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In the adult rat, chronic cadmium intoxication induces nephropathy with Fanconi-like features. This result raises the question of whether intoxication of pregnant rats has any deleterious effects on renal function in their offspring. To test this hypothesis, we measured the renal function of 2- to 60-day-old postnatal offspring from female rats administered cadmium chloride by the oral route (0.5 mg·kg−1·day−1) throughout their entire gestation. Investigations of rat offspring from contaminated pregnant rats showed the presence of cadmium in the kidney at gestational day 20. After birth, the cadmium kidney concentration increased from postnatal day 2to day 60(PND2 to PND60), presumably because of 1) milk contamination and 2) neonatal liver cadmium content release. Although the renal parameters (glomerular filtration, U/P inulin, and urinary excretion rate) were not significantly affected until PND45, renal failure appeared at PND60, as demonstrated by a dramatic decrease of the glomerular filtration rate associated with increased excretion of the main ions. In parallel, an immunofluorescence study of tight-junction protein expression of PND60 offspring from contaminated rats showed a disorganization of the tight-junction proteins claudin-2 and claudin-5, specifically expressed in the proximal tubule and glomerulus, respectively. In contrast, expression of a distal claudin protein, claudin-3, was not affected. In conclusion, in utero exposure of cadmium leads to toxic renal effects in adult offspring. These results suggest that contamination of pregnant rats is a serious and critical hazard for renal function of their offspring.

We have previously shown that K+-selective TASK2 channels and swelling-activated Cl−currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol122: 177–190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol284: F812–F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl−and K+currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K+currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pHi) and increased the external pH (pHe) in wild-type but not in cftr−/−cells. The inhibitory effect of DIDS suggests that the acidification of pHiand the alkalinization of pHeinduced by hypotonicity in wild-type cells could be due to an exit of HCO3−. In conclusion, these results indicate that Cl−influx will be the driving force for HCO3−exit through the activation of the Cl−/HCO3−exchanger. This efflux of HCO3−then alkalinizes pHe, which in turn activates TASK2 channels.

This study investigates the effect in the rat of chronic CdCl2intoxication (500 μg Cd2+/kg, daily ip injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2(500 μg Zn2+/kg) during chronic CdCl2intoxication. Our results indicate that 1) most of the filtered Cd2+is reabsorbed within the kidney; 2) chronic Cd2+intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2exposure; 4) epithelial integrity is altered by chronic Cd2+intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+supplementation may provide a basis for future treatments.

This study investigates the effect in rats of acute CdCl2(5 μM) intoxication on renal function and characterizes the transport of Ca2+, Cd2+, and Zn2+in the proximal tubule (PT), loop of Henle (LH), and terminal segments of the nephron (DT) using whole kidney clearance and nephron microinjection techniques. Acute Cd2+injection resulted in renal losses of Na+, K+, Ca2+, Mg2+, PO4−2, and water, but the glomerular filtration rate remained stable. 45Ca microinjections showed that Ca2+permeability in the DT was strongly inhibited by Cd2+(20 μM), Gd3+(100 μM), and La3+(1 mM), whereas nifedipine (20 μM) had no effect. 109Cd and 65Zn2+microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of 109Cd were taken up. 109Cd fluxes were inhibited by Gd3+(90 μM), Co2+(100 μM), and Fe2+(100 μM) in all nephron segments. Bumetanide (50 μM) only inhibited 109Cd fluxes in LH; Zn2+(50 and 500 μM) inhibited transport of 109Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute Cd2+intoxication are suggestive of proximal tubulopathy; 2) Cd2+inhibits Ca2+reabsorption possibly through the epithelial Ca2+channel in the DT, and this blockade could account for the hypercalciuria associated with Cd2+intoxication; 3) the PT is the major site of Cd2+reabsorption; 4) the paracellular pathway and DMT1 could be involved in Cd2+reabsorption along the LH; 5) DMT1 may be one of the major transporters of Cd2+in the DT; and 6) Zn2+is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1.

Primary cultures of gill cells from freshwater and seawater-adapted trout were compared. These cultures, developed from an explant technique, exhibited a similar growth. Ultrastructural comparison between cultured and in situ cells showed that most of the cells in primary culture resembled the so called 'pavement' cells, whereas chloride cells were not observed in the cultured epithelium. Several other cells types, representing a minority of cells in primary culture, were observed (mucous cells, vesicolar cells, cells with large dense granules and cells containing lysosomes). Morphological observations of cultured pavement cells from freshwater and seawater trout gills were similar, although the density of cellular organelles in cells was less under freshwater conditions. In addition to the morphological comparison, the regulation of intracellular pH in cultured cells from freshwater and seawater gills was examined. Resting pHi was not different for freshwater or seawater gill cells. A sodium-dependent and amiloride-sensitive mechanism was found in cultured cells. Under the experimental conditions used here, this mechanism was most likely a Na+/H+antiporter in pavement cells from freshwater and seawater-adapted trout. The comparison of pHi recovery after acidification of cells from freshwater and seawater gills showed that the activity or the number of antiporters was higher for cells from seawater trout gill.

The purpose of this study was to investigate intracytoplasmic pH (pHi) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pHi was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pHi values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pHi by means of the classic Na+/H+ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pHi recovery was mediated by a Na+/H+ exchange present in the basolateral side. Moreover, at physiological initial pHi values, chloride removal from the apical solution caused the pHi to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pHi recovery was induced in sodium-free media by 15 mM HCO3-in the presence of an outward chloride gradient. This pHi change was completely abolished by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na+-independent Cl-/HCO3-exchanger.

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured, to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occured during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occured only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.

Abstract.: Chloride (Cl−) conductances were studied in primary cultures of the bright part of rabbit distal convoluted tubule (DCTb) by the whole cell patch clamp technique. The bath solution (33°C) contained (in mm): 140 NaCl, 1 CaCl2, 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4 and the pipette solution 140 N-methyl-d-glucamine (NMDG)-Cl, 5 MgATP, 1 ethylene-glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, pH 7.4. We identified a Cl− current activated by 10−5m forskolin, 10−3m 8-bromo adenosine 3′,5′-cyclic monophophosphate (8 Br-cAMP), 10−6m phorbol 12-myristate 13-acetate (PMA), 10−3m intracellular adenosine 3′,5′-cyclic monophophosphate (cAMP) and 10−7m calcitonin. The current-voltage relationship was linear and the relative ion selectivity was Br− > Cl−≫ I− > glutamate. This current was inhibited by 10−3m diphenylamine-2-carboxylate (DPC) and 10−4m 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and was insensitive to 10−3m 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These characteristics are similar to those described for the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− conductance. In a few cases, forskolin and calcitonin induced an outwardly rectifying Cl− current blocked by DIDS. To determine the exact location of the Cl− conductance 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) fluorescence experiments were carried out. Cultures seeded on collagen-coated permeable filters were loaded overnight with 5 mm SPQ and the emitted fluorescence analyzed by laser-scan cytometry. Cl− removal from the apical solution induced a Cl− efflux which was stimulated by 10−5m forskolin, 10−7 calcitonin and inhibited by 10−5m NPPB. In 140 mm NaBr, forskolin stimulated an apical Br− influx through the Cl− pathway. Forskolin and calcitonin had no effect on the basolateral Cl− permeability. Thus in DCTb cultured cells, exposure to calcitonin activates a Cl− conductance in the apical membrane through a cAMP-dependent mechanism.

To examine the intracellular pH (pHi) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pHi measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na+/H+ exchanger and an apical Cl-/HCO3-exchanger [1]. The pHi of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na+/H+ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pHi is the Cl-/HCO3-exchanger. This study confirms the importance of this transporter in pHi regulation within the physiological pHi range and the influence of calcitonin in the regulation of DCTb cell function.

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.

Primary cultures were obtained from microdissected rabbit proximal tubules (S1 segments). The growing epithelia were maintained in culture for up to 30 days. Electron microscopy study revealed that the cells formed a monolayer and showed a morphological polarity with apical microvilli and tight junctions. An immunofluorescence technique using two monoclonal antibodies raised against two apical brush border enzymes of the proximal tubule (LAP, DPP IV) revealed that these enzymes were expressed in the cultured cells. Membrane associated and cytosolic enzyme activities were measured on 12, 20 and 30-day-old cultures. Cultured epithelia exhibited leucine aminopeptidase, ? glutamyl transferase and fructose 1–6 biphosphatase activities that remained constant for up to 30 days, whereas alkaline phosphatase activity decreased in the oldest cultures. Hexokinase activity on the other hand, increased after 12 days of culture. Cyclic AMP synthesis was stimulated by parathyroid hormone at 12, 20 and 30 days of culture and was insensitive to arginine vasopressin. After 20 days of culture the epithelia grown on permeable supports developed a transepithelial potential of -0.13 mV (apical negative) and a transepithelial resistance of 37 O cm2 that increased to -1.13 mV and 60 O cm2 respectively in 30-day-old cultures. The patch clamp technique was applied to the apical membrane of 12–15-day-old cultures. In the whole cell recording configuration, a cellular potential of -61.5 mV was measured, which was mainly due to K+ diffusion. A non-selective cationic channel was present in the apical membrane of the cultured cells. In cell-attached patches the channel carried an inward current and had a conductance of 13 pS. On excised patches the channel discriminated poorly between Na+ and K+ and was impermeant to Cl- and its conductance ranged between 20 and 28 pS. The channel activity was not voltage dependent but required a high calcium concentration (1 mM Ca2+) on the cytoplasmic face.

86Rb+efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeusvenom and charybdotoxin was attributed to the stimulation of Ca++-activated maxi K+channels. Snake venom from Dendroaspis polylepisdid not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca++-activated maxi K+channels were predominently found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca++-activated maxi K+channel present in MDCK cells.

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl−currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl−currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I−= Br−> Cl−≫ glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid and 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 μM LaCl3, 10 μM nifedipine, 100 μM verapamil, 5 μM tamoxifen, and 50 μM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+was without effect on the fully developed Cl−currents. Osmotic shock also activated K+currents in Cl−-free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl−and K+currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.