Marie Leclerc

Testosterone enhances calcium reabsorption by the kidney

The kidney is a target tissue for androgens, but the role of these hormones in the regulation of calcium (Ca2+) reabsorption remains unclear. The present study examines the effects of testosterone on Ca2+ transport by the luminal membranes of proximal and distal nephrons of the rabbit kidney. Tubule suspensions were pre-incubated in the presence or absence of the hormone, and 45C2+ uptake by the luminal membranes was measured using the rapid filtration technique. In the proximal tubules, testosterone did not influence Ca2+ uptake. In the distal tubules, a 5 min incubation with the hormone increased this uptake with a maximal response at 10(-10)M. Ca2+ transport by the distal membranes shows a dual kinetics. Testosterone enhanced the Vmax value of the low affinity component. In an attempt to identify the underlying mechanisms involved in this action, several messenger inhibitors were introduced in the tubule suspension. PD 98059 and U0 126 as well as AG 99 and genistein interfered with the hormone action suggesting the implication of a MEK kinase and a tyrosine kinase. To determine the type of the channels involved in this effect, Ca2+ uptake was measured in the presence of diltiazem, omega-conotoxin MVIIC and mibefradil, i.e. selective inhibitors of the L-type, P/Q type and T-type channels. An inhibition of Ca2+ transport was observed exclusively with mibefradil. These results indicate that testosterone enhances Ca2+ transport by opening a T-type Ca2+ channel in the distal luminal membrane, via MEK kinase and tyrosine kinase dependent mechanisms.

Renal action of progesterone: effect on calcium reabsorption

Recently, the kidney has been reported to be the site of receptors for progesterone. Although the exact segment of the nephron has not been precisely determined, the cortical collecting tubule was suspected, since the hormone displaces bound 3H aldosterone. The aim of the present study was to investigate the effect of progesterone on calcium (Ca(2+)) transport by the renal luminal membranes and to determine the site and mechanisms of action. Incubation of proximal tubules from rabbit kidney with progesterone did not influence Ca(2+) or Na(+) transport by the luminal membranes. In the distal tubules (DT), a 5 min treatment with 10(-11) M of the hormone enhanced 0.5 mM 45Ca uptake from 0.60+/-0.02 to 0.84+/-0.08 pmol/microg per 10 s (P<0.05) in the absence of Na(+) and from 0.26+/-0.02 to 0.41+/-0.02 pmol/microg per 10 s (P<0.01) in the presence of 100 mM Na(+). The dose-response curve showed a biphasic action with a peak at 10(-11) M. Ca(2+) uptake by DT membranes presents dual kinetics. The hormone enhanced the Vmax value of the high affinity component from 0.41+/-0.05 to 0.57+/-0.06 pmol/microg per 10 s (P<0.05). In contrast, incubation of DT with 10(-8) M progesterone decreased 1 mM Na(+) uptake from 0.68+/-0.03 to 0.53+/-0.07 pmol/microg per 10 s (P<0.05). Finally, 10(-11) M progesterone also enhanced Ca(2+) uptake by the DT membranes through a direct nongenomic mechanism.

The paradoxical effect of adrenomedullin on Na+ transport by the renal distal tubule luminal membrane

Adrenomedullin (ADM) is a potent hypotensive and natriuretic peptide which is synthetized in several mammalian tissues including the kidney. The purpose of this study was to investigate whether the natriuresis was due to a change in Na+ transport by either the proximal (PT) or the distal tubule (DT) luminal membrane, and to characterize this effect, if present. PT and DT suspensions were incubated with human ADM for 20 min at 37 degrees C and luminal membranes of these tubules were purified using the Mg2+ precipitation technique. Na+ uptake was measured by the Millipore filtration technique. A volume of 10(-8) M ADM had no effect on Na+ uptake by the PT luminal membranes. In contrast and unexpectedly, the hormone increased Na+ transport by the DT membranes from 0.28 +/- 0.03 to 0.68 +/- 0.06 pmol/microg per 5 s (P < 0.01). The dose-response curve of this effect showed a maximal response with 10(-7) M ADM. The hormone influenced exclusively the Na+/H+ exchanger, leaving the N-ethyl-N-isopropyl-amiloride (EIPA) insensitive transport intact. The addition of Rp cAMPs to the preparations completely abolished the effect of the hormone on Na+ transport suggesting that cAMP was the messenger involved in this action. Finally, incubation of the DT suspensions with aldosterone also stimulated 1 mM Na+ uptake by the luminal membrane and the two hormone actions were not additive. We conclude that, although ADM is a natriuretic hormone probably through its vasodilating action, it increases distal Na+ reabsorption by the stimulation of the Na+/H+ exchanger activity, as does aldosterone at the same site.

Effect of calbindin D 28K on sodium transport by the luminal membrane of the rabbit nephron.

We previously reported that in the rabbit, the vitamin D-dependent calcium binding protein 28K (CaBP 28K) increases calcium (Ca2+) transport in the distal tubule by opening a high affinity Ca2+ channel in the luminal membrane. Since Na+ and Ca2+ transports are interdependent in this membrane, we questioned whether the calbindin has any influence on Na+ transport. Luminal membranes from rabbit proximal and distal tubules were purified and 22Na uptake by the membrane vesicles was measured using the rapid filtration technique. The vesicles were loaded with 280 mM mannitol and 20 mM Tris-Hepes pH 7.4, with either 3 microM CaBP or the carrier. Incubation medium contained 1 mM 22NaCl, 278 mM mannitol, and 20 mM Tris-Hepes pH 7.4. The presence of 3 microM CaBP 28K in the distal luminal membrane vesicles increased the 0.5 mM Ca2+ uptake from 0.91 +/- 0.21 to 1.84 +/- 0.33 pmol/microg/10 s (P < 0.01) and decreased 1 mM Na+ uptake from 0.62 +/- 0.15 to 0.27 +/- 0.08 pmol/microg/10 s (P < 0.05). A similar decrease of Na+ uptake was observed in proximal luminal membrane experiments. The effect on Na+ uptake by the distal membrane was dose-dependent with a IC50 of 4.5 microM. Addition of 2 mM Ca2+ to the incubation medium decreased 1 mM Na + uptake from 0.62 +/- 0.15 to 0.49 +/- 0.12 pmol/microg/10 s (P < 0.05), but did not influence the effect of CaBP 28K on Na+ uptake. Experiments performed in the presence and absence of ethyl isopropyl amiloride (EIPA) suggest that the effect of calbindin involves the Na+/H+ exchanger activity.

Na+ transport by human placental brush border membranes: Are there several mechanisms?

Na+ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na+ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na+ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na+ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na+ uptake was lower but still sensitive to amiloride. The Lineweaver‐Burk plot of Na+ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride‐sensitive Na+ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na+ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na+. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na+ uptake, whereas benzamil, the Na+ channel blocker, had no effect, it was concluded that the amiloride sensitive Na+ uptake was predominantly or exclusively due to a Na+‐H+ exchanger activity. K+ in trans‐position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis‐position had no effect. The amiloride resistant Na+ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10−7 M insulin, 10−10 M angiotensin II, or 10−8 M human parathyroid hormone (PTH) did not influence Na+ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na+‐H+ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co‐substrates such as phosphate and aminoacids, the Na+‐H+ exchange is probably the unique mechanism of Na+ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane.

Characterization of the Na+/H+ Exchanger in the Luminal Membrane of the Distal Nephron

Abstract. In the rabbit as well as the rat, a Na+/H+ exchanger is expressed in the apical membrane of both the proximal and distal tubules of the renal cortex. Whereas the isoform derived from the proximal tubule has been extensively studied, little information is available concerning the distal luminal membrane isoform. To better characterize the latter isoform, we purified rabbit proximal and distal tubules, and examined the ethylpropylamiloride (EIPA)-sensitive 22Na uptake by the luminal membrane vesicles from the two segments. The presence of 100 μm EIPA in the membrane suspension decreased the 15 sec Na+ uptake to 75.70 ± 4.70% and 50.30 ± 2.23% of the control values in vesicles from proximal and distal tubules, respectively. The effect of EIPA on 35 mm Na+ uptake was concentration dependent, with a IC50 of 700 μm and 75 μm for the proximal and distal luminal membranes. Whereas the proximal tubule membrane isoform was insensitive to cimetidine and clonidine up to a concentration of 2 mm, the 35 mm Na+ uptake by the distal membrane was strongly inhibited by cimetidine (IC50 700 μm) and modestly inhibited by clonidine (IC50 1.6 mm).The incubation of proximal tubule suspensions with 1 mm (Bu2) cAMP decreased the 15-sec EIPA-sensitive Na+ uptake by the brush border membranes to 24.1 ± 2.38% of the control values. Unexpectedly, the same treatment of distal tubules enhanced this uptake by 46.5 ± 10.3%. Finally, incubation of tubule suspensions with 100 nm phorbol 12-myristate 13-acetate (PMA) decreased the exchanger activity to 58.6 ± 3.04% and 79.7 ± 3.21% of the control values in the proximal and distal luminal membranes, respectively. In conclusion, the high sensitivity of the distal luminal membrane exchanger to various inhibitors, and its stimulation by cAMP-dependent protein kinase A, indicate that this isoform differs from that of the proximal tubule and probably corresponds to isoform 1.

Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane

Although in LLC‐PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 μM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 ± 0.04 to 0.39 ± 0.08 pM/μg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPγs, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300‐μM‐Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 μM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.

G Proteins Regulate Calcium Channels in the Luminal Membranes of the Rabbit Nephron

The filtered calcium (Ca2+) is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by various hormones implies several steps, including binding of these hormones to the basolateral membrane, interaction with G proteins, liberation of messengers, activation of kinases and finally opening of the channels at the opposite pole of the cells. In the present study, we examined whether the Ca2+ entry through the luminal membranes of proximal and distal tubules is also regulated by G proteins, by a membrane-limited process. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPγs or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPγs significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles from 0.53 ± 0.06 to 0.72 ± 0.06 pmol/µg/10 s (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPγs increased 0.5 mM Ca2+ uptake in the absence (from 0.57 ± 0.02 to 0.79 ± 0.02 pmol/µg/10 s, p < 0.02) and in the presence (from 0.36 ± 0.03 to 0.55 ± 0.03 pmol/µg/10 s, p < 0.02) of Na+. The action of GTPγs, when present, was dose dependent with a half-maximal effect at 20 µM. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPγs increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.