bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of carboxyl group in Na+-entry step at apical membrane of toad urinary bladder
Authors: Park C. S. , Kipnowski J. , Fanestil D. D.
Published: December 2017

Mucosal addition of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and some lipid-soluble carbodiimides, agents which are selective for carboxyl groups, irreversibly inhibited Na+ transport as measured by short-circuit current (SCC) in the urinary bladder of the toad. The inhibition of Na+ transport by EEDQ had the following characteristics: 1) the inhibition was accompanied by a significant increase in the transepithelial electrical resistance; 2) the decrease in SCC was accounted for by a comparable decrease in 22Na+ influx without effect on Na+ efflux; 3) amphotericin B produced complete recovery of SCC inhibited with EEDQ but not with antimycin A or ouabain; 4) mucosal EEDQ decreased the amiloride-sensitive reversal of Na+ current that is induced by serosal nystatin in the absence of mucosal Na+; 5) vasopressin and acid mucosal pH caused an increase in SCC in proportion to the SCC remaining after EEDQ inhibition; and 6) Vmax of the SCC was decreased without alteration in the apparent Km for Na+. Based on these characteristics of EEDQ inhibition of Na+ transport, we infer that a carboxyl group of the Na+ channel is involved in the Na+-entry step across the apical membrane of “tight” epithelia. The inhibition of Na+ transport with EEDQ most likely involves closing the Na+ channel through a chemical reaction involving a carboxyl group of the channel.
Functional groups of the Na+ channel: role of carboxyl and histidyl groups
Authors: Park C. S. , Fanestil D. D.
Published: December 2017

Two titratable groups, with effect on Na+ transport and with apparent acid dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical membrane of toad urinary bladder and are tentatively identified as a carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport in the urinary bladder of toads. The underlying chemical reaction whereby EEDQ produces inhibition through potential modification of carboxyl residues was studied. The inhibitory action of EEDQ on Na+ transport was dependent on pH of reaction media and availability of nucleophile, indicating that formation of a covalent acyl-nucleophile bond is probably involved in the irreversible inhibition of Na+ transport. The kinetics of the inhibition showed a stoichiometry of formation of one acyl-nucleophile bond per closure of one Na+ transport site, presumably the Na+ channel. The nucleophile that appears to be involved in the formation of the acyl-nucleophile bond was tentatively identified as having an apparent pKa of 6.7. Amiloride and two analogues of amiloride added to the mucosal Ringer solution (but not serosal amiloride) protected against inhibition of Na+ transport by EEDQ--a finding consistent with the hypothesis that the EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or near the site of specific binding of amiloride onto the apical membrane, most likely at the Na+ channel. Our findings led us to postulate that amiloride must interact with at least two sites on the Na+ channel in order to block the channel. One of the two sites appears to be an ionic interaction between the anionic carboxyl group at the Na+ channel and the cationic guanidinium group of amiloride.
Flow-dependent water permeability of the rabbit descending limb of Henle's loop
Authors: Miwa T. , Imai M.
Published: December 2017

Because completely opposite results have been reported on the water permeability of the rabbit descending limbs of Henle's loop (DLH), we rigorously examined water permeability of the upper portion of the descending limb of the rabbit long-looped nephron. Even when the double-cannulation method was used in an attempt to reduce the resistance of tubular outflow, the collected fluid-to-perfusate inulin ratio was equal to or very close to the bathing fluid-to-perfusate osmolality ratio, indicating that osmotic equilibration occurred along the tubule by absorption of water. When perfusion rates were controlled by varying the height of the fluid reservoir connected to the perfusion pipette, osmotic (Pf) as well as diffusional (Pdw) water permeability was shown to be correlated with perfusion rate and/or perfusion pressure. Pf and Pdw at zero perfusion rate as determined from the values of the intercept of regression lines were 253 X 10(-3) and 4.54 X 10(-3) cm X s-1, respectively. The maximal values for Pf and Pdw were 737-1,098 X 10(-3) and 18.3 X 10(-3) cm X s-1, respectively. By changing the resistance to perfusion at the tubular outflow, it was shown that changes in Pf paralleled changes in perfusion rate rather than changes in perfusion pressure. Under stop-flow conditions the luminal fluid volume rapidly decreased after the osmolality of the bathing fluid was increased, suggesting that the segment is highly permeable to water even at zero flow rate. Reflection coefficients for urea and NaCl were 1.01 and 0.82, respectively. These data support the view that this segment is highly permeable to water and that increases in osmolality along the DLH in vivo may be accounted for mainly by abstraction of water rather than addition of solutes.
Corticosterone binding sites along the rat nephron
Authors: Lee S. -M. K. , Chekal M. A. , Katz A. I.
Published: December 2017

Contents: the correct title for the article on page F504 by S.-M. K. Lee, M. A. Chekal, and A. I. Katz is “Corticosterone binding sites along the rat nephron.”
The first seven years and plans for the future
Authors: Schafer James A.
Published: December 2017

Editorial by AJP-Renal editor James A. Schafer
Identification of actin-binding protein and villin in toad bladder epithelia
Authors: Ausiello D. A. , Corwin H. L. , Hartwig J. H.
Published: December 2017

Two actin-modulating proteins, actin-binding protein and villin, have been identified and functionally characterized in toad bladder epithelial cells. A 270-kdalton protein from bladder epithelial extracts 1) cross-reacts with antibodies to purified toad oocyte or rabbit macrophage actin-binding protein and comigrates with these proteins on gel electrophoresis, 2) contains all of the actin filament cross-linking activity found in epithelial cell extracts, and 3) its activity is calcium insensitive. A 95-kdalton protein from the same extracts 1) cross-reacts with antibodies to purified toad oocyte villin and comigrates with purified villin from the oocyte and chicken intestine on polyacrylamide gels, and 2) is capable of decreasing the viscosity of actin solutions only in the presence of calcium. These proteins offer potential sites for calcium-dependent and -independent regulation of toad bladder epithelial cell structure.
Luminal hypotonicity: a driving force for fluid absorption from the proximal tubule
Authors: Green R. , Giebisch G.
Published: December 2017

The ability of rat proximal tubules to generate a hypotonic luminal fluid was investigated. Simultaneous perfusion of tubules and peritubular capillaries was performed with simple solutions. When tubules were perfused at 10 nl X min-1 and NaCl was the perfusate for tubules and capillaries, solute and fluid (0.41 nl X min-1 X mm-1) were transported and the luminal fluid became hypotonic (delta osmol = -1.7 mosmol X kg-1). When the same solutions were used but the tubule was perfused at 45 nl X min-1, more fluid (0.89 nl X min-1 X mm-1) was reabsorbed and the fluid became more hypotonic (delta osmol = -3.9 mosmol X kg-1). Bicarbonate in the peritubular capillaries increased the fluid reabsorption (1.21 nl X min-1 X mm-1) but did not generate cryoscopically hypotonic fluid. Cyanide abolished all net movement of fluid and solute. It is concluded that the tubule can generate a hypotonic fluid, that the hydraulic conductivity for proximal tubular epithelium is 3,200-3,400 microns X s-1, and that the reflection coefficient for NaHCO3 is slightly higher than for NaCl.
Effects of low [Ca2+] and La3+ on PAH transport by isolated perfused renal tubules
Authors: Dantzler W. H. , Brokl O. H.
Published: December 2017

Effects of low medium calcium concentration and of lanthanum on p-aminohippurate (PAH) transport by isolated perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Nominal removal of calcium from bath produced rapid, irreversible disruption of cells and depression of net PAH secretion (JPAH). However, nominal removal of calcium from lumen with calcium in bath reversibly depressed JPAH and apparent PAH permeability of luminal membrane without altering net fluid absorption (Jv) or intracellular PAH concentration [( PAH]cell). In the presence of calcium (1.8 mM), lanthanum (2 mM) in perfusate alone, in bath alone, or in both perfusate and bath reversibly depressed JPAH, apparent PAH permeabilities of luminal and peritubular membranes, and initial rate of PAH transport into cells without altering [PAH]cell or Jv. Variations in calcium concentration had little effect on depression of JPAH in sodium-free bath. These data suggest that calcium entry into cells, but possibly not cytosolic calcium concentration alone, is important for PAH transport from bath into cells and from cells into lumen and for normal passive permeability of peritubular membrane to PAH and that increased cytosolic calcium concentration is not primarily responsible for depression of JPAH in sodium-free medium.
Verapamil and quinidine effects on PAH transport by isolated perfused renal tubules
Authors: Dantzler W. H. , Brokl O. H.
Published: December 2017

The effects of verapamil and quinidine on p-aminohippurate (PAH) transport by isolated perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Addition of 5 X 10(-5) M verapamil or 1 X 10(-4) M quinidine to the bath reversibly depressed net PAH secretion (JnetPAH), apparent permeabilities of luminal (PL) and peritubular membranes (Pp) to PAH, and initial rate of PAH transport into cells at the peritubular membrane without affecting net fluid absorption (Jv), but simultaneously produced a threefold increase in cellular PAH concentration ( [PAH]cell). Addition of 5 X 10(-5) M verapamil to the perfusate reversibly depressed JnetPAH and PL without affecting [PAH]cell or Jv. Tetraethylammonium (1 X 10(-3) M) did not alter the effects of verapamil on PAH transport. The data indicate that verapamil and quinidine in the bath inhibit JnetPAH by inhibiting uptake into cells across peritubular membrane and efflux from cells across luminal and peritubular membranes and possibly by producing intracellular binding, verapamil in the perfusate inhibits JnetPAH by inhibiting efflux from cells across the luminal membrane and probably uptake into cells across the peritubular membrane, and the verapamil effects on JnetPAH do not depend on its entry into cells. The effects of verapamil may result primarily from inhibition of calcium entry into cells, but both verapamil and quinidine may have effects on PAH transport independent of effects on calcium.
Body potassium by four-pi 40K counting: an anthropometric correction
Authors: Pierson R. N. , Wang J. , Thornton J. C. , Van Itallie T. B. , Colt E. W.
Published: December 2017

Four-pi whole body counting for the 1.46 meV photon of 40K has apparent advantages over single-crystal or two-pi counters in efficiency and in subject geometry independence. However, our studies of obese populations have disclosed a systematic undermeasurement of 40K, suggesting that nonhomogeneous K distribution results in systematic undercounting of 40K. In the current study 42K, emitting a 1.52 meV photon, was used in 109 volunteers ranging from 50 to 181 kg, and multiregression covariance analysis was applied to develop correction formulas based on anthropometrics. These corrections quantitatively account for the unappreciated loss of 40K and 42K photons in annular adipose tissue that surrounds the lean body, in which most K+ is concentrated. The correction ranges from 1 to 28% and is a linear (although different) function of weight in both sexes. Thus corrected, body potassium measurements, taken in conjunction with exchangeable sodium and water measurements, provide estimates for whole body osmolality that match measured serum values. Such a quantitative accounting for previously "lost" cation in 58 subjects provides independent evidence for the appropriateness and accuracy of the correction. With this correction, body potassium was recalculated in the 1,492 adult members of a previously reported group of 3,083 subjects.
Water-permeable and -impermeable barriers of snake distal tubules
Authors: Beyenbach K. W.
Published: December 2017

Isolated perfused snake distal tubules transport Na from lumen bath via an amiloride-sensitive transport pathway. Elevation of the luminal Na concentration from 16 to 150 mM leads to non-steady-state electrical behavior and cell swelling. To elucidate the mechanism of cell swelling, the water permeabilities of the epithelium and its apical and basolateral membrane were assessed. Distal tubules were found to be virtually impermeable to transepithelial water flow. Hydraulic conductivity measured 1.2 X 10(-7) cm3 X s-1 X cm-2 X atm-1 in the absence or presence of vasopressin. Effects of transepithelial osmotic pressure gradients on epithelial cell volume revealed the luminal membrane as the water-impermeable cellular barrier and the basolateral membrane as a barrier that is freely permeable to water. Epithelial cell swelling was blocked during perfusion with 150 mM Na when the perfusate also contained amiloride (10(-5) M). These results support the hypothesis that, in the case of transepithelial transport in the presence of high luminal Na concentrations, Na entry across the apical membrane exceeds Na extrusion across the basolateral membrane. Hence, the cells accumulate solute: Na from the apical side and some anion from the apical and/or serosal side. Concomitantly, the epithelial cells swell as water enters across the highly permeable basolateral membrane.
Critical role of extracellular calcium in vanadate-induced renal vasoconstriction
Authors: Benabe J. E. , Cruz-Soto M. A. , Martinez-Maldonado M.
Published: December 2017

Intra-arterial infusion of vanadate (VO4) in dogs produces a reduction in renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (V), and the fractional excretion of sodium (FENa+). To evaluate the role of Ca2+ in these changes VO4 was infused into the renal artery in the presence of the calcium antagonists trifluoperazine (TFP), verapamil, or EGTA. TFP inhibited the effect of VO4 on RBF (TFP + VO4:64.1, VO4:38.5 ml/min; P less than 0.05), GFR (TFP + VO4:22.9, VO4:9.3, ml/min; P less than 0.05) and V (TFP + VO4: 0.80, VO4: 0.38 ml/min; P less than 0.05) without changing FENa+ (TFP + VO4: 3.8, VO4: 3.2%). Similar changes were obtained with verapamil as well as with EGTA. Furthermore thyroparathyroidectomy (TPTX) decreased serum calcium (control: 8.78, TPTX: 4.98 mg/100 ml; P less than 0.05) and blunted the effects of VO4 on renal hemodynamics. Reestablishing normal serum Ca2+ by an intra-arterial infusion of CaCl2 elicited the VO4 effects of vasoconstriction and decreased GFR; V was not affected and FENa+ rose. The data support the idea that influx of extracellular calcium into smooth muscle cells mediates the hemodynamic effects of VO4 in the dog.
Direct measurement of potassium leak from single 3 M KCl microelectrodes
Authors: Stoner L. C. , Natke E. , Dixon M. K.
Published: December 2017

Potassium loss was measured from individual microelectrodes filled with 3 M KCl. The potassium content of a distilled-water droplet into which a microelectrode was inserted for a known time interval was analyzed by helium-glow photometry. The rate of potassium loss from single microelectrodes (18-97 M omega) was within the detectable limits of this method. The rate of potassium loss appeared to decrease with increasing input resistance (determined in 0.16 M NaCl) for electrodes having a low resistance (18-50 M omega). Higher resistance electrodes (greater than 50 M omega) appeared to achieve a uniform rate of potassium loss of 3.5 fmol X s-1. Estimates of the rate of potassium loss calculated from the rate of swelling of impaled cells were identical to those measured directly on the same group of electrodes. We conclude the electrolyte loss not only occurs into a distilled-water drop but also during intracellular recording. An alternative method for fabrication of microelectrodes was tested. Of these electrodes, the ones having an input resistance greater than 50 M omega lost potassium at rates below 1 fmol X s-1.
Pathways of K+ permeation across the rabbit cortical collecting tubule: effect of amiloride
Authors: Stokes J. B.
Published: December 2017

These experiments were designed to examine passive K+ transport by the rabbit cortical collecting tubule. Potassium diffusion voltages were used to assess the presence of apical and basolateral K+ conductances. With amiloride (0.1 mM) in the lumen, reproducible K+ diffusion voltages from both the lumen and bath were obtained. Amiloride enhanced the magnitude of these voltage deflections (delta VT). There were time-dependent increases in the K+ diffusion voltage but the steady-state values were highly reproducible in the same tubule. In the amiloride-treated tubules, delta VT induced by raising bath [K+] to 20 mM was larger than that produced by the same increase in lumen [K+]. To evaluate whether the amiloride-treated tubule had, as suggested by the K+ diffusion voltages, substantial K+ permeabilities on both apical and basolateral membranes, the K+ rate coefficient (lumen-to-bath, KK) was measured before and after amiloride treatment. The amiloride-induced increase in KK, from 66 +/- 6 to 205 +/- 35 nm/s, was significantly larger than could be accounted for by the changes in transepithelial voltage or membrane voltages alone. This discrepancy could be due to single-file diffusion across the apical membrane and/or the (secondary) enhancement of K+ permeability following inhibition of Na+ transport.
Cellular K+ permeation across the cortical collecting tubule: effects of Na+-K+ pump inhibition and membrane depolarization
Authors: Stokes J. B.
Published: December 2017

These experiments examined the possibility that alterations in cell cation content and/or membrane voltage could influence cell K+ permeability of the cortical collecting tubule. Using the amiloride-treated isolated perfused rabbit cortical collecting tubule, ouabain or a K+-free bath reduced the magnitude of the K+ diffusion voltage. In addition, both methods of Na+-K+-ATPase inhibition reduced the K+ efflux (lumen-to-bath) rate coefficient (KK) without affecting the Na+ efflux rate coefficient. The magnitude of the reduction of KK could not be explained by a model of simple diffusion across two membranes in series even if the intracellular voltage were abolished. Thus, pump inhibition reduced cell K+ permeability. To determine whether membrane depolarization could induce a change in membrane permeability, [K+] was increased to 20 mM in both perfusate and bath. The reduction in KK was within the range predicted by the three-compartment model (36%). Differential membrane depolarization by raising lumen [K+] or bath [K+] produced disparate results. Apical depolarization reduced KK but basolateral depolarization did not. Taken together these results indicate that intracellular ion content may play a major role in regulating cell K+ permeability independent of voltage-dependent effects. In addition, under these experimental conditions, the apical membrane may be the rate-limiting barrier to cellular transfer.
Modulation of antidiuretic hormone-dependent capacitance and water flow in toad urinary bladder
Authors: Palmer L. G. , Speez N.
Published: December 2017

To test the hypothesis that antidiuretic hormone- (ADH) dependent water permeability is associated with changes in apical membrane area, hormone-dependent water flow and capacitance changes were measured in the toad urinary bladder under a number of different conditions. Dose-response relationships for water flow (Jv) and capacitance increases (delta C) were similar from 1 to 20 mU/ml ADH. At higher concentrations, Jv reached a plateau, while delta C decreased. The decrease in delta C was prevented by elimination of the osmotic gradient across the tissue. Serosal hydrazine (10 mM) increased Jv sevenfold and delta C threefold in the presence of 1 mU/ml ADH. Mucosal NH4Cl, at constant mucosal pH, increased Jv by 50-100%, but did not significantly change delta C. In the absence of an osmotic gradient, mucosal NH+4 increased delta C by 50%. NH4Cl had no effect on hydroosmotic response to 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP). Mucosal CO2 (9%) decreased Jv by greater than 90%, and delta C by 60% with 20 mU/ml ADH. Mucosal CO2 also inhibited the hydroosmotic response to 8-bromo-cAMP. Removal of serosal Na diminished cAMP-dependent Jv and delta C. The results confirmed the close relationship between ADH-dependent water permeability and membrane capacitance. They indicate, however, that under some circumstances membrane may be retrieved from the apical surface without affecting water permeability.
Dual action of aldosterone on toad bladder: Na+ permeability and Na+ pump modulation
Authors: Park C. S. , Edelman I. S.
Published: December 2017

The effects of aldosterone on the functional characteristics of the Na+ entry step across the apical membrane and on the Na+ exit step across the basolateral membrane of the urinary bladder of toads were examined using amiloride and ouabain as probes of the respective surfaces of the cell. Aldosterone stimulated Na+ transport with a concurrent increase in the transepithelial electrical conductance as did two other agents, vasopressin (ADH) and p-chloromercuriphenylsulfonate (PCMPS), primarily active on the apical membrane. Unlike the effects of ADH and PCMPS, however, the effect of aldosterone on Na+ conductance was blocked by actinomycin D and was associated with a decreased sensitivity of the apical Na+ channel to amiloride. In addition, aldosterone increased the sensitivity of the Na+ pump on the basolateral side to ouabain, an effect that was dependent on the metabolic state of the urinary bladder. These results support the inference of coordinate effects on Na+ permeability of the apical membrane and the Na+ pump of the basolateral membrane. Both effects of aldosterone appear to be dependent on the metabolic state of the transporting epithelium.
Magnesium transport in the proximal straight tubule of the rabbit
Authors: Quamme G. A. , Smith C. M.
Published: December 2017

Magnesium transport was evaluated in proximal straight tubules of rabbits by in vitro perfusion. Magnesium transport from lumen to bath was less than the fractional absorption rates of sodium and calcium. Accordingly, the tubule fluid magnesium concentration increased with water absorption. Magnesium transport in proximal straight tubules obtained from rabbits maintained on high dietary magnesium intake was not different from normal animals, which suggests little, if any, change in tubular function. There were no discernible differences in magnesium fluxes between superficial and juxta-medullary proximal straight tubules. To assess bath-to-lumen magnesium flux, tubules were perfused with solutions containing zero magnesium concentration. Magnesium movement from the bath to lumen was small and dependent on the transepithelial magnesium concentration gradient. This magnesium influx may be sufficient to account for net magnesium entry provided the appropriate concentration gradients from interstitium to lumen are available in the intact kidney.
Effects of luminal fluid anions on calcium transport by proximal tubule
Authors: Bomsztyk K. , George J. P. , Wright F. S.
Published: December 2017

To determine whether the anion composition of tubule fluid affects calcium absorption by the renal proximal tubule, in vivo microperfusion techniques were employed in anesthetized rats. Experiments were designed so that total calcium and sodium concentrations were kept constant in fluids entering the tubule. A control solution, in which the main anion was chloride, was modified either by addition of ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetra-acetic acid or by replacing most of the chloride with nitrate, thiocyanate, sulfate, or citrate. Sufficient mannitol was added to the perfusion fluids to reduce net fluid flux to near zero. Net fluxes of calcium and sodium were calculated from measurements of total concentrations in perfused and collected fluids. Electrochemical driving forces across the tubule wall were calculated from measurements of transepithelial voltage and of Ca2+ activity in perfused and collected fluids. Results showed that calcium absorption by the proximal tubule depends on both the luminal Ca2+ activity and the transepithelial voltage. With zero transepithelial electrochemical driving force calcium absorption was significantly different from zero. Calcium and sodium transport rates were seen to vary independently. We conclude that the calcium absorptive mechanism involves active transport and can be dissociated from the sodium transport pathway. Calcium transport is also affected by changes in transepithelial electrochemical driving forces with an apparent permeability similar to values reported for sodium and potassium.
Development of solute transport in rabbit proximal tubule. III. Na-K-ATPase activity
Authors: Schwartz G. J. , Evan A. P.
Published: December 2017

HCO-3 transport (JHCO-3) in early juxtamedullary proximal convoluted tubules isolated from infant rabbits during the 1st 3 wk of life is about one-third that in tubules obtained from adults. A rapid increase in transport ensues during wk 4 through 6, so that near-mature levels are attained by the end of this time. Because the pattern for development of glucose absorption was similar and because both HCO-3 and glucose absorption are driven by the lumen-to-cell Na+ flux, the activity of Na-K-ATPase (the Na+-extruding pump) was considered to be a critical mediator. A kinetic microassay (which couples ATP hydrolysis to NADH oxidation) allowed the measurement of Na-K-ATPase and ouabain-insensitive ATPase on the same tubular segment. Three to nine early juxtamedullary proximal convoluted tubules were obtained after collagenase treatment of the kidney and four to six rabbits were studied at each week of life. The mean activity of Na-K-ATPase during the 1st wk of life was 44.5 +/- 3.5 pmol X min-1 X mm-1, one-third of the adult level. During an interim period of development (2-6 wk), enzyme activity gradually reached 60% of adult levels (76.3 +/- 3.0 at 6 wk), while transport of HCO-3 and glucose, studied previously in other animals, attained mature rates. Only in the 7th wk did the enzyme activity reach that of the adult (106.8 +/- 6.8 in wk 7 vs. 128.4 +/- 14.0 in adult rabbits).(ABSTRACT TRUNCATED AT 250 WORDS)
Volume expansion modulates NaHCO3 and NaCl transport in the proximal tubule and Henle's loop
Authors: Bichara M. , Paillard M. , Corman B. , de Rouffignac C. , Leviel F.
Published: December 2017

The effects of extracellular fluid volume expansion on kidney reabsorption of bicarbonate, chloride, and water were examined in Sprague-Dawley rats by paired micropuncture experiments in which tubular fluid was collected from early distal and end-proximal sites. In group I, rats were studied during a plasma-replete euvolemic state and after 10% body wt colloid-free volume expansion. The filtered loads were similar in euvolemia and expansion; expansion increased end-proximal total CO2 concentration ([tCO2] EP) from 13.0 +/- 0.8 to 18.4 +/- 0.7 mM and inhibited the tCO2 fractional reabsorption 26% in the proximal convoluted tubule and 15% in Henle's loop. Early distal tCO2 delivery therefore increased from 61 +/- 7 to 140 +/- 17 pmol X min-1 X g kidney wt-1. A more pronounced inhibition of fractional reabsorption of chloride (45%) and water (35%) occurred in the proximal convoluted tubule during expansion. In group II, rats were studied during 5% body wt 70 g/liter albumin-containing expansion and after 10% body wt colloid-free expansion. Compared with euvolemia of group I, albumin expansion decreased proximal fractional reabsorption of tCO2 13%, chloride 19%, and water 14% without change in filtered loads, and [tCO2]EP rose to 17.2 +/- 0.7 mM. A 10 g/liter decrease in plasma protein concentration during colloid-free expansion was associated, compared with albumin expansion, with inhibition of proximal fractional reabsorption of tCO2 by 4%, chloride by 18%, and water by 9% without further altering [tCO2]EP. We conclude that 1) expansion specifically inhibits bicarbonate reabsorption in the proximal convoluted tubule and in Henle's loop independent of change in filtered load or in peritubular protein concentration, probably by enhancing bicarbonate backdiffusion and reducing proton secretion; 2) expansion-induced decrease in peritubular protein concentration contributes to the proximal expansion effect probably by specifically inhibiting transcellular sodium chloride reabsorption.
Prostaglandin E2 but not F2 alpha restores the natriuretic response to acetylcholine in indomethacin-treated dogs
Authors: Yun J. C. , Gill J. R. , Ho S. S. , Kelly G. D. , Keiser H. R.
Published: December 2017

Renal arterial infusion of acetylcholine (ACh) (40 micrograms/min) produces a natriuresis, diuresis, and an increase in renal plasma flow (RPF) without a change in glomerular filtration rate (GFR) or renin secretory rate (RSR). The present study was designed to examine the role of prostaglandins in this natriuretic response to ACh. In dogs pretreated with indomethacin (Indo) (5 mg/kg, i.v.), an inhibitor of prostaglandin synthesis, renal arterial infusion of ACh produced an increase and then a decline in urinary flow and sodium excretion accompanied by a progressive fall in GFR and RPF and a progressive increase in RSR. Renal arterial infusion of PGE2 (1.9 micrograms/min) but not PGF2 alpha (1.9 micrograms/min) before and during the infusion of ACh restored the diuretic and natriuretic response to ACh in Indo-treated dogs. Renal arterial infusion of bradykinin (BK) (3 micrograms/min) in Indo-treated dogs produced a diuresis and natriuresis similar to that produced by PGE2; renal arterial infusion of BK, however, did not restore the diuretic and natriuretic response to ACh in Indo-treated dogs. The data suggest that Indo shortens the diuretic and natriuretic response to ACh by inhibiting synthesis of prostaglandins, possibly PGE but not PGF. The data further suggest that PGE2 restores the diuretic and natriuretic response to ACh in Indo-treated dogs through a specific action rather than by its action as a renal vasodilator.
Comparison of body fluid compartment sizes in Brattleboro homozygous and Long-Evans rats
Authors: Robinson D. H. , Conrad K. P. , Edwards B. R.
Published: December 2017

Because of their hypothalamic diabetes insipidus (DI), Brattleboro homozygous rats exhibit profound polyuria and polydipsia with elevated plasma osmolalities. This combination of symptoms has led to the tacit assumption that these animals are chronically volume contracted. However, the few direct measurements that have been published indicate otherwise. In the present study, total body water (TBW), total body lipid content, and plasma volume were measured in conscious DI rats and compared with corresponding values in weight-matched conscious Long-Evans (LE) rats. Fat content of DI rats (5.0 +/- 0.4 g/100 g body wt) was significantly lower than that of LE rats (7.6 +/- 0.6, P less than 0.005). TBW was not significantly different between the two strains whether expressed as milliliters per 100 g wet wt (DI 69.2 +/- 0.4 vs. LE 67.7 +/- 0.7) or as milliliters per 100 g fat-free wet wt (DI 73.4 +/- 0.5 vs. LE 73.3 +/- 0.4). Plasma volume of DI rats (4.4 +/- 0.1 ml/100 g body wt) was significantly higher than that of LE rats (3.9 +/- 0.2, P less than 0.025). Extracellular fluid volume was similar in the two strains although, for this measurement, the animals were neither weight- nor age-matched. Accordingly, the data indicate that under the conditions of this study the DI rat is not chronically volume contracted. It would appear that, given an adequate water supply, water balance can be achieved via the thirst mechanism, perhaps abetted by vasopressin-independent mechanisms of urine concentration.
Ontogenesis of hormone-dependent adenylate cyclase in isolated rat nephron segments
Authors: Imbert-Teboul M. , Chabardes D. , Clique A. , Montegut M. , Morel F.
Published: December 2017

Ontogenesis of hormone-dependent adenylate cyclase (AC) was investigated in rat kidney by single tubule microassay between two days postnatal and adulthood. This approach allowed us to analyze the kinetics of vasopressin-sensitive AC maturation in its tubular target sites, namely the thick ascending limb and collecting tubule. It was also possible to compare in a single segment--the thick ascending limb--the kinetics of AC ontogenesis for three hormones-- vasopressin, calcitonin, and parathyroid hormone. The results show that 1) 2 days after birth AC is still poorly responsive to vasopressin, especially in the thick ascending limb. By contrast, this segment exhibits marked AC responses to calcitonin and parathyroid hormone. 2) For a given hormone, the kinetics of AC ontogenesis depends on the segment in which the receptor-enzyme complex is located. 3) For a given segment, the pattern of AC maturation is specific for each hormone. These data indicate that the process of tubular AC maturation cannot be accounted for simply by an increase in basolateral membrane area or by the synthesis of new catalytic units. More specific mechanisms must be involved that regulate independently the synthesis of each kind of hormone receptor and/or its coupling to cyclase.
Insulin-stimulated phosphorylation and insulin binding in canine renal basolateral membranes
Authors: Hammerman M. R. , Gavin J. R.
Published: December 2017

To characterize the interaction of insulin with the renal proximal tubular cell, we measured insulin-stimulated phosphorylation in basolateral membranes and brush border membranes isolated from canine renal cortex. Insulin stimulated the specific phosphorylation of a 92,000 Mr protein band demonstrable on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of basolateral membranes. Dephosphorylation of the 92,000 Mr band occurred over time. Insulin-stimulated phosphorylation was concentration dependent, being clearly detectable at 10(-9) M insulin and maximal at 10(-6) M. The phosphorylated 92,000 Mr protein band from detergent-solubilized basolateral membranes was immunoprecipitated using serum from a patient with anti-receptor antibodies. No insulin-stimulated phosphorylation was detected in brush border membranes. Binding of insulin to membranes was highly specific for native hormone and was severalfold greater in basolateral membranes than in brush border membranes. These observations are consistent with the asymmetrical distribution of insulin-stimulated protein kinase as well as specific insulin binding sites in the proximal tubular cell. The data suggest that insulin exerts physiological effects on the cell through binding to specific basolateral membrane receptors and phosphorylation of those receptors.
Fluidity and composition of brush border and basolateral membranes from rat kidney
Authors: Hise M. K. , Mantulin W. W. , Weinman E. J.
Published: December 2017

The physical state of rat renal brush border and basolateral membranes was examined using the technique of electron spin resonance. As estimated from the order parameters of both 5- and 12-doxyl spin-labeled phosphatidylcholine, the brush border membrane was significantly more ordered than the basolateral membrane. To confirm these findings, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene was studied. Fluorescence anisotropy at 25 and 37 degrees C was significantly greater in the brush border membrane when compared with the basolateral membrane. The relative contribution of the lipid component to this difference was examined using multilamellar liposomes prepared from lipid extracts of these membranes. Fluorescence anisotropy in the brush border membrane lipids was significantly greater than that in the basolateral membrane lipids at both 25 and 37 degrees C. Compositional determinants of fluidity were examined in both membranes. Factors that may contribute to the more fluid state of the basolateral membrane include 1) a greater lipid-to-protein ratio; 2) a greater ratio of phosphatidylcholine to sphingomyelin; and 3) a tendency toward shorter-length fatty acyl chains.
Bicarbonate-carbon dioxide buffer system: a determinant of the mitochondrial pH gradient
Authors: Simpson D. P. , Hager S. R.
Published: December 2017

The influence of the bicarbonate-carbon dioxide buffer system on the pH gradient (delta pH) across the inner membrane of mitochondria from rabbit renal cortex was studied with and without phosphate in the medium. delta pH with bicarbonate buffer or phosphate in the medium was greater at low than at high medium pH so that the difference (delta delta pH) between delta pH at pH 7.1 and at 7.6 was positive. Varying the concentration of phosphate from 0 to 10 mM had little effect on delta delta pH produced by bicarbonate buffer. Inhibition of the phosphate-hydroxyl carrier with N-ethylmaleimide abolished delta delta pH when phosphate was present in non-bicarbonate-containing media. With bicarbonate buffer present, N-ethylmaleimide increased delta delta pH. Similar effects were observed in mitochondria from liver and heart as well as from kidney. The effects of the bicarbonate buffer system on delta pH may result either from an inner membrane permeable to carbon dioxide but not to bicarbonate ion or from an active carrier for bicarbonate ion in the inner membrane. In intact kidney cells, the influence of the bicarbonate buffer system on delta pH may provide a mechanism for regulating substrate metabolism in response to acid-base changes. It may also serve in many organs to reduce fluctuations in matrix pH when alterations in cytoplasmic pH occur.
Analysis of the determinants of renal cortical PCO2
Authors: Bidani A. , Crandall E. D. , DuBose T. D.
Published: December 2017

Recent studies employing Pco2 microelectrodes demonstrated CO2 tensions in the renal cortex (60-65 mmHg) that are significantly greater than systemic arterial Pco2. Three sources for CO2 generation have been proposed: 1) luminal production from H+ and HCO-3,2) addition of HCO-3 to peritubular plasma with disequilibrium for H+/HCO-3/CO2, and 3) metabolic CO2 production. None of these mechanisms alone can adequately explain the findings. The purpose of this study was to examine mechanisms of elevated renal cortical Pco2 generation and maintenance by developing a mathematical model for the reaction and transport processes involved in proximal tubule HCO-3 reabsorption. Steady-state calculations of pH, Pco2, and [HCO-3] for luminal, cellular, and vascular compartments employing simple mass balance considerations are presented. Besides exploring the role of metabolic CO2 production in the genesis and maintenance of elevated renal cortical Pco2, we also propose and examine the additional mechanism of diffusive transfer of CO2 between renal cortical venular and afferent arteriolar capillaries. Our results show that the required magnitude of either metabolic CO2 production or diffusive CO2 transfer alone is large (3 mmol/liter RBF). However, vascular-vascular exchange of CO2 gas in combination with accepted levels of metabolic CO2 production can adequately explain the findings of an elevated renal cortical Pco2 of approximately 65 mmHg as measured experimentally in vivo.
Stimulation of Na-K-ATPase in the rat collecting tubule by two diuretics: furosemide and amiloride
Authors: Mernissi G. E. , Doucet A.
Published: December 2017

To test whether sodium availability controls the concentration of renal Na-K-ATPase, we evaluated the effect of chronic alterations in apical membrane sodium permeability in specific nephron segments on the maximal activity of Na-K-ATPase. For this purpose Na-K-ATPase activity was determined in nephron segments microdissected from rats treated continuously for 3-8 days with either furosemide or amiloride, two diuretics known to lower the apical permeability to sodium in the thick ascending limb and the collecting tubule, respectively. Unexpectedly, Na-K-ATPase activity was decreased neither in the thick ascending limb nor in the collecting tubule after administration of either drug. In fact, both diuretics paradoxically increased the pump activity by 60-150% in the collecting tubule. This stimulation of Na-K-ATPase activity was accompanied with an enhancement of the collecting tubule diameter. Stimulation of Na-K-ATPase was identical in the collecting tubule of diuretic-treated rats receiving spironolactone. These results suggest that Na-K-ATPase maximal activity is not controlled by sodium availability or by aldosterone under these conditions and that chronic administration of furosemide or amiloride induces Na-K-ATPase activity in the collecting tubule. This effect appears to be independent of aldosterone.
Prostaglandin synthesis inhibitors and vasa recta erythrocyte velocities in the rat
Authors: Lemley K. V. , Schmitt S. L. , Holliger C. , Dunn M. J. , Robertson C. R. , Jamison R. L.
Published: December 2017

Vasa recta erythrocyte velocities (VRBC) in the exposed renal papilla of anesthetized water-loaded rats were determined before and 60 min after intravenous administration of a prostaglandin synthesis inhibitor (indomethacin, meclofenamate) or the inhibitor vehicle alone. The change in VRBC of ascending and descending vasa recta for the inhibitor group [-17 +/- 5% (SE)] was different from that for controls (+12 +/- 4%, P less than 0.002). Erythrocyte velocities were also determined in vasa recta of antidiuretic rats before and 30 min after administration of indomethacin or vehicle alone. Prostaglandin synthesis inhibition was again associated with a significant decrease in VRBC compared with control (-24 +/- 4% vs. +28 +/- 20%, respectively, P less than 0.025). These findings suggest that prostaglandins play a similar role in regulating blood flow in the renal medulla in water diuresis and antidiuresis.
Prostaglandin I2 attenuates ischemic acute renal failure in the rat
Authors: Lifschitz M. D. , Barnes J. L.
Published: December 2017

We gave prostaglandin I2 (PGI2) (8 ng X kg-1 X min-1 i.v.) for 20 min before, during, and 20 min after clamping of the rat left renal artery for 40 min to evaluate the effect of PGI2 in this model of acute renal failure. Control animals were given glycine buffer (PGI2 diluent). Glomerular filtration rate was estimated by the clearance of inulin 24 h later from each kidney. In group I rats (studied during hydropenia) inulin clearance in the control (right) kidney averaged 1.4 ml/min. Inulin clearance in kidneys exposed to 40 min of ischemia was 0.05 (glycine treated) versus 0.22 (PGI2 treated) ml/min. Although PGI2 offered significant protection in the group I animals, the differences were small and many of the glycine-treated ischemic kidneys were anuric. In the group II studies the same protocol was employed except that 5% body wt volume expansion was done with Ringer solution prior to measurement of inulin clearance. In the group II rats inulin clearance in control (right) kidneys averaged 1.5 ml/min. Inulin clearance after 40 min of renal ischemia was 0.04 ml/min in glycine-treated rats versus 0.90 ml/min in PGI2-treated animals. Histological examination of the group II ischemic kidneys revealed cellular necrosis and cast formation in the S3 segments of the glycine-treated animals and significantly less necrosis and cast formation in the PGI2-treated animals. The degree of necrosis and casts was inversely related to inulin clearance. Accordingly, PGI2 significantly attenuated the fall in inulin clearance measured 24 h after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanism of ammonia secretion by cortical collecting ducts of rabbits
Authors: Knepper M. A. , Good D. W. , Burg M. B.
Published: December 2017

The collecting duct system is a major site of ammonia addition to the tubule fluid. To study the mechanisms involved, we measured total ammonia and total CO2 transport in isolated, perfused cortical collecting ducts (CCD) from deoxycorticosterone-(DOC) treated rabbits. Perfusate and bath solutions contained 25 meq/liter HCO3 and 4 mM total ammonia. Net fluid transport was not significantly different from zero. Net secretion of total CO2 occurred in all tubules (mean collected concentration, 44.2 mM). Despite bicarbonate secretion, there was net secretion of total ammonia (mean collected concentration, 6.4 mM). There was no detectable ammonia addition to the collected fluid when ammonia was excluded from the perfusate and bath, ruling out a major contribution from synthesis. Ouabain did not significantly affect net transport of total ammonia or total CO2. To test the hypothesis that an acid pH disequilibrium may lower the luminal pH enough to drive ammonia secretion by nonionic diffusion, we perfused CCD from DOC-treated rabbits with carbonic anhydrase (CA) (0.1 mg/ml). Without CA, there was net total ammonia secretion (-2.2 pmol X min-1 X mm-1) and net total CO2 secretion (-16.6 pmol X min-1 X mm-1). Luminal CA converted the net total ammonia secretion to net absorption (1.0 pmol X min-1 X mm-1) while the bicarbonate secretion persisted (-11.2 pmol X min X mm-1). We conclude that total ammonia secretion in these tubules occurs primarily by diffusion of NH3 and is dependent on a luminal acid pH disequilibrium.
Response of ammoniagenesis to acute alkalosis
Authors: Tannen R. L. , Goyal M.
Published: December 2017

To examine the response of ammonia production to acute alkalosis, isolated rat kidneys were perfused with 5 mM glucose and 0.5 mM glutamine for an initial 45 min at pH 7.7, achieved by raising the perfusate HCO3 concentration or reducing the PCO2, followed by one or more 45-min periods at a normal pH. During the initial high pH period, respiratory alkalosis had no effect on NH3 production in comparison with perfusions at a normal pH. However, during the subsequent 45-min period at a normal pH, kidneys exposed initially to a high pH produced less NH3 than those perfused at a normal pH for the entire experiment. This suppression of ammoniagenesis abated during an additional 45 min of perfusion at a normal pH. Kidneys exposed to metabolic alkalosis produced more NH3 than controls during the initial 45 min of perfusion. However, similar to respiratory alkalosis, NH3 production was lower than controls during the subsequent 45 min of perfusion at a normal pH. Hence a high bicarbonate concentration can rapidly, but transiently, stimulate NH3 production by the isolated rat kidney. However, a high pH, whether produced by metabolic or respiratory manipulations, suppresses ammoniagenesis. This suppressive effect requires 45 min of exposure to an elevated pH to be manifest and at least 45 min of exposure to a normal pH for reversal. The delayed response to alkalosis contrasts strikingly with the immediate effects of acute acidosis on NH3 production by the perfused kidney and suggests that different mechanisms account for the regulation of ammoniagenesis in response to acute decrements and elevations in pH.