Michèle G. Brunette

Micropuncture study of renal phosphorus transport in hypophosphatemic vitamin D resistant rickets mice.

SummaryA micropuncture study of inorganic phosphorus (Pi) transport was performed in 6 mice presenting hypophosphatamic vitamin D resistant rickets (Hyp) and results compared to those obtained in 13 normal (N) mice. The mean plasma (P Pi) and fractional excretion of Pi (FE Pi) in N and Hyp mice were 85.10±2.27 mg/l and 15.81±1.90 vs 48.43±3.29 mg/l and 35.34±5.26%, respectively.In N mice, tubular fluid over plasma Pi ratio (TF/P Pi) progressively decreases along the proximal tubule to reach approximately 0.6 in the late accessible part. The fraction of filtered Pi (% E Pi) remaining in this segment of the nephron is therefore 20–25%. In Hyp mice the TF/P Pi in proximal tubule remains relatively high and does not significantly vary with TF/P inulin (mean TF/P Pi=1.19±0.12 S.E., compared to 0.73±0.04 in N mice). Precise conclusions concerning % E Pi at the end of the proximal tubule of Hyp mice are not available because of the scattering of the data. However, all the values of % E Pi (mean % E Pi: 78.68±7.39 compared to 49.10±4.59 in N mice) are far above the total urinary FE Pi (35.34 ±5.26), suggesting a relatively greater distal fractional Pi reabsorption in Hyp mice than in N mice. Since the P Pi, and therefore the amount of filtered Pi is lower in Hyp mice, it is probable that the absolute amount of Pi reabsorbed distally is comparable in the two series of animals.

Demonstration of an osteoblast defect in two cases of human malignant osteopetrosis. Correction of the phenotype after bone marrow transplant.

Osteopetrosis is an inherited disorder characterized by bone sclerosis due to reduced bone resorption. Here we report that human osteopetrotic osteoblast-like (Ob) cells express a defective phenotype in primary cultures in vitro, and that bone marrow transplant (BMT) corrects osteoblast function. DNA analysis at polymorphic short-tandem repeat loci from donor, recipient, and primary Ob-like cells pre-BMT and 2 yr post-BMT revealed that Ob were still of recipient origin post-BMT. Osteopetrotic Ob-like cells obtained pre-BMT showed normal and abnormal 1,25(OH)2D3-induced alkaline phosphatase (ALPase) and osteocalcin production, respectively, and failed to produce macrophage colony-stimulating factor (M-CSF) in response to IL-1a and TNF-alpha. These parameters were all normalized in primary Ob-like cells prepared 2 yr post-BMT. X-linked clonality analysis at the human androgen receptor (HUMARA) locus revealed that osteoblasts showed a polyclonal and an oligoclonal derivation pre- and post-BMT respectively, indicating that a limited number of progenitor reconstituted this population. Because osteoblasts were still of recipient origin post-BMT, this suggests that functional osteoclasts, due to the replacement of hematopoeitic cells, provided a local microenvironment in vivo triggering the differentiation and/or recruitment of a limited number of functional osteoblasts.

Phosphate uptake by superficial and deep nephron brush border membranes

Dietary phosphate restriction and acute parathyroidectomy in rat are known to be associated with a selective increase in phosphate uptake by renal cortical brush border membranes (BBM). Conversely, phosphate loading and parathyroid hormone (PTH) administration result in a decrease of this uptake.In the present study, we investigated whether the response of the membrane to these various stimuli implies similar or different modifications of the kinetic properties of this membrane, whether these modifications affect one or both of the two systems of phosphate transport previously described, whether both superficial and deep nephron populations are involved, and whether the two stimuli: dietary phosphate, and parathyroid activity, are additive or not.Kinetic studies of phosphate (PO4) uptake by BBM vesicles were performed in seven groups of rats: control (N), acutely thyroparathyroidectomized (TPTX), PTH loaded (PTH), phosphate loaded (P+), phosphate depleted (P−) phosphate depleted with acute thyroparathyroidectomy (P−TPTX), and phosphate depleted-PTH loaded (P−PTH). In each of these experimental conditions, superficial and deep nephrons were investigated.Results indicate that 1. BBM from deep nephrons present a greater capacity for PO4 transport than those from superficial nephrons; 2. Whereas a dual system of PO4 uptake is observed in superficial BBM, deep BBM present only one single system; 3. Phosphate in the diet influences PO4 uptake by BBM to a greater extent in the deep than in the superficial nephrons; 4. PTH status on the contrary, equally influences both populations; 5. TPTX does not significantly enhance PO4 uptake in phosphate depleted rats; however, PTH loading curtails this uptake; 6. In the deep BBM neither the PTH status nor the phosphate content of the diet modify the apparentKm. In the superficial BBM, the apparentKm of the high affinity system (low substrate concentrations) varies with the PTH activity.

Testosterone enhances calcium reabsorption by the kidney

The kidney is a target tissue for androgens, but the role of these hormones in the regulation of calcium (Ca2+) reabsorption remains unclear. The present study examines the effects of testosterone on Ca2+ transport by the luminal membranes of proximal and distal nephrons of the rabbit kidney. Tubule suspensions were pre-incubated in the presence or absence of the hormone, and 45C2+ uptake by the luminal membranes was measured using the rapid filtration technique. In the proximal tubules, testosterone did not influence Ca2+ uptake. In the distal tubules, a 5 min incubation with the hormone increased this uptake with a maximal response at 10(-10)M. Ca2+ transport by the distal membranes shows a dual kinetics. Testosterone enhanced the Vmax value of the low affinity component. In an attempt to identify the underlying mechanisms involved in this action, several messenger inhibitors were introduced in the tubule suspension. PD 98059 and U0 126 as well as AG 99 and genistein interfered with the hormone action suggesting the implication of a MEK kinase and a tyrosine kinase. To determine the type of the channels involved in this effect, Ca2+ uptake was measured in the presence of diltiazem, omega-conotoxin MVIIC and mibefradil, i.e. selective inhibitors of the L-type, P/Q type and T-type channels. An inhibition of Ca2+ transport was observed exclusively with mibefradil. These results indicate that testosterone enhances Ca2+ transport by opening a T-type Ca2+ channel in the distal luminal membrane, via MEK kinase and tyrosine kinase dependent mechanisms.

Phosphate Uptake by Syncytial Brush Border Membranes of Human Placenta

ABSTRACT: Human placental brush border membranes were obtained from full-term normal vaginal deliveries. A sodium dependent transport system for phosphate was identified in these membranes. In the presence of a sodium gradient, at pH 7, T° 35° C, the apparent Km and Vmax were 86.5 ± 24 μM and 1.9 ± 0.18 nmol mg-1 30 s-1. The apparent Km was pH dependent, increasing to 500 ± 55 μM when the pH was elevated to 8.5. At low substrate concentration, the optimal uptake was obtained at pH 7.0. Both monovalent and divalent forms of phosphate were transported so that alteration in the transport by pH is believed to be the result of a direct action on the transport system. Finally, sodium levels in the incubation medium dramatically influenced phosphate uptake by increasing the maximal velocity without modifying the affinity for the substrate. This active transport through the placental brush border membranes is believed to be at the origin of the maternal-fetal gradient of phosphate observed during pregnancy

Renal action of progesterone: effect on calcium reabsorption

Recently, the kidney has been reported to be the site of receptors for progesterone. Although the exact segment of the nephron has not been precisely determined, the cortical collecting tubule was suspected, since the hormone displaces bound 3H aldosterone. The aim of the present study was to investigate the effect of progesterone on calcium (Ca(2+)) transport by the renal luminal membranes and to determine the site and mechanisms of action. Incubation of proximal tubules from rabbit kidney with progesterone did not influence Ca(2+) or Na(+) transport by the luminal membranes. In the distal tubules (DT), a 5 min treatment with 10(-11) M of the hormone enhanced 0.5 mM 45Ca uptake from 0.60+/-0.02 to 0.84+/-0.08 pmol/microg per 10 s (P<0.05) in the absence of Na(+) and from 0.26+/-0.02 to 0.41+/-0.02 pmol/microg per 10 s (P<0.01) in the presence of 100 mM Na(+). The dose-response curve showed a biphasic action with a peak at 10(-11) M. Ca(2+) uptake by DT membranes presents dual kinetics. The hormone enhanced the Vmax value of the high affinity component from 0.41+/-0.05 to 0.57+/-0.06 pmol/microg per 10 s (P<0.05). In contrast, incubation of DT with 10(-8) M progesterone decreased 1 mM Na(+) uptake from 0.68+/-0.03 to 0.53+/-0.07 pmol/microg per 10 s (P<0.05). Finally, 10(-11) M progesterone also enhanced Ca(2+) uptake by the DT membranes through a direct nongenomic mechanism.

Sodium gradient-dependent phosphate transport in placental brush border membrane vesicles

Abstract We recently described a sodium gradient-dependent transport of phosphate through the brush border membrane vesicles from human placenta. In order to characterize this transport carrier further, we studied the influence of temperature and membrane potential on the transport of this electrolyte, the stoichiometry of the sodium-phosphate interaction, and the interrelationship between phosphate uptake and other sodium-dependent systems. Temperature influenced phosphate uptake by changing the maximal velocity and the affinity of the carrier for the substrate. The Arrhenius plot for uptake velocity exhibited an abrupt breakpoint at 28.6°C, suggesting that membrane fluidity is a factor in phosphate uptake. Increasing the sodium concentration in the incubation medium augmented the phosphate uptake according to a sigmoid curve, and the Hill plot analysis of these data indicates that at least two sodium ions are transported with each phosphate radical. The effect of membrane potential on phosphate uptake was studied by inducing potassium diffusion with vafinomycin and by using various sodium salts with different anion conductance in the incubation medium. In both series of experiments, the inside-negative potential significantly enhanced phosphate uptake. We concluded that the phosphate-sodium cotransport is an electrogenic process, a conclusion which is compatible with the observation that at least two sodium ions accompany each phosphate radical. Glycine, alanine and proline all inhibited phosphate uptake according to an uncompetitive type of inhibition. In contrast, the addition of glucose to the incubation medium had no effect.

Hyposthenuria in hypercalcemia

SummaryThe intrarenal blood flow (IRBF) distribution has been studied in dogs during acute and chronic calcium intoxication, using clearance technique,85Kr disappearance curves, autoradiograms, and silicone rubber injections. The purpose of this study was to determine whether a modification of the IRBF could be responsible for the decrease of the cortico-medullary osmotic gradient and the hyposthenuria generally observed in these conditions. Acute hypercalcemia was produced in 12 dogs by infusing a solution of CaCl2 into the left renal artery. 20 min after starting infusion (PCa in renal vein = 7.34 mEq/l), the urinary osmolarity and the TcH2O were slightly decreased on the experimental side (P<0.05). C.PAH was also depressed (P<0.005), while E.PAH remained constant. The85Kr curves and the autoradiograms reflected a redistribution of IRBF toward the deeper region of the kidney. In 10 dogs chronically intoxicated with vitamin D, (PCa=6.68 mEq/l) the U.osm and TcH2O were markedly depressed (P<0.001). Renal tissue analysis showed a significant fall of the cortico-medullary osmotic gradient of Na. In parallel, a decrease in C.PAH was observed.85Kr curves demonstrated a significant redistribution of IRBF with a decrease of the percent of initial radioactivity in the cortex. Autoradiograms and silicone rubber vascular casts also demonstrated a vasodilatation of the vasa recta of the medulla. These data therefore support the conclusion that a redistribution of IRBF contributes to the hyposthenuria observed during hypercalcemia.

Fluorometric method for the determination of magnesium in renal tubular fluid

The fluorescent reaction between the N,N′-bis-salicylidene 2,3, diamino benzofuran (SABF) and magnesium (Mg) can be utilized for determination of Mg in nanoliter volumes of renal tubular fluid. Problems of interference are minimal and have been overcome by adding inorganic phosphorus to the standards. Mg in urine may also be determined by this technique if urines of known Mg content are utilized as standards, and if both samples and standards are diluted 110 in artificial tubular fluid. Microdetermination of Mg in glomerular filtrates of Munich Wistar rats confirms that the UF Mg is isotonically filtered through the glomerular membrane.

Characterization of phosphate binding by alkaline phosphatase in rat kidney brush border membrane

Phosphate binding by rat renal brush border membranes occurs on a single protein, as visualized by SDS polyacrylamide gel electrophoresis. The same protein can also be specifically labelled by γ-32P ATP at 0°C or in the absence of magnesium. The phosphate binding protein co-migrates with monomers of two alkaline phosphatase activity bands previously localized on acrylamide gel. Measurement of binding by TCA precipitation, ion-exchange chromatography and dialysis gave an average of 31.1±5.7 pmol phosphate bound/mg protein. Alkaline phosphatase would then represent 0.23% of total brush border membrane protein.Maximal binding activity is obtained at pH 6.5, but when membranes are phosphorylated at pH 6.5 and the pH increased to 9.4, 50% of the bound radioactivity is released. The binding of phosphate to this protein presents two different apparentKm: one at 40 μM for low and one at 390 μM for high substrate concentrations. The membrane bound phosphate is readily exchangeable with phosphate in the medium. Phosphate binding and phosphate release are complete within 5 s. Alkaline phosphatase substrates and EDTA are potent inhibitors of phosphate binding and produce over 90% inhibition. Characteristics of phosphate binding for kidney membrane bound alkaline phosphatase seem very similar to the soluble form of the enzyme from various sources.