Anion transport regulates intracellular pH in renal cortical tissue.

The regulation of cell pH by anion transport was examined in suspensions of rabbit renal proximal tubules. Values for cell pH were derived from 14C-labeled 5,5-dimethyloxazolidine-2,4-dione distribution. In buffer with 10 mM/l HCO3-- and gassed with 95% O2/5% CO2, the anion transport inhibitors, 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene and furosemide, raised the cell-to-extracellular pH gradient from 0.23 +/- 0.02 to 0.31 +/- 0.02 and 0.31 +/- 0.03, respectively, but in combination their effects were not additive. Replacement of extracellular Cl-- by NO3-- raised the pH gradient from 0.24 +/- 0.04 to 0.37 +/- 0.05. Neither inhibitor raised the pH gradient in Cl-- -free media. Incubation of suspensions in HCO3-- and CO2-free media raised the pH gradient from 0.18 +/- 0.02 to 0.29 +/- 0.03. Removal of Cl-- in addition to HCO3-- and CO2 raised the pH gradient still further, to 0.36 +/- 0.02. The results demonstrate that two different anion transport inhibitors raise cell pH and the cell-to-extracellular pH gradient in proximal tubules and are consistent with the idea that the mechanism for this effect is inhibition of alkali anion exit from the tubule cell. This process appears to depend on extracellular Cl-- and probably occurs primarily by HCO3-- transport. The results support the concept that alkali anion transport, most probably HCO3-- exit from the peritubular cell border, is an important regulator of cell pH in renal proximal tubule.

Clinicopathologic, enzymatic, and genetic features in a case of Fabry's disease.

We report renal lesions and functional alterations in a 32-year-old man with Fabry's disease (ceramidetrihexosidase deficiency). By light microscopy of a renal biopsy specimen, distinctive "foamy" cytoplasmic alterations were observed in renal glomerular, tubular, vascular, and interstitial cells. Histochemical analysis of vacuolated epithelial cells showed glycolipid- and phospholipid-like material. Ultrastructurally, dense osmiophilic as well as stacked and concentric laminated profiles were observed within these epithelial cells. In addition, glomerular endocapillary, parietal, and vascular epithelial cells contained opaque osmiophilic granular deposits with paracrystalline arrays. Renal function studies indicated a glomerular filtration rate of 86.1 mL/min/1.73 sq m, effective renal plasma flow of 415 mL/min/1.73 sq m, tubular reabsorption of glucose of 356 mg/min/100 glomerular filtration rate, and maximal urinary concentrating and diluting ability of 568 and 46 mOsm/kg, respectively. Serum ceramide hexosidase activity was 0.18 nmole/hr/mL (normal, 8 to 15). We conclude that renal dysfunction associated with Fabry's disease is associated mainly with accumulation of glycolipid and phospholipid compounds in the walls of blood vessels and distal nephrons.

Flaking corn: processing mechanics, quality standards, and impacts on energy availability and performance of feedlot cattle.

Based on performance of feedlot cattle, steam flaking increases the value of corn by 18%, considerably more than is suggested by tabular values. Tabular values underestimate the energy availability of flaked corn by failing to account for digestibility of the nonstarch OM that is increased by flaking by the same magnitude (10%) as starch. Correcting for improvement in digestibility of nonstarch OM increases the NEg value of steam-flaked corn to 1.70 Mcal/kg, a value very close to values calculated from cattle performance trials. Digestibility of starch from corn grain is limited by the protein matrix that encapsulates starch granules, and by the compact nature of the starch itself. Disruption of the protein matrix (by shear forces on hot grain during flaking) is the first limiting step toward optimizing starch digestion. Five critical production factors influence the quality of steam-flaked corn: steam chest temperature, steaming time, roll corrugation, roll gap, and roll tension. For optimal shear, it is important that rolls be hot and that kernels be hot when flaked. Steam chests should be designed to allow a steaming time of at least 30 min at maximum roller mill capacity producing a flake of 0.31 kg/L (24 lb/bushel). As little as 5% moisture uptake during steaming appears adequate. The rate of flaking and distribution of kernels across the rolls also are critical. Quality standards for steam-flaked corn include measurements of flake thickness, flake density, starch solubility, and enzyme reactivity. Flake density, the most common quality standard, closely associated with starch solubility (r2 = 0.87) and enzyme reactivity (r2 = 0.79), still explains only 63% of the variability in percentage fecal starch and 52% of the variability in starch digestibility. Direct determination of fecal starch can explain 91% of the variability in starch digestion. The NEg value of corn can be predicted from fecal starch: NEg= 1.78 - 0.0184FS. Starch digestion is a Kappa Curve function of hot flake density, reaching a maximum at a flake density of approximately 0.31 kg/L. Flaking to a density of less than 0.31 kg/L, though increasing starch solubility, may reduce DMI, increase variability of weight gain among animals within a pen, and predispose cattle to acidosis and bloat without increasing starch digestion. We recommend that the steam-flaking process be optimized on the basis of fecal starch analysis.

Influence of dietary urea level on digestive function and growth performance of cattle fed steam-flaked barley-based finishing diets.

Four Holstein steers (282 kg) with cannulas in the rumen and proximal duodenum were used in a 4 x 4 Latin square experiment to evaluate the influence of dietary urea level (0, 0.4, 0.8, and 1.2%, DM basis) in a steam-flaked barley-based finishing diet on digestive function. There were no treatment effects (P > 0.20) on ruminal digestion of OM and ADF. Increasing dietary urea level increased (linear, P < 0.01) ruminal starch digestion. Ruminal degradability of protein in the basal diet (no supplemental urea) was 60%. Increasing dietary urea level did not increase (P > 0.20) ruminal microbial protein synthesis or nonammonia N flow to the small intestine. There were no treatment effects (P > 0.20) on total-tract ADF digestion. Total tract digestion of OM (quadratic, P < 0.01) and starch (linear, P < 0.05) increased slightly with increasing urea level. Urea supplementation increased (linear, P < 0.01) ruminal pH 1 h after feeding; however, by 3 h after feeding, ruminal pH was lower (cubic, P < 0.05) with urea-supplemented diets. Urea supplementation did not affect (P > 0.20) ruminal molar proportions of acetate and propionate. One hundred twenty crossbred steers (252 kg; approximately 25% Brahman breeding) were used in an 84-d feeding trial (five pens per treatment) to evaluate treatment effects on growth performance. Daily weight gain increased (linear, P = 0.01) with increasing urea level, tending to be maximal (1.53 kg/d; quadratic, P = 0.13) at the 0.8% level of urea supplementation. Improvements in ADG were due to treatment effects (linear, P < 0.01) on DMI. Urea supplementation did not affect (P > 0.20) the NE value of the diet for maintenance and gain. Observed dietary NE values, based on growth performance, were in close agreement with expected based on tabular values for individual feed ingredients, averaging 100.4%. We conclude that with steam-flaked barely-based finishing diets, ruminal and total-tract digestion of OM and ruminal microbial protein synthesis may not be increased by urea supplementation. In contrast, ADG was optimized by dietary inclusion of 0.8% urea. Urea supplementation may not enhance the net energy value of steam-flaked barely-based finishing diets when degradable intake protein is greater than 85% of microbial protein synthesis.

Starch digestion by feedlot cattle: predictions from analysis of feed and fecal starch and nitrogen.

To evaluate the utility of N as a digestion marker to predict total tract starch digestion, data from 32 metabolism trials involving 147 steers and 637 individual starch digestibility measurements were compiled. All trials were conducted at the University of California Desert Research and Extension Center. Total tract starch digestibility was determined from concentrations of starch and chromic oxide in feed and feces. In all trials, the steers were adapted to diets for 10 d followed by 4 d for collection of samples of feces. During collection, fecal samples (approximately 200 g, wet basis) were obtained twice daily. Samples from each steer within each collection period were composited for analysis. Diets contained 46.5 +/- 7.4% starch and 1.85 +/- 0.20% N. Apparently digestible N as a percentage of diet DM was closely associated (r(2) = 0.73; P < 0.001) with dietary N concentration. Fecal N concentration (FN, % of DM) explained 35% of the variation in fecal DM excretion (S(y.x) = 4.3; P < 0.001). Incorporating FN into the model, starch digestion was estimated as follows: starch digestion, % of intake = 100 {1 - [(0.938 -0.497FN + 0.0853FN(2)) FS/DS]}, where FS is fecal starch concentration (% of DM) and DS is dietary starch concentration (% of DM; r(2) = 0.94; S(y.x) = 0.68; P < 0.001). Fecal starch concentration alone explained 96% of the variation (S(y.x) = 0.45; P < 0.001) in total tract starch digestion: starch digestion, % = 99.9 - 0.413FS -0.0104FS(2). Omitting cases in our data set in which observed total tract starch digestion was less than 95%, the r(2) between FS and starch digestibility decreased to 0.82 (S(y.x) = 0.26; n = 529). However, estimated starch digestion using the equation incorporating FN remained closely associated with the observed starch digestion (r(2) = 0.90; S(y.x) = 0.22; P < 0.001; n = 529). Equations also were developed to predict NE(m) and NE(g) concentrations of common feed grains based on starch digestibility and FS. Starch digestion can be accurately predicted based on FS. However, incorporation of FN into the model markedly enhanced the estimates of grain quality and the efficacy of processing when total tract starch digestion exceeded 95%.

Influence of surfactant supplementation and maceration on the feeding value of rice straw in growing-finishing diets for Holstein steers.

Two trials were conducted to evaluate the interaction of the maceration process and surfactant (Tween 80) supplementation on feeding value of rice straw. Treatments were steam-flaked, corn-based diets containing 14% forage (DM basis), which was 1) Sudangrass hay; 2) ground rice straw; 3) ground rice straw plus 0.22% Tween 80; 4) macerated rice straw; and 5) macerated rice straw plus 0.22% Tween 80. In the maceration process, rice straw was passed through 2 sequentially placed pairs of corrugated rolls set at zero tolerance under a ram pressure of 62,050 millibars, similar to a conventional grain roller mill, except that the opposing rolls operated at different speeds (12 and 14 rpm, respectively). Sudangrass hay and rice straw (native and macerated) were ground through a 2.6-cm screen before incorporation into complete mixed diets. In trial 1, 125 Holstein steers (292 +/- 1.7 kg of BW) were used in a 188-d evaluation of the treatment effects on growth performance and carcass characteristics. In trial 2, 5 Holstein steers (224 +/- 3.5 kg of BW) with cannulas in the rumen and proximal duodenum were used in a 5 x 5 Latin square design to evaluate the treatment effects on digestion. There were no interactions between maceration and surfactant on growth or carcass characteristics. Tween 80 did not influence the feeding value of rice straw. Compared with grinding alone, maceration of rice straw increased the carcass-adjusted ADG (6%, P < 0.10), G:F (6%, P < 0.05), and dietary NE (5%, P < 0.05); DMI was similar across treatments. Assuming NE(m) and NE(g) of Sudangrass hay are 1.18 and 0.62 Mcal/kg, the NE(m) and NE(g) were 0.61 and 0.13 Mcal/kg for ground rice straw and 1.21 and 0.65 Mcal/kg for macerated rice straw. There were no treatment interactions on characteristics of digestion. Tween 80 did not influence ruminal or total tract digestion of OM, starch, NDF, or N. Compared with grinding alone, maceration of rice straw increased ruminal digestion of OM (7.7%, P < 0.10) and NDF (30.8%, P < 0.05), and total tract digestion of OM (2.3%, P < 0.10), NDF (21.1%, P < 0.01), and N (3.7%, P < 0.05). Total tract digestion of OM, NDF, starch, and N for the Sudangrass diet corresponded closely with that of the macerated rice straw diets. Maceration increases the feeding value of rice straw to a level similar to that of good-quality (flag stage of maturity) Sudangrass hay, which is attributable to increased OM and NDF digestion. Effects of surfactant supplementation on growth performance and digestion are not appreciable.

Sodium gradient-driven transport processes in ATP-depleted renal tubules.

The effects of directed Na+ gradients on proximal tubule cell transport processes were examined in suspensions of rabbit renal tubules depleted of ATP. Cells of high-Na+ content were generated by suspending the tubules in high-Na+ media, whereas low-Na+ cells were produced by incubating tubules in Na+-free media. Resuspension of the high-Na+ tubules in Na+-free media caused a fall in cell pH simultaneous with a fall in cell Na+. Resuspending the low-Na+ cells in Na+-replete media led to a rise in cell pH, in parallel with the rise in cell Na+. Removing HCO-3 and CO2 augmented and amiloride inhibited the increase in cell pH generated by the inward Na+ gradient. Low-Na+ tubules exposed to an inwardly directed Na+ gradient also concentrated the sugar analogue alpha-methylglucoside, and this uptake was blocked by phlorizin. These findings demonstrate the suitability of ATP-depleted renal tubules for the study of linked transport processes by providing evidence for the existence of the proximal luminal transport processes, Na+-H+ exchange, and Na+-sugar cotransport in this preparation.

Proximal tubule hydrogen ion transport processes in diuretic-induced metabolic alkalosis.

Transport systems involved in proximal tubule HCO-3 reabsorption were examined in disaggregated renal cortical tubules from rabbits with metabolic alkalosis. The acid-base disorder was induced by first treating the animals with furosemide, and then maintaining them on low Cl--high HCO-3 diets. On this regimen, the rabbits had increases in blood pH and total CO2 values and decreases in serum K+ concentrations. Urine Cl- concentrations were less than 15 mEq/L in all cases. Na+-H+ exchange was evaluated by incubating tubules in rotenone in an Na+-free medium to deplete them of Na+ and adenosine triphosphate. Then the tubules were resuspended in media containing 65 or 12.5 mEq/L Na+ at either pH 7.1 or pH 7.6. The rise in cell pH estimated by dimethadione distribution was taken as a measure of Na+-H+ exchanger activity. At the high incubation pH, Na+-H+ exchanger activity appeared to be the same in tubules taken from alkalotic rabbits compared with those prepared from normal rabbits. At the low incubation pH, the activity of this transport system appeared to be depressed by 40% to 50% in alkalosis, with kinetics that suggested a decreased Vmax for the exchanger. Na+-independent H+ transport, presumably reflecting activity of an H+-adenosine triphosphatase, was evaluated by preincubating tubules in a Na+-free medium in the presence of ouabain, and then sequentially exposing them to and removing them from a solution containing 20 mmol/L NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein.

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.

Cell pH and acid transport in renal cortical tissue.

Cell pH (pHc) was examined by the [14C]DMO technique in suspensions of proximal tubule fragments from rabbit renal cortex. In buffer with 10 mM HCO3(-), pHc was more alkaline than external pH (pHe) at values of the latter < 7.4. Maximal cell-to-extracellular pH gradients (delta pH) occurred at pHe = 6.8 and below. At pHe > 7.4, pHc was more acid than pHe was. However, pHc was always more alkaline than the electrochemical equilibrium pH. At pHe congruent to 7.0, 60 min of deoxygenation decreased delta pH from 0.22 +/- 0.02 to 0.05 +/- 0.01. Reoxygenation restored delta pH to control values. Incubation with ouabain abolished the delta pH. Both the carbonic anhydrase inhibitor, acetazolamide, and the anion transport inhibitor, 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS), increased delta pH. The studies demonstrate relative intracellular alkalinity in proximal tubule. A fall in pHc occurs with maneuvers that interfere with H+ pumping out of the cells. A rise in pHc occurs with maneuvers that interfere with the disposition of intracellular alkali: slowing of HCO3(-) generation with acetazolamide or blocking of HCO3(-) exit with SITS. The results support a H+-secretory model of proximal tubule acid transport that is dependent on maintenance and dispersal of intracellular alkalinity.