Donald W. Seldin

The Mechanism of Bicarbonate Reabsorption in the Proximal and Distal Tubules of the Kidney

The mechanism of HCO3- reabsorption in proximal and distal tubules was examined in rats undergoing NaHCO3 diuresis. The steady-state intratubular pH was measured with pH-sensitive glass microelectrodes and compared with the equilibrium pH calculated from the HCO3- concentration of the tubular fluid (measured with quinhydrone electrodes) and plasma Pco2. In the proximal tubule the intratubular pH and the equilibrium pH were identical, indicating no accumulation of excess H2CO3. After inhibition of carbonic anhydrase, however, intratubular pH was significantly lower (0.85 pH U) than the equilibrium pH. It was concluded that HCO3- reabsorption in the proximal tubule was mediated by H+ secretion, but that carbonic anhydrase located in the luminal membrane of the cell prevented H2CO3 from accumulating in the tubular fluid. In the distal tubule the intratubular pH was 0.85 U lower than the equilibrium pH. This difference could be obliterated by an intravenous injection of carbonic anhydrase. It was concluded that HCO3- reabsorption in this segment was also accomplished by H+ secretion. The accumulation of excess H2CO3 in the tubular fluid indicated that, in contrast to the proximal tubule, carbonic anhydrase was not located in the luminal membrane of distal tubular cells.

The Site of Action of Furosemide and Other Sulfonamide Diuretics in the Dog

The discovery of the countercurrent multiplier system as the mechanism responsible for the concentration and dilution of the urine has provided a framework for the identification of the site of action of diuretic drugs. Agents that act at a single anatomic site in the nephron can be expected to alter the pattern of urine flow in a predictable way. A drug that acts solely in the proximal convoluted tubule, by causing the delivery of increased amounts of filtrate to the loop of Henle and the distal convolution, would augment the clearance of solute-free water (CH2o) during water diuresis and the reabsorption of solute-free water (TCHO) during water restriction. In contrast, drugs that inhibit sodium reabsorption in Henles loop would impair both CHSo and TCHO. Finally, drugs that act only in the distal tubule would reduce CHSo but not TcH2o (1). It is possible, however, that a diuretic drug might inhibit sodium reabsorption at multiple sites. Under such circumstances, localization of the areas of the nephron where the drug acts is far more difficult, since the effects of action in one segment might alter or obscure the effects on another. A new sulfonamide diuretic, furosemide, 4-chloro-N-(2-furylmethyl)-5-sulfonylanthranilic acid (2), offers several advantages in the study of localization of action: a) it is extremely potent, and therefore produces large effects; and b) its duration of action is brief, thereby facilitating multiple studies during a single experiment. A study * of furosemide was, therefore, undertaken to ex plore its effects on water excretion during hy-dration and hydropenia, and on potassium excre-tion during potassium loading. In addition, the diuretic effect of furosemide was compared to three other sulfonamide diuretics: chlorothiazide, chlor-thalidone, and benzhydroflumethiazide. The conventional sulfonamide diuretics resulted in the excretion of about 10% of filtered sodium and inhibited CQ2o without decreasing TCH2O. In contrast, as much as 38% of filtered sodium was excreted during furosemide diuresis, and both CH20 and TCHSo were inhibited. None of the diu-retics had an inhibitory effect on potassium secretion. The effects of the conventional sulfonamide diuretics can be explained by a single site of action distal to the loop of Henle. In contrast, furose-mide clearly has a potent effect in the ascending limb of Henles loop and probably in the proximal convoluted tubule as well. Methods A total of 50 experiments was performed on 40 dogs. All dogs were fasted overnight before each study. Mild pentothal anesthesia was induced …

Mineralocorticoid modulation of rabbit medullary collecting duct acidification. A sodium-independent effect.

Rabbit medullary collecting duct (MCD) from inner stripe of outer medulla has been identified as a major distal nephron acidification site. The isolated, perfused tubule technique was used to examine the roles of mineralocorticoid and glucocorticoid in regulation of MCD acidification. Surgical adrenalectomy reduced bicarbonate reabsorptive rate (JHCO3, pmol X mm-1 X min-1) from the normal of 9.79 +/- 1.21 to 0.67 +/- 1.1. Chronic administration of deoxycorticosterone acetate (DOCA) increased JHCO3 of MCD significantly to 18.02 +/- 1.62 whereas chronic dexamethasone administration did not affect JHCO3. The direct effects of aldosterone and dexamethasone upon MCD acidification were examined by perfusing tubules harvested from adrenalectomized rabbits in the presence of aldosterone or dexamethasone. Aldosterone, at 5 X 10(-8) M, increased JHCO3 significantly from 1.27 +/- 0.28 to 3.09 +/- 0.34. At 10(-6) M, aldosterone produced a greater increase in JHCO3 from 0.67 +/- 1.1 to 9.39 +/- 1.59. In vitro dexamethasone treatment had no effect on JHCO3. Studies examining the sodium dependence of aldosterone-stimulated acidification demonstrated that JHCO3 in tubules harvested from normal and deoxycorticosterone acetate-treated animals was unaffected by total replacement of sodium with tetramethylammonium. Likewise, luminal amiloride (5 X 10(-5) M) had no effect on JHCO3 in tubules harvested from adrenalectomized and normal animals. Moreover, the acute, in vitro stimulatory effect of aldosterone was seen to occur in the presence of luminal amiloride. These studies define a mammalian distal nephron segment that possesses major acidifying capacity, which is modulated by mineralocorticoid but independent of luminal sodium.

Resting transmembrane potential difference of skeletal muscle in normal subjects and severely ill patients

The resting membrane potential difference (Em) of skeletal muscle was measured in 26 normal human subjects, 7 patients with mild illness, and 21 patients with severe, debilitating medical disorders. A closed transcutaneous approach to the muscle was made by needle puncture and the Em was measured utilizing standard Ling electrodes. Measurements revealed an Em of -88 +/-3.8 mv in healthy subjects and -89 +/-2.1 mv in patients hospitalized for minor medical problems. The mean Em in 21 in-hospital patients, judged to be severely ill clinically from a variety of causes, was -66.3 +/-9.0 mv. Open deltoid muscle biopsies were performed in 7 of the healthy subjects and in 13 of the severely ill group. Estimation of the intra-extracellular water partition was made by calculating the chloride space from the previously measured Em. Analysis of the muscle samples revealed no significant difference in the intra-extracellular potassium ratios of the two groups biopsied. Intracellular Na(+) concentrations were uniformly increased in the muscle samples of the severely ill subjects and averaged 42.3% higher than those of the normal subjects. The mechanisms which might account for the elevation of intracellular Na(+) and a depression of Em independent of changes in intra-extracellular K(+) ratios are discussed and it is suggested that this defect may be a generalized cellular abnormality which is a common quality of serious illnesses.

Localization of diuretic action from the pattern of water and electrolyte excretion.

The response to diuretic agents is principally determined by three factors: the locus in the nephron where the drug exerts its inhibitory effect on sodium reabsorption; the potency of its action; and the nature and magnitude of the internal regulatory influences which augment the renal tubular reabsorption of sodium. The present studies in dogs were undertaken to ascertain the site of action within the nephron of various diuretic agents. The site of action was inferred from the pattern of water and electrolyte excretion during water diuresis and hydropenia. In general, the following criteria were used: An action in the proximal tubule was assumed if the fraction of filtrate delivered out of this portion of the nephron after administration of a diuretic (as estimated from clearance studies) exceeded that normally delivered out of the proximal convolution (as disclosed by micropuncture studies). An action in the ascending limb of Henle’s loop was assumed if free-water clearance was impaired, either alone or together with an impairment of free-water reabsorption. Finally, an action in the distal tubule and collecting duct was assumed if potassium and hydrogen excretion was reduced at any given rate of sodium excretion. Micropuncture studies in dogs, utilizing the inulin tubular fluid to plasma ratio (inulin TF/P) at the end of the proximal convolution as an index of fractional reabsorption of the filtrate, have delineated the magnitude of sodium reabsorption in this portion of the nephron during normal circumstances and during water diuresis, saline diuresis, and contraction of effective blood volume.13 No similar characterization of reabsorption in the thick ascending limb is available, since micropuncture has not been applicable in the dog to this segment of the nephron. It was necessary, therefore, to examine the normal reabsorptive characteristics of the thick ascending limb as a function of delivery of filtrate before the effects of diuretics could be ascertained.

AN EVALUATION OF RADIOSULFATE FOR THE DETERMINATION OF THE VOLUME OF EXTRACELLULAR FLUID IN MAN AND DOGS

Reasonably satisfactory methods have been developed for estimation of plasma volume (1) and total body water (2, 3) in normal subjects. On the other hand, no entirely satisfactory substance for estimating the volume of the extracellular fluid has as yet been found. It is not surprising that the material for this purpose should be more difficult to select, since, in order to have a volume of distribution equal to the extracellular fluid, it must be capable of passing through one membrane, the capillary wall, and yet be effectively excluded by the other, the cell wall. The use of large molecules such as inulin (4, 5) and sucrose (6, 7), which are excluded for the most part from body cells, is restricted by their slow rate of capillary diffusion, particularly in the case of inulin, which generally necessitates a constant infusion and limits the applicability of these methods in edematous states (8). Mannitol, which diffuses more rapidly (9, 10), suffers from the disadvantage of changing extracellular fluid in two ways in the process of measuring it, because the plasma concentration required for accurate analysis exerts an effective osmotic pressure which draws water from cells, thereby expanding extracellular fluid volume, at the same time that a mannitol diuresis is sweeping sodium chloride into the urine. Several ions which diffuse rapidly into the interstitial fluid have been employed for the measurement of extracellular volume, including bromide (11, 12), thiocyanate (7), thiosulfate (13, 14), ferrocyanide (15), and sulfate (7, 16). Bromide (17), thiocyanate (7, 18), and thiosulfate (13) all penetrate cells to a significant extent. Thiosul-

Angiotensin II in arterial and renal venous plasma and renal lymph in the dog.

Angiotensin II was determined by radioimmunoassay in systemic arterial, pulmonary arterial, and renal venous plasma and in renal hilar lymph in dogs. Levels of the peptide were determined prior to and during progressive graded hemorrhage or reduction in renal perfusion pressure. Levels of angiotensin II in plasma consistently rose during transit through the lung indicating pulmonary conversion of angiotensin I to angiotensin II. On the other hand, angiotensin II in the renal vein plasma was less than that in arterial plasma indicating renal extraction of the peptide from plasma. When renal hilar lymph was sampled under similar conditions, angiotensin II in lymph was consistently higher than that in arterial or renal venous plasma. Furthermore, in some experiments angiotensin II in lymph increased at a time when the concentration in plasma was undetectable. No evidence was found to indicate that angiotensin II in plasma entered renal lymph. It was concluded that angiotensin II levels in lymph reflected the concentration of angiotensin II in renal tissue. The data further suggested that angiotensin II is partially removed from arterial plasma by hydrolysis during transit through the kidney.

Influence of Expansion of Extracellular Volume on Tubular Reabsorption of Sodium Independent of Changes in Glomerular Filtration Rate and Aldosterone Activity

The excretion of sodium by the kidney is influenced by changes in glomerular filtration rate (GFR), serum sodium concentration, adrenocortical activity, the quantity of nonreabsorbable solutes in the glomerular filtrate, and the volume of extracellular fluid (ECF) (1-4). Some of these factors alter sodium excretion by affecting the amount of sodium filtered; others modulate the tubular reabsorption of sodium. The mechanism whereby acute changes in the volume of ECF influence sodium excretion is not entirely clear. At least two factors, however, have been identified. Both the secretory rate of aldosterone and the GFR are influenced by alteration in the volume of ECF and may play important roles in the regulation of sodium excretion by volume. Alteration of sodium excretion by patients with Addisons disease in a parallel fashion with sodium intake and without detectable changes in GFR (5, 6) suggests that a third factor may be operative. Furthermore, a number of investigators (7-9) have shown that, under conditions where the volume of ECF was acutely altered in the face of fixed adrenocortical activity, sodium excretion varied independently of GFR, suggesting that changes in volume were in some manner influencing the tubular reabsorption of sodium independent of adrenocortical hormones.

The Mechanism of Suppression of Proximal Tubular Reabsorption by Saline Infusions

The mechanism by which expansion of extracellular fluid volume with isotonic saline suppresses reabsorption in the proximal tubule was studied in rats by examining the relations among glomerular filtration rate (GFR), absolute and fractional reabsorption of filtrate, intrinsic reabsorptive capacity (rate of reabsorption per unit tubular volume), transit time, and tubular volume. Saline infusions reduced the per cent of the glomerular filtrate reabsorbed in the proximal tubule from 50% during antidiuresis to 25% during saline diuresis. The suppression of proximal reabsorption was the result of two factors: 1) a 30% reduction of intrinsic reabsorptive capacity, and 2) a 26% reduction of tubular volume per unit GFR.GFR invariably rose during saline diuresis. However, prevention of the rise in GFR by aortic clamping had no effect on either the inhibition of intrinsic reabsorptive capacity or the reduction in tubular volume per unit GFR produced by saline infusions. Expansion of extracellular fluid volume with isotonic saline, therefore, depressed intrinsic reabsorptive capacity and tubular volume per unit GFR by some mechanism completely independent of GFR. The effects of furosemide administration were contrasted with those of saline infusions. Furosemide inhibited intrinsic reabsorptive capacity by 40% but had no significant effect on proximal fractional reabsorption. The failure to suppress fractional reabsorption was the consequence of a disproportionate rise in tubular volume (relative to GFR) that was sufficient to completely overcome the inhibition of intrinsic reabsorptive capacity. Inhibition of intrinsic reabsorptive capacity alone, therefore, will not result in a net suppression of reabsorption of filtrate in the proximal tubule. We concluded that, although intrinsic reabsorptive capacity was inhibited during saline diuresis, the critical factor responsible for translating this inhibition into effective net suppression of proximal reabsorption was the observed reduction in tubular volume per unit GFR.

The Renal Diluting and Concentrating Mechanism in Hypercalcemia

The effects of acute hypercalcemia produced by infusing calcium lactate were studied in dogs during hydration and hydropenia. This allowed a direct observation of the effect of calcium on renal concen