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.     

Characterization of a mouse cortical collecting duct cell line.

A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 +/- 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 +/- 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 +/- 10.3 microA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 +/- 0.9 and B = 127.4 +/- 0.4; [K+], L = 8.6 +/- 0.6 and B = 2.1 +/- 0.3; [Cl], L = 68.6 +/- 5.8 and B = 101.8 +/- 6.6; [HCO3], L = 15.5 +/- 1.5 and B = 8.6 +/- 1.2; while pH was 7.16 +/- 0.03 at the luminal and 6.94 +/- 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

IGF-1 vs insulin: respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 pathway in a cortical collecting duct cell line

Insulin and insulin-like growth factor 1 (IGF-1) may play a role in the regulation of sodium balance by increasing basal and aldosterone-stimulated transepithelial sodium transport in the aldosterone-sensitive distal nephron (ASDN). As insulin and IGF-1 are capable of binding to each other's receptor with a 50- to 100-fold lower affinity than to their cognate receptor, it is not clear which receptor mediates its respective sodium transport response in the ASDN. The aim of the present study was to characterize the IGF-1 regulation of Na(+) transport in the mCCD(cl1) cell line, a highly differentiated cell line which responds to physiological concentrations (K(1/2)=0.3 nM) of aldosterone. IGF-1 increased basal transepithelial Na(+) transport with a K(1/2) of 0.41+/-0.07 nM. Insulin dose-response curve was displaced to the right 50-fold, as compared to that of IGF-1 (K(1/2)=20.0+/-3.0 nM), indicating that it acts through the IGF type 1 receptor (IGF-1R). Co-stimulation with IGF-1 (0.3 nM) (or 30 nM insulin) and aldosterone (0.3 nM), either simultaneously or by pretreating the cells for 5 h with aldosterone, induced an additive response. The phosphatidylinositol-3' kinase (PI3-K) inhibitor LY294002 completely blocked IGF-1 and aldosterone induced and co-induced currents. As assessed by Western blotting, protein levels of the serum-, and glucocorticoid-induced kinase (Sgk1) were directly and proportionally related to the current induced by either or both IGF-1 and aldosterone, effects also blocked by the PI3-K inhibitor LY294002. IGF-1 could play an important physiological role in regulating basal sodium transport via the PI3-K/Sgk1 pathway in ASDN.     

Mineralocorticoid versus glucocorticoid receptor occupancy mediating aldosterone-stimulated sodium transport in a novel renal cell line

Aldosterone controls sodium balance by regulating an epithelial sodium channel (ENaC)-mediated sodium transport along the aldosterone-sensitive distal nephron, which expresses both mineralocorticoid (MR) and glucocorticoid receptors (GR). Mineralocorticoid specificity is ensured by 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes cortisol or corticosterone into inactive metabolites that are unable to bind MR and/or GR. The fractional occupancy of MR and GR by aldosterone mediating the sodium transport response in the aldosterone-sensitive distal nephron cannot be studied in vivo. For answering this question, a novel mouse cortical collecting duct cell line (mCCD(cl1)), which expresses significant levels of MR and GR and a robust aldosterone sodium transport response, was used. Aldosterone elicited a biphasic response: Low doses (K(1/2) = approximately 0.5 nM) induced a transient and early increase of sodium transport (peaking at 3 h), whereas high doses (K(1/2) = approximately 90 nM) entailed an approximately threefold larger, long-lasting response. At 3 h, the corticosterone dose-response curve was shifted to the right compared with that of aldosterone by more than two log concentrations, an effect that was fully reverted in the presence of the 11beta-hydroxysteroid dehydrogenase type 2 inhibitor carbenoxolone. Low doses of dexamethasone (0.1 to 1 nM) failed to induce an early response, but high doses elicited a long-lasting response (K(1/2) = approximately 8 nM), similar to that observed for high aldosterone concentrations. Equilibrium binding assays showed that both aldosterone and corticosterone bind to a high-affinity, low-capacity site, whereas dexamethasone binds to one site. Within the physiologic range of aldosterone concentrations, sodium transport is predicted to be controlled by MR occupancy during circadian cycles and by MR and GR occupancy during salt restriction or acute stress.     

Water transport in the immature rabbit collecting duct

 Immature animals have limited ability to concentrate the urine. This is in part the result of end-organ resistance to arginine vasopressin (AVP). To characterize this response, we measured water absorption in microperfused cortical collecting ducts (iCCD) and outer medullary CD (iOMCD) derived from 2- to 12-day-old rabbits. The roles of adenosine 3′,5′-cyclic monophosphate (cAMP) and prostaglandins were investigated. Baseline osmotic water permeability (L_p, 10⁻⁷ cm/atm per s) in the iCCD (20.3±2.4) and iOMCD (19.7±5.6) was not different from mature CCD (mCCD) (14.6±3.1). After AVP, L_p in the iCCD (46.7±10.0) was significantly lower than in the mCCD (114.3±21.8). Neither stimulation with cAMP (85.6±51.3) nor inhibition of endogenous prostaglandin production with indomethacin (57.6±29.8) abolished the blunted response to AVP in the iCCD. We conclude that AVP-stimulated water transport in the iCCD is impaired. The disruption in AVP response is, at least in part, localized distal to cAMP, and is not mediated by prostaglandins. Received: 29 December 1997 / Revised: 13 April 1998 / Accepted: 14 April 1998     

Effects of chemical chaperones on partially retarded NaCl cotransporter mutants associated with Gitelman's syndrome in a mouse cortical collecting duct cell line.

BACKGROUND: Epithelial cells lining the distal convoluted tubule express the thiazide-sensitive Na-Cl cotransporter (NCC) that is responsible for the reabsorption of 5-10% of the filtered load of Na(+) and Cl(-). Mutations in NCC cause the autosomal recessive renal disorder Gitelman's syndrome (GS). GS mutations give rise to mutant transporters that are either fully (class I) or partially (class II) retarded. Recent evidence indicates that class II mutations do not alter the intrinsic transport activity of NCC. These findings suggest that in GS caused by class II NCC mutations, pharmacological chaperones may be useful in treatment. METHODS: Initial attempts using 4-phenylbutyrate and glycerol to increase Na(+) uptake in Xenopus laevis oocytes expressing the class II mutant L215P were unsuccessful. To study the effect of the chaperones in a more physiological setting, we next expressed hNCC in the polarized epithelial cell line of distal tubular origin, mpkCCD. RESULTS: mpkCCD cells readily expressed the class II mutant R955Q, but not the class I mutant G741R. Wild-type hNCC was predominantly present in the approximately 120-1403 kD complex glycosylated form. In contrast, the R955Q mutant was predominantly present in a lower molecular weight form of approximately 100 kD. Pretreatment of R955Q expressing cells with 4-phenylbutyrate (5 mM, 16 h), but not thapsigargin (1 microM, 90 min), dimethyl sulfoxide (1%, 16 h) or glycerol (4%, 16 h), increased the expression of the complex glycosylated form and in parallel the number of hNCC positive cells. CONCLUSIONS: Taken together, the data indicate that 4-phenylbutyrate is a promising candidate for rescuing partially retarded but otherwise functional class II GS mutants.     

Mechanism of iodide transport in the rabbit cortical collecting duct

Background Pendrin, an anion exchanger known to participate in iodide transport in the apical membrane of follicular cells of the thyroid gland, has recently been shown to exist in the apical membrane of the β- and γ-intercalated (β/γ-IC) cells of the cortical collecting duct (CCD). We examined mechanisms of iodide transport in the CCD. Methods Rabbit CCD was perfused in vitro, and lumen-to-bath flux coefficients for both ¹²⁵I⁻ (K_{I (lb)}) and ³⁶Cl⁻ (K_{Cl (lb)}) were measured simultaneously. The intracellular pH (pHi) of β/γ-IC cells in the perfused CCD was measured by microscopic fluorometory, by loading 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxy methylester (BCECF-AM), a fluorescent marker for pHi. The effects on pHi of the replacement of NaCl with Na cyclamate, NaI, or NaBr in the lumen or bath were observed. Results K_{I (lb)} was comparable to or slightly higher than K_{Cl (lb)}. Both iodide and chloride in the lumen caused self- and cross-inhibitions to both fluxes. The addition of 5-nitro-2-(-3-phenylpropylamino)-benzoate (NPPB), a Cl⁻ channel inhibitor, to the bath significantly reduced K_{Cl (lb)}, but not K_{I (lb)}. Replacement of luminal fluid NaCl with Na cyclamate, NaI, or NaBr caused alkalization of pHi, no change in pHi, and slight acidification of pHi, respectively. Replacement of bath NaCl with Na cyclamate, NaI, or NaBr caused alkalization, alkalization, and acidification of pHi, respectively. Luminal NaI prevented the acidification of pHi caused by bath Na cyclamate. Conclusions The data are consistent with the model that iodide is transported via the Cl⁻/HCO₃⁻ exchanger in the apical membrane of β/γ-IC cells and exits the basolateral membrane via an electroneutral transporter that is distinct from the Cl⁻ channel. We could not, however, identify which type of β/γ-IC cell was mainly responsible.     

Oxalate toxicity in cultured mouse inner medullary collecting duct cells

PURPOSE: Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin. However, to our knowledge it is unknown whether inner medullary collecting duct (IMCD) cells, which are physiologically exposed to higher concentrations of oxalate, also behave in a similar manner. MATERIALS AND METHODS: A line of IMCD cells was exposed to oxalate (0.2 to 10 mM) for various time points. Trypan blue, and hematoxylin and eosin stains were used to assess cell morphology and membrane integrity. The production of reactive oxidative species was determined using the nitro blue tetrazolium reaction and crystal violet staining was used to measure cell density. RESULTS: Exposure of IMCD cells to oxalate produced time and concentration dependent changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in alterations in cell viability with net cell loss following exposure to concentrations of 2 mM and greater. Free radical production was time and concentration dependent. Crystal formation occurred in less than 1 hour and cells in proximity to crystals lost membrane integrity. Compared to IMCD cells LLC-PK1 and HK2 cells showed significant toxicity starting at lower oxalate concentrations (0.4 mM and above). CONCLUSIONS: To our knowledge the results provide the first direct demonstration of toxic effects of oxalate in IMCD cells, a line of renal epithelial cells of the inner medullary collecting duct, and suggest that cells lining the collecting duct are relatively resistant to oxalate toxicity.     

Basolateral Na+/H+ exchange maintains potassium secretion during diminished sodium transport in the rabbit cortical collecting duct

Stimulation of the basolateral Na(+)/K(+)-ATPase in the isolated perfused rabbit cortical collecting duct by raising either bath potassium or lumen sodium increases potassium secretion, sodium absorption and their apical conductances. Here we determined the effect of stimulating Na(+)/K(+)-ATPase on potassium secretion without luminal sodium transport. Acutely raising bath potassium concentrations from 2.5 to 8.5 mM, without luminal sodium, depolarized the basolateral membrane and transepithelial voltages while increasing the transepithelial, basolateral and apical membrane conductances of principal cells. Fractional apical membrane resistance and cell pH were elevated. Net potassium secretion was maintained albeit diminished and was still enhanced by raising bath potassium, but was reduced by basolateral ethylisopropylamiloride, an inhibitor of Na(+)/H(+) exchange. Luminal iberitoxin, a specific inhibitor of the calcium-activated big-conductance potassium (BK) channel, impaired potassium secretion both in the presence and absence of luminal sodium. In contrast, iberitoxin did not affect luminal sodium transport. We conclude that basolateral Na(+)/H(+) exchange in the cortical collecting duct plays an important role in maintaining potassium secretion during compromised sodium supplies and that BK channels contribute to potassium secretion.