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Biomedical subjects

M Barac-Nieto

Publications and source records attributed to M Barac-Nieto.

At least 37 records · Page 2Linked to original sources

Insulin-responsive glucose transporter expression in renal microvessels and glomeruli.

The insulin-responsive glucose transporter (GLUT4) is expressed at high levels in fat and skeletal muscle, which account for the majority of insulin-stimulated glucose uptake. However, GLUT4 is also expressed at lower levels in kidney and several other tissues. We have used a variety of protein and mRNA detection techniques to determine the sites of renal GLUT4 expression. Indirect immunofluorescence experiments with two specific anti-peptide antisera detected GLUT4 in the smooth muscle cells of the rat renal microvasculature, in renal glomerulus, and in cultured glomerular mesangial and epithelial cells. PCR amplification of cDNA derived from microdissected renal glomeruli, microvessels and tubules corroborated this distribution of GLUT4, and Northern blotting demonstrated GLUT4 mRNA in cultured glomerular mesangial cells. Both the immunofluorescence and PCR data suggested that GLUT4 is most highly expressed in renal microvessels. Our results show that certain renal cells, such as renal microvascular smooth muscle cells, express the insulin-responsive glucose transporter and therefore may demonstrate altered glucose uptake and metabolism in diabetes mellitus.

Animals↗

Na+ alters the affinity for glucose and phosphate in rat renal brush-border membranes: a study of NMR relaxation rates.

Transport of D-glucose (Glc) and Pi across renal microvilli brush-border membrane vesicles (BBMV) is coupled to Na+.Na+ may alter substrate binding and dissociation or the mobility of the translocator and its complexes. To evaluate substrate binding in NaCl or KCl solutions containing BBMV, we measured nuclear magnetic resonance transverse relaxation rates (1/T2) of [1-13C]Glc and 31P(i), as well as their temperature, magnetic field, and pH dependencies. The data indicate the following: 1) the alpha-anomer, but not the beta-anomer, of Glc binds to BBMV, more in the presence of Na+ than of K+; 2) interactions of P(i) with BBMV that increase its 1/T2 are significant in the presence of K+ but not of Na+; 3) temperature and magnetic field dependencies of the 1/T2 of P(i) bound to BBMV are consistent with more rapid exchange with free P(i) in the presence of Na+ than of K+; and 4) in the presence of Na+, [H+] promoted interactions of P(i) with BBMV that increase its 1/T2, probably by interfering with formation of rapidly dissociating Na(+)-P(i)-carrier complexes. In conclusion, Na+ promotes association of alpha-Glc to BBMV but promotes rapid exchange and dissociation of P(i) from BBMV. In both cases, Na+ effects coupling by altering the affinity of the cotransporters for the substrate.

Animals↗

Renal protein synthesis in diabetes mellitus: effects of insulin and insulin-like growth factor I.

Is increased synthesis of proteins responsible for the hypertrophy of kidney cells in diabetes mellitus? Does the lack of insulin, and/or the effect of insulin-like growth factor I (IGFI) on renal tubule protein synthesis play a role in diabetic renal hypertrophy? To answer these questions, we determined the rates of 3H-valine incorporation into tubule proteins and the valine-tRNA specific activity, in the presence or absence of insulin and/or IGFI, in proximal tubule suspension isolated from kidneys of streptozotocin diabetic and control rats. The rate of protein synthesis increased, while the stimulatory effects of insulin and IGFI on tubule protein synthesis were reduced, early (96 hours) after induction of experimental diabetes. Thus, hypertrophy of the kidneys in experimental diabetes mellitus is associated with increases in protein synthesis, rather than with decreases in protein degradation. Factor(s) other than the lack of insulin, or the effects of IGFI, must be responsible for the high rate of protein synthesis present in the hypertrophying tubules of diabetic rats.

Animals↗

Changes in NMR-visible kidney cell phosphate with age and diet: relationship to phosphate transport.

To test the hypothesis that growth and dietary Pi affect the intracellular concentration of Pi ([Pi]i) as well as its renal reabsorption, we measured nuclear magnetic resonance (NMR)-visible [Pi]i in isolated perfused kidneys of less than 1- and greater than 4-wk-old guinea pigs fed various amounts of Pi. Changes in [Pi]i were correlated with those in fractional Pi reabsorption (FRPi) in vivo and in capacity (Vmax) for Na(+)-Pi cotransport in microvilli derived from animals of similar age and fed the same diets. In animals fed normal (0.76% Pi) diet, [Pi]i was lower (0.91 +/- 0.14 vs. 1.85 +/- 0.23 mM, P less than 0.05), whereas FRPi was higher (0.90 +/- 0.02 vs. 0.70 +/- 0.03, P less than 0.01) in less than 1- than in greater than 4-wk-old guinea pigs. Pi deprivation decreased [Pi]i in mature animals to 0.74 +/- 0.29 mM, P less than 0.05, and increased FRPi to 0.99 +/- 0.01. Excess dietary Pi increased [Pi]i in immature animals to 1.67 +/- 0.56 mM, P less than 0.05, and decreased FRPi to 0.55 +/- 0.03. Diet-induced changes in [Pi]i were associated with reciprocal changes in Vmax of similar absolute magnitude in immature and mature animals. However, diets that resulted in comparable [Pi]i at the two ages were associated with higher (P less than 0.05) Vmax in less than 1- than in greater than 4-wk-old animals. The reciprocal nature of the relationship between [Pi]i and renal Pi transport indicates that [Pi]i is primarily determined by Pi efflux from the cells or Pi organification rather than Pi influx through Na(+)-Pi cotransport. Findings indicate that changes in [Pi]i with growth or diet may be a cause but cannot be the consequence of changes in abundance or maximal mobility of Na(+)-Pi cotransporters. Data also indicate that factors in addition to low [Pi]i contribute to the high Na(+)-Pi cotransport capacity observed in renal microvilli of growing animals.

Aging↗

NMR studies of phosphate metabolism in the isolated perfused kidney of developing rats.

During growth, the capacity for renal phosphate (Pi) reabsorption varies as a function of Pi demand. These changes occur in the absence of changes in extracellular concentration of Pi and are also observed in renal cells cultured in defined media. These findings suggest a direct regulatory effect of intracellular Pi on its transport systems. We postulate that a low intracellular Pi concentration [( Pi]i) occurs in the developing kidney as a consequence of differences in Pi metabolism between growing and mature cells and that a low [Pi]i, in turn, leads to adaptive changes in renal Pi transport. In order to assess this hypothesis, we used 31P-nuclear magnetic resonance (NMR) to measure the intracellular concentrations of NMR-visible Pi and phospho-metabolites and the rates of Pi turnover due to adenosine triphosphate (ATP) synthesis, in isolated perfused kidneys of 3- to 4-week-old and 12- to 13-week-old rats. The [Pi]i was lower (1.7 +/- 0.1 vs 2.7 +/- 0.1 mM, P less than 0.05) in kidneys of growing than of adult rats, while the ATP (2.9 +/- 0.3 vs 2.8 +/- 0.5 mM) and adenosine diphosphate (ADP) (-0.2 mM) concentrations were similar at the two ages, consistent with a high phosphorylation potential in the kidneys of the younger animals. Radiofrequency irradiation of the gamma-P of ATP resulted in reduction in the intensity of the Pi resonance of 62 +/- 5% in the newborn and 38 +/- 3% in the adult (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

Glomerular and tubular alpha 1- and alpha 2-adrenoceptors in the rat kidney: distribution in basolateral and brush border membranes of tubular cells.

Rat kidney glomeruli and cortical tubules were obtained by a combination of sieving and differential centrifugation technique. [3H]Prazosin and [3H]rauwolscine were used to identify and quantify the alpha 1- and alpha 2-adrenergic receptors, respectively. In the glomeruli, the alpha 1-adrenoceptor concentration was 27% and alpha 2-adrenoceptor concentration was 33% of the corresponding values in the tubules. Further localization of the tubular alpha-adrenoceptors was undertaken by studies in the isolated basolateral membrane and comparison with values in the crude plasma membrane. alpha 1-Adrenoceptors were enriched 1.54 +/- 0.1 times and alpha 2-adrenoceptors were enriched 1.73 +/- 0.04 times in the basolateral membrane as compared to crude plasma membrane. However, these values were significantly (p less than 0.05) less than the enrichment value of 2.77 +/- 0.3 obtained for the basolateral membrane marker (Na+ + K+)-ATPase. These results suggested the possibility that alpha 1- and alpha 2-adrenoceptors may also be distributed in the brush border membrane. Direct-binding studies in the purified renal brush border membrane indicated alpha 1-adrenoceptor concentration of 82.1 +/- 3.8 and alpha 2-adrenoceptor concentration of 108.2 +/- 9.3 fmol/mg protein. These values were 32 and 17% of the corresponding values in the basolateral membrane. Overall, our results suggest that alpha 1- and alpha 2-adrenoceptors are present both in the basolateral and brush border membranes analogous to what has been reported for angiotensin and insulin receptors; their primary concentration, however, is in the basolateral membranes.

Alkaline Phosphatase↗

Renal reabsorption of phosphate during development: transport kinetics in BBMV.

The mechanism responsible for enhanced reabsorption of phosphate (Pi) in growing animals was assessed in renal brush-border membrane vesicles (BBMV) prepared from 3- to 14-day-old and greater than 57-day-old guinea pigs. On standard diet, Vmax (pmol.mg-1.s-1) of Na+-Pi cotransport was higher (P less than 0.001) in newborn (650 +/- 77) than in adult (144 +/- 17) but Vmax of Na+-glucose cotransport did not differ with age. Low dietary Pi did not affect significantly Vmax of Na+-Pi cotransport in the newborn (P greater than 0.8) but increased it in the adult (to 318 +/- 32, P less than 0.05). A high Pi intake resulted in a smaller relative decrease in Vmax in the newborn than in the adult (27 vs. 44%, P less than 0.05). In the newborn, the serum Pi (mM) decreased on a low-Pi diet (from 1.8 +/- 0.1 to 0.8 +/- 0.1, P less than 0.001) and rose by twofold on the high-Pi diet (to 3.5 +/- 0.2, P less than 0.001). In the adult, there were no significant changes in serum Pi with changes in Pi intake (P greater than 0.5). Thus in the newborn the Na+-Pi cotransport system is characterized by high transport capacity but low adaptability to changes in dietary Pi.

Aging↗

NMR measurements of intra- and extravesicular sodium in renal microvilli.

Previous attempts to separate the nuclear magnetic resonances of intra- and extravesicular Na+ in brush-border membrane vesicles (BBMV) were unsuccessful and led to the proposal of rapid exchange of Na+ via sodium channels in BBMV. However, passive conductance of Na+ in this membrane has been found to be relatively small. This inconsistency prompted us to use a different shift reagent to reassess the issue. In guinea pig renal BBMV (15-30 mg protein/ml) equilibrated with Na+ (130 mequiv. 1), using the impermeant Na+ shift reagent dysprosium tripolyphosphate (3 mM), the resonances of intra- (3.3%) and extravesicular (96.7%) Na+ were resolved by 6 ppm. Increases in Na+ conductance induced by gramicidin D did not alter the characteristics of intra- and extravesicular Na+ resonances. By contrast, addition of glucose caused a transient increase in the area of the intravesicular Na+ resonance. The clear separation between the intra- and the extravesicular Na+ resonances allowed us to measure the relaxation times of Na+, which depend on its interactions with its immediate environment. The longitudinal relaxation time of intravesicular Na+ (13 +/- 1 ms) was much shorter than that of the extravesicular Na+ (44.0 +/- 0.4 ms). Thus, in intact renal BBMV, as well as in membranes treated with the cationophore gramicidin D, the exchange of Na+ between the intra- and the extravesicular compartments is slow on the NMR time scale, consistent with the low Na+ channel density of this membrane. In contrast, the increase in intravesicular Na+ induced by glucose, is consistent with a significant contribution of the glucose cotransport pathway to Na+ flux across these membranes. The short longitudinal relaxation time of Na+ in the intravesicular space indicates interaction of Na+ with BBMV binding sites or ordering of these ions in the intravesicular compartment.

Alkaline Phosphatase↗

The relationship between renal metabolism and proximal tubule transport during ontogeny.

The proximal tubules of newborn and adult animals reabsorb a similar fraction of the filtered load of Na+ and H2O (65%-70%). In tubules from adult animals, transcellular, active Na+ reabsorption accounts for one-third of the total, while two-thirds occur passively through the paracellular pathway, driven by hydrostatic and oncotic forces (one-third) and by cell-generated effective osmotic and ionic gradients (one-third). Since two-thirds of the Na+ is reabsorbed passively and does not require energy, the mature proximal tubule has a high Na+/O2 molar ratio (48 Eq of Na+/mol of O2). Measurements of ouabain-sensitive oxygen consumption in suspensions of proximal tubules indicate that in newborn, aerobic metabolism can support about 50% of the net Na+ transport rate compared with the 33% in tubules from adult animals. Independent confirmation of the direct and proportional relationship between active Na+ transport and ouabain-sensitive O2 consumption exists for the adult but not for the newborn. However, measurements of epithelial conductances and of transepithelial hydrostatic and oncotic pressure differences indicate that passive paracellular fluxes can account for the remaining 50% of the proximal Na+ reabsorption in newborn. The high permeability of the proximal tubules of newborn animals to small molecular weight solutes suggests that cell-generated osmotic and ionic transepithelial gradients are minimal in the tubules of newborn animals. Yet in the newborn, the osmolality of the end proximal tubule fluid was found to exceed that in plasma. This indicates that osmotic gradients due to differences in reflection coefficients for preferentially reabsorbed solutes and Cl- do exist across the proximal tubules of the newborn and suggests that these gradients may contribute to Na+ and H2O reabsorption. If this is indeed the case, then the contribution of active and of hydrostatic and oncotic pressure-driven flows to the overall reabsorption of Na+ and fluid has been overestimated. Resolution of this discrepancy requires measurements of the reflection coefficients for HCO3- and Cl- in the proximal tubule of the newborn. The metabolic processes by which energy is supplied to renal proximal cells during development are also incompletely characterized. There is evidence that maturation of aerobic metabolism, Krebs cycle enzymes activity, and of the mitochondrial membrane surface area precede the development of net reabsorptive transport (Na+, H2O, HCO3, glucose). By contrast, maturation of Na(+)-K(+)-ATPase activity at the basolateral cell membrane follows that in reabsorptive transport and does not limit its development.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

D(-)3-hydroxybutyrate cotransport with Na in rat renal brush border membrane vesicles.

Which are the driving forces for D(-)3-hydroxybutyrate (HB) transport in rat renal brush border membranes (RBB)? Sodium, even in the absence of gradients, accelerates the unidirectional (1-5 s) flux of HB into rat RBB vesicles. Valinomycin (and Ki = Ko) does not significantly alter the NaCl gradient driven HB influx. Thus, the Na-dependent HB influx is driven by the chemical Na+ gradient but it is not driven by changes in the transmembrane electrical potential. Indeed, in valinomycin-treated membranes, vesicle-inside more negative potentials (K-gluconate in-Na-gluconate out) sufficient to accelerate Na-glucose cotransport, did not stimulate HB influx, in the presence of inwardly directed Na+ gradients, and did not significantly inhibit when in the absence of Na+. Thus, cotransport of HB with Na in rat RBB membranes does not involve the net transfer of positive charge and the passive conductance of this membrane for HB- is not large. However, vesicle inside more negative potentials (induced by inwardly directed NaNO3 gradients or by outwardly directed K+ gradients and valinomycin in the presence of inwardly directed Na+ gradients) inhibited HB influx, suggesting that another potential sensitive mechanism, perhaps redistribution of intramembrane charges, may influence HB influx. Acidification (pHi = pHo = 6.4 vs. 7.4) or inwardly directed H+ gradients (pHo/pHi = 6.4/7.4) did not alter HB influx, in the absence of Na+. Thus there is no evidence for a H+ driven HB influx. HB influx is significantly inhibited by high (100 mEq/l) trans concentration of Na+. Also, influx of 2.25 mM 14C-HB was significantly increased by 5-10 mM intravesicular HB under Na-equilibrated conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxybutyric Acid↗

Marginal malnutrition in school-aged Colombian boys: metabolic rate and estimated daily energy expenditure.

Total daily energy expenditure (TDEE) and energy expenditure in activity (EAc) were estimated in 114 free-ranging, nutritionally normal, and undernourished boys 6-16 yr of age by measuring basal and resting metabolic rates, average daily heart rate while awake, and oxygen consumption and heart rate during exercise on a treadmill. Mean daily heart rates were in the range of exercising heart rates and gave reasonable estimates of TDEE and EAc. TDEE increased with age (p less than 0.001) and was reduced in undernourished boys (p = 0.011). Results indicate that nutritional group differences in TDEE were due to differences in body size. EAc increased with age but did not show significant differences between nutritional groups, indicating that in the marginal malnutrition of school-aged children, reduced growth and associated economy of energy expenditure in locomotion is sufficient physiological adaptation. Peer pressure in school and play activities may interfere with the protective mechanism of reduced activity.

Adolescent↗

Renal reabsorption and utilization of hydroxybutyrate and acetoacetate in starved rats.

Are there increases in the renal reabsorption (R) and utilization (Q) of ketoacids during starvation? R and Q of D-3-hydroxybutyrate and acetoacetate were measured in anesthetized normally fed (C) and 3-day starved (S) rats, using clearance and arteriovenous extraction methods. R was nearly complete in both groups at low filtered ketoacids loads (less than 2mumol/g X min). At higher filtered loads, R of the two ketoacids increased in proportion to their filtered loads (r2 greater than 0.95) but with significantly steeper slopes in the S than in the C group; fractional R of the two ketoacids were significantly higher in the S than in the C animals. Saturable and nonsaturable components for ketoacid reabsorption were evident, but only the nonsaturable component was significantly increased with starvation. Increased R of ketoacids during starvation was in the absence of changes in the initial rates of hydroxybutyrate influx into isolated rat renal brush-border membrane vesicles. Thus the rate-limiting step for ketoacid reabsorption through the nonsaturable pathway may not be located at the luminal membrane of the proximal cells. The increases in R of ketoacids in the S group were associated with simultaneous decreases in the net rates of renal utilization of the two ketoacids. Thus increased renal conservation of ketoacids is a transport and not a metabolic event. Overall, during complete starvation, both the increased R and the decreased renal Q of the ketoacids contribute to increase the availability of these metabolites to other organs.

Absorption↗

Renal hydroxybutyrate and acetoacetate reabsorption and utilization in the rat.

Is the renal conservation of ketones effected through saturable transport systems? Does the renal reabsorption of ketoacids limit their utilization? The reabsorptive transport (T) and metabolic utilization (Q) of D-3-hydroxybutyrate (HB) and acetoacetate (AA) in the kidney were measured in anesthetized rats by use of clearance and arteriovenous extraction determinations. THB and TAA increased in proportion with increasing HB and AA filtered loads. Fractional THB decreases from 0.97 to 0.72 upon increases in the plasma HB. Fractional TAA also decreases from 0.89 to 0.73, at a constant filtered AA load, when the filtered HB load increased. Thus both saturable and nonsaturable components effect the reabsorption of HB and AA. Renal QHB increased from 0.4 to 4 mumol X g-1 X min-1 on increase in the plasma HB. THB always exceeded the simultaneously measured QHB. However, QHB was significantly correlated with THB, suggesting that QHB may be limited by delivery of HB to the renal cells through reabsorptive pathways. There was no net renal conversion of HB and AA. Both HB and AA were utilized by the kidney. Thus, utilized ketoacids must have entered pathways for complete oxidation or for synthesis (lipid?) in the kidney.

3-Hydroxybutyric Acid↗

Marginal malnutrition in school-aged Colombian boys: efficiency of treadmill walking in submaximal exercise.

Measurements of oxygen consumption (VO2) were made during submaximal treadmill exercise (3.5 mph) in 658 boys 6 to 16 yr of age classified as nutritionally normal, low weight for age, and low weight for height. Gross efficiency (at 15% grade) increased with age and was significantly lower in low weight for age and height boys than in normal children but delta efficiency showed no statistically significant differences between nutritional groups. It is concluded that marginal malnutrition has no effect on the efficiency of submaximal work during treadmill walking.

Adolescent↗

Marginal malnutrition in school-aged Colombian boys: body composition and maximal O2 consumption.

Do changes in body composition account for the changes in O2 consumption during maximal exercise (VO2max) in nutritionally deprived boys? School age urban and rural boys with no deficits (N) or with significant deficits (W-H) in weight for age and for height were studied. Percentage lean body mass was higher in W-H than in N boys. By contrast percentage intracellular water, the creatinine excretion coefficient and percentage arm muscle mass were not higher in W-H boys. Thus nutritional compromise decreased more the cellular than the support tissues of the body. VO2max per unit body weight was higher in W-H than in N boys. In the rural group, this was accounted for by the higher percentage lean body mass in W-H boys. In the urban group, VO2max/lean body mass was also higher in most W-H boys. Thus W-H urban boys had higher levels of aerobic training than N urban boys, reflecting perhaps a relative increase in daily physical activity as the same tasks have to be performed with less muscle by W-H boys.

Adolescent↗

Effects of pH, calcium, and succinate on sodium citrate cotransport in renal microvilli.

Sodium-dependent citrate uptake (v) by rabbit renal brush border vesicles was studied as a function of pH and Ca2+, citrate, and succinate concentrations to evaluate the hypothesis that factors which alter renal citrate reabsorption also alter v. At pH 8 a 10-fold increment in citrate concentration (up to 3.3 mM) was required to achieve the same v observed at pH 7. This provides proof that the protonated citrates are the effective substrates for v. At higher citrate concentrations (5-10 mM) and pH 8, substrate inhibition of v occurred. Studies of the substrate concentration dependency of v at various pH levels and calcium concentrations indicated that v was competitively inhibited by citrate3-. Changes in calcium concentrations altered v by altering the protonated citrate and citrate3- concentrations; v showed countertransport with succinate. Sodium-dependent succinate uptake (vs) was not pH dependent but was more inhibited by 1 mM citrate at pH 7 than at pH 8, indicating that the protonated citrates are inhibitors of vs. However, citrate3- was also found to inhibit vs. Thus, changes in v at the brush border membrane due to the effects of pH or calcium concentration on the protonated citrate concentration and to competition between polycarboxylates may be relevant to changes in renal citrate reabsorption in vivo.

Animals↗