Prevalence and densitometric characteristics of incomplete distal renal tubular acidosis in men with recurrent calcium nephrolithiasis

The aim of this study was to assess the prevalence of incomplete distal renal tubular acidosis (idRTA) in men with recurrent calcium nephrolithiasis and its potential impact on bone mineral density. We conducted a retrospective analysis of 150 consecutive, male idiopathic recurrent calcium stone formers (RCSFs), which had originally been referred to the tertiary care stone center of the University Hospital of Berne for further metabolic evaluation. All RCSFs had been maintained on a free-choice diet while collecting two 24-h urine samples and delivered second morning urine samples after 12 h fasting. Among 12 RCSFs with a fasting urine pH >5.8, a modified 3-day ammonium chloride loading test identified idRTA in 10 patients (urine pH >5.32, idRTA group). We matched to each idRTA subject 5 control subjects from the 150 RCSFs, primary by BMI and then by age, i.e., 50 patients, without any acidification defect (non-RTA group) for comparative biochemistry and dual energy X-ray absorptiometry (DEXA) analyses. The prevalence of primary idRTA among RCSFs was 6.7% (10/150). Patients with idRTA had significantly higher 2-h fasting and 24-h urine pH (2-h urine pH: 6.6 ± 0.4 vs. 5.2 ± 0.1, p = 0.001; 24-h urine pH: 6.1 ± 0.2 vs. 5.3 ± 0.3, p = 0.001), 24-h urinary calcium excretion (7.70 ± 1.75 vs. 5.69 ± 1.73 mmol/d, p = 0.02), but significantly lower 24-h urinary urea excretion (323 ± 53 vs. 399 ± 114 mmol/d, p = 0.01), urinary citrate levels (2.32 ± 0.82 vs. 3.01 ± 0.72 mmol/d, p = 0.04) and renal phosphate threshold normalized for the glomerular filtration rate (TmPO(4)/GFR: 0.66 ± 0.17 vs. 0.82 ± 0.21, p = 0.03) compared to non-RTA patients. No significant difference in bone mineral density (BMD) was found between idRTA and non-RTA patients for the lumbar spine (LS BMD (g/cm(2)): 1.046 ± 0.245 SD vs. 1.005 ± 0.119 SD, p = 0.42) or femoral neck (FN BMD (g/cm(2)): 0.830 ± 0.135 SD vs. 0.852 ± 0.127 SD). Thus, idRTA occurs in 1 in 15 male RCSFs and should be sought in all recurrent calcium nephrolithiasis patients. Bone mineral density, however, does not appear to be significantly affected by idRTA.

Synthesis, maturation, and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Cerebral formation in situ of S-carboxymethylcysteine after ifosfamide administration to mice: a further clue to the mechanism of ifosfamide encephalopathy

The clinical use of the alkylating oxazaphosphorine ifosfamide is hampered by a potentially severe encephalopathy. S-carboxymethylcysteine (SCMC), a metabolite of ifosfamide (IF), activates the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor, causes neuronal acidification, and could thus be responsible for the encephalopathy. Since the presence of SCMC in brain has not been documented following administration of IF, SCMC was measured in the brain of mice following both the individual i.p. administration of IF and SCMC. SCMC was found in a concentration of 108.2 +/- 29.7 nmol/g following IF, but was detectable at much lower levels following the administration of SCMC (21.1 +/- 21.2 nmol/g). Together with the observation that the concentration of SCMC was 10-fold higher in liver than in brain 1h after administration of SCMC, these findings suggest that the SCMC found after IF was formed in the brain in situ. The concentration of glutamic acid was similar in IF and SCMC treated animals. Methylene blue, which is used clinically to treat and to prevent IF encephalopathy, did not decrease the formation of SCMC in brain. By inhibiting monoamine oxidase activity it did, however, markedly increase the concentration of serotonin in brain which could modulate the effects of SCMC on AMPA/kainate receptors. Thus, SCMC is present in brain following the administration of IF and could contribute to the IF-associated encephalopathy by activation of AMPA/kainate receptors.

Effects of experimental nitrogen additions on plant diversity in an old-growth tropical forest

Response of plant biodiversity to increased availability of nitrogen (N) has been investigated in temperate and boreal forests, which are typically N-limited, but little is known in tropical forests. We examined the effects of artificial N additions on plant diversity (species richness, density and cover) of the understory layer in an N saturated old-growth tropical forest in southern China to test the following hypothesis: N additions decrease plant diversity in N saturated tropical forests primarily from N-mediated changes in soil properties. Experimental additions of N were administered at the following levels from July 2003 to July 2008: no addition (Control); 50 kg N ha−1 yr−1 (Low-N); 100 kg N ha−1 yr−1 (Medium-N), and 150 kg N ha−1 yr−1 (High-N). Results showed that no understory species exhibited positive growth response to any level of N addition during the study period. Although low-to-medium levels of N addition (≤100 kg N ha−1 yr−1) generally did not alter plant diversity through time, high levels of N addition significantly reduced species diversity. This decrease was most closely related to declines within tree seedling and fern functional groups, as well as to significant increases in soil acidity and Al mobility, and decreases in Ca availability and fine-root biomass. This mechanism for loss of biodiversity provides sharp contrast to competition-based mechanisms suggested in studies of understory communities in other forests. Our results suggest that high-N additions can decrease plant diversity in tropical forests, but that this response may vary with rate of N addition.

The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction

Mutations in the B1 subunit of the multisubunit vacuolar ATPase cause autosomal-recessive distal renal tubular acidosis and sensorineural deafness. Here, we report a novel frameshift mutation that truncates the C-terminus of the human B1 subunit. This mutant protein failed to assemble with other subunits in the cytosol to form the complex that can be targeted to vesicular structures in mammalian cells. Loss of proton pump activity was demonstrated in a functional complementation assay in B-subunit null yeast. The mutation caused loss of a discreet C-terminal region critical for subunit interaction not related to the C-terminal PDZ motif. Co-expression studies failed to demonstrate dominant negative effects of this truncated mutant over wild-type B1. Analysis of 12 reported B1 subunit missense mutations showed one polymorphic allele had intact pump function, two point mutants had intact assembly but defective proton pumping, and the remaining nine had disrupted assembly with no pump function. One presumed polymorphic allele was actually an inactivating mutation. Our study shows that multiple mechanisms of pump dysfunction result from B1 subunit mutations with a common outcome being defective assembly. Polymorphisms of the B1 subunit in the general population may affect renal acidification and urinary chemistry.

Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1(-/-) skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K(1/2)) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K(1/2) for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K(1/2) for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K(1/2) for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in "parallel" at alkaline pH. In the second "serial" model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.

Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton

The annexins are a family of Ca(2+)- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca(2+)](i) or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca(2+) entry (SOCE), but did not influence the rates of Ca(2+) extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca(2+) entry as long as [Ca(2+)](i) was below the threshold of annexin A6-membrane translocation. However, when [Ca(2+)](i) reached the levels necessary for the Ca(2+)-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca(2+) entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca(2+) entry, with consequences for the rates of cell proliferation.

Vergleich der umweltrelevanten Faktoren von Holzfurnierlagenverbundwerkstoffen (WVC – Wood Veneer Composite) und metallischen Werkstoffen am Praxisbeispiel eines Skidfördersystems

Für eine Beurteilung von Produkten bzw. Produktsystemen im Maschinenbau spielen neben technischen Kennwerten immer mehr die Umweltauswirkungen der Systeme eine wichtige Rolle. Diese Anforderungen haben die Nachfrage für nachhaltige und umweltfreundliche Konstruktionswerkstoffe im Maschinenbau erhöht. Eine Möglichkeit für solche ökologisch vorteilhaften Werkstoffe stellen ausgewählte Holzwerkstoffe dar. Mit diesen Holzwerkstoffen sollen technische Produkte entwickelt werden, welche den Unternehmen die Möglichkeit eröffnet, ihren unternehmerischen Beitrag zur Nachhaltigkeit zu steigern und wirtschaftliche Vorteile zu erzielen. Durch diesen Ansatz ist ein gewisses Maß an Ressourcen- und Energieeffizienz verbunden, dass sich kurzfristig und / oder langfristig wirtschaftlich lohnt. Ein damit verbundener gesellschaftlicher Imagegewinn erzeugt einen zusätzlichen Nutzen. Als sogenannte GLP (Green Logistics Plant) wird diese Art der Holzkonstruktion gegenwärtig im Bereich der Fördertechnik entwickelt und angewendet. Ein Anwendungsbeispiel innerhalb der GLP stellt das Gestellsystem für einen Skidförderer dar. Um die ökologische Wirkung der Konstruktionswerkstoffe transparent und nachvollziehbar zu untersuchen, werden vordergründig die Kategorien des Treibhauspotenzials und des (Primär-) Energieaufwandes genutzt. Weiterhin werden die Wirkungskategorien Versauerung, Eutrophierung, Sommersmog und Ozonabbau analysiert. Ergänzend zu bestehenden Untersuchungen soll die ökologische Vorteilhaftigkeit von Holzfurnierlagenverbundwerkstoffe (Wood Veneer Composite – WVC), Baustahl, verzinktem Stahl und Aluminiumlegierungen in der Lebensphase Produktion untersucht werden. Anschließend werden die Ergebnisse auf das Gestell eines Skid-Fördersystems aus WVC und Baustahl übertragen.

Status and Causal Pathway Assessments Supporting River Basin Management

The European Water Framework Directive (WFD) requires a status assessment of all water bodies. If that status is deteriorated, the WFD urges the identification of its potential causes in order to be able to suggest appropriate management measures. The instrument of investigative monitoring allows for such identification, provided that appropriate tools are available to link the observed effects to causative stressors, while unravelling confounding factors. In this chapter, the state of the art of status and causal pathway assessment is described for the major stressors responsible for the deterioration of European water bodies, i.e. toxicity, acidification, salinisation, eutrophication and oxygen depletion, parasites and pathogens, invasive alien species, hydromorphological degradation, changing water levels as well as sediments and suspended matter. For each stressor, an extensive description of the potential effects on the ecological status is given. Secondly, stressor-specific abiotic and biotic indicators are described that allow for a first indication of probable causes, based on the assessment of available monitoring data. Subsequently, more advanced tools for site-specific confirmation of stressors at hand are discussed. Finally, the local status assessments are put into the perspective of the risk for downstream stretches in order to be able to prioritise stressors and to be able to select appropriate measures for mitigation of the risks resulting from these stressors.

Proton flux across artificial vesicle bilayers

The study of proton conductance across artificial membranes has revealed a surprisingly high permeability for H+, (Pnet H+). A high Pnet H+ is difficult to reconcile with the biological requirement for the maintenance of pH gradients across the plasma membranes of cells, organellar study was undertaken to examine the role played by cholesterol and phospholipid fatty acid side chain composition in determining how well a membrane will function as a barrier to acid. The effects of counter-ion movement on acidification rates were examined in order to interpret the data obtained from variations in membrane composition. In phosphate buffered saline solutions, vesicle membranes composed of unsaturated fatty acid phosphatidylcholines proved to be poorer barriers to acid than membranes composed of saturated fatty acids. The barrier properties of these membranes could be ranked in the following order: DPL, (palmitic) $>$ Egg PC, (mixed chains) $>$ DLL, (linoleic), with DPL being the most effective in maintaining a one pH unit gradient near neutrality. Cholesterol decreased acidification rates of membranes made from the unsaturated phosphatidylcholines Egg PC and DLL, but enhanced acidification rates in vesicle membranes composed of the saturated phospholipid DPL. The cholesterol and fatty acid side chain effects were mediated by changes in membrane fluidity, with more rigid bilayers forming better barriers to acid. Experimental evidence was obtained which confirmed the Pnet H+ is very high relative to the permeabilities of other ions. Counter-ion controlled acidification rates depended on the size and charge of the ion which was moving in order to maintain electroneutrality. The biological relevance of a high intrinsic Pnet H+ and the possible role of counter-ion controlled acidification were discussed. ^

Comment on “The phase relation between atmospheric carbon dioxide and global temperature” Humlum et al. [Glob. Planet. Change 100: 51–69.]: Isotopes ignored

A recent study relying purely on statistical analysis of relatively short time series suggested substantial re-thinking of the traditional view about causality explaining the detected rising trend of atmospheric CO2 (atmCO2) concentrations. If these results are well-justified then they should surely compel a fundamental scientific shift in paradigms regarding both atmospheric greenhouse warming mechanism and global carbon cycle. However, the presented work suffers from serious logical deficiencies such as, 1) what could be the sink for fossil fuel CO2 emissions, if neither the atmosphere nor the ocean – as suggested by the authors – plays a role? 2) What is the alternative explanation for ocean acidification if the ocean is a net source of CO2 to the atmosphere? Probably the most provocative point of the commented study is that anthropogenic emissions have little influence on atmCO2 concentrations. The authors have obviously ignored the reconstructed and directly measured carbon isotopic trends of atmCO2 (both δ13C, and radiocarbon dilution) and the declining O2/N2 ratio, although these parameters provide solid evidence that fossil fuel combustion is the major source of atmCO2 increase throughout the Industrial Era.

Twentieth Century Increase of Atmospheric Ammonia Recorded in Mount Everest Ice Core

An NH4+ record covering the period A.D. 1845-1997 was reconstructed using an 80.4 m ice core from East Rongbuk Glacier at an elevation of 6450 m on the northern slope of Mount Everest. Variations in NH4+ are characterized by a dramatic increase since the 1950s. The highest NH4+ concentrations occur in the 1980s. They are about twofold more than those in the first half of twentieth century. Empirical orthogonal function (EOF) analysis on the eight major ion (Na+,K+,Mg2+,NH4+,Ca2+,NO3-,SO42- and Cl-) series from this core indicates that NH4+ is loaded mainly on EOF3 (60% of NH4+ variance), suggesting that NH4+ has a unique signature. Instrumental sea level pressure (SLP) and regional temperatures are used to explore the relationship between NH4+ variations and both atmospheric circulation and natural source strength over Asia. Higher NH4+ concentrations are associated with an enhanced winter Mongolian High and a deepened summer Mongolian Low. A positive relationship also exists between NH4+ concentrations and regional temperature changes of the GIS Box 36 (Indian subcontinent), indicating that an increase in temperature may contribute to the strengthening of natural ammonia emissions (e. g., from plants and soils). A close positive correlation between NH4+ and acidic species (SO42- plus NO3-) concentrations suggests that a portion of the increase in NH4+ concentrations could be contributed by enhanced atmospheric acidification. Anthropogenic ammonia emissions from enhanced agricultural activities and energy consumption over Asia in concert with population increase since the 1950s appear also to be a significant factor in the dramatic increase of NH4+ concentrations during the last few decades.

Long-Term and Seasonal Variations in CO2: Linkages to Catchment Alkalinity Generation

As atmospheric emissions of S have declined in the Northern Hemisphere, there has been an expectation of increased pH and alkalinity in streams believed to have been acidified by excess S and N. Many streams and lakes have not recovered. Evidence from East Bear Brook in Maine, USA and modelling with the groundwater acid-base model MAGIC (Cosby et al. 1985a,b) indicate that seasonal and yearly variations in soil PCO2 are adequate to enhance or even reverse acid-base (alkalinity) changes anticipated from modest decreases of SO4 in surface waters. Alkalinity is generated in the soil by exchange of H+ from dissociation of H2CO3, which in turn is derived from the dissolving of soil CO2. The variation in soil PCO2 produces an alkalinity variation of up to 15 mu eq L-1 in stream water. Detecting and relating increases in alkalinity to decreases in stream SO4 are significantly more difficult in the short term because of this effect. For example, modelled alkalinity recovery at Bear Brook due to a decline of 20 mu eq SO4 L-1 in soil solution is compensated by a decline from 0.4 to 0.2% for soil air PCO2. This compensation ability decays over time as base saturation declines. Variable PCO2 has less effect in more acidic soils. Short-term decreases of PCO2 below the long-term average value produce short-term decreases in alkalinity, whereas short-term increases in PCO2 produce shortterm alkalization. Trend analysis for detecting recovery of streams and lakes from acidification after reduced atmospheric emissions will require a longer monitoring period for statistical significance than previously appreciated.

Increasing Silicon Concentrations in Bohemian Forest Lakes

Long-term trends of dissolved silicon (Si) concentrations in five glacial lakes in the Bohemian Forest, Czech Republic, recovering from acidification show higher mobility of Si from the soil to surface waters despite lower atmospheric deposition of acids. Si increased by 0.95 to 1.95 mu mol yr(-1) (36 to 51%) from 1986-2004 and with increasing pH. A change in soil solution conditions because of a sharp decrease in acidic deposition has led to marked decline in Al mobility and to considerable decreases in dissolved Al, especially Al3+. The increase in Si may be related to: (1) unblocking of the inhibitory effect of dissolved Al on weathering of alummosilicates, (2) biogenic opal (phytoliths) dissolving faster, and/or (3) lower Si precipitation as secondary alummosilicates in soil. The change in Al speciation on the dissolution rate of biogenic silica is critical. A lack of change in Si at sites outside central Europe may be explained by small or no decline in mobility of dissolved Al. The effect of a long-term increase in temperature was probably minor.

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.