Mechanism of sodium and chloride transport in the thin ascending limb of Henle.

Our previous in vitro studies have disclosed that the thin ascending limb of Henle (tALH) possesses some unique membrane characteristics. In those studies we failed to demonstrated active transport of sodium chloride by the tALH, although it was shown that the isotopic permeability to sodium and chloride was unusually high. However, we did not examine the mechanisms by which the apparent high permeation of sodium chloride occurs. Thus the purpose of the present studies was to elucidate the mechanism of sodium chloride transport across the isolated tALH of the rabbit by conducting four different types of studies: (1) comparison of the observed chloride and sodium flux ratios to those predicted by Ussing's equation under imposed salt concentration gradients; (2) kinetic evaluation of chloride and sodium fluxes; (3) examination of the effect of bromide on the kinetics of chloride transport; and (4) experiments to test for the existence of exchange diffusion of chloride. In the first set of studies the predicted and the theoretical flux ratios of sodium were identical in those experiments in which sodium chloride was added either to the perfusate or to the bath. However, the observed chloride flux ratio, lumen-to-bath/bath-to-lumen, was significantly lower than that predicted from Ussing's equation when 100 mM sodium chloride was added to the bath. In the second set of experiments the apparent isotopic permeability for sodium and for chloride was measured under varying perfusate and bath NaCl concentrations. There was no statistical change in the apparent sodium permeability coefficient when the NaCl concentration was raised by varying increments from 85.5 to 309.5 mM. However, permeation of 36Cl decrease significantly with an increase in Cl from 73.6 to 598.6 mM. These events could be explained by a two component chloride transport process consisting of simple diffusion and a saturable facilitated diffusion process with a Vmax = 3.71 neq mm-1 min-1. In the third set of studies it was shown that bromide inhibits transport of chloride and that the magnitude of inhibition is dependent on chloride concentrations. The fourth set of studies ruled out the existence of exchange diffusion. In conclusion, these studies indicate that sodium transport across tALH is by simple passive diffusion, while chloride transport across tALH involves at least two mechanisms: (1) simple passive diffusion; and (2) a specific membrane interaction process (carrier-mediated) which is competitively inhibited by bromide.     

Regulation by adrenal corticosteroids of sodium and potassium transport in loop of Henle and distal tubule of rat kidney.

Studies were conducted to examine the effects of adrenalectomy (ADX) and selective, physiological adrenal corticosteroid replacement on sodium and potassium transport by the superficial loop of Henle and distal tubule of rat kidney in vivo. In the loop of Henle, ADX inhibited sodium reabsorption by 33%. Whereas dexamethasone had no effect on reabsorption, aldosterone increased sodium transport to control levels. Thus, physiological levels of mineralocorticoids, but not glucocorticoids, control a fraction of sodium reabsorption in the loop of Henle. ADX also inhibited potassium reabsorption in the loop of Henle. Both dexamethasone and aldosterone reversed the inhibition, although only aldosterone increased reabsorption to control levels. In the distal tubule, ADX reduced sodium reabsorption by 44%. Both aldosterone and dexamethasone stimulated reabsorption: however, only aldosterone increased transport to control. Potassium secretion by the distal tubule was also reduced 34% by ADX. Aldosterone, but not dexamethasone, stimulated secretion. Thus, physiological levels of aldosterone regulate a fraction of sodium reabsorption and potassium secretion in the distal tubule.     

Protein intake and cation transport in the loop of Henle

High-protein intake enhances maximal urinary concentrating ability and suppresses tubuloglomerular feedback activity in a manner that correlates with enhanced salt reabsorption in the loop of Henle. In this article we describe studies designed to localize the site at which protein intake alters loop sodium uptake (JNa) in rats fed diets containing either 6% or 40% protein for approximately 8 to 10 days. In vivo microperfusion demonstrated that luminal bumetanide (10(-5) mol/L) fully reversed the stimulation of JNa by high-protein intake, thus suggesting that high-protein intake stimulates salt transport in the thick ascending limb. In vitro studies supported this possibility, showing that high-protein intake significantly increased sodium-potassium adenosine triphosphatase (NaK ATPase) activity in homogenates of outer renal medulla (68%) and in dissected medullary thick ascending limbs (87%). This effect was partly selective, since high-protein intake did not alter NaK ATPase activity in superficial renal cortex, had a smaller and statistically insignificant effect on NaK ATPase activity in dissected pars rectae, and did not affect magnesium ATPase activity in any tissue. Furthermore, this effect did not appear to require hypertrophy, since high-protein intake for approximately 8 days did not detectably alter the relative amounts of tissue protein and DNA in either medulla or cortex. A last series of studies demonstrated that high-protein intake increased plasma aldosterone levels. We conclude that increased protein intake stimulates salt reabsorption predominantly in the thick ascending limb, an effect that is partly selective; does not appear to require hypertrophy; and may be related to increased plasma aldosterone levels.     

Bicarbonate transport along the loop of Henle. I. Microperfusion studies of load and inhibitor sensitivity.

We microperfused the loop of Henle (LOH) to assess its contribution to urine acidification in vivo. Under control conditions (Na HCO3- = 13 mM, perfusion rate approximately 17 nl/min-1) net bicarbonate transport (JHCO3-) was unsaturated, flow- and concentration-dependent, and increased linearly until a bicarbonate load of 1,400 pmol.min-1 was reached. Methazolamide (2 x 10(-4) M) reduced JHCO3 by 70%; the amiloride analogue ethylisopropylamiloride (EIPA) (2 x 10(-4) M) reduced JHCO3 by 40%; neither methazolamide nor EIPA affected net water flux (Jv). The H(+)-ATPase inhibitor bafilomycin A1 (10(-5) M) reduced JHCO3 by 20%; the Cl- channel inhibitor 5-nitro-2'-(3-phenylpropylamino)-benzoate (2 x 10(-4) M) and the Cl(-)-base exchange inhibitor diisothiocyanato-2,2'-stilbenedisulfonate (5 x 10(-5) M), had no effect on fractional bicarbonate reabsorption. Bumetanide (10(-6) M) stimulated bicarbonate transport (net and fractional JHCO3-) by 20%, whereas furosemide (10(-4) M) had no effect on bicarbonate reabsorption; both diuretics reduced Jv. In summary: (a) the LOH contributes significantly to urine acidification. It normally reabsorbs an amount equivalent to 15% of filtered bicarbonate; (b) bicarbonate reabsorption is not saturated; (c) Na(+)-H+ exchange and an ATP-dependent proton pump are largely responsible for the bulk of LOH bicarbonate transport.     

Bicarbonate transport along the loop of Henle. II. Effects of acid-base, dietary, and neurohumoral determinants.

The loop of Henle contributes to renal acidification by reabsorbing about 15% of filtered bicarbonate. To study the effects on loop of Henle bicarbonate transport (JHCO3) of acid-base disturbances and of several factors known to modulate sodium transport, these in vivo microperfusion studies were carried out in rats during: (a) acute and chronic metabolic acidosis, (b) acute and chronic (hypokalemic) metabolic alkalosis, (c) a control sodium diet, (d) a high-sodium diet, (e) angiotensin II (AII) intravenous infusion, (f) simultaneously intravenous infusion of both AII and the AT1 receptor antagonist DuP 753, (g) acute ipsilateral mechanicochemical renal denervation. Acute and chronic metabolic acidosis increased JHCO3; acute metabolic alkalosis significantly reduced JHCO3, whereas chronic hypokalemic alkalosis did not alter JHCO3. Bicarbonate transport increased in animals on a high-sodium intake and following AII administration, and the latter was inhibited by the AII (AT1) receptor antagonist DuP 753; acute renal denervation lowered bicarbonate transport. These data indicate that bicarbonate reabsorption along the loop of Henle in vivo is closely linked to systemic acid-base status and to several factors known to modulate sodium transport.     

Simple method for preparing a concentration gradient of serum components by freezing and thawing.

We created a simple method for obtaining a series of successively more-concentrated samples from a serum without changing the ratio of its components. We froze a pooled serum and then allowed it to thaw undisturbed. The serum components formed a gradient of increasing concentration from the top of the sample to the bottom. We found that (a) in test results, each fraction of serum in the gradient showed almost the same relative concentrations of components (i.e., inorganic and organic compounds, proteins, metals, and hormones), irrespective of atomic or molecular mass; (b) the concentration gradient depended on the thawing temperature but not on the freezing temperature; (c) when we thawed the frozen sample with centrifugation, the slope of the concentration gradient increased with increasing centrifugal force; (d) when the thawed sample was fractionated into 10 fractions from the top to the bottom, the original serum concentration was always maintained between the sixth and seventh fractions from the top; and (e) the concentration gradient became steeper with repeated freezing and thawing. By using this method, one can easily prepare serum samples at gradients of concentration useful in the clinical laboratory, although the mechanism of gradient formation is still unclear.     

Lactate concentration gradient from right atrium to pulmonary artery: a commentary

Inadequate myocardial performance is a common complication of severe sepsis. Studies in humans strongly argue against a decrease in coronary blood flow in the pathogenesis of this sepsis-induced cardiac injury. Moreover, regional myocardial ischemia may well be present in sepsis patients with coexistent coronary artery disease. Nevertheless, the diagnosis of myocardial ischemia remains difficult in patients with sepsis, since elevation of troponin in these patients can be the result of a variety of conditions other than acute myocardial ischemia. The use of the right atrium to pulmonary artery lactate gradient could perhaps help the clinician in detecting myocardial ischemia in patients with sepsis.     

Modulation of Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle. Effects of mineralocorticoids and sodium.

This study investigates the effect of variations in mineralocorticoid as well as cell sodium delivery and uptake on Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle (mTALH). Pharmacologic doses of the mineralocorticoid deoxycorticosterone acetate (DOCA) resulted in a 28% increase of Na-K-ATPase activity. Furosemide-induced inhibition of sodium uptake by the mTALH cell also resulted in Na-K-ATPase activity reduction (45%). Sodium deprivation did not cause a clear change in enzyme activity, either at 3 d or 2 wk, likely reflecting the result of the opposing influences of decreased sodium delivery and increased endogenous aldosterone. Finally, the behavior of Na-K-ATPase activity at 3 d of sodium deprivation in the mTALH contrasted with a 60% increase in activity observed in the cortical collecting tubule, a nephron segment known to be responsive to mineralocorticoid, and this heterogeneity of response may suggest an important role for the mTALH in maintaining salt homeostasis.     

Calcium transport in the pars recta and thin descending limb of Henle of the rabbit, perfused in vitro.

Unidirectional calcium flux (JCa) in the superficial pars recta and thin descending limb of Henle (DLH) was examined by the isolated tubule microperfusion technic using 45Ca as the isotopic tracer. In the pars recta sequential measurements of lumen-to-bath flux (JlbCa) and bath-to-lumen flux (JblCa) revealed: JlbCa 22.4 +/- 4.18, JblCa 7.97 +/- 1.95, and calculated net efflux of calcium (JnetCa 13.0 +/- 1.74 peq min-1 mm-u. To measure JnetCa directly, 45Ca of identical specific activity was used to bathe and perfuse the tubule. These studies revealed: JlbCa 14.1 +/- 1.33, JnetCa 11.2 +/- 1.15, and calculated JblCa 2.91 +/- 0.49 peq min-1 mm-1. The addition of ouabain (10 microM) resulted in a rise in potential difference and a fall in water absorption, but not a statistically significant change in JnetCa. Tubules studies at 25 degrees C bath temperature, showed no significant JnetCa, and upon heating the bath to 37 degrees C, showed JnetCa of 3.75--5.00 peg min-1 mm-1. Unidirectional and net efflux studies in six DLH showed no significant transport of calcium. These studies demonstrate substantial active absorption of calcium by the superficial pars recta, which is not inhibitable by ouabain but is inhibited by lowering bath temperature to 25 degrees C. No significant calcium transport was found in the DLH using identical technics.     

Localization of diuretic effects along the loop of Henle: an in vivo microperfusion study in rats

In order to clarify the effects on sodium reabsorption in the loop of Henle of methazolamide (a carbonic anhydrase inhibitor), chlorothiazide and the loop diuretics frusemide and bumetanide, superficial loops were perfused in vivo in anaesthetized rats and the individual diuretics were included in the perfusate. Differentiation between effects in the pars recta and in the thick ascending limb of Henle (TALH) was achieved by comparing responses to the diuretics when using a standard perfusate, designed to mimic native late proximal tubular fluid, and a low-sodium perfusate, designed to block net sodium reabsorption in the pars recta. With the standard perfusate, methazolamide caused decreases in sodium reabsorption (J(Na)) and water reabsorption (J(V)); with the low-sodium perfusate, a modest effect on J(Na) persisted, suggesting that carbonic anhydrase inhibition reduces sodium reabsorption in both the pars recta and the TALH. The effects of chlorothiazide were very similar to those of methazolamide with both the standard and low-sodium perfusates, suggesting that chlorothiazide also inhibits sodium reabsorption in the pars recta and TALH, perhaps through inhibition of carbonic anhydrase. With the standard perfusate, both frusemide and bumetanide produced the expected large decreases in J(Na), but J(V) was also lowered. With the low-sodium perfusate, the inhibitory effects of the loop diuretics, particularly those of frusemide, were substantially reduced, while net potassium secretion was found. These observations indicate that a significant component of the effect of frusemide (and possibly of bumetanide) on overall sodium reabsorption is located in the pars recta, and that loop diuretics induce potassium secretion in the TALH.     

Estimation of scatterer volume density near a concentration gradient

A general formulation of the echo received from a random scatterer ensemble illuminated by a short electromagnetic or sonic signal is developed. It is shown theoretically and confirmed experimentally with ultrasound, that a gradient in scatterer density will return an echo which is partially spatially coherent (i.e. specular). The surface of a random ensemble of uniform scatterers is shown to produce an echo from whose degree of coherence, the scatterer density and cross section can be calculated.     

Glucose reabsorption from bile. Evidence for a biliohepatic circulation

Glucose is absent from human bile and present in low concentrations in bile from the rat. To study the mechanisms of this blood-bile glucose concentration difference, infusions of glucose were administered i.v. to 300-400 g male Sprague-Dawley rats with ligated renal pedicles and to two postcholecystectomy patients with indwelling t-tubes. Glucose was assayed in plasma, bile, and rat liver by a hexokinase method specific for D-glucose. In man, glucose was detected in bile when plasma glucose increased above 350 mg/100 ml. In animals studies, low concentrations of bile glucose were observed at plasma levels between 100 and 300 mg/100 ml. However, when plasma concentrations increased between 400 and 900 mg/100 ml, glucose appeared more rapidly in bile, defining by extrapolation an apparent plasma glucose threshold of 280 mg/100 ml. Intraportal phlorizin, a competitive inhibitor of glucose transport, significantly increased bile glucose concentrations. Plasma-bile concentration differences were also observed in rats after i.v. [3-14C]O-methyl glucose (3-O-MG) but not after [3H]mannitol. Hepatic glucose levels were never lower than plasma levels and liver-plasma 3-O-MG ratios were 0.92 +/- 0.22 indicating that entry of glucose and 3-O-MG into hepatocyte water was not limiting. Furthermore, when sodium dehydrocholate augmented canalicular secretion, biliary glucose excretion increased proportionally suggesting that glucose entry into bile was not impeded. When estimates of hepatic glucose secretion were compared with biliary glucose excretion, the latter increased progressively when estimated secretion rates exceeded 50 micrograms/min or when phlorizin was given. Finally, during bile stop-flow experiments, [3-14C]O-MG and [14C]glucose were selectively removed from bile compared with [3H]mannitol. The findings suggest that glucose and 3-O-MG are reabsorbed from bile after entry at the hepatocyte, accounting for their low bile-plasma ratio. The biliary glucose transport process may be described by Michaelis-Menten kinetics and is analogous to recently defined kinetics for renal tubular reabsorption of glucose. These studies provide evidence that certain products of bile secretion may undergo a "biliohepatic" circulation.     

Ouabain-uninhibited sodium transport in human erythrocytes. Evidence against a second pump

Others have concluded that a second Na "pump" (active Na outflux) exists in human erythrocytes. This second pump was said to be ouabain-insensitive, unlike the classic ouabain-sensitive Na-K pump. An alternative explanation is that "pump II" is Na exchange diffusion. These hypotheses were examined in the present experiments, utilizing (22)Na influx and outflux measurements, net Na fluxes, and ATPase determinations. Ouabain-uninhibited Na outflux was reduced 0.58+/-0.05 mmol/liter cells per h when extracellular Na (Na(o)) was replaced by Mg. Ethacrynic acid or furosemide produced similar decrements of outflux (0.50 mmol) in the presence of ouabain and Na(o). However, these diuretics had minimal inhibitory effects on outflux in the absence of Na(o) suggesting that they inhibited principally the Na(o)-dependent outflux. Whereas this ouabain-uninhibited portion of outflux was dependent on Na(o), it was independent of K(o). Contrary to expectations, Na influx did not change when intracellular Na was altered. No uphill, net Na transport (ouabain-uninhibited) could be demonstrated under a variety of circumstances. Furosemide at high concentrations inhibited ATPase, reducing both ouabain-sensitive and ouabain-insensitive enzyme at 1.0 mM concentration while showing no effect on ATPase at 0.05-0.1 mM concentration. The effects of furosemide on ATPase and on Na flux were dissociable on a dose-response curve. Energy depletion for 22 h practically eliminated the Na(o)-dependent, diuretic-inhibited Na outflux. Activation energies and temperature coefficients for the diuretic-inhibited outflux were one-half the values for the classic ouabain-inhibited pump. These data are interpreted as evidence against a second Na pump. Exchange diffusion accounts adequately for most of these observations; however, the ouabain-insensitive fluxes may be complex and composed of several processes.     

Direct evidence for the absence of active Na+ reabsorption in hamster ascending thin limb of Henle's loop.

The mechanisms of Na+ transport across cell membranes were investigated in the in vitro microperfused hamster ascending thin limb (ATL) of Henle's loop using a fluorescent Na+ indicator sodium-binding benzofuran isophthalate. The intracellular Na+ concentration ([Na+]i) of the ATL cells was 17.1 +/- 1.7 mM (n = 22) when the ATL was microperfused in vitro with Hepes-buffered solution containing 204 mM Na+. Elimination of metabolites such as glucose and alanine from the basolateral solution increased [Na+]i. Applying either 5 mM cyanide or 5 mM iodoacetic acid to the bath also increased [Na+]i. The elimination of K+ and the addition of 10(-4) M ouabain in the bath increased [Na+]i by 25.0 +/- 5.0 mM (n = 5) in 3 min and by 10.7 +/- 2.4 mM (n = 4), respectively. The elimination of luminal and basolateral Na+ resulted in a decrease in [Na+]i, indicating Na+ permeability of both the luminal and basolateral cell membranes. The luminal Na+ permeability was not affected by furosemide. The presence of luminal Na+ permeability and the basolateral Na+/K+ ATPase suggests the presence of net active reabsorption of Na+, which is not a physiologically important amount, in our estimation.     

Evidence for enhanced distal tubule sodium reabsorption in chronic salt-depleted dogs.

In order to assess the renal tubular site(s) at which sodium reabsorption is enhanced in chronic sodium-depletion, seven normal dogs, six salt-depleted dogs, and three normal dogs receiving aldosterone were studied during a steady-state water diuresis under Pentothal anesthesia and during progressive hypotonic saline diuresis. For both maintenance of the water diuresis and progressive hypotonic saline diuresis 0.45% NaCl was used. During the steady state water diuresis delivery of sodium to the diluting segment of the nephron as approximated by solute-free water clearance + sodium clearance/glomerular filtration rate (CH2O + CNa/GFR) was significantly lower in salt-depleted dogs compared to normal dogs with or without aldosterone. During progressive hypotonic saline infusion fractional free water excretion (CH2O/GFR) was similar in all three groups as CH2O + CNa/GFR increased up to 12-14 ml/min-100 ml GFR. Thereafter, CH2O/GFR continued to rise in virtually a straight line in salt-depleted dogs but leveled off in normal dogs with or without aldosterone. These data demonstrate that enhanced sodium reabsorption in the diluting segment of the nephron is an important determinant of the renal sodium retention in chronic extracellular volume contraction in dogs in addition to confirming the presence of increased proximal tubule sodium reabsorption in these animals.     

Loop of Henle bicarbonate accumulation in vivo in the rat.

We have carried out perfusion studies on hydropenic and bicarbonate-loaded rats to provide direct in vivo observations on bicarbonate accumulation in the short loops of Henle. Analysis of early distal tubular fluid was made during bicarbonate-free saline perfusion from the end proximal to the early distal site, documenting accumulation of "new" bicarbonate. During perfusion in hydropenic rats, steady-state bicarbonate concentrations were suggested by early distal values of approximately equal to mM, which were independent of perfusion rate and virtually indistinguishable from bicarbonate concentration measured during free flow when filtered bicarbonate was allowed to enter the loop. Thus, loop bicarconate accumulation was apparently sufficient to allow new bicarbonate to enter at a rate comparable to that delivered to the early distal site during free flow, recognizing of course that free-flow delivery rates are the result of complex components of filtration and bidirectional fluxes. In bicarbonate-loaded rats, however, bicarbonate accumulation rates although higher than in hydropenia, were much lower than free-flow delivery rates. Furthermore, early distal bicarbonate concentrations during bicarbonate loading fell as perfusion rate increased, presumably because of a limitation to increasing ionic bicarbonate entry.     

Decrease in the transmembrane sodium activity gradient in ferret papillary muscle as a prerequisite to the calcium paradox.

Sodium-dependent calcium exchange may be an important mediator of calcium reperfusion damage during the calcium paradox phenomenon. We measured intracellular sodium activity with ion-selective electrodes during a 15-min period of calcium reperfusion in isolated ferret papillary muscles. During the calcium-free period, alpha Nai increased from 9.0 +/- 0.9 to 18.9 +/- 4.3 mM. With reinstitution of calcium there was a significant contracture. The amount of contracture after calcium reinstitution was related to sodium loading during the calcium-free period. We were unable to block sodium entry during the calcium-free period with either nitrendipine, tetrodotoxin, or low concentrations of amiloride. 10(-3) M amiloride or lithium for sodium substitution in the calcium-free period, however, obliterated the increase in alpha Nai activity and the subsequent paradox. These data suggest that sodium loading is a necessary prerequisite for the calcium paradox and that one mechanism of sodium entry is through Na+/Ca2+ exchange. Under these conditions, no increase in the rest force is seen without previous sodium gains, suggesting that sodium-dependent calcium exchange is an important trigger for the calcium reflow, the calcium paradox.