Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

A study of xlcaax-1: Patterns of localization, possible function and usefulness as a developmental marker

Morphogenesis is the process by which the 3-dimensional structure of the developing embryo takes shape. We are studying xlcaax-1, a gene whose product can be used as a molecular marker for several morphogenetic events. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole mount immunocytochemistry and immunoperoxidase staining of tissue sections showed that during development the xlcaax-1 protein accumulation was coincident with the differentiation of the epidermis, pronephros and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein was localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein served as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. The xlcaax-1 protein was most abundant in kidney and immunogold EM analysis showed that the xlcaax-1 protein was highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. The xlcaax-1 protein was also localized in other ion transporting epithelia. The localization pattern and preliminary functional assays of xlcaax-1 suggest that the protein may function in association with an ion transport channel or pump.^ Cell migration and cell-cell interactions play important roles in numerous processes during morphogenesis. One of these is the formation of the pronephric (wolffian) duct (PD), which connects the pronephros to the cloaca. It is currently accepted that in most amphibians the pronephric duct is formed by active migration of the pronephric duct rudiment (PDR) cells along a pre-determined pathway. However, there is evidence that in Xenopus, the PD may be formed entirely by in situ segregation of cells out of the lateral mesoderm. In this study, we showed, using PDR ablation and X. laevis - X. borealis chimeras, that PD elongation in Xenopus required both active cell migration and an induced recruitment of cells from the posterior lateral plate mesoderm. We also showed that PDR cell migration was limited to only a few stages during development and that this temporal control is due, at least in part, to changes in the competence of the PD pathway to support cell migration. In addition, our data suggested that an alkaline phosphatase (APase) adhesion gradient may be involved in determining this competence. ^

Spontaneous peristaltic airway contractions propel lung liquid through the bronchial tree of intact and fetal lung explants

Spontaneous contractions of the fetal airways are a well recognized but poorly characterized phenomenon. In the present study spontaneous narrowing of the airways was analyzed in freshly isolated lungs from early to late gestation in fetal pigs and rabbits and in cultured fetal mouse lungs. Propagating waves of contraction traveling proximal to distal were observed in fresh lungs throughout gestation which displaced the lung liquid along the lumen. In the pseudoglandular and canalicular stages (fetal pigs) the frequency ranged from 2.3 to 3.3 contractions/min with a 39 to 46% maximum reduction of lumen diameter. In the saccular stage (rabbit) the frequency was 10 to 12/min with a narrowing of approximately 30%. In the organ cultures the waves of narrowing started at the trachea in whole lungs, or at the main bronchus in lobes (5.2 +/- 1.5 contractions/min, 22 +/- 8% reduction of lumen diameter), and as they proceeded distally along the epithelial tubes the luminal liquid was shifted toward the terminal tubules, which expanded the endbuds. As the tubules relaxed the flow of liquid was reversed. Thus the behavior of airway smooth muscle in the fetal lung is phasic in type (like gastrointestinal muscle) in contrast to that in postnatal lung, where it is tonic. An intraluminal positive pressure of 2.33 +/- 0.77 cm H(2)O was recorded in rabbit fetal trachea. It is proposed that the active tone of the smooth muscle maintains the positive intraluminal pressure and acts as a stimulus to lung growth via the force exerted across the airway wall and adjacent parenchyma. The expansion of the compliant endbuds by the fluid shifts at the airway tip may promote their growth into the surrounding mesenchyme.

Furosemide inhibits 11beta-hydroxysteroid dehydrogenase type 2.

11Beta-hydroxsteroid dehydrogenase 2 (11beta-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11beta-OHSD2 might enhance urinary loss of potassium. As diuretics influence the renal handling of potassium, we analyzed the impact of 13 commonly used diuretics on 11beta-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 micromol when extracts from COS-1 cells transfected with human 11beta-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11beta-OHSD2 and 11beta-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11beta-OHSD2 in intact distal tubules and 11beta-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide i.p., and the ratio of tetrahydrocorticosterone plus 5alpha-tetrahydrocorticosterone to 11-dehydrotetrahydrocorticosterone was determined in urine. This ratio increased after the administration of furosemide in all animals, indicating inhibition of the oxidative activity of 11beta-OHSD. Thus, furosemide inhibits the 11beta-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11beta-hydroxyglucocorticoids.

The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

AIMS As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

Detection and quantification of leptospires in urine of dogs: a maintenance host for the zoonotic disease leptospirosis.

Leptospirosis is a global zoonotic disease. Pathogenic Leptospira species, the causative agent of leptospirosis, colonize the renal tubules of chronically infected maintenance hosts such as dogs, rats and cattle. Maintenance hosts typically remain clinically asymptomatic and shed leptospires into the environment via urine. In contrast, accidental hosts such as humans can suffer severe acute forms of the disease. Infection results from direct contact with infected urine or indirectly, through contaminated water sources. In this study, a quantitative real-time PCR specific for lipL32 was designed to detect the urinary shedding of leptospires from dogs. The sensitivity and specificity of the assay was evaluated using both a panel of pathogenic Leptospira species and clinical microbial isolates, and samples of urine collected from experimentally infected rats and non-infected controls. The lower limit of detection was approximately 3 genome equivalents per reaction. The assay was applied to canine urine samples collected from local dog sanctuaries and the University Veterinary Hospital (UVH) at University College Dublin. Of 525 canine urine samples assayed, 37 were positive, indicating a prevalence of urinary shedding of leptospires of 7.05%. These results highlight the need to provide effective canine vaccination strategies and raise public health awareness.

Polycystic Kidneys and GM2 Gangliosidosis-Like Disease in Neonatal Springboks (Antidorcas marsupialis)

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 μm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 μm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies.

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo. However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Direct observation of molecular arrays in the organized smooth endoplasmic reticulum.

BACKGROUND Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. RESULTS Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. CONCLUSION Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.

Human 3β-hydroxysteroid dehydrogenase deficiency seems to affect fertility but may not harbor a tumor risk: lesson from an experiment of nature.

CONTEXT 3β-hydroxysteroid dehydrogenase deficiency (3βHSD) is a rare disorder of sexual development and steroidogenesis. There are two isozymes of 3βHSD, HSD3B1 and HSD3B2. Human mutations are known for the HSD3B2 gene which is expressed in the gonads and the adrenals. Little is known about testis histology, fertility and malignancy risk. OBJECTIVE To describe the molecular genetics, the steroid biochemistry, the (immuno-)histochemistry and the clinical implications of a loss-of-function HSD3B2 mutation. METHODS Biochemical, genetic and immunohistochemical investigations on human biomaterials. RESULTS A 46,XY boy presented at birth with severe undervirilization of the external genitalia. Steroid profiling showed low steroid production for mineralocorticoids, glucocorticoids and sex steroids with typical precursor metabolites for HSD3B2 deficiency. The genetic analysis of the HSD3B2 gene revealed a homozygous c.687del27 deletion. At pubertal age, he showed some virilization of the external genitalia and some sex steroid metabolites appeared likely through conversion of precursors secreted by the testis and converted by unaffected HSD3B1 in peripheral tissues. However, he also developed enlarged breasts through production of estrogens in the periphery. Testis histology in late puberty revealed primarily a Sertoli-cell-only pattern and only few tubules with arrested spermatogenesis, presence of few Leydig cells in stroma, but no neoplastic changes. CONCLUSIONS The testis with HSD3B2 deficiency due to the c.687del27 deletion does not express the defective protein. This patient is unlikely to be fertile and his risk for gonadal malignancy is low. Further studies are needed to obtain firm knowledge on malignancy risk for gonads harboring defects of androgen biosynthesis.

Evaluation of the Morphological Characteristics of Laser-Irradiated Dentin

OBJECTIVE The aim of this study was to investigate the effect of different energy settings of Er:YAG laser irradiation on dentin surface morphology with respect to the number of opened dentinal tubules. BACKGROUND DATA An ideally prepared dentin surface with opened dentinal tubules is a prerequisite for adhesive fixation. No study, however, has yet compared the numbers of opened dentinal tubules with regard to statistical differences. METHODS Conventional preparations using a bur with or without additional acid etching acted as control groups. Dentin specimens were prepared from human third molars and randomly divided into eight groups according to the energy settings of the laser (1, 1.5, 4, 6, 7.5, and 8 W) and two controls (bur and bur plus acid etching). After surface preparation, dentin surfaces were analyzed with a scanning electron microscope, and the number of opened dentinal tubules in a defined area was counted. RESULTS The control groups showed smooth surfaces with (bur plus acid etching) and without opened dentinal tubules (bur), whereas all laser-irradiated surfaces showed rough surfaces. Using the energy setting of 4 W resulted in significantly more opened dentinal tubules than the conventional preparation technique using the bur with additional acid etching. In contrast, the energy setting of 8 W showed significantly fewer opened dentinal tubules, and also exhibited signs of thermal damage. CONCLUSIONS The Er:YAG laser with an energy setting of 4 W generates a dentin surface with opened dentinal tubules, a prerequisite for adhesive fixation.

Analysis of Wnt/beta-catenin signaling in kidney tubulogenesis

The β-catenin/Lef/Tcf-mediated Wnt pathway is central to the developmental of all animals, stem cell renewal, and cancer progression. Prior studies in frogs and mice have indicated that the ligand Wnt-4 is essential for the mesenchyme to epithelial transition that generates tubules in the context of kidney organogenesis. More recently, Wnt-9b in mice, was likewise found to be required. Yet despite the importance of Wnt signals in renal development, the corresponding Frizzled receptor(s) and downstream signaling mechanim(s) are unclear. My work addresses these knowledge gaps using in vitro (Madin-Darby Canine Kidney cells) and in vivo (Xenopus laevis and zebrafish pronephros) tubulogenic kidney model systems. Employing established reporter constructs of Wnt/β-catenin pathway activity, I have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions. I have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/β-catenin pathway using β-Engrailed and dnTCF-4, constructs that suppress this pathway. I have further found that MDCK cells express the Frizzled-6 receptor, and that Wnt-4 forms a biochemical complex with Frizzled-6, yet does not appear to transduce Wnt-4's canonical signal. Additionally, I demonstrate that standard Hepatocyte Growth Factor (HGF)-mediated (non-physiologic) induction of MDCK tubulogenesis in collagen matrices is not altered by activation or suppression of β-catenin signaling activity; however, β-catenin signaling maintains cell survival in this in vitro system. Using a Wnt/β-catenin signaling reporter in Xenopus laevis, I detect β-catenin signaling activity in the early pronephric epithelial kidney tubules. By inhibiting the Wnt/β-catenin signaling pathway in both zebrafish and Xenopus , a significant loss of kidney tubulogenesis is observed with little or no effect on adjoining axis or somite development. This inhibition also leads to the appearance of severe edema that phenocopies embryos depleted for Wnt-4. Tubulogenic loss does not appear to be caused by increased cell death in the Xenopus pronephric field, but rather by lessened expression of tubule epithelium genes associated with cellular differentiation. Together, my results show that Wnt/β-catenin signaling is required for renal tubule development and that Wnt-4 is a strong candidate for activating this pathway. ^

Composition and age of bottom sediments from the Southern Ocean

Geomorphology, geology, stratigraphy, lithology and geochemistry of bottom sediments in the South Ocean are under consideration. Regularities of distribution of iron-manganese nodules, features of occurrence of ore components in the nodules, nodule abundance in bottom sediments have been studied.

Complex gangliosides are essential in spermatogenesis of mice: Possible roles in the transport of testosterone

Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC 2.4.1.94) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides in vivo.