Absence of hexose-6-phosphate dehydrogenase results in reduced overall glucose consumption but does not prevent 11β-hydroxysteroid dehydrogenase-1-dependent glucocorticoid activation

Hexose-6-phosphate dehydrogenase (H6PD) is thought to be the major source of NADPH within the endoplasmic reticulum (ER), determining 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) reaction direction to convert inert 11-oxo- to potent 11β-hydroxyglucocorticoids. Here, we tested the hypothesis whether H6pd knock-out (KO) in primary murine bone marrow-derived macrophages results in a switch from 11β-HSD1 oxoreduction to dehydrogenation, thereby inactivating glucocorticoids (GC) and affecting macrophage phenotypic activation as well as causing a more aggressive M1 macrophage phenotype. H6pdKO did not lead to major disturbances of macrophage activation state, although a slightly more pronounced M1 phenotype was observed with enhanced proinflammatory cytokine release, an effect explained by the decreased 11β-HSD1-dependent GC activation. Unexpectedly, ablation of H6pd did not switch 11β-HSD1 reaction direction. A moderately decreased 11β-HSD1 oxoreduction activity by 40-50% was observed in H6pdKO M1 macrophages but dehydrogenation activity was undetectable, providing strong evidence for the existence of an alternative source of NADPH in the ER. H6pdKO M1 activated macrophages showed decreased phagocytic activity, most likely a result of the reduced 11β-HSD1-dependent GC activation. Other general macrophage functions reported to be influenced by GC, such as nitrite production and cholesterol efflux, were altered negligibly or not at all. Importantly, assessment of energy metabolism using an extracellular flux analyzer and lactate measurements revealed reduced overall glucose consumption in H6pdKO M1 activated macrophages, an effect that was GC independent. The GC-independent influence of H6PD on energy metabolism and the characterization of the alternative source of NADPH in the ER warrant further investigations. ENZYMES: 11β-HSD1, EC 1.1.1.146; H6PD, EC 1.1.1.47

Adult nephron-specific MR-deficient mice develop a severe renal PHA-1 phenotype

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR$^{Pax8/LC1}$) using a doxycycline-inducible system. Under standard diet, MR$^{Pax8/LC1}$ mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR$^{Pax8/LC1}$ mice survive but develop a severe PHA-1 phenotype with higher urinary Na^+ levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na^+/Cl^- co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR$^{Pax8/LC1}$ knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na^+ and low in K^+ does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state

11β-Hydroxysteroid dehydrogenase 1: Regeneration of active glucocorticoids is only part of the story

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is an endoplasmic reticulum membrane enzyme with its catalytic site facing the luminal space. It functions primarily as a reductase, driven by the supply of its cosubstrate NADPH by hexose-6-phosphate dehydrogenase (H6PDH). Extensive research has been performed on the role of 11β-HSD1 in the regeneration of active glucocorticoids and its role in inflammation and metabolic disease. Besides its important role in the fine-tuning of glucocorticoid action, 11β-HSD1 is a multi-functional carbonyl reductase converting several 11- and 7-oxosterols into the respective 7-hydroxylated forms. Moreover, 11β-HSD1 has a role in phase I biotransformation reactions and catalyzes the carbonyl reduction of several non-steroidal xenobiotics. Recent observations from experiments using selective inhibitors and studies with transgenic mice indicated a role for 11β-HSD1 in oxysterol metabolism and in bile acid homeostasis, with evidence for glucocorticoid-independent effects on gene expression. This review focuses on the promiscuity of 11β-HSD1 to accept structurally distinct substrates and discusses recent progress mainly on non-glucocorticoid substrates. This article is part of a Special Issue entitled 'Enzyme Promiscuity and Diversity'

Inhibition of 11β-hydroxysteroid dehydrogenase 2 by the fungicides itraconazole and posaconazole

Impaired 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2)-dependent cortisol inactivation can lead to electrolyte dysbalance, hypertension and cardiometabolic disease. Furthermore, placental 11β-HSD2 essentially protects the fetus from high maternal glucocorticoid levels, and its impaired function has been associated with altered fetal growth and a higher risk for cardio-metabolic diseases in later life. Despite its important role, 11β-HSD2 is not included in current off-target screening approaches. To identify potential 11β-HSD inhibitors among approved drugs, a pharmacophore model was used for virtual screening, followed by biological assessment of selected hits. This led to the identification of several azole fungicides as 11β-HSD inhibitors, showing a significant structure-activity relationship between azole scaffold size, 11β-HSD enzyme selectivity and inhibitory potency. A hydrophobic linker connecting the azole ring to the other, more polar end of the molecule was observed to be favorable for 11β-HSD2 inhibition and selectivity over 11β-HSD1. The most potent 11β-HSD2 inhibition, using cell lysates expressing recombinant human 11β-HSD2, was obtained for itraconazole (IC50 139±14nM), its active metabolite hydroxyitraconazole (IC50 223±31nM) and posaconazole (IC50 460±98nM). Interestingly, experiments with mouse and rat kidney homogenates showed considerably lower inhibitory activity of these compounds towards 11β-HSD2, indicating important species-specific differences. Thus, 11β-HSD2 inhibition by these compounds is likely to be overlooked in preclinical rodent studies. Inhibition of placental 11β-HSD2 by these compounds, in addition to the known inhibition of cytochrome P450 enzymes and P-glycoprotein efflux transport, might contribute to elevated local cortisol levels, thereby affecting fetal programming

Deletion of the serine protease CAP2/Tmprss4 leads to dysregulated renal water handling upon dietary potassium depletion

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Mutual recognition of parental and F1 lymphocytes. Selective abrogation of eytotoxic potential of FI lymphoeytes by parental lymphocytes.J. Exp. Med

The injection of F1 hybrid animals with parental spleen or lymph node cells leads to the activation of specific parental lymphocytes which recognize major histocompatibility complex (MHC) 1-coded antigens of the other parental haplotype expressed by the F1 host (1). Such recognition can result in graft-vs.-host (GVH) reactions which are frequently associated with depressed in vivo cell-mediated immune functions, such as resistance to bacterial infection (2), skin graft rejection (2, 3), T-helper cell dysfunction (4), as well as antibody responses to thymic-dependent and-independent antigens (5-8). The depression of T-cell-mediated lympholysis (CML) responses by cells from F1 hybrid mice injected with parental lymphocytes has not been reported but could provide a useful approach for investigating T-cell receptors because self MHC-restricted as well as allogeneic CML responses can be analyzed. This report describes an experimental system in which intravenous injection of F1 hybrid mice with parental T-splenic lymphocytes can result in the abrogation or severe depression of CML potential. This loss of CML activity did not appear to be specific for self determinants because responses to alloantigens, trinitrophenyl (TNP)modified F1 cells (TNP-self), and TNP-modified parental cells were all affected. However, an unexpected finding was the observation that the parental-induced CML depression was dependent upon the H-2 type of the injected parental lymphocytes. Thus, the injection of H-2 a, H-2 k, or H-2 a, but not H-2 6, parental lymphocytes resulted in depressed CML potential. These findings are discussed with respect to (a) the selective resistance of F1 mice to H-2 b parental lymphocytes, and (b) the possibility that Fa lymphocytes recognize idiotypic determinants specific for non-H-2 b antigens on H-2 b lymphocytes, but not those for H-2 b antigens on non-H-2 b lymphocytes

Lack of Renal Tubular Glucocorticoid Receptor Decreases the Thiazide-Sensitive Na+/Cl– Cotransporter NCC and Transiently Affects Sodium Handling

International audienceChronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1Pax8/LC1). Upon standard salt diet, Nr3c1Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11βHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na+ and/or K+ balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na+ restriction renal tubular GR-deficiency transiently affects Na+ handling and transport pathways. Overall, upon standard, low Na+ and high Na+ diet exposure Na+ and K+ balance is maintained as evidenced by normal plasma and urinary Na+ and K+ and aldosterone concentrations