Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.

LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na(+) transport in A6 epithelia.

Blocker-induced noise analysis of epithelial Na(+) channels (ENaCs) was used to investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na(+) transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na(+) transport rates (I(Na)) of 4.0 +/- 0.1 (unstimulated) and 9.1 +/- 0.9 microA/cm(2) (aldosterone), 10 microM LY-294002 caused, following a relatively small initial increase of transport, a completely reversible inhibition of transport within 90 min to 33 +/- 6% and 38 +/- 2% of respective baseline values. Initial increases of transport could be attributed to increases of channel open probability (P(o)) within 5 min to 143 +/- 17% (unstimulated) and 142 +/- 10% of control (aldosterone) from baseline P(o) averaging near 0.5. Inhibition of transport was due to much slower decreases of functional channel densities (N(T)) to 28 +/- 4% (unstimulated) and 35 +/- 3% (aldosterone) of control at 90 min. LY-294002 (50 microM) caused larger but completely reversible increases of P(o) (215 +/- 38% of control at 5 min) and more rapid but only slightly larger decreases of N(T). Basolateral exposure to LY-294002 induced no detectable effect on transport, P(o) or N(T). We conclude that an LY-294002-sensitive PI 3-kinase plays an important role in regulation of transport by modulating N(T) and P(o) of ENaCs, but only when presented to apical surfaces of the cells.

Characterization of the ion transport responses to ADH in the MDCK-C7 cell line.

The Madin-Darby canine kidney (MDCK) cell line expresses many characteristics of the renal collecting duct. The MDCK-C7 subclone forms a high-resistance, hormone-responsive model of the principal cells, which are found in distal sections of the renal tubule. The electrophysiological technique of short-circuit current measurement was used to examine the response to antidiuretic hormone (ADH) in the MDCK-C7 clone. Three discrete electrogenic ion transport phenomena can be distinguished temporally and by the use of inhibitors and effectors. Initially the cells exhibit anion secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). The presence of CFTR was confirmed by immunoprecipitation followed by Western blotting. The CFTR-mediated anion secretion is transient and is followed, in time, by a verapamil- and Ba(+)-sensitive anion secretion or cation absorption and, finally, by Na+ reabsorption via epithelial Na+ channels (ENaC). In contrast to other studies of MDCK cells, we see no indication that the presence of CFTR functionally inhibits ENaC. The characterization of the various ion transport phenomena substantiates this cell line as a model renal epithelium that can be used to study the hormonal and metabolic regulation of ion transport.

Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption.

Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+ reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.

Hormonal regulation of ENaCs: insulin and aldosterone.

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+ transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current (iNa), channel open probability (Po), and functional channel density (NT) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10-30 min the density of the pool of apical membrane ENaCs (NT) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of iNa (10-20%) and decreases of p.o. (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of iNa, p.o., and NT were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.

To study and define the early time-dependent response (< or = 6 h) of blocker-sensitive epithelial Na+ channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current (iNa) channel open probability (Po), and channel density (NT) under transient conditions of transport as measured by macroscopic short-circuit currents (Isc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 microA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. NT varied linearly over a 70-fold range of transport (0.5-35 microA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of Po occurred during control (10-20% over 2 h) and aldosterone experimental periods (10-30% over 6 h). When the Po of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, Po was observed to vary inversely with Isc, falling from approximately 0.5 to approximately 0.15 at the highest rates of Na+ transport or approximately 25% per 3-fold increase of transport. Because decreases of Po from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane NT.

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells.

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 approximately 6 microM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

Protein prenylation is required for aldosterone-stimulated Na+ transport.

Aldosterone stimulation of transcellular Na+ flux in polarized epithelial cells is dependent on at least one transmethylation reaction, but the substrate of this signaling step is unknown. Because it is clear that the majority of cellular protein methylation occurs in conjunction with protein prenylation, we examined the importance of prenylation to aldosterone-stimulated Na+ transport in the A6 cell line. Lovastatin, an inhibitor of the first committed step of the mevalonate pathway, inhibits the natriferic effect of aldosterone but does not inhibit insulin-stimulated Na+ flux. The addition of a farnesyl group does not appear to be involved in aldosterone's action. Neither alpha-hydroxyfarne-sylphosphonic acid, an inhibitor of farnesyl:protein transferase, nor N-acetyl-S-farnesyl-L-cysteine, an inhibitor of farnesylated protein methylation, inhibits the hormone-induced increase in Na+ transport. In contrast, N-acetyl-S-geranyl-geranyl-L-cysteine, an inhibitor of geranylgeranyl protein methylation, completely abolishes the aldosterone-induced increase in Na+ flux with no effect on insulin-mediated Na+ transport or cellular protein content. These data indicate that methylation of a geranylgeranylated protein is involved in aldosterone's natriferic action.

The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities.

The amiloride-sensitive Na+ channel found in many transporting epithelia plays a key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and quantitate this important channel. Among biophysical methods, there is agreement as to the single-channel conductance and gating kinetics of the highly selective Na+ channel found in native epithelia. Amiloride and its analogs inhibit transport through the channel by binding to high-affinity ligand-binding sites. This characteristic of high-affinity binding has been used biochemically to quantitate channel densities and to isolate presumptive channel proteins. Although the goals of biophysical and biochemical experiments are the same in elucidating mechanisms underlying regulation of Na+ transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and characterize the channel proteins.

Aldosterone-induced proteins in primary cultures of rabbit renal cortical collecting system.

Primary cultures of immunodissected cells from rabbit kidney connecting tubule and cortical collecting duct were used to study aldosterone's action on transcellular Na+ flux. Incubation with 10(-7) M aldosterone stimulated transcellular Na+ transport which was detected as an increase in benzamil-sensitive short-circuit current. The stimulatory response was consistently noted after 2 h of incubation and stabilized after 6 h. 2D-PAGE was used to identify proteins which were induced concurrently with the increase in transcellular Na+ flux after an aldosterone incubation of 15 h. Three aldosterone-induced proteins (AIPs; M(r) = 100, 70-77 and 46-50 kDa) were found in the membrane and microsomal fractions. Two of these appeared to have more than one isoform. A single heterogeneous AIP (M(r) = 77 kDa) was detected in the soluble fraction.

Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms.

The individual effects of aldosterone and insulin on amiloride-sensitive Na+ transport in model renal epithelia have been well characterized. However, in the physiological state, many hormones are present concurrently and their interactions need to be addressed. We have found that, over 5 h, the effects of insulin and aldosterone are additive. This indicates that the biochemical pathways are largely independent. To delineate the signaling pathways, we examined the requirement for tyrosine kinases by using genistein, a tyrosine kinase inhibitor. Genistein blocks basal (constitutive) Na+ transport and inhibits insulin- and aldosterone-stimulated Na+ transport. From these results, we conclude that a tyrosine phosphorylation is an important component of amiloride-sensitive Na+ transport.

Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line.

The Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na+ transport.

Differential effects of brefeldin A on hormonally regulated Na+ transport in a model renal epithelial cell line.

Na+ transport in renal epithelia is regulated by a wide variety of endogenous and exogenous cellular factors. Although most natriferic agents have an action on the amiloride-sensitive Na+ channel, the biochemical pathways which precede activation of the channel remain incompletely defined. One approach to dissecting such intricate pathways is to perturb a specific cellular process and determine its importance in the postulated mechanism. The current studies examine the effect of brefeldin A (BFA), an inhibitor of the central vacuolar system, on basal as well as aldosterone-, insulin-, and forskolin-stimulated Na+ transport. In the A6 cell line, BFA had a time-dependent effect on basal transport. Aldosterone-induced Na+ transport was sensitive to BFA while insulin's action was only partially blocked and forskolin-stimulated Na+ transport was relatively resistant to the action of the inhibitor. These studies highlight differences as well as points of convergence in the natriferic pathways.

Insulin and IGF1 receptors in a model renal epithelium: receptor localization and characterization.

Insulin and IGF1 stimulate transepithelial Na+ transport in the urinary bladder of the toad Bufo marinus, a model renal epithelium. The signal transduction mechanisms for the natriferic action are unknown. Ultrastructural techniques were used to localize both receptors and ligands in the epithelium. Electron microscopy using gold-labelled anti-insulin or anti-IGF1 receptor antibodies demonstrated the majority of the insulin receptors were associated with the basolateral membrane while IGF1 receptors were found on basolateral and apical membranes. Both insulin and IGF1 receptors were found in endosomes and on the membranes surrounding subapical granules. In intact tissues incubated with iodinated IGF1 or insulin, both ligands were associated with the basolateral membrane. IGF1 was internalized to a greater extent than insulin and only IGF1 accumulated in cell nuclei.

Aldosterone-mediated Na+ transport in renal epithelia: time-course of induction of a potential regulatory component of the conductive Na+ channel.

To correlate synthesis of aldosterone-induced proteins (AIPs) with the hormone's effect on transepithelial Na+ transport, protein synthesis in two model renal epithelia was examined during the natriferic response to aldosterone. Newly synthesized epithelial cell proteins were pulse-labelled with [35S]-methionine, separated by 2D-PAGE and detected by autoradiography. In both toad urinary bladder and A6 cells, a constellation of polypeptides which are a component of the renal epithelial conductive Na+ channel first appeared prior to, or concordant with, the increase in Na+ transport. The early synthesis of these proteins taken together with the fact that they appear to be the only aldosterone induced component of the multimeric Na+ channel, is compatible with the hypothesis that they are responsible for regulating Na+ channel activity during aldosterone-stimulated epithelial Na+ transport.

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.

Insulin and IGF I receptor-mediated Na+ transport in toad urinary bladders.

We compared the concentration dependence of insulin- and insulin-like growth factor I (IGF I)-stimulated Na+ transport with ligand-receptor affinities in the urinary bladder of the toad Bufo marinus. Threshold, half-maximal, and maximal natriferic concentrations of both peptides were approximately 0.1, 1, and 10 nM, respectively. Amiloride, but not ethyl isopropyl amiloride, (10(-5) M), abolished Na+ transport. Maximal responses to either peptide rendered the tissue insensitive to challenge with the other. Separate insulin and IGF I receptors were identified by equilibrium binding and polyacrylamide gel electrophoresis of cross-linked ligand-receptor complexes. For both peptides, half-maximal binding occurred at 3-10 nM; crossover binding to the other receptor occurred with 10- and 100-fold lower affinity. Thus, in this model "high-resistance" renal epithelium, 1) ligand binding to specific insulin and IGF I receptors stimulates transcellular Na+ flux, 2) the natriferic effects of insulin and IGF I apparently depend on activation of apical Na+ channels rather than Na+-H+ antiporters, and 3) the natriferic pathways activated by insulin and IGF I appear to converge subsequent to ligand-receptor binding but before the final transport ("effector") step(s).

Insulin-like growth factor 1 stimulates renal epithelial Na+ transport.

Insulin-like growth factor 1 (IGF1) stimulates vectorial Na+ transport in a classical model of the mammalian distal nephron, the toad urinary bladder. Net mucosal to serosal Na+ flux is stimulated by concentrations of IGF1 as low as 0.1 nM, and the response is maximal at 10 nM. Na+ transport increases within minutes of the serosal addition of IGF1, reaches a maximum in 2-3 h, and is sustained for at least 5 h. Neither the initial nor the sustained response to IGF1 is dependent on a new protein synthesis. The IGF1 response is inhibited by a concentration of amiloride (10(-5) M) that is known to specifically block the conductive apical Na+ channel but that has little effect on the Na+-H+ antiporter. Further studies will be necessary to establish a role for this growth factor in normal renal epithelial function, but it is possible that the natriferic and growth-stimulatory effects of IGF1 are intimately related.

Affinity purified immunoglobulin G transfers immediate hypersensitivity to guinea pig colonic epithelium in vitro.

Our aim was to establish whether the epithelial type 1 hypersensitivity reaction in response to a dietary antigen, as we have previously described, had an immunologic basis. Using serum from guinea pigs fed with milk proteins we examined the passive transfer of sensitivity to beta-lactoglobulin. Biophysical methods were used to measure electrogenic chloride secretion across colonic epithelia. This response has been shown previously to be the cellular function that is altered after antigen challenge. Affinity column chromatography was used to separate immune serum into immunoglobulin G-(IgG)-rich and immunoglobulin G-depleted fractions and the purity of these fractions was established. Passive transfer of sensitivity was possible with the immunoglobulin G-rich, but not the immunoglobulin G-depleted, fraction. We have proven, therefore, that the reaction is mediated, at least in part, by immunoglobulins of the G class. This finding may have implications for the study of phenomena of food allergy.