INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

EPITHELIAL CELLS SHOW extensive regulation of cell volume during 1) developmental or regenerative proliferation, 2) transepithelial transport when cell solutes are accumulated or lost, and 3) adaptation to large changes in environmental osmolarity, which, for instance, occurs periodically in the renal medulla (14). In embryonic development, proliferating cells exhibit large changes in cell volume and shape dependent on cell cycle and cell migration (14). The collecting duct epithelium of the metanephric kidney develops by sequential branching and outgrowth of the embryonic ureteric bud (UB) (21). In rat kidney, this branching morphogenesis begins around embryonic day E14 and lasts until about postnatal day P6. During this time, cells of the UB proliferate and, most importantly, express an apolar epithelial phenotype as defined by the symmetrical, apolar distribution of the Na-K-ATPase -subunit in both basolateral and apical plasma membrane (16). After morphogenesis has been completed, functional epithelial cell polarization occurs by the acquisition of different ion channel types in the apical membrane of the cortical collecting duct (CCD) cell (10, 20). Mature, nonepithelial cells, as well as mature, epithelial cells (e.g., CCD principal cells, see Ref. 15) downregulate their cell volume after hypotonic stress-evoked swelling. This regulatory volume decrease (RVD) utilizes several channel types, e.g., volume-sensitive, organic osmolyte and anion channels (VSOAC) (23), probably in concert with ClC-2 Cl-selective channels (24). The present study investigates for the first time the development-dependent expression of hypotonic swelling-activated ion conductances in nephrogenesis. For this purpose, embryonic UB (day E17) and perinatal UB (P1-6) and postnatal CCD (P9-14) were compared in primary monolayer culture. These cultures of microdissected UBs and CCDs had been shown previously to conserve their stage-dependent phenotypes (10). The data imply that the mature type of hypotonic swelling-activated Cl conductance may evolve already in UB cells between embryonic day E17 and perinatal day P1.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Primary monolayer cultures. Electrical recordings were made in renal epithelial cells of defined segmental origin in primary culture, as previously described (10). Specifically, branching UB and CCD were microdissected from the outermost cortex of embryonic (UB day E17), perinatal (UB days P1-6), and postnatal (CCD days P9-14) rat kidney (Sprague-Dawley), respectively, in Ca²⁺-free and Mg²⁺-free, phosphate-buffered solution at 4°C without enzymatic digestion. UB and CCD were explanted on dishes coated with several thin layers of newborn rat tail collagen, and attached to the matrix. Cells migrated from the tubular epithelium at those sites where the basal lamina had been injured mechanically, proliferated, and formed confluent monolayers within 1-3 days of culture in nephron culture medium (9) supplemented with 10% FCS and bovine pituitary extract (50 µg/ml; Sigma, Deisenhofen, Germany). Culture medium was exchanged daily and was replaced 24 h before an experiment by medium containing 1 µM aldosterone (Sigma) instead of FCS.

Ultrastructure. For standard scanning electron microscopy, postconfluent monolayers were fixed at room temperature using modified Karnovsky reagent (2% paraformaldehyde and 2.5% glutaraldehyde in 80 mM phosphate buffer pH 7.4), postfixed with 1% OsO₄ (in 100 mM phosphate buffer, pH 7.4), dehydrated by increasing concentrations of ethanol, and critical-point dried.

Uncoupling. Epithelial cells were uncoupled to achieve free access of bath solutions to the basolateral membrane and to isolate electrically the recorded cell from the monolayer. For this purpose, dishes were rinsed with uncoupling solution and mounted on the stage of an inverted microscope equipped with differential-interference contrast optics (Zeiss, Oberkochen, Germany), and cells were superfused (see below) with the uncoupling solution (in mM: 150 NaCl, 10 D-glucose, 10 HEPES, 5 KCl, 3 EGTA, 0.91 MgCl₂, and 0.171 CaCl₂, titrated with NaOH to pH 7.2). Superfusion was continued for ~30 min until the morphology of the cobblestone-like epithelial cells had changed to spherical shape and well-defined cell borders and intercellular spaces had appeared (see RESULTS).

Patch-clamp recordings. Experiments were performed at 37°C on uncoupled cells from postconfluent monolayers that were morphologically highly differentiated. Continuous superfusion (0.5 ml/min) was applied through a flow system inserted into the dish, which reduced bath volume to ~50 µl. The bath was earthed via a 2% agarose bridge filled with pipette solution. Borosilicate glass pipettes (2-5 M tip resistance, model GC 150 TF-10; Clark Medical Instruments, Pangbourne, UK) manufactured by a microprocessor-driven puller (Zeitz, Augsburg, Germany) were used in combination with a water hydraulic micromanipulator (model WR-88; Narishige, Tokyo, Japan). Currents were recorded in the fast whole cell voltage-clamp mode and were 1-kHz low-pass filtered by an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Whole cell currents (series resistances of some 10 M) were evoked by a pulse protocol, clamping the voltage in 11 successive 400-ms square pulses from the 0 mV holding potential to potentials between 100 mV and +100 mV. Pulse protocols were applied and data were sampled with a rate of 5 kHz by a microcomputer, using pClamp software and a TL1 DMA-Interface (Axon Instruments). Whole cell currents were normalized between individual cells by reference to the membrane capacitance as determined from the capacitive current transient evoked by a 10-mV voltage step. The current transient was fitted by exponential regression after 10-kHz low-pass filtering. For the whole cell recordings, membrane capacitance and series resistance were compensated to 100 and 80%, respectively. Whole cell slope conductance (in nS/10 pF, means ± SE) of the outward currents between +20 and +80 mV command voltage was approximated by linear regression.

Experimental protocol. Whole cell currents were recorded first in isotonic and then in hypotonic bath solutions (containing in mM: 150 and 100 NaCl, 10 D-glucose, 10 HEPES, 5 KCl, 1.6 CaCl₂, and 0.8 MgCl₂, titrated with NaOH to pH 7.2, 315 and 215 mosmol/kgH₂O, respectively). The pipettes were filled with (in mM) 100 potassium D-gluconate, 33 KCl, 3 EGTA, 1.82 MgCl₂ (0.8 free Mg²⁺), 1 dipotassium ATP, 0.171 CaCl₂ (10⁸ M free Ca²⁺), and 10 mM HEPES, titrated to pH 7.2 with KOH, 270 mosmol/kgH₂O. For isotonic conditions, an osmolarity of 270 mosmol/kgH₂O in the pipette was combined with 315 mosmol/kgH₂O in the bath to repress spontaneous cell swelling, which has been reported during the use of isosmotic bath and pipette solutions (26). Some experiments were performed using a pipette solution in which 133 mM potassium was replaced by 133 mM N-methyl-D-glucamine (NMDG⁺), additionally containing (in mM) 3 EGTA, 1.82 MgCl₂, 1 Tris-ATP, 0.171 CaCl₂, and 10 mM HEPES, titrated to pH 7.2 with 33 HCl and D-gluconic acid, 270 mosmol/kgH₂O. Cl currents were identified and characterized under isotonic control conditions and during cell swelling by substituting equimolar amounts of sodium D-gluconate, NaI, NaSCN, and NaBr for bath NaCl, or by application of DIDS (100 µM; Sigma) into the bath. Offset potentials between both electrodes were zeroed before sealing, whereby the liquid junction potential between agarose bridge (filled by pipette solution) and bath was equal and opposite to that between pipette and bath. After sealing, the remaining liquid junction potentials (E = E^S  E^P) between the bath (S) and the agarose bridge (P) were estimated according to Ref. 4 by the equation where where F is Faraday's constant; R is the gas constant; T is the absolute temperature; and u, a, and z represent the mobility, activity, and valency (including sign) of each ion species (i). Relative u values of u_K = 1.0, u_Na = 0.682, u_NMDG = 0.5, u_Cl = 1.0388, u_I = 1.045, u_SCN = 0.9, u_Br = 1.063, and u_gluconate = 0.5 were assumed. Data were corrected for the estimated E values.

Data analysis and statistics. Data are means ± SE. Differences between mean values were defined by unpaired t-test or Welch's approximation (25) (two-tailed) using InStat (GraphPad Software, San Diego, CA). P  0.05, P  0.01, and P  0.001 are indicated by single, double, and triple asterisks, respectively.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Embryonic (E17) and perinatal (P1-6) UBs as well as postnatal (P9-14) CCDs in identical primary monolayer culture expressed the morphological phenotype of the principal cell. This phenotype was defined in scanning electron micrographs by smooth apical membranes with scarce, stubby microvilli and a prominent central cilium (Fig. 1). Cells from the three developmental stages exhibited similar mean whole cell capacitances after uncoupling by EGTA (~15 pF; Fig. 2), which suggested similar cell sizes.

View larger version (160K): [in this window] [in a new window]   Fig. 1.   Morphology of embryonic and mature collecting duct epithelium in primary culture. Scanning electron microscopic view of the apical surface of two monolayers grown from microdissected ureteric buds (UB, embryonic day E17, left) and from cortical collecting ducts (CCD, day P14, right), respectively. The different ontogenetic stages, i.e., UB E17 and UB P1-6 (not shown) and CCD (P9-14), homogeneously express only one cell type, which is characterized as a principal cell-like cell by the central cilium and the smooth microvillous aspect.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Effect of EGTA treatment on cell-cell contact. Light micrographs show UB monolayers (A) before and (B) during superfusion with EGTA solution (see METHODS). C: mean whole cell capacitance (means ± SE; n = number of cells) of primary cultured embryonic (E17), perinatal (P1-6) UB, and postnatal (P9-14) CCD cells as determined after uncoupling by EGTA.

Whole cell current densities and derived specific conductances (G, expressed in nS/10 pF), recorded with a KCl/K-D-gluconate pipette and isotonic NaCl bath solution, differed substantially between embryonic UB (G = 4.0 ± 0.5; n = 31), perinatal UB (G = 2.0 ± 0.1; n = 16), and postnatal CCD cells (G = 1.3 ± 0.2 nS/10 pF; n = 12; Fig. 3, A-C). In all three developmental stages, current/voltage (I/V) curves of the mean, whole cell current density rectified outwardly. The reversal potentials (V_rev) were more positive than the chloride equilibrium potential (E_Cl = 38 mV), suggesting a high fractional, nonselective cation or Na-selective conductance (Fig. 3B). A characteristic fraction of whole cell current in most of the embryonic UB cells (22 of 31 cells) activated time dependently at high positive voltages (time constant  = 35 ± 5 ms at +100 mV) and inactivated at high negative voltages ( = 17 ± 2 ms; see Fig. 3A and 4A). Perinatal UB and postnatal CCD cells, in contrast, did not exhibit this activating current (Fig. 3D). The main fraction of whole cell conductance G in embryonic UB cells (G = 2.8 ± 0.7, G_control = 5.3 ± 0.6 nS/10 pF; n = 12) was sensitive to DIDS (100 µM), whereas conductance of postnatal CCD (G = 0.05 ± 0.1, G_control = 1.26 ± 0.06 nS/10 pF; n = 7) cells was not sensitive to DIDS, and that of perinatal UB cells was sensitive only by ~25% (G = 0.53 ± 0.07, G_control = 1.9 ± 0.1 nS/10 pF; n = 9; Fig. 3E).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Development-dependent changes in whole cell currents under isotonic control conditions. A: original, whole cell current traces of UB (E17 and P1-6; left and middle, respectively) and CCD (right), recorded with KCl/K-D-gluconate pipette and isotonic NaCl bath solution. Current traces, evoked by eleven 400-ms square pulses from 0 mV holding potential to voltages between 100 mV and +100 mV, are superimposed. B: current density/voltage relation and corresponding specific conductance (C) of whole cell currents in UB (E17 and P1-6) and CCD cells, recorded as in A. Specific conductances are calculated for outward currents 5 ms after the beginning of each voltage step. D: time-dependently activating fraction of specific conductance, determined by subtracting outward currents at 5 ms from those at 395 ms (same experiments as in C and D). E: DIDS (100 µM)-sensitive fraction of specific conductances (calculated as in C). Values are means ± SE; n = number of cells.

The main fraction of whole cell current of embryonic UB cells in isotonic NaCl bath solution was Cl selective. This conclusion is based on the following observations. 1) Substitution of bath chloride by gluconate markedly decreased the outward currents as determined with standard KCl/gluconate pipette buffer at the end of each square pulse (G = 4.5 ± 0.6, G_control = 7.2 ± 0.7 nS/10 pF; n =12). As a result of this Cl replacement, reversal potentials shifted by V_rev = +23 ± 5 mV (Fig. 4B; see also Fig. 4, D and E). 2) Outward currents, recorded with NMDG as the impermeant cation in the pipette, were in the range of the above current fraction responsive to bath chloride-by-gluconate replacement (Fig. 4B). 3) Also in this range was a current fraction that was inhibited by the Cl channel blocker DIDS, suggesting DIDS sensitivity for most of the Cl-selective current in embryonic (E17) UB cells. In these cells a minor fraction of the whole cell outward currents activated time dependently (Fig. 4, A and C). These activating outward currents were inhibited by DIDS (Fig. 4C). Bath anion substitution in embryonic UB cell experiments suggested a conductance rank order of I  Cl  Br  gluconate for the overall Cl-selective currents (calculated for the outward currents). With SCN in the bath, conductance tended to be higher (although it was not quite significantly different) compared with those recorded with the halides (Fig. 4D). The permselectivity, as deduced from the anion replacement-induced shift in V_rev (due to competition of the various anions with Cl at the channel pore binding sites) was I  Cl  Br  SCN  gluconate (Fig. 4E).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Characterization of control whole cell currents in embryonic (E17) UB cells. A: original records obtained with KCl/K-D-gluconate pipette solution and isotonic NaCl bath solution before (chloride) and after application of DIDS (100 µM) and after washout (chloride) or after substitution of bath chloride by gluconate. B: mean current density/voltage-relations measured with N-methyl-D-glucamine (NMDG)-Cl/NMDG-D-gluconate pipette and isotonic NaCl bath solution (solid squares; n = 9) or with KCl/K-D-gluconate pipette and isotonic NaCl- or Na-D-gluconate bath solution, respectively (open symbols; n = 12). Currents were determined at the end (395 ms) of each square pulse. C: relation of different fractions of current densities and applied membrane potentials as calculated from the experiments recorded with KCl/K-D-gluconate pipette solution in B. The decrease in current density as evoked by replacing bath chloride by gluconate (gluconate-responsive) or following bath application of DIDS (100 µM), as well as its increase by time-dependent activation in the absence (activating) and presence (activating DIDS) of DIDS (100 µM), are depicted. The activating fraction of current density was defined as the current at 395 ms minus that at 5 ms of each square pulse. D: whole cell conductance as calculated for the outward currents with 150 mM NaCl or 150 mM sodium salt of various anions in the bath. Conductances are expressed as ratio (G/G0) of the NaCl control. E: changes in reversal potential (Vrev) evoked by replacing 150 mM chloride in the isotonic NaCl bath solution by 150 mM of various anions. Vrev values are corrected for estimated liquid junction potentials. Estimated liquid junction potentials between KCl/K-D-gluconate pipette and NaX bath solution are 11.25, 11.55, 11.35, 9.40, and 3.30 mV for X = Cl, Br, I, SCN, and gluconate, respectively. Values are means ± SE; n = number of cells.

When bath osmolarity was reduced by ~100 mosmol/kgH₂O, a large increase in whole cell conductance was evoked in perinatal UB and in postnatal CCD cells within 5-12 min (see Fig. 6A). This evoked conductance typically inactivated time dependently at positive voltages (greater than or equal to +40 mV) and activated at negative voltages (less than or equal to 80 mV). The majority of embryonic UB cells (15 of 25), in contrast, either did not respond or responded only weakly to hypotonic stress by an increase in the time-dependently activating (at positive voltages) Cl conductance (Fig. 5, A and B). Only 10 cells generated small, time-dependently inactivating outward currents similar to those observed in perinatal UB and postnatal CCD cells. Interestingly, 7 of these 10 cells did not have any time-dependently activating outward currents during the isotonic control phase. The mean hypotonic swelling-activated conductance increase was strikingly lower in embryonic UB compared with perinatal UB and to postnatal CCD cells (Fig. 5C).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Development-dependent changes in whole cell current evoked by hypotonic swelling. A: original traces recorded with KCl/K-D-gluconate pipette solution and with either isotonic bath (left) or hypotonic NaCl bath buffer (right) in a UB at E17 (top), a UB at P1-6 (middle), and a CCD (P9-14, bottom) cell. B: current density-voltage relations of whole cell currents (at 5 ms of the 400 ms square pulse) in isotonic control solution and after hypotonic swelling recorded as in A. C: increase in specific conductance evoked by hypotonic swelling as calculated from the experiments in B. Data are means ± SE; n = number of cells.

Hypotonic swelling-activated whole cell currents in perinatal UB and postnatal CCD cells were mainly Cl selective, since replacement of bath Cl by gluconate decreased the outward currents (G = 15.4 ± 1.5, G_{hypotonic control} = 23.3 ± 1.9 nS/10 pF; n =15) and shifted the reversal potentials by V_rev = +29 ± 3 mV (Fig. 6C; see also Fig. 6, E and F). Inward currents recorded with gluconate in the bath were lower compared with those recorded with Cl (Fig. 6C), which pointed to both an inhibitory action of the poorly permeating anion gluconate and the presence of Cl-selective inward currents.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Characterization of the hypotonic swelling-activated Cl conductance in perinatal UB (P1-6) and postnatal CCD (P9-14) cells. A: time course of changes in whole cell currents during superfusion with hypotonic NaCl bath and KCl/K-D-gluconate pipette solution. Original traces from a UB (P1-6) cell. B: original traces from a UB (P1-6) cell after exposure to hypotonic NaCl bath solution. Currents were recorded before (chloride) and during inhibition by DIDS (100 µM) and after washout (chloride) or after substitution of bath chloride by gluconate (top traces). The DIDS-sensitive and time-dependently inactivating current fractions (bottom traces) were calculated by subtracting the "DIDS" from the "chloride" traces and the "gluconate" from the "DIDS" traces, respectively (outward currents only are shown). C: current density/voltage relations recorded in isotonic NaCl and in hypotonic NaCl or sodium gluconate bath solution (n = 15 UB and CCD cells). D: relation of different fractions of current densities and applied membrane potentials as calculated from the experiments recorded in C. Decrease in current density as evoked by replacing bath chloride by gluconate (gluconate responsive), by bath application of DIDS (100 µM), and by time-dependent inactivation in the absence (inactivating) and presence (inactivating DIDS) of DIDS (100 µM) are shown. The latter two were defined as the current at 5 ms minus that at 395 ms of each square pulse. E: whole cell conductance as calculated for the outward currents with 100 mM NaCl or 100 mM sodium salt of various anions in the bath. Conductances are expressed as ratio (G/G0) of the NaCl control. F: changes in reversal potential (Vrev) following substitution of 100 mM chloride in the hypotonic NaCl bath solution by 100 mM of various anions. Vrev values are corrected for estimated liquid junction potentials. Estimated liquid junction potentials between KCl/K-D-gluconate pipette and hypotonic NaX bath solution are 11.30, 11.55, 11.35, 9.90, and 5.25 mV for X = Cl, Br, I, SCN, and gluconate, respectively. Data are means ± SE; n = 5.

DIDS (100 µM) inhibited the whole cell currents during hypotonic swelling (G = 9.6 ± 1.1 nS/10 pF; n = 15). Interestingly, this DIDS blockade occurred only at positive holding potentials (Fig. 6D), suggesting either voltage dependence of inhibition or voltage dependence of the DIDS-sensitive current fraction itself. The I/V curve of the total Cl-selective outward current, as calculated by subtracting currents recorded with bath sodium gluconate from those in NaCl, rectified outwardly and exhibited a V_rev close to E_Cl (E_Cl = 27 mV; Fig. 6D). The outward currents comprised two components, a DIDS-sensitive one and an insensitive (at positive voltages), time-dependently inactivating one (Fig. 6B, bottom; and Fig. 6D). Time constants were  = 73 ± 13 ms (n = 9) for inactivation at +100 mV and  = 40 ± 4 ms for activation at 100 mV holding potential, respectively. Inactivating outward currents were never seen with sodium gluconate bath medium (Fig. 6B), indicating that this current fraction is Cl selective. Conductances of overall whole cell outward currents recorded with Cl, Br, I, or SCN in the bath solution were higher in comparison to that recorded with gluconate (Fig. 6E). Although these differences were not significant, conductance generated by halides and SCN tended to be in the rank order of Cl  Br  I  SCN. The permselectivity, in contrast, was SCN  I  Br > Cl  gluconate (Fig. 6F).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

This study for the first time investigates the early ontogenetic development of renal Cl conductances by comparing data in primary monolayer cultures of embryonic UB, of perinatal UB, and of postnatal CCD. Conclusions drawn from this in vitro approach can be extrapolated to developmental phenomena in vivo under the condition that 1) these cells, in culture, do not dedifferentiate and do not continue their developmental program; and 2) the experimental protocol, i.e., uncoupling of cells, does not introduce artifactual changes in cell volume and thereby in membrane conductance, which, moreover, could be different in embryonic, perinatal, and postnatal stages.

In vitro, these cultures conserve their in vivo developmental stage-dependent differentiation. This conclusion is based on the following observations. 1) CCD cells, cultured as in the present study, had been shown previously to express the characteristics of in vivo sodium-reabsorbing collecting duct principal cells, such as apical amiloride-sensitive Na channels and apical low-conductance K channels (10). 2) Identically cultured embryonic UB cells, in contrast, exhibited membrane conductance properties entirely different from those of cultured CCD but, again, similar to those of cells of acutely dissected embryonic UBs. The major whole cell current fraction of the latter activates/inactivates time dependently at positive/negative potentials with time constants and current densities similar to those observed in the embryonic UB cultures of the present study (unpublished observations).

As discussed for the culture techniques, the procedure of uncoupling cells in the monolayer before each patch clamp experiment did not alter membrane conductance artifactually. The current densities of large apical patches excised from uncoupled and from intact (i.e., non-uncoupled) perinatal UB monolayers did not differ either in the amplitude or in the composition of fractional currents (not shown). Therefore, a significant change in cell volume and a subsequent alteration in membrane conductance caused by the uncoupling procedure is not likely. Even a loss of cell polarity does not occur in uncoupled cells. CCD cultures, uncoupled as in the present study, have been demonstrated previously to continue to express their characteristic distribution of Na and Cl conductances in the apical and basolateral membrane, respectively (10).

The collecting duct tree develops by sequential branching and outgrowth of UBs (until about P6 in the rat). This branching morphogenesis is maintained by proliferation of UB cells, which resemble morphologically mature CCD principal cells (7). As mentioned, the functional phenotype of UB cells is entirely different from that of mature CCD cells. UB cells thus represent early stages of embryonic epitheliogenesis. They have been shown to express Na-K-ATPase in both basolateral and apical plasma membranes (16). This incomplete epithelial polarization is suggested also by patch-clamp experiments in which perinatal UB cells express identical specific ion conductances in apical and in whole plasma membrane (10).

In the present study, primary monolayer cultures of embryonic UB cells were found to have a large time-dependently activating (at positive voltages), outwardly rectifying, DIDS-sensitive whole cell Cl conductance. Its apparent anion permselectivity of I  Cl  Br  gluconate differs from that reported for the secretory cystic fibrosis transmembrane conductance regulator (CFTR) channel (2) and for the volume-regulatory channels ClC-2 (24) and VSOAC (23), but resembles that of a type of intermediate-conductance, outwardly rectifying Cl channel (13). Intermediate- conductance Cl channels have been observed in embryonic UB cells in vivo and in vitro (unpublished observations), but their contribution to the macroscopic Cl current cannot be deduced.

The macroscopic whole cell current of embryonic UB cultures resembled a subtype of Ca-activated Cl conductance by its time-dependent activation at positive voltages and inactivation at negative voltages (1, 6). This subtype of Ca-activated Cl channel, as well as the intermediate-conductance, outwardly rectifying Cl channel, are typical for nonepithelial cells or for epithelia grown in monolayer on impermeable supports but not for differentiated, i.e., highly polarized cells (1, 3, 17). A time-dependently activating Cl conductance (at positive voltages) was not observed in perinatal UB cultures, suggesting downregulation of its expression between embryonic day E17 and birth. Remarkably, over this same time span, a hypotonic swelling-activated Cl conductance appears to be upregulated. This conductance exhibited anion permselectivity (SCN  I  Br > Cl  gluconate), voltage dependence, sensitivity to DIDS, and single channel properties (unpublished observations) identical to those reported for the macroscopic VSOAC current (23). VSOAC-type currents that mediate cell swelling-evoked RVD in many mature epithelial and nonepithelial cells are probably generated by time-dependent inactivating (at positive potentials), intermediate-conductance, and outwardly rectifying Cl channels (5, 15) that are possibly encoded or regulated by the I_Cln gene (19, 23).

In the present study, DIDS inhibited only a non-inactivating fraction of outward Cl currents in volume-regulating perinatal UB and postnatal CCD cultures. This might be explained either by voltage dependence of the DIDS blockade or by the presence of a further volume-sensitive Cl conductance that is inwardly rectifying and DIDS insensitive, as reported for ClC-2 Cl channels (24). ClC-2 channels inactivate slowly at positive and activate at highly negative holding potentials. They have a permselectivity of Cl > I and a low conductance of 2-3 pS (12). In many different cell types, ClC-2 mediates RVD in concert with the organic osmolyte permeant (VSOAC) Cl channels (23). A specific embryonic function of ClC-2 has been attributed to ClC-2 in the developing lung, because ClC-2 is highly expressed in embryonic and perinatal respiratory epithelium and downregulated after birth (18). In the collecting duct system, which develops by branching morphogenesis homologously to the lung, development-dependent ClC-2 mRNA expression shows an identical temporal pattern (11). Therefore, a contribution of ClC-2 to the hypotonic swelling-activated Cl conductance, at least in perinatal UB cultures, appears likely. This is further supported by the identification of slowly inactivating 3-pS Cl channels in outside-out patches of perinatal UB cultures (unpublished results).

Rat CCD principal cells neither exhibit DIDS-sensitive conductances under isotonic control conditions, as seen in the present in vitro study, nor do they transport Cl transcellularly (22); a specific embryonic function, therefore, could be suggested for the DIDS-sensitive Cl conductance in UB. Involvement of this conductance in vectorial fluid secretion is very unlikely because of the apolar phenotype of the UB cells in vitro and in vivo (10, 16). The embryonic Cl currents were recorded under putative physiological, isotonic control conditions. Thus, to contribute to cell volume regulation, it must be postulated that net Cl flux via this apparently constitutively active Cl conductance is driven by the activity of cation channels. Cells at the tip of acutely microdissected embryonic UBs (rat E17) express two types of K channels (unpublished observations) that resemble those identified in the basolateral membrane of mature rat principal CCD cells (8). Embryonic cells might utilize these K channels to regulate KCl and water efflux.

In summary, cultured embryonic UB cells expressed a high, apparently constitutively active whole cell Cl conductance. Cultured perinatal UB and postnatal CCD cells, in contrast, expressed a hypotonic swelling-activated Cl conductance. Both types of Cl conductance differ in their voltage dependence, their activation/inactivation kinetics, and their anion permselectivity. The data suggest that in CCD ontogeny, a VSOAC-like Cl conductance evolves between embryonic day E17 and postnatal day P1, i.e., even before morphogenesis is completed and vectorial transports begin. Simultaneously, an embryonic type of Cl conductance is downregulated.