Effect of acute metabolic acidosis on transmembrane electrolyte gradients in individual renal tubule cells

We studied the effect of acute metabolic acidosis on potassium, sodium and chloride gradients across the apical membrane of proximal and distal tubule cells by determining electrolyte concentrations in individual cells and in tubule fluid employing electron microprobe analysis. Cellular measurements were performed on freeze-dried cryosections of the renal cortex, analysis of tubule fluid electrolyte concentrations on freeze-dried microdroplets of micropuncture samples obtained from proximal and from early and late distal collection sites. Acidosis (NH₄Cl i.v. and i.g.) induced a substantial rise in plasma potassium concentration without significant effects on cell potassium concentrations. Potassium concentrations along the surface distal tubule were also unaltered; thus the chemical driving force for potassium exit from cell to lumen was not affected by acidosis. In all but intercalated cells acidosis markedly increased cell phosphorus concentration and cell dry weight indicating cell shrinkage and thus diminution of cell potassium content. Because the increase in intracellular chloride concentration exceeded the increase in plasma chloride concentration, the chemical chloride gradient across the contraluminal membrane was markedly depressed by acidosis.     

Effect of potassium adaptation on the distribution of potassium,sodium and chloride across the apical membrane of renal tubular cells

To assess the effect of K adaptation on the electrolyte concentrations of renal tubular cells and on the concentration gradients across the luminal membrane, electron microprobe analysis was employed on freeze-dried cryosections of the renal cortex and on freeze-dried samples of tubular fluid in control and high-K rats. The measurements were performed in individual cells of the proximal and superficial distal tubule and on samples of tubular fluid obtained by free flow micropuncture from proximal and early and late distal collection sites. The ingestion of a potassium-rich diet for at least 10 days together with an acute potassium load of 0.4 mmol/kg/h led to a small increase in potassium concentration of about 7 mmol/kg wet weight (w.w.) in all cell types analysed. In distal convoluted tubule, connecting tubule and principal cells sodium concentration was markedly decreased by 4, 4, and 6 mmol/kg w.w., respectively, while no significant changes in sodium concentration were found in proximal tubule and intercalated cells. No consistent changes in cell chloride could be observed under K adaptation. Analysis of the tubular fluid samples showed that the K concentration gradient across the apical cell membrane of all distal tubular cell types investigated was diminished in the high-K rats. The concentration gradient for sodium entry, however, was clearly enhanced in the distal convoluted tubule, connecting tubule and principal cells. These data suggest that an increment in cell potassium concentration is not a major functional determinant for the increased distal K secretion observed in high-K rats and that the enhanced distal sodium absorption under this condition may be due to a stimulation of the Na exit step across the basolateral cell membrane in excess of the luminal entry step in most distal tubular cell types.     

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.     

Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct

Transmembrane sodium transport pathways were studied in principal and intercalated cells of the isolated perfused rabbit cortical collecting duct. Intracellular electrolyte concentrations in individual collecting duct cells were measured by electron microprobe analysis during blockage of basolateral Na-K-ATPase by ouabain and simultaneous inhibition of sodium entry across the apical and/or basolateral cell membrane. In principal cells the ouabain-induced rise in cell sodium concentration could only partially be blocked by amiloride (10^–4mol/l) in the perfusion fluid. Amiloride (10^–3mol/l) added to the bathing solution produced a further, significant reduction of sodium influx. In principal cells the ouabain-induced increase in sodium concentration was completely prevented by amiloride in the perfusion solution in combination with omission of sodium from the peritubular bathing solution. In intercalated cells ouabain caused a less pronounced increase in sodium concentration than in principal cells. Neither amiloride in the perfusate, nor amiloride in both bathing and perfusion solution, significantly reduced the ouabain-induced rise in intercalated cell sodium concentration. These results indicate that in principal cells amiloride-sensitive sodium channels constitute the predominant pathway for sodium entry across the apical cell membrane. In addition, substantial amounts of sodium enter principal cells across the basolateral cell membrane, probably via Na-H exchange. Finally, the data suggest that in intercalated cells sodium channels and the Na-H exchange are sparse or even absent.     

Electron microprobe analysis of proximal tubule cellular Na,Cl and K element concentrations during acute mannitol-saline volume expansion in rats: evidence for inhibition of the Na pump

It has previously been shown that during mannitol-saline volume expansion (VE) Na transport was inhibited 50% by harvested proximal tubular fluid without a change in paracellular shunt pathway permeability to Na. To determine whether this inhibition was due to changes in cellular entry step or an effect on the pump itself, intracellular element concentrations were measured by electron microprobe X-ray ranalysis in proximal tubular cells of control (non-expanded, NE) and VE rats. Na _i , Cl _i and phosphorus _i were increased (mean±S.E.) from 19.3±0.8 to 23.4±0.6, 15.8±0.4 to 21.3±0.4 and 124.3±2.6 to 138.0±1.8 mmol · kg^–1 wet weight (P<0.001) respectively while K _i remained unchanged: 122.9±2.2 and 124.2±1.3 mmol · kg^–1 wet weight. The increases in Na _i and Cl _i were in excess of cell shrinkage produced by the hyperosmolal peritubular environment while the unchanged K _i in the face of cell shrinkage indicates and actual loss. It is concluded that mannitol-saline VE inhibits the Na pump producing a rise in Na _i and a fall in K _i .