Incomplete Distal Renal Tubular Acidosis from a Heterozygous Mutation of the V-ATPase B1 Subunit

Congenital distal renal tubular acidosis (dRTA) from mutations of the B1 subunit of the V-ATPase is considered an autosomal recessive disease. We analyzed a dRTA kindred with a truncation-mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of the V-ATPase. All heterozygous carriers in this kindred have normal plasma bicarbonate concentrations, thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria are present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also have inappropriate urinary acidification with acute ammonium chloride loading and impaired urine-blood pCO2 gradient during bicarbonaturia indicating presence of H+ gradient and flux defects. In normal human renal papillae, wild type B1 is located primarily on the plasma membrane but papilla from one of the heterozygote who had kidney stones had renal tissue secured from surgery showed B1 in both plasma membrane as well as a diffuse intracellular staining. Titrating increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+-pump activity of the wild type B1 in mammalian HEK293 cells and in V-ATPase-deficient S. cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of mutant B1 subunit; which cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.

Associations of Urinary Uromodulin with Clinical Characteristics and Markers of Tubular Function in the General Population

BACKGROUND AND OBJECTIVES Allelic variants in UMOD, the gene coding for uromodulin, are associated with rare tubulointerstitial kidney disorders and risk of CKD and hypertension in the general population. The factors associated with uromodulin excretion in the normal population remain largely unknown, and were therefore explored in this study. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Urinary uromodulin excretion was measured using a validated ELISA in two population-based cohorts that included more than 6500 individuals. The Swiss Kidney Project on Genes in Hypertension study (SKIPOGH) included 817 adults (mean age±SD, 45±17 years) who underwent renal ultrasonography and performed a 24-hour urine collection. The Cohorte Lausannoise study included 5706 adults (mean age, 53±11 years) with fresh spot morning urine samples. We calculated eGFRs using the CKD-Epidemiology Collaboration formula and by 24-hour creatinine clearance. RESULTS In both studies, positive associations were found between uromodulin and urinary sodium, chloride, and potassium excretion and osmolality. In SKIPOGH, 24-hour uromodulin excretion (median, 41 [interquartile range, 29-57] mg/24 h) was positively associated with kidney length and volume and with creatinine excretion and urine volume. It was negatively associated with age and diabetes. Both spot uromodulin concentration and 24-hour uromodulin excretion were linearly and positively associated (multivariate analyses) with eGFR<90 ml/min per 1.73 m(2). CONCLUSION Age, creatinine excretion, diabetes, and urinary volume are independent clinical correlates of urinary uromodulin excretion. The associations of uromodulin excretion with markers of tubular functions and kidney dimensions suggest that it may reflect tubule activity in the general population.

Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

Synaptic Vesicles Form by Budding from Tubular Extensions of Sorting Endosomes in PC12 Cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15°C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37°C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.

Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger AE1 have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in AE1. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in AE1 were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to AE1. In contrast, heterozygous mutations in AE1 were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a de novo mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in AE1 cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.

Biochemical evolution II: Origin of life in tubular microstructures on weathered feldspar surfaces

Mineral surfaces were important during the emergence of life on Earth because the assembly of the necessary complex biomolecules by random collisions in dilute aqueous solutions is implausible. Most silicate mineral surfaces are hydrophilic and organophobic and unsuitable for catalytic reactions, but some silica-rich surfaces of partly dealuminated feldspars and zeolites are organophilic and potentially catalytic. Weathered alkali feldspar crystals from granitic rocks at Shap, north west England, contain abundant tubular etch pits, typically 0.4–0.6 μm wide, forming an orthogonal honeycomb network in a surface zone 50 μm thick, with 2–3 × 106 intersections per mm2 of crystal surface. Surviving metamorphic rocks demonstrate that granites and acidic surface water were present on the Earth’s surface by ∼3.8 Ga. By analogy with Shap granite, honeycombed feldspar has considerable potential as a natural catalytic surface for the start of biochemical evolution. Biomolecules should have become available by catalysis of amino acids, etc. The honeycomb would have provided access to various mineral inclusions in the feldspar, particularly apatite and oxides, which contain phosphorus and transition metals necessary for energetic life. The organized environment would have protected complex molecules from dispersion into dilute solutions, from hydrolysis, and from UV radiation. Sub-micrometer tubes in the honeycomb might have acted as rudimentary cell walls for proto-organisms, which ultimately evolved a lipid lid giving further shelter from the hostile outside environment. A lid would finally have become a complete cell wall permitting detachment and flotation in primordial “soup.” Etch features on weathered alkali feldspar from Shap match the shape of overlying soil bacteria.

Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.

The goal of this study was to determine whether sphingosine and ceramide, second messengers derived from sphingolipid breakdown, alter kidney proximal tubular cell viability and their adaptive responses to further damage. Adult human kidney proximal tubular (HK-2) cells were cultured for 0-20 hr in the presence or absence of sphingosine, sphingosine metabolites (sphingosine 1-phosphate, dimethylsphingosine), or C2, C8, or C16 ceramide. Acute cell injury was assessed by vital dye exclusion and tetrazolium dye transport. Their subsequent impact on superimposed ATP depletion/Ca2+ ionophore-induced damage was also assessed. Sphingosine (> or = 10 microM), sphingosine 1-phosphate, dimethylsphingosine, and selected ceramides (C2 and C8, but not C16) each induced rapid, dose-dependent cytotoxicity. This occurred in the absence of DNA laddering or morphologic changes of apoptosis, suggesting a necrotic form of cell death. Prolonged exposure (20 hr) to subtoxic sphingosine doses (< or = 7.5 microM) induced substantial cytoresistance to superimposed ATP depletion/Ca2+ ionophore-mediated damage. Conversely, neither short-term sphingosine treatment (< or = 8.5 hr) nor 20-hr exposures to any of the above sphingosine/ceramide derivatives/metabolites or various free fatty acids reproduced this effect. Sphingosine-induced cytoresistance was dissociated from the extent of cytosolic Ca2+ loading (indo-1 fluorescence), indicating a direct increase in cell resistance to attack. We conclude that sphingosine can exert dual effects on proximal renal tubular viability: in high concentrations it induces cell necrosis, whereas in low doses it initiates a cytoresistant state. These results could be reproduced in human foreskin fibroblasts, suggesting broad-based relevance to the area of acute cell injury and repair.

The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

Avaliação do crescimento de Ankistrodesmus braunii em reator tubular empregando diferentes concentrações de nitrato em diferentes condições de cultivo

Microalgas são organismos unicelulares, eucariontes, fotossintetizantes e eficientes fixadores de gás carbônico que apresentam grande potencial para produção de ácidos graxos além de pigmentos, como os carotenóides e a clorofila, de interesse nas indústrias de alimentos, química, farmacêutica e de cosméticos. Dentre as microalgas, o microrganismo Ankistrodesmus braunii vem sendo citado como capaz de produzir grandes quantidades de lipídios, podendo corresponder a até 73% de sua massa seca, com produção de ácidos graxos insaturados, como o ácido linolênico. Esse microrganismo se destaca do ponto de vista industrial por poder ser conduzido em reatores e em meios de cultivo complexos. As fontes de nitrogênio, as concentrações empregadas destes nutrientes, bem como o tipo de processo de cultivo interferem na composição de biomassas fotossintetizantes. O uso de reatores tubulares tem sido estudado e tem se apresentado interessante por permitir a obtenção de altas concentrações celulares. Nesse sentido, este trabalho teve a finalidade de estudar o crescimento de Ankistrodesmus braunii em reator tubular com uso de diferentes quantidades de nitrato de sódio por processos descontínuo, descontínuo alimentado e semi-contínuo. Nos cultivos descontínuos, a máxima concentração celular (Xm) encontrada foi de 1588 ± 11 mg.L-1 com uso de 20 mM de NaNO3. O uso do processo descontínuo alimentado, o qual teve adição de 20 mM de NaNO3 feito num intervalo a cada 48 horas sendo iniciada a adição no primeiro dia, permitiu a obtenção de Xm = 2753 ± 7 mg.L-1; porém não foi possível eliminar a fase lag do cultivo, levando a uma produtividade em células (Px) de 351 ± 1 mg.L-1.dia-1. O processo semi-contínuo foi eficiente para eliminar a fase lag do cultivo, permitindo a obtenção de Xm = 2399 ± 5 mg.L-1 e um aumento de até 50% em Px, que chegou a valores de 525 ± 1 mg.L-1.dia-1 em cultivos com uso de 20 mM de NaNO3. Nesta condição os teores de proteínas e lipídios nas biomassas foram de 34,8 ± 0,2% e 38,6 ± 0,2%, respectivamente. Foi observado que, independentemente do tipo de processo empregado, há um decréscimo do valor do fator de conversão de nitrogênio em células (YX/N) com o aumento da adição de NaNO3. O maior valor de YX/N foi obtido no experimento com processo semi-contínuo e uso de 2 mM de NaNO3 no meio de cultivo, com valor médio de 29,1 ± 0,1 mg mg-1 ao final do segundo ciclo. Porém, nesta condição, o teor de proteínas da biomassa foi de 17,3 ± 0,4%. Já os maiores valores de YX/N encontrados nos processos descontínuo e descontínuo alimentado foram, respectivamente, de 22,5 ± 1,6 e 7,1 ± 0,1 mg mg-1. Os resultados obtidos neste trabalho evidenciam o potencial de Ankistrodesmus braunii como fonte de proteínas e lipídios para uso industrial.

Influência da densidade tubular em diferentes profundidades dentinárias na estabilidade de união de cimentos de iônomero de vidro

O objetivo do estudo foi avaliar a influência da densidade tubular em diferentes profundidades dentinárias na estabilidade de união de dois cimentos de ionômero de vidro (CIV) de alta viscosidade. Vinte terceiros molares foram alocados em 6 grupos experimentais, de acordo com a profundidade da dentina - proximal, oclusal superficial ou oclusal profunda, e os CIVs - Fuji IX (GC Corp.) e Ketac(TM) Molar Easy Mix (3M/ESPE). Inicialmente os dentes foram cortados a fim de se obter fatias de aproximadamente 1 mm de espessura de dentina proximal, oclusal superficial e profunda. Em seguida, foi realizado uma análise topográfica das secções das diferentes superfícies e profundidades em microscopia confocal a laser (100X) para obtenção das médias da densidade tubular em cada profundidade. Cânulas de polietileno foram então posicionadas sobre as secções de dentina pré-tratadas e preenchidas pelos CIVs. Os espécimes foram armazenados em água destilada por 24 h e 12 meses a 37°C, em seguida foram submetidos ao ensaio de microcisalhamento (0,5 mm/min). Após o ensaio, foi realizada a análise do padrão de fratura em estereomicroscópio (400X). Os dados obtidos foram submetidos à Análise de Variância para dados repetidos, seguido do teste de Tukey (?=5%). Verificamos que a densidade dos túbulos dentinários, em diferentes profundidades de molares permanentes, é inversamente proporcional a resistência de união de cimentos de ionômero de vidro de alta viscosidade. Foi ainda observado em todos os grupos que a resistência de união após 24 horas é maior do que em 12 meses, indicando degradação da interface adesiva ao longo do tempo.

Modelagem estocástica da dispersão axial: aplicação em um reator tubular de polimerização.

Reatores tubulares de polimerização podem apresentar um perfil de velocidade bastante distorcido. Partindo desta observação, um modelo estocástico baseado no modelo de dispersão axial foi proposto para a representação matemática da fluidodinâmica de um reator tubular para produção de poliestireno. A equação diferencial foi obtida inserindo a aleatoriedade no parâmetro de dispersão, resultando na adição de um termo estocástico ao modelo capaz de simular as oscilações observadas experimentalmente. A equação diferencial estocástica foi discretizada e resolvida pelo método Euler-Maruyama de forma satisfatória. Uma função estimadora foi desenvolvida para a obtenção do parâmetro do termo estocástico e o parâmetro do termo determinístico foi calculado pelo método dos mínimos quadrados. Uma análise de convergência foi conduzida para determinar o número de elementos da discretização e o modelo foi validado através da comparação de trajetórias e de intervalos de confiança computacionais com dados experimentais. O resultado obtido foi satisfatório, o que auxilia na compreensão do comportamento fluidodinâmico complexo do reator estudado.

Thermal Runaway in Tubular Reactor Systems with n-th Order Kinetics

This paper addresses the problem of predicting the critical parameters that characterize thermal runaway in a tubular reactor with wall cooling, introducing a new view of the n-th order kinetics reactions. The paper describes the trajectories of the system in the temperature-(concentration)n plane, and deduces the conditions for the thermal risk.

Wastewater demineralization by tubular reverse osmosis process /

Mode of access: Internet.