Increased adrenal steroidogenesis and suppressed corticosteroid responsiveness in critical COVID-19.

BACKGROUND: The effect of coronavirus disease 2019 (COVID-19) on adrenal endocrine metabolism in critically ill patients remains unclear. This study aimed to investigate the alterations in adrenal steroidogenic activity, elucidate underlying mechanisms, provide in situ histopathological evidence, and examine the clinical implications. METHODS: The comparative analyses of the adrenal cortices from 24 patients with fatal COVID-19 and 20 matched controls were performed, excluding patients previously treated with glucocorticoids. SARS-CoV-2 and its receptors were identified and pathological alterations were examined. Furthermore, histological examinations, immunohistochemical staining and ultrastructural analyses were performed to assess corticosteroid biosynthesis. The zona glomerulosa (ZG) and zona fasciculata (ZF) were then dissected for proteomic analyses. The biological processes that affected steroidogenesis were analyzed by integrating histological, proteomic, and clinical data. Finally, the immunoreactivity and responsive genes of mineralocorticoid and glucocorticoid receptors in essential tissues were quantitatively measured to evaluate corticosteroid responsiveness. FINDINGS: The demographic characteristics of COVID-19 patients were comparable with those of controls. SARS-CoV-2-like particles were identified in the adrenocortical cells of three patients; however, these particles did not affect cellular morphology or steroid synthesis compared with SARS-CoV-2-negative specimens. Although the adrenals exhibited focal necrosis, vacuolization, microthrombi, and inflammation, widespread degeneration was not evident. Notably, corticosteroid biosynthesis was significantly enhanced in both the ZG and ZF of COVID-19 patients. The increase in the inflammatory response and cellular differentiation in the adrenal cortices of patients with critical COVID-19 was positively correlated with heightened steroidogenic activity. Additionally, the appearance of more dual-ZG/ZF identity cells in COVID-19 adrenals was in accordance with the increased steroidogenic function. However, activated mineralocorticoid and glucocorticoid receptors and their responsive genes in vital tissues were markedly reduced in patients with critical COVID-19. INTERPRETATION: Critical COVID-19 was characterized by potentiated adrenal steroidogenesis, associated with increased inflammation, enhanced differentiation and elevated dual-ZG/ZF identity cells, alongside suppressed corticosteroid responsiveness. These alterations implied the reduced effectiveness of conventional corticosteroid therapy and underscored the need for evaluation of the adrenal axis and corticosteroid sensitivity.

Biological sex modifies aldosterone's secretion at a cellular level.

Inconsistencies have been reported on the effect of sex on aldosterone (ALDO) levels leading to clinical confusion. The reasons for these inconsistencies are uncertain but include estrogen and/or its receptor modulating target gene responses to mineralocorticoid receptor activation and ALDO secretagogues' levels. This study's goal was to determine whether ALDO's biosynthesis also differed by sex. Two approaches were used. First, plasma renin activity and aldosterone were measured in rats. Both were significantly higher in males. Secondly, using rat zona glomerulosa (ZG) cells, we assessed three ex vivo areas: (1) activity/levels of early steps in ALDO's biosynthesis (StAR and CYP11A1); (2) activity/levels of a late step (CYP11B2); and (3) the status of the mineralocorticoid receptor (MR)-mediated, ultrashort feedback loop. Females had higher expression of CYP11A1 and StAR and increased CYP11A1 activity (increased pregnenolone/corticosterone levels) but did not differ in CYP11B2 expression or activity (ALDO levels). Activating the ZG's MR (thereby activating the ultrashort feedback loop) reduced CYP11B2's activity similarly in both sexes. Exvivo, these molecular effects were accompanied, in females, by lower ALDO basally but higher ALDO with angiotensin II stimulation. In conclusion, we documented that not only was there a sex-mediated difference in the activity of ALDO's biosynthesis but also these differences at the molecular level help explain the variable reports on ALDO's circulating levels. Basally, both in vivo and ex vivo, males had higher ALDO levels, likely secondary to higher ALDO secretagogue levels. However, in response to acute stimulation, ALDO levels are higher in females because of the greater levels and/or activity of their StAR/CYP11A1.

Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

Ultrastructural Mechanisms of Impaired Aldosterone Synthesis in Rats Exposed to DDT during Prenatal and Postnatal Development.

The study is aimed at elucidation of ultrastructural mechanisms underlying impaired aldosterone synthesis by glomerulosa cells in Wistar rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal development. Analysis of rat zona glomerulosa histology and function during the pubertal and postpubertal periods showed that exposure to endocrine disrupter DDT disturbs its development and reduced the production of aldosterone. Electron microscopy showed that changes in the aldosterone synthesis are related to impaired reorganization of the mitochondrial apparatus, one of the leading factors in the regulation of steroidogenesis, in glomerulosa cells in DDT-exposed rats during puberty.

Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.

Long-term triphenyltin exposure disrupts adrenal function in adult male rats.

Triphenyltin is an organotin, which is widely used as a fungicide in agriculture. Here, we reported the effects of triphenyltin on adrenal function in adult male rats. Adult male Sprague Dawley rats were daily gavaged with triphenyltin (0, 0.5, 1, and 2 mg/kg body weight) from postnatal day 56-86. Triphenyltin significantly decreased serum corticosterone levels at 1 and 2 mg/kg without affecting serum levels of aldosterone and adrenocorticotropic hormone. Triphenyltin increased thickness of zona glomerulosa without affecting that of zona fasciculata. Triphenyltin did not affect cell number in zona fasciculata and zona glomerulosa. Triphenyltin down-regulated the expression of Scarb1, Star, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, and Hsd11b1 at 1 and/or 2 mg/kg while it up-regulated the expression of At1, Nr4a2, and Hsd11b2 at 2 mg/kg. Triphenyltin activated the phosphorylation of AMPKα while suppressed the phosphorylation of AKT1 and SIRT1/PGC-1α in rat adrenals in vivo and H295R cells in vitro. In vitro, triphenyltin also induced ROS production in H295R cells at 100 nM, a concentration at which no apoptosis was induced. In conclusion, triphenyltin disrupts glucocorticoid synthesis in rat adrenal cortex via several mechanisms: 1) lowering AKT1 phosphorylation and SIRT1/PGC-1α levels; 2) activating AMPKα; and 3) possibly inducing ROS production.

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Changes in Canonical ß-Catenin/Wnt Signaling Activation in the Adrenal Cortex of Rats Exposed to Endocrine Disruptor Dichlorodiphenyltrichloroethane (DDT) during Prenatal and Postnatal Ontogeny.

Prenatal and postnatal exposure to low doses of the endocrine disruptor dichlorodiphenyltrichloroethane (DDT) leads to delayed activation of the canonical ß-catenin/Wnt signaling in zona glomerulosa and zona reticularis of the adrenal cortex in rats, which changed the rate of their postnatal development. Suppression of the Wnt pathway in zona fasciculata promotes its regeneration after DDT-induced blood circulation disorders and cell death.

The angiotensin type 2 receptor in the human adrenocortical zona glomerulosa and in aldosterone-producing adenoma: low expression and no functional role.

The angiotensin II (Ang II) type 2 receptor (AT2R) and the angiotensin-(1-7) (Ang-(1-7)) receptor (MasR) play a cardiovascular protective role by counter-regulating Ang II type 1 receptor (AT1R)-mediated effects, but whether this involves blunting of adrenocortical hormone secretion is unknown. We investigated the presence of AT1R, AT2R, and MasR in aldosterone-producing adenoma (APA), a condition featuring hyperaldosteronism, and in APA-adjacent tissue. The effect of Compound 21 (C21), an AT2R agonist, on CYP11B1 (cortisol synthase) and CYP11B2 (aldosterone synthase) gene expression in NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, was also assessed using the AT1R antagonist irbesartan to ascertain the specificity of C21 effect. We found that the AT1R, AT2R, and MasR were expressed in APA and APA-adjacent tissue, albeit heterogeneously. The gene expression of AT1R and AT2R was lower, and that of the MasR higher in APAs than in APA-adjacent tissue. In steroid-producing NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, C21 was ineffective at nanomolar concentrations, but increased CYP11B1 and CYP11B2 gene expression at micromolar concentrations through AT1R, as this effect was blunted by irbesartan. The scant expression of the AT2R, along with the lack of any effect of C21 at low concentrations on CYP11B2, do not support the contention that the protective arm of renin-angiotensin system (RAS) blunts aldosterone synthase in the normal adrenal cortex and primary aldosteronism.

Targeted disruption of the Kcnj5 gene in the female mouse lowers aldosterone levels.

Aldosterone is released from adrenal zona glomerulosa (ZG) cells and plays an important role in Na and K homoeostasis. Mutations in the human inwardly rectifying K channel CNJ type (KCNJ) 5 (KCNJ5) gene encoding the G-coupled inwardly rectifying K channel 4 (GIRK4) cause abnormal aldosterone secretion and hypertension. To better understand the role of wild-type (WT) GIRK4 in regulating aldosterone release, we have looked at aldosterone secretion in a Kcnj5 knockout (KO) mouse. We found that female but not male KO mice have reduced aldosterone levels compared with WT female controls, but higher levels of aldosterone after angiotensin II (Ang-II) stimulation. These differences could not be explained by sex differences in aldosterone synthase (Cyp11B2) gene expression in the mouse adrenal. Using RNAseq analysis to compare WT and KO adrenals, we showed that females also have a much larger set of differentially expressed adrenal genes than males (395 compared with 7). Ingenuity Pathway Analysis (IPA) of this gene set suggested that peroxisome proliferator activated receptor (PPAR) nuclear receptors regulated aldosterone production and altered signalling in the female KO mouse, which could explain the reduced aldosterone secretion. We tested this hypothesis in H295R adrenal cells and showed that the selective PPARα agonist fenofibrate can stimulate aldosterone production and induce Cyp11b2. Dosing mice in vivo produced similar results. Together our data show that Kcnj5 is important for baseline aldosterone secretion, but its importance is sex-limited at least in the mouse. It also highlights a novel regulatory pathway for aldosterone secretion through PPARα that may have translational potential in human hyperaldosteronism.

Sonic Hedgehog and WNT Signaling Promote Adrenal Gland Regeneration in Male Mice.

The atrophy and hypofunction of the adrenal cortex following long-term pharmacologic glucocorticoid therapy is a major health problem necessitating chronic glucocorticoid replacement that often prolongs the ultimate return of endogenous adrenocortical function. Underlying this functional recovery is anatomic regeneration, the cellular and molecular mechanisms of which are poorly understood. Investigating the lineage contribution of cortical Sonic hedgehog (Shh)+ progenitor cells and the SHH-responsive capsular Gli1+ cells to the regenerating adrenal cortex, we observed a spatially and temporally bimodal contribution of both cell types to adrenocortical regeneration following cessation of glucocorticoid treatment. First, an early repopulation of the cortex is defined by a marked delamination and expansion of capsular Gli1+ cells, recapitulating the establishment of the capsular-cortical homeostatic niche during embryonic development. This rapid repopulation is promptly cleared from the cortical compartment only to be supplanted by repopulating cortical cells derived from the resident long-term-retained zona glomerulosa Shh+ progenitors. Pharmacologic and genetic dissection of SHH signaling further defines an SHH-dependent activation of WNT signaling that supports regeneration of the cortex following long-term glucocorticoid therapy. We define the signaling and lineage relationships that underlie the regeneration process.

Obligatory Metabolism of Angiotensin II to Angiotensin III for Zona Glomerulosa Cell-Mediated Relaxations of Bovine Adrenal Cortical Arteries.

Hyperaldosteronism is associated with hypertension, cardiac hypertrophy, and congestive heart failure. Steroidogenic factors facilitate aldosterone secretion by increasing adrenal blood flow. Angiotensin (Ang) II decreases adrenal vascular tone through release of zona glomerulosa (ZG) cell-derived vasodilatory eicosanoids. However, ZG cell-mediated relaxation of bovine adrenal cortical arteries to Ang II is not altered by angiotensin type 1 or 2 receptor antagonists. Because traditional Ang II receptors do not mediate these vasorelaxations to Ang II, we investigated the role of Ang II metabolites. Ang III was identified by liquid chromatography-mass spectrometry as the primary ZG cell metabolite of Ang II. Ang III stimulated ZG cell-mediated relaxation of adrenal arteries with greater potency than did Ang II. Furthermore, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by aminopeptidase inhibition, and Ang III-stimulated relaxations persisted. Ang IV had little effect compared with Ang II. Moreover, ZG cell-mediated relaxations of adrenal arteries by Ang II were attenuated by an Ang III antagonist but not by an Ang (1-7) antagonist. In contrast, Ang II and Ang III were equipotent in stimulating aldosterone secretion from ZG cells and were unaffected by aminopeptidase inhibition. Additionally, aspartyl and leucyl aminopeptidases, which convert Ang II to Ang III, are the primary peptidase expressed in ZG cells. This was confirmed by enzyme activity. These data indicate that intra-adrenal metabolism of Ang II to Ang III is required for ZG cell-mediated relaxations of adrenal arteries but not aldosterone secretion. These studies have defined an important role of Ang III in the adrenal gland.

Effects of acrolein on aldosterone release from zona glomerulosa cells in male rats.

A positive correlation between smoking and hypertension has been well established. Acrolein is a major toxic volatile compound found in cigarette smoke. Human exposure to low levels of acrolein is unavoidable due to its production in daily activities, such as smoke from industrial, hot oil cooking vapors, and exhaust fumes from vehicles. The toxicity and the action mechanism of acrolein to induce apoptosis have been extensively studied, but the effects of acrolein on hypertension are still unknown. The present study aimed to examine the effects of acrolein on aldosterone release both in vivo and in vitro. Male rats were divided into three groups, and intraperitoneally injected with normal saline, or acrolein (2mg/kg) for 1 (group A-1) or 3 (group A-3) days, respectively. After sacrificing, rat blood samples were obtained to measure plasma aldosterone and angiotensin II (Ang II) levels. Zona glomerulosa (ZG) cells were prepared from rat adrenal cortex, and were incubated with or without stimulants. We found that the serum aldosterone was increased by 1.2-fold (p<0.05) in A-3 group as compared to control group. Basal aldosterone release from ZG cells in A-3 group was also increased significantly. Moreover, acrolein enhanced the stimulatory effects of Ang II and 8-bromo-cyclic AMP on aldosterone secretion from ZG cells prepared in both A-1 and A-3 groups. Furthermore, the enzyme activity of P450scc, the rate-limiting step of aldosterone synthesis, was elevated after acrolein injection. Plasma level of Ang II was increased in both A-1 and A-3 groups. These results suggested that acrolein exposure increased aldosterone production, at least in part, through elevating the level of plasma Ang II and stimulating steroidogenesis pathways.

Angiotensin II up-regulates monocarboxylate transporters expression in the rat adrenal gland.

Angiotensin II (Ang II) is a major regulator of aldosterone secretion in the adrenal zona glomerulosa because it up-regulates the expression of a large number of genes involved in aldosterone biosynthesis. The transport of acetate across adrenocortical cells is a crucial step in the de novo synthesis of cholesterol, the steroid precursor of aldosterone. However, whether Ang II can affect this transport remains unknown. The current study aims to investigate the effect of in vivo infusion of Ang II on monocarboxylate transporters (MCT1, MCT2, and MCT4) gene expression in the rat adrenal gland. Immunohistochemical analysis and real-time PCR were used to examine the expression of MCTs at the protein and mRNA levels, respectively. The immunohistochemical analysis showed that higher numbers of cells expressed MCT1, MCT2, and MCT4 proteins in the zona glomerulosa and zona fasiculata of the adrenal cortex of Ang II-infused rats. Furthermore, real-time PCR indicated that in vivo infusion of Ang II increased the mRNA levels of MCT1, MCT2, and MCT4 in the rat adrenal gland. MCT up-regulation might maximize the intracellular transport of acetate in response to the stimulatory effect of Ang II on aldosterone secretion by the adrenal zona glomerulosa..

Stimulus-selective induction of the orphan nuclear receptor NGFIB underlies different influences of angiotensin II and potassium on the human adrenal gland zona glomerulosa-specific 3ß-HSD isoform gene expression in adrenocortical H295R cells.

In the adrenal, the type I 3ß-hydroxysteroid dehydrogenase (HSD3B1) is expressed exclusively in the zona glomerulosa (ZG), where aldosterone is produced. Angiotensin II (AngII) and potassium (K(+)) are the major physiological regulators of aldosterone synthesis. However, their respective roles in regulation of aldosterone synthesis are not fully defined, particularly in terms of transcriptional regulation of steroidogenic enzyme genes. We previously showed that AngII can stimulate expression of HSD3B1. But, K(+) responsiveness of this gene has remained unexplored. Here, we report that K(+) stimulation lacks the ability to induce HSD3B1 expression in human adrenocortical H295R cells. Both AngII and K(+) were able to enhance transcription of the aldosterone synthase gene (CYP11B2). Promoter analysis revealed that although both AngII and K(+) activate transcription from the Ca(2+)/cAMP-responsive element (CRE) located in the CYP11B2 promoter, the orphan nuclear receptor NGFIB-responsive element (NBRE) located in the HSD3B1 promoter fails to respond to K(+), being only able to enhance transcription after AngII treatment. We found that induction of de novo protein synthesis of NGFIB occurs only after AngII treatment. This sharply contrasts with the phosphorylation that occurs in response to both AngII and K(+) on the CREB/ATF family transcription factor ATF2. Chromatin immunoprecipitation assay confirmed that the NGFIB protein occupies the HSD3B1 promoter only after AngII, while ATF2 binds to the CYP11B2 promoter in response to both AngII and K(+). These data provide evidence that downstream signals from AngII and K(+) can be uncoupled in the regulation of HSD3B1 in the human adrenocortical H295R cells.

Effect of swimming on the production of aldosterone in rats.

It has been demonstrated that exercise is one of the stresses known to increase the aldosterone secretion. Both potassium and angiotensin II (Ang II) levels are shown to be correlated with aldosterone production during exercise, but the mechanism is still unclear. In an in vivo study, male rats were catheterized via right jugular vein (RJV), and divided into four groups namely water immersion, swimming, lactate infusion (13 mg/kg/min) and pyruvate infusion (13 mg/kg/min) groups. Each group was treated for 10 min. Blood samples were collected at 0, 10, 15, 30, 60 and 120 min from RJV after administration. In an in vitro study, rat zona glomerulosa (ZG) cells were challenged by lactate (1-10 mM) in the presence or absence of Ang II (10(-8) M) for 60 min. The levels of aldosterone in plasma and medium were measured by radioimmunoassay. Cell lysates were analyzed by immunoblotting assay. After exercise and lactate infusion, plasma levels of aldosterone and lactate were significantly higher than those in the control group. Swimming for 10 min significantly increased the plasma Ang II levels in male rats. Administration of lactate plus Ang II significantly increased aldosterone production and enhanced protein expression of steroidogenic acute regulatory protein (StAR) in ZG cells. These results demonstrated that acute exercise led to the increase of both aldosterone and Ang II secretion, which is associated with lactate action on ZG cells and might be dependent on the activity of renin-angiotensin system.

Angiotensin II-induced protein kinase D activates the ATF/CREB family of transcription factors and promotes StAR mRNA expression.

Aldosterone synthesis is initiated upon the transport of cholesterol from the outer to the inner mitochondrial membrane, where the cholesterol is hydrolyzed to pregnenolone. This process is the rate-limiting step in acute aldosterone production and is mediated by the steroidogenic acute regulatory (StAR) protein. We have previously shown that angiotensin II (AngII) activation of the serine/threonine protein kinase D (PKD) promotes acute aldosterone production in bovine adrenal glomerulosa cells, but the mechanism remains unclear. Thus, the purpose of this study was to determine the downstream signaling effectors of AngII-stimulated PKD activity. Our results demonstrate that overexpression of the constitutively active serine-to-glutamate PKD mutant enhances, whereas the dominant-negative serine-to-alanine PKD mutant inhibits, AngII-induced StAR mRNA expression relative to the vector control. PKD has been shown to phosphorylate members of the activating transcription factor (ATF)/cAMP response element binding protein (CREB) family of leucine zipper transcription factors, which have been shown previously to bind the StAR proximal promoter and induce StAR mRNA expression. In primary glomerulosa cells, AngII induces ATF-2 and CREB phosphorylation in a time-dependent manner. Furthermore, overexpression of the constitutively active PKD mutant enhances the AngII-elicited phosphorylation of ATF-2 and CREB, and the dominant-negative mutant inhibits this response. Furthermore, the constitutively active PKD mutant increases the binding of phosphorylated CREB to the StAR promoter. Thus, these data provide insight into the previously reported role of PKD in AngII-induced acute aldosterone production, providing a mechanism by which PKD may be mediating steroidogenesis in primary bovine adrenal glomerulosa cells.

Chronic hyperaldosteronism in cryptochrome-null mice induces high-salt- and blood pressure-independent kidney damage in mice.

Although aldosterone has an essential role in controlling electrolyte and body fluid homeostasis, aldosterone also exerts certain pathological effects on the kidney. Several previous studies have attempted to examine these deleterious effects. However, the majority of these studies were performed using various injury models, including high-salt treatment and/or mineralocorticoid administration, by which the kidney changes observed were not only due to aldosterone but also due to prior injury caused by salt and hypertension. In the present study, we investigated aldosterone's pathological effect on the kidney using a mouse model with a high level of endogenous aldosterone. We used cryptochrome-null (Cry 1, 2 DKO) mice characterized by high aldosterone levels and low plasma renin activity and observed that even under normal salt exposure conditions, these mice showed increased albumin excretion and kidney tubular injury, decreased nephrin expression and increased reactive oxygen species production in the absence of hypertension. Exposure to high salt levels exacerbated the kidney damage observed in these mice. Moreover, we noted that decreasing blood pressure without blocking aldosterone action did not provide beneficial effects to the kidney in high-salt-treated Cry 1, 2 DKO mice. Thus, our findings support the hypothesis that aldosterone has deleterious effects on the kidney independent of high-salt exposure and high blood pressure.

Regulation of aldosterone biosynthesis by the Kir3.4 (KCNJ5) potassium channel.

The G-protein-activated inwardly rectifying potassium channel Kir3.4 is expressed in the zona glomerulosa cell membrane and transports potassium out of the cell. Angiotensin II stimulation of aldosterone secretion is mediated, in part, by suppression of the transcription of KCNJ5, the gene coding for Kir3.4, and blocking channel activity. This results in membrane depolarization, mobilization of intracellular calcium, activation of the calcium-calmodulin pathway and increasing gene transcription of steroidogenic enzymes required for aldosterone secretion. In 40-60% of aldosterone-producing adenomas there is a somatic mutation in the region of the KCNJ5 gene that codes for the selectivity filter that decreases potassium selectivity, allowing sodium to leak into the cells, thus depolarizing the membrane and initiating events that result in increased aldosterone synthesis. The mechanism by which mutated KCNJ5 induces cell proliferation and adenoma formation remains unclear.

Pharmacological characterization of FE 201874, the first selective high affinity rat V1A vasopressin receptor agonist.

BACKGROUND AND PURPOSE: Distinct vasopressin receptors are involved in different physiological and behavioural functions. Presently, no selective agonist is available to specifically elucidate the functional roles of the V1A receptor in the rat, one of the most widely used animal models. FE 201874 is a new derivative of the human selective V1A receptor agonist F180. In this study, we performed a multi-approach pharmacological and functional characterization of FE 201874 to determine whether it is selective for V1A receptors. EXPERIMENTAL APPROACH: We modified an available human selective V1A receptor agonist (F180) and determined its pharmacological properties in cell lines expressing vasopressin/oxytocin receptors (affinity and coupling to second messenger cascades), in an ex vivo model (aorta ring contraction) and in vivo in rats (proliferation of adrenal cortex glomerulosa cells and lactation). KEY RESULTS: FE 201874 exhibited nanomolar affinity for the rat V1A receptor; it was highly selective towards the rat V1B and V2 vasopressin receptors and behaved as a full V1A agonist in all the pharmacological tests performed. FE 201874 bound to the oxytocin receptor, but with moderate affinity, and behaved as an oxytocin antagonist in vitro, but not in vivo. CONCLUSIONS AND IMPLICATIONS: On functional grounds, all the data demonstrate that FE 201874 is the first selective agonist of the rat V1A receptor isoform available. Hence, FE 201874 may have potential as a treatment for the vasodilator-induced hypotension occurring in conditions such as septic shock and could be the most suitable compound for discriminating between the behavioural effects of arginine vasopressin and oxytocin.