F. Papavassiliou

Coupling between proximal tubular transport processes

SummaryThe rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (α-methyl-d-glycoside), H+ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (Δc) of these substances. In the case of calcium the electrochemical potential differenceΔc +zFciΔϕ/RT) was the criterion employed. The rate of isotonic Na+-absorption (JNa) 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-4′isothiocyanatostilbene 2,2′-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited JNa (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, α-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 JNa 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 HCO3− 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, α-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 HCO3− from the cell and secondary to that, the luminal H+-Na+ exchange and consequently the Na+ reabsorption. In the absence of HCO3− 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 onPi reabsorption observed under these conditions might be explained by possible variations in intracellular pH.

Bicarbonate reabsorption in the papillary collecting duct of rats

Using the technique of capillary perfusion and simultaneous luminal stop flow microperfusion the reabsorption of bicarbonate and glycodiazine from the papillary collecting duct was evaluated. Starting with equal H14CO3− and3H-glycodiazine concentrations in the luminal and peritubular perfusates, the decrease in the luminal concentration at 10 and 45 s contact time was measured.In control rats with 25 mmol/l HCO3− in the perfusates the rate of HCO3− reabsorption calculated from the 10 s values was 0.34 nmol cm−2s−1. In acute metabolic acidosis, the rate of bicarbonate reabsorption was 2,3 times higher. In metabolic alkalosis, the rate of bicarbonate absorption dropped to 13% of the control values. Also the 45 s values of acidotic and alkalotic animals differed significantly from each other. With 25 mmol/l glycodiazine in both perfusates the rate of biffer reabsorption as calculated from the 10 s values was 0.76 nmol cm−2s−1 in control rats and did not deviate significantly from this value in acidotic and alkalotic animals.In control rats the bicarbonate reabsorption in % was the same, no matter whether both luminal and capillary perfusate contained 25 mmol/l bicarbonate or 10 mmol/l. In acidotic rats the rate of HCO3− reabsorption did not change significantly if all Na+ in the perfusates was replaced by choline (0.88 versus 0.79 nmol cm−2s−1 at 25 mmol/l HCO3−). When in acidotic rats 0.1 mmol/l acetazolamide or 1 mmol/l SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid) was added to both perfusates the rate of HCO3− reabsorption dropped by 75 and 58%, respectively. A potassium deficient diet for one week and DOCA administration had no influence on the bicarbonate reabsorption of rats which were on standard diet.The data indicate that (1) the buffer reabsorption from the papillary collecting duct is rather due to H+ ion secretion than to buffer anion reabsorption. (2) The adaptation to metabolic acidosis and alkalosis is specific for bicarbonate and not seen with glycodiazine. (3) Within the concentration range tested the HCO3− reabsorption rises linearly with the HCO3t- concentration. (4) The HCO3− reabsorption in the papillary collecting duct is Na+-independent, it can be inhibited by acetazolamide and SITS, but is not influenced by K+-deficient diet plus DOCA.

Contraluminal transport of organic cations in the proximal tubule of the rat kidney

In order to study the characteristics of contraluminal organic cation transport from the blood site into proximal tubular cells the stopped-flow capillary perfusion method was applied. The disappearance of N1-[3H]methylnicotinamide (NMeN+) and [3H]tetraethylammonium (TEA+) at different concentrations and contact times was measured and the following parameters evaluated: Km,NMeN = 0.54 mmol/l, Jmax,NMeN = 0.4 pmol s−1 cm−1; Km,TEA = 0.16 mmol/l, Jmax,TEA = 0.8 pmol s−1 cm−1. TEA+ inhibited NMeN+ transport and NMeN+ the uptake of TEA+. Thereby, the Ki values for inhibition correspond closely to the Km values for uptake. Similar inhibitory potencies of ten organic cation against TEA+ and NMeN+ transport provide further evidence for a common transport system. Omission of HCO3−, or Na+ and addition of K+ (with or without Ba2+) reduce NMeN+ transport, while omission of K+ (with or without valinomycin) or addition of thiocyanate has no effect. Since the manoeuvres that depolarize contraluminal electrical potential difference reduce NMeN+ transport, cell-negative electrical potential difference is suggested as a driving force for contraluminal organic cation transport from the interstitium into the cell. Furthermore, the inhibitory potency (app. Ki values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquarternary ammonium compounds against NMeN+ transport was tested. The inhibitory potency increased in the sequence methyl < ethyl < propyl < butyl and primary < secondary < tertiary amines < quarternary ammonium compounds. With the amines a reversed correlation between Ki,NMeN and the octanol/water partition coefficient (log octanol) is seen. With quarternary ammonium compounds the inhibitory potency decreases with increasing molecular size: tetrabutyl- > tetrapentyl- > tetrahexyl- > tetraheptyl > tetraoctylammonium. Introducing two OH groups into triethylamine reduces the inhibitory potency while introduction of two OH groups into diethylamine or three OH groups into triethylamine abolishes the inhibitory potency as a result of reduced hydrophobicity. With choline (trimethylethanolamine) and its analogues the reversed correlation between Ki,NMeN and log octanol was also seen. Molecules with a similar hydrophobic moiety to those of the monoammonium compounds, but with two ammonium groups, showed only a small or no inhibitory potency against NMeN+ transport. The data indicate that (a) hydrophobic moieties are important for the interaction with the contraluminal organic cation transporter, and (b) the size of the molecule can be a limiting factor. The reduced or missing interaction of the bisquarternary compound might be caused either by the second charge and/or reduced hydrophobicity and/or too large size of a molecule.

Renal proximal tubular buffer-(glycodiazine) transport

SummaryUsing the stop flow microperfusion technique with simultaneous capillary perfusion the secretory rate of H+ ions in the proximal tubule was evaluated by measuring the level flow reabsorption as well as the static head concentration difference of3H labelled glycodiazine. At ambient glycodiazine concentration of 21 mmol/l the level flow reabsorption is in the same range as that of bicarbonate. In the early proximal loops the reabsorption is 20% greater than in the late proximal loops. The carbonic anhydrase inhibitors acetazolamide and 3,4-methylenedioxyphenyl-sulfonamide (both 10−4 M) as well as furosemide (10−3 M) inhibit the glycodiazine reabsorption 43%, 27% and 22% respectively. Thiocyanate (2 · 10−2 M), however, exerted only an insignificant inhibition (12%). When Na+ in the ambient perfusion solutions was replaced by Li+ or choline+ the glycodiazine transport was strongly reduced. Ouabain (5 · 10−2 M) inhibited too, but amiloride (10−3 M) had no effect on glycodiazine transport.The glycodiazine transport was 28% reduced in metabolic alkalosis and to a smaller although significant extent (17%) in metabolic acidosis; it was unchanged in chronic hypercapnia. In chronic K+ depletion the glycodiazine reabsorption was accelerated by 12% only in the early proximal loops. Chronic parathyroidectomy as well as acute substitution with parathyroid hormone had no effect on the glycodiazine absorption. The main conclusions are: Proximal H+ transport proceeds with suitable buffers. Although independent of HCO3− and carbonic anhydrase, it could be partially inhibited by CA inhibitors. H+ transport is supposed to proceed as countertransport with Na+ ions. In chronic alkalosis the H+ transport is reduced.

pH dependence of phosphate reabsorption in the proximal tubule of rat kidney

SummaryEarly loops of the proximal convoluted tubule of parathyroidectomized rats (PTX-rats) were microperfused with a phosphate (4 mM) containing perfusate. With a perfusion solution of pH around 7.45 as estimated as anion deficit thePireabsorption was two times greater than with a perfusion solution of pH around 6.85. ThePireabsorption is reduced in PTX-rats made chronic alkalotic (PTX-cA-rats) but the same pH dependence ofPireabsorption was found. The data indicate that the divalent phosphate is preferentially reabsorbed.

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney

AbstractUsing the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent Ki values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E2, and prostaglandin D2 was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. Ki, PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. Ki,PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N1-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [3H]cAMP and [3H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: Km=1.5 mmol/l, Jmax=0.34 pmol s−1 cm−1, r (extracellular/intracellular amount at steady state)=0.91; for cGMP: Km=0.29 mmol/l, Jmax=0.31 pmol s−1 cm−1, r=0.55. Comparison of app. Ki, cGMP with app. Ki, PAH of ten substrates gave a linear relation with a ratio of 1.83±0.5. All prostanoids applied inhibited the contraluminal PAH transport; the prostaglandins E1, F1α, A1, B1, E2, F2α, D2, A2 and B2 with an app. Ki, PAH between 0.08 and 0.18 mmol/l. The app. Ki of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1α (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. Ki, PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1α (0.77 mmol/l), and 2,3-dinor-6-keto-PGF1α (0.57 mmol/l) as well as that of thromboxane Bin2 (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB1, E2 and D2 inhibited contraluminal sulphate transport (app.

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

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A cytotoxin of pseudomonas aeruginosa acts directly on the cortical thick ascending limb of henle's loop of rabbit kidney

5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed Km=0.61 mmol/l and Jmax of 4.26 pmol s−1 cm−1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD2, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E2 a significant uptake through the sulphate transporter occurs in addition. The hypothesis that prostaglandins as well as 8-bromo and dibutyryl cyclic nucleotides permeate cell membranes by simple diffusion because of their lipid solubility must be considered with reservation.