Physical exercise in patients with severe kidney disease.

Patients with advanced chronic kidney disease (CKD), especially those on long-term dialysis, often suffer from muscle wasting and excessive fatigue. It is known that inactivity, muscle wasting and reduced physical functioning are associated with increased mortality in CKD. Known causes include uraemic myopathy and neuropathy, inactivity, and anaemia. Exercise in patients receiving regular dialysis treatment for end-stage renal disease was first introduced 3 decades ago, but is still only offered in a minority of renal units around the world, despite a significant body of evidence to support its use. Work is needed to increase awareness of the potential benefits of increased physical activity for patients with advanced CKD. This review summarizes the mechanisms of exercise intolerance and debility in advanced CKD patients, the methods used for the estimation of functional capacity, the options currently available for exercise training, and their influence on the well-being of this group of patients.

Geomorphic and geologic controls of geohazards induced by Nepal's 2015 Gorkha earthquake.

The Gorkha earthquake (magnitude 7.8) on 25 April 2015 and later aftershocks struck South Asia, killing ~9000 people and damaging a large region. Supported by a large campaign of responsive satellite data acquisitions over the earthquake disaster zone, our team undertook a satellite image survey of the earthquakes' induced geohazards in Nepal and China and an assessment of the geomorphic, tectonic, and lithologic controls on quake-induced landslides. Timely analysis and communication aided response and recovery and informed decision-makers. We mapped 4312 coseismic and postseismic landslides. We also surveyed 491 glacier lakes for earthquake damage but found only nine landslide-impacted lakes and no visible satellite evidence of outbursts. Landslide densities correlate with slope, peak ground acceleration, surface downdrop, and specific metamorphic lithologies and large plutonic intrusions.

Regulation of phosphate metabolism in human red cells following prolonged incubation to steady state in vitro.

Human red cells were incubated aseptically in vitro for 24 or 48 h to allow the cellular concentrations of orthophosphate (Pi) and organic phosphates to attain steady state. In plasma at pH 7.0-8.0, the transmembrane Pi concentration ratio R (cellular Pi/plasma Pi) decreased with increasing pH, with a slope which was 2.7-times greater than that predicted if Pi simply distributed passively across the cell membrane. The concentration of 2,3-bisphosphoglycerate (2,3-BPG), the most abundant cytosolic organic phosphate, decreased at acidic pH and increased at alkaline pH, but stabilised at these values after 24 h. Therefore, while net generation or consumption of Pi by 2,3-BPG may initially have contributed to the steep dependence of R on pH, some other factor must have maintained this anomaly after 24 h. In plasma in which the Pi concentration was increased from 1 to 2.5 mM, the cellular Pi concentration increased from 0.6 to only 1.0 mmol/l cells, and 2,3-BPG increased by less than 20%. Thus, cellular Pi and 2,3-BPG concentrations seemed to be buffered or regulated in the face of changes in extracellular Pi. However, this regulation failed in a Pi-free balanced salt solution, as the 2,3-BPG concentration declined to half that observed in freshly drawn blood, although cell Pi remained at about 0.3 mM. Incubation in Pi-free solution with ouabain for 24 h to decrease the transmembrane sodium gradient, or incubation for 2 h in the absence of sodium, decreased this residual cellular Pi by about 20%, but did not abolish it. In Pi-free solution, but not with 1 mM Pi, cellular Pi increased when passive transmembrane Pi leakage was inhibited with 4-acetamido-4'-iso-thiocyanatostilbene-2,2'-disulphonate (SITS). We conclude that red cell Pi concentration cannot be explained fully by passive transmembrane distribution of Pi, nor by changes in 2,3-BPG, and that part of the anomaly may arise from sodium-linked active Pi transport.

Net fluxes of orthophosphate across the plasma membrane in human red cells following alteration of pH and extracellular Pi concentration.

Even though net fluxes of Pi (orthophosphate) across the cell membrane may be important in clinical disorders involving the abnormal extracellular Pi concentration, in acid-base disturbances, and in the responses of some cells to hormones, relatively few studies have been made of these fluxes, owing to the complexities of interpretation. Here we have studied net fluxes in response to changes in extracellular pH and Pi concentration in the simple case of the human red cell. The permeability of the cell membrane to net Pi fluxes was described in terms of a first-order rate constant, epsilon. By means of a mathematical model, it was possible to discriminate between transmembrane Pi movement, net intracellular generation or consumption of Pi by organic phosphates, and extracellular generation of Pi from the cells lysing during the experiment. We show that net Pi influx into the cell during experimental alkalosis was probably driven by net consumption of Pi by organic phosphates, and that this was reversed during acidosis. Inhibition of net Pi influx by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) suggests that, like Pi self-exchange, net influx is at least partly mediated by the band 3 transport protein. Unexpectedly, epsilon increased from 2 h-1 at extracellular pH 7.4 to approx. 7 h-1 at pH 7.8. From the value of epsilon at pH 7.4, we conclude that the apparent buffering or regulation of steady-state Pi concentrations, previously reported in red cells in vitro, was not an artifact of intracellular generation of Pi from organic phosphates.

Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralization: mineralization by betaglycerophosphate in the absence of osteoblasts.

It has been shown in several laboratories that addition of beta-glycerophosphate (beta GP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM beta GP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone explants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from beta GP in all wells. beta GP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in beta GP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular phosphate metabolism in patients receiving bisphosphonate therapy.

Patients with Paget's disease of bone were treated with oral disodium dihydrogen ethylidene-1-hydroxy-1,1-bisphosphonate (EHBP), a drug that is known to stimulate renal tubular reabsorption of orthophosphate (Pi). After 2 weeks of treatment, plasma Pi rose from 1.02 to 1.67 mmol/l. No increase in Pi was observed with the related drug, dichloromethylene bisphosphonate, which also reduces bone turnover in Paget's disease. Intravenous EHBP caused a more rapid increase in plasma Pi, but maximum hyperphosphatemia was not observed until 7-11 days after treatment commenced. It is therefore unlikely that this effect is due to an immediate action of EHBP on the luminal face of the renal brush border Pi transporter. After 2 weeks of oral EHBP, the increase in the Pi concentration in patients' erythrocytes was 31% compared with 64% in plasma. In blood platelets and leukocytes, negligible changes in cellular Pi occurred. The concentrations of 2,3-diphosphoglycerate, adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) were unaltered, indicating that these organic phosphates were not offsetting a potential change in cellular Pi. The decrease in erythrocyte/plasma distribution ratio for Pi was also observed in patients receiving intravenous EHBP. However, no change occurred in cell/plasma distribution of chloride, suggesting that this apparent regulation of cellular Pi did not arise from changes in erythrocyte membrane potential, pH, or water content.

Phosphorus-31 nuclear magnetic resonance studies of pig adrenal glands.

We have used phosphorus-31 nuclear magnetic resonance to study the secretion of adenosine 5'-triphosphate (ATP) from the medulla of perfused pig adrenal glands. The resonances of the nucleotide pools in the chromaffin granules and cytoplasm are clearly resolved and therefore the intragranular and cytoplasmic processes involving ATP can be monitored simultaneously in the gland during secretion. Secretion of nucleotide during a 3 h continuous stimulation by infusion of acetylcholine chloride was monitored by the decrease in intensity of the intragranular ATP resonances. Up to 40% of the total intragranular nucleotide was released under these conditions. The rate of secretion decreased with duration of stimulation. No significant changes in the steady-state levels of cytoplasmic ATP or in oxygen consumption were observed. The intragranular pH in ischaemic glands was 5.52 + 0.15, while in glands which had been perfused until their cytoplasmic nucleotide levels had recovered, the intragranular pH was 5.76 +/- 0.16. These results provide the first estimate of the internal pH of the chromaffin granules in intact perfused adrenal glands and show that no net acidification occurs in the presence of cytoplasmic ATP. However, the isolated chromaffin granule possesses a proton-pumping adenosine 5'-triphosphatase which, in the presence of a permeant counter-ion, such as chloride, acidifies the granule interior. It is, therefore, suggested that in the intact cell, the cytoplasmic concentration of permeant counter-ions is too low to allow electrically neutral proton accumulation in the granules.

Enhanced oxygen consumption in adrenal medulla on stimulation with acetylcholine.

When perfused cortex-free ox adrenal medulla was stimulated to secrete catecholamine by infusion of 0.1 mM acetylcholine for 4 min, the oxygen consumption increased to a value which was 0.15 +/- 0.07 mumole O2/min/g wet weight (+/- S.D., N = 12) above the pre-stimulation value of 0.49 +/- 0.15 (P less than 0.001). 1.4 +/- 0.9 (+/- S.D., N = 12) mole of catecholamine was secreted per mole of enhanced O2 consumption in the 16 min following the start of the stimulation. The rate of ATP hydrolysis by the proton-translocating Mg-ATPase of the chromaffin granule may increase on fusing with the plasma membrane of the chromaffin cell during exocytosis. However, from the amount of catecholamine secreted, this was estimated to account for less than 17% of the oxygen consumption increase. The amount of catecholamine secreted by 4 min 0.1 mM acetylcholine stimulations correlated with the enhancement of oxygen consumption (r = 0.82, P less than 0.001) but, on stimulation with 60 microM veratridine for 4 min, O2 consumption enhancement was anomalously low. This dependence on mode of stimulation suggests that ATP consumption in exocytosis itself is an inadequate explanation. Ouabain-sensitive oxygen consumption rose from undetectable levels to 18 +/- 8% (+/- S.D., N = 4) of the basal respiration during prolonged 0.1 mM acetylcholine stimulation in the absence of Ca, indicating that Na,K-ATPase was not responsible for all of the oxygen consumption enhancement.

Declining catecholamine secretion in adrenal medulla on prolonged stimulation with acetylcholine.

Perfused pig adrenal glands and cortex-free ox adrenal medullae were stimulated by continuous infusion of 10(-4) M acetylcholine (ACh). Secretion of adrenaline rose to a maximum in approximately 5 min but, after a further 15 min, declined to 36 +/- 19% (+/- S.D.) of maximum for pig (N = 5) and 27 +/- 10% of maximum for ox (N = 3), in spite of continued infusion of ACh. After 20 min, no further significant decline was detectable. Nevertheless, in ox medullae, oxygen consumption measured after stimulation showed no significant change relative to the pre-stimulation value, indicating that the decline in secretion did not arise from a failure of oxidative energy metabolism. In 4 pig adrenal glands subjected to a 1 hr infusion of ACh, adrenaline secreted in the last 20 min was only 52 +/- 10% of that secreted in the first 20 min but, after a 2 hr rest interval, recovered to 74 +/- 18% (P less than 0.05) in the first 20 min of a second 1 hr stimulation. In the same glands, no reproducible recovery was detectable for noradrenaline and, by the last 20 min of the second 1 hr stimulation, noradrenaline secretion had declined to 36 +/- 20% of the initial value, even though only 15% of the noradrenaline originally in the gland had been secreted. It is concluded that, while decline and recovery of adrenaline secretion may, in part, have arisen from desensitization and resensitization of the ACh receptor; the decline in noradrenaline secretion arose mainly from depletion of a readily secreted pool which was considerably smaller than the total in the gland.

Leucine suppresses acid-induced protein wasting in L6 rat muscle cells.

BACKGROUND: Metabolic acidosis induces protein wasting in skeletal muscle cells, accompanied by decreased glycolysis and compensatory increased consumption of other metabolic fuels, implying that protein wasting arises from fuel starvation and might be rectified by fuel supplements. Design To test this hypothesis, total protein and protein degradation (release of 14C-phenylalanine) were measured in L6 skeletal muscle cells cultured in Eagle's Minimum Essential Medium at pH 7.1-7.5 for 3 days with metabolic inhibitors or metabolic fuel supplements. RESULTS: Inducing metabolic fuel starvation with inhibitors (1 mmol L(-1) 2-deoxyglucose or 0.1 mmol L(-1) KCN [potassium cyanide]) failed to stimulate protein degradation or net protein wasting under nonacidaemic conditions (pH 7.5). Conversely metabolic fuel supplements (1 mmol L(-1) octanoate, pyruvate or alanine) failed to increase the protein content of the cultures at any pH tested, in spite of significant consumption of the fuels by the cells. Only leucine (1-3 mmol L(-1)) increased protein content and suppressed protein degradation in opposition to the catabolic effect of acidaemia (pH 7.1). Conclusion Leucine exerts a beneficial anabolic effect on cultured skeletal muscle cells in the face of metabolic acidaemia. The failure of other metabolic fuels to do this, and of the metabolic inhibitors to exert a catabolic effect, suggests that leucine acts as a specific modulator of protein turnover and not as a nonspecific source of carbon for oxidation as a fuel.

Hypertrophy of proximal tubular epithelial cells induced by low pH in vitro is independent of ammoniagenesis.

Metabolic acidosis can lead to tubular hypertrophy in vivo. This is thought to arise from stimulation of renal production of ammonia, a known hypertrophic agent. To examine this effect in vitro, confluent opossum (OK) proximal tubular epithelial cells were cultured at acidic pH (7.21 +/- 0.02) or at control pH (7.37 +/- 0.01) for 4 days. Protein content was 9% higher at acidic pH whereas DNA content was unaffected. The resulting increase in mean cell size (protein/DNA ratio) was 10% but correlated inversely with the mass of cells in control wells, varying from +48% at low cell mass to -14% at high cell mass. In contrast, low pH decreased 3H-thymidine incorporation by 9%. However, ammonia production was unaffected. These changes in protein/DNA ratio and 3H-thymidine incorporation cannot therefore be attributed to acid-induced ammoniagenesis and imply that low pH exerts a more direct effect on tubular cell growth than previously envisaged.

Regulation of intracellular creatine in erythrocytes and myoblasts: influence of uraemia and inhibition of Na,K-ATPase.

The regulation of intracellular creatine concentration in mammalian cells is poorly understood, but is thought to depend upon active sodium-linked uptake of creatine from extracellular fluid. In normal human erythrocytes, creatine influx into washed cells was inhibited by 40 per cent in the absence of extracellular sodium. In washed cells from uraemic patients, sodium-independent creatine influx was normal, whereas the sodium-dependent component of creatine influx was 3.3 times higher than normal, possibly reflecting the reduced mean age of uraemic erythrocytes. In spite of this, the intracellular creatine concentration was no higher than normal in uraemic erythrocytes, implying that some factor in uraemic plasma in vivo inhibits sodium-dependent creatine influx. Both in normal and uraemic erythrocytes, the creatine concentration was 10 times that in plasma, and the concentration in the cells showed no detectable dependence on that in plasma, suggesting that the intracellular creatine concentration is controlled by an active saturable process. Active sodium-dependent accumulation of creatine was also demonstrated in L6 rat myoblasts and was inhibited when transport was measured in the presence of 10(-4) M ouabain or digoxin, implying that uptake was driven by the transmembrane sodium gradient. However, when creatine influx was measured immediately after ouabain or digoxin had been washed away, it was higher than in control cells, suggesting that Na,K-ATPase and/or sodium-linked creatine transport are up-regulated when treated with inhibitors of Na,K-ATPase.

Theoretical interpretation of isotope labelling experiments in cells in which the label is chemically incorporated: the example of orthophosphate.

Studies of transport across the plasma membrane in intact cells frequently involve measuring the incorporation of a labelled extracellular species into the cells. Unfortunately, if the labelled species is metabolized in the cell, the kinetics of labelling are made more complicated. Using the example of the incorporation of 32P-labelled orthophosphate into cells, we describe a mathematical model which allows for this complication, and show how this may alter the interpretation of experiments. The analysis is widely applicable to cellular labelling studies with any species that undergoes chemical exchange with a large cellular pool.

Creatine and creatinine metabolism in uraemia.

Measurements of serum creatinine to monitor the progression of chronic renal failure may be misleading and influenced by factors other than GFR. Creatinine is produced by the degradation of creatine which is stored in muscle. The effect of biochemical changes observed in uraemia on the transport of [1-14C]creatine were examined in a cultured muscle model. In a Na-free medium flux of [1-14C]creatine was reduced (p < 0.001), however the addition of various uraemic toxins, insulin, parathyroid hormone and the alteration of pH had no effect on creatine influx. The variance of red cell creatine levels in uraemic patients was significantly different from controls (p < 0.02) and the plasma to red cell creatine gradient was low in some patients.

Erythrocyte phosphate metabolism and pH in vitro: a model for clinical phosphate disorders in acidosis and alkalosis.

When whole blood from 9 normal donors was incubated at 37 degrees C for 90 min under 95%, O2/5% CO2, over a range of pH from 7.1 to 7.9, the concentrations of orthophosphate (Pi) in plasma, in whole blood and in the cells decreased with increasing pH. At all pHs the ratio of cell to plasma concentrations of Pi was higher than that predicted from the cell to plasma distribution ratio for chloride, on the assumption that the mono- and di-anionic forms of Pi distributed passively in response to the membrane potential. Both the observed and predicted distribution ratios for Pi, and the difference between them, decreased with increasing pH. It is concluded that the observed distribution of Pi between erythrocytes and plasma is not consistent with a steady-state passive distribution, and that small changes of pH in vitro can lead to marked alterations in cellular Pi concentration. These measurements provide a direct example of the redistribution of Pi between cells and plasma, which has been postulated to occur in glycolyzing cells, leading to Pi depletion, during pH disturbances in vivo.

Inhibition of protein synthesis by acid in L6 skeletal muscle cells: analogies with the acute starvation response.

Impaired protein synthesis (PS) occurs in skeletal muscle during acute starvation. Even though it is well established that uraemic metabolic acidosis (MA) stimulates protein degradation (PD) and is a major contributor to skeletal muscle wasting in chronic renal failure, the accompanying effects of MA on PS are much less clear. Previous work has shown that, in cultured L6 skeletal muscle cells, PD and leucine oxidation are stimulated by acid. The aim of the present study was to determine whether acid (like acute starvation) can also inhibit PS. PS (14C-phenylalanine incorporation) was measured in L6 cells in MEM + 2% serum at acid pH (7.1) or control pH (7.5). After 24 h, acid inhibited PS (7.7 +/- 0.2 vs. 8.9 +/- 0.1 nmol Phe/4 h/35-mm culture well in controls, p = 0.01) and this was maintained at 72 h. In vitro this could arise because acid only inhibits the rapid PS occurring in dividing cells. However, when division was abolished with 10(-5) mol/l cytosine arabinoside, PS inhibition by acid still occurred (6.9 +/- 0.1 vs. 8.3 +/- 0.2 at control pH, p < 0.05). Acid also had no effect on the specific radioactivity of cellular phenylalanine, suggesting that the impaired PS was not a consequence of inadequate labelling of this pool. Elevated PD and impaired PS together led to loss of 7% of the total protein in only 28 h (-21 +/- 3 microg/well, p = 0.004). This combination of impaired PS with increased PD and increased leucine oxidation in response to acid resembles the response of skeletal muscle to acute starvation. These superficial similarities between the starvation state and MA suggest that fundamental metabolic signals may occur which are common to both states.

Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast-like cells: effect of Pi, Na+ and K+.

Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106-01 and UMR 106-06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106-01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [32Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular 32P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106-01 and DET, but the decrease was smaller than that in [32Pi]-uptake. Ouabain decreased [32Pi]-uptake and cellular [Pi] in DET, but not in UMR 106-01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts.

Metabolic acidosis is a potent stimulus for cellular inorganic phosphate generation in uraemia.

1. During metabolic acidosis, significant fluxes of inorganic phosphate (Pi) may occur from cellular to extracellular fluid. In this study Pi was measured in erythrocytes of uraemic patients before and after haemodialysis and was related to their plasma pH (acidosis), plasma Pi (hyperphosphataemia) and cellular organic phosphate concentrations. 2. Before dialysis, the ratio of cellular to extracellular Pi concentration correlated inversely with plasma pH, increasing 2.5-fold as pH fell from 7.4 to 7.2. 3. An increase in cellular Pi similar to that seen in the patients was observed within 90 min of adding acid to normal erythrocytes in vitro. 4. The total Pi content of the cell suspension increased 25% on decreasing plasma pH from 7.4 to 7.2, largely as a result of generation of Pi from 2,3-bisphosphoglycerate in the cells. This was accompanied by net efflux of Pi into plasma. 5. In addition, the increase in the steady-state cellular Pi concentration on adding a constant extracellular Pi load was 50% greater at pH 7.2 than at 7.4, implying that alterations in the regulation of the transmembrane Pi gradient also contribute to the rise in cellular Pi observed at low pH. 6. At normal plasma Pi concentration (1 mM), glycolytic flux (lactate production) was inhibited by 20% when pH was lowered from 7.4 to 7.2. However, this inhibition was blocked when cellular Pi was increased by adding Pi to the plasma in vitro. 7. Metabolic acidosis is therefore a potent stimulus for Pi generation in erythrocytes, and this Pi may serve to stimulate glycolysis which is normally inhibited by low pH.

32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.