Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.

Characterization of the sodium/hydrogen exchanger NHA2

Cation/proton exchange has been recognized for decades in mammalian mitochondria, but the exchanger proteins have eluded identification. In this study, a cDNA from a human brain library, previously designated NHA2 in the genome, was cloned and characterized. The NHA2 transcript bears more similarity to prokaryotic than known eukaryotic sodium/proton exchangers, but it was found to be expressed in multiple mammalian organs and cultured cells. A mAb to NHA2 was generated and found to label an approximately 55-kD native protein in multiple tissues and cell lines. The specificity of this antibody was confirmed by demonstrating the loss of the native NHA2 band on immunoblots when cultured cells were treated with NHA2-specific small interfering RNA. Although NHA2 protein was detected in multiple organs, within each, its expression was restricted to specific cell types. In the kidney, co-localization with calbindin 28k and reverse transcription-PCR of microdissected tubules revealed that NHA2 is limited to the distal convoluted tubule. In cell lines, native NHA2 was localized both to the plasma membrane and to the intracellular compartment; immunogold electron microscopy of rat distal convoluted tubule demonstrated NHA2 predominantly but not exclusively on the inner mitochondrial membrane. Furthermore, co-sedimentation of NHA2 antigen and mitochondrial membranes was observed with differential centrifugation, and two mitochondrial markers co-localized with NHA2 in cultured cells. Regarding function, human NHA2 reversed the sodium/hydrogen exchanger-null phenotype when expressed in sodium/hydrogen exchanger-deficient yeast and restored the ability to defend high salinity in the presence of acidic extracellular pH. In summary, NHA2 is a ubiquitous mammalian sodium proton/exchanger that is restricted to the distal convoluted tubule in the kidney.

Velocity recovery cycles of human muscle action potentials and their sensitivity to ischemia

This study was undertaken to test whether recovery cycle measurements can provide useful information about the membrane potential of human muscle fibers. Multifiber responses to direct muscle stimulation through needle electrodes were recorded from the brachioradialis of healthy volunteers, and the latency changes measured as conditioning stimuli were applied at interstimulus intervals of 2-1000 ms. In all subjects, the relative refractory period (RRP), which lasted 3.27 +/- 0.45 ms (mean +/- SD, n = 12), was followed by a phase of supernormality, in which the velocity increased by 9.3 +/- 3.4% at 6.1 +/- 1.3 ms, and recovered over 1 s. A broad hump of additional supernormality was seen at around 100 ms. Extra conditioning stimuli had little effect on the early supernormality but increased the later component. The two phases of supernormality resembled early and late afterpotentials, attributable respectively to the passive decay of membrane charge and potassium accumulation in the t-tubules. Five minutes of ischemia progressively prolonged the RRP and reduced supernormality, confirming that these parameters are sensitive to membrane depolarization. Velocity recovery cycles may provide useful information about altered muscle membrane potential and t-tubule function in muscle disease. Muscle Nerve, 2008.

GABARAP deficiency modulates expression of NaPi-IIa in renal brush-border membranes

Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.

Dentin hypersensitivity: pain mechanisms and aetiology of exposed cervical dentin

OBJECTIVES The paper's aim is to review dentin hypersensitivity (DHS), discussing pain mechanisms and aetiology. MATERIALS AND METHODS Literature was reviewed using search engines with MESH terms, DH pain mechanisms and aetiology (including abrasion, erosion and periodontal disease). RESULTS The many hypotheses proposed for DHS attest to our lack of knowledge in understanding neurophysiologic mechanisms, the most widely accepted being the hydrodynamic theory. Dentin tubules must be patent from the oral environment to the pulp. Dentin exposure, usually at the cervical margin, is due to a variety of processes involving gingival recession or loss of enamel, predisposing factors being periodontal disease and treatment, limited alveolar bone, thin biotype, erosion and abrasion. CONCLUSIONS The current pain mechanism of DHS is thought to be the hydrodynamic theory. The initiation and progression of DHS are influenced by characteristics of the teeth and periodontium as well as the oral environment and external influences. Risk factors are numerous often acting synergistically and always influenced by individual susceptibility. CLINICAL RELEVANCE Whilst the pain mechanism of DHS is not well understood, clinicians need to be mindful of the aetiology and risk factors in order to manage patients' pain and expectations and prevent further dentin exposure with subsequent sensitivity.

Sp1 trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.

The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro.

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.