Uptake and phosphorylation of thiamine in rat kidney cortical slices. I. Effect of ethanol.

Processes involved in the disposition of thiamine within the kidney were studied in rat kidney slices. Uptake of [14C]thiamine and its metabolism to [14C]thiamine phosphate were measured with and without the presence of ethanol. Whereas slice to medium ratios of 3.41 +/- 0.11 indicated uphill movement of [14C]thiamine, metabolism to [14C]thiamine phosphate provided a metabolic sink for movement of thiamine into the cell. Accumulation was saturable and associated, in part, with the formation of thiamine phosphate. Ethanol, at 25 mM, a concentration compatible with alcohol abuse, significantly (P < .001) decreased the maximal accumulation of [14C]thiamine from 210 +/- 12.7 to 115 +/- 4.2 nmol/g and the production of thiamine phosphate from 0.44 to 0.041 nmol/g. These data indicate a facilitated uptake of thiamine and a conversion to thiamine phosphate by the kidney. The effect of ethanol to decrease thiamine accumulation in kidney tissue is suggested to be at the phosphorylating step.

Uptake and phosphorylation of thiamine in rabbit primary proximal tubule cells and Madin Darby canine kidney cells. II. Effect of ethanol.

Uptake of [14C]thiamine was studied in renal primary proximal tubule cells and in Madin Darby Canine Kidney cells in culture. Findings were compared with data for the accumulation of [14C]thiamine and its phosphorylation in renal cortical slices. There was a saturable component for thiamine uptake in all cell types. When normalized for milligrams of protein, renal cortical slices accumulated 82% less 14C than renal primary proximal tubule cells and 51% less than distal tubule cells. Maximal [14C]thiamine levels accumulated by the saturable component was 1.33 nmol/g in slices and 7.02 nmol/g in proximal tubule cells. Ethanol, at 25 mM, inhibited thiamine uptake and phosphorylation in the cell cultures similar to its effect in the kidney tissue.

Small animal magnetic resonance imaging: a means of studying the development of structural pathologies in the rat brain.

Small animal magnetic resonance imaging (SAMRI) was developed to detect structural tissue changes associated with disease states in animal models. The disease state of particular interest here is that associated with long-term alcohol abuse. The small animal model used for this study was the thiamine-deficient Sprague-Dawley rat, a model that provides a relatively rapid means of mimicking the ventriculomegaly frequently found in human chronic alcohol abusers. A custom-designed coil tuned to the magnetic field of a 1.5 Tesla clinical magnetic resonance imager provided the technology necessary to delineate discreet regions of the rat brain with clarity. Adult, male rats were imaged, placed on a thiamine-deficient pellet diet for approximately 6 weeks, and then reimaged. Treatment associated enlargement of the lateral ventricles identified in the images was verified by posttreatment histological analysis of the brains of these rats. The results demonstrated that SAMRI is capable of providing dramatic and reliable visual evidence of pathological structural changes in small tissue volumes with high resolution and reproducibility. Furthermore, the noninvasiveness of SAMRI allowed for imaging of the same animals over time, thereby reducing the numbers of animals needed for convincing documentation of the changes in ventricular size.

Regulation of Na+, K(+)-ATPase by chronic ethanol exposure of PC 12 cells.

The effects of chronic ethanol exposure on Na+, K(+)-ATPase were investigated in PC 12 cells. Inclusion of ethanol in the Na+, K(+)-ATPase assay (i.e. in vitro addition of ethanol) inhibited enzyme activity. Conversely, intrinsic Na+, K(+)-ATPase activity was increased after chronic ethanol exposure of the cells. This increase in Na+, K+ pumps occurred without any alteration in the inhibitory effects of in vitro ethanol. A similar response was observed when the chronic treatments were carried out using serum-free defined medium. The effects of other agents, which like ethanol decrease membrane order, were investigated. The addition of ketamine and tert-butanol in vitro caused a concentration-dependent inhibition of Na+, K(+)-ATPase activity. However, chronic exposure of the PC 12 cells to tert-butanol or ketamine did not alter either intrinsic Na+, K(+)-ATPase activity or the inhibitory effects of ethanol in vitro. Maintenance of PC 12 cells in medium containing ethanol resulted in an increase in the intracellular content of Na+ without any change in the K+ levels. In contrast, maintenance of the cells in medium containing tert-butanol did not alter intracellular levels of Na+ or K+. The present study shows that the ethanol-induced increase in Na+, K+ pumps involved an increase in the intracellular content of Na+. This increase in Na+ content did not appear to be secondary to an inhibition of Na+, K(+)-ATPase activity.

Brain osmoregulation during extreme and moderate dehydration in a rat model of severe DKA.

To determine if osmoprotective molecules accumulate in the brain during severe DKA with extreme (DKA-E) and moderate (DKA-M) dehydration, Fischer 344 rats (250-350g) were given STZ 45 mg/kg (i.p.) and allowed food and water ad lib. DKA-M received NaCl 77 mmol/L 60 ml/kg (i.p.) q 4 hrs. on day 2. All rats were anesthetized and sacrificed at 48 hrs. Half of each brain was used to measure water content (BWC) and half to measure Na+, K+, and organic osmoles by HPLC. Just prior to expiration, values for mean concentration of serum glucose (mmol/L) percent weight loss and median blood pH for DKA-E were 33.4, 19%, 6.98; for DKA-M, 16.8, 7.5% and 6.84, respectively. Means +/- SEM were compared by Student's t-test. Percent BWC was 76.3, 77.3 and 77.6 in DKA-E, DKA-M and normal controls, respectively (NS). Brain Na+ and K+ were increased in DKA-M compared to controls (p < .05) but not significantly different in DKA-E compared to controls. Of organic osmoles measured (umol/g wet weight) taurine was significantly increased (p < .01) in DKA-E and DKA-M (8.04 +/- .39 and 9.73 +/- .78, respectively) as compared to controls (5.92 +/- .35) as was myoinositol in DKA-E compared to controls (9.96 +/- .39 vs. 8.87 +/- .28) (p < .05) and urea in DKA-E as compared to controls (14.24 +/- 3.9 vs. 4.14 +/- .52) (p < .01). DKA-M were not significantly different for brain myoinositol or urea as compared to control animals. There was no significant difference in brain glutamine between either study group and controls. Preservation of brain water despite systemic dehydration can be partly explained by increased brain concentrations of osmoprotective molecules. Such adaption in the clinical setting of DKA warrants a cautious repair of dehydration and hyperosmolality.

MR imaging of normal rat brain at 0.35 T and correlated histology.

A custom-built small-animal transceiver was used for in vivo imaging of normal rat brain at 0.35 T, with the objective of identifying anatomic components by comparison of images with corresponding histologic sections. The cerebrum, cerebellum, brain stem, ventricles, hippocampus, and subarachnoid space were identified and cerebrospinal fluid (CSF) was differentiated from gray matter and white matter on coronal and transaxial magnetic resonance (MR) images. These images compare favorably with those obtained by others at higher field strengths in regard to delineating major neuroanatomic structures. It is concluded that this technique will be useful for investigating small-animal models of human neurologic disease involving morphologic and morphometric changes in gray matter, white matter, and CSF-filled spaces.

Osmoregulatory betaine uptake by rat renal medullary slices.

Betaine is an osmolyte present in high concentrations in renal medullary cells. Betaine and other organic osmolytes, such as glycerophosphorylcholine, myo-inositol, and sorbitol, have been shown to increase in concentration during antidiuresis when the inner medullary extracellular osmolality rises. Its concentration may increase in renal cells either by betaine uptake or by choline metabolism to betaine. These studies measured the uptake of (14C)betaine into cortical, outer medullary and inner medullary slices from rat kidney. The tissue-to-medium ratio of (14C) betaine increased with increasing osmolality up to 450 mosmol/kg in outer medullary and inner medullary slices, but not in cortical slices. Betaine uptake increased when the osmolality was raised with NaCl or mannitol, but not with urea. When LiCl was substituted for NaCl in a medium of 300 mosmol/kg, there was significant inhibition of betaine uptake, although the tissue-to-medium ratios remained greater then unity. Thus, increases in osmolality stimulate betaine uptake in rat renal medullary slices and this uptake occurs by both sodium-dependent and sodium-independent betaine transport.

Effect of vanadate on renal hypertrophy and sorbitol accumulation in streptozotocin induced diabetes in rats.

Vanadate has been previously shown to normalize blood glucose in streptozotocin-induced diabetic (STZ-DM) rats. The effect of a previously studied dose of vanadate (0.8 mg/ml) in drinking water on blood glucose, renal hypertrophy, and whole kidney polyol accumulation was studied in STZ-DM rats. Rats with diabetes of 5 weeks duration had higher blood glucose, greater urinary output, higher kidney weight, lower body weight, and higher kidney to body weight ratios than controls. Whole kidney sorbitol concentrations were significantly increased in diabetes but myo-inositol levels were unchanged vs control animals. After four weeks of oral vanadate treatment, blood glucose, urine volume, and kidney weights were similar to control values. Kidney to body weight ratios fell below that of the STZ-DM animals, but because body weights remained decreased, the kidney to body weight ratios were not normalized. Renal sorbitol levels returned to control values and renal myo-inositol levels remained unchanged in STZ-DM and normal animals treated with vanadate. These results provide evidence that vanadate therapy may result in regression of the hypertrophy and polyol accumulation characteristic of diabetic nephropathy in STZ-DM rats. This effect is most likely due to normalization of blood glucose by the insulin-mimetic activity of vanadate treatment.

Diuretic and natriuretic effects of sorbinil, an aldose reductase inhibitor.

The renal effects of sorbinil, an aldose reductase inhibitor that interferes with the conversion of glucose to sorbitol, were studied in rats and rabbits before and after fluid deprivation. The intracellular osmolar solute, sorbitol, is found in increasing concentrations from cortex to medulla in the kidney and may be involved in the urinary concentrating mechanism. Oral administration of sorbinil in the rabbit resulted in significant increases in urine flow rate and sodium excretion with a tendency toward decreased urine osmolality and increased potassium excretion both before and after water deprivation. When fluid intake was controlled in the rat study, significant increases in urine flow rate and sodium and potassium excretion and a significant decrease in urine osmolality occurred only in response to fluid deprivation. Thus, sorbinil has diuretic and natriuretic properties and may prevent the normal concentration of urine in the antidiuretic animal.

Magnetic resonance imaging (MRI) and pathophysiology of the rat kidney in streptozotocin-induced diabetes.

Proton magnetic resonance imaging was performed on rats before induction of diabetes with streptozotocin (STZ) and at 2 and 12 days postinduction. Images revealed an increase in maximal longitudinal and axial dimensions of the kidneys at 2 days and a further increase at 12 days. Similarly, an increase in the size of the remaining kidney was seen in a rat which underwent uninephrectomy as a positive control. Two major differences were observed between the kidney undergoing compensatory hypertrophy and those developing diabetic nephropathy: (i) Expansion of the renal vasculature was seen only in images of the diabetic rat; (ii) A loss in conspicuity of the normal corticomedullary junction was seen in the T2-weighted images of the diabetic rat but not in the uninephrectomized rat. Histologic examination revealed that the medulla increased to a size greater than the cortex during diabetic nephropathy whereas the medullary volume was less than that of the cortex during compensatory hypertrophy. In vitro T1 relaxation times in cortex, outer medulla and inner medulla of kidneys from control rats were measured and compared with the same respective regions in diabetic rats. When these values were correlated with tissue water content, a linear increase in relaxation rate versus percent water content from cortex to inner medulla was found in the control kidneys, but this correlation was absent in diabetic nephropathy. These studies demonstrate that MRI is an effective noninvasive tool for studying the course of renal hypertrophy and hydration changes in the development of renal disease in STZ-induced diabetes in the rat.

Magnetic resonance imaging and histopathology of hydronephrosis in the rat.

Magnetic resonance imaging revealed conspicuously hyperintense regions in the papillary area of kidneys of three untreated rats. When the kidneys were examined histologically, a hydronephrosis associated with the presence of bacteria was found. This study relates magnetic resonance images of an early stage of hydronephrosis to its histological picture.

Effect of dimethylaminoethanol, an inhibitor of betaine production, on the disposition of choline in the rat kidney.

The choline metabolite betaine has been shown to be an important organic osmoregulatory solute in the kidney. The isolated perfused rat kidney and kidney slice incubations were used to investigate the effect of 2-dimethylaminoethanol (DMAE), a choline oxidase inhibitor, on the renal excretion and metabolism of choline. In the isolated perfused kidney, [14C]choline, at an initial perfusate concentration of 300 microM, was effectively removed from the perfusate over 25 min, with nearly all the 14C in the perfusate accounted for by betaine during the remainder of the 90-min perfusion. DMAE at concentrations of 3.0 or 5.0 mM significantly decreased the rate of removal of [14C]choline from the perfusate and the rate of addition of [14C]betaine to the perfusate, yet [14C]betaine remained the only metabolite of choline in perfusate and urine. In kidney tissue slice experiments, conversion of [14C]choline to [14C]betaine was found in cortical, outer medullary and inner medullary regions of rat kidney. DMAE at 5.0 mM significantly inhibited [14C]betaine production in each of the three regions studied. These data show that DMAE is an effective inhibitor of betaine production by the kidney and, as such, may be an important agent for the study of osmoregulation by the kidney.

Excretion and metabolism of nicotinic acid by the avian kidney.

The renal tubular transport and metabolism of nicotinic acid (NA) were investigated using the Sperber technique in unanesthetized hens. Infusion of [14C]NA into the avian renal portal circulation at 10(-10) mol/kg/min revealed that the 14C label was actively transported into urine at a rate 74% that of simultaneously infused tetraethylammonium. Increases in NA infusion rates enhanced 14C label transport so that it eventually equalled the excretion rate of tetraethylammonium. At a NA infusion rate of 10(-8) mol/kg/min, this transport was not affected by probenecid, pyrazinoic acid or p-aminohippurate. Above infusion rates of 10(-7) mol/kg/min, saturation of 14C label transport was reached. Electrophoretic analysis of the 14C label excreted in urine at NA infusion rates less than 10(-7) mol/kg/min revealed a single, unidentified metabolite. At infusion rates greater than 10(-7) mol/kg/min, both the radiolabeled metabolite and [14C]NA were found in the urine. We conclude that NA is transported by a specific mechanism into the avian tubule cell where it is metabolized. As the body's load of this vitamin increases, NA is excreted first in the form of a metabolite and then as both metabolite and unutilized NA.

Effect of acute and chronic hypernatremia on myoinositol and sorbitol concentration in rat brain and kidney.

In animal models of hypernatremia, increases in brain electrolyte content account for the entire increase in osmolality in acute but not chronic hypernatremia, suggesting that there is generation of additional intracellular solutes ("idiogenic osmoles") in chronic hypernatremic states. In the present study, the concentration of the polyols myoinositol and sorbitol and water content were determined in the brain and kidneys of rats made acutely (2 hours) and chronically (72 hours) hypernatremic by intraperitoneal injection of NaCl and water restriction. Both the brain and the kidney responded to chronic hypernatremia with increased levels of myoinositol. Sorbitol levels increased in the kidney in response to both acute and chronic hypernatremia. Water content dropped in acute hypernatremia, but remained unchanged during chronic hyperosmolar challenge. We conclude that the polyols, myoinositol and sorbitol, may play a significant role in cellular osmoregulation in brain and kidney during chronic hypernatremia in the rat.

Renal effects of prednisolone in the chicken.

The effects of prednisolone on renal tubular transport of organic ions, urine flow and glomerular filtration rate were studied using the Sperber preparation in chickens. Low infusion rates of 1 and 5 nmole/min prednisolone slightly enhanced organic ion excretion. At higher infusion rates up to 1,000 nmol/min, no change in organic ion transport was observed. Urine flow and glomerular filtration rate increased at prednisolone infusion rates of 50 nmole/min and higher, independent of changes in renal blood flow.

Probenecid inhibition of the renal excretion of dyphylline in chicken, rat and man.

The effect of probenecid on the renal excretion of dyphylline was studied in chicken, rat and man. Dyphylline was found to be actively excreted when measured by the Sperber preparation in hens, the isolated perfused kidney of the rat and clearance studies in man. In each study probenecid significantly decreased dyphylline excretion demonstrating that dyphylline occupied the renal organic anion transport system. This same drug interaction, at the level of the renal excretory system, in these three species occurred at comparable concentrations of dyphylline and probenecid.

Renal effects of atrial natriuretic factor in domestic fowl.

The renal hemodynamic and tubular effects of ANF were investigated using the Sperber technique in chickens. This technique takes advantage of the unique portal circulation of the avian kidney and permits direct access to the renal peritubular space independent of renal arterial blood flow and glomerular filtration. Infusion of ANF into the avian renal portal system increased urine flow rate and sodium excretion by as much as 300% and 100%, respectively. These changes occurred in the absence of significant alterations in glomerular filtration rate or renal plasma flow. There was no significant difference in urine flow, sodium excretion or glomerular filtration rate between the ANF-infused kidney and the contralateral, non-infused kidney. We conclude that the diuretic and natriuretic effects of ANF do not depend on changes in glomerular filtration rate and that the site of action of ANF is the renal medulla.

Relation between transport maxima and inhibition of organic cation excretion in the chicken kidney.

The ability of several organic cations to inhibit differentially the renal excretion of two prototypical organic cations, tetraethylammonium (TEA) and N1-methylnicotinamide (NMN), was investigated using the Sperber technique in chickens. TEA and NMN excretion were inhibited by the following organic cations in order of decreasing potency: quinine, TEA and NMN. The respective competitive potency of these substances was related inversely to their maximum tubular transport rates (Tm). Regardless of inhibitor used (quinine, TEA or NMN), NMN excretion was always inhibited more easily than TEA excretion. In addition, TEA excretion was suppressed more easily than cimetidine excretion by the competitive inhibitor quinine. The Tm of cimetidine was determined to be less than the Tm of TEA, which in turn is less than that of NMN. These results indicate that the Tm of an organic cation is related inversely to its inhibitory potency and related directly to its susceptibility to inhibition, reflecting different affinities of organic cations for the same carrier-mediated transport system.

Thiazide diuretics inhibit chloride absorption by rabbit distal colon.

In order to investigate the cellular mechanisms of action of thiazide diuretics, the effect of diuretic and nondiuretic thiazide compounds on Cl- absorption across rabbit distal colon was assessed in tissues mounted in Ussing chambers. This epithelium absorbs Cl- via an active electroneutral transport process. Net 36Cl- absorption across short-circuited tissues was decreased 53, 36 and 20% after addition of 10(-4) M trichlormethiazide, bendroflumethiazide or hydrochlorothiazide, respectively, to the mucosal bathing solution. This inhibitory effect was a result of a decrease in the mucosa-to-serosa unidirectional Cl- flux (P less than .02). Neither the serosa-to-mucosa Cl- flux nor Isc was affected by the thiazides. Thiazide diuretics may exert their effect on Cl- transport across rabbit distal colon through inhibition of a Cl(-)-HCO-3 exchange mechanism. The nondiuretic thiazide, diazoxide, had no effect on Cl- transport. The similarity between the diuretic potency of these compounds and their potency as inhibitors of Cl- absorption by rabbit colon suggests that the thiazides have a similar mechanism of action in renal epithelia.