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Biomedical subjects

L G Fine

Publications and source records attributed to L G Fine.

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

Adaptation of proximal tubular structure and function: insights into compensatory renal hypertrophy.

Hypertrophy of the renal tubular cells, especially those of the proximal tubule (PT), accounts for the majority of the increase in kidney size that follows partial removal of renal mass. The propensity of PTs to enlarge appears to be closely linked to an elevation in glomerular filtration rate and may be related to altered tubular fluid flow rate. Hypertrophied PTs reabsorb fluid at an increased rate in vitro, which indicates an intrinsic adaptation of their transport capacity. The hypertrophied cells demonstrate a predominant increase in basolateral membrane area with little change in luminal surface area. This asymmetric structural hypertrophy does not, however, appear to be accompanied by functional asymmetry, for basolateral Na+-K+ pump activity increases roughly in proportion to the increase in cell protein. The activity of the Na+-H+ antiporter, on the other hand, is increased in the brush-border membrane of proximal tubules derived from animals with reduced renal mass. In view of the reported association of Na+-H+ antiport stimulation and mitogenesis in a variety of cell types, the increased activity of this transporter, possibly induced by an increase in tubular fluid flow rate, could be the local stimulus that initiates hypertrophy and determines the organ specificity of the response.

Adaptation, Physiological↗
PubMed1985

Hypertrophy of basolateral Na-K pump activity in the proximal tubule of the remnant kidney.

Reduction of renal mass leads to an increase in the filtration rates of the remaining glomeruli and an increased rate of sodium and water reabsorption by the proximal tubules. To define the basis for this increased tubular reabsorptive capacity, the authors studied the relationship of basolateral sodium pump activity to the process of hypertrophy in the proximal tubule. They wished to determine whether the growth of the cell is associated with an increase in the number of basolateral Na-K pumps and whether basolateral membrane hypertrophy is symmetrical with respect to overall cell growth. Normal and subtotally nephrectomized rabbits (remnant kidneys) were studied. Ouabain-sensitive potassium uptake was measured in a highly purified suspension of cortical proximal tubules using 86Rb as a tracer. In normal kidneys Km was 0.99 +/- 0.30 mM and Vmax 83.1 +/- 13.7 nmoles X mg-1 X minute-1; in remnant kidneys Km was 0.63 +/- 0.10 mM and Vmax 49.2 +/- 10.9 nmoles X mg-1 X minute-1. These values are not significantly different from each other. In a suspension of isolated cortical proximal tubular cells, protein per cell was 172 +/- 23 pg in normal kidney and 450 +/- 56 pg in remnant kidneys, representing a 2.6-fold increase. The extrapolated Vmax for K uptake per cell was thus increased approximately 2.6-fold in the remnant kidney. This was confirmed by measuring the number of specific ouabain-binding sites in proximal tubular cells. This was also found to be approximately 2.5 to 3 times greater in the remnant kidney cells, the increase being proportional to the increase in cell protein. Histomorphometric analysis of S2 proximal convoluted tubules, which comprise the bulk of the cortical tissue, revealed that basolateral membrane area per cross-sectional area of tubule was increased in the remnant kidney. The mean absolute surface area per cross-section of tubule and the surface density (surface/volume ratio) of the basolateral membrane increased by 110 and 26%, respectively, whereas these changes in the luminal membrane were only 38 and -9%, respectively. Thus, the membrane areas of the proximal tubular cell hypertrophy asymmetrically. Although mitochondrial density does not increase in remnant tubules, mitochondrial volume increases significantly, possibly providing a source for the increased ATP required by the hypertrophied basolateral Na-K pump activity. In summary, the cells of the proximal convoluted tubule of the remnant kidney undergo functional and structural hypertrophy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Transport and metabolism of glucose by renal proximal tubular cells in primary culture.

A highly purified suspension of rabbit proximal tubules was cultured in a hormone-supplemented serum-free medium. This primary culture yielded a homogeneous population of cells that demonstrated functional and morphological polarity in mono-layers. The characteristics of the Na-dependent glucose transporter in the luminal membrane were studied by measuring the uptake of alpha-methylglucoside (AMG). The kinetics of Na-dependent AMG uptake were consistent with a single saturable system with an apparent Km of 0.8 mM and Jmax of 0.14 nmol X mg-1 X min-1. AMG permeability was 0.10 microliter X mg-1 X min-1. Uptake was inhibited 95% by 0.1 mM phlorizin and by removal of sodium. The stoichiometry of Na/glucose interaction with the carrier was 2:1. These characteristics are typical of the characteristics described for the late proximal tubule. To examine whether the glucose that enters the cell across the luminal membrane is incorporated into the metabolic pool of the cell, we studied the oxidation of [14C]glucose to 14CO2 in the absence and presence of phlorizin. Significant decarboxylation of [1-14C]glucose and [6-14C]glucose was observed, consistent with the existence of aerobic metabolism and a hexose monophosphate shunt. In the presence of 0.1 mM phlorizin, uptake and oxidation of D-glucose were inhibited to an identical degree, suggesting that luminal uptake is a rate-limiting step in the oxidation of glucose by these proximal tubular cells. These studies indicate that proximal tubular cells in primary culture utilize glucose as an energy source and that the glucose derived from transport across the luminal membrane is incorporated into the metabolic pool of the cell.

Animals↗
PubMed1984

Mechanisms of Na+ uptake into renal brush border membrane vesicles.

Brush border membrane vesicles were used to investigate the pathways for Na+ uptake across the apical membrane of the renal proximal tubular cell. The kinetics of uptake in the absence of organic solutes were consistent with parallel saturable and nonsaturable pathways. At pH equilibrium (pHin = pHout = 7.5), the Jmax and Kt for saturable uptake were 41 +/- 15 (+/- SE) nmol X mg-1 X min-1 and 33 +/- 9, respectively, and the apparent permeability coefficient, P'Na, was 0.27 +/- 0.02 microliters X mg-1 X min-1. As the equilibrium pH was varied between 6.0 and 8.0, no consistent trend for Kt or P'Na was observed; Jmax varied up to twofold. In contrast, in the presence of an outward H+ gradient (pHin = 6.0 vs. pHout = 7.5), the Kt decreased by an order of magnitude, with little change in Jmax. At low sodium concentrations (1 mM) external Li+ and NH+4, and to a lesser extent K+, Rb+, and Cs+, inhibited Na+ uptake. Amiloride (10(-3) M) inhibited 1 mM Na+ uptake by 80% even in the absence of a H+ gradient. Uptake also varied with the anion composition at high sodium concentrations (100 mM), as predicted from the anion permeabilities. Sodium uptake was more sensitive to variations in membrane potential at high sodium concentrations than at low concentrations. On the basis of these experiments we suggest that the saturable Na+ uptake occurs via an electroneutral Na+-H+ antiporter and that the diffusive flux occurs through a conductive pathway.

Amiloride↗
PubMed1983

Functional profile of the isolated uremic nephron: intrinsic adaptation of phosphate transport in the rabbit proximal tubule.

The maintenance of phosphate homeostasis in uremia appears to be governed both by parathyroid hormone (PTH) and by PTH-independent adaptations in renal tubular function. The relative contributions of these two mechanisms that control phosphate excretion by the diseased kidney have been difficult to define in intact animals. The present study was designed to examine the nature of the adaptation of phosphate handling by the proximal tubule of subtotally nephrectomized, uremic rabbits in vitro. Euparathyroid and hyperparathyroid uremic rabbits were studied. Tubular sensitivity to PTH was examined in vitro. The dose-response relationship between bath PTH concentration and inhibition of lumen-to-bath phosphate flux (Jp-lb) in isolated perfused proximal straight tubules (PSTs) revealed that PTH sensitivity was increased in the euparathyroid uremic rabbits and was decreased in hyperparathyroid uremic rabbits. The dose-response to dibutyryl cAMP was the same as normal in both uremic groups. These data strongly suggest the existence of a receptor-mediated adaptation in the effects of PTH on the uremic proximal tubule. In addition to an altered PTH-sensitivity the uremic PST also exhibited an alteration in the basal rate of phosphate transport studied in the presence of normal rabbit serum. Although net volume flux across the PST increased in both uremic groups as a possible consequence of hypertrophy, net phosphate flux per unit length was unchanged. Considering the increase of luminal area in these tubules, net phosphate flux per unit reabsorptive surface area was actually decreased. This dissociation is supportive of the existence of an intrinsic tubular adaptation which is independent of the size of the tubule per se. These studies indicate that there is an intrinsic adaptation of the basal rate of phosphate transport by the uremic rabbit proximal tubule and that the sensitivity of the tubule to PTH is altered. The data are strongly suggestive of an increase in the number of PTH receptors in the proximal tubule of the euparathyroid uremic rabbit and suggest that "down regulation" or persistent occupancy of these receptors occurs when hyperparathyroidism supervenes.

Adaptation, Physiological↗
PubMed1983

Nucleotide inhibition of phosphate transport in the renal proximal tubule.

The observation that NAD inhibits sodium-dependent phosphate (P) uptake by the luminal brush border membrane (BBM) of the proximal tubule prompted us to examine the specificity and mechanism of this process. Addition of 10(-5) M NAD to the perfusate of isolated perfused rabbit proximal straight tubules inhibited lumen-to-bath P flux by approximately 50%. ADP-ribose had an identical effect, whereas nicotinamide had no effect. ADP and 5'-AMP (10(-5) M) also inhibited P flux. Na-dependent uptake of 32P by rabbit BBM vesicles was inhibited by 0.1-0.3 mM NAD, ADP-ribose, ADP, ATP, 5'-AMP, and GDP, which were preincubated with the vesicles for 30 min. The kinetics of inhibition showed an apparent increase in the Km for P but no change in Vmax. These findings are consistent with "competitive inhibition." The nucleotides inhibited P uptake even when BBM alkaline phosphatase was inhibited by 96% with 10 mM theophylline. Evidence of nonspecific phosphatase activity was present, since incubation of BBM with 0.1 mM solution of nucleotides for 30 min resulted in an elevation of free P in the medium of approximately 0.15-0.22 mM. Correction of 32P specific activity for this change resulted in values for Km and Vmax that were not significantly different from control. The "competitive inhibition" could thus be ascribed to an isotope-dilution effect. There was no evidence to suggest that NAD caused ADP-ribosylation of the luminal membrane. These studies indicate that adenine and guanine nucleotides do not inhibit P transport by a direct action on the luminal membrane of the proximal tubule but do inhibit lumen-to-bath P flux in isolated perfused proximal tubules at concentrations of 10(-5) M. Since there is no direct inhibitory effect of these compounds at the level of the BBM, it is possible that they inhibit P transport by altering some event subsequent to the transfer of P across the luminal membrane.

Adenine Nucleotides↗
PubMed1982

Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium.

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Regulation of vasopressin action by prostaglandins. Evidence for prostaglandin synthesis in the rabbit cortical collecting tubule.

The present studies examined whether vasopressin increases prostaglandin biosynthesis in isolated rabbit cortical collecting tubules (CCT) and whether endogenous prostaglandin biosynthesis plays a role in modulating the response of this nephron segment to vasopressin. Three groups of studies were performed. In the first group, CCT and proximal straight tubules (PST) were incubated with [(3)H]arachidonic acid, and metabolites were separated and identified using silica gel thin-layer chromatography. CCT were capable of producing all of the major prostaglandins (PG) (PGE(2) > thromboxane B(2)[TxB(2)] > PGF(2alpha) > PGI(2)). PST produced significantly lesser quantities of these lipids. In the second group, radiolabeled arachidonic acid was incorporated into the phospholipid pool of both CCT and PST, vasopressin was added to the incubation medium, and metabolities were separated and identified as above. Vasopressin stimulated the release of all of the major prostaglandins in CCT but had no effect on PST. PGE release into the incubation medium, as assessed by a radioreceptor assay, increased 108%, and a vasopressin analogue, 1-desamino-8-d-arginine vasopressin, had a quantitatively similar effect. In the third group, a submaximal dose of vasopressin was administered to isolated, perfused CCT studied in the presence and absence of indomethacin to assess whether endogenous prostaglandins play a role in modulating the antidiuretic response to vasopressin. Studies were performed in rabbits on a normal diet and in desoxycorticosterone acetate (DOCA)- or KCl-loaded animals. In the state of mineralocorticoid excess, basal prostaglandin synthesis was 63% lower, and vasopressin-stimulated prostaglandin synthesis 76% lower, than the synthesis observed in rabbits on a normal diet. Cyclooxygenase inhibition exposed a significant hydroosmotic response to a submaximal dose of vasopressin in CCT from DOCA- or KCl-loaded animals. With arachidonic acid in the bath, the same dose of vasopressin failed to elicit a hydroosmotic response in CCT from rabbits on a normal diet even in the presence of a cyclooxygenase inhibitor. However, removal of exogenous arachidonic acid, with a consequently lower rate of prostaglandin synthesis, allowed the cyclooxygenase inhibitor to enhance the hydroosmotic response to vasopressin in these tubules.We conclude from these studies that the rabbit CCT has the capacity to synthesize all of the major prostaglandins and that the rate of synthesis of these lipids is enhanced by vasopessin. Prostaglandin synthesis by the CCT is postulated to modulate the antidiuretic action of vasopressin via a closed feedback loop. The effectiveness of this feedback regulation is dependent upon the mineralocorticoid status of the animal, which determines the level of basal and vasopressin-stimulated prostaglandin synthesis by the CCT.

Animals↗
PubMed1981

Effects of vasopressin on the isolated perfused human collecting tubule.

Cortical collecting tubules (CCT) were dissected from the surviving normal tissue of human kidneys removed at operation for either carcinoma or calculus. These CCT's were perfused in vitro shortly after the nephrectomy was performed. Transtubular potential differences in different tubules varied from +3.2 to -2.0 mV and were reduced towards zero by lowering the temperature or by adding ouabain to the bath. In the absence of vasopressin, tubules were essentially impermeable to water with extremely low net water fluxes even in the presence of a transtubular osmotic gradient. Addition of vasopressin to the bath caused the transtubular osmotic water permeability coefficient to increase to values of 125, 175, and 155 X 10(-4) cm/sec in three tubules thus studied. These results demonstrate close similarities between the human CCT and the more extensively studied rabbit CCT.

Cell Membrane Permeability↗
PubMed1981

Functional profile of the isolated uremic nephron. Evidence of proximal tubular "memory" in experimental renal disease.

In experimental models of glomerular and nonglomerular renal disease, single nephron filtration rate and proximal tubular reabsorption of fluid decrease or increase in parallel in the same nephron. To assess whether intrinsic adaptations in proximal tubular function, i.e., changes that are independent of the peritubular or humoral milieu, contribute to this phenomenon, segments of rabbit late superficial proximal convoluted tubules (PCT) were studied by in vitro perfusion. PCT were obtained from normal kidneys, from remnant kidneys, and from kidneys embolized with microspheres. Single nephron filtration rates are increased in the remnant and decreased in the embolized kidneys. Whereas the embolized-kidney rabbits were nonazotemic (the contralateral kidney was left in situ), the remnant-kidney animals were uremic. In order to study a nonazotemic model of increased single nephron filtration rate, PCT were also obtained from uninephrectomized rabbits. Significant compensatory hypertrophy occurred in the PCT of the remnant kidney. Net fluid reabsorption (Jv) per unit length was increased by approximately 60%; Jv per unit luminal surface area was the same as in the normal PCT. Transepithelial potential difference (PD) was significantly greater than normal. This was associated with a reversal of the normal permselective properties (P(Cl) > P(Na)) of the late superficial PCT so that P(Na) exceeded P(Cl). The changes could not be ascribed to some undetermined effect of the uremic state in vivo, since increases in tubule size, Jv per unit length, and PD also occurred in PCT from nonazotemic uninephrectomized rabbits. In contrast, Jv, per unit length or per unit luminal surface area, was decreased by approximately 50% in PCT from embolized kidneys and PD was also reduced. In these tubules, the normal permselective properties were also reversed. Tubule size, however, was not significantly different from normal. The increases or decreases in Jv that occurred in the different disease models were not dependent on tubular fluid flow rate or the uremic milieu in vitro. These studies indicate that intrinsic proximal tubular function is modified by the disease state in vivo and that the "memory" of this adaptation is expressed in the in vitro situation. The changes in Jv observed in vitro parallel the increases or decreases in single nephron filtration rates that occur in vivo. Compensatory hypertrophy, with an attendant increase in luminal surface area, could explain the increased Jv per millimeter in the remnant kidneys, but the adaptation observed in the embolized kidneys cannot be ascribed to changes in tubule size.

Animals↗
PubMed1979

Functional profile of the isolated uremic nephron: potassium adaptation in the rabbit cortical collecting tubule.

As a renal function declines in patients and experimental animals with chronic renal disease, potassium homeostasis is maintained by a progressive increase in potassium secretion by the surviving nephrons, a phenomenon known as potassium adaptation. To determine the nephron site and the underlying mechanisms responsible for this phenomenon, studies were performed on normal and 75% nephrectomized rabbits maintained on normal or high-potassium diets. Cortical collecting tubules (CCT) were dissected from the normal and remnant kidneys and perfused in vitro in an artificial solution. In normal CCT mean (+/- SE) net K secretion, JK, (peq/cm per s) was 1.26 +/- 0.43 (normal diet) and 3.27 +/- 0.66 (high-K diet). In uremic CCT, JK was 3.55 +/- 0.60 (normal diet) and 6.83 +/- 0.58 (high-K diet). By reducing the dietary intake of potassium in proportion to the reduction of renal mass in these uremic animals, the adaptation in K secretion was prevented (JK: 1.22 +/- 0.40). Transepithelial potential difference was similar in CCT from normal and uremic animals on a normal diet despite the fact that JK was significantly greater in the latter group. However, in both normal and uremic CCT, the increase in JK caused by potassium loading was associated with an increase in luminal negativity. Uremic CCT underwent significant compensatory hypertrophy regardless of the dietary intake or potassium secretory rates. Plasma aldosterone levels were elevated only in the uremic-high potassium rabbits suggesting that a mineralocorticoid effect on the CCT may be exaggerated when potassium loading is superimposed upon decreased excretory capacity. The activity of Na-K ATPase was comparable in normal and uremic CCT from rabbits on either normal or high-K diets indicating that potassium adaptation may occur independently of changes in the activity of this enzyme. Intracellular potassium content measured chemically and by 42K exchange, was not significantly altered in either normal or uremic CCT when dietary potassium intake was increased, despite the fact the JK was increased under these circumstances. These data indicate that the CCT is an important site of potassium adaptation in the surviving nephrons of animals with reduced renal mass. This adaptation is an intrinsic property of the CCT and is expressed in the absence of a uremic milieu. Potassium adaptation by the uremic CCT is not fixed according to the degree of compensatory hypertrophy but varies according to the excretory requirements of the animal. Transepithelial potential difference and circulating aldosterone levels contribute to the adaptation but neither factor can entirely account for the phenomenon. Potassium adaptation by the CCT occurs in the absence of changes in Na-K ATPase activity and intracellular potassium content.

Animals↗