Alex Odermatt

Hexose-6-phosphate dehydrogenase determines the reaction direction of 11β-hydroxysteroid dehydrogenase type 1 as an oxoreductase

The impact of hexose‐6‐phosphate dehydrogenase (H6PDH) on 11β‐hydroxysteroid dehydrogenase (11β‐HSD) type 1 activity was investigated upon coexpression in HEK‐293 cells. Confocal microscopy analysis indicated colocalisation of both enzymes at the lumenal side of the endoplasmic reticulum (ER) membrane. Functional analysis in intact cells revealed fivefold stimulation of 11β‐HSD1 oxoreductase activity and sixfold decrease of dehydrogenase activity upon coexpression with H6PDH, without changing kinetic parameters in cell lysates. Thus, H6PDH directly determines the reaction direction of 11β‐HSD1 in intact cells as an oxoreductase without changing intrinsic catalytic properties of 11β‐HSD1 by regenerating NADPH in the ER‐lumen.

The N-terminal anchor sequences of 11beta-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane.

11β-Hydroxysteroid dehydrogenase enzymes (11β- HSD) regulate the ratio of active endogenous glucocorticoids to their inactive keto-metabolites, thereby controlling the access of glucocorticoids to their cognate receptors. In this study, the topology and intracellular localization of 11β-HSD1 and 11β-HSD2 have been analyzed by immunohistochemistry and protease protection assays ofin vitro transcription/translation products. 11β-HSD constructs, tagged with the FLAG epitope, were transiently expressed in HEK-293 cells. The enzymatic characteristics of tagged and native enzymes were indistinguishable. Fluorescence microscopy demonstrated the localization of both 11β-HSD1 and 11β-HSD2 exclusively to the endoplasmic reticulum (ER) membrane. To examine the orientation of tagged 11β-HSD enzymes within the ER membrane, we stained selectively permeabilized HEK-293 cells with anti-FLAG antibody. Immunohistochemistry revealed that the N terminus of 11β-HSD1 is cytoplasmic, and the catalytic domain containing the C terminus is protruding into the ER lumen. In contrast, the N terminus of 11β-HSD2 is lumenal, and the catalytic domain is facing the cytoplasm. Chimeric proteins where the N-terminal anchor sequences of 11β-HSD1 and 11β-HSD2 were exchanged adopted inverted orientation in the ER membrane. However, both chimeric proteins were not catalytically active. Furthermore, mutation of a tyrosine motif to alanine in the transmembrane segment of 11β-HSD1 significantly reducedV max. The subcellular localization of 11β-HSD1 was not affected by mutations of the tyrosine motif or of a di-lysine motif in the N terminus. However, residue Lys5, but not Lys6, turned out to be critical for the topology of 11β-HSD1. Mutation of Lys5 to Ser inverted the orientation of 11β-HSD1 in the ER membrane without loss of catalytic activity. Our results emphasize the importance of the N-terminal transmembrane segments of 11β-HSD enzymes for their proper function and demonstrate that they are sufficient to determine their orientation in the ER membrane.

Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.

A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (0.25 h), followed by a significant rise of plasma aldo (0.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about 0.5 h). The functional changes were accompanied by a rapid and sustained (0.25-6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldo-independent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron.

Prevalence of cam-type deformity on hip magnetic resonance imaging in young males: a cross-sectional study.

To determine the prevalence of cam‐type deformities on hip magnetic resonance imaging (MRI) in young males.

The Western-style diet: a major risk factor for impaired kidney function and chronic kidney disease

The Western-style diet is characterized by its highly processed and refined foods and high contents of sugars, salt, and fat and protein from red meat. It has been recognized as the major contributor to metabolic disturbances and the development of obesity-related diseases including type 2 diabetes, hypertension, and cardiovascular disease. Also, the Western-style diet has been associated with an increased incidence of chronic kidney disease (CKD). A combination of dietary factors contributes to the impairment of renal vascularization, steatosis and inflammation, hypertension, and impaired renal hormonal regulation. This review addresses recent progress in the understanding of the association of the Western-style diet with the induction of dyslipidemia, oxidative stress, inflammation, and disturbances of corticosteroid regulation in the development of CKD. Future research needs to distinguish between acute and chronic effects of diets with high contents of sugars, salt, and fat and protein from red meat, and to uncover the contribution of each component. Improved therapeutic interventions should consider potentially altered drug metabolism and pharmacokinetics and be combined with lifestyle changes. A clinical assessment of the long-term risks of whole-body disturbances is strongly recommended to reduce metabolic complications and cardiovascular risk in kidney donors and patients with CKD.

Tissue-specific modulation of mineralocorticoid receptor function by 11β-hydroxysteroid dehydrogenases: an overview.

In the last decade significant progress has been made in the understanding of mineralocorticoid receptor (MR) function and its implications for physiology and disease. The knowledge on the essential role of MR in the regulation of electrolyte concentrations and blood pressure has been significantly extended, and the relevance of excessive MR activation in promoting inflammation, fibrosis and heart disease as well as its role in modulating neuronal cell viability and brain function is now widely recognized. Despite considerable progress, the mechanisms of MR function in various cell-types are still poorly understood. Key modulators of MR function include the glucocorticoid receptor (GR), which may affect MR function by formation of heterodimers and by differential genomic and non-genomic responses on gene expression, and 11β-hydroxysteroid dehydrogenases (11β-HSDs), which determine the availability of intracellular concentrations of active glucocorticoids. In this review we attempted to provide an overview of the knowledge on MR expression with regard to the presence or absence of GR, 11β-HSD2 and 11β-HSD1/hexose-6-phosphate dehydrogenase (H6PDH) in various tissues and cell types. The consequences of cell-specific differences in the coexpression of MR with these proteins need to be further investigated in order to understand the role of this receptor in a given tissue as well as its systemic impact.

Glucocorticoid-mediated mineralocorticoid receptor activation and hypertension.

Purpose of reviewTraditionally, the mineralocorticoid receptor was thought to be activated by the mineralocorticoid hormone aldosterone, and to exhibit its main action on epithelia by promoting renal sodium retention, potassium excretion and inducing hypertension upon excessive activation. Recently, evidence appeared that mineralocorticoid receptors are expressed in nonepithelial cells and activated by endogenous glucocorticoids including cortisol. Therefore, the prereceptor regulation of cortisol access to the mineralocorticoid receptors by 11β-hydroxysteroid dehydrogenase enzymes (11β-HSDs), a mechanism absent in most nonepithelial cells, appears to be relevant for disease states with cortisol-induced mineralocorticoid action. The present review focuses on direct and indirect effects attributable to mineralocorticoid receptor activation by glucocorticoids. Recent findingsThe determination of the intracellular topology of 11β-HSD1, facing the endoplasmic reticulum lumen, and 11β-HSD2, facing the cytoplasm, suggests that 11β-HSD1 acts as a prereceptor mechanism in the local activation of glucocorticoid receptors, whereas 11β-HSD2 controls mineralocorticoid receptors by interacting with the receptor in the absence of aldosterone. Downregulation of 11β-HSD2 was observed with various stimuli including hypoxia, shear stress, angiotensin II and tumor necrosis factor α. The corresponding signal transcription pathways and some relevant transcription factors have been identified. Renal sodium retention in liver cirrhosis, nephrotic syndrome and hypoxia have been linked to 11β-HSD2 reduced activity. Overexpression of 11β-HSD1 specifically in adipose tissue in mice caused central obesity, a metabolic syndrome and hypertension due to increased intracellular cortisol concentrations. Peroxisome proliferator-activated receptor γ agonists reduce 11β-HSD1 activity and diminish the intracellular availability of cortisol, an effect accompanied by a decline in blood pressure. Three individuals with loss-of-function mutations of peroxisome proliferator-activated receptor γ developed early hypertension. A potential mechanism might be glucocorticoid dependent mineralocorticoid receptor-mediated downregulation of endothelial nitric oxide synthase. SummaryRecently, mineralocorticoid receptor antagonists have been used in the randomized aldactone evaluation study (RALES) with spironolactone, the eplerenone post-AMI heart failure efficacy and survival study (EPHESUS), and in severe and postmyocardial infarct heart failure, respectively. These investigations cannot be understood on the basis of the present physiological knowledge and underscore the relevance of focusing on mineralocorticoid receptor activation by ligands other than aldosterone.

Copper pumping ATPases: Common concepts in bacteria and man

Recently, four genes encoding putative copper pumping ATPases have been cloned from widely different sources: two genes from Enterococcus hirae that are involved in copper metabolism and two human genes that are defective in the copper‐related Wilson and Menkes disease. The predicted gene products are P‐type ATPases. They exhibit extensive sequence similarity and appear to be members of a new class of ATP driven copper pumps involved in the regulation of cellular copper.

An ATPase Operon Involved in Copper Resistance by Enterococcus hiraea

Many microorganisms exhibit resistance to heavy metal ions such as mercury, cadmium, copper, arsenite, or selenite. Tolerance to these compounds has probably evolved in response to growth in adverse environments containing normally toxic levels of such metal ions. We recently discovered that the Gram-positive bacteria Enterococcus hirae can tolerate relatively high levels of copper. Although copper resistance genes have been identified in other little is known about the actual mechanism of copper resistance. We here describe an operon containing two ATPase genes that appear to be involved in conferring copper resistance to E. hirae.

Endoplasmic reticulum : reduced and oxidized glutathione revisited

Summary The reducing power of glutathione, expressed by its reduction potential EGSH, is an accepted measure for redox conditions in a given cell compartment. In the endoplasmic reticulum (ER), EGSH is less reducing than elsewhere in the cell. However, attempts to determine EGSH(ER) have been inconsistent and based on ineligible assumptions. Using a codon-optimized and evidently glutathione-specific glutaredoxin-coupled redox-sensitive green fluorescent protein (roGFP) variant, we determined EGSH(ER) in HeLa cells as −208±4 mV (at pH 7.0). At variance with existing models, this is not oxidizing enough to maintain the known redox state of protein disulfide isomerase family enzymes. Live-cell microscopy confirmed ER hypo-oxidation upon inhibition of ER Ca2+ import. Conversely, stressing the ER with a glycosylation inhibitor did not lead to more reducing conditions, as reported for yeast. These results, which for the first time establish the oxidative capacity of glutathione in the ER, illustrate a context-dependent interplay between ER stress and EGSH(ER). The reported development of ER-localized EGSH sensors will enable more targeted in vivo redox analyses in ER-related disorders.