Mineralocorticoid Receptor Expression Research Articles

391-P: Targeted Deletion of Rho-Associated Coiled-Coil–Containing Protein Kinase 2 in Kidney Tubules Suppresses Expression and Function of Mineralocorticoid Receptor in Mice

391-P: Targeted Deletion of Rho-Associated Coiled-Coil–Containing Protein Kinase 2 in Kidney Tubules Suppresses Expression and Function of Mineralocorticoid Receptor in Mice

#417 Targeting parietal epithelial cell activation with mineralocorticoid receptor antagonism controls FSGS and crescentic glomerulonephritis

Abstract Background and Aims We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury [1]. Accumulating evidence has indicated the potential contributions of the mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is expressed in endothelial cells, glomerular mesangial cells, podocytes, and distal tubular cells. Previous studies have shown that administering mineralocorticoid receptor antagonists has beneficial effects in various renal injury animal models, the role of the MR in extracapillary glomerulopathies is still elusive and mechanistically unclear. Methods To investigate the cell-specific role of the MR in PEC in the course of focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (GN), we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Maladaptive FSGS was induced by combining a high-salt diet and deoxycorticosterone acetate (DOCA) with unilateral nephrectomy (DOCA-UniN). Crescentic glomerulonephritis (GN) was induced using the anti-glomerular basement membrane nephrotoxic serum (NTS) model. Results At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged by DOCA-UniN, PEC-selective Nr3c2 targeting significantly inhibited the expression of PEC activation markers Ki67, CD44, and FSGS lesions. Importantly, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function compared to Pec Cre Nr3c2 wt/wt counterparts. Likewise, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function in the NTS-induced CGN model. Crescents were also more numerous and organised in Pec Cre Nr3c2wt/wt mice than in Pec Cre Nr3c2lox/lox animals. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less expression of the CD44 activation marker on PECs. Thus, genetic disruption of MR in PEC and pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC within FSGS and crescents. We next evaluated PEC migration under various conditions (DMSO, HB-EGF, Eplerenone, or HB-EGF+Eplerenone) for 24 hours. Eplerenone fully abrogated HB-EGF-induced PEC migration and Pcna, Snai1, and Vim mRNA expressions. Conclusion Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis, along with the recently discovered CD9/EGFR/PDGFR [1] and CLDN1 pathways. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in FGSS and, more surprisingly, in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

Both endothelial mineralocorticoid receptor expression and hyperleptinemia are required for clinical characteristics of placental ischemia in mice.

One of the initiating events in preeclampsia (PE) is placental ischemia. Rodent models of placental ischemia do not present with vascular endothelial dysfunction, a hallmark of PE. We previously demonstrated a role for leptin in endothelial dysfunction in pregnancy in the absence of placental ischemia. We hypothesized that placental ischemia requires hyperleptinemia and endothelial mineralocorticoid receptor (ECMR) expression to induce PE-associated endothelial dysfunction in pregnant mice. We induced placental ischemia via the reduced uterine perfusion pressure (RUPP) procedure in pregnant ECMR-intact (ECMR+/+) and ECMR deletion (ECMR-/-) mice at gestational day (GD) 13. ECMR+/+ RUPP pregnant mice also received concurrent leptin infusion via miniosmotic pump (0.9 mg/kg/day). RUPP increased blood pressure via radiotelemetry and decreased fetal growth in ECMR+/+ pregnant mice. Both increases in blood pressure and reduced fetal growth were abolished in RUPP ECMR-/- mice. Placental ischemia did not decrease endothelial-dependent relaxation to acetylcholine (ACh) but increased phenylephrine (Phe) contraction in mesenteric arteries of pregnant mice, which was ablated by ECMR deletion. Addition of leptin to RUPP mice significantly reduced ACh relaxation in ECMR+/+ pregnant mice, accompanied by an increase in soluble FMS-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PLGF) ratio. In conclusion, our data indicate that high leptin levels drive endothelial dysfunction in PE and that ECMR is required for clinical characteristics of hypertension and fetal growth restriction in placental ischemia PE. Collectively, we show that both ECMR and leptin play a role to mediate PE.NEW & NOTEWORTHY Leptin is a key feature of preeclampsia that initiates vascular endothelial dysfunction in preeclampsia characterized by placental ischemia. Endothelial mineralocorticoid receptor (ECMR) deletion in placental ischemia protects pregnant mice from elevations in blood pressure and fetal growth restriction in pregnancy. Increases in leptin production mediate the key pathological feature of endothelial dysfunction in preeclampsia in rodents. ECMR activation contributes to the increase in blood pressure and fetal growth restriction in preeclampsia.

High Salt Diet Induces Aldosterone Synthase Elevation in Adrenal and Visceral Adipose Tissue - A Potential Culprit for Unsuppressed Aldosterone Levels in Female BALB/c Mice

Women are more salt-sensitive than men, irrespective of age, menopausal status, or ethnic background. Nonetheless, the mechanisms predisposing women to salt sensitivity of blood pressure (SSBP) are poorly understood, notably due to the diffculty in finding appropriate animal models. Previously, we demonstrated that female BALB/c mice spontaneously develop SSBP, likely through an inability to reduce the expression of adrenal aldosterone synthase (CYP11B2) appropriately, which leads to unsuppressed aldosterone levels in response to high salt intake. Herein, we aim to decipher the mechanism involved in SSBP in female BALB/c mice by comparing them to female salt-resistant C57BL/6 mice. Eleven-week-old female BALB/c and C57BL/6J mice were fed either a normal (0.4%, NSD) or a high salt diet (4%, HSD) for seven days. HSD significantly reduced adrenal aldosterone synthase transcript and protein expression in female C57BL/6 mice compared to those on a NSD. Conversely, HSD substantially increased CYP11B2 levels in BALB/c female mice when compared to strain-matched NSD and C57BL/6 NSD mice (2-way ANOVA, p<0.05). HSD induced a more significant aldosterone drop in C57BL/6 versus BALB/c mice (C57BL/6: 72% vs. BALB/c: 49%). Remarkably, HSD led to an increase in CYP11B2 in the visceral adipose tissue (VAT) of BALB/c mice in comparison to strain-matched NSD mice, while no such alteration was observed between C57BL/6 NSD and HSD. We previously showed that leptin regulates CYP11B2 expression. We report here that HSD increased leptin receptor (lepR) expression in adrenal and VAT from BALB/c mice in comparison to both strain-matched NSD and C57BL/6 NSD. Baseline levels of leptin were similar in C57BL/6 and BALB/c mice. However, BALB/c mice on HSD displayed increased levels of leptin compared to its strain-matched NSD mice, while no such alteration was observed in C57BL/6 mice. Furthermore, female BALB/c mice on HSD displayed elevated endothelial cell-specific mineralocorticoid receptor (ECMR) expression compared to female C57BL/6 NSD and HSD mice (2-way ANOVA, p<0.05). Endothelium-dependent relaxation to acetylcholine was significantly reduced in the female BALB/c mice on HSD compared to strain-matched NSD and C57BL/6 NSD (n = 3, 2-way ANOVA, P < 0.05). Interestingly, on NSD, C57BL/6 mice present a better endothelial function than BALB/c, which was not altered by HSD. Collectively, our data supports an overactivation of the leptin-CYP11B2-aldosterone axis in both adrenals and VAT, leading to inappropriate aldosterone levels, which, associated with higher ECMR expression, induce endothelial dysfunction in BALB/c mice. Together, these mechanisms are likely the cause of SSBP in female BALB/c mice. Funding: R01HL130301, R01HL155265 (E.J.B.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metabolic effects of aldosterone

Currently, increasing evidence shows the mutual influence of aldosterone and adipose tissue. Aldosterone excess has been reported in patients with obesity and metabolic syndrome. Aldosterone has a direct effect on adipose tissue increasing anabolic activity and expression of mineralocorticoid receptors. In turn, excessive activation of MCR leads to stimulation of adipogenesis and an increase in the volume of adipose tissue. Aldosterone excess can be considered an independent cardiovascular risk factor that affects such processes as cardiac fibrosis, nephrosclerosis, and arteriosclerosis. There is convincing evidence of higher prevalence and severity of impaired glucose homeostasis and lipid metabolism disorders among patients with primary hyperaldosteronism. Similar pathological changes are also observed in patients with obesity and metabolic syndrome. This review presents scientific data on the metabolic effects of aldosterone, in particular its effect on adipose tissue function, glucose and lipid metabolism. Treatment with mineralocorticoid receptor antagonists may provide substantial benefit in the management of metabolic syndrome, contribute to the stabilisation of glucose and lipid metabolism, improve clinical status of patients with cardiovascular diseases and reduce the risk of complications. However, available evidence from the conducted studies is not sufficient to justify introduction of such therapy into clinical practice.

In your CORT: Corticosterone and its receptors in the brain underlie mate choosiness in female Cope's gray treefrogs (Hyla chrysoscelis)

Selecting an attractive mate can involve trade-offs related to investment in sampling effort. Glucocorticoids like corticosterone (CORT) are involved in resolving energetic trade-offs. However, CORT is rarely studied in the context of mate choice, despite its elevated levels during reproductive readiness and the energetic transitions that characterize reproduction. Few systems are as well suited as anuran amphibians to evaluate how females resolve energetic trade-offs during mate choice. Phonotaxis tests provide a robust bioassay of mate choice that permit the precise measurement of inter-individual variation in traits such as choosiness–the willingness to pursue the most attractive mate despite costs. In Cope's gray treefrogs (Hyla chrysoscelis), females exhibit remarkable variation in circulating CORT as well as choosiness during mate choice, and a moderate dose of exogenous CORT rapidly (<1 h) and reliably induce large increases in choosiness. Here we measured the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors in the brains of females previously treated with exogenous CORT and tested for mate choosiness. We report a large decrease in GR expression in the hindbrain and midbrain of females that were treated with the moderate dosage of CORT–the same treatment group that exhibited a dramatic increase in choosiness following CORT treatment. This association, however, does not appear to be causal, as only forebrain GR levels, which are not affected by CORT injection, are positively associated with variation in choosiness. No strong effects were found for MR. We discuss these findings and suggest future studies to test the influence of glucocorticoids on mate choice.

Effects of the mineralocorticoid receptor antagonist eplerenone in experimental autoimmune encephalomyelitis

There is growing evidence indicating that mineralocorticoid receptor (MR) expression influences a wide variety of functions in metabolic and immune response. The present study explored if antagonism of the MR reduces neuroinflammation in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). Eplerenone (EPLE) (100 mg/kg dissolved in 30% 2-hydroxypropyl-β-cyclodextrin) was administered intraperitoneally (i.p.) daily from EAE induction (day 0) until sacrificed on day 17 post-induction. The MR blocker (a) significantly decreased the inflammatory parameters TLR4, MYD88, IL-1β, and iNOS mRNAs; (b) attenuated HMGB1, NLRP3, TGF-β mRNAs, microglia, and aquaporin4 immunoreaction without modifying GFAP. Serum IL-1β was also decreased in the EAE+EPLE group. Moreover, EPLE treatment prevented demyelination and improved clinical signs of EAE mice. Interestingly, MR was decreased and GR remained unchanged in EAE mice while EPLE treatment restored MR expression, suggesting that a dysbalanced MR/GR was associated with the development of neuroinflammation. Our results indicated that MR blockage with EPLE attenuated inflammation-related spinal cord pathology in the EAE mouse model of Multiple Sclerosis, supporting a novel therapeutic approach for immune-related diseases.

Potential of Modulating Aldosterone Signaling and Mineralocorticoid Receptor with microRNAs to Attenuate Diabetic Kidney Disease.

Diabetic Kidney Disease (DKD) is a significant complication of diabetes and primary cause of end-stage renal disease globally. The exact mechanisms underlying DKD remain poorly understood, but multiple factors, including the renin-angiotensin-aldosterone system (RAAS), play a key role in its progression. Aldosterone, a mineralocorticoid steroid hormone, is one of the key components of RAAS and a potential mediator of renal damage and inflammation in DKD. miRNAs, small noncoding RNA molecules, have attracted interest due to their regulatory roles in numerous biological processes. These processes include aldosterone signaling and mineralocorticoid receptor (MR) expression. Numerous miRNAs have been recognized as crucial regulators of aldosterone signaling and MR expression. These miRNAs affect different aspects of the RAAS pathway and subsequent molecular processes, which impact sodium balance, ion transport, and fibrosis regulation. This review investigates the regulatory roles of particular miRNAs in modulating aldosterone signaling and MR activation, focusing on their impact on kidney injury, inflammation, and fibrosis. Understanding the complex interaction between miRNAs and the RAAS could lead to a new strategy to target aldosterone signaling and MR activation using miRNAs. This highlights the potential of miRNA-based interventions for DKD, with the aim of enhancing kidney outcomes in individuals with diabetes.

First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.

Post-stress expression of genes involved in neuroplasticity in the hippocampus and blood of rats with different level of nervous system excitability

The causes and mechanisms of most mental disorders remain unclear. While stress is known to trigger depressive and anxiety disorders, the role of genetically determined individual differences in forming vulnerability to post-stress neurochemical disorders, including those that affect neuroplasticity, remains unresolved. Early studies suggest that acute and chronic stress lead to elevated glucocorticoid levels in the bloodstream and alter the expression of glucocorticoid and mineralocorticoid receptors. Additionally, both forms of stress can decrease the levels of brain-derived neurotrophic factor (BDNF), which plays a role in neuronal growth, development, and synapse building [1]. However, current studies insufficiently explain the complete mechanism of the stress response as they only explore variations in post-stress alteration of gene expression for proteins related to differing nervous system functional characteristics. The strains of rats from the Biocollection of the Pavlov Institute of Physiology, RAS, are a convenient model for studying the impact of genetically determined nervous system features on stress reactivity. The strains, selected based on the threshold of nervous system excitability [2], include HT (high threshold of excitability, low excitability) and LT (low threshold of excitability, high excitability) strains. These strains display contrasting responses to extended emotional and painful stress and exhibit unique changes in brain structures implicated in emotional regulation and stress reactivity at the molecular, cellular, and epigenetic levels [2]. The aim of this study is to examine the mRNA levels of genes encoding glucocorticoid and mineralocorticoid receptors (NR3C1, NR3C2) and brain-derived neurotrophic factor (BDNF) in the hippocampus and blood of rat strains that exhibit contrasting nervous system excitability. The study investigates interstrain variations in animals under non-stressful conditions and changes in response to prolonged exposure to emotional and painful stimuli. The experiment utilized 96 animals, with 6 animals in both the control and experimental groups at each time point. The stress model utilized was long-term emotional and painful stress as per K. Gecht [2]. The study assessed the expression of genes for glucocorticoid receptors and neurotrophic factor by isolating RNA from the hippocampus and blood of rats of two strains decapitated at 1, 7, 24, and 60 days post-stress. Gene expression analysis was conducted through real-time PCR and underwent further data processing using the ΔΔCt method. The statistical tests employed included the Kruskal–Wallis and Mann–Whitney tests. The analysis of the expression of the observed genes in intact animals demonstrates that highly excitable LT rats have a significantly lower level of expression of the nr3c2 gene, which encodes the mineralocorticoid receptor in the hippocampus, when compared to the low excitable HT strain. When studying the short-term and long-term effects of stress on the expression of the observed genes (nr3c1, nr3c2, bdnf), a decline was observed in the level of bdnf mRNA in the hippocampus of the LT strain 60 days after stress. The study suggests that the genetically determined characteristics of highly excitable animals affect the weakened adaptive abilities of their nervous system. One of the markers of this weakening is the reduced level of mRNA for the nr3c2 gene in the hippocampus under normal conditions, as well as a decrease in the expression of the bdnf gene in response to stress.

Decreased sympathetic nerve activity in young hypertensive rats reared by normotensive mothers

AimsEarly postnatal development can be significantly compromised by changes in factors provided by the mother, leading to increased vulnerability to hypertension in her offspring. TGR(mRen-2)27 (TGR) mothers, characterised by an overactivated renin-angiotensin system, exhibit altered ion composition in their breast milk. Therefore, we aimed to analyse the impact of cross-fostering on cardiovascular parameters in hypertensive TGR and normotensive Hannover Sprague-Dawley (HanSD) offspring. Materials and methodsWe measured cardiovascular parameters in 5- to 10-week-old male offspring by telemetry. The expression of proteins related to vascular function was assessed by western blotting in the aortic samples obtained from 6- to 12-week-old male offspring. Plasma renin activity and plasma angiotensin II (Ang II) levels were evaluated by radioimmunoassay (RIA). Key findingsThe development of hypertension was in TGR accompanied by increased low-to-high frequency ratio (LF/HF; a marker of sympathovagal balance; 0.51 ± 0.16 in week 10). Furthermore, TGR exhibited increased aortic expression of mineralocorticoid receptor (MR; p < 0.05) and transforming growth factor beta type 1 (TGF-β1; p = 0.002) compared to HanSD offspring. Fostering significantly decreased sympathovagal balance (0.23 ± 0.10 in week 10) and, transiently, plasma Ang II levels and MR expression in TGR offspring reared by HanSD mothers. SignificanceThese findings highlight the importance of understanding the complex interplay between early life experiences, maternal factors, and later cardiovascular function. Understanding the mechanisms behind the observed effects may help to identify potential interventions to prevent the development of hypertension later in life.

Expression of functional mineralocorticoid receptor (MR) and G-protein coupled estrogen receptor (GPER) in human T lymphocytes

Aldosterone plays a key role in controlling blood pressure (BP) values by maintaining body salt, water, and fluid homeostasis. Excess aldosterone production is associated with arterial hypertension, cardiovascular and metabolic diseases, partly via generation of an inflammatory state followed by fibrotic changes in the organs that are target of hypertension. Aldosterone exerts genomic effects that are known to involve activation of the mineralocorticoid receptor (MR). Other aldosterone effects, including those usually defined as ‘rapid’ or ‘non genomic’, involve additional receptors as the G-protein coupled estrogen receptor (GPER). To date, the receptor(s) implicated in the inflammatory action of aldosterone in cells of the innate and adaptive immunity are unknown. Considering the potential role of T-lymphocytes in adaptive immunity in arterial hypertension and related hypertension-mediated organ damage (HMOD), we herein investigated and quantified the expression of the MR and GPER in human CD4+ and CD8+ T-cells. Results provided compelling evidence for the presence at the mRNA and protein level and suggest a functional role of these receptors in the two T-lymphocyte subtypes, thus indicating that they can represent a potential target for modulation of steroid hormone-induced inflammation and ensuing HMOD.

FRI163 Central Role Of Mirnas During Kidney Development And Establishment Of The Mineralocorticoid Signaling Pathway Establishment Of The Mineralocorticoid Signaling Pathway

Abstract Disclosure: I. Hani: None. T. Vu: None. R. Brillet: None. J. Perrot: None. J. Bouligand: None. J. Guegan: None. N. Cherradi: None. P. Kamenicky: None. M. Lombes: None. L. Martinerie: None. S. Viengchareun: None. In tight epithelia, aldosterone controls sodium and water homeostasis by binding to the Mineralocorticoid Receptor (MR), a steroid nuclear receptor. We have identified a specific temporal window during renal development in which the mineralocorticoid signaling pathway is ineffective due to downregulation of MR expression, explaining physiological sodium wasting observed in human newborns during first days of life. However, underlying molecular mechanisms controlling MR expression remain unknown. Recently, we demonstrated in adult renal KC3AC1 cell line that MR expression is controlled by microRNAs (miR-30c-2-3p and miR-324-5p) in response to variations of tonicity. These regulators bind to 3'-untranslated region (3'-UTR) of MR transcripts to modulate their stability and translation. Therefore, we hypothesized that these miRNAs may be responsible for low expression level of MR at birth, where variations in tonicity are observed due to the transition from intra-amniotic to extra-uterine life. Using primary cultures of renal epithelial cells from neonatal mouse kidneys, harvested at birth (D0) and at Day 8 postnatal (D8), we showed that only miR-30c-2-3p regulates MR expression at D8. For this reason, we performed a complete transcriptomic analysis (miRNA-Seq and RNA-Seq) of kidneys collected at D0 and D8 to identify other miRNAs specifically involved in regulation of MR expression in perinatal period. Bioinformatics analysis allowed us to identify deregulated miRNAs and transcripts between D0 and D8 and to specify their involvement in biological processes and/or signaling pathways. We mainly focused on deregulated miRNAs that may modulate MR expression and affect mineralocorticoid signaling. miR-Seq identified 221 differentially expressed miRNAs. First, we selected 3 over- (miR-431-5p, miR-409-3p, and miR-92a-1-5p) and 3 under-expressed (miR-30a-5p, miR-30e-5p, and miR-802-5p) candidate miRNAs (FDR&amp;lt;0.05 and log2FC&amp;gt;1) having putative binding sites in MR 3'-UTR. Paradoxically, we showed in primary kidney cultures that overexpression of miR-30a-5p, miR-30e-5p, or miR-802-5p increased MR expression (from 50 to 100%, P&amp;lt;0.05) at D0, suggesting that these miRNAs may either positively modulate MR expression or may involve an intermediate regulator. On the opposite, over-expression of miR-92a-1-5p, miR-431-5p, and miR-409-3p at D8 led to a decrease (from 30-40%, P&amp;lt;0.05) in MR expression, suggesting that these miRNAs may modulate MR expression in postnatal period. Currently, we are evaluating whether they could be used as prognostic or severity biomarkers of water loss in neonates by quantifying these candidate miRNAs in urine exosomes of preterm and term infants. A better understanding of mechanisms regulating MR expression in the perinatal period will undoubtedly have a major impact on the management of premature infants with sodium balance maladjustment at birth. Presentation Date: Friday, June 16, 2023

FRI126 Autoreactive T Cells To Angiotensin II Type 1 Receptor In Salt-dependent Hypertension Due To Primary Aldosteronism

Abstract Disclosure: R. Scarpa: None. M. Piazza: None. B. Caroccia: None. S. Carraro: None. F. Vianello: None. F. Cinetto: None. C. Agostini: None. T.M. Seccia: None. G. Rossi: None. Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but the pathogenic role of this acquired immunity is still unknown. Primary Aldosteronism, the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary (‘essential’) hypertension and by high titer of autoantibodies against the type 1 angiotensin receptor (AT1R). As T cells are broadly recognised as HMOD-mediators, we aim to investigate: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of know aldosterone receptors on T lymphocytes. The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number by droplet digital PCR and immunoblotting from healthy donors (HD) subjects and PA patients. Peripheral blood mononuclear cells (PBMCs) from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFNɣ release with flow cytometry. PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR&amp;gt;MR&amp;gt;&amp;gt;GPER. Aldosterone significantly increased IFNɣ release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-γ production by CD8+ T cells by acting via the MR, as it was prevented (p &amp;lt; 0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p &amp;lt; 0.003), but also by G36 (p &amp;lt; 0.02), and mimicked by G1. These results showed the presence of AT1R-autoreactive T cells in patients with PA. Moreover, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs. Presentation: Friday, June 16, 2023

Crosstalk between Aldosterone and Glycation through Rac-1 Induces Diabetic Nephropathy.

Background: Advanced glycation end products (AGEs) interaction with its receptor (RAGE) and aldosterone (Aldo) through the mineralocorticoid receptor (MR) activates Rac-1 and NF-κB independently in diabetic nephropathy (DN). However, the crosstalk of Aldo with AGEs-RAGE is still unresolved. Our study examined the impact of the AGEs-Aldo complex on renal cells and its effect on the RAGE-MR interaction. Methods and results: Glycation of human serum albumin (HSA) (40 mg/mL) with methylglyoxal (10 mM) in the presence of Aldo (100 nM) and aminoguanidine (AG) (100 nM) was performed. Glycation markers such as fructosamine and carbonyl groups and fluorescence of AGEs, pentosidine, and tryptophan followed by protein modification were measured. Renal (HEK-293T) cells were treated with the glycated HSA-Aldo (200 μg/mL) along with FPS-ZM1 and spironolactone antagonists for RAGE and Aldo, respectively, for 24 h. Glycation markers and esRAGE levels were measured. Protein and mRNA levels of RAGE, MR, Rac-1, and NF-κB were estimated. Glycation markers were enhanced with Aldo when albumin was only 14-16% glycated. AGEs-Aldo complex upregulated RAGE, MR, Rac-1 and NF-κB expressions. However, FPS-ZM1 action might have activated the RAGE-independent pathway, further elevating MR, Rac-1, and NF-κB levels. Conclusion: Our study concluded that the presence of Aldo has a significant impact on glycation. In the presence of AGEs-Aldo, RAGE-MR crosstalk exerts inflammatory responses through Rac-1 in DN. Insights into this molecular interplay are crucial for developing novel therapeutic strategies to alleviate DN in the future.

Sex-specific behavioral and physiological changes during single parenting in a biparental species, Columba livia

Many species exhibit biparental care to maximize fitness. When a partner is lost, the surviving partner may alter their behavior to compensate offspring. Whether both sexes use the same physiological mechanisms to manifest their change in behavior remains elusive. We investigated behaviors and mechanisms associated with the alteration of parental care post-partner removal in a biparental avian species, the rock dove (Columba livia). We hypothesized that rock dove single parents experience sex-biased changes in neural genomic transcription and reproductive behaviors, and these changes are related to chick development. We manipulated parental partner presence and measured parental attendance, offspring growth, gene expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) in the pituitary, and GR, MR, and estrogen receptor beta (ER-β) in the hypothalamus. We also measured circulating plasma concentrations of the stress-associated hormone corticosterone and the parental care-associated hormone prolactin. We also quantified prolactin gene (PRL) expression changes in the pituitary, as well as prolactin receptor (PRLR) expression in the hypothalamus and pituitary. We found that single mothers and fathers maintained similar provisioning levels as paired parents, but spent less cumulative time brooding chicks. Chicks of single parents were smaller than paired-parented chicks after three days post-hatch. Mothers in both treatment groups experienced higher expression of hypothalamic GR as compared to fathers. Single parents experienced lower PRL gene expression in the pituitary as compared to paired parents. No significant differences were found for the circulating hormones or other genes listed.

Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males.

AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na+ channel) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-hypertension. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na+ reabsorption and BP increase in female rats with AngII-hypertension. We employ a combination of physiological, pharmacological, biochemical, and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats. MR blockade markedly attenuates AngII-hypertension in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and HSD11β2 (11β-hydroxysteroid dehydrogenase type 2) that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is ≈2× lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2, targeting ENaC for degradation. MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-hypertension, but not in males.

Negative feedback regulation in the hypothalamic-pituitary-interrenal axis of rainbow trout subjected to chronic social stress

The formation of dominance hierarchies in pairs of juvenile rainbow trout (Oncorhynchus mykiss) results in subordinate individuals exhibiting chronically elevated plasma cortisol concentrations. Cortisol levels reflect a balance between cortisol production, which is coordinated by the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish, and negative feedback regulation and hormone clearance, which act to lower cortisol levels. However, the mechanisms contributing to the longer-term elevation of cortisol levels during chronic stress are not well established in fishes. The current study aimed to determine how subordinate fish maintain elevated cortisol levels, by testing the prediction that negative feedback and clearance mechanisms are impaired by chronic social stress. Plasma cortisol clearance was unchanged by social stress based on a cortisol challenge trial, hepatic abundance of the cortisol-inactivating enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11βHSD2), and tissue fate of labelled cortisol. The capacity for negative feedback regulation in terms of transcript and protein abundances of corticosteroid receptors in the preoptic area (POA) and pituitary appeared stable. However, changes in 11βHSD2 and mineralocorticoid receptor (MR) expression suggest subtle regulatory changes in the pituitary that may alter negative feedback. The chronic cortisol elevation observed during social subordination likely is driven by HPI axis activation and compounded by dysregulated negative feedback.

AUTOREACTIVE T CELLS TO ANGIOTENSIN II TYPE 1 RECEPTOR IN SALT-DEPENDENT HYPERTENSION DUE TO PRIMARY ALDOSTERONISM

Objective: Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but its pathogenic role is still unknown. Primary Aldosteronism (PA), the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary hypertension and by high titer of autoantibodies against the angiotensin type-1 receptor (AT1R). As T cells are broadly recognised as HMOD-mediators we investigated: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of aldosterone receptors on T lymphocytes. Design and Methods: The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number and immunoblotting from healthy donors and PA patients. PBMCs from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFN? release with flow cytometry. Results: PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR&gt;MR&gt;&gt;GPER. Aldosterone significantly increased IFN? release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-? production by CD8+ T cells by acting via the MR, as it was prevented (p&lt;0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p&lt;0.003), but also by G36 (p&lt;0.02), and mimicked by G1. Conclusion: These results showed the presence of AT1R-autoreactive T cells in patients with PA and demonstrates that, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs.

Neutral Effect of Skeletal Muscle Mineralocorticoid Receptor on Glucose Metabolism in Mice.

The mineralocorticoid receptor (MR) is able to regulate the transcription of a number of genes in the myotube, although its roles in skeletal muscle (SM) metabolism still await demonstration. SM represents a major site for glucose uptake, and its metabolic derangements play a pivotal role in the development of insulin resistance (IR). The aim of this study was to investigate the contribution of SM MR in mediating derangements of glucose metabolism in a mouse model of diet-induced obesity. We observed that mice fed a high-fat diet (HFD mice) showed impaired glucose tolerance compared to mice fed a normal diet (ND mice). Mice fed a 60% HFD treated with the MR antagonist Spironolactone (HFD + Spiro) for 12 weeks revealed an improvement in glucose tolerance, as measured with an intraperitoneal glucose tolerance test, compared with HFD mice. To investigate if blockade of SM MR could contribute to the favorable metabolic effects observed with pharmacological MR antagonism, we analyzed MR expression in the gastrocnemius, showing that SM MR protein abundance is downregulated by HFD compared to ND mice and that pharmacological treatment with Spiro was able to partially revert this effect in HFD + Spiro mice. Differently from what we have observed in adipose tissue, where HDF increased adipocyte MR expression, SM MR protein was down-regulated in our experimental model, suggesting a completely different role of SM MR in the regulation of glucose metabolism. To confirm this hypothesis, we investigated the effects of MR blockade on insulin signaling in a cellular model of IRin C2C12 myocytes, which were treated with or without Spiro. We confirmed MR protein downregulation in insulin-resistant myotubes. We also analyzed Akt phosphorylation upon insulin stimulation, and we did not observe any difference between palmitate- and palmitate + Spiro-treated cells. These results were confirmed by in vitro glucose uptake analysis. Taken together, our data indicate that reduced activity of SM MR does not improve insulin signaling in mouse skeletal myocytes and does not contribute to the favorable metabolic effects on glucose tolerance and IR induced by systemic pharmacological MR blockade.