Mario Barac-Nieto

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. The extent to which age-related changes in reabsorptive fluxes are due to the development of luminal membrane transport systems, to the decrease in paracellular permeability, or both remains to be determined. The high activity of enzymes in the hexosemonophosphate pathway and the high NADH/NAD ratio present during the first few weeks of extrauterine life poise the proximal tubules for high rates of biosynthesis of membrane lipids, glycoproteins, nucleic acids, and transporter proteins necessary for final differentiation.

20-HETE mediates the effect of parathyroid hormone and protein kinase C on renal phosphate transport

Parathyroid hormone (PTH) is a major inhibitor of renal proximal tubule (PT) sodium-dependent phosphate (Na+-Pi) cotransport. PTH is thought to exert its effect on Pi transport in the PT via the protein kinase A (PKA) and C (PKC) intracellular signalling pathways. PKC-dependent phosphorylation of phospholipase A2 stimulates arachidonic acid (AA) release, the latter a potent inhibitor of Pi transport. In turn, AA is metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE) in the PT. In addition, 20-HETE production is stimulated by PTH. We therefore explored the possibility that 20-HETE may mediate the PTH/PKC inhibition of renal Na+-Pi cotransport. To this end, we tested the effect of 20-HETE on Na+-Pi cotransport in proximal tubule-like cells. Exposure of opossum kidney (OK) cells for 4 h to 20-HETE (10(-7) M) decreased Na+-dependent uptake of 32Pi (from 0.26 +/- 0.02 to 0.19 +/- 0.01 nmol/mg protein.min) by approximately 25% (P < 0.001). The inhibition was due to a reduction in Vmax. 20-HETE had no significant effect on either the apical amiloride-sensitive and insensitive 22Na uptakes or on basolateral ouabain-sensitive 86Rb uptake, and was specific for Pi. These results indicate that 20-HETE specifically inhibits Na+-dependent Pi transport in OK cells and that it may be a mediator of PTH action in the PT.

Study of renal metabolic disturbances related to renal lithiasis at school age in very-low-birth-weight children

We studied 34 asymptomatic children who were born with a very-low-birth-weight (VLBW) and had no perinatal history of acute renal failure nor treatment with furosemide. The study was done at preschool or school age, looking for echographic changes and renal tubular disturbances which are known to predispose to renal lithiasis. The results were compared with those of a control group of 18 children who had been born at term with a body weight >2,500 g. One or more renal tubular disturbances were found in 64.70% of the VLBW children. Most frequently found were decreased ammonium excretion in response to furosemide (38.23%), enhanced N-acetylglucosaminidase excretion (35.29%), hypercalciuria (26.47%), and hypocitraturia (23.53%). Echography revealed renal cortical hyperechogenicity (17.65%) and renal lithiasis (8.82%) in some of the VLBW children. We found a significant positive correlation (r = 0.7) between the perinatal level of plasma phosphate and the total amount of H+ excreted in response to furosemide at preschool or school age. Because these renal tubular anomalies may be precursors of future lithiasis, and the renal function and echography tests are not invasive, we suggest that renal tubular function be measured and followed up in every VLBW child, particularly when perinatal hypophosphatemia has occurred.

Role of intracellular phosphate in the regulation of renal phosphate transport during development

Direct correlations have been observed between the renal intracellular concentration of phosphate ([Pi]i) and postnatal age (3–13 weeks in rats, 1–4 weeks in guinea pigs), as well as between the dietary supply of Pi and [Pi]i. In turn, [Pi]i was found to be inversely correlated with the renal tubular transport of phosphate (TRPi). However, age- and diet-related differences in [Pi]i alone do not explain the high capacity of Na+-Pi cotransport present in the kidney of the neonate. Therefore, we explored whether changes in TRPi induced by altering Pi demand (whole body growth or bone mineralization) are mediated by factors other than changes in [Pi]i. TRPi was measured in vivo and nuclear magnetic resonance-visible [Pi]i in perfused kidneys of 8-week-old genetically growth hormone (GH)-deficient and GH-treated dwarf rats and in 8-week-old thyroparathyroidectomized (TPTX) Sprague-Dawley (SD) rats treated or untreated with etidronate (EHDP), an inhibitor of bone mineralization. In dwarf rats, [Pi]i was 1.2±0.2 mM and TRPi 2.4±0.2 μmol/ml glomerular filtrate. In TPTX SD rats, [Pi]i was 1.6±0.2 mM and TRPi 4.2±0.3 μmol/ml glomerular filtrate. Administration of GH to dwarf rats resulted in increases in Pi transport of 38%±8% (P<0.05), while administration of EHDP to TPTX SD rats decreased TRPi by 52%±7% (P<0.05). Neither GH nor EHDP significantly affected [Pi]i. Thus, in the rat changes in TRPi due to alterations in Pi demand occur in the absence of significant changes in [Pi]i. Consequently at least two complementary but independent regnlatory factors, GH and low [Pi]i, account for the high rates of TRPi observed in the neonate.

23Na, 19F, 35Cl and 31P Multinuclear Nuclear Magnetic Resonance Studies of Perfused Rat Kidney

The concentration of intracellular sodium [Na+]i has been measured in the perfused rat kidney using 23Na nuclear magnetic resonance (NMR) in combination with the extracellular shift reagent Dy(PPPi)7-(2). The data show 100% visibility of Na+ in interstitial spaces. A measurement of the resonance intensities of intra- and extracellular 23Na ions along with a knowledge of the extracellular space as a fraction of the total kidney water space yielded an average [Na+]i of 27 +/- 2 mM for the kidney at 37 degrees C. After prolonged ischemia [Na+]i rose to approach that in the external medium. In the absence of 5% albumin in the perfusion medium, the linewidth of the 35Cl resonance of an adult kidney (45 Hz) was about twofold larger than that of the medium alone (25 Hz). In contrast, the linewidth of 35Cl resonance of an adult kidney perfused with an albumin-containing medium (82 Hz) was only about 27% of that from the medium alone (300 Hz). We interpret this effect to be due to compartmentation of albumin in the extracellular space such that the interstitial space is not freely accessible to albumin. However, for a developing, immature kidney from a growing animal, perfused with an albumin-containing medium, the linewidth of the 35Cl resonance (233 Hz) was only slightly less than that of the medium alone (300 Hz), indicating a much greater albumin permeability of the capillary walls. 19F NMR of a perfused adult kidney, loaded with the membrane-impermeant intracellular calcium indicator 5FBAPTA, yielded a value of 256 nM for [Ca2+]i. Induction of ischemia for 10 min caused the [Ca2+]i to rapidly rise to 660 nM, which could not be fully reversed by reperfusion, suggesting irreversible injury.

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules

Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca2+ concentration ([Ca2+]i), cell integrity, and transport functions in micro-dissected proximal convoluted tubules (PCT) of <3-week-old (newborn) and >12-week-old (adult) rabbits. Tubules were loaded with 10 ώM fura-2 AM by incubation for 60 min at 37° C,and then superfused with isosmotic saline solution gassed with either 95%O2-5%CO2) control group) or 95%N2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubules (from resting values of 245±10 to 975±100 nM, P<0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137±5 to 165±5 nM, P<0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca2+]i from baseline, to 920±90 nM in tubules from adult and to 396±16 nM in those from newborn rabbits (P<0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-depedent uptakes of a glucose analogue (14C-α-methyl-glucopyranoside) and phosphate (32Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca2+ rises to critical levels in PCTs of adults compared with those of <3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+]o, presumably voltage-sensitive Ca2+ channels or reversal of Na+-Ca2+ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits.

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.

Hormonal Regulation of Sodium-Dependent Phosphate Transport in Opossum Kidney Cells

There are multiple regulators of renal proximal tubule sodium-dependent phosphate (Na+-Pi) transport, including 1,25-dihydroxyvitamin D (1,25-Vit. D), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), and arachidonic acid (AA) and/or its metabolites. The purpose of our studies was to determine whether the effect of these factors on Pi transport is synergistic or antagonistic. The control solution or the substances were added independently or coincidentally to opossum kidney (OK) cells before incubation for 4 h. 1,25-Vit. D (10–8M) had no significant effect on Pi transport (↑6.8%; p = 0.8). PTH (10–7M) significantly inhibited Pi transport by 39.6% (p < 0.0001). IGF-1 (10–8M) stimulated Pi transport by 19.6% (p < 0.0001). The AA metabolite 20-HETE (10–7M) had no significant impact on Pi transport (⇓6.4; p = 0.3). The combined effect of 1,25-Vit. D and PTH was no different from PTH alone (p = 0.2). Likewise, addition of either 1,25-Vit. D or 20-HETE to IGF-1 failed to affect the magnitude of the increase on Pi transport induced by IGF-1 alone (p = 0.4, p = 0.6, respectively). The combination of 20-HETE and PTH was not different from that observed with PTH alone (p = 0.9). We conclude that in OK cells, PTH inhibits whereas IGF-1 stimulates Pi transport into OK cells. The effects of each of these hormones are independent and unaffected by either 1,25-Vit. D or 20-HETE.

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 used31P-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<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 γ-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<0.05). The corresponding 1.6-fold higher fractional turnover rate of the Pi pool in the younger than in the older rats accounts for the similar rates of ATP synthesis at the two ages (30±7 vs 35±4 μmol/min per g,P>0.3), despite the smaller intracellular Pi pool present in the younger than in the older animals. The low [Pi]i may stimulate the synthesis of 1,25 hydroxivitamin D3 and the expression of Pi transport related proteins. The high phosphorilation potential drives the ATP flux necessary for growth related transport and biosynthetic processes.

d(-)3-Hydroxybutyrate cotransport with Na in rat renal brush border membrane vesicles

Which are the driving forces ford(-)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 (andKi=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-gluconatein-Na-gluconateout) 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 mM14C-HB was significantly increased by 5–10 mM intravesicular HB under Na-equilibrated conditions. Thus, the rate of translocation of the free carrier appears to limit HB influx through the cotransport system.