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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 rag. [kg.sup.-1] [day.sup.-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 2 to clay 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. heavy metals; renal failure; newborn; claudins

We have previously shown that [K.sup.+]-selective TASK2 channels and swelling-activated [Cl.sup.-] 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 Physiol 122: 177-190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: 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.sup.-] and [K.sup.+] 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.sup.+] currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH ([pH.sub.i]) and increased the external pH ([pH.sub.e]) in wild-type but not in [cftr.sup.-/-] cells. The inhibitory effect of DIDS suggests that the acidification of [pH.sub.i] and the alkalinization of pile induced by hypotonicity in wild-type cells could be due to an exit of HC[O.sup.-.sub.3]. In conclusion, these results indicate that [Cl.sup.-] influx will be the driving force for HC[O.sup.-.sub.3] exit through the activation of the [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger. This efflux of HC[O.sup.-.sub.3] then alkalinizes pile, which in turn activates TASK2 channels. regulatory volume decrease; CFTR; KCNE5; potassium and chloride channels; external and internal pH

This study investigates the effect in the rat of chronic Cd[Cl.sub.2] intoxication (500 [micro]g [Cd.sup.2+]/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 Zn[Cl.sub.2] (500 [micro]g [Zn.sup.2+]/kg) during chronic Cd[Cl.sub.2] intoxication. Our results indicate that 1) most of the filtered [Cd.sup.2+] is reabsorbed within the kidney; 2) chronic [Cd.sup.2+] 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 CD[Cl.sub.2] exposure; 4) epithelial integrity is altered by chronic [Cd.sup.2+] intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with [Zn.sup.2+] protects against the renal toxic effects of [Cd.sup.2+], preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that [Cd.sup.2+] toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, [Zn.sup.2+] supplementation may provide a basis for future treatments. heavy metals; kidney; protective treatment; tight junction

Acute study of interaction among cadmium, calcium, and zinc transport along the rat nephron in vivo. Am J Physiol Renal Physiol 287: F1067-F1075, 2004. First published July 27, 2004; doi:10.1152/ajprenal.00120.2004.--This study investigates the effect in rats of acute Cd[Cl.sub.2] (5 [micro]M) intoxication on renal function and characterizes the transport of [Ca.sup.2+], [Cd.sup.2+], and [Zn.sup.2+] 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 [Cd.sup.2+] injection resulted in renal losses of [Na.sup.+], [K.sup.+], [Ca.sup.2+], [Mg.sup.2+], P[O.sup.-2.sub.4], and water, but the glomerular filtration rate remained stable. [sup.45]Ca microinjections showed that [Ca.sup.2+] permeability in the DT was strongly inhibited by [Cd.sup.2+] (20 [micro]M), [Gd.sup.3+] (100 [micro]M), and [La.sup.3+] (1 mM), whereas nifedipine (20 [micro]M) had no effect, [sup.109]Cd and [sup.65][Zn.sup.2+] microinjections showed that each segment of nephron was permeable to these metals. In the PT, 95% of injected amounts of [sup.109]Cd were taken up. [sup.109]Cd fluxes were inhibited by [Gd.sup.3+] (90 [micro]M), [Co.sup.2+] (100 [micro]M), and [Fe.sup.2+] (100 [micro]M) in all nephron segments. Bumetanide (50 [micro]M) only inhibited [sup.109]Cd fluxes in LH; [Zn.sup.2+] (50 and 500 [micro]M) inhibited transport of [sup.109]Cd in DT. In conclusion, these results indicate that 1) the renal effects of acute [Cd.sup.2+] intoxication are suggestive of proximal tubulopathy; 2) [Cd.sup.2+] inhibits [Ca.sup.2+] reabsorption possibly through the epithelial [Ca.sup.2+] channel in the DT, and this blockade could account for the hypercalciuria associated with [Cd.sup.2+] intoxication; 3) the PT is the major site of [Cd.sup.2+] reabsorption; 4) the paracellular pathway and DMT1 could be involved in [Cd.sup.2+] reabsorption along the LH; 5) DMT1 may be one of the major transporters of [Cd.sup.2+] in the DT; and 6) [Zn.sup.2+] is taken up along each part of the nephron and its transport in the terminal segments could occur via DMT1. heavy metals; epithelial calcium channel; divalent metal transporter 1; kidney

The ionic currents from cell swelling were described in primary cultures of rabbit distal clamp method using the whole cell patch-clamp method. Chlorine ion currents were instantaneously generated by whole cell recording utilizing an isotonic pipette solution. The results revealed that the initial ion currents was observed to have outwardly rectifying current-voltage relationship. The currents were prevented by 0.1 millimole 5-nitro-2-(3-phenylpropylamino)benzoic acid and severely blocked by one millimole 4,4'-diisothiocyadiphelamine-2-carboxylate.

An experiment was performed to study the role of ion channels involved in the movement of ions across the apical membranes epithelial gill cells of cultures of the sea bass. Chloride channels that show inward rectification and slow open kinetics were observed in the primary cultures. The similarity of properties seen in the experiment and in other experiments of other types of fish implies that respiratory cells have an important role in ion movement control in the gill of seawater fish.

The chloride permeability of cultured rabbit distal 'bright' convoluted tubules (DCTb) was analyzed by whole cell clamp and fluorescence methods. Extracellular application of ionomycin increased the levels of Cl- currents in a time- and voltage-dependent manner with steady-state outward rectification. The apical membrane of the cultured DCTb cells exhibited K+ conductance channels and small-conductance Cl- channels that are regulated by Ca2+.

Duranton, C., E. Mikulovic, M. Tauc, M. Avella, and P. Poujeol. Potassium channels in primary cultures of seawater fish gill cells. II. Channel activation by hypotonic shock. Am J Physiol Regulatory Integrative Comp Physiol 279: R1659-R1670, 2000.--Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated [K.sup.+] channels with a conductance of 122 pS. The present report examines the involvement of [K.sup.+] channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, [K.sup.+] currents were produced by exposing cells to a hypotonic solution or to 1 [micro] M ionomycin. These K+ currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using [sup.86][Rb.sub.+] as a tracer to indicate potassium movements. Apical and basolateral membrane [sup.86]Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of [sup.86]Rb efflux on the apical side of the monolayer was inhibited by 500 [micro] M quinidine or 100 [micro] M gadolinium but was insensitive to scorpion venom [Leirus quinquestriatus hebraeus (LQH)]. An increased [sup.86]Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane [sup.86]Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of [K.sup.+] channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology. whole cell; patch clamp; [K.SUP.+] and [Cl.sup.-] ion fluxes; gill epithelium

Duranton, C., E. Mikulovic, M. Tauc, M. Avella, and P. Poujeol. Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated [K.sup.+] channels. Am J Physiol Regulatory Integrative Comp Physiol 279: R1647-R1658, 2000.--Previous studies using the patchclamp technique demonstrated the presence of a small conductance [Cl.sup.-] channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A [K.sup.+] channel of 123 [+ or -] 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for [K.sup.+] over [Na.sup.+] and [Ca.sup.2+]. The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 [micro] M), and gadolinium (500 [micro] M). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on [K.sup.+] channel activity is consistent with the hypothesis that stretch-activated [K.sup.+] channels could mediate an increase in [K.sup.+] conductance during cell swelling. mechanosensitive [K.sup.+] channels; patch clamp; gill epithelium

Background and Purpose For decades, inhibitors of the cystic fibrosis transmembrane conductance regulator ( CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh-172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl−-conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non- CFTR-expressing cells. Experimental Approach Using patch-clamp technique, we tested the effects of CFTRinh-172 and GlyH-101 inhibitors on three distinct types of Cl− currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl− conductance ( VSORC) and finally the Ca2+-dependent Cl− conductance ( CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines. Key Results We confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl− conductance. However, GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh-172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations. Conclusions and Implications Both inhibitors affected Cl− conductances apart from CFTR. Our results provided insights into their use in mouse models. [ABSTRACT FROM AUTHOR]

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 Physiol 122: 177-190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: 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 pHi and the alkalinization of PHe induced 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. [ABSTRACT FROM AUTHOR]

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 microM LaCl3, 10 microM nifedipine, 100 microM verapamil, 5 microM tamoxifen, and 50 microM 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. [ABSTRACT FROM AUTHOR]

Studies calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule. Composition of solutions used in whole cell clamp experiments; Primary cultures; Fluorescence experiments; Intracellular calcium measurements; Basolateral membranes; Representative tracings of effects of forskolin and ionomycin.