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Background Cyclosporin A (CyA) may induce acute nephrotoxicity. The question has been raised of the possible long-term unfavorable course of CyA-induced lesions. Advantage was taken of a large cohort of diabetic patients treated for several months using moderate CyA dosage to evaluate the long-term evolution of renal function in such patients. Methods Two hundred and eighty five recently diagnosed type 1 diabetic patients having received CyA for a mean of 19.9 months were monitored for 13 years, in parallel with 100 similar patients treated with insulin alone. Results In the CyA-treated group, a transient increase in creatininemia levels occurred during the first 18 months of treatment associated with a transient increase in renal vascular resistance. Both effects disappeared later on: creatininemia levels then remained normal. Inulin and p-aminohippurate (PAH) clearances remained normal throughout follow-up. Neither permanent renal failure nor progressive deterioration of renal function occurred in either group or in individual patients. A 10 to 12% increase in inulin and PAH clearance was elicited by IV amino acid infusion at 7 to 10 years, a finding consistent with a normal renal functional reserve. Patients with moderate kidney lesions on biopsy at 1 year had normal and stable clearance values at 7 to 13 years. The prevalence of arterial hypertension and retinopathy was lower in the CyA-treated group than in the control group, possibly because of the tighter metabolic control obtained in the CyA group. Conclusion These results suggest that low-dose CyA treatment combined with thorough monitoring does not result in long-term renal dysfunction. Copyright © 2002 John Wiley & Sons, Ltd.

Hearing loss, vertigo, and tinnitus have been related to arterial hypertension. The aim of the present work was to study the permeability of the blood-perilymph and of the labyrinthine barrier, between endolymph and perilymph, to small molecules during chronic and acute hypertension. Experiments were performed in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Acute hypertension was induced by phenylephrine. Perilymph was sampled from the first turn of the scala vestibuli and the Na, K, urea, and radioactive concentrations ((14)C-urea and (3)H-mannitol) were measured. In another experimental set, the endocochlear potential was recorded from the basal turn of scala media, before and after phenylephrine injection. The composition of the perilymph and the kinetic constants for (14)C-urea and (3)H-mannitol were similar in WKY and SHR, and not modified after acute hypertension. In endolymph, the endocochlear potential in SHR (+80+/-2.7 mV, n=24) was lower (P

In Meniere's disease, an inner ear disorder related to an endolymphatic hydrops, an alteration of the functioning of the endolymphatic sac has been proposed. The endolymphatic sac is assumed to be involved in the secretion/resorption of endolymph. The epithelial transport systems have been indirectly studied by the recording of the endolymphatic sac transepithelial potential (ESP) in control conditions and after the local injection of drugs such as diuretics that have been proposed in the treatment of Meniere's disease. The ESP was recorded, in vivo, in guinea pigs up to 150 minutes after the perisaccular injection of 5 μL of a 150 mmol/L (mM) NaCl solution containing various drugs known to inhibit ionic transport systems. The initial ESP was +8.4 ± 0.3 mV (mean ± SEM, n = 78). The basolateral injection of 5 μL of 150 mM NaCl induced an ESP decrease of 64% ± 6.0% (n = 12), 5 minutes after the end of the injection. Then ESP increased, returning to its initial value at 60 minutes and surpassing it at 120 minutes. Diuretics such as acetazolamide (10 -3 mol/L [M]), an inhibitor of carbonic anhydrase, and amiloride (10 -4 M), an inhibitor of Na channel or Na/H exchanger, decreased the ESP recovery. At variance, bumetanide (10 -6 M, 10 -4 M), the Na-K-Cl cotransport inhibitor, and chlorothiazide (10 -4 M), a Na-Cl cotransporter inhibitor, failed to alter the ESP as compared with the control group. Ouabain (10 -3 M), the Na + ,K + -adenosine triphosphatase (ATPase) inhibitor, prevented the ESP recovery otherwise observed 60 minutes after the NaCl injection. Bafilomycin A 1 , the inhibitor of the vacuolar-type H + -ATPase, prevented the recovery of the ESP with a log-dose/effect (10 -5 M, 10 -6 M, 10 -9 M). Disulfonic acid stilbene (DIDS) (10 -4 M), an inhibitor of transporters involving HCO 3 - , also prevented the ESP recovery. These results suggest that the genesis of the ESP was highly dependent on acid-base transport systems including carbonic anhydrase, a vacuolar-type H + -ATPase, and an anionic transport system blocked by DIDS. Further studies are needed to confirm the alteration of the acid-base balance in this epithelium and its possible involvement in the pathogenesis of Meniere's disease.

Platelet activating factor inhibits Cl and K transport in the medullary thick ascending limb. Since the kidney medulla was reported to generate platelet activating factor (PAF), we investigated a possible effect of this agent on the reabsorptive function of in vitro microperfused medullary thick ascending limbs from mouse kidney (mTAL). PAF, 10 -7 M in the bath, significantly decreased the net chloride flux (JCl) from 48.8 ± 7.1 to 27.4 ± 5.7 pmol/min. This effect was reversible, blocked by the antagonist BN 50730, and not reproduced by the inactive metabolite lyso-PAF. PAF inhibited the transepithelial potential difference with a threshold at 10 -9 M. In the presence of isoproterenol, the PAF-induced decrease of JCl was not significantly different from that observed in basal conditions; moreover, PAF did not modify the adenylate cyclase activity in isolated mTALs, either in basal condition or under stimulation by isoproterenol. The effect of PAF on JCl was not prevented by mepacrine, NDGA associated with proadifen, or adenosine desami-nase. When the apical Na-K-2Cl cotransport was blocked by furosemide or bumetanide, a net K secretion occurred (-1.1 ± 0.2 pmol/min), which was significantly decreased by PAF (-0.06 ± 0.3 pmol/min). Moreover, it was verified on isolated mTALs that PAF did not modify the Na,K-ATPase activity. It is concluded that PAF inhibits the reabsorptive function of the mTAL, as indicated by the decrease of Cl reabsorption and K secretion. This effect could not be accounted for by adenosine or arachidonic acid metabolite action, and was not mediated by an inhibition of the adenylate cyclase activity.

In order to evaluate the influence of membrane fluidization on three apical transport systems and on a basolateral enzyme, and to analyse the mechanisms involved, we studied, in cultured rabbit proximal tubular cells, the effect of increasing concentrations of the local anesthetic drug benzyl alcohol on Na(+)-dependent uptakes of phosphate (Pi), methyl alpha-D-glucopyranoside (MGP), and L-alanine, as well as on basal and stimulated cyclic AMP content. At 10 mM, benzyl alcohol increased the Vmax of Pi uptake by 31%, decreased that of MGP uptake by 24%, and did not affect alanine uptake. Km values were not affected. Benzyl alcohol, up to 40 mM, increased in a concentration-dependent manner basal, PTH-stimulated, and cholera toxin-stimulated, but not forskolin-stimulated cyclic AMP accumulation. In the presence of 40 mM benzyl alcohol, the magnitude of PTH-induced inhibition of Pi uptake was enhanced from 11% to 24%. It is concluded that: (i) fluidization of apical membranes affected differently Na+/Pi, Na+/MGP, and Na+/alanine cotransports, reflecting differences in the lipidic environments of these transport system; (ii) fluidization of basolateral membranes enhanced PTH-stimulated cyclic AMP generation through improved coupling between the receptor-GS complex and the catalytic subunit of adenylate cyclase; (iii) these variations may result in physiological and pathophysiological modulation of the renal handling of solutes and of the phosphaturic effect of PTH.

The involvement of dopamine (DA) receptor subtypes in regulation of renal phosphate transport by DA, either exogenous or locally synthesized from L-dihydroxyphenylalanine (L-dopa) was evaluated in opossum kidney (OK) cells with proximal tubular phenotype. DA synthesis from L-dopa by OK cells was abolished by carbidopa and benserazide, two dissimilar inhibitors of aromatic L-amino acid decarboxylase. L-Dopa stimulated cyclic AMP generation and inhibited Na-dependent Pi uptake, and these effects were abolished by carbidopa and benserazide. The effects of L-dopa or DA on cyclic AMP generation and on Na-Pi co-transport were mimicked by SKF 38393, a DA1 receptor agonist, and were potentiated by S-sulpiride, a DA2 receptor antagonist. Bromocriptine, a DA2 receptor agonist, blunted in a pertussis toxin-dependent manner parathyroid hormone (PTH)-induced cyclic AMP generation and inhibition of Pi uptake. In contrast with PTH, neither L-dopa nor DA affected significantly the cytosolic calcium concentration. These results support the involvement of DA1 and DA2 receptors, positively and negatively coupled into adenylate cyclase respectively, in modulation of renal phosphate transport.

PTH-induced phosphaturia is exerted in part by cAMP added to the renal tubular lumen under the influence of the hormone. Modulation of renal phosphate transport by luminal cAMP requires degradation of the nucleotide into adenosine by brush-border membrane ectoenzymes, among them ecto-5'-nucleotidase (5'-NU). Hormonal modulation of 5'-NU activity was evaluated in cultured opossum kidney cells. PTH (1-100 nM) stimulated 5'-NU in a time-, concentration-, and protein synthesis-dependent manner. The effect of PTH-(1-34) was mimicked by PTH-(3-34), which does not activates adenylate cyclase, and by phorbol 12-myristate 13-acetate (PMA), but not by forskolin or (Bu)2cAMP. Down-regulation or pharmacological inhibition of protein kinase-C (PKC) abolished the effect of PTH fragments and PMA. PTH fragments increased intracellular Ca2+ and translocated PKC activity to the membrane. PTH or PMA did not affect 5'-NU messenger RNA content. Inhibition of sodium-phosphate cotransport by extracellular cAMP was decreased by 5'-NU inhibition and was magnified by PTH. These results indicate that 1) PTH stimulates 5'-NU activity in renal proximal tubular cells in a manner involving PKC activation and de novo protein synthesis; and 2) this effect participates in PTH modulation of renal phosphate transport.

In the cochlea, endolymph is hyperosmotic to plasma and perilymph. To test the hypothesis that antidiuretic hormone is involved in the modulation of endolymph secretion, the electrochemical composition of cochlear fluids, endolymph and perilymph, was studied in three groups of anaesthetized rats: control Long Evans rats, homozygous Brattleboro rats that are genetically deprived of antidiuretic hormone, and Brattleboro rats that were treated with antidiuretic hormone (dDAVP, 0.5 microgram/100 g body weight/24 h during 8 days). Endolymph was sampled from the scala media at each turn of the cochlea and perilymph from the scala vestibuli. In Long Evans rats, the endocochlear potential, the endolymphatic K+ and Cl- concentrations decreased from base to apex of the cochlea as previously reported in guinea pigs and Sprague Dawley rats. In Brattleboro rats, the endocochlear potential and the Cl- concentration gradients were still present, whereas the K+ concentration gradient were still present, whereas the K+ concentration gradient was absent. This K+ gradient was restored by the administration of dDAVP, which increased the K+ concentration at the base of the cochlea. This work indicates that the K+ secretion in endolymph, and thus the osmolality, may be locally modulated by the antidiuretic hormone, probably via V2 receptors.

The electrochemical changes of the inner ear fluids were studied in the guinea pig during the development of endolymphatic hydrops in an experimental model of Meniere's disease obtained by the blockage of the vestibular aqueduct. The endocochlear potential (first and third turns) was recorded, and the sodium, potassium, and chloride concentrations, and osmolality of the endolymph (first and third turns) and perilymph were determined at different intervals from 2 to 24 weeks after the induction of the hydrops. The development of hydrops was monitored by the compound action potential once a week during the observation period. In normal, nonoperated guinea pigs, longitudinal endolymphatic gradients of endocochlear potential, potassium and chloride concentrations, and osmolality, increasing from the apex to the base of the cochlea, were observed. After 2 weeks of hydrops, no alteration of this pattern was detected. After 6 and 9 weeks of hydrops, a progressive decrease of endocochlear potential, potassium and chloride concentrations, and osmolality was noticed at the first turn (6 and 9 weeks) and then at the third turn (9 weeks) which resulted in the disappearance of longitudinal gradients. At 24 weeks, the endocochlear potential was still diminished by 60%, whereas potassium and chloride concentrations and osmolality increased as compared to 9-week values but remained lower than in controls. The changes in composition of endolymph induced by the development of the hydrops could be related to the progressive alteration of the ionic permeability of the cochlear epithelium, which should be localized at the distended Reissner's membrane.

The term "renal functional reserve" (RFR) refers commonly to the reserve of glomerular filtration rate (GFR) and renal blood flow. RFR can be elicited by an oral protein load or by infusion of aminoacids, glucagon, or dopamine. The increase in GFR which follows aminoacid administration results from a cascade of events including at least pancreatic release of glucagon, involvement of an hepatic step yet unidentified, and renal synthesis of vasodilatory prostaglandins. RFR represents a constant fraction of baseline GRF as long as the latter is above 40-50 l/min. It has been suggested tha permanent challenge of RFR, which occurs in protein-rich diet or during the hyperfiltration phase of diabetic nephropathy, might lead to and accelerate impairment of renal function. The relevance of RFR measurement as a tool to predict the evolution of renal function in various types of renal diseases remains to be evaluated.

Since doxorubicin (Adriamycin) is known to bind on membranous negative surface charges, its effect on the electrochemical composition of the cochlear fluids was studied in rats. Doxorubicin was infused into the cerebrospinal fluid via the lateral cerebral ventricle. The endocochlear resting potential was recorded, and endolymph and perilymph of the scala vestibuli were collected from the basal cochlear turn before and 1, 2, and 4 h after the drug application. Na+, K+, and Cl- concentrations and osmolality of the endolymph and perilymph were measured in 1 nl aliquots. In perilymph, Cl- concentration increased 4 h after doxorubicin treatment to reach a concentration 8 mM higher than recorded initially. In endolymph, the endocochlear potential decreased by 5 mV/h while its K+ concentration and osmolality increased by about 3 mM/h and 4 mosmol/kg H2O per hour, respectively. These results suggest that the negative surface charges demonstrated on Reissner's membrane may play a role in the homeostasis of endolymph.

The effect of parathyroid hormone (PTH) on transepithelial Na+, Cl−, K+, Ca2+ and Mg2+ transport was investigated in isolated perfused cortical thick ascending limbs (cTAL) and that of human calcitonin (hCT) was tested in both cortical and medullary thick ascending limbs (mTAL) of the mouse nephron. The transepithelial ion net fluxes (J x) were determined by electron probe analysis of the perfused and collected fluids. Simultaneously, the transepithelial voltage (PDte) and resistance (R te) were recorded. In cTAL segments, PTH and hCT significantly stimulated the reabsorption of Na+, Cl−, Ca2+ and Mg2+. hCT generated a net K+ secretion towards the lumen and PTH tended to exert the same effect. Neither PDte nor R te were significantly altered by either PTH or hCT. However, in the post-experimental period a significant decrease in PDte was noted. Time control experiments carried out under similar conditions revealed a significant decrease in PDte with time, which could have masked the hormonal response. In mTAL segments, Mg2+ and Ca2+ transport was close to zero. hCT did not exert any detectable effect on either PDte or J Cl −, J Na + J K +, J Mg 2+ and J Ca 2+ in these segments. In conclusion, our data demonstrate that PTH and hCT stimulate NaCl reabsorption as well as Mg2+ and Ca2+ reabsorption in the cTAL segment of the mouse. These data are in agreement with and extend data obtained in vivo in the rat.

The role of glucagon as a mediator of aminoacid-induced alteration of renal haemodynamics was evaluated in man in three different protocols. In the first it was shown that the increase in glomerular filtration rate (GFR) and renal plasma flow (RPF) observed during an aminoacid infusion was prevented by the additional infusion of somatostatin (SRIF), but reproduced by a glucagon infusion in the presence of SRIF. In the second protocol it was shown that, at variance with normal subjects, six totally pancreatectomised patients, thus deprived of pancreatic glucagon secretion, did not increase their GFR and RPF when infused with amino-acids, whereas they exhibited the expected hyperfiltration when infused with glucagon. In the third protocol it was shown that glucagon infusion in a renal artery did not alter the homolateral renal haemodynamics. It is concluded that glucagon secretion is a mandatory step in the cascade of events linking the infusion of aminoacids to the renal hyperfiltration. Other steps beyond glucagon secretion are necessarily involved because glucagon has no direct renal effect.

To the Editor: Dipyridamole, a widely used vasodilatory drug, enhances renal tubular reabsorption of phosphate by decreasing adenosine uptake by tubular cells.1 In rats, dipyridamole prevents phosphate excretion induced by cyclic AMP and decreases the phosphaturic effect of parathyroid hormone.1 This demonstrates that adenosine, a local product of the degradation of nephrogenous cyclic AMP, is associated with phosphaturia. We sought to determine whether dipyridamole might decrease urinary phosphate excretion in humans. We evaluated the short-term effect of dipyridamole in 48 people (22 men and 26 women) with a mean age of 52 years (range, 26 to 74). Twelve were normal . . .

The composition of endolymph and perilymph was studied in the guinea pig cochlea after 2 and 6 weeks of blockage of the vestibular aqueduct in an experimental model of hydrops. Compound action potential was monitored several times in the observation period. The endocochlear potential was measured and the endolymph was sampled at the first and third turns of the scala media. The Na, K, and Cl concentrations were determined in nanolitre aliquots of endolymph and of perilymph, the latter sampled from the basal scala vestibuli. After 2 weeks, no change in endolymphatic electrochemical composition was observed. After 6 weeks, endocochlear potential was decreased by 25% at both cochlear turns; K concentration was decreased in endolymph of the basal turn and Cl concentration was decreased in both turns; the calculated osmolality (Na + K + Cl) was decreased in both turns. These results indicate that the blockage of the vestibular aqueduct induced early auditory dysfunction whereas alterations of the electrochemical composition of endolymph occurred later after a time lag of more than 2 and less than 6 weeks.

To evaluate a possible modulation by membrane fluidity of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of the neutral local anesthetic, benzyl alcohol, on membrane fluidity and on basal and stimulated intracellular cAMP content in intact MDCK cells. Benzyl alcohol induced a dose-dependent decrease of lipid order which was measured by steady-state fluorescence anisotropy using trimethylammonium-diphenylhexatriene and propionyl-diphenylhexatriene as fluorescent probes. Benzyl alcohol induced a 2-fold increase in basal cAMP content, likely as a consequence of increased prostaglandin synthesis since this effect was abolished by indomethacin. The effect of benzyl alcohol on stimulated cAMP synthesis depended on the nature of the ligand: 10 mM benzyl alcohol increased significantly the stimulatory effect of prostaglandin E2, glucagon and forskolin but not of vasopressin. At higher concentrations (40 mM), benzyl alcohol did not affect significantly the glucagon-stimulated cAMP content, while it inhibited significantly the prostaglandin E2-, forskolin- and vasopressin-stimulated cAMP synthesis. The 40 mM benzyl alcohol-induced inhibition was reversed by 1 mM Mn2+, which is known to block the inhibitory GTP-binding protein Ni. These results suggest that: (i) the various components of the adenylate cyclase-cAMP system and their coupling are affected differently by changes in membrane fluidity, which might reflect differences in their lipid environment, (ii) changes in membrane fluidity can modulate responses of renal tubular cells to hormones, and thus tubular functions.

The effects of glucagon on transepithelial Na+, Cl−, K+, Ca2+ and Mg2+ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (JNa+,JCl−,JK+,JCa2+,JMg2+) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PDte) and the transepithelial resistance (Rte) were recorded. In cTAL-segments (n=8), glucagon (1.2×10−8 mol · l−1) stimulated significantly the reabsorption of Na+, Cl−, Ca2+ and Mg2+∶JNa+ increased from 204±20 to 228±23 pmol · min−1 · mm−1,JCl− from 203±18 to 234±21 pmol · min−1 · mm−1,JCa2+ from 0.52±0.13 to 1.34±0.30 pmol · min−1 · mm−1 andJMg2+ from 0.51±0.08 to 0.84±0.08 pmol · min−1 · mm−1.JK+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min−1 · mm−1. Neither PDte (16.3±1.5 versus 15.9±1.4 mV) norRte (22.5±3.0 versus 20.3±2.6 Ωcm2) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PDte and increase inRte were noted. In mTAL-segments (n=9), Mg2+ and Ca2+ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na+ and Cl−, however, were strongly stimulated:JNa+ increased from 153±17 to 226±30 pmol · min−1 · mm−1 andJCl− from 151±23 to 243±30 pmol · min−1 · mm−1. The rise in NaCl transport was accompanied by an increase in PDte from 10.3±1.1 to 12.3±1.2 mV and a decrease inRte from 19.1±2.7 to 17.8±2.0 Ωcm2. No net K+ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na+, Cl−, K+, Mg2+ and Ca2+ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca2+ and Mg2+ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.

The effect of somatostatin and alpha 2-adrenergic agonists on cyclic AMP accumulation was examined in MDCK cells, grown in defined medium. These hormones inhibited vasopressin-induced cyclic AMP formation, without affecting either the basal or the glucagon- and prostaglandin E2-stimulated level. Pretreating the cells with pertussis toxin, or incubating them with MnCl2 at a low concentration reversed the effect of somatostatin and alpha 2-agonists. These results suggest that somatostatin and norepinephrine could selectively modulate the renal effect of vasopressin, via the inhibitory regulatory subunit (Ni) of adenylate cyclase.

The Ca++ messenger system is nearly universally present in the control of cell functions by extracellular messengers. It elicits different kinds of responses: either brief, as in neurotransmission and in skeletal muscle contraction, or sustained, as in smooth muscle contraction and in the regulation of transepithelial transport systems. In this latter function the Ca++ messenger system interacts with other cellular modulation systems such as cAMP production and arachidonic acid cascade. In the vestibular apparatus, the Ca++ messenger system is involved in the transduction processes in that it controls both the release of neurotransmitter at the basal pole of the hair cell and the electrical resonance of the cell. It also figures in the contractile properties of cochlear outer hair cells. The Ca++ messenger system as well as adenylate cyclase and prostaglandins may play an important role in the modulation of endolymph secretion.

The origin of the endolymph was elucidated by kinetic studies of the entry of water and electrolytes into endolymph and perilymph after intravenous administration of radioactive tracers in rats. The compartmental analysis of the data and the comparison of this study with the results of Konishi and associates (Acta Otolaryngol (Stockh) 86, 22-34 and 176-184, 1978), using perilymphatic perfusion of tracers, indicate that perilymph rather than plasma may be considered the precursor of endolymph. Since the cochlear epithelium was found to be freely permeable to water, an alteration of electrolyte transportation across the membranous labyrinth may be involved in the pathophysiology of Ménière's disease. Chloride transport across the cochlear epithelium was investigated using acetazolamide, a specific carbonic anhydrase inhibitor.

The recent localization, in the rat, of a glucagon-sensitive adenylate cyclase in these segments where the bulk of calcium and magnesium is reabsorbed suggests an effect of this hormone on calcium and magnesium tubular transport. Renal tubular handling of calcium and magnesium as well as of sodium and phosphate was therefore studied by clearance methods in anesthetized rats, either intact or thyroparathyroidectomized (TPTX), infused with glucagon at a rate of 25 ng.min-1/100 g bw just after a priming dose of 2.5 micrograms. The hormone administration resulted in a significant decrease of absolute and fractional magnesium excretion (from 16.3 +/- 0.7% to 9.7 +/- 1.7% for intact rats and from 20.9 +/- 1.8% to 6.9 +/- 1.0% for TPTX rats), associated with the well-known increase in sodium and phosphate fractional excretion. Moreover, a small and transient decrease of calcium fractional excretion was observed concomitantly with a decrease of plasma calcium concentration. The significant increase in magnesium absolute reabsorption, observed whatever the filtered load and independently of PTH and calcitonin, may be an evidence for a direct tubular effect of glucagon.

Phosphate handling by the rat nephron during saline diuresis. Inorganic phosphate handling by the nephron was investigated using renal micropuncture techniques in fasted rats undergoing sustained saline diuresis and in controls. The early distal and the late accessible proximal convolutions of the same nephron were micropunctured. Despite the presence of decreased fractional volume reabsorption at the end of the proximal tubule during saline diuresis, the tubular fluid to plasma phosphate ratio was unaffected. Fractional phosphate delivery at this site was not significantly higher. Fractional phosphate delivery at the early distal and late proximal segments of the same nephron were significantly correlated in both groups. The slopes of the regression lines were not different. The delivery of phosphate to the early distal tubule, for a given value of delivery from the late proximal, was lower during saline diuresis. Mean fractional phosphate reabsorption between the late proximal and the early distal tubules was twice as high during saline diuresis as in controls. This observation may account for the decreased fractional phosphate excretion in ureteral urine observed in fasting animals. Clearance studies indicated a different behavior between fed and fasted rats undergoing saline diuresis. Fractional phosphate excretion increased in the former and decreased in the latter.

The pivotal role of nucleotides in cell function and metabolism has been acknowledged for more than three decades since the milestone work of Sutherland [1]. Intracellular nucleotides are involved in each and every event of cell life including synthesis of nuclear material, phosphorylation processes, or transduction of extracellular signals. As regards to this last point, a huge number of studies has been devoted to identifying the key role of GTP-binding proteins or G proteins, on the one hand, and of cyclic nucleotides cAMP and cGMP, on the other hand, in the early steps of cell signaling. In epithelial cells such as renal tubular cells, generation of these mediators as a hallmark of cell-hormone interaction has been investigated in detail [reviewed in 2]. More recently, experimental evidence was provided showing that extracellular nucleotides are able to affect virtually every organ and/or tissue system. These aspects have been extensively reviewed [3—7] and will not be discussed here. Instead, attention will be focused on the effect of extracellular nucleotides, primarily cAMP, on renal tubular transport of inorganic phosphate (Pi). The initial observations that systemic infusion of cAMP to parathyroidectomized rats induced phosphaturia, thus mimicking the proximal effect of parathyroid hormone (PTH), drew attention on the effects of extracellular cAMP on renal function [8, 9]. A striking feature was that other renal effects of PTH (modulation of calcium or magnesium excretion), which take place beyond the proximal tubule, or renal effects of other peptidic hormones that act through the cAMP-protein kinase A pathway in distal parts of the nephron, such as antidiuteric hormone, were not reproduced by cAMP infusion [9]. Along the same line, glucagon, infused or injected at large pharmacological doses that induced a rise of plasma cAMP concentration from hepatic origin and urine excretion of the nucleotide [8, 10], was shown to increase phosphate excretion [8]. These data raised the possibility that handling (metabolism and/or transport) of extracellular cAMP by proximal cells was involved in the effect of the cyclic nucleotide on Pi transport. In contrast with the extreme experimental conditions described above, physiological situations are characterized by a fairly constant systemic cAMP concentration, while luminal cAMP concentration varies with the parathyroid status [10—12]. These observations suggest that luminal cAMP may act as local paracrine modulator of Pi transport and raise three questions: (a) How does cAMP reach the extracellular space under normal situations; (b)

Localization of parathyroid-hormone-independent sodium bicarbonate inhibition of tubular phosphate reabsorption. We investigated phosphate transport along superficial nephrons in two groups of acutely parathyroidectomized (APTX) rats. Animals of group 1 were infused with 0.5 M sodium chloride; and those of group 2, with 0.5 M sodium bicarbonate at the same rates. Compared to the sodium chloride infusion, the sodium bicarbonate infusion was associated with a significant increase in urinary excretion of phosphate: the fractional phosphate excretion was 2.3 ± (SD) 1.3% in the sodium chloride group and 14.4 ± 3.2% in the sodium bicarbonate group, P < 0.01, whereas the fractional sodium excretion was identical, 7.4 ± 0.60% and 7.5 ± 0.50%. Micro-puncture studies performed at the late accessible proximal and early accessible distal sites of the same superficial nephrons indicate that the reabsorptive capacity for phosphate (absolute reabsorption/absolute delivered phosphate per nephron segment) is decreased during sodium bicarbonate infusion in the convoluted proximal tubule as well as in the loop (segment located between late proximal and early distal accessible convolutions) and the terminal nephron. Such an effect is independent of both parathyroid hormone secretion and extracellular fluid volume expansion.Localisation de l'inhibition par le bicarbonate de sodium, indépendamment de l'hormone parathyroidiene de la réabsorption tubulaire du phosphate. Le transport de phosphate le long des néphrons superficiels a été étudié dans deux groupes de rats en situation de parathyroïdectomie aiguë (APTX). Les animaux du groupe 1 ont été perfuses avec 0,5 M chlorure de sodium et ceux du groupe 2 avec 0,5 M bicarbonate de sodium au même débit. Par comparaison avec l'effet du chlorure de sodium, la perfusion de bicarbonate de sodium est contemporaine d'une augmentation significative de l'excrétion urinaire de phosphate: l'excrétion fractionnelle est de 2,3 ± 1,3% (moyenne ± déviation standard) dans le groupe chlorure de sodium et 14,4 ± 3,2% dans le groupe bicarbonate de sodium, P < 0.001, alors que l'excrétion fractionnelle du sodium est identique, 7,4 ± 0,60% et 7,5 ± 0,50%. Les microponctions réalisées à la fin du tube proximal et au début du tube distal accessibles indiquent que la capacité de réabsorption pour le phosphate (réabsorption absolue/débit absolu délivré à chaque segment) est diminuée au cours de la perfusion de bicarbonate de sodium dans le tube contourné proximal de même que dans l'anse (segment compris entre la fin du proximal et le début du distal accessibles) et dans le néphron terminal. Cet effet est indépendant de la sécrétion d'hormone parathyroïdienne et de l'expansion des liquides extra-cellulaires.

To evaluate a possible modulation by protein kinase C of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of protein kinase C activators and of bradykinin on intracellular cAMP accumulation in MDCK cells. A 15-min pretreatment of cells with phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetylglycerol induced a dose-dependent inhibition of vasopressin-stimulated cAMP synthesis, but not of basal or glucagon-, prostaglandin E2-, and forskolin-stimulated cAMP generation. 4 alpha-Phorbol 12,13-didecanoate, inactive on protein kinase C, did not affect cAMP accumulation. Bradykinin (0.1-10 microM) also inhibited the stimulatory effect of vasopressin on cAMP synthesis in a concentration-dependent manner, but affected neither basal cAMP content, nor its stimulation by glucagon, prostaglandin E2 and forskolin. The effect of activators of protein kinase C and of bradykinin occurred while renal prostaglandin synthesis was blocked with indomethacin. The inhibitory effect of protein kinase C activators and bradykinin on cAMP generation was reversed by the protein kinase C inhibitor H7, was enhanced by monensin, one effect of which is to block the recycling of membrane receptors, and persisted when the GTP-binding protein N1 was blocked with 1 mM Mn2+. Our data suggest that: protein kinase C can modulate the tubular effects of vasopressin by inhibiting cAMP generation; this effect is not mediated by renal prostaglandins, and might result from a direct action on the vasopressin receptor, or on its coupling with Ns; the modulation by bradykinin of vasopressin effects are likely to be exerted, at least partly, through activation of protein kinase C.

The effects of glucagon and PTH on electrolyte reabsorption in the distal tubule were investigated in rats deprived of vasopressin, calcitonin, PTH, and glucagon. Micropunctures of distal tubule, at a late and an early site of a same nephron, have been performed in 23 rats, nine control, seven infused with glucagon (5 ng X min-1 X 100 g-1 b.w.) and seven with PTH (5 mU X min-1 X 100 g-1 b.w.). The Ca and Mg reabsorptive capacity of the distal segment was increased by glucagon and by PTH. Moreover, fractional Na and Cl reabsorption was significantly higher than in control during PTH administration. A K secretion appeared during the administration of both hormones. No phosphate net transport was observed in any group. Finally, the data presented here, together with those previously reported, indicate that the increase of Ca and Mg renal reabsorption observed with glucagon and PTH results from an effect located in both Henle's loop, where the bulk of Ca and Mg is reabsorbed, and the distal tubule.

The entry of arginine-vasopressin (AVP) and sucrose into cochlear endolymph, perilymph of scala vestibuli (PLV), perilymph of scala tympani (PLT), and cisternal cerebrospinal fluid (CSF) was studied, in anesthetized rats, after the administration into the cerebral lateral ventricle of a 10μ1 solution containing the radioactive tracers. Both tracers were detected in PLV, PLT, and CSF but not in endolymph. Monoexponential decay curves were calculated for PLV, PLT, and CSF, and for each tracer no difference was found between the regression lines calculated for the different fluids. These results indicate that (i) injection into the cerebral lateral ventricle is a useful tool to study the permeability of the cochlear epithelium to different solutes and (ii) no specific transport system exists for AVP across the cochlear epithelium, suggesting that AVP may exert its effect via the perilymphatic side of the stria vascularis.

Antiarrhythmic drugs with local anesthetic properties modify the physical state of membrane phospholipids and could change adenylate cyclase activity and, thus, influence cardiac ischemic arrhythmias. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cardiomyocytes cultured from newborn rat and fluorescence anisotropy of sarcolemma-enriched membranes were investigated in the presence of a neutral anesthetic drug benzyl alcohol and of a cationic anesthetic drug lidocaine. Benzyl alcohol increased in a dose-dependent manner both sarcolemma fluidity and isoproterenol- or cholera toxin-stimulated cAMP accumulation. In contrast, benzyl alcohol inhibited cAMP accumulation in forskolin-stimulated cells. Lidocaine induced a dose-related inhibition of isoproterenol-, forskolin-, and cholera toxin-stimulated cAMP accumulation without eliciting any change in sarcolemma fluidity. The inhibitory effect of lidocaine on isoproterenol-stimulated cAMP accumulation was reversed when cells were pretreated with pertussis toxin. These data suggest that the inhibitory effect of lidocaine on cAMP synthesis might involve a polar interaction with the Gi regulatory subunit of adenylate cyclase. Such an effect could contribute, in vivo, to both the antiarrhythmic and the negative inotropic effect of lidocaine.

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na+, Cl-, K+, Ca2+ and Mg2+ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (JNa+, JCl-, JK+, JCa2+, JMg2+) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PDte) and transepithelial resistance (Rte) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO3- containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO3- free Ringer solutions. In cTAL segments, AVP (10(-10) mol.l-1) significantly increased JMg2+ and JCa2+ from 0.39 +/- 0.08 to 0.58 +/- 0.10 and from 0.86 +/- 0.13 to 1.19 +/- 0.15 pmol.min-1 mm-1 respectively. Neither JNa+ nor JCl-, (JNa+: 213 +/- 30 versus 221 +/- 28 pmol.min-1 mm-1, JCl-: 206 +/- 30 versus 220 +/- 23 pmol.min-1 mm-1) nor PDte (13.4 +/- 1.3 mV versus 14.1 +/- 1.9 mV) or Rte (24.6 +/- 6.5 omega cm2 versus 22.6 +/- 6.4 omega cm2) were significantly changed by AVP. No significant effect of AVP on net K+ transport was observed. In mTAL segments, Mg2+ and Ca2+ net transports were close to zero and AVP (10(-10) mol.l-1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone, JNa+ increased from 107 +/- 33 to 148 +/- 30 and JCl- from 121 +/- 33 to 165 +/- 32 pmol.min-1 mm-1.(ABSTRACT TRUNCATED AT 250 WORDS)