mid-narocortical receptor antagonists in Cardio-Renal Medicine. I have nothing to disclose. As you might know, following several experimental studies that show that aldosterone had a deleterious role by promoting cardiac fibrosis, the RALES trial was the first major trial to show that in patients with heart failure with reduced ejection fraction, spironolactone was able to reduce mortality. In this study, spironolactone, given 25 milligram per day, was able to reduce mortality by 30% as compared to placebo. However, as we saw in the previous talk, spironolactone was associated with a risk of hyperkalemia and gynecomastia. With the development of a plurinone, a more specific, sorry, a less specific, but a more specific but less potent non-steroidal mid-narocortical receptor antagonist, in the FESUS trial, it was shown that a plurinone was able to reduce by 15% the risk of all-cause mortality compared with placebo in patients with left ventricle reduced ejection fraction. And in the emphasis heart failure, a plurinone was able to reduce the risk of mortality in hospitalization for heart failure by 37%. However, the risk of hyperkalemia was evidenced in this study that showed that following the publication of the RALES trial, the rate of hospital admission for hyperkalemia was increased. And in fact, the fear for hyperkalemia has led to a reduced prescription of mineralocorticoid receptor antagonist. Even though MRAs are indicated for the treatment of patients with chronic symptomatic heart failure with reduced ejection fraction, according to this study, only 33% of the patients are adequately treated with spironolytin, whereas 69% of the patients that had no contraindication are not treated with mineralocorticoid receptor antagonist. This has motivated the search for novel non-steroidal mineralocorticoid receptor antagonist that had a better therapeutic index and a better safety profile. Among them, we find KBP5074, ACID9977, and Aparerenon, which are in clinical development. And Esaxerenon and Finerenon, that as we saw in the previous talk, are already approved for different pathologies in Japan, United States, and Europe. There are differences between steroidal and non-steroidal mineralocorticoid receptor antagonist. As we saw, the structure of these antagonists are different, being Finerenon, Abulki, non-steroidal mineralocorticoid receptor antagonist that prevents the binding of cofactors and leading to a different gene translational profile. Finerenon maintains its high potency towards mineralocorticoid receptor and high selectivity. It does not penetrate the central nervous system and it doesn't have the sexual side effects that steroidal mineralocorticoid receptor antagonists have. There's a short high life and the absence of active metabolites and the effect on blood pressure is very moderate. In addition, several research in the recent years has suggested that mineralocorticoid receptor activation in different tissues can lead to pathological effects in multiple pathologies, including obesity, as we saw in the previous talk, cardiovascular disease, skin disease, eye disease, inflammation, and cardio-renal injury. In particular, for this talk, we will focus on the cardio-renal injury that is mediated by MR activation in non-classical tissues. In the kidney, mineralocorticoid receptor activation in non-epithelial tissues that includes MR activation in fibroblasts, podocytes, myeloid cells, smooth muscle cells, and endothelial cells may lead to the activation of different targets that finally lead to oxidative stress, inflammation, and fibrosis, which are main drivers of kidney disease. Therefore, the blockade of the action of mineralocorticoid receptor in these non-epithelial cells might be responsible for the organ protective effect observed when mineralocorticoid receptor antagonists are used. To prove that this novel non-steroidal mineralocorticoid receptor antagonist has had similar beneficial effects as spironolactone or pleurenone, we use a mice model of chronic kidney disease induced by ischemia reperfusion. In this case, we use C57 mice that were underwent bilateral kidney ischemia of 22.5 minutes and received placebo or finerenone and were followed for four weeks to evaluate for AKI to say kidney transition. As you can see, the mice that were not treated developed chronic kidney disease evidenced by renal dysfunction and increasing plasma creatinine, plasma urea, and developed tubal interstitial fibrosis as evidenced in the serious red staining and in the quantification of the fibrotic area. In contrast, the mice that were treated with finerenone where we were able to observe a protection from chronic kidney disease transition as evidenced by the prevention in renal dysfunction and in the development of fibrosis. We evaluated some inflammatory mechanisms that could be responsible for the protection observed by finerenone in this mouse model of AKI to say kidney transition. For this purpose, we isolated macrophages from the kidneys of the mice that underwent bilateral renal ischemia reperfusion and we evaluated the expression of several markers of macrophage activation towards a M1 pro-inflammatory phenotype or a M2 anti-inflammatory phenotype. And as you can see, in these macrophages that were isolated from the kidneys of these mice, in the macrophages from mice treated with finerenone, there was a decrease in the expression of some of the markers of M1 polarization and an increase in the markers of M2 polarization, suggesting that MR activation in macrophages modulates M1 to M2 polarization in kidney disease and that this could be modulated also with the treatment of finerenone. In addition, there's been several studies that have shown that mineralocorticoid receptor antagonist has beneficial effects in diabetic kidney disease. Either in mice models of type 1 diabetes or mice models of type 2 diabetes, a plurinone has been able to show a protective effect by reducing albuminuria, glomerular injury, and glomerular inflammation. As you can see here, the infiltration of macrophages was reduced in the glomerulus of these mice, showing the impact of mineralocorticoid receptor antagonist on renal inflammation. Following this study, there's been a lot of studies that have shown protective effects and here I present you a summary of most of the findings that have been shown as a protective mechanism of mineralocorticoid receptor antagonism in preclinical diabetic kidney disease. Mineralocorticoid receptor antagonist has been shown to reduce oxidative stress, the production of profiabiotic and proinflammatory mediators that ultimately lead to prevention of tubal interstitial fibrosis, to prevent tubular alteration as well as glomerular alterations that include mesangial expansion, glomerulosclerosis, collagen deposition in the glomeruli, and infiltration of inflammatory cells. But the protective effects of mineralocortico-receptor antagonists in diabetic kidney disease are not restricted to the kidney in these preclinical models. It has also been shown that MRAs are able to prevent left ventricle hypertrophy, remodeling, collagen deposition, and cardiovascular effects. All this preclinical evidence has led to many small clinical trials in which steroidal mineralocortico-receptor antagonists, such as spironolactone or eplerenone, have consistently shown an antiproteinuric effect in patients with kidney disease associated or not to diabetes. However, the testing of the efficacy of mineralocortico-receptor antagonists in heart renal outcomes was not possible due to the contraindication of steroidal mineralocortico-receptor antagonists due to the risk of hyperkalemia in patients with reduced renal function. This was only possible with the development of the novel non-steroidal mineralocortico-receptor antagonist, and it was recently shown in the Fidelio de AQD and Figaro de AQD trials. The Fidelio de AQD, as we saw, was a clinical trial that was focused on a primary renal composite outcome of time-to-onset of kidney failure, sustained decrease of EGFR of more than 40% from baseline, or renal death. And in the case of Figaro de AQD, it was a cardiovascular-focused trial in which the primary endpoint was time-to-cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. It is important to mention that these two trials had complementary outcomes, and this could facilitate the pooled analysis of more than 13,000 patients that were included in these two trials. It is also important to note that in the Fidelio de AQD, patients with more severe chronic kidney disease that are highlighted in blue, with a more reduced renal function and higher proteinuria were included as compared to the Figaro de AQD, which was more focused on cardiovascular outcomes and also on patients with less severe renal dysfunction. For both trials, the patients were treated with a maximum RAS inhibitor dose. The serum potassium levels at the baseline had to be 4.8 millimole per liter. And Fineranone was started at 10 milligram per day and was up to 20 milligrams after one month of treatment. In the Fidelio de AQD, the primary composite renal outcome was reduced by 18%, whereas the secondary composite outcome was reduced by 14%. Mild hyperkalemia that was defined as plasma potassium of more than 5.5 millimole per liter was observed in 4.5% of the patients in the Fineranone arm versus 1.4% in placebo. And the rate of this continuation was 2.3% in Fineranone versus 0.9 in the placebo group. To start exploring the cardiovascular effect of Fineranone in patients with diabetic kidney disease, in a pre-specified analysis of Fidelio de AQD, it was shown that Fineranone was associated with a 29% risk reduction in the new onset of atrial fibrillation. Next, the figure of AQD show that Fineranone was able to reduce the primary composite cardiovascular outcome by 13%, whereas in this trial, the secondary composite renal outcome did not reach a statistical significance. This might be explained by the fact that patients with less albuminuria and less severe reduction in chlomerular filtration were included in this trial as compared to Fidelio de AQD. Hyperkalemia-related discontinuation in figure of AQD was 1.2% in Fineranone group versus 0.4% in placebo. A pre-specified analysis of this trial continued to show the beneficial effects of Fineranone in diabetic kidney disease patients. In this case, it was observed that Fineranone was associated with a 32% risk reduction in hospitalization for heart failure. And when analyzed in the Fidelity pooled analysis, the efficacy of Fineranone to reduce the cardiovascular composite outcome was 14%, 22% for the reduction in hospitalization for heart failure, whereas for the kidney composite outcome, the efficacy of Fineranone was 23%, and it reduced also 20% the risk of dialysis. Based on this trial, the FDA approved Fineranone and is currently indicated to reduce the risk of sustained EGFR decline, end-stage kidney disease, cardiovascular death, nonfatal myocardial infarction, and hospitalization for heart failure in adult patients with chronic kidney disease associated with type 2 diabetes. However, Fineranone is not the only non-steroidal mineralocorticoid receptor antagonist that is exploring the potential beneficial effects of its use in diabetic kidney disease. Esaxeranone has also shown beneficial effects in animal models of kidney disease, as you can see here. In this case, I present you results from hypertensive rats that were treated with tocasalt and were treated with esaxeranone at increasing doses. And as you can see here, there was a beneficial effect in organ damage, in this case, in renal injury, as evidenced by the prevention on kidney hypertrophy, glomerulosclerosis, a reduction in systolic blood pressure, and proteinuria. In a first phase 2 trial with esaxeranone that included type 2 diabetic patients with chronic kidney disease and microalbuminuria, in which the patients were treated for 52 weeks, esaxeranone was initiated at 1.5 milligram per day and was deteriorated to 2.5 milligram per day, all of these on the basis of serum potassium monitoring. Esaxeranone was associated with a 22% remission in albuminuria, as compared to 4% in the placebo group. With esaxeranone, the treatment discontinuation due to hyperkalemia was 4% in esaxeranone group versus 1% in placebo. Similar beneficial effects in type 2 diabetes with chronic kidney disease patients have been observed also with aparerenone. As you can see here, this other non-steroidal mineralocorticoid receptor antagonist was able to reduce albuminuria when given to these patients for 24 weeks, and it was not associated with clinically significant events of hyperkalemia. Next, the KBP5074 non-steroidal mineralocorticoid receptor antagonist has also been evaluated for its efficacy in resistant hypertension in patients with advanced chronic kidney disease. In this case, as you can see here, KBP5074 was efficient to reduce uncontrolled hypertension in this kind of patients and was not associated with severe hyperkalemia cases, acute kidney injury, or hospitalization due to hyperkalemia. So on all of these trials, there's been a lot of beneficial effects that have been reported with this non-steroidal mineralocorticoid receptor antagonist in patients with type 2 diabetes and chronic kidney disease. However, there's still a lot to do in this area to continue exploring what could be the beneficial effect of mineralocorticoid receptor antagonist in cardiorenal benefits in non-diabetic chronic kidney disease patients, and also in the pediatric population in which it has not been explored so far. The mechanism of action in the clinical trials in patients to explore if the protective effect is related to the anti-fibrotic, anti-inflammatory, or hemodynamic effects of mineralocorticoid receptor antagonist, and if there could be an additive effect with no other treatments that have been developed in the recent years, such as SGLT2 inhibitors or GLPR1 receptor antagonist. Finally, I would like to take the last slides of my presentation to highlight also that MR activation elicits pathological mechanisms in the heart. As you can see here, this is a summary of many preclinical studies that have shown that mineralocorticoid receptor activation in different cell types in the heart can lead to pathological mechanisms, such as fibroblast proliferation, the increase in the expression of fibrotic mediators that ultimately lead to extracellular matrix accumulation and fibrosis, an increase in inflammatory mediators, adhesion molecules, which modulates M1 macrophage activation and T cell activation, oxidative stress via increase in the activity of NADPH oxidase, which leads to DNA and protein damage, and also modulates the expression of different vasoactive factors, calcium channels, that can modulate contractility leading to arrhythmia and calcification. So based on these findings, there's been also recent studies that have been exploring the effect of non-steroidal mineralocorticoid receptor antagonist in preclinical cardiac models of disease. In this case, transgenic mice with cardiac-specific overexpression of RAC1, which leads to cardiac fibrosis, was used, and it was shown that finerenon treatment in these mice was able to reduce cardiac fibrosis, which was associated with a reduction in the expression of fibrotic factors, such as TGF-beta, CTGF, LOX, and finally, preventing cardiac fibrosis and remodeling. In this other study, we used the transgenic soccer rat, which is a mouse rat model of obesity and metabolic syndrome-induced cardio-renal damage, and in this rat, we showed that finerenon treatment was able to show cardio-renal benefits, including the prevention on cardiac hypertrophy, collagen deposition in the left ventricle, and this was associated with improved cardiac tissue perfusion. Moreover, there was an antiproteinuric effect and a reduction in N-gal expression as kidney injury marker. Finally, saxarenon has also shown cardioprotective effects, in this case, in the cell-sensitive hypertensive rats. In this case, saxarenon treatment was associated with the prevention on interstitial and perivascular fibrosis and a reduction in inflammatory mediators in the cardiac tissue, including TNF-alpha, interleukin-6, and CXCL-8. So all of these studies are showing that these non-steroidal mineralocorticoid receptor antagonists have beneficial effects both in cardiac and renal damage, and there are future ongoing major trials that will evaluate if this can also be translated into the patient. In the case of Finereart's heart failure trial, it's a phase three trial in which Finerenon will be evaluated on heart failure with preserved ejection fraction and its efficacy to prevent stroke, hospitalization for heart failure, or cardiovascular death. The Miracle trial will be one of the first trials to evaluate the possible additive effect of combining a mineralocorticoid receptor antagonist, in this case, ACD9977, with dapaglifrazine and SGLT2 inhibitor on heart failure with reduced ejection fraction and chronic kidney disease. And the FINDES-AKD trial will evaluate the efficacy of Finerenon on the EGFR slope reduction from baseline to 32 months in non-diabetic patients with chronic kidney disease, whereas the FIONA trial will evaluate the efficacy of Finerenon in pediatric population. So there's a lot of work to do on mineralocorticoid receptor antagonism, and to continue evaluating its potential beneficial effects in different settings in the clinical practice. With that, I would like to finish, and thank you for your attention. Thank you. Thank you, Jonathan. We are now open for questions. Hi, Suchitra Nanchella from Northern Virginia. So we may have found the magic pill, combine SGLT2 inhibitor with Finerenon, and these patients will live for a long, long time. So my question is, since SGLT2 inhibitors work like a loop diuretic, in a sense, you cause more sodium excretion. Would that also cause more potassium wasting and counteract the effect of these agents? Yes, yes. Actually, there's a lot of exciting recent data showing that SGLT2 inhibitor is associated with potassium reduction, and this could enhance the combination of SGLT2 with an MRA antagonist, because that will prevent the risk of hyperkalemia in this kind of patient. So yes, definitely it's something that could be working in that way, and that it's being explored in these days. Great, okay. My second question is, with spironolactone and the steroidal MRAs, do you see a reduction in cardiac fibrosis? Yes. Well, in the animal models, we have shown that with spironolactone, there's reduction in cardiac fibrosis in different models of direct cardiac failure or cardiac failure associated to chronic kidney disease. And you expect to see the same in humans? Yes. Yeah, thank you. Are there questions? Everyone? I've got one, too. Oh, you go first, you go first. No, no, you go first, you go first, please. Lovely presentation, thank you so much. I wanted to ask about the pediatric patients. Are there special, are you going to do this? You too, you're the pediatrician. No, go on. Are there special considerations for treating pediatric patients with mineralocorticoid receptor antagonists in dose reduction, or do you just use exactly the same dosing as for adult patients, just reduce according to their body weight? Yeah, the dose will be adjusted to the body weight. Sure, but no other specific? Not other, no. Okay. And strict potassium monitoring as it was used in epithelial AQD and figaro AQD. And do you find that it's just, it's well tolerated or tolerated similarly to adults using it? Well, that will be a question to follow up because it's still, yeah, it's part of the ongoing trials that are just starting, so that will be in the future. Very interesting, yeah, thank you. Sorry, you were too focused. Thank you for the question. We have one online. Oh, yes, you can go. Any trials looking at Finrenone utility in primary aldosteronism? Sorry, sorry. The utility of Finrenone in primary aldo? Oh, I think there's recently been registered one trial that will evaluate the effect of Finrenone on primary aldosteronism. Yeah, but it's not so far. There's not so far anything done. No more questions. Thank you, Jonathan. Thank you. Great presentation. Thank you. Thank you. Great, thank you so much. And our last speaker today is Lauren Bewer, who is here, Dr. Lauren Bewer, I should say, excuse me, who is here from Tufts Medical Center from the Molecular Cardiology Research Institute. And welcome, we're glad to have you here. And we look forward to your presentation entitled Interaction Between Mineralocorticoid and Estrogen Receptors in Endothelial Function. Thank you. I'm going to do progressives, and then it's the nice one. Don't touch the middle. Don't touch the middle. Okay, okay, got it. All right, thank you everyone for sticking with us. I also want to thank the organizers for the invitation to speak today. I'm really excited to tell you a little bit about what I've been doing as a postdoc fellow in Iris Jaffe's lab at Tufts. And I have no financial relationships to disclose. So what I want to talk about first is obesity. And we know obesity is defined as a BMI greater than 30. This is just a prevalence map across the world. You can see roughly a third of the world's population is affected by obesity. And importantly, it's a major risk factor for cardiovascular disease, which is what we study. And what we know is that women bear greater burden of obesity than men. So this is a study that looked at the male prevalence of obesity and the female prevalence of obesity. And you can see each dot represents a country. And if there was no sex difference, you would expect all of the dots to fall along this line here. However, we see that females are more affected by obesity than males in most countries. And the problem with obesity is that it's negating premenopausal protection from cardiovascular disease. So this is a study that looked at over 38,000 adults. You can see age 25 to 45, so premenopausal women are included here. And they grouped people based off of their BMI. So you can see that as blood pressure, or as BMI increases, blood pressure increases. And this is happening in men and women, but you see a sex difference at the lower BMIs that is negated in the higher BMIs. And this is also true for other things like heart attacks. And I think as Gail may have alluded to earlier, aldosterone levels in women are directly correlated with obesity. So this was a study looking at visceral adipose tissue and plasma aldosterone in women before and after weight loss. So before weight loss, women with more adipose tissue had higher plasma aldosterone levels. When they lost weight, they tended to have lower aldosterone levels. And we know that excess aldosterone can promote cardiovascular disease by activating the MR, as we've heard a lot about. And again, citing Gail here, but they took obese diabetic patients and randomized them to spironolactone, hydrochlorothiazide, or placebo, as we saw earlier. And what we took away from this is that coronary flow reserve is increased with MR inhibition. And this suggests that inhibiting MR is really beneficial in the microvasculature. And just so we're all on the same page here, we study microvascular arteries. The arteries are lined by endothelial cells, which are kind of the first defense from the first layer in between the blood and the rest of the blood vessel. And we know that endothelial cells maintain vascular homeostasis, they're anti-inflammatory and anti-thrombotic, and they can control blood pressure, which is what we're ultimately interested in. And the problem with endothelial cell dysfunction is it's the first hallmark of, endothelial dysfunction's the first hallmark of vascular dysfunction, and that's why we're interested in studying them. So here we have an endothelial cell, and like I mentioned, they can modulate blood pressure, and one of the ways they do this is by promoting vasodilation. So here we have endothelial nitric oxide synthase, or ENOS. When it is phosphorylated, it can become active, produce nitric oxide, promote vasodilation. And we'll be coming back to a little bit more on ENOS later. And so as I told you, women are disproportionately affected by obesity. One of the ways that this might be happening is by sex differences in MR expression. And so this was a study with a cooperation between our lab and Eric Belinda Shenton-Mel's lab, looking at MR expression in healthy males and females, their adipose endothelial cells. What we see is that there's a greater MR expression, specifically in endothelial cells of females versus males. And this also is exacerbated in obesity. Okay, and so previous studies in our lab have looked at microvascular function using mice with or without the endothelial MR. And so I'm gonna show you a lot of graphs that look like this. So hopefully you can follow along with me. So what we do is we take out arteries and we measure their relaxation to this molecule acetylcholine. And so this shows how healthy the endothelial cells are. So as an example, we took male mice, exposed them to obesity or hyperlipidemia. And we can see that hyperlipidemia in the male mice caused a decrease in relaxation. So they had endothelial dysfunction. However, when we removed the endothelial MR, we still saw that dysfunction. And this indicated that endothelial MR does not play a large role in males. However, when we repeated this study in females, you can see that both obesity and hyperlipidemia affected the females and their vasodilation. But really interestingly, endothelial MR knockout protected against this dysfunction. And so this led us to the question, what is mediating this improved dilation in the obese female endothelial MR knockouts? And that's kind of where I came into the story. And our first thought was that perhaps ER-alpha could be playing a role. So ER-alpha is also a hormone-activated transcription factor. But we know in endothelial cells, both ER-alpha and MR can be localized to the membrane. They can bind to striatin. And when ER-alpha is activated, it promotes ENOS phosphorylation and NO production. So MR can also be there, binding to striatin, but seems to have opposite effects, and it promotes reactive oxygen species production. And so we first just took endothelial cells and exposed them to an MR inhibitor, and we looked at ER-alpha expression. And we saw that when you put spironolactone on these endothelial cells in culture, they have an increased amount of ER-alpha expression. So we went back to our mouse model, where we focused on females, again, because they were most impacted by obesity, and they were rescued with the endothelial MR deletion. And we had lean mice, either MR intact or MR knockout, specifically in the endothelial cells. And then we had mice fed a high-fat diet, and the same, MR intact or knockout. And we saw with the high-fat diet feeding that we had increased body weight, increased fasting glucose. This was not affected by ECMR deletion. And what I did then was I took these small mesenteric arteries from the mice and even harvested them for RNA or protein. And the other thing was measure this endothelial-dependent vasodilation. So the way we do that is just put two small wires through the lumen of the artery, and then we can measure how much it dilates to that molecule acetylcholine. And what we found first is that in these small arteries ground up, we found that the knocking out endothelial MR, regardless of obesity, was increasing ER alpha expression, which we thought was really interesting. And this is mRNA. And we saw similar effects when we looked at protein expression. So increased ER alpha with knockout, and maybe even more so in the obese knockout mice. So we conclude that ECMR knockout is upregulating the vascular ER alpha expression. And we wondered if we went to some of the functional studies, does inhibiting ER alpha maybe remove that protective effect we saw with the endothelial MR knockout? So I repeated the study that I had showed you previously in the females that were obese, either wild type or knockout, and we see obesity causes this impairment in vasodilation, whereas the knockout obese females had preserved vasodilation. And this is all in the context of not affecting ER alpha or ER beta signaling. But what I can do is I can add different inhibitors to the bath with these arteries, and then measure the resultant dilation. So first we took ICI, which should inhibit and degrade ER, and repeated the vasodilation. And now you see that where this knockout was once dilating down here, it now looks like the obese wild type. And so we think that ER is acutely mediating these effects as we've inhibited both ER alpha and ER beta. And so we wanted to get a little bit more specific, so we repeated the experiment with an ER alpha specific inhibitor, and we didn't see an effect on the obese wild type mice. They pretty much still had impairment in vasodilation, and nothing was changed. But when we repeated the studies by inhibiting ER alpha in our knockout mice, we saw a significant impairment in vasodilation in these mice that were lacking endothelial MR. So again, suggesting that ER alpha is acutely mediating this improved vasodilation. And so then the question was, well how is ER alpha potentially mediating this improvement in dilation? And so we went to cell culture, we took endothelial cells. Again, I've told you ENOS produces nitric oxide. When ENOS is phosphorylated, it's activated, so we looked at the ratio of phosphorylated to total ENOS in our endothelial cells. And we stimulated the cells with either ALDO to stimulate MR, estradiol to stimulate ER, and then we combined the two. And so what we see is that, as we might expect, when you add estradiol to endothelial cells, you get an increase in phosphorylated ENOS. But when you also activate MR, you get a significant decrease in that phosphorylated ENOS. And to see if this was dependent on MR, we did the experiment in the presence of MR siRNA, so we knocked out MR from these endothelial cells, and you no longer see that decrease in phosphorylation. So kind of what I've told you about the vascular function studies is that we have impaired dilation in these obese females, we have preserved vasodilation in the obese knockout females, but we also have a double knockout mouse model that we wanted to look at. So that's right here, so we already know this mouse is basically the same as this mouse, so they have impaired vasodilation. But what about removing ER-alpha on top of MR, specifically in endothelial cell? So this is just what I showed you earlier, the obese MR knockout mouse, impaired dilation, improved with the knockout. But when we overlay our double knockout on top of that, we see that the mice that lack both ER-alpha and MR have even worse dilation in response to acetylcholine. And so this made us wonder about MR and ER-alpha's binding to striatin in the endothelial cell. And so like I alluded to earlier, we know ER-alpha can bind to striatin, that produces enose phosphorylation and nitric oxide beneficial effects. And we know that MR can also bind to striatin and produce kind of the opposite effect of this reactive oxygen species. We wonder maybe if they're competing for binding. So I'll walk you through this slowly. So we repeated, or we did EAHY, endothelial cell line that stably express ER-alpha, grew them in culture, then we IP'd them for striatin, Western blotted for striatin as a control to show that we've pulled down striatin, and then we Western blotted for ER-alpha. And so what we see is that when we add estradiol to these cells, we increase this ER-alpha-striatin interaction, which is what we might expect. However, when you also add aldosterone to the mix, you get an impairment in that ER-alpha-striatin interaction. And when we look at the opposite, when we look at MR-striatin interaction, we would expect to see that aldo increases MR and striatin's binding, which we do. However, when you add estradiol, you see less MR binding to striatin. So this is really interesting and showed us that upon hormone activation, these two different receptors are both binding to striatin. However, when you have both on board, they're inhibiting each other from binding. So then we wonder, well, maybe they're binding at the same location on striatin. And so we have this nice mimetic peptide that we can transfect the cells with, and we overexpress it, and it's basically a piece of ER-alpha that binds to striatin. And so our lab has shown before that when that's expressed in the cells, ER-alpha can no longer bind to striatin. And that's what we see here. So this is kind of control. So we again took these cells and we IPed for striatin, Western bought it for striatin. But the interesting thing is this, that the peptide is working, and it's blocking ER-alpha's binding to striatin as we would expect. And now the novel part is that we looked at MR and striatin's binding, and what we see, again, as we've shown before and what we would expect, aldo is increasing this MR-striatin interaction. However, the peptide is decreasing the, oh, I think my microphone went out. Right at the. Hello, hello? Sorry, guys. I can also try to yell. Hello, hello, hello? Not working. I can also just. Hello? Okay, there we go. Thank you, sorry. Okay, so basically when we look at the MR-striatin interaction, it's blocked by this peptide that blocks ER-alpha-striatin interaction. So conclusion with all of this is that MR is binding to striatin at the same location as ER-alpha. Whoa. Okay. Almost done. All right. So just in summary, what I've shown, well, what I've shown you today, can you guys hear me if I just do this? Yeah. What I've shown you today is that in obese female endothelial cells, there may be more MR that's found in striatin, and less ENOS phosphorylation that's resulting in decreased nitric oxide, impaired vasodilation. However, when we get rid of endothelial MR, remember we see increased ER-alpha expression in the vasculature, which means more ER-alpha bound to striatin, more ENOS phosphorylation. And we didn't test this, but potentially more transcription of ER-alpha target genes. And this is all resulting in increased nitric oxide, restored vasodilation. So this might be a mechanism of particularly why MR inhibition in obese females might be protective. And when we remove both ER-alpha and MR, we've now negated our beneficial effects that we saw by removing MR, and we had a further impairment in vasodilation, decreased nitric oxide. So all of this is kind of a preclinical rationale to give obese women MR antagonists, and see if that could improve their endothelial function. And I just want to thank you all for your attention, and I want to thank my mentor Iris Jaffe, and Ching Liu, and Bridget Carvajal, and Josh Mann, who all helped with these experiments, and my funding sources. So I'll take any questions if you have them. Thank you. Thank you so much for that lovely presentation. We'll open, yes, Gail, first question. That was great, I really liked it. I have a question. When you knocked out MR from the male, you didn't see the improvement in vasodilation. Did you see an increase in the ER in the male, and they just don't have estrogen, and that's why you don't see it, or did you not look? That's a great question. So when we started this, we actually see opposite effects, but we haven't quite followed up on it. Part of this is like, you know, we have so many different groups, and we had to kind of choose something. But I think some of the initial studies that we looked at, we see basically the opposite effect, like less ER alpha, but I'm not sure, you know, what that means just yet. That's a great question. Thank you. Please, oh, okay. So along the same lines, in a post-menopausal woman, there's no more estrogen coming, so would it not make sense to continue the MR antagonist? Yeah, yeah, I mean, that's a great point. We've mainly focused on, you know, the negation of the pre-menopausal protection. There might be different mechanisms, but presumably, I mean, MR inhibition seems to be beneficial for a lot of populations. Okay, thank you. This is Xu from Emory. Very nice talk. Would you mind to tell me a little bit about the ER mimetic peptides? Where does peptide from? Which domain? Oh, you're testing me here. More particularly, like the one, even though you showed that kind of in your summary slide, so what you talked about today has a little to do with transcription activities, most like not? The majority of things we focused on, obviously, are the non-genomic signaling, so obviously, it gets complicated. The peptide is blocking the non-genomic signaling, and I think maybe 20 years ago, Qing from our lab published, you know, more specific information about that peptide, but I believe it's amino acids 176 to 253. I'm sorry, I don't remember what other questions you asked. So this is not too much to do with this receptor go to the nucleus, but you still need the ligand there to release them from the chaperone, then go to the sub-cell memory region instead of going to a nuclear? When that, so the striatium is kind of a transmembrane protein there, right? Yeah, I guess there's two kind of different, or at least the way I look at it is like there's two different functions, right, of both MR and ER alpha, and we focus mainly on this non-genomic where it doesn't meet, like it's already tagged to the membrane, if you will, by striatin, so upon ER alpha binding to, or estrogen binding to ER alpha in that membrane pool, it's already there, it wouldn't be moving is my understanding. Okay, thanks. Does that answer your question? Well, because the classical view, we think about always in the plasmas kind of sequestered by the chaperone, then the estradiol, or those that come here to release the MR or ER, then go to the nuclear. Yeah. It seems that what you talk about today is not in the nuclear. Yeah, and that's kind of like the difference we're not focused on that pathway, but more on the endothelial cell membrane signaling. I see, thanks. June. June from Hudson Institute, Australia. Thanks for the great talk, Lauren. I was just curious about the mechanism for increase in ER alpha expression following MR knockout. Yeah, I am too. We don't have a lot of insight on that just yet. There was a paper maybe about eight years ago now from Iris' lab before I was there. They looked on cell culture, but saw that genomic effects of, I wanna say they got rid of ER alpha, or they saw ER alpha would block MR transcription, but not the reverse. But yeah, we haven't really delved into that mechanism. It could be really interesting. So I guess I'm just wondering, in patients who are taking MR antagonist, would you expect that the women have a slightly different effect? Would they also experience some change in their ER alpha expression? I mean, that's what I would hypothesize based on this. Obviously mice are not humans, but potentially that might be why MR antagonism is beneficial in females, is it's increasing ER alpha, which then estrogen binds to it, whereas males have the unliganded version, which actually might cause some issues. Have you looked at that in humans? No, no. Thank you. Adina. Congrats, this was really beautiful work. I have a quick question. Another hormone that is different in women, especially reproductive age versus males is progesterone, and progesterone is an MR antagonist. Have you looked, have you controlled for that? We didn't look at progesterone in any of our studies or the progesterone receptor. That paper I cited with the increased female expression of ECMR, they looked at progesterone receptor as maybe a driver of that increased MR expression causing the sex differences, but yeah, we haven't looked at it in our model. We have some online questions for you as well. Great talk. Do you think that women on aromatase inhibitors would benefit from mineralocorticoid receptor antagonists? That is a great question. I'm not familiar with why someone would be on an aromatase inhibitor. Oh, it's as breast cancer preventative treatment because it blocks conversion to estrogen from... So I guess that might, I mean... So you have the aromatase inhibitors long-term preventing further breast cancer. So you're basically reducing estrogen. Yeah, I mean, I feel like anything that's reducing estrogen is probably not ideal for endothelial function in our females, but I don't know much about that to say. There's another target for this approach, for MR therapy. Yeah. And then secondly, would you expect a biophasic effect with mineralocorticoid receptor blockers in pre and post-metapausal women? Biophasic effect. So I guess in the presence of estrogen and then presumably in non-obese, I'm guessing from looking at this. So where there is estrogen present in pre-metapausal women, then loss of estrogen production, would you expect differential responses to MR antagonists? Yeah, I mean, I would definitely hypothesize that obviously we didn't see it, at least in vascular function-wise, we didn't see a benefit of ECMR deletion in males, which not the exact same thing as a post-metapausal woman, but might be similar. But obviously there's other benefits of MR antagonism beyond just the vasodilation. And I also think that, or it's a good point to make, is that lean females, and we haven't looked in age in this context, but in general, lean females don't have this decrease in vascular function. So the MR doesn't really play a role in that situation. Not sure if I can fully answer that. No, no, that's fine. No, no, that sounds great. I have one question. What about in the context of inflammation? What happens to striatum? And do you have any insights into what might happen for the striatum estrogen receptor interaction or the striatum MR interaction in the context of inflammation? Yeah, that's a great question. We have not focused on striatum. That's more, you know, Gail and Gordon and Jose's group, but we're currently looking at the role of inflammation in terms of atherosclerosis and the impact of, you know, endothelial MR and the ER alpha in, you know, promoting or decreasing atherosclerosis in that context. Okay. Stay tuned. Yes, yes, stay tuned is the short answer. Okay, so if there are no further questions, thank you so much for your lovely presentation. Thank you, everyone. And we'd like to thank, just like to thank all of our speakers today and our audience here and all of our listeners online as well. Thank you for a wonderful session and safe travels home. Thank you.

symposium

mineralocorticoid receptor antagonism

health conditions

non-steroidal mineralocorticoid receptor antagonists

finerenone

cardiovascular outcomes

renal outcomes

chronic kidney disease

type 2 diabetes

obesity

endothelial cells

vascular homeostasis