Rat Cortical Collecting Duct Cell Research Articles

Hydrogen sulfide induces Ca2+ influx in the principal cells of rat cortical collecting ducts

Hydrogen sulfide (H2S) acts as a gas-signaling agent in various tissues. Although it has been reported that endogenous enzymes that generate H2S are expressed abundantly in the kidney, few reports examine cellular responses to H2S in renal tubular epithelial cells. In this study, we investigated the effects of NaHS, an H2S donor, and l-cysteine, a substrate for H2S production, on the principal cells of rat cortical collecting ducts (CCDs). NaHS increased the intracellular Ca2+ concentration ([Ca2+]i) in the principal cells. The removal of extracellular Ca2+ largely attenuated the [Ca2+]i response. The TRPV4 channel blocker significantly inhibited the effect of NaHS. Extracellular administration of l-cysteine also elicited a rise in [Ca2+]i. Prior treatment of CCDs with AOAA, an inhibitor of H2S production enzyme, l-cysteine-induced [Ca2+]i response was significantly reduced. These results suggest that not only exogenous H2S but also endogenously produced H2S triggers the extracellular influx pathway of Ca2+ in the principal cells of rat CCDs.

AQP2 can modulate the pattern of Ca2+ transients induced by store-operated Ca2+ entry under TRPV4 activation.

There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the TRPV4 Ca2+ channel. Ca2+ release from intracellular stores and plasma membrane hyperpolarization due to opening of Ca2+ -activated potassium channels (KCa) are events that have been proposed to take place downstream of TRPV4 activation. A major mechanism for Ca2+ entry, activated after depletion of intracellular Ca2+ stores and driven by electrochemical forces, is the store-operated Ca2+ entry (SOCE). The consequences of the interplay between TRPV4 and AQPs on SOCE have not been yet investigated. The aim of our study was to test the hypothesis that AQP2 can modulate SOCE by facilitating the interaction of TRPV4 with KCa channels in renal cells. Using fluorescent probe techniques, we studied intracellular Ca2+ concentration and membrane potential in response to activation of TRPV4 in two rat cortical collecting duct cell lines (RCCD1 ), one not expressing AQPs (WT-RCCD1 ) and the other transfected with AQP2 (AQP2-RCCD1 ). We found that AQP2 co-immunoprecipitates with TRPV4 and with the small-conductance potassium channel (SK3). We also showed that AQP2 is crucial for the activation of SK3 by TRPV4, leading to hyperpolarization of the plasma membrane. This seems to be relevant to modulate the magnitude of SOCE and is accompanied by TRPV4 translocation to the plasma membrane only in AQP2 expressing cells. These findings open the perspective to further investigate whether the interplay between different AQPs with TRPV4 and KCa channels can be an important mechanism to modulate SOCE with physiological relevance.

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca2+]i response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging.Reduction of extracellular osmolality from 305 (control) to 195mosmol/kgH2O (hypotonic) evoked transient increase in [Ca2+]i of principal cells of rat CCDs. The [Ca2+]i increase was markedly attenuated by the removal of extracellular Ca2+. The application of a P2 purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2+]i increase. The [Ca2+]i increase in response to extracellular hypotonicity was not influenced by application of Gd3+ and ruthenium red. On the other hand, a voltage-gated Ca2+ channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca2+]i increase in the principal cells. In order to assess Ca2+ entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn2+. The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn2+, indicating that a Ca2+-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn2+ quenching was significantly inhibited by nicardipine.These results strongly suggested that a nicardipine-sensitive Ca2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+]i elevation of principal cells in rat CCD.

Role of AQP2 in activation of calcium entry by hypotonicity: implications in cell volume regulation

We previously reported in a rat cortical collecting duct cell line (RCCD(1)) that the presence of aquaporin 2 (AQP2) in the cell membrane is critical for the rapid activation of regulatory volume decrease mechanisms (RVD) (Ford et al. Biol Cell 97: 687-697, 2005). The aim of our present work was to investigate the signaling pathway that links AQP2 to this rapid RVD activation. Since it has been previously described that hypotonic conditions induce intracellular calcium ([Ca(2+)](i)) increases in different cell types, we tested the hypothesis that AQP2 could have a role in activation of calcium entry by hypotonicity and its implication in cell volume regulation. Using a fluorescent probe technique, we studied [Ca(2+)](i) and cell volume changes in response to a hypotonic shock in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. We found that after a hypotonic shock only AQP2-RCCD(1) cells exhibit a substantial increase in [Ca(2+)](i). This [Ca(2+)](i) increase is strongly dependent on extracellular Ca(2+) and is partially inhibited by thapsigargin (1 muM) indicating that the rise in [Ca(2+)](i) reflects both influx from the extracellular medium and release from intracellular stores. Exposure of AQP2-RCCD(1) cells to 100 muM gadolinium reduced the increase in [Ca(2+)](i) suggesting the involvement of a mechanosensitive calcium channel. Furthermore, exposure of cells to all of the above described conditions impaired rapid RVD. We conclude that the expression of AQP2 in the cell membrane is critical to produce the increase in [Ca(2+)](i) which is necessary to activate RVD in RCCD(1) cells.

DHEA Induces 11β-HSD2 by Acting on CCAAT/Enhancer-Binding Proteins

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) type 1 and type 2 catalyze the interconversion of inactive and active glucocorticoids. Impaired regulation of these enzymes has been associated with obesity, diabetes, hypertension, and cardiovascular disease. Previous studies in animals and humans suggested that dehydroepiandrosterone (DHEA) has antiglucocorticoid effects, but the underlying mechanisms are unknown. In this study, DHEA treatment markedly increased mRNA expression and activity of 11beta-HSD2 in a rat cortical collecting duct cell line and in kidneys of C57BL/6J mice and Sprague-Dawley rats. DHEA-treated rats tended to have reduced urinary corticosterone to 11-dehydrocorticosterone ratios. It was found that CCAAT/enhancer-binding protein-alpha (C/EBP-alpha) and C/EBP-beta regulated HSD11B2 transcription and that DHEA likely modulated the transcription of 11beta-HSD2 in a phosphatidylinositol-3 kinase/Akt-dependent manner by increasing C/EBP-beta mRNA and protein expression. Moreover, it is shown that C/EBP-alpha and C/EBP-beta differentially regulate the expression of 11beta-HSD1 and 11beta-HSD2. In conclusion, DHEA induces a shift from 11beta-HSD1 to 11beta-HSD2 expression, increasing conversion from active to inactive glucocorticoids. This provides a possible explanation for the antiglucocorticoid effects of DHEA.

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

Transition from antidiuresis to diuresis exposes cortical collecting duct cells (CCD) to asymmetrical changes in environment osmolality, inducing an osmotic stress, which activates numerous membrane-associated events. The aim of the present work was to investigate, either in the presence or not of AQP2, the transepithelial osmotic water permeability (P(osm)) following cell exposure to asymmetrical hyper- or hypotonic gradients. For this purpose, transepithelial net volume fluxes were recorded every minute in two CCD cell lines: one not expressing AQPs (WT-RCCD(1)) and another stably transfected with AQP2 (AQP2-RCCD(1)). Our results demonstrated that the rate of osmosis produced by a given hypotonic shock depends on the gradient direction (osmotic rectification) only in the presence of apical AQP2. In contrast, hypertonic shocks elicit P(osm) rectification independently of AQP2 expression, and this phenomenon may be linked to modulation of basolateral membrane permeability. No asymmetry in transepithelial resistance was observed under hypo- or hypertonicity, indicating that rectification cannot be attributed to a shunt through the tight junction path. We conclude that osmotic rectification may be explained in terms of dynamical changes in membrane permeability probably due to activation/incorporation of AQPs or transporters to the plasma membrane via some mechanism triggered by osmolality.

Functional and Molecular Adaptation of Cl-/HCO3- Exchanger to Chronic Alkaline Media in RenalCells

The Cl^-/HCO₃^- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO₃^- exchangers in a rat cortical collecting duct cell line (RCCD1). pH_i was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pH_i to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pH_i became alkaline and was not regulated, after 48 h adaptation; steady-state pH_i reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl^-/HCO₃^- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pH_i and that AE2 would play a key role in cell homeostasis.

Basolateral translocation by vasopressin of the aldosterone-induced pool of latent Na-K-ATPases is accompanied by alpha1 subunit dephosphorylation: study in a new aldosterone-sensitive rat cortical collecting duct cell line.

The regulation of plasma membrane Na(+)-K(+)-ATPases (NKA) expression by aldosterone and arginin vasopressin (AVP) in the cortical collecting duct (CCD) has been examined in a new rat CCD cell line, designated as RCCD(2). This cell line has maintained many characteristics of the CCD-in particular, the expression of the mineralocorticoid receptor. Mineralocorticoid receptor is expressed at the protein level and binds (3)H-aldosterone (approximately 15 to 20 fmol/mg protein). Short-circuit current (Isc) experiments showed approximately a twofold increase in Isc associated with a decrease in transepithelial resistance when cells were treated with aldosterone concentrations as low as 10(-9) M. This effect on Isc was significant 2 h after aldosterone addition and was still present after 24 h. It was accompanied by an increase in the amount of mRNA encoding for the alpha subunit of the epithelial sodium channel (sixfold) and the alpha1 subunit of NKA (fourfold) after 24 h of hormone treatment. In addition, mRNA expression of the serum- and glucocorticoid-induced kinase (Sgk) was increased by 10(-9) M aldosterone treatment as early as 45 min after hormone addition. As had already been documented in native CCD obtained by microdissection, incubation of RCCD(2) cells for 24 h with aldosterone resulted in the constitution of a latent pool of NKA that could be rapidly recruited by AVP (15 min). NKA biotinylation experiments and preparation of membrane fractions show that this latent pool of NKA is present in the intracellular compartment of the cells and is recruited by AVP in the basolateral membrane through a translocation process. This mechanism is accompanied by dephosphorylation of the alpha(1) catalytic subunit of NKA.

Vasopressin Regulates Water Flow in a Rat Cortical Collecting Duct Cell Line Not Containing Known Aquaporins

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD(1)). Transepithelial net water fluxes (J(w)) were recorded every minute in RCCD(1) monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10(-5) m), apical glibenclamide (10(-4) m) and apical BaCl(2) (5 x 10(-3) m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD(1) cells. AVP stimulates cAMP production and sodium reabsorption in RCCD(1) cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I(sc)) was paralleled by a simultaneous modification of the observed J(w): both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J(w) induced by serosal hypertonicity. We can conclude that: (i) transepithelial J(w) occurs in RCCD(1) cells in the absence of known renal aquaporins; (ii) the "water secretory component" observed could be linked to Cl- and K = secretion; (iii) the natriferic response to AVP, preserved in RCCD(1) cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost.

Vasopressin stimulates long-term net chloride secretion in cortical collecting duct cells

The classical short-term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long-term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long-term increase in net chloride secretion. In the RCCD 1 rat cortical collecting duct cell line, 10 −8 M AVP induced, after several hours, an increase in net 36Cl − secretion. This delayed effect of AVP was inhibited by basal addition of 10 −4 M bumetanide and apical addition of 10 −4 M glibenclamide, suggesting chloride entry at the basal membrane through a Na +/K +/2Cl − and apical secretion through a chloride conductance. An original acute cell permeabilization method was developed to allow for entry of antibodies directed against the regulatory region (R) of the cystic fibrosis transmembrane regulator (CFTR) into the cells. This procedure led to a complete and specific blocking of the long-term net chloride secretion induced by AVP. Finally, it was observed that CFTR transcripts steady-state level was significantly increased by AVP treatment. Besides the well-documented short-term effect of AVP on chloride transport, these results provide evidence that in RCCD 1 cells, AVP induces a delayed increase in transepithelial net chloride secretion that is mediated by a Na +/K +/2Cl − co-transporter and CFTR.

Transcriptional Regulation of Sodium Transport by Vasopressin in Renal Cells

We have examined whether arginine vasopressin (AVP) can induce a long-term modulation of transepithelial ion transport in addition to its well known short-term effect. In the RCCD1 rat cortical collecting duct cell line, an increase in both short-circuit current and 22Na transport was observed after several hours of 10(-8) M AVP treatment (a concentration above the in vivo physiological range). This delayed effect was partially prevented by apical addition of 10(-5) M amiloride and was blocked by 10(-6) M actinomycin D and 2 x 10(-6) M cycloheximide. The amounts of mRNA encoding the alpha1 (not beta1) subunit of Na+/K+-ATPase and the beta and gamma (not alpha) subunits of the amiloride-sensitive epithelial Na+ channel were significantly increased by AVP treatment. The increase in mRNA was blocked by actinomycin D, not by amiloride, suggesting a Na+-independent increase in the rate of transcription of these subunits. The translation rates of the alpha1 subunit of Na+/K+-ATPase and the beta and gamma subunits of the rat epithelial sodium channel increased significantly, whereas the translation rates of the other subunits remained unchanged. Finally, the number of Na+ channels present in the apical membrane of the cells increased, as demonstrated by enhanced specific [3H]phenamil binding.

Effects of sodium nitroprusside in the rat cortical collecting duct are independent of the NO pathway

Recently we described K+ channels in the basolateral membrane of principal cells of rat cortical collecting duct (CCD) which are regulated by a cGMP-dependent protein kinase (Pflugers Arch 429:338-344, 1995). We examined the effects of the NO-liberator sodium nitroprusside (SNP) on single channel activity and membrane voltage (Vm) in principal cells of rat CCD, and on transepithelial voltage, lumen-to-bath Na+ fluxes, and osmotic water permeability in isolated perfused rat CCD tubules. While in patch clamp experiments SNP (10 microM) hyperpolarized principal cells from -54 +/- 10 mV to -71 +/- 5 mV (N = 5) and increased the activity of the described K+ channels from 0.05 +/- 0.03 to 0.45 +/- 0.14 (N = 5) in cell-attached and from 0.04 +/- 0.02 to 0.25 +/- 0.05 (N = 4) in excised patch clamp experiments, it had no effect on basal or AVP-dependent transepithelial voltage, Na+ fluxes, or the osmotic water permeability. In addition, neither 50 microM SIN-1, another liberator of NO, nor 1 mM L-NAME, an inhibitor of the NO-synthase, changed Vm significantly. Furthermore, in cGMP-assays SNP failed to increase intracellular cGMP in CCD segments. Thus, we conclude that in the rat CCD transport is not regulated via the NO-pathway and that SNP acts as an cGMP independent activator of K+ channels in the basolateral membrane of these cells.

CGMP-activating peptides do not regulate electrogenic electrolyte transport in principal cells of rat CCD.

K+ channels in the basolateral membrane of rat cortical collecting duct (CCD) are regulated by a cGMP-dependent protein kinase (J. Hirsch and E. Schlatter. Pfluegers Arch. 429: 338-344, 1995). Conflicting data exist on the effects of cGMP-activating agonists on Na+ transport in these cells. Thus we tested members of the family of peptides that increase intracellular cGMP [cardiodilatin/atrial natriuretic peptide (CDD/ANP), brain natriuretic peptide, C-type natriuretic peptide, urodilatin, guanylin, and uroguanylin], as well as bradykinin +/- CDD/ANP on membrane voltages (Vm) of principal cells of isolated rat CCD using the slow whole cell patch-clamp technique (E. Schlatter, U. Fröbe, and R. Greger. Pfluegers Arch. 421: 381-387, 1992). None of the agonists tested changed Vm significantly. There was also no effect of dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) on AVP-dependent lumen-to-bath Na+ flux, transepithelial voltage, or osmotic water permeability in isolated perfused rat CCD. Finally, CDD/ANP increased intracellular cGMP only in glomeruli but not in CCD. Thus the findings provide no evidence for control of electrogenic electrolyte transport by these natriuretic peptides in principal cells of rat CCD, and the agonist that physiologically regulates the cGMP-dependent K+ channels remains to be identified.

Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance

This study describes the establishment of a rat kidney cortical collecting duct (CCD) clonal cell line (RCCD1 cells) that maintains high transepithelial resistance and specific hormonal sensitivities. Immortalized cells were obtained by infection of primary cultured CCD cells with the wild-type simian virus 40. Grown on Petri dishes, RCCD1 cells are organized as monolayers of cuboid cells separated by tight junctions and form domes. Grown on permeable filters, confluent RCCD1 cells exhibit high transepithelial resistance (Rt: 2390 +/- 140 omega. cm2), transepithelial potential difference (PD) of -10.5 +/- 1.2 mV lumen negative, an associated short-circuit current (Isc) of 4.3 +/- 0.5 microA/cm2, and generated significant Na+, K+, H+ and HCO3- gradients, reflecting Na+ and H+ reabsorption and K+ and HCO3- secretion. RCCD1 cells exhibit features of both principal (PC) and intercalated (IC) cells. Consistent with PC phenotype, about 50% of the cells were positively stained by a PC-specific agglutinin. In situ hybridization studies revealed the presence of alpha, beta and gamma subunit mRNAs of the amiloride-sensitive epithelial Na+ channel and alpha 1 and beta 1 subunits of Na(+)-K(+)-ATPase. Moreover, Na(+)-K(+)-ATPase was immunolocalized at the basolateral side of the cells. Arginine vasopressin (AVP) induced a significant increase in both cellular cAMP content and Isc. Amiloride decreased in a dose-dependent manner Isc from untreated and AVP-treated RCCD1 cells. In addition, a barium-sensitive K+ conductance was evidenced in the apical side of the cells. Consistent with IC phenotype, isoproterenol (ISO) provoked a large increase in cellular cAMP and stimulated Isc. The effect of ISO on Isc was blocked by 5 x 10(-3) M DPC, a chloride channel blocker. Finally, AVP plus ISO had additive effect on Isc. Taken together, these results provide evidence that the RCCD1 cell line has maintained many of the original properties of rat CCD from which they were derived.