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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.

Effect of Renin-Angiotensin System Blockade on the Expression of the Angiotensinogen Gene and Induction of Hypertrophy in Rat Kidney Proximal Tubular Cells

Studies have shown that high levels of glucose and angiotensin II (Ang II) stimulate hypertrophy and the expression of matrix protein genes in mouse proximal tubular cells in vitro. The present study tested the hypothesis that blockade of the renin-angiotensin system (RAS) inhibits the stimulatory effect of high levels of glucose on the expression of the renal angiotensinogen (ANG) gene and the formation of Ang II and subsequently attenuates the induction of hypertrophy in kidney proximal tubular cells. Immortalized rat proximal tubular cells (IRPTC) were cultured in monolayer. The levels of expression of rat ANG and ANG mRNA in the IRPTC were quantified by specific radioimmunoassays for rat ANG (RIA-rANG) and by a reverse-transcription polymerase chain reaction (RT-PCR) assay, respectively. Hypertrophy of IRPTC was analyzed by flow cytometry (FACScan) and cellular protein assay. Our studies showed that losartan (an Ang II (AT1)-receptor blocker), perindopril and captopril (inhibitors of angiotensin-converting enzyme) blocked the stimulatory effect of a high level of glucose (i.e. 25 mM) on the expression of the rat ANG gene and hypertrophy in IRPTC but not by the Ang II (AT2)-receptor blocker. Our studies indicate that the blockade of RAS is effective in inhibiting the stimulatory effect of hyperglycemia on the expression of the ANG gene and hypertrophy in IRPTC, supporting the notion that the local formation of intrarenal Ang II may play a role in the development of renal hypertrophy during early diabetes.

Composition and physical properties of lipids from plasma membranes of dog kidney

Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have been determined in plasma membranes of the dog kidney. The lipid composition of brush-border membranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospholipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44 degrees C approximately. In the range of temperature studied (5-55 degrees C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37 degrees C which compares with that of other biological membranes.

Molecular cloning and expression of the rat angiotensinogen gene.

To identify tissue- and hormonal-specific DNA controlcis-elements in the rat gene, we have constructed fusion genes consisting of various lengths of the 5′-flanking region of the rat angiotensinogen gene linked to a human growth hormone (hGH) reporter gene and have introduced them into a subclone of rat pancreatic islet tumor cell line (1056A) which expresses the highest level of angiotensinogen mRNA. As a negative control, we have also introduced them into a human choriocarcinoma cell line (JEG-3), which does not express the endogenous angiotensinogen gene. The level of the expression of these fusion genes in these cells was determined by the level of immunoreactive hGH secreted into the culture medium. The expression of angiotensinogen-growth hormone (ANG-GH) fusion genes, pOGH (ANG N-1498/+18), pOGH (ANG N-688/+18), pOGH (ANG N-110/+18), pOGH (ANG N-53/+18), and pOGH (ANG N-35/+18) was 1.0, 1.8, 1.5, 12.0 and 3.0-fold higher, respectively, than the promoterless growth hormone expression vector (pOGH). The addition of dexamethasone (10−6 M), aldosterone (10−5 M), and thyroid hormone, L-T3 (10−7 M), stimulated the expression of pOGH (ANG N-1498/+18) by 4.0-, 2.5-, and 2.0-fold above the control level, respectively. Combination of dexamethasone (10−6 M), L-T3 (10−7 M), and ethinyl-estradiol (10−6 M) stimulated the expression of the pOGH (ANG N-1498/+18) to greater than 10-fold over the control. Ethinyl-estradiol (10−6 M) or progesterone (10−6 M) alone had no effect on the expression of the pOGH (ANG N-1498/+18). These studies demonstrate that the induction of expression of the angiotensinogen gene by dexamethasone and L-T3 in 1056A cells is due to a transcriptional mechanism and the 1056A cells could be useful for studying angiotensinogen gene regulation and for identifying the glucocorticoid and L-T3-responsivecis-regulatory elements in the angiotensinogen gene.

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.

Catecholamine sulfates: end products or metabolic intermediates?

Dopamine-beta-hydroxylase catalyzes the beta-oxidation of dopamine to noradrenaline while phenylethanolamine-N-methyltransferase converts noradrenaline to adrenaline. Since catecholamine sulfates represent the predominant form of catecholamines in human tissues, we have studied the role of dopamine sulfate and noradrenaline sulfate as alternate substrates for dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase, respectively. Dopamine 3-sulfate, dopamine 4-sulfate and noradrenaline 3-sulfate were chemically synthesized and exhaustively purified by ion-exchange chromatography. Dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase were partially purified from human adrenals. Using tyramine as substrate, dopamine-beta-hydroxylase is slightly inhibited by dopamine 3-sulfate according to some irreversible or mixed mechanisms. When dopamine-beta-hydroxylase was incubated with dopamine 3-sulfate or dopamine 4-sulfate, we were not able to find any synthesis of either noradrenaline sulfate or free noradrenaline. Using phenylethanolamine as substrate, the enzymatic activity of phenylethanolamine-N-methyltransferase remains unchanged with addition of dopamine 3-sulfate, dopamine 4-sulfate or noradrenaline 3-sulfate. It was concluded that dopamine sulfate is not an alternate substrate for either dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase nor is noradrenaline 3-sulfate an alternate substrate for phenylethanolamine-N-methyltransferase.

Partial purification and characterization of two isoenzymes involved in the sulfurylation of catecholamines

Abstract To elucidate the specificity of the enzymatic system involved in the sulfurylation of catecholamines, the purification of the enzyme(s) from canine liver was undertaken. Ion-exchange chromatography led to the resolution of two sulfotransferases A and B with different pH optima for dopamine (6 for A and 9.5 for B). The apparent K m values for 3′-phosphoadenosine 5′-phosphosulfate and dopamine were 1.7 μM and 17.7 μM for enzyme A and 26 μM and 6.2 μM for enzyme B. Each enzyme has a molecular weight of 60 000 while their isoelectric points differ; 5.7 for A and 4.7 for B. The enzyme B catalyzes the sulfurylation of a wider range of substrates than A which is preferentially active with dopamine. These results suggest the presence of two isoenzyme forms of sulfotransferase in canine liver.

Magnesium handling by the papilla of the young rat

In a recent study it was found that in Mg loaded rats, the fraction of filtered Mg (% E Mg) recovered in the bend of the loop of Henle of papilla was greater than the filtered load. However, the site of this Mg addition was unspecified and could be either the juxtamedullary proximal tubule, the pars recta, or in the papilla, the descending limb of the loop of Henle.In order to investigate the movement of Mg in the various structures of the papilla, we have studied: 1. The transport of this electrolyte along the collecting duct. 2. Its relative concentration in the loop of Henle and in the adjacent vasa recta. The experiments have been performed in hydropenic and Mg loaded rats.In the collecting duct, the inulin and Mg concentrations increase proportionally, indicating an absence of any transport of Mg along this part of this nephron.In the vasa recta of the accessible papilla, the capillary over peripheral plasma Mg ratio (C/UF Mg) in hydropenia and after Mg loading [1.88±0.15 (ES) and 2.89±0.24] were significantly lower than the corresponding TF/UF Mg in the adjacent loops of Henle (2.90±0.17 and 4.04±0.37). This finding reduces the possibilities of a Mg passive diffusion from the capillaries to the tubular lumen, unless the electrical potential of the descending limb is more negative than −5 mV. The hypothesis of an active secretion, or a passive diffusion of Mg in the deep proximal tubule, in the pars recta, or in the early non accessible descending limb constitutes the other alternative.

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells.

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5′-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a β1/β2-adrenergic receptor (AR) agonist) and iodoclonidine (an α2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (α1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (β-AR blocker), atenolol (β1-AR blocker), yohimbine (α2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (β2-AR blocker) and prazosin (α1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP 254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5′-flanking region of the ANG gene and subsequently stimulates the gene expression.

Indapamide for out-patient treatment of hypertension: modifications in serum catecholamine levels

A study was carried out to evaluate the efficacy of indapamide in the treatment of 20 patients with mild hypertension and to determine whether a favourable response to treatment was related to initial hyperadrenergic status, to changes in serum catecholamine levels or modified by orthostatic stress. After 4 weeks on placebo, patients received 2.5 mg indapamide per day for 4 weeks and, if diastolic pressure was controlled below 100 mmHg, this regimen was continued for a further 8 weeks. Those not adequately controlled with indapamide alone were treated additionally with nadolol (mean dose 140 mg per day) for a period of 12 weeks. The results showed that indapamide alone produced a significant reduction in blood pressure both in the recumbent and upright position in 12 (60%) of the patients and that addition of the beta-blocker augmented the antihypertensive effect. No relationship was demonstrated between initial high serum catecholamine levels and a favourable response to indapamide. Recumbent serum norepinephrine levels after indapamide alone or with nadolol for 4 to 8 weeks remained comparable with placebo levels. However; the amplitude of the norepinephrine increase due to orthostatic stress was significantly reduced after 8 weeks of indapamide treatment. Possible explanations for this are discussed.