A search for a defect of proximal transport in denervated kidneys of conscious dogs

The influence of renal nerves on proximal Na+ reabsorption was studied in clearance experiments with unilaterally renal-denervated conscious dogs prepared by surgical bladder division. Two types of experiments were made : A. maximal water diuresis, and B. Total blockade of distal NaCl reabsorption with ethacrynic acid and chlorothiazide. In maximal water diuresis CH2O + CNa was used as a measure of fluid delivery to the distal nephron. At similar GFR on both sides, the proximal reabsorption estimated as GFR--(CH2O + CNa) was 38.4 +/- 5.6 ml/min for the intact and 35.9 +/- 4.2 ml/min for the denervated kidney (n = 6, difference NS). After distal tubular blockade, proximal Na+ reabsorption calculated as filtered load minus urinary excretion was 3.84 +/- 0.43 mmol/min for the intact and 3.91 +/- 0.36 mmol/min for the denervated kidney (n = 6, difference NS). The fractional reabsorption of NA+ was 64.9 +/- 1.0% for the intact and 66.9 +/- 1.1% for the denervated kidney (difference NS). In contrast to data from renal denervation studies with anaesthetized animals, the present experiments did not show any difference in proximal reabsorption between the innervated- and denervated kidney. We conclude that in absence of anaesthesia renal efferent nerves have no major effect on NaCl transport in dog proximal tubule.     

Nonsaturable amino acid reabsorption in kidneys of normal and mercury-poisoned rabbits

At high plasma concentrations, a high-capacity, low-affinity or nonsaturable flux (Jhc) accounts for a residual fractional reabsorption of cycloleucine, aspartate, and AIB of approximately 50% of the filtered load in rabbits; Jhc in micromoles per milliliter glomerular filtrate is reduced in Hg-poisoned animals. The nonsaturable flux of cycloleucine is characterized by a transepithelial transit time (TET) of approximately 2 min in control animals; it was consistently much longer in Hg-poisoned animals. The clearance ratio of creatinine/inulin averaged 1.0, and no Jhc could be demonstrated for glucose. We conclude that Jhc is a high-capacity, low-affinity amino acid flux which passes through an intracellular solute pool, and which is sensitive to Hg at both the brush border and the basolateral cell membrane. If calculation of the saturation constants of aspartate reabsorption is restricted to experiments in which U/P less than 1.0, i.e. where Jhc is unlikely to contribute greatly to reabsorption, values some 20% lower than those previously reported are obtained; the Hg inhibition still is apparently uncompetitive in nature.     

Substrate utilization by the distal nephron in dogs with chronic metabolic acidosis: studies with ethacrynic acid

Glutamine and lactate oxidations provide the bulk of ATP required for sodium reabsorption in the dog kidney during chronic metabolic acidosis. Indirect evidence has suggested that glutamine is oxidized in the proximal convoluted tubule; if this is true, lactate should be the major fuel of the more distal nephron sites. The purpose of these experiments was to determine which substrates were metabolized by the acidotic dog kidney when a significant proportion of sodium chloride reabsorption was inhibited in the thick ascending limb of the loop of Henle. Ethacrynic acid, a loop diuretic, caused the fractional excretion of sodium to increase from 1 to 34%. The glomerular filtration rate declined somewhat, but there was no significant change in the renal blood flow rate. Renal oxygen consumption declined in conjunction with the natriuresis. However, when the data were examined at a constant filtered load of sodium (a constant rate of ATP turnover), there was no reduction in glutamine uptake or glutamine conversion to ATP in the presence of this natriuretic agent. The major change observed concerned lactate metabolism, in the presence of ethacrynic acid, there was no longer a significant rate of lactate extraction. These data are best explained by assuming that glutamine is the fuel of the proximal convoluted tubule of the acidotic dog kidney, whereas lactate oxidation occurs principally in the nephron sites where sodium reabsorption was inhibited by ethacrynic acid.     

Study of the effect of seven-day immersion on the excretory function of the kidneys

The excretion of total protein, creatinine, urea, uric acid, glucose, potassium, sodium, calcium, phosphorus, and magnesium, as well as the uroproteinogram (12 groups of proteins) and uroenzymogram (five enzymes) parameters, was studied in the experiment with seven-day immersion in eight men aged 21–26 years. The results of the study allow a conclusion that seven-day immersion did not lead to any unfavorable changes in the renal function. The study of the uroproteinogram showed the absence of shifts in either glomerular filtration or tubular reabsorption, which agrees with the absence of significant changes in the uroenzymogram values. Even in significantly increased diuresis, the physiological norm of protein and glucose excretion was not exceeded; the electrolyte excretion was normalized quickly enough. An increased excretion of creatinine, urea, calcium, magnesium, and phosphorus appears to reflect activation of catabolic processes in skeletal muscles.     

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s?1) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.     

Effects of L-glucose on sodium reabsorption in the isolated perfused rat kidney.

In the isolated perfused rat kidney, sodium reabsorption is enhanced in the presence of 5.5 mM D-glucose. However, it is unclear whether this effect is metabolic or whether it is due to a requirement for sodium transport in the process of glucose reabsorption. A third possibility is solvent drag. In an attempt to differentiate between these possibilities, kidneys were perfused with the D-glucose isomer, L-glucose (L-G), a nonmetabolizable hexose. At a perfusate concentration of 5.5 mM L-G, per cent L-G reabsorption was approximately 30. Inhibition of L-G reabsorption by D-glucose suggests carrier-mediated transport. In the presence of 5.5 mM L-G, sodium reabsorption approximated 92% during the course of perfusion. When L-G was omitted from perfusate, sodium reabsorption ultimately declined to 85%. Since significant metabolism of L-G was not observed, the results are compatible with the hypothesis that enhanced sodium reabsorption may be brought about by some still to be defined aspect of glucose transport.     

Altered reabsorption of protein by the renal cortex in rats treated with hypertonic saline or mannitol.

The reabsorption of horseradish peroxidase (HRP) by the proximal tubule cells of rat kidneys was investigated by measuring the concentration of HRP in total particulate fractions of the cortex 1/4 and 1 hr after intravenous injection, and by correlated cytochemical observations. When compared to the corresponding values of the control animals, the concentration of HRP 1 hr after injection was decreased approximately 10-fold in the renal cortex of rats which had received an intravenous injection of hypertonic saline or two subcutaneous injections of mannitol. The plasma clearance and the urinary excretion of HRP were not altered significantly after injection of hypertonic saline, but the plasma clearance was decreased and the urinary excretion increased after injection of mannitol. When the dose of injected HRP was varied, the reabsorption of HRP by the renal cortex was proportional to the dose in the experimental and the control animals. Cytochemical staining for peroxidase activity also showed that the phagosomes and phagolysosomes of the proximal tubule cells contained much less peroxidase in the experimental rats than in the control rats. After injection of mannitol, large vacuoles appeared in the proximal tubule cells. The vacuoles often contained peroxidase-positive granules (phagosomes) which varied in diameter from the limit of microscopic visibility up to several microns. Most of the vacuoles did not react for acid phosphatase activity, but lysosomes were often aggregated around the vacuoles and seemed to release acid phosphatase into the cytoplasm. Certain analogies between the reabsorption of protein and that of water by the proximal tubule cells are discussed.     

Methane production from glucose in vitro by mixed rumen bacteria

1. Methane was produced in vitro by incubating cell suspensions of rumen bacteria with glucose, under nitrogen. The amount of methane produced varied considerably and was lowered by high glucose concentrations. Carbon dioxide, acetic acid, propionic acid, butyric acid and lactic acid were also produced. An oxidation–reduction balance of near unity could be calculated, although carbon recovery was low. Under the experimental conditions, rumen bacteria used most of the metabolic hydrogen produced during the oxidation of glucose to form lactic acid. 2. Lower methane production at high glucose concentrations was balanced by higher lactic acid production. Low pH values due to a high production rate of lactic acid might explain the inhibition of methane production. 3. No lactic acid, less methane, but considerably more propionic acid were formed when nitrogen was replaced by carbon dioxide in the incubation system.     

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of α-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released α-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.     

Changes in the blood chemistry of rainbow trout, Salmo gairdneri Rich, in relation to dietary protein level, and an anabolic steroid hormone, ethylestrenol

The effect of feeding isocaloric test diets containing 35, 45 and 55% protein, with and without inclusion of the anabolic steroid hormone, ethylestrenol, was studied in rainbow trout, Salmo gairdneri , in relation to blood chemistry. The control (no hormone) and experimental diets (hormone-supplemented), were fed for a total of 60 days, after which time all groups of fish were fed an identical commercial diet with no hormone for a further 30 days (withdrawal period). After 60 days, the body weights offish fed the 35 and 45% experimental diets were significantly greater than their respective controls, and after hormone withdrawal, increased growth was still apparent in the 35% experimental group.
No significant changes in serum amino acid nitrogen (AAN), protein, or glucose in relation to the dietary protein level, or inclusion of steroid, were observed after 30 days. Serum creatinine, however, increased with an increase in dietary protein, and was significantly higher in the 35% experimental group than the respective control. After 60 days, the most significant observation was the marked increase in serum glucose with an increase in dietary protein, but respective control and experimental values were not significantly different at this time. Following a 30-day withdrawal period, serum AAN in the 55% experimental group was significantly higher than the control, whereas serum protein, creatinine, and glucose stabilized to similar concentrations in all groups. Over the 90-day period of feeding, in both control and experimental groups, serum AAN and protein tended to increase, serum creatinine and glucose to decrease, whilst haematocrit remained constant. It is concluded that addition of ethylestrenol to trout diets has apparently little effect on serum metabolite concentrations and haematocrit, the most significant variations being related more to diet composition and duration of study.     

Modelling of simultaneous production of polygalacturonase and exopolysaccharide by Aureobasidium pullulans ATHUM 2915

The polymorphic fungus Aureobasidium pullulans ATHUM 2915, produced significant quantities of extracellular polygalacturonase and polysaccharide when grown, under controlled conditions, in liquid medium with pectin and glucose as carbon sources and nitrogen source as limited factor. Growth, substrate consumption and products formation were simulated by a structured mathematical model, which was compared with the experimental data from batch culture in a chemostat. This model was applied successfully in the study of some essential parameters influenced the process at various pH values.     

Optimization of Culture Conditions for Fermentation of Soymilk Using Lactobacillus casei by Response Surface Methodology

Soymilk was fermented with Lactobacillus casei, and statistical experimental design was used to investigate factors affecting viable cells of L. casei, including temperature, glucose, niacin, riboflavin, pyridoxine, folic acid and pantothenic acid. Initial screening by Plackett-Burman design revealed that among these factors, temperature, glucose and niacin have significant effects on the growth of L. casei. Further optimization with Box-Behnken design and response surface analysis showed that a second-order polynomial model fits the experimental data appropriately. The optimum conditions for temperature, glucose and niacin were found to be 15.77 °C, 5.23 and 0.63 g/L, respectively. The concentration of viable L. casei cells under these conditions was 8.23 log₁₀ (CFU/mL). The perfect agreement between the observed values and the values predicted by the equation confirms the statistical significance of the model and the model’s adequate precision in predicting optimum conditions.     

Application of a fiber-matrix model to transport in renal tubules

The effects of tight junction structure on water and solute fluxes across proximal tubular epithelium were examined with fiber-matrix equations previously derived by Curry and Michel (1980. Microvascular Research. 20:96-99). Using plausible estimates of tight junction fiber length and width the model predicts solute (Ps) and water permeability (Lp) coefficients that agree with the measured values. When fiber-matrix and pore models were compared for physiologically relevant ranges of matrix void fraction (80-98%) and pore radii (0-20 A), the fiber-matrix model predicted a 10-fold higher Lp/Ps ratio. Lp/Ps was most sensitive to small changes in tight junction structure when void fractions exceeded 90%. Void fractions of 96.5% and 97.1% predicted previously measured values for Lp and solute permeabilities in rat and rabbit proximal tubules. These values are consistent with void fractions and permeabilities of artificial membranes. The fiber-matrix tight junction model was incorporated into a model of reabsorption from the rat proximal tubule developed by Weinstein (1984). American Journal of Physiology. 247:F848-F862.) A void fraction of 98% predicted the experimental results for isosmotic reabsorption driven by active transport. Changing void fraction over the range of 97-99% produced a 50-75% change in predicted volume reabsorption with active transport. According to the fiber-matrix model: (a) solute permeabilities alone cannot be used to predict Lp, (b) previously measured solute permeabilities in the proximal tubule are compatible with significant water reabsorption through a water-permeable tight junction, and (c) hydraulic and solute permeabilities may be sensitive to small changes in tight junction fiber length and diameter or ionic strength within the tight junction.     

A pH Control System Based on Malate Decarboxylation for the Cultivation of Lactic Acid Bacteria

Most species of lactic acid bacteria decarboxylate l-malate to lactate and CO(2) if an energy source such as glucose is present. A proton is taken up in the reaction, which prevents pH decreases in the growth medium caused by lactic acid production from glucose fermentation. MRS broth (pH 7.0) (Difco Laboratories) containing 10 mM glucose and various concentrations of l-malate (0, 25, 50, 75, and 100 mM) was used to cultivate Lactobacillus plantarum. After 72 h at 37 degrees C, all malate was decarboxylated and all glucose was fermented, with resultant final pH values of 4.5, 6.3, 6.9, 7.3, and 7.5, respectively. When d-malate (which cannot be decarboxylated) was substituted for l-malate, the final pH values were 4.5, 5.2, 5.6, 5.8, and 5.9. By varying the ratios of glucose to l-malate in the growth medium, it was possible to obtain pH values which were lower, the same, or higher than the initial pH values. In contrast, buffers such as phosphate only retard decreases in pH. l-Malate, when compared with K(2)PO(4) on an equal molar basis, provided greater resistance to decreases in pH. Higher specific growth rates were observed for L. plantarum and Leuconostoc mesenteroides when l-malate rather than K(2)PO(4) was incorporated into the growth medium.     

Alveolar fluid reabsorption is impaired by increased left atrial pressures in rats

Cardiogenic pulmonary edema results from increased hydrostatic pressures across the pulmonary circulation. We studied active Na(+) transport and alveolar fluid reabsorption in isolated perfused rat lungs exposed to increasing levels of left atrial pressure (LAP; 0--20 cmH(2)O) for 60 min. Active Na(+) transport and fluid reabsorption did not change when LAP was increased to 5 and 10 cmH(2)O compared with that in the control group (0 cmH(2)O; 0.50 +/- 0.02 ml/h). However, alveolar fluid reabsorption decreased by approximately 50% in rat lungs in which the LAP was raised to 15 cmH(2)O (0.25 +/- 0.03 ml/h). The passive movement of small solutes ((22)Na(+) and [(3)H]mannitol) and large solutes (FITC-albumin) increased progressively in rats exposed to higher LAP. There was no significant edema in lungs with a LAP of 15 cmH(2)O when all active Na(+) transport was inhibited by hypothermia or amiloride (10(-4) M) and ouabain (5 x 10(-4) M). However, when LAP was increased to 20 cmH(2)O, there was a significant influx of fluid (-0.69 +/- 0.10 ml/h), precluding the ability to assess the rate of fluid reabsorption. In additional studies, LAP was decreased from 15 to 0 cmH(2)O in the second and third hours of the experimental protocol, which resulted in normalization of lung permeability to solutes and alveolar fluid reabsorption. These data suggest that in an increased LAP model, the changes in clearance and permeability are transient, reversible, and directly related to high pulmonary circulation pressures.     

Selecting the right blood glucose monitor for the determination of glucose during the enzymatic hydrolysis of corncob pretreated with different methods

In order to assess their accuracy for the determination of glucose during the enzymatic hydrolysis of pretreated lignocellulosic biomass, four different blood glucose monitors (BGMs), each utilizing a different enzymatic mechanism for the determination of glucose, were utilized in an experimental setup, which compares the efficiency of ionic liquid pretreatment with dilute acid and alkaline pretreatments applied on corncob. Among the tested devices, Optium Xceed was found to be the most accurate device for the determination of glucose where Accu-Chek Active was the least accurate BGM, yielding similar results to those obtained with DNS method. Based on the HPLC results, the % error values for Optium Xceed ranged between 3.9-10.5% for the determination of glucose concentration. Upon enzymatic hydrolysis, ionic liquid and alkaline pretreatments gave similar glucose yields, which were slightly higher than the dilute acid pretreatment, which were 31.9%, 31.0% and 27.8%, respectively, based on untreated corncob.     

Pyelonephritis alters the reabsorption of nutrients and brush border membrane enzymes of rat kidney

The uptake of nutrients and activities of membrane enzymes in the kidney were investigated using renal brush border membrane (BBM) vesicles in acute pyelonephritis in rats. A significant decrease (P less than 0.001) in the uptake of D-glucose and L-phenylalanine was observed in both the unobstructed right and obstructed left kidney, while there was a significant increase (P less than 0.001) in the uptake of L-alanine in the left kidney of pyelonephritic rats, demonstrating disturbances in the reabsorption of the glucose and aminoacids in the kidneys. Vmax of alkaline phosphatase, leucine-amino-peptidase and maltase was found to be decreased in the left kidney, suggesting that there was a reduction in the active enzyme molecule number. Km of alkaline phosphatase and leucine-aminopeptidase remained unchanged, while km of maltase decreased in both the right and left kidneys. An increase in the Vmax of alkaline phosphatase and leucine-aminopeptidase and substrate affinity of the maltase in the right kidney demonstrated a compensatory phenomenon for the malfunctioning of the left kidney. This is the first report demonstrating alterations in reabsorption of nutrients and BBM enzymes in experimental pyelonephritis.     

Blood changes in brook trout induced by infection with Aeromonas salmonicida.

Furunculosis was induced in brook trout, Salvelinus fontinalis, by experimental inoculation with Aeromonas salmonicida. Total protein, hemoglobin, sialic acid, fatty acids, triglycerides, cholesterol, inorganic-phosphorus, acid-soluble phosphorus, and lipid-phosphorus decreased in the blood of the infected fish while amino acids, urea, total creatinine, ammonia, and glucose increased. Pyruvic acid, lactic acid, and ascorbic acid values showed no significant change.     

Lack of D-amino-acid oxidase activity causes a specific renal aminoaciduria in the mouse

Thin-layer chromatography and amino acid analysis showed that urine of mutant ddY/DAO- mice lacking D-amino-acid oxidase activity contained more serine, proline, alanine and methionine than that of normal ddY/DAO+ mice. Among these four, an increase in alanine was conspicuous. However, the urinary levels of 11 other amino acids and glucose were not different between the ddY/DAO- and ddY/DAO+ mice. Amino acid analysis showed that the plasma levels of serine, proline and methionine were not elevated in the ddY/DAO- mice, though a slight increase in alanine was observed. Genetic crosses showed that aminoaciduria and lack of D-amino-acid oxidase activity were concomitantly transmitted as a set through generations. These results indicated that the lack of enzyme activity caused a specific renal aminoaciduria. Whether this enzyme merely diminishes the D-amino acid load presented for reabsorption, or actually participates catalytically in the reabsorption process, remains undetermined.     

Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens

Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic, acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during efficient succinic acid production from glucose. These models thus can be employed for the development and optimization of biobased succinic acid production processes.