The Renal Physiology of Pendrin-Positive Intercalated Cells.

Intercalated cells (ICs) are found in the connecting tubule and the collecting duct. Of the three IC subtypes identified, type B intercalated cells are one of the best characterized and known to mediate Cl- absorption and HCO3- secretion, largely through the anion exchanger pendrin. This exchanger is thought to act in tandem with the Na+-dependent Cl-/HCO3- exchanger, NDCBE, to mediate net NaCl absorption. Pendrin is stimulated by angiotensin II and aldosterone administration via the angiotensin type 1a and the mineralocorticoid receptors, respectively. It is also stimulated in models of metabolic alkalosis, such as with NaHCO3 administration. In some rodent models, pendrin-mediated HCO3- secretion modulates acid-base balance. However, of probably more physiological or clinical significance is the role of these pendrin-positive ICs in blood pressure regulation, which occurs, at least in part, through pendrin-mediated renal Cl- absorption, as well as their effect on the epithelial Na+ channel, ENaC. Aldosterone stimulates ENaC directly through principal cell mineralocorticoid hormone receptor (ligand) binding and also indirectly through its effect on pendrin expression and function. In so doing, pendrin contributes to the aldosterone pressor response. Pendrin may also modulate blood pressure in part through its action in the adrenal medulla, where it modulates the release of catecholamines, or through an indirect effect on vascular contractile force. In addition to its role in Na+ and Cl- balance, pendrin affects the balance of other ions, such as K+ and I-. This review describes how aldosterone and angiotensin II-induced signaling regulate pendrin and the contribution of pendrin-positive ICs in the kidney to distal nephron function and blood pressure.

Toll-like receptor 4-dependent innate immune responses are mediated by intracrine corticosteroids and activation of glycogen synthase kinase-3ß in astrocytes.

Reactive astrocytes are important pathophysiologically and synthesize neurosteroids. We observed that LPS increased immunoreactive TLR4 and key steroidogenic enzymes in cortical astrocytes of rats and investigated whether corticosteroids are produced and mediate astrocytic TLR4-dependent innate immune responses. We found that LPS increased steroidogenic acute regulatory protein (StAR) and StAR-dependent aldosterone production in purified astrocytes. Both increases were blocked by the TLR4 antagonist TAK242. LPS also increased 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and corticosterone production, and both were prevented by TAK242 and by siRNAs against 11ß-HSD1, StAR, or aldosterone synthase (CYP11B2). Knockdown of 11ß-HSD1, StAR, or CYP11B2 or blocking either mineralocorticoid receptors (MR) or glucocorticoid receptors (GR) prevented dephosphorylation of p-Ser9GSK-3ß, activation of NF-κB, and the GSK-3ß-dependent increases of C3, IL-1ß, and TNF-α caused by LPS. Exogenous aldosterone mimicked the MR- and GSK-3ß-dependent pro-inflammatory effects of LPS in astrocytes, but corticosterone did not. Supernatants from astrocytes treated with LPS reduced MAP2 and viability of cultured neurons except when astrocytic StAR or MR was inhibited. In adrenalectomized rats, intracerebroventricular injection of LPS increased astrocytic TLR4, StAR, CYP11B2, and 11ß-HSD1, NF-κB, C3 and IL-1ß, decreased astrocytic p-Ser9GSK-3ß in the cortex and was neurotoxic, except when spironolactone was co-injected, consistent with the in vitro results. LPS also activated NF-κB in some NeuN+ and CD11b+ cells in the cortex, and these effects were prevented by spironolactone. We conclude that intracrine aldosterone may be involved in the TLR4-dependent innate immune responses of astrocytes and can trigger paracrine effects by activating astrocytic MR/GSK-3ß/NF-κB signaling.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC ß- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Aldosterone and aldosterone synthase inhibitors in cardiorenal disease.

Modulation of the renin-angiotensin-aldosterone system is a foundation of therapy for cardiovascular and kidney diseases. Excess aldosterone plays an important role in cardiovascular disease, contributing to inflammation, fibrosis, and dysfunction in the heart, kidneys, and vasculature through both genomic and mineralocorticoid receptor (MR)-mediated as well as nongenomic mechanisms. MR antagonists have been a key therapy for attenuating the pathologic effects of aldosterone but are associated with some side effects and may not always adequately attenuate the nongenomic effects of aldosterone. Aldosterone is primarily synthesized by the CYP11B2 aldosterone synthase enzyme, which is very similar in structure to other enzymes involved in steroid biosynthesis including CYP11B1, a key enzyme involved in glucocorticoid production. Lack of specificity for CYP11B2, off-target effects on the hypothalamic-pituitary-adrenal axis, and counterproductive increased levels of bioactive steroid intermediates such as 11-deoxycorticosterone have posed challenges in the development of early aldosterone synthase inhibitors such as osilodrostat. In early-phase clinical trials, newer aldosterone synthase inhibitors demonstrated promise in lowering blood pressure in patients with treatment-resistant and uncontrolled hypertension. It is therefore plausible that these agents offer protection in other disease states including heart failure or chronic kidney disease. Further clinical evaluation will be needed to clarify the role of aldosterone synthase inhibitors, a promising class of agents that represent a potentially major therapeutic advance.

Dietary Na+ depletion up-regulates NKCC1 expression and enhances electrogenic Cl- secretion in rat proximal colon.

The corticosteroid hormone, aldosterone, markedly enhances K+ secretion throughout the colon, a mechanism critical to its role in maintaining overall K+ balance. Previous studies demonstrated that basolateral NKCC1 was up-regulated by aldosterone in the distal colon specifically to support K+ secretion-which is distinct from the more well-established role of NKCC1 in supporting luminal Cl- secretion. However, considerable segmental variability exists between proximal and distal colonic ion transport processes, especially concerning their regulation by aldosterone. Furthermore, delineating such region-specific effects has important implications for the management of various gastrointestinal pathologies. Experiments were therefore designed to determine whether aldosterone similarly up-regulates NKCC1 in the proximal colon to support K+ secretion. Using dietary Na+ depletion as a model of secondary hyperaldosteronism in rats, we found that proximal colon NKCC1 expression was indeed enhanced in Na+-depleted (i.e., hyperaldosteronemic) rats. Surprisingly, electrogenic K+ secretion was not detectable by short-circuit current (ISC) measurements in response to either basolateral bumetanide (NKCC1 inhibitor) or luminal Ba2+ (non-selective K+ channel blocker), despite enhanced K+ secretion in Na+-depleted rats, as measured by 86Rb+ fluxes. Expression of BK and IK channels was also found to be unaltered by dietary Na+ depletion. However, bumetanide-sensitive basal and agonist-stimulated Cl- secretion (ISC) were significantly enhanced by Na+ depletion, as was CFTR Cl- channel expression. These data suggest that NKCC1-dependent secretory pathways are differentially regulated by aldosterone in proximal and distal colon. Development of therapeutic strategies in treating pathologies related to aberrant colonic K+/Cl- transport-such as pseudo-obstruction or ulcerative colitis-may benefit from these findings.

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.

Bicarbonate is the primary inducer of KCC3a expression in renal cortical B-type intercalated cells.

A primary function of intercalated cells in the distal tubule of the kidney is to maintain pH homeostasis. For example, type B intercalated cells secrete bicarbonate largely through the action of the apical Cl-/HCO3- exchanger, pendrin, which helps correct metabolic alkalosis. Since both the K-Cl cotransporter, KCC3a and pendrin colocalize to the apical region of type B and non-A, non-B intercalated cells and since both are upregulated in models of metabolic alkalosis, such as with dietary NaHCO3 loading, we raised the possibility that apical KCC3a facilitates pendrin-mediated bicarbonate secretion, such as through apical Cl- recycling. The purpose of this study was to determine if KCC3a abundance changes through intake of bicarbonate alone or through bicarbonate plus its accompanying cation, and if it requires a direct interaction with pendrin or the renin-angiotensin-aldosterone system. We observed that KCC3a protein abundance, but not mRNA, increases in a mouse model of metabolic alkalosis, achieved with dietary NaHCO3 or KHCO3 intake. Bicarbonate ion increases KCC3a abundance, both in vivo and in vitro, independently of the accompanying cation. Moreover, bicarbonate intake upregulates KCC3a independently of aldosterone or angiotensin II. Since NaHCO3 intake increased KCC3a abundance in wild-type as well as in pendrin knockout mice, this KCC3a upregulation by bicarbonate does not depend on a direct interaction with pendrin. We conclude that increased extracellular bicarbonate, as observed in models of metabolic alkalosis, directly raises KCC3a abundance independently of angiotensin II, aldosterone, or changes in KCC3a transcription and does not involve a direct interaction with pendrin.NEW & NOTEWORTHY KCC3a expression is stimulated in alkalemia. This paper shows that bicarbonate itself is mediating this effect through a posttranscriptional mechanism. The paper also shows that this phenomenon is not mediated by aldosterone or angiotensin II.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 µM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 µM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

Vasoactive mediators of hypertension in obesity.

Obesity is associated with hypertension. However, the mechanisms involved are not fully understood. Therefore, we investigated the relationship between obesity and vasoactive mediators. In this cross-sectional study, blood pressure (BP) and vasoactive mediators of hypertension are compared among 135 adults in the nonobese, obese, and morbidly obese body mass index (BMI) ranges (BMI ≤27, 30-40, and >40 kg/m2, respectively). Angiotensinogen, angiotensin II, renin, aldosterone, endothelin-1 (ET-1), neprilysin, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cyclic guanosine monophosphate (cGMP), and cyclic adenosine monophosphate (cAMP) levels were measured and their relationship to BP, BMI, race, and gender were investigated. Systolic and diastolic BP (SBP and DSP) were significantly higher in subjects with obesity and morbid obesity compared with nonobese. Angiotensin II, ET-1, and neprilysin were significantly higher in subjects with morbid obesity while BNP was lower. Levels of angiotensinogen, renin, aldosterone, ANP, cGMP, and cAMP did not differ between the groups. BMI was positively related to SBP, DBP, angiotensin II, ET-1, and neprilysin, and inversely related to cGMP and BNP. Age, male gender, and African-American race were associated with higher SBP. SBP was positively related to angiotensin II and ET-1 and inversely related to aldosterone, renin, and cGMP. On multivariate analyses, age, BMI, gender, and race were the main determinants of SBP, and excluding these variables, angiotensin II, aldosterone, renin, and ET-1 accounted for 21.1% ability to predict SBP. Obesity, especially morbid obesity, is associated with higher BP, higher angiotensin II and ET-1 (vasoconstrictors), and lower levels BNP and cGMP (vasodilators). SBP variability can be partly explained by angiotensin II, aldosterone, renin, and ET-1.NEW & NOTEWORTHY Our data show that obesity, especially morbid obesity, is associated with higher blood pressure levels and increases angiotensin II and endotherlin-1 (ET-1) (vasoconstrictors) and lower levels BNP and cGMP (vasodilators) and that systolic blood pressure variability can be partly explained by levels of angiotensin II, aldosterone, renin, and ET-1. The effect of these mediators on blood pressure is in addition to the effects of other known factors related to age, male gender, and AA race.

Dual deletion of guanylyl cyclase-A and p38 mitogen-activated protein kinase in podocytes with aldosterone administration causes glomerular intra-capillary thrombi.

Natriuretic peptides exert not only blood-lowering but also kidney-protective effects through guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. Signaling through GC-A has been shown to protect podocytes from aldosterone-induced glomerular injury, and a p38 mitogen-activated protein kinase (MAPK) inhibitor reduced glomerular injury in aldosterone-infused podocyte-specific GC-A knockout mice. To explore the role of p38 MAPK in podocytes, we constructed podocyte-specific p38 MAPK and GC-A double knockout mice (pod-double knockout mice). Unexpectedly, aldosterone-infused and high salt-fed (B-ALDO)-treated pod-double knockout mice resulted in elevated serum creatinine, massive albuminuria, macrophage infiltration, foot process effacement, nephrin and podocin reduction, and additionally, intra-capillary fibrin thrombi, indicating endothelial injury. Microarray analysis showed increased plasminogen activator inhibitor-1 (PAI-1) in glomeruli of B-ALDO-treated pod-double knockout mice. In B-ALDO-treated pod-double knockout mice, PAI-1 increased in podocytes, and treatment with PAI-1 neutralizing antibody ameliorated intra-capillary thrombus formation. In vitro, deletion of p38 MAPK by the CRISPR/Cas9 system and knockdown of GC-A in human cultured podocytes upregulated PAI-1 and transforming growth factor- ß1 (TGF-ß1). When p38 MAPK knockout podocytes, transfected with a small interfering RNA to suppress GC-A, were co-cultured with glomerular endothelial cells in a transwell system, the expression of TGF-ß1 was increased in glomerular endothelial cells. PAI-1 inhibition ameliorated both podocyte and endothelial injury in the transwell system signifying elevated PAI-1 in podocytes is a factor disrupting normal podocyte-endothelial crosstalk. Thus, our results indicate that genetic dual deletion of p38 MAPK and GC-A in podocytes accelerates both podocyte and endothelial injuries, suggesting these two molecules play indispensable roles in podocyte function.

Differential Functional Contribution of BK Channel Subunits to Aldosterone-Induced Channel Activation in Vascular Smooth Muscle and Eventual Cerebral Artery Dilation.

Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory ß1 subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: ß1 provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone's cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3-1 µM and ≥100 µM for ß1. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from ß1-/- vs. wt mice. Data showed that ß1 leftward-shifted aldosterone-induced BK activation, rendering EC50~3 µM and ECMAX ≥ 10 µM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in ß1-/- mice. Therefore, ß1 enables BK activation and MCA dilation by low µM aldosterone.

Novel 1,4-Dihydropyridine Derivatives as Mineralocorticoid Receptor Antagonists.

The mineralocorticoid receptor (MR) belongs to the steroid receptor subfamily of nuclear receptors. MR is a transcription factor key in regulating blood pressure and mineral homeostasis. In addition, it plays an important role in a broad range of biological and pathological conditions, greatly expanding its interest as a pharmacological target. Non-steroidal MR antagonists (MRAs) are of particular interest to avoid side effects and achieve tissue-specific modulation of the receptor. The 1,4-dihydropyridine (1,4-DHP) ring has been identified as an appropriate scaffold to develop non-steroidal MRAs. We report the identification of a novel series of 1,4-DHP that has been guided by structure-based drug design, focusing on the less explored DHP position 2. Interestingly, substituents at this position might interfere with MR helix H12 disposition, which is essential for the recruitment of co-regulators. Several of the newly synthesized 1,4-DHPs show interesting properties as MRAs and have a good selectivity profile. These 1,4-DHPs promote MR nuclear translocation with less efficiency than the natural agonist aldosterone, which explains, at least in part, its antagonist character. Molecular dynamic studies are suggestive of several derivatives interfering with the disposition of H12 in the agonist-associated conformation, and thus, they might stabilize an MR conformation unable to recruit co-activators.

Differential Role of Aldosterone and Transforming Growth Factor Beta-1 in Cardiac Remodeling.

Angiotensin II, a major culprit in cardiovascular disease, activates mediators that are also involved in pathological cardiac remodeling. In this context, we aimed at investigating the effects of two of them: aldosterone (Ald) and transforming growth factor beta-1 (TGF-ß1) in an in vivo model. Six-week-old male wild-type (WT) and TGF-ß1-overexpressing transgenic (TGF-ß1-TG) mice were infused with subhypertensive doses of Ald for 2 weeks and/or treated orally with eplerenone from postnatal day 21. Thehearts' ventricles were examined by morphometry, immunoblotting to assess the intracellular signaling pathways and RT qPCR to determine hypertrophy and fibrosis marker genes. The TGF-ß1-TG mice spontaneously developed cardiac hypertrophy and interstitial fibrosis and exhibited a higher baseline phosphorylation of p44/42 and p38 kinases, fibronectin and ANP mRNA expression. Ald induced a comparable increase in the ventricular-heart-weight-to-body-weight ratio and cardiomyocyte diameter in both strains, but a less pronounced increase in interstitial fibrosis in the transgenic compared to the WT mice (23.6% vs. 80.9%, p < 0.005). Ald increased the phosphorylation of p44/42 and p38 in the WT but not the TGF-ß1-TG mice. While the eplerenone-enriched chow partially prevented Ald-induced cardiac hypertrophy in both genotypes and interstitial fibrosis in the WT controls, it completely protected against additional fibrosis in transgenic mice. Ald appears to induce cardiac hypertrophy independently of TGF-ß1, while in the case of fibrosis, the downstream signaling pathways of these two factors probably converge.

Esaxerenone Inhibits Renal Angiogenesis and Endothelial-Mesenchymal Transition via the VEGFA and TGF-ß1 Pathways in Aldosterone-Infused Mice.

Renal fibrosis is an inevitable process in the progression of chronic kidney disease (CKD). Angiogenesis plays an important role in this process. Vascular endothelial cells are involved in renal fibrosis by phenotypic transformation and secretion of extracellular matrix. Aldosterone stimulates mineralocorticoid receptor (MR) activation and induces inflammation, which is important for angiogenesis. Clinically, MR blockers (MRBs) have a protective effect on damaged kidneys, which may be associated with inhibition of angiogenesis. In this study, we used aldosterone-infused mice and found that aldosterone induced angiogenesis and that endothelial-mesenchymal transition (EndMT) in neovascular endothelial cells was involved in renal fibrosis. Notably, aldosterone induced inflammation and stimulated macrophages to secrete vascular endothelial growth factor (VEGF) A to regulate angiogenesis by activating MR, whereas EndMT occurred in response to transforming growth factor-ß1 (TGF-ß1) induction and participated in renal fibrosis. These effects were antagonized by the MRB esaxerenone. These findings suggest that reducing angiogenesis may be an effective strategy for treating renal fibrosis.

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm.

The cation channel of sperm (CatSper) is the principal entry point for calcium in human spermatozoa and its proper function is essential for successful fertilization. As CatSper is potently activated by progesterone, we evaluated a range of steroids to define the structure-activity relationships for channel activation and found that CatSper is activated by a broad range of steroids with diverse structural modifications. By testing steroids that failed to elicit calcium influx as inhibitors of channel activation, we discovered that medroxyprogesterone acetate, levonorgestrel, and aldosterone inhibited calcium influx produced by progesterone, prostaglandin E1, and the fungal natural product l-sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K+ and pH. In contrast to these steroid antagonists, we demonstrated for the first time that the T-type calcium channel blocker ML218 acts similarly to mibefradil, blocking CatSper channels activated by both ligands and alkalinization/depolarization. These T-type calcium channel blockers produced an insurmountable blockade of CatSper, whereas the three steroids produced antagonism that was surmountable by increasing concentrations of each activator, indicating that the steroids selectively antagonize ligand-induced activation of CatSper rather than blocking channel function. Both the channel blockers and the steroid antagonists markedly reduced hyperactivated motility of human sperm assessed by computer-aided sperm analysis, consistent with inhibition of CatSper activation. Unlike the channel blockers mibefradil and ML218, which reduced total and progressive motility, medroxyprogesterone acetate, levonorgestrel, and aldosterone had little effect on these motility parameters, indicating that these steroids are selective inhibitors of hyperactivated sperm motility. SIGNIFICANCE STATEMENT: The steroids medroxyprogesterone acetate, levonorgestrel, and aldosterone selectively antagonize progesterone- and prostaglandin E1-induced calcium influx through the CatSper cation channel in human sperm. In contrast to T-type calcium channel blockers that prevent all modes of CatSper activation, these steroid CatSper antagonists preferentially reduce hyperactivated sperm motility, which is required for fertilization. The discovery of competitive antagonists of ligand-induced CatSper activation provides starting points for future discovery of male contraceptive agents acting by this unique mechanism.

Paracrine and endocrine regulation of renal K+ secretion.

The seminal studies conducted by Giebisch and coworkers in the 1960s paved the way for understanding the renal mechanisms involved in K+ homeostasis. It was demonstrated that differential handling of K+ in the distal segments of the nephron is crucial for proper K+ balance. Although aldosterone had been classically ascribed as the major ion transport regulator in the distal nephron, thereby contributing to K+ homeostasis, it became clear that aldosterone per se could not explain the ability of the kidney to modulate kaliuresis in both acute and chronic settings. The existence of alternative kaliuretic and antikaliuretic mechanisms was suggested by physiological studies in the 1980s but only gained form and shape with the advent of molecular biology. It is now established that the kidneys recruit several endocrine and paracrine mechanisms for adequate kaliuretic response. These mechanisms include the direct effects of peritubular K+, a gut-kidney regulatory axis sensing dietary K+ levels, the kidney secretion of kallikrein during postprandial periods, the upregulation of angiotensin II receptors in the distal nephron during chronic changes in K+ diet, and the local increase of prostaglandins by low-K+ diet. This review discusses recent advances in the understanding of endocrine and paracrine mechanisms underlying the modulation of K+ secretion and how these mechanisms impact kaliuresis and K+ balance. We also highlight important unknowns about the regulation of renal K+ excretion under physiological circumstances.

ROMK channels are inhibited in the aldosterone-sensitive distal nephron of renal tubule Nedd4-2-deficient mice.

We used whole cell recording to examine the renal outer medullary K+ channel (ROMK or Kir1.1) and epithelial Na+ channel (ENaC) in the late distal convoluted tubule (DCT2)/initial connecting tubule (iCNT) and in the cortical collecting duct (CCD) of kidney tubule-specific neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2) knockout mice (Ks-Nedd4-2 KO) and floxed neural precursor cell-expressed developmentally downregulated 4-like (Nedd4l) mice (control). Tertiapin Q (TPNQ)-sensitive K+ currents (ROMK) were smaller in both the DCT2/iCNT and CCD of Ks-Nedd4-2 KO mice on a normal diet than in control mice. Neither high dietary salt intake nor low dietary salt intake had a significant effect on ROMK activity in the DCT2/iCNT and CCD of control and Ks-Nedd4-2 KO mice. In contrast, high dietary K+ intake (HK) increased, whereas low dietary K+ intake (LK) decreased TPNQ-sensitive K+ currents in floxed Nedd4l mice. However, the effects of dietary K+ intake on ROMK channel activity were absent in Ks-Nedd4-2 KO mice since neither HK nor LK significantly affected TPNQ-sensitive K+ currents in the DCT2/iCNT and CCD. Moreover, TPNQ-sensitive K+ currents in the DCT2/iCNT and CCD of Ks-Nedd4-2 KO mice on HK were similar to those of control mice on LK. Amiloride-sensitive Na+ currents in the DCT2/iCNT and CCD were significantly higher in Ks-Nedd4-2 KO mice than in floxed Nedd4l mice on a normal K+ diet. HK increased ENaC activity of the DCT2/iCNT only in control mice, but HK stimulated ENaC of the CCD in both control and Ks-Nedd4-2 KO mice. Moreover, the HK-induced increase in amiloride-sensitive Na+ currents was larger in Ks-Nedd4-2 KO mice than in control mice. Deletion of Nedd4-2 increased with no lysine kinase 1 expression and abolished HK-induced inhibition of with no lysine kinase 1. We conclude that deletion of Nedd4-2 increases ENaC activity but decreases ROMK activity in the aldosterone-sensitive distal nephron and that HK fails to stimulate ROMK, but robustly increases ENaC activity in the CCD of Nedd4-2-deficient mice.NEW & NOTEWORTHY We demonstrate that renal outer medullary K+ (ROMK) channel activity is inhibited in the late distal convoluted tubule/initial connecting tubule and cortical collecting duct of neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2)-deficient mice. Also, deletion of Nedd4-2 abolishes the stimulatory effect of dietary K+ intake on ROMK. The lack of high K+-induced stimulation of ROMK is associated with the absence of high K+-induced inhibition of with no lysine kinase 1.

High baseline ROMK activity in the mouse late distal convoluted and early connecting tubule probably contributes to aldosterone-independent K+ secretion.

The renal outer medullary K+ channel (ROMK) is colocalized with the epithelial Na+ channel (ENaC) in the late distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical collecting duct (CCD). ENaC-mediated Na+ absorption generates the electrical driving force for ROMK-mediated tubular K+ secretion, which is critically important for maintaining renal K+ homeostasis. ENaC activity is aldosterone dependent in the late CNT and early CCD (CNT/CCD) but aldosterone independent in the DCT2 and early CNT (DCT2/CNT). This suggests that under baseline conditions with low plasma aldosterone, ROMK-mediated K+ secretion mainly occurs in the DCT2/CNT. Therefore, we hypothesized that baseline ROMK activity is higher in the DCT2/CNT than in the CNT/CCD. To test this hypothesis, patch-clamp experiments were performed in the DCT2/CNT and CNT/CCD microdissected from mice maintained on a standard diet. In single-channel recordings from outside-out patches, we detected typical ROMK channel activity in both the DCT2/CNT and CNT/CCD and confirmed that ROMK is the predominant K+ channel in the apical membrane. Amiloride-sensitive and tertiapin-sensitive whole-cell currents were determined to assess ENaC and ROMK activity, respectively. As expected, baseline amiloride-sensitive current was high in the DCT2/CNT (∼370 pA) but low in the CNT/CCD (∼60 pA). Importantly, tertiapin-sensitive current was significantly higher in the DCT2/CNT than in the CNT/CCD (∼810 vs. ∼350 pA). We conclude that high ROMK activity in the DCT2/CNT is critical for aldosterone-independent renal K+ secretion under baseline conditions. A low-K+ diet significantly reduced ENaC but not ROMK activity in the DCT2/CNT. This suggests that modifying ENaC activity in the DCT2/CNT plays a key regulatory role in adjusting renal K+ excretion to dietary K+ intake.NEW & NOTEWORTHY ROMK-mediated renal K+ secretion is essential for maintaining K+ balance and requires a lumen negative transepithelial potential critically dependent on ENaC activity. Using microdissected distal mouse tubules, we demonstrated that baseline apical ROMK activity is high in the DCT2/CNT. Aldosterone-independent baseline ENaC activity is also high in the DCT2/CNT and downregulated by a low-K+ diet, which highlights the important role of the DCT2/CNT in regulating K+ secretion in an aldosterone-independent manner.