Joachim Greven

Effects of dopamine on whole kidney function and proximal transtubular volume fluxes in the rat

SummaryThe renal effects of dopamine were studied using clearance and micropuncture techniques in rats. Intravenous infusion of dopamine (4.7 · 10−6 mol · kg−1 · h−1) increased glomerular filtration rate and renal blood flow. Renal blood flow was measured by an electromagnetic flowmeter. The increase in filtered fluid and sodium was nearly completely matched by increased tubular reabsorption. Thus, only a small rise in urine flow and in urinary sodium excretion was observed.The micropuncture experiments using the split oil droplet method of Gertz demonstrated a stimulation of the transepithelial fluid transfer after injection of dopamine (10−4M) into the proximal tubular lumen. This effect was abolished by simultaneous injection of propranolol (10−3 M) which, by its own, did not affect transtubular volume fluxes. It is concluded that dopamine, by stimulation of β-adrenoceptors, may increase reabsorptive capacity of the proximal tubular epithelium independent of changes in renal hemodynamics.

Basolateral transport of glutarate in proximal S2 segments of rabbit kidney: kinetics of the uptake process and effect of activators of protein kinase A and C.

Abstractu2002The kinetics of tubular glutarate uptake, the coupling of glutarate to p-aminohippurate (PAH) transport and the effect of activators of protein kinase A and C on glutarate uptake were studied using isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of [14C]glutarate reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30xa0s glutarate uptake measurements were performed. In a first set of experiments it could be shown that preloading proximal S2 segments with glutarate (10–3xa0M) stimulated [3H]PAH uptake indicating that glutarate may be a substrate of the PAH /dicarboxylate exchanger. The kinetic data revealed a Km value of 0.62xa0mM and a Vmax value of 84.1xa0pmol nl–1min–1 for tubular [14C]glutarate uptake across the basolateral cell membrane. In contrast to basolateral PAH transport (previous studies from this laboratory), tubular 30xa0s [14C]glutarate uptake was not affected by either the phorbol ester phorbol 12-myristate 13-acetate (PMA, 10–7xa0M), an activator of protein kinase C, or by the membrane-permeant analogues of cAMP, dibutyryl cyclic AMP (db-cAMP, 10–4xa0M) and 8-bromoadenosine 3′,5′-cyclic monophosphate (Br-cAMP, 10–4xa0M). The results indicate that the protein kinases A and C have no function in the regulation of the renal basolateral dicarboxylate transporter. This finding agrees well with the structural feature of the recently cloned rabbit renal dicarboxylate transporter which does not contain any putative phosphorylation sites for protein kinase C or cAMP-dependent kinase.

Role of the calcium/calmodulin-dependent protein kinase II in the regulation of the renal basolateral PAH and dicarboxylate transporters.

Abstract— The aim of the present study was to investigate whether the activities of the renal basolateral organic anion transporter (PAH transporter) and the sodium‐dependent dicarboxylate transporter are modulated by the calcium/calmodulin‐dependent multifunctional protein kinase II (CaM kinase II). The studies were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H‐PAH was used as marker substance of the anion transporter, and 14C‐glutarate as a marker of the sodium/dicarboxylate cotransporter. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of the marker substances reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s tubular uptake measurements were performed. The results show that a selective inhibitor of CaM kinase II, KN93, inhibited tubular PAH uptake. The smallest effective dose was 10−7 M. An inactive analogue of KN93, KN92, was without effect, even at the high concentration of 105 M. In contrast to PAH transport, tubular 14C‐glutarate uptake was not affected by KN93 (10−5 M). PAH transport was also inhibited after elevation of intracellular Ca2+ by the Ca2+‐ionophore A 23187 and by the polycationic antibiotic neomycin, but not by the intracellular Ca2+ modulators thapsigargin and ryanodine. The effect of the Ca2+‐ionophore could be abolished by KN93, but not by Rp‐cAMPs, an inhibitor of protein kinase A, indicating that this event was mediated by CaM kinase II, but not by PKA. The results provide the first evidence that, in addition to the protein kinases A and C (previous studies from this lab), CaM kinase II has a role in the regulation of the renal basolateral PAH transporter, whereas the renal basolateral dicarboxylate transporter does not depend on CaM kinase II activity.

Effect of activation of protein kinase A and of protein kinase C on the kinetics of the renal basolateral PAH transporter.

Summary— The aim of the present study was to examine the effect of activation of the protein kinase A (PKA) and protein kinase C (PKC) pathways on 3H‐p‐aminohippurate (PAH) uptake of isolated S2 segments of proximal tubules, microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of 3H‐PAH reflects transport across the basolateral membrane. The phorbol ester phorbol 12‐myristate 13‐acetate (PMA) (10 7 M), an activator of PKC, significantly increased tubular 3H‐PAH uptake with steady state conditions (by 115%), whereas dibutyryl cyclic adenosine monophosphate (db‐cAMP) (10‐4 M) and forskolin (10‐4 M) significantly inhibited it (by 42% and 52%, respectively). Kinetic data, which were based on 15 sec PAH uptake measurements, revealed that PMA, after a 10 min incubation period, significantly enhanced Km and Vmax of the PAH transporter (Km from 174 ± 22 to 447 ± 91 μM, Vmax from 2.76 ± 0.24 to 16.67 ± 1.85 pmol nL‐1 min‐1), whereas db‐cAMP significantly decreased Vmax (from 2.76 ± 0.24 to 1.82 ± 0.19 pmol nL‐1 min‐1). The Km value was also numerically lowered by dibutyryl‐cAMP (from 174 ± 22 to 139 ± 21 μM), but this change did not reach statistical significance. The data provide evidence that short time activation of the PKC pathway 1) enhances the effectiveness of PAH transport into proximal S2 segments across the basolateral cell membrane, 2) increases the maximum transport rate of the PAH transporter and 3) decreases its affinity for PAH. Activation of the cAMP/PKA pathway induces the opposite effects.

Regulation of Taurocholate Transport in Freshly Isolated Proximal Tubular Cells of the Rat Kidney by Protein Kinases

Background/Aims: The bile acids filtered through the glomeruli nearly completely escape urinary excretion due to an efficient tubular reabsorption process. Reabsorption is mediated mainly by the sodium-dependent bile acid transporter (ASBT) which is located in the brush border membranes of proximal tubular cells. The present study addresses the question whether this transporter is subject to short-term regulation by protein kinases. Methods: The effects of specific activators or inhibitors of eight different protein kinases (PKs) on 3H-taurocholate uptake of proximal tubular cells were investigated. The cells were freshly isolated from rat kidneys by nycodenz density gradient centrifugation. Results: Activation of the cAMP/PKA system by forskolin, 8-Br-cAMP, or the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine significantly diminished cellular 3H-taurocholate uptake whereas 8-Br-cGMP had no effect. Also the MEK1/2 inhibitors PD98059 and U0126, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 decreased 3H-taurocholate uptake. Phorbol myristate acetate and dioctanolglycerol, activators of PKC, and chelerythrine, a selective inhibitor of PKC, did not affect 3H-taurocholate uptake. Likewise the phosphatidylinositol-3 kinase inhibitor wortmannin and the tyrosine kinase inhibitor genistein induced no significant change of cellular 3H-taurocholate uptake. In a sodium-free medium forskolin and PD98059 did not affect 3H-taurocholate uptake but SB203580 significantly decreased it. Conclusion: It is concluded that PKA and MAP kinases are involved in the regulation of the ASBT-mediated taurocholate uptake into proximal tubular cells. p38 MAP kinase may have an additional effect on a sodium-independent tubular taurocholate transporter.

Site of action of moxonidine in the rat nephron

Abstract. Moxonidine is a centrally acting antihypertensive agent which has been found to exert its blood pressure lowering effect by interaction with α2-adrenoceptors and imidazoline receptors of the I1-type. These receptors have also been demonstrated to be present in the rat kidney. In the present study, clearance and micropuncture techniques were applied to anaesthetized rats to localize the site of action of moxonidine within the nephron. The clearance data show that moxonidine (0.25xa0mg/kg i.v., followed by a continuous i.v. infusion of 0.25xa0mg/h) induced a marked increase in urine flow and urinary excretion of sodium, chloride and potassium. The changes in urine flow and urinary solute excretion were accompanied by an enhanced glomerular filtration rate. The micropuncture experiments revealed that moxonidine significantly increased glomerular filtration rate of superficial nephrons, and significantly inhibited fractional reabsorption of fluid, sodium, potassium and chloride by similar amounts (by 9.0%–9.8%) in superficial proximal tubules. Regarding fluid and sodium reabsorption, the proximal effect of moxonidine was continuously weakened by a compensatory increase of reabsorption in the loop of Henle and the subsequent distal nephron segments. The inhibitory effect of moxonidine on fractional proximal potassium reabsorption was completely compensated in the loop of Henle, but the drug induced a net secretion of potassium into the segments lying beyond the early distal tubule, probably as a consequence of the increased tubule fluid and sodium load delivered to them. The experiments have identified the proximal tubule as the principal nephron site where the diuretic action of moxonidine arises. The proximal effect may be related to the increased glomerular filtration rate and to a direct inhibitory interaction of moxonidine with the proximal Na+/H+ exchanger.

Potassium-Sparing Renal Effects of Trimethoprim and Structural Analogues

Background/Aims: The antibiotic antagonists of folic acid trimethoprim, tetroxoprim, pyrimethamine and their antineoplastic analogue methotrexate have structural characteristics in common with the potassium-sparing diuretic triamterene. They may, therefore, share with triamterne potassium-sparing renal effects. Methods: Clearance studies were performed on anesthetized male Sprague-Dawley rats, and the drug effects on glomerular filtration rate and on urinary excretion of sodium, chloride, and potassium were studied. Results: Trimethoprim and tetroxoprim, injected intravenously at doses ranging from 0.3 to 30 mg/kg, induced dose-dependent natriuretic and antikaliuretic renal effects, whereas pyrimethamine at doses ranging from 1 to 10 mg/kg and methotrexate at doses ranging from 10 to 50 mg/kg did not affect urinary sodium and potassium excretion. An antikaliuretic effect was also observed after application of the structurally related antiprotozoal compound pentamidine at doses ranging from 3 to 10 mg/kg. In contrast to trimethoprim and tetroxoprim, the antikaliuresis was accompanied by a marked decrease of urinary sodium and chloride excretion at all of the doses tested. At 10 mg/kg, pentamidine induced a pronounced fall of the glomerular filtration rate (by 43.5%). Conclusions: Trimethoprim and tetroxoprim share with potassium-sparing diuretics natriuretic and antikaliuretic renal effects which may be caused by similar mechanisms in the distal nephron, whereas pyrimthamine and methotrexate do not. A depression of renal hemodynamics is an important factor involved in the antikaliuretic effect of pentamidine.

Involvement of protein phosphatases in differential regulation of renal proximal tubular PAH and sodium-dependent dicarboxylate transport*

Abstract— It has been demonstrated that the basolateral organic anion (PAH) transporter and the sodium‐dependent dicarboxylate transporter of rabbit renal proximal tubules are regulated differentially. A variety of protein kinases has been shown to be involved in the regulation of organic anion transport while dicarboxylate uptake, to which the first is coupled functionally, is not inlluenced by these kinases. This study was undertaken to elucidate whether respective transporter activities are modulated differentially by protein phosphatases as well. The experiments were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H‐PAH was used as marker substance of the PAH transporter. 14C‐glularate as a marker of the sodium/dicarboxylate cotransporter. 30 s tubular uptake measurements were performed. Vanadate (10−3M). a selective inhibitor of tyrosine phosphatase, did not reduce PAH uptake significantly, while inhibitors of the serine/threonine phosphatases I and 2A. okadaic acid and calyculin A (10 6M. each) induced a significant decrease of 30 s PAH uptake (by 32.3% ± 7.9% and 25.6% ± 6.4%) but not a change in dicarboxylate transport. These findings indicate that, in addition to a variety of protein kinases, serine/threonine phosphatases have a role in the regulation of renal basolateral PAH transport. There is no effect of these phosphatases on basolateral 30s glularate transport. Thus, additional evidence for differential regulation of short‐time activity of the transporters for PAH and dicarboxylales is provided.

Renal effects of mannitol in the early stage of glycerol-induced acute renal failure in the rat.

The effects of mannitol on the hemodynamics and excretory function of the kidney in control rats and in rats with acute renal failure (ARF), induced by glycerol, were studied using clearance, micropuncture and flowmeter techniques. In control animals, 20% mannitol significantly increased renal blood flow (RBF), urinary volume and sodium excretion and decreased urinary sodium concentration and fractional tubular sodium reabsorption. Glomerular filtration rate (GFR) and effective filtration pressure remained unchanged. 50% glycerol (10 ml/kg bw) was injected intramuscularly in other groups of rats to induce ARF. Functional impairment of the glycerol-treated animals consisted in a significant decrease in urinary sodium concentration, RBF, GFR and effective filtration pressure of superficial nephrons. Urea concentration in arterial plasma and urine volume increased significantly. Infusion of 20% mannitol into these animals led to a significant further decrease in GFR and net filtration pressure, but not in RBF. Urine volume increased to the same degree as in controls.

Hydrostatische Drucke in proximalen und distalen Konvoluten und in peritubulären Capillaren von Rattennieren nach Furosemid und Acetazolamid

SummaryHydrostatic pressures were measured in proximal and distal tubules and in peritubular capillaries before and after the administration of acetazolamide and furosemide. In controls, proximal pressure averaged 11.5 mm Hg and distal pressure 4.9 mm Hg. The pressures rose after acetazolamide to 13.4 mm Hg in proximal and to 10.8 mm Hg in distal tubules. The difference between proximal and distal pressures was significant in both, controls and after acetazolamide. After furosemide the intratubular pressure increased in the proximal tubule to 32.7 mm Hg and in the distal tubule to 29.5 mm Hg, the difference being not significant.The correlation between the proximal and distal intratubular pressures and urine volume was linear and significant after both acetazolamide and furosemide. The increase of intratubular pressures relative to the urine volume is less when the renal capsula was removed. The quotient, intratubular pressure/urine volume is higher some 10 min after the injection of furosemide than subsequently. The pressure in peritubular capillaries was greater than the pressure in proximal convolutions in controls but was found to be lower than intratubular pressure after furosemide.It is assumed that the flow resistance of the loop of Henle is reduced by increasing urine volume whereas intratubular flow is hindered by a resistance at the distal end of the nephron which increases both intratubular pressures and tubular diameters.ZusammenfassungAn Rattennieren wurden hydrostatische Drucke in proximalen und distalen Tubuli und in peritubulären Capillaren unter Kontrollbedingungen und nach Gabe von Acetazolamid und Furosemid gemessen. Die Kontrollwerte betrugen 11,5 mm Hg proximal und 4,9 mm Hg distal. Die Drucke stiegen nach Acetazolamid proximal auf 13,4 mm Hg und distal auf 10,8 mm Hg. Der Unterschied zwischen proximalen und distalen Drucken war auch nach Acetazolamid signifikant. Nach Furosemid nahm der proximale Druck im Mittel auf 32,7 mm Hg zu, der distale auf 29,5, die Differenz zwischen beiden war nicht mehr signifikant.Es bestand eine signifikante lineare Korrelation zwischen Drucken in proximalen und distalen Tubuli und Harnvolumen sowohl nach Acetazolamid als auch nach Furosemid. Der Druckanstieg bei zunehmendem Harnvolumen ist nach Entfernen der Nierenkapsel relativ geringer. Er ist im Vergleich zum Harnvolumen in den ersten 10 min nach Gabe von Furosemid größer als in der Folgezeit, obwohl das Harnvolumen später stärker zunimmt. Die Drucke in den peritubulären Capillaren lagen unter Kontrollbedingungen um 3,1 mm über den Drucken in proximalen Konvoluten, nach Furosemid dagegen um etwa denselben Betrag darunter.Es wird angenommen, daß der Strömungswiderstand der Henleschen Schleife mit zunehmendem Harnfluß stärker vermindert wird, während ein Widerstand am Ende des Nephrons zum Aufstau der intratubulären Flüssigkeit und zur Druckerhöhung führt, die die Erweiterung der Tubulusquerschnitte erzwingt.