Electron-probe analysis of cultured cells.
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Animals↗
Biomedical subjects
C Lechene
Publications and source records attributed to C Lechene.
Studies on final differentiation of rat renal proximal tubular cells in culture.
The ontogeny of effective Na and K permeability has been studied in renal epithelial cells isolated from the outermost superficial cortex from adult and young (10-15 days) rats. The cells were cultured for 2-4 days and exhibited phloridzin-inhibitable alpha-methylglucoside uptake, characteristic of renal proximal tubular cells (RPTC). Intracellular concentrations of K, Na, Cl, and P and kinetics of changes in intracellular ionic content after inhibition of Na-K-ATPase with 1 mM ouabain (or by incubation in low-K medium) were measured in individual cells using electron probe analysis. Intracellular concentrations of K, Na, Cl, and P were equivalent in young and adult rat RPTC. Adult rat and young rat cells preincubated in K-free medium rapidly recovered normal intracellular K and Na contents when returned to 5.5 mM K medium. The recovery was almost immediately blocked by ouabain. Effective permeabilities measured as half time of K efflux and Na influx after ouabain inhibition of Na-K-ATPase were higher in adult than in young RPTC cultured for less than 4 days. Effective K and Na permeabilities decreased significantly with increasing time in culture in adult but not in young rat RPTC. Among young rat RPTC, half times of Na and K fluxes were significantly correlated to age. Effective K and Na permeabilities were lower in both young and adult rat RPTC that had been serum deprived for 24 h than in cells that had been continuously cultured in serum. In cells cultured for 3 days and serum deprived for 1 day, the addition of serum significantly increased K and Na permeability both in young and adult RPTC, but the effect was more pronounced in young RPTC where permeability reached the same high values as in adult RPTC continuously cultured in serum. In conclusion, effective Na and K permeabilities and serum activation of "permeability units" change during ontogeny. These ontogenic changes might be blunted after a few days in culture due to dedifferentiation of adult rat RPTC.
Aging↗
Coupling of Na-H exchange and Na-K pump activity in cultured rat proximal tubule cells.
Na-H exchange was studied using electron probe analysis and microfluorescent pH analysis of individual cells, in 3-day primary cultures of rat proximal tubule cells (RPTC) obtained from 40- to 50-day-old Sprague-Dawley rats. After Na-K pump inhibition, the initial rate of net Na influx was inhibited 87% by 1 mM amiloride. K influx, an estimate of Na-K pump activity, was increased approximately three times in cells containing high Na (0.114 mM K X mM P-1 X min-1) compared with control cells containing low Na (0.038 mM K X mM P-1 X min-1). Single cell measurements of RPTC loaded with the cytoplasmic pH indicator 5- (and -6) carboxy-4',5'-dimethylfluorescein indicated that there was reversible intracellular acidification in the absence of external Na or in the presence of amiloride. When intracellular acidification was induced by the addition and subsequent removal of NH4Cl, recovery of intracellular pH was inhibited in the absence of external Na or in the presence of amiloride. Using a similar protocol, it was found that after intracellular acidification, the rate of Na influx increased at least 5.9 times, and intracellular Na content was increased 3.15 +/- 0.64 times at 60 s. There was an initial 50% inhibition of Na-K pump activity within the first 60 s compared with control (nonacidified) RPTC, secondarily followed by an increase in Na-K pump activity. Amiloride (0.5 mM) inhibited the acidification-induced increase in Na influx, and persistent acidification led to a persistent inhibition of Na-K pump activity compared with control. In summary, these results demonstrate that Na-H exchange mediates the majority of net Na influx into RPTC under our basal conditions and is necessary for maintenance of intracellular pH homeostasis. In RPTC, Na-H exchange is further activated by intracellular acidification, leading to a net increase in intracellular Na content, which secondarily stimulates Na-K pump activity. The initial inhibition of Na-K pump activity may be due to a direct effect of intracellular acidification.
Amiloride↗
Role of renal prostaglandins in the fall in urine osmolality after papillary micropuncture.
The effect of micropuncture of the renal papilla through an intact ureter on urinary concentrating ability of rats was examined. Micropuncture of the renal papilla caused a fall in urine osmolality in the punctured kidney from 1718 +/- 106 to 1035 +/- 79 mosmol/kg X H2O. In order to investigate the role of renal prostaglandins in this process, PGE2 excretion was measured and found to increase from 63.4 +/- 14.0 to 205.5 +/- 57.1 pg/min. Urine osmolality and PGE2 excretion from the contralateral kidney were not significantly altered. In animals given meclofenamate (2 mg/kg X hr), renal PGE2 excretion was reduced to 22.3 +/- 5.1 pg/min prior to micropuncture and it remained low at 8.9 +/- 1.8 pg/min after papillary micropuncture. Meclofenamate also blocked the fall in urine osmolality caused by micropuncture of the renal papilla, with urine osmolality averaging 1940 +/- 122 before and 1782 +/- 96 mosmol/kg X H2O after the micropuncture. These results indicated that papillary micropuncture through an intact ureter increased renal PGE2 excretion and that a rise in renal production of PGE2 or some other prostanoid is associated with a fall in urine concentrating ability.
Animals↗
Cellular volume and cytoplasmic gel.
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Animals↗
Preparation of individual human diploid fibroblasts and study of ion transport.
A method for analyzing individual mammalian cells with electron probe microanalysis has been developed using human diploid fibroblasts. Cells were grown on the same support that is used for experimental manipulations and analysis. Steady-state cation and anion concentrations and kinetic processes during experimental perturbations could be measured on populations of less than 1,000 cells. Human diploid fibroblasts in normal tissue culture medium had the following intracellular concentrations (in mM): K, 168; Na, 25.0; Cl, 51.2; P, 84.1; S, 16.5; Ca, 6.04; and Mg, 10.0. The ratios of K to Na were equivalent when measured in the nuclear or cytoplasmic area of the cells. Serum in the incubation medium was found to increase the cellular effective permeability to Na by a factor of 2.5, while leaving the effective permeability to K unchanged. When returned to control medium after 7 h of incubation in K-free medium, the cells recovered normal K/Na in less than 1 h. In some experiments the coupling ratio of the ouabain-inhibitable cellular transport of Na to K was 3:2 and the ratio of Cl to K was 1:2. The sum of intracellular content (Na + K) (an estimate of cellular volume) did not change when the cells were placed in K-free medium and increased by less than 30% after ouabain treatment. After 5-7 h of ouabain treatment or of incubation in K-free medium, long after the intracellular K had been replaced by Na, the cellular chloride content had not changed significantly.
Biological Transport↗
Nucleotides in a single mammalian ovum or preimplantation embryo.
ATP, ADP, and AMP have been measured jointly on a single mouse ovum or preimplantation embryo using an ultramicrofluorescence technique. The method uses the traditional approach of enzymatic analysis based on changes in the concentrations of nucleotide cofactors, but eliminates the need for enzymatic recycling. It permits the measurement of as little as 10 fmol, and may be adapted for numerous metabolites.
Adenine Nucleotides↗
Prostaglandin-vasopressin interactions on the renal handling of calcium and magnesium.
The role of prostaglandins in the regulation of sodium and water excretion has been widely studied, but little is known about the influence of prostaglandins (PGs) on the tubular handling of calcium, magnesium or phosphorus. Recent observations have suggested that PGE2 and vasopressin may interact and influence reabsorption of calcium and phosphorus in the cortical collecting duct. The present study investigated the effect of meclofenamate (2 mg/kg), and inhibitor of PG synthesis, on the excretion of calcium, magnesium and phosphorus. Experiments were performed in antidiuretic and water diuretic rats to examine potential PG-vasopressin interactions on the reabsorption of these ions by renal tubules. In antidiuretic rats given meclofenamate, urine osmolality increased whereas urine flow and the fractional excretion of water, urea, sodium, calcium and magnesium decreased by 30 to 50%. In water diuretic animals, urine osmolality and urea excretion were unaltered after meclofenamate administration. Fractional excretion of sodium, water, calcium and magnesium declined approximately 50% in water diuretic rats given meclofenamate. Urinary excretion of PGE2 was not significantly different in water diuretic and antidiuretic rats averaging 262 +/- 78 vs. 167 +/- 35 pg/min, respectively. Meclofenamate significantly reduced urinary excretion of PGE2 in both groups. The results indicate that renal PGs modulate renal tubular reabsorption of calcium and magnesium, as well as sodium and water.
Absorption↗
An NADH-coupled assay for femtogram or nanogram quantities of chymotrypsin.
Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.
Animals↗
Effect of phosphate deprivation on phosphate reabsorption in rat nephron: role of PTH.
The sites of enhanced phosphate (PO4) reabsorption after PO4 deprivation were investigated before and after infusion of parathyroid hormone (PTH) in acutely thyroparathyroidectomized rats. Animals were fed either a control PO4 diet (1.6% P) or a low PO4 diet (0.025% P) for 2 days or 7-10 days. In control rats, PTH decreased PO4 reabsorption in the proximal tubule, loop of Henle, and distal convolution. PO4 reabsorption in the proximal tubule was enhanced after 2 days of PO4 deprivation. In this group, proximal PO4 reabsorption was decreased by PTH but remained greater than in control rats (70 +/- 6 vs. 45 +/- 6 pmol/min; P less than 0.025). After PTH, PO4 reabsorption increased in the loop of Henle from 3 +/- 0.5 to 13 +/- 2 pmol/min (P less than 0.005), whereas it was unaltered in the distal convolution in PO4-deprived rats. PTH markedly increased fractional excretion of PO4 in control rats but not in PO4-deprived rats. After prolonged PO4 deprivation, PO4 reabsorption along the nephron was unaltered by PTH. These results demonstrate that acute PO4 deprivation enhances PO4 reabsorption in the proximal tubule, although the phosphaturic effect of PTH in this segment is not abolished. Resistance to the inhibitory effect of PTH on PO4 reabsorption in some portion of the loop of Henle and possibly also in the distal convolution accounts for the absence of a significant phosphaturic effect of the hormone in acutely PO4-deprived rats. Prolongation of PO4 deprivation results in unresponsiveness to PTH extending to the proximal tubule.
Animals↗
Electron probe analysis of small populations of single cells.
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Animals↗
Fluorescence assay for picomole quantities of ammonia.
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Ammonia↗
Analysis of standing droplets in rat proximal tubules.
Volume, osmolality, and concentrations for Na, Cl, and raffinose have been measured as a function of time in standing droplets within rat intermediate and late proximal tubules. Standing droplet reabsorption proceeds without the development of a measurable osmotic difference across the epithelium. After 140 s of tubular exposure, droplet-to-plasma concentration differences are observed for raffinose, Na, and Cl with the observed Na concentration difference, usually referred to as limiting gradient, being approximately 9 mM. It is possible that a smaller or even no limiting difference would be attained with longer exposure times. Previous values measured for the limiting Na concentration in the rat proximal tubule were determined before the attainment of constant concentrations. Assuming that the Na concentration we measured is the limiting value, we estimate that active NaCl transport accounts for a very small fraction, less than 6%, of the volume reabsorption; using an alternative approach of fitting a theoretical model to our experimental data, active NaCl transport is again estimated to account for only 6% of the total reabsorbate. The previous interpretation that a limiting Na concentration gradient constitutes the most direct evidence for active Na transport may be in error; the gradient we measure can be modeled without incorporating active NaCl transport.
Animals↗
Meclofenamate and urine concentration with and without exposure of the renal papilla.
The recent finding that inhibitors of prostaglandin synthesis prevent the fall in urine concentration produced by papillary exposure challenges the hypothesis that contact between the pelvic urine and papilla is essential to the renal concentrating process. The present study examines the change in urine osmolality produced by exposure of the renal papilla in rats given meclofenamate. In control animals urine osmolality(Uosmol) decreased 57% after 2 h of exposure of the renal papilla. In rats given meclofenamate 4 mg/kg urine osmolality increased 16%, urine flow decreased 30%, and glomerular filtration rate was unchanged in the nonexposed kidney. Meclofenamate, however, did not alter the decrease in Uosmol seen in the kidney with the exposed papilla. Meclofenamate 10 mg/kg was also ineffective in preventing the fall in urine osmolality produced by papillary exposure, although this higher dose decreased glomerular filtration rate and arterial blood pressure. These results are consistent with the finding that pelvic urine urea is important to the urinary concentrating process and with the hypothesis that urine osmolality falls after papillary exposure because contact between pelvic urine and papilla is interrupted.
Animals↗
Prostaglandin E2 and F2 alpha reduces urea reabsorption from the rat collecting duct.
The present study examines the possibility that prostaglandins affect the renal tubular handling of urea. Meclofenamate (1 mg.kg-1.h-1), an inhibitor of prostaglandin synthesis, decreased fractional urea clearance from 86 to 67%, increased urine osmolality, and decreased the fractional excretion of water in rats undergoing a hypertonic sodium chloride diuresis. The percentage of [14C]urea microinjected into distal convoluted tubules that was recovered in urine fell from 75 to 64% (P less than 0.01) after meclofenamate. The fraction of injected urea excreted like inulin (direct recovery) was reduced from 20 to 8% (P less than 0.0001) by meclofenamate. Addition of PGE2 (1.2 or 89 pmol) or PGF2 alpha (1.4 pmol) to the microinjectate returned the urinary recovery of the microinjected [14C]urea to the control level, but PGA2 (3 pmol) did not. Direct urea recovery was doubled by PGE2 or PGF2 alpha. These results indicate that prostaglandin E2 and F2 alpha inhibit the reabsorption of urea in the collecting duct. Prostaglandins may participate in the renal concentrating mechanism by altering the inner medullary influx of urea.
Absorption↗
ADH-PGE2 interactions in cortical collecting tubule. I. Depression of sodium transport.
Antidiuretic hormone (ADH) (200 microunits/ml Pitressin or synthetic arginine vasopressin) causes a transitory increase followed by a sustained decrease in the potential difference (PD) and in the net fluxes of sodium and chloride across rabbit cortical collecting tubules perfused in vitro. The inhibitory action of vasopressin is reversible; removal of the hormone from the bath promotes recovery in the PD and in the transport of sodium and chloride to the level of the controls. After 70 min of incubation with ADH, 10(-5) M meclofenamate, an inhibitor of the synthesis of prostaglandins, was added to the bath of some tubules. Despite the presence of ADH, the PD and ionic fluxes increased to control levels. The introduction of exogenous prostaglandin E2 (10(-5) M PGE2) to the bathing medium containing ADH and meclofenamate mimicked the inhibitory action of ADH, decreasing the PD and the reabsorption of sodium and chloride. Pretreatment of collecting tubules with meclofenamate prevented the inhibitory effect of ADH. These findings show that vasopressin exerts a prolonged inhibitory action on PD and on net reabsorption of Na and Cl and that this action may be exerted through stimulating the biosynthesis of prostaglandin E2 by the cortical collecting tubule.
Animals↗
ADH-PGE2 interactions in cortical collecting tubule. II. inhibition of Ca and P reabsorption.
In the absence of ADH, microperfused cortical collecting tubules of rabbits reabsorb calcium and phosphorus. Antidiuretic hormone (ADH) (200 microunits/ml Pitressin or synthetic arginine vasopressin) inhibits the reabsorption and may promote the secretion of calcium and phosphorus. At 5 min after incubation with ADH, there was a transitory increase in the potential difference and the reabsorption of sodium. The fluxes of calcium and phosphorus, however, showed no significant change from the control values. At 30-50 min after treatment with ADH, the reabsorption of calcium and phosphorus was inhibited and in some tubules calcium and phosphorus were secreted. The removal of vasopressin from the bath or the addition of 10(-5) M meclofenamate in vitro prevented ADH from inhibiting the reabsorption of calcium and phosphorus. Treatment of tubules with 10(-5) M prostaglandin E2 (PGE2) subsequent to incubation in a medium containing ADH and meclofenamate inhibited the reabsorption or even promoted the secretin of calcium and phosphorus, as did the prolonged incubation with ADH alone. We conclude that cortical collecting tubules reabsorb calcium and phosphorus in the absence of vasopressin and that ADH inhibits calcium and phosphorus reabsorption. Endogenous synthesis of PGE2 may mediate the inhibitory action of ADH, since meclofenamate (an inhibitor of the synthesis of prostaglandins) opposes and exogenous PGE2 mimics ADH.
Absorption↗
Acute metabolic acidosis augments collecting duct acidification rate in the rat.
It is generally assumed that at least part of distal acidification occurs along the collecting duct. Complete and direct evaluation of acidification along this nephron segment is unavailable, however. The purpose of these experiments was to quantify the net acidification rate along the inner medullary collecting duct (IMCD) and to measure the effect of acute HCl acidosis. In 13 control rats (arterial pH, 7.39 +/- 0.01; PCO2, 39 +/- 1 mmHg) and 11 HCl-infused rats (arterial pH 7.18 +/- 0.01; PCO2, 40 +/- 1 mmHg) we obtained four to eight IMCD samples by a modified microcatheterization technique that also permitted measurement of in situ and in vitro pH. Tubular fluid pH decreased along the IMCD in both groups and was more acidic by 0.2-0.4 pH units in the acid-infused rats. Bicarbonate reabsorption was noted in both groups as delivery along the IMCD decreased from 205 +/- 127 to 26 +/- 6 nmol/min in control rats and from 219 +/- 118 to 17 +/- 4 nmol/min in the acidotic group. Ammonia delivery to and addition along the IMCD was significantly greater in the acidotic rats--from 193 +/- 59 to 462 +/- 53 nmol/min in control and from 887 +/- 126 to 1,396 +/- 90 nmol/min in acidotic rats. No significant change in total or titrated phosphate was seen. Net acid addition along the IMCD was over twice as great in acidotic rats, 450 vs. 970 nmol/min. Our results indicate that significant net acid addition occurs along the IMCD and that during acidosis this acidification rate increases.
Acid-Base Equilibrium↗