Single nephron filtration rates (SNGFR) in the trout,Salmo gairdneri

1. Trout kidney slices incubated in the presence of¹⁴C-sodium ferrocyanide accumulated radioactivity. Uptake by individual nephroi increased with time, stabilizing after 30 min. The greater the concentration of radioactivity the greater the uptake per mm of tubule, while reduced specific activity inhibited¹⁴C uptake.
2. The renal excretory pattern of¹⁴C-sodium ferrocyanide was compared with that of³H-inulin in fresh water adapted trout. The renal clearance rates of the substances were similar and linearly related over a range or urine flows, providing plasma ferrocyanide concentrations were between 1.5 and 2.5 mM.¹⁴C-ferrocyanide is thus a reliable marker for glomerular activity in this species.
3. A protocol for the determination of single nephron glomerular filtration rates (SNGFR) in trout is described. The method exploits the ability to visualize ferrocyanide as Prussian Blue within nephroi after a single pulse injection superimposed upon a constant infusion of¹⁴C-ferrocyanide. The effects of surgical procedure and extraluminal contamination are assessed.
4. The SNGFR of freshwater trout, 1.31±0.2 nl/min is approximately 65% less than that observed in sea water adapted fish (3.74±1.12 nl/min). The overall, total kidney glomerular filtration rate, (GFR) is 142.6±17.4 μl/min/kg in fresh water animals and 20.1±0.9 μl/min/kg in sea water fish.
5. It is concluded that whilst SNGFRs of sea water animals are higher than those of fresh water animals, filtration is distributed to nephron populations selected to meet the homeostatic demands of the organism.

     

The early phase of experimental acute renal failure

Summary Experiments were designed to determine whether leakage of substances across the tubular epithelium, which are impermeant in the normal kidney, falsifies the measurement of glomerular filtration rate in acute renal failure. Permeability to those substances most commonly used for filtration rate determination, polyfructosan, inulin and ferrocyanide, was estimated by measuring their recoveries following perfusion through various nephron segments in haeme pigment, ischaemic and nephrotoxic models of actue renal failure. Late proximal recovery of¹⁴C ferrocyanide was only marginally decreased compared to controls, by a maximum of 6%. Distal recovery of polyfructosan,¹⁴C and³H inulin were depressed somewhat more, by a maximum of 11%. Urinary recovery of¹⁴C inulin was reduced by only 15% in kidneys showing severely restricted renal function. It is concluded that tubular leakage is not a feature of significance in the early phase of moderate acute renal failure, that ferrocyanide and inulin are reliable markers for the determination of nephron filtration rate and water reabsorption, and that the reduction in whole kidney inulin or polyfructosan clearance reflects primarily a reduction in glomerular filtration rate.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Effect of procaine on potassium permeability of canine tracheal smooth muscle

Procaine depolarized the cell membrane, and initiated oscillations and spike-like potentials in canine tracheal smooth muscle, at concentrations between 1 and 5 mM, while higher concentrations of this drug suppressed the spontaneous activities. Inhibitory effects of procaine on⁸⁶Rb-efflux were not evident in normal solution. In high K solution, procaine decreased the rate of⁸⁶Rb-efflux, in a dose dependent manner, and a Scatchard plot suggested two sites of action for procaine. The site with a higher affinity (KD=0.26 mM) may directly regulate the K permeability and interact with procaine in a one to one manner. In high concentrations (10 mM), procaine interacted at another site and the tracheal smooth muscle which contracted in high K solution relaxed almost completely. Thus, the decrease in⁸⁶Rb-efflux by high concentrations of procaine may partly result from a decrease in intracellular Ca concentration. It is proposed that procaine has the dual effect by inhibiting the K conductance: (1) depolarization of cell membrane, (2) increase in membrane excitability. These actions would explain the spontaneous electrical activity induced by procaine in canine tracheal smooth muscle.     

Evidence for Na+ dependent rheogenic HCO 3 − transport in fused cells of frog distal tubules

The mechanism of HCO ₃ ^– transport was studied applying microelectrodes in giant cells fused from single epithelial cells of the diluting segment of frog kidney. A sudeen increase of extracellular HCO ₃ ^– concentration from 10 to 20 mmol/l at constant pH hyperpolarized the cell membrane potential of the fused cell. This cell-voltage response was totally abolished by 10^–3 mol/l SITS and significantly reduced by 10^–4 mol/l acetazolamide or by omission of Na⁺ from the extracellular perfusate. Removal of Na⁺ from the perfusate caused a transient depolarization. Reapplication of Na⁺ induced a transient hyperpolarization. 10^–3 mol/l SITS abolished the cell-voltage response to removal and reapplication of Na⁺. In the intact diluting segment of the isolated perfused frog kidney peritubular perfusion of 10^–4 mol/l acetazolamide reduced the limiting transepithelial electrochemical gradient for H⁺ significantly from 30±4 mV to 14±3 mV. The results suggest: (i) In the diluting segment of the frog kidney a Na⁺-dependent rheogenic HCO ₃ ^– transport system exists across the peritubular cell membrane. (ii) This rheogenic peritubular Na⁺/HCO ₃ ^– cotransporter cooperates with a Na⁺/H⁺ exchanger in the luminal membrane, thus driving HCO ₃ ^– reabsorption. (iii) Reabsorption of HCO ₃ ^– and secretion of H⁺ depend upon the presence of carbonic anhydrase.     

New Li+-selective ionophores with the potential ability to mediate Li+-transport in vivo

A series of structurally-related Li⁺-selective ionophores were studied in planar lipid bilayer membranes, to assess their potential ability to act as Li⁺-selective carriers in vivo. The ionophores are acyclic, neutral molecules of similar structure: N,N-diheptyl-N,N-diR-5,5-dimethyl-3,7-dioxanonane diamide. The structural differences among them are the N-amide substituents (the R residues) as follows: an aliphatic ether (AS 701), tetrahydrofuran (AS 706), furan (AS 708), an ester (AS 702) and an amide (AS 704)For each ionophore, the steady-state, single salt, membrane conductances and conductance-voltage behaviors were determined in the presence of LiCl, NaCl and MgCl₂. Membrane zero-current potentials were measurd for NaCl/LiCl and MgCl₂/LiCl mixtures.All five ionophores were found to operate as equilibrium-domain carriers of monovalent ions. All select lithium over sodium, but with different magnitudes of selectivity, ranging from P_Li/P_Na of 13 (for AS 701) to P_Li/P_Na of 2 (for AS 708). The ionophores also differ in their ability to mediate Li⁺ membrane permeation, the order of decreasing potency being: AS 701AS 706>AS 702>AS 704AS 708.Of the five molecules studied, the AS 701 molecule was found to have the best Li⁺ over Na⁺ selectivity and highest potency. These findings indicate that this molecule has the best potential for mediating lithium-selective membrane permeation in vivo, among the group studied.     

Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO 3 − (OH−) exit

Intracellular pH (pH_c) was measured on surface loops of rat kidney proximal tubules under free-flow conditions in vivo using fine tip double-barrelled pH microelectrodes based on a neutral H⁺ ligand. The microelectrodes had Nernstian slopes and a resistance of the order of 10¹² . By using a driven shield feed back circuit the response time to pH jumps was lowered to around 1 s. At a peritubular pH of 7.42 and a luminal pH of 6.68 ± 0.13 (n=27), pH_c was 7.17 ± 0.08 (n=19). Perfusing the peritubular capillaries suddenly with bicarbonate Ringer solutions of plasma-like composition which were equilibrated with high or low CO₂ pressures, acidified or respectively alkalinized the cells rapidly as expected from the high CO₂ permeability of the cell membranes. Such data allowed us to calculate the cytoplasmic buffering power of the tubular cells. Sudden peritubular perfusion with Ringer solution containing only 3 mmol/l of HCO ₃ ^– at constant physiological CO₂ pressure led to a similar fast cell acidification which indicated that the peritubular cell membrane is also highly permeable for bicarbonate or OH^– (H⁺). The latter response was completely blocked by the stilbene derivative SITS at the concentration of 10^–3 mol/l. The observations indicate first that pH_c of rat proximal tubule is more acidic than was previously thought on the basis of distribution studies of weak acids, second that intracellular bicarbonate concentration is around 13 mmol/l and third that bicarbonate exit across the peritubular cell membrane is a passive rheogenic process via a conductive pathway which can be inhibited by SITS. The latter point confirms the conclusion which we had derived previously from membrane potential measurements in response to changing peritubular bicarbonate concentrations.     

Short-term and long-term stimulation of Na+−H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney

The effect of unilateral nephrectomy on Na⁺–H⁺ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70–75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted toV _max, whereas theK _m for Na⁺ was similar in control and compensatory growing kidney. The increase of Na⁺–H⁺ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na⁺–H⁺ exchange. The late, but not the initial stimulations of Na⁺–H⁺ exchange were associated with elevated activities of cortical (Na⁺+K⁺)-ATPase indicating that changes in antiporter activity precede those in the (Na⁺+K⁺)-pump. The early stimulation of Na⁺–H⁺ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na⁺–H⁺ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na⁺–H⁺ exchanger may be regulated by phosphorylation-dephosphorylation events. In support, the concentration of a well known protein kinase C activator, 1,2-diacylglycerol, in the cortical tissue of the remnant kidney increased up to 100% within 5 min following unilateral nephrectomy and preceded the increase in Na⁺–H⁺ exchange. The early stimulation of Na⁺–H⁺ exchange may be a trigger in initiating the kidney growth.     

Incomplete and flow dependent extraction of86Rb in the rat kidney

Renal uptake of⁸⁶Rb, lasting for 30 s after intravenous bolus injection, and uptake of 15 m radioactive microspheres (Ms) simultaneously injected in the left ventricle were measured in rats. Calculated from renal blood flow (RBF) determined by Ms and⁸⁶Rb clearance, the renal extraction ratio of⁸⁶Rb (E_Rb) was 0.81±0.07 (SD). With increasing RBF, E_Rb decreased significantly. Total body⁸⁶Rb extraction ratio nearly equalled E_Rb as determined 30 s after injection. In another group of rats⁸⁶Rb was injected into an extracorporal circuit from the carotid artery perfusing the left kidney only. Renal recovery of⁸⁶Rb averaged 0.76±0.13 15–20s after injection. As in the first group, E_Rb was inversely related to RBF. At increased RBF, calculated local cortical flow is seriously underestimated unless E_Rb as well as E_Rb flow dependency are taken into account. However, the relative local flow rates calculated for cortical zones are not appreciably affected by E_Rb and its flow dependency.     

Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons

K⁺ and Cl^– homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K⁺ and Cl^– efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl^– channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl^– current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C₂-ceramide, or serum deprivation. Cl^– channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl^– channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K⁺ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl^– and K⁺ homeostasis in regulating different apoptotic events.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

Electrophysiological analysis of rat renal sugar and amino acid transport

Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion ofd-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na⁺ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brushborder membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition ofd-glucose transport by various inhibitors, such as phlorizin, harmaline and oubain. The latter experiments suggest that the active Na⁺ pump in the peritubular cell membrane, which establishes the Na⁺ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane.     

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current (= transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 · 10^–7 mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from –78 to –81 mV and from –72 to –81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 · 10^–5 mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 cm², by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse.The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na⁺ 2Cl^–K⁺ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide. The weaker effect of torasemide from the peritubular side can, at least in part, be explained as an interference with chloride channels present in the basolateral membrane. Torasemide is also structurally related to chloride channel blockers such as diphenylamine-2-carboxylate.Supported by Deutsche Forschungsgemeinschaft DFG Gr 480/6-4 and 6-5     

Determination of intracellular K+ activity in rat kidney proximal tubular cells

The intracellular K⁺ activity of rat kidney proximal tubular cells was determined in vivo, using intracellular microelectrodes. In order to minimize damage from the impaling electrodes, separate measurements on separate cells, were performed with single-barrelled KCl-filled non-selective electrodes and single-barrelled, K⁺-sensitive microelectrodes, which were filled with a liquid K⁺-exchanger resin that has also a small sensitivity to Na⁺. Both electrodes had tip diameters of 0.2 m or below. The proper intracellular localization of the electrodes was ascertained by recording the cell potential response to intermittent luminal perfusions with glucose. The membrane potential measured with the non-selective microelectrodes was –76.3±8.1 mV (n=81) and the potential difference measured with the K⁺-sensitive microelectrode was –7.2±5.8 mV (n=32). Based on the activity of K⁺ in the extracellular fluid of 3 mmol/l the intracellular K⁺ activity was estimated to be 82 mmol/l. Assuming equal K⁺-activity coefficients to prevail inside and outside the cell, this figure suggests that the intracellular K⁺ concentration is 113 mmol/l which must be considered as a lower estimate, however. The data indicate that the K⁺-ion distribution between cytoplasm and extracellular fluid is not in equilibrium with the membrane potential, but that K⁺ is actively accumulated inside the cell. This result provides direct evidence for the presence of an active K⁺ pump in the tubular cell membranes, which in view of other observations, must be envisaged as a (not necessarily electroneutral) Na⁺/K⁺-exchange pump which operates in the peritubular cell membrane and is eventually responsible for the major part of the tubular solute and water absorption.     

Coupling between proximal tubular transport processes

Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (-methyl-d-glycoside), H⁺ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (c) of these substances. In the case of calcium the electrochemical potential differencec +zFc_i /RT) was the criterion employed. The rate of isotonic Na⁺-absorption (J_Na) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4isothiocyanatostilbene 2,2-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited J_Na (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, -methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H⁺ (glycodiazine) transport and PAH transport was only slightly affected.When SITS (1 mM) was applied from both sides of the cell it inhibited H⁺ (glycodiazine) transport by 72% and reduced J_Na by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H⁺ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied.When the HCO ₃ ^– buffer or physically related buffers were omitted from the perfusate the absorption of Na⁺ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected.The data are consistent with the hypothesis that the active transport processes of histidine, -methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na⁺-Ca²⁺ countertransport located at the contraluminal cell side. The residual Na⁺ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H⁺ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO ₃ ^– from the cell and secondary to that, the luminal H⁺-Na⁺ exchange and consequently the Na⁺ reabsorption. In the absence of HCO ₃ ^– buffer in the perfusates the luminal H⁺-Na⁺ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP _i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.     

14C-histamine release during vasodilatation induced by lumbar ventral root stimulation

Summary The vascularly isolated hindlimbs of dogs under chloralose anesthesia and neuromuscular blockade were perfused at constant flow with the animal's own arterial blood. Stimulation of the spinal ventral roots from L₅ to L₇ induced an antihistamine sensitive vasodilatation (VD) in the ipsilateral limb [Graham, Lioy: Histaminergic vasodilatation in the hindlimb of the dog. Pflügers Arch.342, 307–318 (1973)]. One hundred C of¹⁴C-histidine were injected into the limb perfusion line. Endogenously formed¹⁴C-histamine increased in the femoral venous blood from 59.2±17.0 to 190.3±41.9 cpm/ml of blood (P<0.005) during the VD induced by ventral root stimulation, while¹⁴C-histidine kept decreasing during the periods of observation. A significant increase of labeled histamine was also present upon ventral root stimulation after the VD had been drastically reduced or abolished by mepyramine maleate. It is concluded that the hindlimb VD under study is due to the nervously mediated release of histamine.     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.     

Luminal transport system for H+/organic cations in the rat proximal tubule

The efflux of radiolabelled organic cations from the tubular lumen into proximal tubular cells was investigated by using the stop-flow microperfusion method. The efflux rate increased in the sequence: N ¹-methylnicotinamide (NMeN⁺) < cimetidine < tetraethylammonium (TEA⁺) < N-methyl-4-phenylpyridinium (MPP⁺). Preloading the animals by i.v. infusion or pre perfusion of the peritubular capillaries with NMeN⁺ increased the efflux rate of MPP⁺. Luminal efflux was also augmented when the tubular solution was made alkaline with HCO ₃ ^– or phosphate, whereby HCO ₃ ^– is more effective than phosphate. Replacement of Na⁺ by Cs⁺ showed no effect. With i.v. preloading the animals with NMeN⁺ and with 25 mM HCO ₃ ^– in the luminal perfusate the 2-s efflux follows kinetics with a Michaelis constant K _m=0.21 mmol/l and maximal flux J _max=0.42 pmol · cm^–1 · s^–1 and a permeability term with P=37.7 m² · s^–1. Comparing the apparent luminal inhibitory constant values for MPP⁺ with the apparent contraluminal values of substrates of homologous series, it was found that (1) limitation by molecular size occurs at the contraluminal cell side earlier than at the luminal cell side; (2) affinity increases with hydrophobicity of the substrates at the luminal cell side, with a steeper or equal slope than at the contraluminal cell side; (3) affinity increases with basicity (i.e. pKa values) at the luminal cell side with a steeper slope than at the contraluminal cell side. Taken together, substrates with low hydrophobicity and low basicity interact at the luminal cell side more weakly than at the contraluminal cell side. On the other hand large, hydrophobic substrates have, at the luminal cell side, a higher affinity than at the contraluminal cell side. Many substrates, however, have equal affinity at the luminal and contraluminal cell sides.     

Kinetics ofl-histidine transport in the proximal convolution of the rat nephron studied using the stationary microperfusion technique

Summary The kinetics ofl-histidine reabsorption by the proximal convolution of the rat nephron have been studied by stationary microperfusion with simultaneous perfusion of peritubular capillaries. Steady-state concentrations (C ) and transepithelial concentration differences (c ) were determined over a wide range of peritubular bistidine concentrations. It was found that c increased hyperbolically with increase in luminal and peritubular histidine concentrations suggesting saturation transport kinetics. Furthermore c declined linearly along the convolution suggesting that nett active transport was not constant throughout the tubule. Using an expression to describe the rate of attainment of steady-state concentration in terms of lummal and peritubular histidine concentrations, histidine permeability coefficient (P), the maximum rate of active histidine transport (J _max) and the half saturation constant of the transport reaction (K _m ), we were able to determine the cause of the tubule inhomogeneity. We find thatP (14.1×10^–5 cm/s) andJ _max (45×10^–10 mol/cm²· s) are constant along the convolution but thatK _m increases markedly from about 5.4 mmol/kg 26% of the way along the convolution to 40 mmol/kg at 86%. These findings suggest that the histidine reabsorptive mechanism would be relatively inefficient with histidinuria occurring at all plasma concentrations but it would have enormous reserve capacity so that saturation would not readily occur. This prediction accords with available data on histidine clearance in the rat.A preliminary report of this work was presented to a Regional Meeting of the International Union of Physiological Sciences, held at the University of Sydney in Auguat 1972 [7]. The project was supported by the National Health and Medical Research Council of Australia, The Australian Kidney Foundation, The Max-Planck-Gesellschaft and by the Post-Graduate Medical Foundation of the University of Sydney.Mrs. Lingard was the recipient of an Australian Commonwealth Post-Graduate Studentship.     

Basolateral uptake and tubular metabolism of L-citrulline in the isolated-perfused non-filtering kidney of the African clawed toad (Xenopus laevis)

The kidney forms arginine (Arg) by using citrulline (Cit) as precursor, and is the main source of Arg for systemic protein synthesis. Even if the filtered and reabsorbed load (in rats) is sufficient for normal Arg synthesis, the following questions remain. (a) Can Cit be taken up across the contraluminal membrane of the tubule cells also? If so, (b) by what kind of mechanism? And (c) is this Cit, entering the cell from the peritubular side, metabolized to Arg and ornithine (Orn)? Although these questions are raised mainly in connection with mammals, we used the amphibian kidney, which is especially suitable because of its double blood supply, for an initial approach to the problem. After the toad was decapitated, the portal vein, the caval vein and the ureters were catheterized, and the kidneys were perfused through the portal vein (Ringer solution +l- or d-Cit+inulin+p-aminohippurate +l-aspartate). Exclusive peritubular perfusion was assured by showing that inulin perfused into the portal vein did not appear in the urine. During perfusion of the portal vein with l-Cit in a physiological concentration (65 μmol/l), an initial peritubular net uptake of l-Cit of 170±27 (n=10) nmol·h^?1·g kidney^?1 (wet weight) was observed, whereas the value for d-Cit (65 μmol/l) was only 18±7 (n=6) nmol·h^?1·g^?1. After perfusion for 50 min, the uptake of l-Cit reached a steady state with an uptake rate of 108±5 nmol·h^?1·g^?1. Adding l-phenylalanine (l-Phe; 20 mmol/l) to the solution or substituting mannitol for NaCl in the perfusate, decreased this l-Cit uptake to values similar to those for d-Cit. During perfusion with 65 μmol/l l-Cit, the Arg delivery into the venous blood was 40±4.8nmol·h^?1·g^?1, corresponding to 36% of the peritubular Cit uptake in the steady state, and the Orn delivery was 49.6±3.2nmol·h^?1·g^?1, corresponding to 46% of peritubular Cit uptake in the steady state. The venous Arg delivery increased to 51 nmol·h^?1·g^?1 while the kidney was perfused with 1 mmol/l l-Cit. At higher l-Cit concentrations no further increase of venous Arg delivery could be observed. During perfusion with d-Cit (65 μmol/l) or when adding l-Phe (20 mmol/l) no Arg or Orn could be detected in the venous outflow. In conclusion, the amphibian kidney perfused in situ is a suitable model for studying peritubular amino acid uptake and metabolism in the kidney. In the toad kidney, a peritubular uptake mechanism for l-Cit exists, which is stereospecific, saturable, NaCl-dependent and can be inhibited by l-Phe. In the tubule cells, l-Cit is transformed into Arg. Part of the Arg is metabolized further to Orn and urea, and Arg and Orn are released into the venous outflow.