Potassium permeability of luminal and peritubular membranes in the proximal tubule of bullfrog kidneys

Using double-barreled K(+)-selective or conventional voltage microelectrode, K+ permeability of the luminal membrane of renal proximal tubule was investigated in comparison with that of the peritubular membrane in doubly-perfused bullfrog kidneys. A decade change in K+ concentration of the luminal perfusate from 3.5 (control) to 35 mM (high K+) induced depolarization in the luminal membrane potential by 11.2 mV with a slight elevation of intracellular K+ activity, (K)i, while those of the peritubular perfusate depolarized peritubular membrane potential by 31.6 mV with a moderate rise of (K)i by 8.0 mEq. The transport number for K+ (tK) in the luminal membrane was about one-third of that in the peritubular membrane. Both luminal and peritubular administrations of Ba2+ (4 mM) additively depolarized membrane potential. The magnitude of Ba2(+)-induced depolarization in the luminal membrane was about one-fourth of that in the peritubular membrane. The high K(+)-induced depolarization elicited from one-side perfusion was hardly affected by Ba2+ which was given from the other side. From these results we conclude that the transport number of K+ in the luminal and peritubular membranes of the proximal tubular epithelium is in the range of 0.17-0.32 and 0.52-0.66, respectively.     

The effect of cAMP on ion transport in the proximal tubular cells in bullfrog kidney

Using a direct cellular micropuncture technique with double-barreled ion-selective microelectrodes, we investigated the effect of dibutyryl-cyclic AMP (db-cAMP) on the membrane potential and the transport of Na+, K+, and H+ in doubly-perfused bullfrog proximal tubules. The peritubular membrane potential difference (EM) and the intracellular K+, Na+ activities ((K)i, (Na)i) or intracellular and luminal pH were monitored continuously after peritubular administration of db-cAMP (10(-3)-10(-4)M). Results: 1) db-cAMP hyperpolarized the EM by 8.0 mV with an increase of (K)i by 4.8 mEq/l; 2) the peritubular administration of high K+ (13.5 and 50 mM) solutions depolarized the EM by 11.5 and 41.5 mV, respectively. The high K+ perfusate with db-cAMP produced a depolarization to the same level as that in simple high K+ perfusion without db-cAMP; 3) db-cAMP transiently blocked the luminal acidification concomitantly with a cellular alkalinization by about 0.1 pH; and 4) db-cAMP caused a decrease of (Na)i by 5.0 mEq/l. Conclusions: 1) cAMP causes an increase of K+ permeability in the peritubular membrane; 2) cAMP induces a cytosol alkalosis by enhancing incorporation of H+ into the subcellular vesicles, thus favoring the activation of Na+/K+ pump; and 3) cAMP, in effect, suppresses the Na+/H+ exchange mechanism in the luminal membrane and transiently blocks the proximal urinary acidification.     

The effect of loop diuretics on fluid reabsorption from the rat proximal convoluted tubule

The effects of the 'loop diuretics' bumetanide, furosemide and piretanide on fluid reabsorption from the rat proximal convoluted tubule have been tested to assess whether a Na(+)-Cl- cotransport mechanism can support fluid reabsorption from this nephron segment. Proximal convoluted tubules were perfused in vivo at 25 nl min-1 with chloride Ringer solutions containing [3H]inulin. Fluid reabsorptive rate (Jv) was 2.05 +/- 0.07 nl mm-1 min-1 (n = 115) from control chloride Ringer. Bumetanide at 10(-6) and 10(-5) M reduced Jv by about 40%. Bumetanide at 10(-7) M, furosemide (10(-4) and 10(-5) M) and piretanide (10(-4) and 10(-5) M) had no effect on Jv. At 10(-3) M, furosemide and piretanide reduced Jv by about 45%. The results show that luminal application of loop diuretics lowers fluid reabsorption from the proximal convoluted tubule and the order of potency for inhibiting reabsorption is bumetanide greater than furosemide identical to piretanide. The specificity of the loop diuretics at their effective concentrations needs to be confirmed, however, before attribution of a role for Na(+)-Cl- co-transport in the support of proximal tubule fluid reabsorption.     

Correlation between luminal hydrostatic pressure and proximal tubular fluid reabsorption in the rat kidney

Summary Split-drop experiments were performed to evaluate the effect of changes in luminal hydrostatic pressure on net fluid reabsorption in proximal convoluted tubules of the rat kidney. While hydrostatic pressure in control droplets averaged 28.9±1.03 mm Hg, it increased to a mean of 65.2±3.3 mm Hg during pressure elevation and fell to 10.8±1.04 mm Hg during pressure reduction. In paired measurements in identical tubules net fluid absorption changed from a control value of 2.96±0.14 nl/min·mm to 3.88±0.14 nl/min·mm when luminal pressure was elevated. During pressure reduction net fluid absorption fell from a control of 2.98±0.09 nl/min·mm to 2.26±0.13 nl/min·mm (P<0.001). This dependency of fluid absorption upon hydrostatic pressure was not greatly affected by the finding that microphotography overestimated the true intradroplet volume by 31% during control and by 30.2% and 50% during elevated and reduced pressure respectively. From the relation between the changes of net absorption and luminal hydrostatic pressure an apparent hydraulic conductance of 0.04 nl/min·mm Hg was estimated.     

The effect of phenylalanine on the electrical properties of proximal tubule cells in the frog kidney

The present study was designed to elucidate the effects of sodium-coupled transport on the electrical properties of proximal tubule cells in the isolated perfused frog kidney. Cable analysis techniques have been employed to determine the resistance of the luminal and peritubular cell membranes in parallel (R _m) and the apparent ratio of the luminal over the peritubular cell membrane resistance (VDR). Furthermore, the sensitivity of the potential difference across the peritubular cell membrane (PD_pt) to 6-fold increases of peritubular potassium concentration (PD_k) was taken as a measure of the relative potassium conductance of this membrane. In the absence of luminal phenylalanine, PD_pt amounts to –60±1 mV (n=90),R _m to 36±3 k cm (n=22), VDR to 1.81±0.14 (n=20), and PD_k to 15.0±0.9 mV (n=25). The application of 10 mmol/l phenylalanine replacing 10 mmol/l raffinose leads to a rapid (within 30 s) depolarisation of PD_pt to 50±5% of its control value and to a delayed (within 12 min) recovery to 95±5% of control. The rapid depolarisation is associated with a decline ofR _m and VDR, indicating a decrease mainly of the luminal cell membrane resistance. During recovery of PD_pt there is a parallel increase of VDR and a further decline ofR _m pointing to a decline of the basolateral cell membrane resistance. PD_k is decreased during rapid depolarisation but increases again during the recovery phase. Thus, phenylalanine initially decreases but then increases above control the apparent potassium conductance. Removal of phenylalanine leads to a transient hyperpolarisation and increased apparent potassium conductance. If a cell is depolarised by current injection into a neighbouring cell, a similar decrease of PD_k is observed which shows also a similar recovery (partial repolarisation) despite continued injection of constant current. The data point to a potential-dependent peritubular K⁺-conductance (of the inwardly rectifying type) and to a regulatory increase within some ten minutes, when the cell is depolarised either by sodium entry across the luminal cell membrane or by current injection into a neighbouring cell.     

Effects of ouabain on potassium transport in the perfused bullfrog kidney.

We studied the effect of ouabain on transepithelial and cellular potassium transport in the isolated perfused bullfrog kidney. We used recently developed techniques for estimating the unidirectional reabsorptive and secretory K fluxes (Jr and Js) and measuring the kinetics of cellular K transport. Two hours of perfusion with 1 X 10(-6) M ouabain did not affect GFR, reduced fractional Na reabsorption 57%, increased K excretion 41%, and inhibited Jr 34%. Js rose 68% at 60 min and then returned to the control level. Ninety minutes of perfusion with 5 X 10(-6) M ouabain reduced GFR 28% and fractional Na reabsorption 76%. K excretion rapidly increased 71% within 30 min and then fell to 60% of the control level, while Jr fell 64%. Js rose 42% in 30 min and then fell to 23% of the control level. Both doses reduced K uptake into cellular pools from the circulation and increased the rate coefficients for efflux into tubular fluid. The data indicate that ouabain inhibited reabsorption and transiently accelerated the rate of loss of K from the cells into the tubular fluid. This initially masked the ultimate inhibition of K secretion from the circulation into the tubular fluid.     

Correlation between fluid reabsorption and proximal tubule ultrastructure during development of the rat kidney

Parallel functional and ultrastructural studies were performed in maturing rats in order to elucidate factors determining the development of proximal tubular fluid reabsorption. Three groups of hydropenic animals, which were 22 to 24, 28 to 32 and 40 to 45 days old, were studied. Nephron function was evaluated at the single nephron level by micropuncture technique. The ultrastructure of the developing proximal tubules was analysed by morphometric techniques following fixation of single nephrons. Kidney weight, proximal convoluted tubule length and diameter increased during postnatal development. SNGFR increased from 2.98 to 8.57 and to 20.5 nl/min in respective group of rats whereas proximal tubular fluid reabsorption Jv (a) increased from 0.15 to 0.22 and 0.34 μm³μm^_2-s ^_1. Parallel to the functional development the relative area of lateral and basal cell membrane increased, resulting in a constant relationship between net fluid reabsorption and the lateral and basal cell membrane area during the fourth postnatal week and then only a slight increase in this relation during the further development. The results suggest that net fluid transport during hydropenia is determined by the amount of available lateral and basal cell membranes where the transporting enzyme for sodium is located.     

Peritubular buffering power and luminal acidification in proximal convoluted tubules of the rat

Proximal tubular acidification was studied varying peritubular buffer concentration as well as the nature of the main peritubular buffer system. Two buffer systems were used: phosphate which varied between 1 and 20 mM, and glycodiazine, at 5 and 20 mM. Luminal perfusate was always 20 mM phosphate Ringer's. Acidification half times increased as peritubular buffer concentration decreased, independently of the nature of the buffer. At 1 mM phosphate, net H-ion flux (JH +) was 0.53 nmol . cm-2 . s-1; at 5 mM it was 0.73 nmol . cm-2 . s-1 and at 20 mM, 0.97 nmol . cm-2 . s-1. When the peritubular buffer was glycodiazine, JH + was 0.77 nmol . cm-2 . s-1 at 5 mM peritubular buffer concentration and 0.99 nmol . cm-2 . s-1 at 20 mM. Acetazolamide (10(-4) M) and DIDS (10(-4) M) both abolished the effect of peritubular buffer concentration changes on acidification half times. It was shown that these effects were related to the capacity of the peritubular buffer to attenuate changes in peritubular pH as consequence of base transfer by the peritubular membrane. Peritubular buffering power could act limiting intracellular pH increments consequent to luminal H-ion secretion.     

Regulatory mechanism of cell pH in the renal proximal tubule of bullfrog nephron

To examine the cellular mechanism of urinary acidification in detail, micropuncture studies were performed on the in situ bullfrog proximal tubule with nigericin-based pH microelectrodes. Pencil-type double-barreled antimony microelectrodes were also used for monitoring pHs of the tubular fluids. Luminal perfusion of 10(-3) M cyanide caused a biphasic change in cell pH (pHi): i.e., early acidification by 0.04 pH unit in 2 min and later alkalinization by 0.04. A profound depolarization of 30-35 mV was observed in the peritubular membrane potential (EM Peri), although the tubular fluid pH (pHTF) was elevated by 0.11 unit. Luminal substitution of 100 mM Na+ by Li+ acidified the cell by 0.06 pH unit with a depolarization of EM Peri by 8 mV and an alkalinization of pHTF by 0.10 unit. It is a fact that cellular acidification and luminal alkalinization are in good agreement with the depression of luminal H+ secretory mechanism. Perfusion of 10(-4) M SITS from the peritubular side caused a rise in pHi by 0.04 without appreciable changes in EM Peri in the short period application. Peritubular perfusion of 10(-4) M ouabain lowered the pHi by 0.07 with a resulting depolarization of EM Peri by 15.4 mV, meanwhile, the pHTF, while initially lowered by 0.07 unit, was elevated 4 min later by 0.12. Inhibitions of the peritubular ion transport mechanism caused some pH changes in the same direction, both in the cell interior and the tubular fluid. Further, from the ouabain experiment, it is inferred that some linkages, mediated by Na+ and H+(or HCO3-), would exist between the peritubular and luminal membranes.     

Correlation between fluid reabsorption and proximal tubule ultrastructure during development of the rat kidney.

Parallel functional and ultrastructural studies were performed in maturing rats in order to elucidate factors determining the development of proximal tubular fluid reabsorption. Three groups of hydropenic animals, which were 22 to 24, 28 to 32 and 40 to 45 days old, were studied. Nephron function was evaluated at the single nephron level by micropuncture technique. The ultrastructure of the developing proximal tubules was analysed by morphometric techniques following fixation of single nephrons. Kidney weight, proximal convoluted tubule length and diameter increased during postnatal development. SNGFR increased from 2.98 to 8.57 and to 20.5 nl/min in respective group of rats whereas proximal tubular fluid reabsorption Jv (a) increased from 0.15 to 0.22 and 0.34 micron3.micron-2.s-1. Parallel to the functional development the relative area of lateral and basal cell membrane increased, resulting in a constant relationship between net fluid reabsorption and the lateral and basal cell membrane area during the fourth postnatal week and then only a slight increase in this relation during the further development. The results suggest that net fluid transport during hydropenia is determined by the amount of available lateral and basal cell membranes where the transporting enzyme for sodium is located.     

Biionic potentials in the proximal tubule of Necturus kidney

1. Biionic potentials were studied in the proximal tubule of the doubly perfused Necturus kidney and subsequently analysed by means of an equivalent electrical circuit.2. Luminal membrane resistance was found to be at least 3 times greater than peritubular membrane resistance.3. Potassium contribution to peritubular membrane conductance amounts to at least 75%. Replacement of chloride in peritubular circulation by benzene sulphonate or acetyl glycinate hyperpolarized peritubular membrane.4. The peritubular membrane is more permeable to choline than to sodium. Indirect evidence suggests that the opposite may apply at the luminal border.5. The shunt pathway discriminates poorly, if at all, between sodium, potassium and choline, in short term experiments.6. Chloride permeability across the shunt pathway is about three times as great as combined cationic permeabilities.     

The effect of phenylalanine on intracellular pH and sodium activity in proximal convoluted tubule cells of the frog kidney

The present study was performed to test the influence of sodium coupled transport of neutral substrates on intracellular pH and sodium activity in proximal tubules of the amphibian kidney. To this end, kidneys of rana esculenta have been isolated and perfused both through the portal vein (peritubular capillaries) and the aorta (luminal perfusate). The potential difference across the peritubular membrane of proximal tubule cells has been redorced with conventional (PD_pt) as well as with sodium (PD_na) and hydrogen ion (PD_h) selective microelectrodes continuously before during and after the luminal application of 10 mmol/l phenylalanine, replacing 10 mmol/l raffinose. PD_b and PD_na allowed the calculation of intracellular pH (pH_i) and sodium activity (Na_i), respectively. In the absence of phenylalanine in the tubule lumen, PD_pt approximates –57.5±2.3 mV (n=27), pH_i 7.73±0.04 (n=14, extracellular pH 7.77), and Na_i 13.3±0.9 mmol/l (n=13, extracellular sodium activity 74 mmol/l). Within 1 min the luminal application of phenylalanine leads to a depolarisation of PD_pt by +32±2 mV, as well as an increase of pH_i by 0.24±0.04 and of Na_i by 5.2±1.0 mmol/l. At 8 min from luminal application of phenylalanine, Na_i plateaus 5±1 mmol/l above control value, PD_pt increases again to a value of +12±2 mV below and pH_i decreases to a value 0.04±0.07 above their respective control values. All changes are fully reversed after removal of phenylalanine from the tubule lumen. The steady state of intracellular sodium activity might be explained by an extrusion of sodium via the sodium/potassium-ATPase, which approaches the entry across the luminal membrane, the intracellular alkalinisation is probably due to the reduced exit of bicarbonate across the peritubular cell membrane following the depolarisation of PD_pt.