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N Farman

Publications and source records attributed to N Farman.

At least 91 records · Page 5Linked to original sources
PubMed1985

1,25(OH)2D3 binding along the rat nephron: autoradiographic study in isolated tubular segments.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding sites were studied along the nephron of rats. The animals were pretreated with the diphosphonate EHDP at doses that inhibit the endogenous production of 1,25(OH)2D3. A dry film autoradiographic technique was applied to tubular segments isolated by microdissection from kidneys incubated in vitro with various concentrations (0.2-12 nM) of [3H]1,25(OH)2D3 in the presence or absence of an excess unlabeled hormone (X200) in order to determine specific binding. Total, nonspecific, and specific labeling were quantified by silver grain counting over cytoplasmic and nuclear areas. Specific nuclear labeling appeared in the cortical ascending limb and papillary collecting tubule at 1 nM. In the distal tubule and, to a lesser extent, in the cortical collecting tubule a specific nuclear labeling was also present, but only at higher concentrations. No specific nuclear labeling was detected in the proximal tubule. All along the nephron, a significant and nonspecific labeling was observed in the cytoplasm, either alone or superimposed over the specific nuclear labeling. In conclusion 1,25(OH)2D3 specific binding sites appear to be localized mainly in the cortical ascending limb of the loop of Henle, in the distal and cortical collecting duct, and in the papillary collecting duct.

Animals↗
PubMed1985

Aldosterone binding in isolated tubules. IV. Autoradiography along the nephron of the spontaneously hypertensive rat.

The binding of aldosterone was studied in tubular segments isolated by microdissection from kidneys of spontaneously hypertensive (SHR, n = 8), Kyoto normotensive (KWR, n = 8), and normal Wistar (NWR, n = 6) rats with an autoradiographic technique on dry film. All animals had been previously adrenalectomized. Kidney pyramids were incubated in vitro before microdissection with collagenase and 2 X 10(-9) M [3H]aldosterone in the presence or absence of an excess of unlabeled aldosterone. In addition, the displacement of the binding by 10 times excess dexamethasone or aldosterone was examined in the cortical and medullary collecting tubule of SHR and KWR to assess the specificity of binding sites. In the three groups, no specific nuclear labeling was detectable in the proximal tubule. The highest specific nuclear labeling was found in the distal portions of the nephron, and intermediate values were present along the loop of Henle. In the cortical collecting tubule, the most specific mineralocorticoid segment, the specific nuclear binding, expressed in silver grains per unit surface, was significantly elevated in SHR (16.1 +/- 1.5) and KWR (13.7 +/- 1.5) as compared with NWR (10.2 +/- 0.8, P less than 0.001 and less than 0.05, respectively). The difference between SHR and KWR did not reach statistical significance. In the medullary collecting tubule, binding was higher in SHR (14.3 +/- 1.6) than in both KWR (8.7 +/- 1.0, P less than 0.005) and NWR (10.1 +/- 0.9, P less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1985

Binding of aldosterone metabolites in isolated tubular segments.

High-performance liquid chromatography (HPLC) analysis was performed on tissue extracts from whole kidney cortex (after in vivo injection or in vitro incubation with [3H]-aldosterone ([3H]A], from products bound to aldosterone receptors in cytoplasmic and nuclear fractions, and from the convoluted (PCT) and straight (PR) portion of the proximal tubule and cortical collecting tubule (CCT) isolated by micro-dissection after in vitro incubation with [3H]A (10 and 80 nM). After in vitro incubation of whole tissue, about 10% of the radioactivity corresponded to three groups of metabolites, two polar ones, which eluted earlier than aldosterone, and one less polar, which eluted later than aldosterone. When HPLC analysis was performed on bound products, after removal of free [3H]A, only one group of polar metabolites was detected (about 8% of the radioactivity) in addition to native [3H]A in the cytoplasmic and nuclear fractions. The binding of polar metabolites was nonspecific (nondisplaceable) and did not vary with incubation time and aldosterone concentration. In isolated tubules, in addition to [3H]A, an early peak of polar metabolites, corresponding to that observed in bound fractions, was present in PCT (10%) and in PR and CCT (20-25%). Other metabolites were not found in isolated tubules. We conclude the following: 1) The kidney is able to form at least three groups of aldosterone metabolites in small amounts. 2) Only one of them (more polar) was detected, as a nonspecifically bound product, in cellular fractions containing hormone-receptor complexes after removal of free [3H]A. Other metabolites were removed with free [3H]A. 3) These polar metabolites were found in both mineralocorticoid target segments such as CCT and in classically nontarget segments such as the proximal tubule. 4) The nonspecificity of the binding of polar metabolites and the absence of selective localization along the nephron suggest that they have no major mineralocorticoid action.

Aldosterone↗
PubMed1985

[Binding of steroid hormones in the kidney].

The localization of mineralocorticoid and glucocorticoid binding sites along the nephron has been investigated by using an autoradiographic technique (dry film) and biochemical methods, applied on tubular segments of rabbit and rat kidney, isolated by microdissection, after in vitro incubation of kidney pyramids with tritiated aldosterone (Aldo) or dexamethasone (Dex). Based on analysis of dose-dependent specific nuclear binding values and competition studies, cortical collecting tubule (CCT) appears as the most specific mineralocorticoid segment; the distal tubule exhibits also high binding values, which are less specific for Aldo than in CCT; Aldo binding was lower in the ascending limb of Henle's loop and medullary collecting tubule and of mixed (gluco and mineralocorticoid) specificity. Aldo receptors were not present in proximal tubule and glomerulus. Specific nuclear binding of Dex was high all along the nephron, except in proximal tubules, and somewhat lower in glomeruli and its arterioles than in the distal parts of the nephron. Binding of 3H-Aldo did not differ drastically along the nephron of the rat and the rabbit.

Adrenal Cortex Hormones↗
PubMed1984

Autoradiographic study of nuclear localization of aldosterone binding sites in intact renal cells: lack of temperature dependency.

We examined by autoradiography on intact target cells the nucleocytoplasmic distribution of aldosterone-receptor complexes and the thermodependency of the nuclear translocation process. Autoradiographs (dry films) were performed on cortical collecting tubules isolated by microdissection, after incubation of rabbit kidney pyramids with [3H]aldosterone (2 X 10(-9)M) in the presence or absence of an excess unlabelled aldosterone (X100). Paired experiments were done at 30 degrees C (1 h) or 4 degrees C (2 h). In both cases, the specific labelling was exclusively nuclear. Values were higher at 30 degrees C (14.5 +/- 1.5 specific silver grains per 100 micron2) than at 4 degrees C (5.4 +/- 0.6), with no concommitant cytoplasmic labelling (4 degrees C:0.7 +/- 0.3; 30 degrees C: -0.04 +/- 0.6). At 30 degrees C, addition of unlabelled spirolactone (X200) prevented the observed autoradiographic nuclear accumulation of aldosterone-receptor complexes, suggesting that cytoplasmic binding preceded the nuclear entry. The results suggest that, in intact cells, almost all aldosterone receptor complexes accumulate in nuclei, and that this process does not depend on temperature. In parallel biochemical-binding series we found the classical distribution of aldosterone receptor complexes both in cytoplasm and nuclei, and the classical thermodependency of nuclear translocation. The present autoradiographic results, together with similar observations reported for sex steroids (Martin P.M. and Sheridan P. J., J. steroid Biochem. 16 (1982) 215-229), question the classical model of thermodependent nuclear translocation, based on biochemical experiments.

Aldosterone↗
PubMed1984

Autoradiographic study of thermo-dependent nucleo-cytoplasmic distribution of aldosterone binding sites in intact target cells.

Autoradiographic studies of [3H]aldosterone [( 3H-A] and [3H]dexamethasone binding sites in intact target cells (isolated collecting tubules of rabbit and rat kidney) revealed an almost exclusive nuclear localization of the hormone-receptor complexes. In the present work we compared the nucleo-cytoplasmic repartition of [3H]A-receptor complexes studied in parallel by biochemical and autoradiographic methods. In addition, the thermo-dependency of the nuclear translocation was examined. Kidney pyramids were incubated in vitro with [3H]A (2 X 10(-9) M) in the presence or absence of a 100-fold excess unlabelled A, at 30 degrees C for 1 h or 4 degrees C for 2 h. Then tissue was processed for isolation of nuclear and cytoplasmic fractions, on the one hand, or for obtention of microdissected tubular segments on which autoradiographs on dry films were performed. Autoradiographs showed that the specific labelling was almost exclusively nuclear without significant cytoplasmic labelling, at both 30 or 4 degrees C. This indicates that almost all binding sites migrated rapidly into nuclei, and that this translocation did not depend on temperature. In contrast, parallel biochemical experiments yielded classical results, that is, at 30 degrees C, the presence of specific binding sites in both cytoplasm and nuclei with a predominance in cytoplasm. At 4 degrees C, the cytoplasmic binding was unchanged, but nuclear binding was drastically reduced, indicating thermodependency of nuclear translocation, when studied by biochemical methods including cell disruption. Autoradiographic results thus questioned the classical notion of thermo-dependent nuclear translocation of aldosterone-receptor complexes, based on results obtained by biochemical methods.

Aldosterone↗
PubMed1984

Distribution of N-acetyl-beta-D-glucosaminidase isoenzymes along the rabbit nephron.

Total N-acetyl-beta-D-glucosaminidase (NAG) activity has been measured in microdissected glomeruli (G) and tubular segments [proximal convoluted tubule (PCT), pars recta (PR), medullary thick ascending limb (MAL), and cortical collecting tubule (CCT)] of the rabbit kidney, by a fluorimetric method using synthetic substrate. Selective activity of the isoenzyme NAG B was also determined. Isoenzyme profiles of NAG were obtained by electrofocusing on each segment. Characterization of the isoenzymes was performed by chromatofocusing and thermosensitivity experiments on PCT. Total NAG activity, mainly composed of NAG A, was low in glomeruli and two and one-half to four times higher in PCT than in other segments, in which comparable activities were found. NAG B was detectable all along the nephron. It represented a very small fraction of total NAG, except in PCT where it was more abundant (20 to 30%). Electrofocusing revealed the presence of a minor form (NAG I) all along the nephron. Chromatofocusing and thermosensitivity studies indicated that NAG I could represent imperfectly solubilized NAG A rather than a well defined entity. From these results, it could be suggested that the reported increase in urinary excretion of NAG B after renal injury may reflect the intensity of proximal tubular lesions.

Acetylglucosaminidase↗
PubMed1984

Heterogeneity of uridine incorporation along the rabbit nephron. I. Autoradiographic study.

We present here an autoradiographic study of uridine labeling in tubular segments micro-dissected from the rabbit kidney. Kidney pyramids were incubated for 60 min with low (66 nM) and high (66 microM) [3H]-uridine concentration. At the two concentrations studied the labeling was almost exclusively nuclear in all segments studied. At the low concentration, labeling predominated in the macula densa (MD = 63.88 +/- 6.15 silver grains/100 micron2, n = 11), cortical ascending limb (CAL = 19.65 +/- 1.65, n = 15), and initial distal tubule (DCTa = 24.31 +/- 2.70, n = 6). It was minimal in the proximal tubule (PCT2 = 9.14 +/- 1.61, n = 16) and in the cortical (CCT = 5.23 +/- 0.75, n = 18) and medullary (MCT = 5.52 +/- 1.10, n = 12) collecting ducts. At a high concentration, the profile of labeling was roughly similar except for a relative increase in labeling much more pronounced in collecting ducts (CCT = +373, MCT = +323%) than in the other structures (MD = -14, CAL = +66, DCTa = +49, PCT = +9%). Pulse-chase experiments do not show evidence for differences in turnover or degradation rates of RNA between segments, at least in the PCT and the connecting part of the CCT. Analysis of the results at low and high concentration suggests that the observed heterogeneity in uridine labeling depends on both variable endogenous nucleoside pools and different rates of uridine incorporation into RNA from one segment to another.

Animals↗
PubMed1984

[Binding of aldosterone to its receptors along the nephron of the normal and spontaneously hypertensive rat].

The specific binding of aldosterone has been investigated using an autoradiographic method, on isolated tubular segments of kidneys from spontaneously hypertensive (SHR) and normotensive (KWR) Okamoto rats, and normotensive Wistar (NWR) rats. A low, mineralocorticoid, concentration of aldosterone (2 X 10(-9) M) was used. In proximal tubules, almost no specific labelling was found in the three groups. In the cortical ascending limb, a specific nuclear labelling was present and equivalent in the three groups. In the distal (DCT) and cortical collecting (CCT) tubule, the specific labelling was double in SHR and KWR than in NWR. This higher binding capacity occurs in target segments for aldosterone, and coincides with the increased sodium reabsorption in the distal structures of Okamoto rats, previously reported by our laboratory. The relations between these observations and the altered membrane permeability described in hypertensive disease is discussed.

Aldosterone↗
PubMed1983

Autoradiographic determination of dexamethasone binding sites along the rabbit nephron.

Specific binding sites of tritiated dexamethasone ([3H]dex) along the tubule of rabbit kidney were investigated using an autoradiographic method (dry film) on isolated tubular segments. After in vitro incubation of kidney pyramids with [3H]dex (0.15-53 nM) in the presence or absence of an excess (X200) of unlabeled dexamethasone, tubular segments were microdissected and processed for autoradiography. A quantitative analysis of specific labeling over cytoplasm and nuclei was performed. Specific nuclear binding was observed in all tubular segments beyond the pars recta. This binding was dose dependent and reached much higher values than those reported for aldosterone. In the proximal tubule, the specific labeling was also high but remained mostly cytoplasmic. The meaning of these drastically different intracellular localizations is still open to interpretation. Autoradiography was performed after in vivo injection of [3H]dex and [3H]aldosterone. The results were not different from those described here for dexamethasone and from those previously reported for aldosterone after in vitro incubation. We conclude that specific nuclear binding sites for dexamethasone range over the nephron except for proximal tubule, with no great difference among segments, in contrast to specific sites for aldosterone, which are restricted to distal and cortical collecting tubules. The exact significance of the proximal cytoplasmic specific binding of [3H]dex remains to be determined.

Aldosterone↗
PubMed1983

Aldosterone binding in isolated tubules. III. Autoradiography along the rat nephron.

Aldosterone binding was investigated along the nephron of adrenalectomized Wistar rats. Autoradiographs of microdissected tubular segments were performed on dry film after incubation with [3H]aldosterone [( 3H]A) at 2 X 10(-10), 2 X 10(-9), and 2 X 10(-8) M in the presence or absence of an excess unlabeled hormone. Competition studies were done with a 10 times excess A, dexamethasone (D), and estradiol in the presence of 2 X 10(-8) [3H]A. Specific nuclear labeling predominated in the cortical collecting tubule (CCT) and A specificity of the binding was assessed by the much better displacing potency of unlabeled A as compared with D. Labeling in the distal tubule (DCT) resembled that of CCT, but D was nearly as good a competitor as A. In the loop and medullary collecting tubule, labeling was lower, with mixed steroid specificity. No specific nuclear labeling was detectable in the proximal tubule. We conclude that the CCT and DCT present the highest binding capacity for aldosterone. Competition studies revealed that the CCT is probably the more specific mineralocorticoid target segment. In other segments beyond the pars recta (loop of Henle, medullary collecting tubule), aldosterone probably binds to both mineralocorticoid and glucocorticoid binding sites. No major difference appeared between the profile of aldosterone binding along the rat tubule and the one previously described in the rabbit.

Adrenalectomy↗
PubMed1982

Aldosterone binding in isolated tubules I. Biochemical determination in proximal and distal parts of the rabbit nephron.

Microbiochemical methods were applied to proximal tubules (PCT) and a mixture of distal and cortical collecting tubules (D + C) of rabbit kidney in order to define aldosterone binding sites. For each experiment, after incubation of kidney pyramids with [3H]aldosterone ([3H]A), either alone or in the presence of an excess unlabeled A, 100-150 mm of both categories of tubules were microdissected using collagenase. Specific binding was determined on the nuclear fraction of each sample. Aldosterone concentrations ranged from 2 X 10(-9) to 4.5 X 10(-8) M. No specific binding was detectable in PCT. Specific binding in D + C increased rapidly as a function of [3H]A concentration up to 5 X 10(-9) M and then more slowly. No plateau was reached. Both the absence of saturation of the binding curve and the curvilinear aspect of the Scatchard plot suggested the presence of two binding sites, one of high affinity, presumably a mineralocorticoid site, and the other of lower affinity, possibly a glucocorticoid site. These experiments suggest that the distal structures of the nephron, located in the cortex, are the main sites of binding of aldosterone and contain a high number of specific binding sites for this hormone.

Aldosterone↗
PubMed1982

Aldosterone binding in isolated tubules II. An autoradiographic study of concentration dependency in the rabbit nephron.

Specific binding of aldosterone along the rabbit nephron was investigated using an autoradiographic method on microdissected tubules within a large range of [3H]aldosterone ([3H]A) concentrations (3 X 10(-10) to 2 X 10(-8) M). Paired incubations with [3H]A in the presence or absence of unlabeled A (100-fold excess) were done to determine the specific binding. In addition, competition studies (10 times excess) with A, dexamethasone (Dex), and estradiol (E) were performed. No specific nuclear binding was detected in the proximal convoluted tubule and pars recta. In the cortical collecting tubule (CCT), a high specific labeling occurred at concentrations of A as low as 3 X 10(-10) M that plateaued at 1.5 X 10(-9) M. Aldosterone was the best competitor. In the bright and granular distal tubule, the binding resembled that of CCT, but Dex was nearly as potent a competitor as A. All along the loop of Henle and in the medullary collecting duct, the binding of A was weak at low concentrations and gradually rose with concentration without reaching a plateau. An almost equivalent displacement was obtained by A and Dex in 10-fold excess. We conclude that aldosterone binds mostly to mineralocorticoid sites in the collecting tubule, whereas the binding appears to be to both mineralo- and glucocorticoid sites in the distal tubule, in the different parts of the loop of Henle, and in the medullary collecting tubule.

Aldosterone↗
PubMed1982

Autoradiographic study of aldosterone and dexamethasone binding in isolated glomeruli of rabbit kidney.

Specific binding of tritiated aldosterone ([3H]A) and dexamethasone ([3H]Dex) has been investigated in glomeruli isolated from rabbit kidney by microdissection, using an autoradiographic method on dry film. At low physiological concentrations of [3H]A (0.2-2 nM), no specific binding was detected. At high concentration (24 nM), a low specific binding was found in cytoplasm (2.5 specific silver grains/100 micrometers2) and nuclei (3.7), which was about 10 times lower than in distal tubule. No difference was found between superficial and juxtamedullary glomeruli. At 2.4 nM [3H]A, no aldosterone binding was present in glomerular arterioles. Nuclear binding of [3H]Dex was low at 3 nM (6.4 specific silver grains/100 micrometers2) and reached 23.6 at 100 nM without significant cytoplasmic binding. Here again, this binding was much lower than in distal structures of the nephron. In the few arterioles obtained, a clear specific nuclear binding was observed. We conclude that the glomerulus is probably not a target structure for aldosterone. The existence of a specific binding of dexamethasone, although lower than in the distal tubule, raises the problem of its physiologic significance.

Aldosterone↗
PubMed1981

Gentamicin incorporation along the nephron: autoradiographic study on isolated tubules.

Gentamicin is a nephrotoxic antibiotic of the aminoglycoside group, which accumulates within the renal cortex. The present study is an attempt to localize precisely the sites of gentamicin accumulation along isolated tubular segments. We performed autoradiography of 3 H-gentamicin (3H-G) uptake on isolated tubules from kidneys of 6 rabbits previously treated by a single dose of this drug (125 muCi/kg of body wt; 140 microgram/kg of body wt). Isolated tubules were obtained by microdissection following collagenase incubation, 4 hours after 3H-G administration. Autoradiography of single isolated tubular segments was performed according to a dry-film technique. Results were as follows. Almost no gentamicin incorporation (less than 2 silver grains per 150 micrometer2) takes place along the distal parts of the nephron, from the beginning of the loop of Henle to the end of the medullary collecting duct. No differences were visible along these parts of the nephron, whatever their localization, cortical or medullary, In the proximal tubule (PT), we observed a gradual regular increase in 3H-G accumulation, from the glomerulus to the end of the pars recta. The silver grain density progressively increased along this structure from the very early PT (5 per 150 micrometer2) to the last millimeter of the pars recta (40 per 150 micrometers). No clear difference between superficial and juxtamedullary nephrons was detected. The possible mechanisms that could account for this observed variation in 3H-G cellular uptake along the PT are discussed.

Animals↗
PubMed1981

Binding of aldosterone to cytoplasmic and nuclear receptors of the rabbit kidney.

The binding characteristics of [3H]aldosterone ([3H]A) in the rabbit kidney were compared with those described in other species, and experimental conditions suitable for both the isolation of renal tubules and the retention of aldosterone receptors were examined. Specific binding of [3H]A to cytoplasmic (C) and nuclear (N) receptors was determined on kidney slices incubated at +30 degrees C in rabbits fed a low K+-high Na+ diet. Results indicate the presence of at least two binding sites in N fractions, one of high affinity (Kd (I)) 1.8 X 10(-8) M) and low capacity (Nmax (I) 4.6 X 10(-14) mol/mg DNA), called type I sites, and the other of lower affinity (Kd (II) 8.9 X 10(-8) M) and more numerous sites (Nmax (II) 13.1 X 10(-14) mol/mg DNA). Competition studies showed that type I sites have higher affinity for [3H]A than for other steroids. Incubation at +37 degrees C resulted in a loss of type I sites. In adrenalectomized rabbits the C binding was improved but not the N binding (Kd (I) 1.2 X 10(-8) M; Kd (II) 5.6 X 10(-8) M). In rabbits treated with aminoglutethimide, an inhibitor of steroid synthesis, the N binding was lower than in other series. It was concluded that 1) [3H]A binding in the rabbit kidney does not differ from that described in other species; 2) a low K+-high Na+ diet is a satisfactory condition for study of [3H]A binding; and 3) incubation at +30 degrees C, a temperature necessary for microdissection procedures, is suitable for aldosterone binding studies.

Adrenal Cortex Hormones↗
PubMed1981

Aldosterone binding along the rabbit nephron: an autoradiographic study on isolated tubules.

The sites of action of aldosterone (A) along the tubule of rabbit kidney were studied by autoradiographic localization of mineralocorticoid-binding sites on microdissected tubular segments. Kidney pyramids were incubated at 30 degrees C for 1 h in a collagenase solution with [3H]aldosterone at a concentration of 1.5 X 10(-9) M with and without an excess unlabeled A. Tubular segments were then microdissected and transferred onto dry film; fixation and staining were done only after exposure of the film 4 mo later in order to avoid diffusion. Specific nuclear labeling was 19.0 +/- 1.3 silver grains/100 micrometers2 in distal convoluted tubules (n = 28) and 21.0 +/- 1.8 in cortical collecting ducts (n = 18). No difference between these two structures was observed (P greater than 0.1, paired t test, n = 15). No specific binding was found in the proximal tubule (0.5 +/- 0.4, n = 17). In the thick ascending limb of Henle's loop, the labeling was low (3.9 +/- 0.9, n = 16). We conclude that, in the rabbit kidney, nuclear mineralocorticoid-binding sites, presumably receptors, are present in the distal and cortical collecting tubule.

Aldosterone↗