Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and Pi. In rat, biliary Piconcentration (0.01 mM) is ∼100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary Pi. We aimed to functionally characterize canalicular Pireabsorption in rat liver and to identify the involved Pitransport system(s). Pitransport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative Pitransporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent Piuptake. Initial Piuptake rates were saturable with increasing Piconcentrations, exhibiting an apparent Kmvalue of ∼11 μM. Pitransport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of Pifrom primary hepatic bile and thus might play an important role in the regulation of biliary Piconcentration.

The Retention of Amino Acids in the Haemolymph During Diuresis in Rhodnius

The haemolymph of Rhodnius is rich in amino acids. During the rapid diuresis after a blood meal, no more than trace amounts of amino acids are lost in the urine. There is no significant reabsorption of amino acids in the excretory system. That they escape elimination can instead be attributed to a combination of the low permeability of the Malpighian tubules to amino acids, the very high rate of fluid secretion by the tubules, and the dilution of the haemolymph by an expansion in its volume after feeding. Amino acid losses are low in spite of the fact that the tubules actively accumulate high concentrations of amino acids in their cells and passive losses from these stores augment to some extent the flux of amino acids into the lumen. At times other than during diuresis, fluid secretion by the Malpighian tubules is slow. Calculations show that haemolymph solutes can then passively reach the higher concentrations in the lumen that are required for the operation of the excretory system (which relies on unselective passive entry and active reabsorption of useful substances). An advantage of the extraordinarily high rate of fluid secretion during diuresis is that fluid excretion can be rapidly completed. There is then little time for significant amounts of haemolymph solute to be lost passively.

Prolylhydroxyproline Absorption in Hamsters

The intestinal transport of the dipeptide prolylhydroxyproline was investigated using hamsters and segments of hamster small intestines. The feeding of prolylhydroxyproline to hamsters resulted in the urinary excretion of 2% of the ingested dipeptide after 3 h or 7% after 24 h. In vitro perfusions of prolylhydroxyproline through the hamster small intestine resulted in the movement of the dipeptide across the intestinal wall. The rate of absorption was proportional to the concentration of prolylhydroxyproline on the mucosal side (0.17 μmol Pro∙Hyp/mM in lumen/g tissue/h). Anoxia, 2,4-dinitrophenol, cyanide, or an excess of proline or hydroxyproline did not change this absorption rate. These results suggest that prolylhydroxyproline is absorbed by "simple passive diffusion", that it reaches the site of intestinal absorption because of its relative resistance to enzymatic hydrolysis, and that it is excreted by glomerular filtration without subsequent active reabsorption.

Nitrate, thiocyanate, and perchlorate clearance in relation to chloride clearance

Simultaneous clearances of inulin, chloride, and nitrate, thiocyanate, or perchlorate were measured in salt-depleted dogs and/or dogs undergoing diuresis induced by mannitol, saline, or sulfate. A method for perchlorate determination in body fluids using methylene blue is given. Plots of excreted/filtered NO3, SCN, or ClO4 against excreted/filtered Cl were made for the present data and for data taken from the literature. All of the available nitrate data conform to a power function between these two variables with an exponent of .36–.38. Thiocyanate clearance was nearly equal to chloride clearance; during infusion of other foreign anions it was usually higher than chloride clearance. Perchlorate clearance was much higher than chloride clearance but varied with it. It is concluded that all of these anions may be reabsorbed passively and noncompetitively in the same portions of the nephron; distal active reabsorption may also play a role.

THE PROXIMAL RENAL TUBULAR TRANSPORT OF α-KETOGLUTARIC ACID

The transport of α-ketoglutaric acid along the length of the renal tubule was studied by the stop-flow method in the dog during infusion of sodium α-ketoglutarate. No deliberate attempts to change the acid–base balance were made. Under these conditions this acid was found to be excreted by a combined process of glomerular filtration and tubular reabsorption. This reabsorption was confined only to the proximal tubule. No tubular secretion was seen in any part of the nephron. The concentration gradients between the luminal urine, cell water and the plasma were measured. It was seen that the gradients between the lumen and the cell and between the lumen and the plasma were against the direction of the transport. The gradient between the plasma and the cell was favorable to the uptake of the acid by the cell. Upon consideration of these gradients, the spontaneous pH changes and the physicochemical characteristics of the molecule, the hypothesis was forwarded that an active reabsorption of α-ketoglutarate must occur in the proximal tubule from the lumen into the cell. The possibility of active transfer of this acid from the blood into the cell was also suggested.

THE PROXIMAL RENAL TUBULAR TRANSPORT OF α-KETOGLUTARIC ACID

The transport of α-ketoglutaric acid along the length of the renal tubule was studied by the stop-flow method in the dog during infusion of sodium α-ketoglutarate. No deliberate attempts to change the acid–base balance were made. Under these conditions this acid was found to be excreted by a combined process of glomerular filtration and tubular reabsorption. This reabsorption was confined only to the proximal tubule. No tubular secretion was seen in any part of the nephron. The concentration gradients between the luminal urine, cell water and the plasma were measured. It was seen that the gradients between the lumen and the cell and between the lumen and the plasma were against the direction of the transport. The gradient between the plasma and the cell was favorable to the uptake of the acid by the cell. Upon consideration of these gradients, the spontaneous pH changes and the physicochemical characteristics of the molecule, the hypothesis was forwarded that an active reabsorption of α-ketoglutarate must occur in the proximal tubule from the lumen into the cell. The possibility of active transfer of this acid from the blood into the cell was also suggested.

Reabsorption and secretion of ι-malic acid in kidney proximal tubule

The ι-malic excretion pattern was studied in the dog using the "stop flow" method. During ι-malic and fumaric infusions, a net reabsorption of ι-malic acid was seen only in the proximal tubule. This reabsorption occurred against a massive concentration gradient and, therefore, was justifiably termed active reabsorption. Infusion of succinate produced a net secretion of ι-malic. This was also confined to the proximal tubule. The secretion, however, occurred in the direction of the concentration gradient and, therefore, was probably a diffusion phenomenon. The secretion could be inhibited by malonate. It was concluded that the renal transports of ι-malic acid are intimately related to the operation of the tricarboxylic acid cycle.

Magnesium excretion in the dog studied by stop-flow analysis

Stop-flow analysis performed in the dog using Mg-28 demonstrated active reabsorption of magnesium in the proximal portion of the distal nephron. ‘Interrupted’ stop-flow analysis failed to document the existence of a renal tubular secretory mechanism for magnesium. During magnesium-loading reabsorption of potassium occurs in the distal nephron despite the presence of hyperkalemia.

Localization of renal calcium transport; effect of calcium loads and of gluconate anion on water, sodium and potassium

We have performed a first series of experiments using our stop flow technique to localize renal transport of calcium in dogs. Calcium is found to be actively reabsorbed in a far distal area but does not appear to be actively lowered in concentration in proximally derived samples. In a second series of dogs, elevation of plasma calcium by infusion of the salt CaCl2 caused a 43% reduction in the additional volume of water reabsorbed by the proximal tubule during stop flow. Calcium does not interfere with the active reabsorption of sodium in the distal area and does not cause significant increases in sodium excretion. In contrast calcium causes a considerable increase in potassium secretion at a far distal site. When calcium gluconate was employed, severe impairment of distal sodium and calcium reabsorption occurred in addition to the increased potassium secretion. We suggest that the gluconate anion restricts the movement of sodium, calcium and potassium electrostatically thus obligating cations to remain in the tubular urine depending upon their reabsorption potential.