Rat Proximal Convoluted Tubule Research Articles

Insulin-induced stimulation of Na+,K(+)-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha-subunit at Tyr-10.

Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Protein kinase C-dependent stimulation of Na(+)-K(+)-ATP epsilon in rat proximal convoluted tubules.

In rat proximal convoluted tubule (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) was previously reported to inhibit Na(+)-K(+)-ATPase, a paradoxical finding in view of the known stimulatory effect of PKC on Na+ reabsorption. Because this inhibition occurs via phospholipase A2 activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on PKC action on Na(+)-K(+)-ATPase. Results confirmed that PDBu inhibited PCT Na(+)-K(+)-ATPase activity under usual conditions. In contrast, when oxygen supply was increased, PDBu had no effect on Na(+)-K(+)-ATPase hydrolytic activity, but it dose-dependently stimulated ouabain-sensitive 86Rb+ uptake. This latter effect, which was abolished by PKC inhibitors, resulted from an increment of the Na+ sensitivity of Na(+)-K(+)-ATPase. Thus, in oxygenated rat PCTs, activation of PKC primarily stimulated Na(+)-K(+)-ATPase. This likely contributes to increase solute reabsorption. Inhibition of Na(+)-K(+)-ATPase was observed only under hypoxic conditions. It may represent an adaptation to protect PCTs against deleterious effects of hypoxia.

Effects of barium and 5-(N-ethyl-N-isopropyl)-amiloride on proximal tubule ammonia transport

Ionic NH+4 transport is an important mode of ammonia transport in the proximal convoluted tubule (PCT). NH+4 transport via the Na-H exchanger has been previously demonstrated. Potassium channels are present in the proximal tubule, but their role in ammonia transport has not been evaluated. We studied rat PCTs perfused in situ at 30 nl/min with solutions containing 5 mM HCO3; ammonia was measured with a fluorometric method. When perfused with the potent amiloride analogue 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 500 microM) or with BaCl2 (10 mM), ammonia entry (14.3 +/- 1.7 and 11.8 +/- 1.5 pmol.min-1.mm-1, respectively) was unaffected compared with control (12.8 +/- 1.0 pmol.min-1.mm-1). However, the combination of EIPA and barium inhibited entry (7.4 +/- 1.0 pmol.min-1.mm-1, P less than 0.02 vs. other groups). Also, when perfused with 10 mM ammonia, neither EIPA nor BaCl alone blocked ammonia loss (70.5 +/- 9.1 and 60.5 +/- 5.9 pmol.min-1.mm-1, respectively) compared with control (53.4 +/- 6.0 pmol.min-1.mm-1). However, the combination inhibited ammonia loss (29.2 +/- 6.3 pmol.min-1.mm-1, P less than 0.025 vs. other groups). Thus blocking both the Na-H exchanger and potassium channels decreases PCT ammonia transport. As the combination was required, this implies that multiple pathways exist for NH+4 transport in the PCT. This is the first demonstration that a mode of NH+4 transport other than via the Na-H exchanger is important in this segment, and the data are most consistent with transport of ammonia via potassium channels.

Dopamine-1 receptors in rat proximal convoluted tubule: regulation by intrarenal dopamine

Dopamine (DA) is synthesized in the renal proximal convoluted tubule (PCT) and may act as a paracrine substance at tubular DA-1 receptors to decrease sodium transport. Although DA-1 receptors have been identified in rabbit renal PCT by use of nonselective dopaminergic radioligands, DA-1 receptors have not been localized in specific nephron segments with the use of selective DA-1 radioligands. In these studies we used a novel DA-1 dopaminergic ligand 125I-SCH-23982, which has been shown to have a high affinity for brain and renal DA-1 receptors, to identify DA-1 receptors in the rat renal PCT and distal convoluted tubule (DCT). DA-1 receptors in the microdissected PCT and DCT were studied by a quantitative autoradiographic technique and by measuring adenylate cyclase (AC) activity. No specific 125I-SCH-23982 binding could be measured in the DCT indicating the absence of DA-1 receptors in this segment. Binding of 125I-SCH-23982 to PCT was saturable with time and radioligand concentration and was stereoselective. Saturation isotherm analysis in control rats yielded a dissociation constant (Kd) of 7.5 +/- 0.14 nM (n = 4) and a maximum receptor density (Bmax) of 0.69 +/- 0.04 pmol/mg protein (n = 4). The rank-order potency for agonist and antagonist displacement of 125I-SCH-23982 binding was consistent for DA-1 receptors: SCH-23390 greater than fenoldopam = SKF 38393 greater than SCH-23388. The stimulatory effect of the DA-1 agonist fenoldopam (10 microM) on AC activity was blocked by the DA-1 antagonist SCH-23390 (10 microM) but not by the beta-adrenergic antagonist (-)-propranolol (10 microM), indicating specificity. The DA-beta-hydroxylase blocker, SKF 102698, increased renal DA concentrations threefold, reduced the PCT DA-1 receptor Bmax by 33%, and abolished the stimulatory effect of 10 microM fenoldopam on AC activity in the PCT but had no effect on Kd. It is concluded that DA-1 receptors are present in rat PCT but not DCT and can be regulated by renal DA.

Angiotensin II stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate.

These studies explored the hypothesis that angiotensin II increases bicarbonate absorption in the proximal convoluted tubule (PCT) by decreasing intracellular cAMP. In vivo microperfusion was performed in rat PCT with measurements of bicarbonate absorption and of tubular fluid cAMP delivery, as a reflection of intracellular cAMP. Intravenous angiotensin II potently increased S1 PCT bicarbonate absorption (348 +/- 11 to 588 +/- 8 peq/min.min, P less than 0.001) and decreased tubular fluid cAMP (18 +/- 2 to 12 +/- 2 fmol/mm.min, P less than 0.05). Parathyroid hormone had the expected opposite effects, which were additive to those of angiotensin II. Over a wide range of hormonal activities, there was an excellent inverse relationship between hormonally modulated bicarbonate absorption and cAMP delivery. Pertussis toxin pretreatment significantly attenuated (by 35-45%) the angiotensin-induced increase in bicarbonate absorption and decrease in cAMP delivery, indicating Gi-protein intermediation. Luminal dibutyryl cAMP abolished the transport response to angiotensin II. In conclusion, these in vivo results suggest angiotensin II stimulates bicarbonate absorption in the S1 PCT by a G1-mediated depression in intracellular cAMP.

Axial heterogeneity of intracellular pH in rat proximal convoluted tubule.

In the proximal convoluted tubule (PT), the HCO3- reabsorptive rate is higher in early (EPS) compared with late proximal segments (LPS). To examine the mechanism of this HCO3- reabsorption profile, intracellular pH (pHi) was measured along the superficial PT of the rat under free-flow and stationary microperfusion using the pH-sensitive fluorescence of 4-methylumbelliferone (4MU). With 4MU superfusion, pHi was found to decline along the PT. Observation with 365-nm excitation revealed that EPS were brightly fluorescent and always emerged away from their star vessel. Midproximal segments were darker and closer to the star vessel which was surrounded by the darkest LPS. Decreasing luminal HCO3- from 15 to 0 mM lowered pHi in both EPS and LPS, but pHi remained more alkaline in EPS with both perfusates. Thus the axial decline in pHi along the PT is due to both luminal factors and intrinsic differences in luminal H+ extrusion in PT cells.

Dopamine causes inhibition of Na+-K+-ATPase activity in rat proximal convoluted tubule segments.

We studied the effect of dopamine (DA) on Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments dissected from perfused rat kidneys. DA inhibited Na+-K+-ATPase activity in a dose-dependent manner. Inhibition was significant with 10(-7) M DA and maximal with 10(-4) M DA. The inhibition was reversible. Enzyme inhibition occurred in the presence of DA and a DA antagonist, metoclopramide, but not when 10(5) M of the DA1 and DA2 agonists fenoldopam mesylate and LY 171555 were added in the absence of DA. In PCT segments incubated with the DA precursor dopa, Na+-K+-ATPase activity was also inhibited. However, dopa did not inhibit the sodium pump if dopa decarboxylase activity was blocked with benserazide. These findings suggest an intracellular site of action of DA. In tubules incubated in different K concentrations, 10(-5) DA decreased the maximal activity (Vmax) and increased the Km. DA 10(-5) M caused an almost immediate swelling of PCT segments. Swelling did not occur in the presence of both DA and 10(-5) M amiloride. The DA-induced tubular swelling was probably due to inhibition of Na+-K+-ATPase-mediated Na+-transport. We conclude that in rat PCT segments, DA causes a rapid and reversible inhibition of apparent Na+-K+-ATPase activity and an apparent reduction in the affinity for K. The site of action appears to be intracellular.