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M Paillard

Publications and source records attributed to M Paillard.

At least 55 records · Page 3Linked to original sources
PubMed1997

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.

Acidosis↗
PubMed1997

[Extraparathyroid hypercalcemias. Physiopathological and therapeutic aspects].

An increase in plasma calcium concentration is always the consequence of at least one of the following events: an increase in the net calcium input in extracellular fluid, a decrease in glomerular filtration rate, and an increase in the tubular reabsorption of the filtered calcium. In parathyroid hormone-related hypercalcemia, that is typically stable with time, the main determinant is the rise in parathyroid hormone-induced tubular calcium reabsorption. By contrast, in parathyroid hormone-independent hypercalcemia, usually steadily progressive (the main cause being hypercalcemia of cancer), the primary event is almost always a rapid increase in the net calcium input in extracellular fluid. The attendant hypercalcemia is commonly poorly tolerated and induces a renal sodium leak and a decrease in extracellular fluid volume. The latter leads to a fall in glomerular filtration rate and a rise in tubular calcium reabsorption which, in turn, worsen hypercalcemia. Treatment includes a reexpansion of extracellular fluid volume and the inhibition of bone calcium release.

Calcium↗
PubMed1996

Calciuric response to an acute acid load in healthy subjects and hypercalciuric calcium stone formers.

Excessive animal protein consumption is associated with a greater risk of occurrence of renal calcium stone, presumably because of the attendant endogenous acid production. Indeed, chronic acid load enhances urinary calcium excretion possibly through an increased bone calcium release. Because acute studies are best designed to elucidate the mechanism, renal or extra renal, underlying hypercalciuria in the setting of enhanced acid load, we examined the response of 9 healthy adults (8 males, 1 female, aged 38 +/- 3 years, weight 67 +/- 2 kg) and 34 hypercalciuric recurrent calcium stone formers (31 males, 3 females, aged 44 +/- 2 years, weight 72 +/- 2 kg), without any associated disease, to an oral acid load (NH4Cl 2 mmol/kg body wt). After an overnight fast, each patient and control was studied during one one-hour period before and three two-hour periods after their intake of the acid load. An additional group of four time-control subjects (4 males, aged 33 +/- 2 years, weight 66 +/- 2 kg) was studied as the experimental groups except that they did not receive the acid load. On baseline, the three groups exhibited similar glomerular filtration rates, net acid excretions, and plasma calcium and magnesium concentrations. However, fasting urine calcium and magnesium excretions were higher in hypercalciuric calcium stone formers than in healthy control or time-control subjects. In time-control subjects, plasma acid base status, net acid excretion, filtered loads of calcium and magnesium, and urinary calcium and magnesium excretions remained unchanged all over the study. By contrast, after the oral acute acid load, net acid excretion increased and urinary pH decreased similarly in patient and control groups; glomerular filtration rate did not change, as well as plasma calcium and magnesium concentrations. Nevertheless, urinary calcium and magnesium excretions markedly increased, in both groups, independently of changes in tubular sodium handling and in plasma parathyroid hormone concentration. The increase in urinary calcium and magnesium excretions that occurred in the absence of any change in the filtered load of calcium and magnesium was therefore mediated by a decrease in tubular calcium and magnesium reabsorption, independent of PTH, but dependent on changes in net acid excretion. A positive linear relationship between urinary calcium and magnesium excretions suggested that the target tubular site for acid load was the thick ascending limb of Henle's loop. Finally, a negative linear relationship was demonstrated between the acid load-induced increase in urinary calcium excretion and fasting urinary calcium excretion; indeed, the lowest calciuric responses were observed in patients with the highest fasting urinary calcium excretion. Thus there was no additional effect of the acid load-induced inhibition on intrinsic defect in tubular calcium reabsorption which suggests that the tubular target site for acid load and the site of calcium transport defect in idiopathic hypercalciuria may be the same.

Acidosis↗
PubMed1996

Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tubule.

Low concentrations of angiotensin II (Ang II) increase, whereas high concentrations inhibit the apical Na/H antiporter activity in the proximal tubule, but the respective roles of the different signaling pathways in mediating these effects remains unsettled. We studied the effects of both low and high doses of Ang II in the presence of selective signaling pathway inhibitors, on the apical Na/H antiport activity of rat proximal tubule. Experiments were carried out in intact cells of freshly prepared tubule fragments obtained from the outer third of cortex, that is, devoid of basolateral Na/H antiport activity in the absence of bicarbonate transport and H(+)-ATPase activity. In tubules acid-loaded by an NH4Cl prepulse, Na/H antiport activity was assessed by the initial rate of intracellular pH recovery (dpHi/dt), measured with BCECF. When tubules were preincubated with low dose Ang II (10(-11) M for 3 min), dpHi/dt increased by 25 +/- 8%, whereas incubation with high dose Ang II (10(-7) M for 3 min) decreased dpHi/dt by 30 +/- 4%, compared to control (P < 0.01 in both cases). Both effects were abolished in the presence of 2.10(-3) M amiloride. Low dose Ang II-induced increase in dpHi/dt was not affected by preincubation with a specific PKA inhibitor, Rp-CPT-cAMP 10(-4) M, and was completely abolished by preincubation with PKC inhibitors, staurosporine 10(-7) M, sphingosine 5.10(-6) M, or calphostin 10(-6) M. In addition, pretreatment of rats with pertussis toxin led to a partial inhibition of the effect of low dose Ang II. The high dose-Ang II-induced decrease in dpHi/dt was not affected by pretreatment with a calcium-calmodulin kinase inhibitor W-7 10(-4) M. Conversely, pretreatment with the cytochrome P-450 inhibitor econazole 10(-5) M reversed the inhibitory effect of high dose Ang II to a stimulatory effect (24 +/- 8%, P < 0.01), quantitatively similar to the effect of low dose Ang II. In addition, arachidonate was found to exert an econazole-sensitive dose-dependent inhibitory effect on dpHi/dt, and 5,6-EET 10(-6) M, a cytochrome P-450 derived-arachidonic acid metabolite, induced a 38 +/- 9% inhibition, similar to that observed with high dose Ang II alone. There was no additive effect of 5,6-EET and high dose Ang II. Finally, pretreatment with two PLA2 inhibitors (BromoPhenacylBromide, 6.10(-6) M, and oleyloxyethyl phosphorylcholine, 5.10(-6) M) reversed the inhibitory effect of high dose Ang II to a stimulatory effect (32 +/- 11% and 25 +/- 11%, respectively, P < 0.05 for both inhibitors). We conclude that, in intact rat proximal cells, low dose Ang II stimulates the apical Na/H antiport through a pertussis toxin-sensitive G protein-dependent PKC pathway, whereas high dose Ang II inhibits the Na/H antiport activity through the PLA2- and cytochrome P-450-dependent metabolites of arachidonate.

Ammonia↗
PubMed1996

Oral calcium tolerance test in the early diagnosis of primary hyperparathyroidism and multiple endocrine neoplasia type 1 in patients with the Zollinger-Ellison syndrome. Groupe de Recherche et d'Etude du Syndrome de Zollinger-Ellison.

BACKGROUND: In patients with the Zollinger-Ellison syndrome, the exclusion of multiple endocrine neoplasia type 1 is of important clinical relevance. Its diagnosis often relies on the existence of primary hyperparathyroidism. AIM AND METHODS: To investigate the parathyroid function of patients with the Zollinger-Ellison syndrome by use of an oral calcium tolerance test to identify both hypercalcaemic and normocalcaemic primary hyperparathyroidism, and, accordingly, multiple endocrine neoplasia type 1. PATIENTS: Among 51 consecutive patients with the Zollinger-Ellison syndrome referred to us between 1988 and 1994, 28 had not been investigated for parathyroid function and were prospectively studied. RESULTS: The investigation of calcium metabolism was abnormal in nine patients. One displayed characteristic features of humoral hypercalcaemia of malignancy. The diagnosis of primary hyperparathyroidism was biologically established in eight patients (29%) and subsequently confirmed by the presence of hyperplasia of the parathyroid glands in the seven patients who underwent neck exploration. Three patients with primary hyperparathyroidism had fasting hyper-calcaemia but the other five had normal fasting serum total calcium concentration and the diagnosis of primary hyperparathyroidism was established by means of the oral calcium tolerance test. Primary hyperparathyroidism was demonstrated in the five patients in whom the diagnosis of multiple endocrine neoplasia type 1 had been previously established on other criteria than primary hyperparathyroidism. By contrast, in three patients, primary hyperparathyroidism, either hypercalcaemic (one patient) or normocalcaemic (two patients) was the sole criteria for the diagnosis of multiple endocrine neoplasia type 1. These results also suggest that primary hyperparathyroidism is present before or close to the time of Zollinger-Ellison syndrome diagnosis. CONCLUSION: Complete investigation of the parathyroid function with calcium calcium and parathyroid hormone concentrations.

Administration, Oral↗
PubMed1996

Interactions of external and internal K+ with K(+)-HCO3- cotransporter of rat medullary thick ascending limb.

We studied [K+]i and [K+]o, where subscripts i and o refer to intracellular and extracellular, respectively, concentration dependency of the kinetic properties of the electroneutral K(+)-HCO3-cotransport, using suspensions of rat medullary thick ascending limb (mTAL). With the use of nigericin and monensin, [K+]i was clamped at various values, while maintaining [Na+]i = [Na+]o = 37 mM, [HCO3-]i = [HCO3-]o = 23 mM, and pHi = pHo = 7.4. As indicated by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein HCO3(-)-dependent rates of change in pHi, at constant [K+]i, increasing the magnitude of the outward K+ gradient by varying [K+]o saturated HCO3-efflux with a Michaelis-Menten curve (apparent Michaelis constant for [K+]o = 2 mM, Hill coefficient = 1). On the other hand, increasing [K+]i from 30 to 140 mM, while either [K+]o or the magnitude of the K+ concentration gradient was fixed, saturated HCO3- efflux with a sigmoidal curve and yielded a Hill coefficient of 3.4 and 50% of maximum velocity at 70 mM [K+]i. These results indicate that [K+]i, independent of its role as a transportable substrate for the cotransport with HCO3-, has a role as an allosteric activator of the K(+)-HCO3- cotransporter. Such an allosteric modulation may contribute to the maintenance of net HCO3- absorption despite large in vivo physiological variations of K+ concentration in the medullary interstitium.

Animals↗
PubMed1996

Na(+)-K+(NH4+)-2Cl- cotransport in medullary thick ascending limb: control by PKA, PKC, and 20-HETE.

Cell pH was monitored in suspensions of medullary thick ascending limbs (MTALs) of rat kidney to determine possible effects of various transduction pathways on apical Na(+)-K+ (NH4+)-2Cl- cotransport, the activity of which was measured as the bumetanide-sensitive component of cell acidification caused by abrupt exposure to 4 mM NH4Cl. 8-Bromoadenosine 3',5'-cyclic monophosphate stimulated cotransport activity through activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), since the cAMP effect was abolished by N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89); stimulation by cAMP (P < 0.02) was observed even when other Na+, Cl-, and K+ carriers were blocked by ouabain, diphenylamine-2-carboxylate, and barium, which indicates that cotransport was directly affected by PKA. Phorbol 12,13-dibutyrate also stimulated cotransport activity (P < 0.03), which was abolished by protein kinase C (PKC) blockade by staurosporine. In contrast, cotransport activity was reduced (P < 0.001) by arachidonic acid or 20-hydroxyeicosatetraenoic acid (20-HETE), as well as by an ionomycin-induced rise in cytosolic Ca2+ ([Ca2+]i). Inhibition by arachidonic acid or ionomycin was abolished by econazole and SKF-525A that inhibit cytochrome P-450-dependent monoxygenase, which produces 20-HETE from arachidonic acid in the MTAL, and the ionomycin effect was prevented when phospholipase A2 (PLA2) was blocked by 4-bromophenacyl bromide or oleyloxyethyl phosphorylcholine. The results demonstrate that MTAL apical Na(+)-K+(NH4+)-2Cl- cotransport is stimulated by PKA and PKC and inhibited by 20-HETE that may be produced after a rise in [Ca2+]i through PLA2 activation.

Ammonia↗
PubMed1996

[Molecular aspects of Na+/H+ exchange in the renal tubule: localization and adaptation to the acid-base state].

Na+/H+ exchangers (NHE) are plasma transmembrane proteins that exchange extracellular Na+ for intracellular H+. Several isoforms of these antiporters belonging to the same gene family have been cloned and four of them (NHE1 to NHE4) are expressed in the kidney. In the kidney, NHEs isoforms display different tubular and membrane (apical vs basolateral) localization and are involved in different functions: regulation of pH and cell volume, NH4+ secretion and NaHCO3 and NaCl reabsorption. NHE3, which is the apical isoform of the proximal tubule and thick ascending limb of Henle, is involved in bicarbonate reabsorption and displays activation during metabolic acidosis. These recent data showing the acid-activation of NHE3 suggest that NHEs isoforms could be involved in the pathogeny of tubular acidosis.

Acid-Base Imbalance↗
PubMed1995

Protein kinase C isoforms in rat kidney proximal tubule: acute effect of angiotensin II.

The present study examined the effect of phorbol esters, Ca2+, and angiotensin II (ANG II) on protein kinase C (PKC) isoforms in the rat proximal tubule. The immunoblot analysis of PKC isoforms of particulate and cytosolic fractions of proximal tubules revealed immunoreactive proteins when antibodies against PKC-alpha, -delta, -epsilon, and -zeta, but not -beta and -gamma were used. Phorbol dibutyrate (PDBU) induced the translocation of PKC-alpha, -delta, and -epsilon, whereas an inactive phorbol ester had no effect. PDBU and ionomycin increased particulate PKC specific activity from 0.67 +/- 0.09 to 1.56 +/- 0.18 and 0.96 +/- 0.04 pmol.microgram protein-1.2 min-1, respectively. ANG II (10(-7) M) induced a time-dependent increase in particulate PKC-alpha immunoreactivity observed after 2 min and maintained for 12 min. Particulate PKC-epsilon immunoreactivity increased after 4 min. Meanwhile, PKC-delta and -zeta were not modified by ANG II. Accordingly, ANG II elicited a rise in the specific activity of the particulate PKC, which increased to 0.89 +/- 0.09 pmol.micrograms protein-1.2 min-1 after 2 min. This was inhibited by a preincubation in the presence of 10(-5) M losartan, specific inhibitor of angiotensin subtype 1 receptors. These data indicate that PKC-alpha and -epsilon are potential candidates to regulate the activity of Na+/H+ and Na(+)-HCO3- transporters because they are translocated with a time course fitting with that of the reported effect of ANG II on those transporters.

Angiotensin II↗
PubMed1995

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb.

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

Animals↗
PubMed1994

NH4+ transport pathways in cells of medullary thick ascending limb of rat kidney. NH4+ conductance and K+/NH4+(H+) antiport.

To characterize NH4+ transport in the renal medullary thick ascending limb (MTAL), cell pH was monitored with the use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in suspensions of rat MTAL tubules in CO2-free media. Abrupt exposure to NH4Cl led to initial cell alkalinization (NH3 entry) followed by cell acidification due to NH4+ influx. The latter had 1 microM amiloride- and barium-sensitive components; the barium effect was unchanged when K+ conductances were completely blocked by quinidine, as assessed with the use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine. NH4+ entry-induced depolarization was abolished by 1 microM amiloride, but it was unaffected by barium. In NH(4+)-free media, barium, verapamil, and raising of the extracellular K+ concentration alkalinized MTAL cells; imposing an outward directed K+ gradient in Na(+)-free medium induced cellular acidification, which was abolished by barium and verapamil but not by other K+ channel and Na+/H+ antiport inhibitors (quinidine and 2 mM amiloride). As measured with a K(+)-selective extracellular electrode, a component of K+ efflux (in presence of furosemide, ouabain, and quinidine) was stimulated by decreasing the extracellular pH from 7.4 to 7.0 and inhibited by barium and verapamil. It was also demonstrated that the K+/H+ antiporter transports NH4+ better than H+ at physiological NH4+ and H+ concentrations. These results demonstrate the presence in MTAL cells of two novel NH4+ transport pathways, amiloride-sensitive NH4+ conductance and barium- and verapamil-sensitive K+/NH4+(H+) antiport.

Amiloride↗
PubMed1994

Enzymatic and functional evidence for adaptation of the vacuolar H(+)-ATPase in proximal tubule apical membranes from rats with chronic metabolic acidosis.

The present work examined the effects of chronic metabolic acidosis on the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) activity both in rat renal cortical homogenates and in their luminal membranes. Moreover, to assess the effect of acidosis on H+ transport by the apical H(+)-ATPase, we have developed a detergent-dilution procedure, resulting in the formation of sealed vesicles having this enzyme at their external surface. NH4Cl loading for 4 days had no effect on homogenates H(+)-ATPase activity, estimated with either N-ethylmaleimide or bafilomycin A1. In contrast, H(+)-ATPase activities were increased significantly by about 30% in both native apical membranes prepared by Ca2+ aggregation and detergent-treated luminal vesicles from acidotic animal. Kinetic analysis revealed that this stimulation was solely through changes in the Vmax for ATP. In membranes prepared by Mg2+ aggregation, acidosis also caused significant stimulation of the H(+)-ATPase activity. In addition, the initial rate of ATP-induced intravesicular acidification was 25% higher in reoriented H(+)-ATPase vesicles from acidotic rats, whereas passive proton permeability was identical in both groups. Finally, both vesicle enrichments and yields of luminal markers and de-enrichments and yields of intracellular membrane markers were identical in the two groups. These results provide enzymatic and functional evidence suggesting that chronic acidosis induces an adaptative change in the rat brush border H(+)-ATPase.

Acidosis, Renal Tubular↗
PubMed1994

Control of H(+)-HCO3- plasma membrane transporters by urea hyperosmolality in rat medullary thick ascending limb.

Hyperosmolality inhibits bicarbonate absorption by the rat medullary thick ascending limb (MTAL) by unknown mechanisms. Intracellular pH (pHi) was monitored with use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in rat MTAL tubule suspensions to specify the H(+)-HCO3- membrane transporters affected by hyperosmolality. Measurements were made after > or = 15-min incubation of the cells in media rendered hypertonic by urea to avoid any change in cell volume. Na(+)-H+ antiport activity, estimated from the Na(+)-induced initial rate of pHi recovery of Na(+)-depleted acidified cells in the presence of 0.1 mM furosemide to inhibit Na(+)-K(+)-2Cl- cotransport, was inhibited by 300 mM urea and 10(-8) M arginine vasopressin (AVP) in an additive manner. Na(+)-H+ antiport inhibition by urea hyperosmolality was maximal at 300 mM urea with a half-maximal inhibitory concentration of 75 mM and was due to a 28% decrease in maximum velocity (Vmax) with no effect on the Michaelis constant for sodium. Urea hyperosmolality (300 mM) did not affect steady-state intracellular calcium concentration ([Ca2+]i), assessed with use of fura 2 fluorescence, and still inhibited Na(+)-H+ antiport in MTAL cells loaded with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to minimize any transient change in [Ca2+]i during the preincubation in urea medium. Furthermore, 300 mM urea did not stimulate basal or AVP-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Plasma membrane H(+)-adenosinetriphosphatase (ATPase) activity and HCO3- transport, assessed by appropriate experimental protocols, were unaltered by 300 mM urea.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗