Impaired 11 beta-hydroxysteroid dehydrogenase contributes to renal sodium avidity in cirrhosis: hypothesis or fact?

Exaggerated renal sodium retention with concomitant potassium loss is a hallmark of cirrhosis and contributes to the accumulation of fluid as ascites, pleural effusion, or edema. This apparent mineralocorticoid effect is only partially explained by increased aldosterone concentrations. I present evidence supporting the hypothesis that cortisol confers mineralocorticoid action in cirrhosis. The underlying molecular pathology for this mineralocorticoid receptor (MR) activation by cortisol is a reduced activity of the 11 beta-hydroxysteroid dehydrogenase type 2, an enzyme protecting the MR from promiscuous activation by cortisol in healthy mammalians.

Eicosanoids in exhaled breath condensates in the assessment of childhood asthma

The value of measurements of eicosanoids in exhaled breath condensate (EBC) for the evaluation of childhood asthma is still inconclusive most likely because of the limited value of the methods used. In this case-control study in 48 asthmatic and 20 healthy children, we aimed to characterize the baseline profile of the inflammatory mediators cysteinyl leukotrienes (cysLTs), 9(alpha)11(beta)PGF(2), PGE(2), PGF(2alpha), 8-isoprostane (8-iso-PGF(2alpha)) within EBC in asthmatic compared with healthy children using new methods. In addition, we investigated their relation to other inflammatory markers. The assessment included collection of EBC, measurement of fractional exhaled nitric oxide (FE(NO)) and evaluation of urinary excretion of leukotriene E(4.) cysLTs were measured directly in EBC by radioimmunoassay and prostanoids were measured using gas chromatography negative-ion chemical ionization mass spectrometry. Only cysLT levels were significantly higher in asthmatic compared with healthy children (p = 0.002). No significant differences in cysLTs were found between steroid naïve and patients receiving inhaled corticosteroids. In contrast, FE(NO) was significantly higher in steroid naïve compared with steroid-treated asthmatic and healthy children (p = 0.04 and 0.024, respectively). The diagnostic accuracy of cysLTs in EBC for asthma was 73.6% for the whole group and 78.2% for steroid-naïve asthmatic children. The accuracy to classify asthmatic for FE(NO) was poor (62.9%) for the whole group, but improved to 79.9% when only steroid-naïve asthmatic children were taken into consideration. cysLTs in EBC is an inflammatory marker which distinguishes asthmatics, as a whole group, from healthy children.

DHEA induces 11 -HSD2 by acting on CCAAT/enhancer-binding proteins

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) type 1 and type 2 catalyze the interconversion of inactive and active glucocorticoids. Impaired regulation of these enzymes has been associated with obesity, diabetes, hypertension, and cardiovascular disease. Previous studies in animals and humans suggested that dehydroepiandrosterone (DHEA) has antiglucocorticoid effects, but the underlying mechanisms are unknown. In this study, DHEA treatment markedly increased mRNA expression and activity of 11beta-HSD2 in a rat cortical collecting duct cell line and in kidneys of C57BL/6J mice and Sprague-Dawley rats. DHEA-treated rats tended to have reduced urinary corticosterone to 11-dehydrocorticosterone ratios. It was found that CCAAT/enhancer-binding protein-alpha (C/EBP-alpha) and C/EBP-beta regulated HSD11B2 transcription and that DHEA likely modulated the transcription of 11beta-HSD2 in a phosphatidylinositol-3 kinase/Akt-dependent manner by increasing C/EBP-beta mRNA and protein expression. Moreover, it is shown that C/EBP-alpha and C/EBP-beta differentially regulate the expression of 11beta-HSD1 and 11beta-HSD2. In conclusion, DHEA induces a shift from 11beta-HSD1 to 11beta-HSD2 expression, increasing conversion from active to inactive glucocorticoids. This provides a possible explanation for the antiglucocorticoid effects of DHEA.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.

Targeted alpha-radionuclide therapy of functionally critically located gliomas with 213Bi-DOTA-[Thi8,Met(O2)11]-substance P: a pilot trial

Functionally critically located gliomas represent a challenging subgroup of intrinsic brain neoplasms. Standard therapeutic recommendations often cannot be applied, because radical treatment and preservation of neurological function are contrary goals. The successful targeting of gliomas with locally injected beta radiation-emitting (90)Y-DOTAGA-substance P has been shown previously. However, in critically located tumours, the mean tissue range of 5 mm of (90)Y may seriously damage adjacent brain areas. In contrast, the alpha radiation-emitting radionuclide (213)Bi with a mean tissue range of 81 microm may have a more favourable toxicity profile. Therefore, we evaluated locally injected (213)Bi-DOTA-substance P in patients with critically located gliomas as the primary therapeutic modality.

Atorvastatin added to interferon beta for relapsing multiple sclerosis: a randomized controlled trial

Statins have anti-inflammatory and immunomodulatory properties in addition to lipid-lowering effects. The present study evaluated the effect of atorvastatin added to interferon beta-1b in multiple sclerosis (MS) in a multicenter, randomized, parallel-group, rater-blinded study performed in eight Swiss hospitals. Seventy-seven patients with relapsing-remitting MS started interferon beta-1b every other day. After 3 months, they were randomized 1:1 to receive atorvastatin 40 mg/day or not in addition to interferon beta-1b until month 15. The primary endpoint was the proportion of patients with new lesions on T2-weighted images at month 15 compared to baseline at month three. At study end, the proportion of patients with new lesions on T2-weighted images was equal in both groups (odds ratio 1.14; 95 % CI 0.36-3.56; p = 0.81). All predefined secondary endpoints including number of new lesions and total lesion volume on T2-weighted images, total number of new Gd-enhancing lesions on T1-weighted images, total brain volume, volume of grey matter, volume of white matter, EDSS, MSFC, relapse rate, time to first relapse, number of relapse-free patients and neutralizing antibodies did not show any significant differences (all p values >0.1). Transient elevations of liver enzymes were more frequent with atorvastatin (p = 0.02). In conclusion, atorvastatin 40 mg/day in addition to interferon beta-1b did not have a beneficial effect on relapsing-remitting MS compared to interferon beta-1b monotherapy over a 12-month period.

Uninephrectomy reduces 11β-hydroxysteroid dehydrogenase type 1 and type 2 concomitantly with an increase in blood pressure in rats

Renal allograft donors are at risk of developing hypertension. Here, we hypothesized that this risk is at least in part explained by an enhanced intracellular availability of 11β-hydroxyglucocorticoids due to an increased 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1), an intracellular prereceptor activator of biologically inactive 11-ketocorticosteroids in the liver, and/or a diminished 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an inactivator of 11β-hydroxyglucocorticoids in the kidney. To test this hypothesis, uninephrectomized (UNX) (n=9) and sham-operated (n=10) adult Sprague-Dawley rats were investigated. Mean arterial blood pressure and heart rate were measured continuously by telemetry for 6 days in week 5 after UNX. The mRNA of 11β-Hsd1 and 11β-Hsd2 in liver and kidney tissues were assessed by RT-PCR and the 11β-HSD activities were directly quantified in their corresponding tissues by determining the ratios of (tetrahydrocorticosterone+5α-tetrahydrocorticosterone)/tetrahydrodehydrocorticosterone ((THB+5α-THB)/THA) and of corticosterone/dehydrocorticosterone (B/A) by gas chromatography-mass spectrometry. The apparent total body activities of 11β-HSD1 and 11β-HSD2 were estimated using the urinary and plasma ratios of (THB+5α-THB)/THA and B/A. Mean arterial blood pressure was increased after UNX when compared with sham operation. Hepatic mRNA content of 11β-Hsd1 and hepatic, plasma, and urinary ratios of (THB+5α-THB)/THA were decreased after UNX, indicating diminished access of glucocorticoids to its receptors. In renal tissue, 11β-Hsd2 mRNA was reduced and B/A ratios measured in kidney, plasma, and urine were increased, indicating reduced 11β-HSD2 activity and enhanced access of glucocorticoids to mineralocorticoid receptors. Both 11β-HSD1 and 11β-HSD2 are downregulated after UNX in rats, a constellation considered to induce hypertension.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Distribution of amyloid precursor protein and amyloid-beta immunoreactivity in DBA/2J glaucomatous mouse retinas

PURPOSE: Evidence suggests that altered metabolism of amyloid precursor protein (APP) may play a role in the pathophysiology of retinal ganglion cell (RGC) death in the etiology of glaucoma. The authors sought to determine the distribution of APP and amyloid-beta (Abeta) in DBA/2J glaucomatous mouse retinas. METHODS: The retinas of 3- and 15-month-old DBA/2J mice and C57/BL-6 mice (control group) were fixed with 4% paraformaldehyde and processed for immunohistochemistry. Antibodies used included a polyclonal antibody to the C terminus of Abeta 40 and a polyclonal antibody to the APP ectodomain. Immunohistochemically stained tissue was graded using light microscopy. Distribution and semiquantitative expression of APP and Abeta in young and old glaucomatous and normal retinas were determined and compared. RESULTS: Strong APP and Abeta immunoreactivity was found in the RGC layer, optic nerve, and pia/dura of old DBA/2J retinas, with considerably higher intensity found in the old compared with the young DBA/2J mice. In contrast to glaucomatous mice, the control group did not show any notable age-related difference. CONCLUSIONS: Disruption of the homeostatic properties of secreted APP with consecutive Abeta cytotoxicity might be a contributing factor of ganglion cell loss in glaucomatous mouse retinas.

Immunohistochemical demonstration of interleukin-1 beta induced changes in acute-phase proteins and albumin in rat liver

Interleukin-1 beta is a potent mediator of the acute-phase response. However, the effects of interleukin-1 beta administration on the topic in vivo production of acute-phase proteins and albumin are so far not well understood. Overnight fasted rats were subcutaneously injected with 0.2 mL 0.9% NaCl (control group) or 6.25 micrograms recombinant human interleukin-1 beta, and rectal temperature was measured at intervals up to 48 h. Livers were perfused-fixed in vivo prior to injection (base-line), and at 9, 24, and 48 h following the interleukin-1 beta injection. Fibrinogen, orosomucoid (alpha 1-acid glycoprotein) and albumin were immunostained using a streptavidin-biotin-immunoperoxidase technique. Rectal temperature peaked 5 h after the single interleukin-1 beta injection, and fell gradually to base-line values by 24 h. Prior to injection only a few hepatocytes, randomly scattered throughout the liver lobule, stained positive for fibrinogen and orosomucoid. In contrast, all hepatocytes stained uniformly positive for fibrinogen and orosomucoid 9 h after interleukin-1 beta injection, whereas at 24 h a predominant centrilobular staining pattern occurred. Due to fasting, albumin positive hepatocytes were already reduced at base-line in both groups. Interleukin-1 beta induced a further significant loss of albumin positive cells in the periportal zone (35 +/- 21%) at 9 h when compared with controls (58 +/- 11%, p = 0.037). In conclusion, subcutaneous interleukin-1 beta (probably by stimulation of interleukin-6) strongly induces fibrinogen and orosomucoid expression in rat liver, and suppresses immunohistochemically stainable albumin in a heterogenous way, mainly in the periportal zone.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation

The signals and molecular mechanisms that regulate the replication of terminally differentiated beta cells are unknown. Here, we report the identification and characterization of transmembrane protein 27 (Tmem27, collectrin) in pancreatic beta cells. Expression of Tmem27 is reduced in Tcf1(-/-) mice and is increased in islets of mouse models with hypertrophy of the endocrine pancreas. Tmem27 forms dimers and its extracellular domain is glycosylated, cleaved and shed from the plasma membrane of beta cells. This cleavage process is beta cell specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic beta cells exhibit increased beta cell mass. Our results identify a pancreatic beta cell transmembrane protein that regulates cell growth of pancreatic islets.

Cadherin-11 up-regulation in overactive bladder suburothelial myofibroblasts

PURPOSE: We investigated whether the adherens junction proteins cadherin-11 and beta-catenin can be immunohistochemically visualized in the human bladder using commercially available antibodies and, if so, whether there are differences between patients with overactive bladder and refractory detrusor overactivity, and controls without lower urinary tract symptoms. MATERIALS AND METHODS: In a prospective, nonrandomized single center study 32 patients with overactive bladder and refractory detrusor overactivity, and 8 controls without lower urinary tract symptoms underwent cystoscopic bladder biopsy. Quantitative immunohistochemistry was performed. The primary outcome was cadherin-11 and beta-catenin expression in the human bladder using commercially available antibodies. The secondary outcome was differences in cadherin-11 and beta-catenin in patients with overactive bladder and refractory detrusor overactivity, and controls. RESULTS: Double labeling experiments showed co-localization of cadherin-11 and connexin 43 in the suburothelium. There was also strong co-localization of cadherin-11 and beta-catenin in the suburothelium and detrusor. Significant 2-fold up-regulation of cadherin-11 was found in the suburothelium of patients with overactive bladder compared with that in controls (p = 0.018), whereas beta-catenin was similar in the groups (p = 0.6). In the detrusor cadherin-11 and beta-catenin expression was comparable in patients with overactive bladder and controls (each p = 0.5). No difference was observed in cadherin-11 and beta-catenin in patients with overactive bladder with idiopathic vs neurogenic detrusor overactivity in the suburothelium and the detrusor (p >0.3 and >0.2, respectively). CONCLUSIONS: Using commercially available antibodies cadherin-11 and beta-catenin expression in human bladder suburothelial myofibroblasts and detrusor smooth muscle cells was noted. Cadherin-11 up-regulation in suburothelial myofibroblasts in patients with overactive bladder may be significant in overactive bladder pathogenesis.