Appearance of specific proteins in the apical plasma membrane of cultured renal collecting duct principal cell epithelium after chronic administration of aldosterone and arginine vasopressin

Principal cell epithelium of renal collecting duct from neonatal rabbit kidney was cultured in the presence of aldosterone (1 x 10(-6) M) and arginine vasopressin (AVP; 1 x 10(-6) M) for 10 days to investigate, by immunohistochemical methods using specific monoclonal antibodies, whether the hormones influence the expression and insertion of plasma membrane proteins. The experiments demonstrated that aldosterone alone or aldosterone plus AVP significantly increased the number of epithelial cells reacting at the luminal and lateral plasma membrane with the antibodies CD 2 and 3, specific for renal collecting duct, as we have shown in the kidney. In cultures treated with aldosterone and aldosterone plus AVP, nearly all epithelial cells were labelled by the antibodies, while controls or AVP treatment showed 41% and 24% unreactive cells, respectively. These findings were complemented with electrophysiological experiments, in which epithelia pretreated by aldosterone or aldosterone plus AVP showed significantly hyperpolarized transepithelial voltage (Vte) and higher resistance (Rte) than controls or AVP-treated specimens. The experiments demonstrated that chronic administration of aldosterone or of aldosterone plus AVP to increase Na+-transport was paralleled by the appearance of collecting-duct-specific proteins in the epithelium. Consequently, this result indicates that aldosterone influences the functional maturity of the cultured epithelium.     

Action of aldosterone on cultured renal collecting duct cells during the latent period.

The effects of aldosterone on protein synthesis in the latent period were investigated on cultured renal collecting duct cells from neonatal rabbit kidneys. Tissue was incubated with radioactively labelled uridine and amino acids and then precipitated with trichloroacetic acid in order to determine the intracellular precursor pool and identify new synthesis of RNA and protein. During the latent period, aldosterone increased the intracellular radioactive uridine pool and total radioactive RNA content already 20 and 60 min after its application; conversely 40 min after aldosterone introduction, no stimulation was found. Further experiments revealed that the intracellular radioactive amino acid pool was generally increased by aldosterone after 20, 40 and 60 min, while a distinct increased radioactive protein content was found to be induced by aldosterone only after 40 min. This indicates that aldosterone increases the uptake of RNA and protein precursors and the new synthesis of RNA and proteins. These events seem to to be regulated not continuously but intermittently. The induced proteins possibly take part in the mediation of the early hormone response. Experiments with the aldosterone antagonist, spironolactone, provide evidence for the specificity of the described hormone effects. The results after application of the Na+ channel blocker, amiloride, and the Na+/K(+)-ATPase inhibitor, G-strophanthin, indicate that the aldosterone effects are controlled by Na+ channels and Na+ pumps and therefore by the intracellular Na+ content. The inhibitory effect of cycloheximide on the aldosterone-induced protein synthesis indicates the role of these proteins on the hormone-stimulated Na+ transport.     

Methylation of cytosolic proteins may be a possible biochemical pathway of early aldosterone action in cultured renal collecting duct cells

The common model of aldosterone-dependent sodium transport is that the hormone increases sodium transport during the "early" and "late" response phases by inducing specific proteins (AIPs). However, in actual biochemical studies, AIPs were mostly detected 6-24 h after aldosterone application. Regarding the physiological early response phase, this implies temporal dissociation of the physiological and biochemical events. The discrepancy raises the question as to whether other biochemical events, such as protein modifications, may be involved in addition to the novo protein synthesis. Labelling of cultured renal collecting duct epithelia for 1-5 h with a radioactive methylgroup donor, S-adenosyl methionine (SAM), following tissue fractionation, resulted in progressive methylations of specific cytosolic proteins. Aldosterone-dependent methylations increased consistently with time, and accounted for a 60% increase in total cytosolic protein content as compared to controls after 5 h labelling. The different methylated proteins showed a molecular weight of 220, 97 and 75 kd and comprised groups of proteins with an isoelectric point of 5.1-5.7 and 6.0-7.5. Methylation of identical proteins was obtained by incubation of the epithelia with unlabelled SAM instead of aldosterone. SAM-induced as well as aldosterone-induced methylation of proteins with an isoelectric point of 6.0-7.5 could be inhibited by the methylation inhibitor S-adenosylhomocysteine. The results indicate that aldosterone may influence the SAM cycle in cultured collecting-duct epithelia during increase of the Na+-transport.     

Mineralocorticoid-specificity of aldosterone-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders.

We have identified a group of proteins (Mr approximately 70000-80000; pI approximately 5.8-6.4) in giant-toad (Bufo marinus) urinary-bladder epithelial cells whose synthesis appears to be related to aldosterone-stimulated Na+ transport. To define this relationship further, we examined whether submaximal natriferic concentrations of aldosterone induced these proteins and whether spironolactone (a specific mineralocorticoid antagonist in renal epithelia) inhibited their synthesis. Short-circuit current was used to measure Na+ transport and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. Submaximal natriferic concentrations of aldosterone (1.4 X 10(-8) M) induced the same proteins as maximal concentrations of the hormone (1.4 X 10(-7) M). In contrast, in previous experiments, similar proteins were not induced by subnatriferic concentrations (5.0 X 10(-8) M) of cortisol, a glucocorticoid. A spironolactone/aldosterone molar ratio of 2000:1 was required to inhibit aldosterone-stimulated Na+ transport completely; ratios of 200:1 and 500:1 produced partial inhibition. Concentrations of spironolactone that abolished aldosterone-stimulated Na+ transport also inhibited aldosterone-induced protein synthesis. We conclude that the synthesis of the proteins we have identified is specifically related to activation of the mineralocorticoid pathway.     

Cell associated glycoproteins synthesized by cultured renal tubular cells

Summary Thin cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's MEM containing 10% fetal bovine seru. Within 24 h the explants formed globular bodies which were completely covered by a monolayered epithelium. The cells show polar differentiation and resemble the renal collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with d-valine instead of l-valine additionally a monolayer of renal collecting duct cells was obtained. For the study of glycoprotein synthesis the globular bodies and the collecting duct monolayers were incubated with various labelled carbohydrates, protein and collagen precursors and then fractionated into coarse membrane pellets. The synthesized glycoproteins were regained in 600×g and 12,000×g coarse membrane fractions and extracted with Triton X 100 buffer for column chromatography and SDS-polyacrylamide electrophoresis in 6 M urea. In addition to a 85,000 d glycoprotein, a carbohydrate rich collagen like protein (apparent molecular weight in column chromatography 200,000 d, in the SDS-polyacrylamide electrophoresis 150,000 d) was found. The 150,000 d glycoprotein incorporates favorably radioactive proline, sulfate, and smaller amounts of lysine, and leucine. Compared to the 85,000 d glycoprotein a double amount of glucosamine and galactose and four fold amount of fucose was detected. The 85,000 d protein has to be ascribed as a usual glycoprotein, in contrast the 150,000 d protein shows an unusual combination of characteristics and has to be considered as a new type of renal glycoprotein.     

Thiazolidinediones expand body fluid volume through PPARgamma stimulation of ENaC-mediated renal salt absorption

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-gamma (PPARgamma, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct-specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg (flox/flox) mice. Deletion of collecting duct Pparg decreased renal Na(+) avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na(+) absorption and Scnn1g mRNA (encoding the epithelial Na(+) channel ENaCgamma) expression through a PPARgamma-dependent pathway. These studies identify Scnn1g as a PPARgamma target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.     

Action of aldosterone on citrate synthase in cultured renal collecting duct cells

Cultured renal collecting duct cells from neonatal rabbit kidney were used to examine the influence of aldosterone on enzymatic activity of citrate synthase during increase in Na+ transport. Control epithelia showed citrate synthase activity of 71 +/- 3 mU/mg protein (n = 28), while after aldosterone treatment citrate synthase activity was significantly increased to 79 +/- 6 mU/mg at 1 h (n = 5), to 88 +/- 6 mU/mg at 2 h (n = 6) and to 93 +/- 8 mU/mg protein at 3 h (n = 5). Citrate synthase activity subsequently decreased to basal values. Spironolactone fully blocked the aldosterone-induced increase in citrate synthase activity. The time course of enzyme stimulation after aldosterone administration indicates that the hormone activates citrate synthase during the physiological early response phase.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600 X g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum. Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.     

Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD(c14) cortical collecting duct cell line to determine effects of ATP on sodium (Na(+)) and chloride (Cl(-)) transport with an Ussing chamber system. ATP, at a concentration of 10(-6) M or less, did not inhibit ENaC-mediated short-circuit current (I(sc)) but instead stimulated a transient increase in I(sc). The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V(te)) is clamped to less than -10 mV. We hypothesized that this negative V(te) might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD(c14) cells with aldosterone (10(-6) M) and then clamped the V(te) to -50 mV, the V(te) of aldosterone-stimulated cells under open-circuit conditions. ATP (10(-6) M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl(-) absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V(te), may dictate the direction of ATP-stimulated Cl(-) transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.     

Aldosterone upregulates vascular endothelial growth factor expression in mouse cortical collecting duct epithelial cells through classic mineralocorticoid receptor

Accumulating evidence shows that aldosterone plays an important role in the pathogenesis of renal fibrosis but its mechanism has not been completely defined. Recently, exogenous administration of aldosterone significantly alleviated ischemic states in a model of femoral artery ligated rats, accompanied by an obvious enhancement of VEGF upregulation. We hypothesized that aldosterone may also regulate the expression of VEGF in the kidney. To confirm this, cultured cortical collecting duct epithelial cells (M-1 cell line) were incubated with aldosterone and control media, respectively. The pathway by which aldosterone regulates VEGF expression was tested by the administration of spironolactone, a specific mineralocorticoid receptor (MR) antagonist. VEGF expression was detected by immunofluorescence staining, ELISA, Western blot and RT-PCR. Aldosterone induced an elevation of VEGF excretion in a time- and dose-dependent manner. Western blotting showed a 1.4-fold elevation in cytosolic VEGF expression following aldosterone (10(-8) M) incubation for 48 h (p<0.01). After aldosterone (10(-7) M) incubation for 48 h, the mRNA level of VEGF164 and VEGF120 showed 1.8- and 1.7-fold increases, respectively (p<0.01). This upregulation was almost completely blocked by spironolactone as shown both by mRNA levels and cytosolic protein levels. In addition, the mRNA of aldosterone receptor was detected in M-1 cells. We demonstrated for the first time that aldosterone induced VEGF expression in M-1 cells, an effect mediated by classic mineralocorticoid receptor. This finding provides experimental evidence for the local non-hemodynamic action of aldosterone.     

AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells

Our previous work in 293T cells and AF17(-/-) mice suggests that AF17 upregulates expression and activity of the epithelial Na(+) channel (ENaC), possibly by relieving Dot1a-AF9-mediated repression. However, whether and how AF17 directly regulates Dot1a cellular distribution and ENaC function in renal collecting duct cells remain unaddressed. Here, we report our findings in mouse cortical collecting duct M-1 cells that overexpression of AF17 led to preferential distribution of Dot1a in the cytoplasm. This effect could be blocked by nuclear export inhibitor leptomycin B. siRNA-mediated depletion of AF17 caused nuclear accumulation of Dot1a. AF17 overexpression elicited multiple effects that are reminiscent of aldosterone action. These effects include 1) increased mRNA and protein expression of the three ENaC subunits (α, β and γ) and serum- and glucocorticoid inducible kinase 1, as revealed by real-time RT-qPCR and immunoblotting analyses; 2) impaired Dot1a-AF9 interaction and H3 K79 methylation at the αENaC promoter without affecting AF9 binding to the promoter, as evidenced by chromatin immunoprecipitation; and 3) elevated ENaC-mediated Na(+) transport, as analyzed by measurement of benzamil-sensitive intracellular [Na(+)] and equivalent short circuit current using single-cell fluorescence imaging and an epithelial Volt-ohmmeter, respectively. Knockdown of AF17 elicited opposite effects. However, combination of AF17 overexpression or depletion with aldosterone treatment did not cause an additive effect on mRNA expression of the ENaC subunits. Taken together, we conclude that AF17 promotes Dot1a nuclear export and upregulates basal, but not aldosterone-stimulated ENaC expression, leading to an increase in ENaC-mediated Na(+) transport in renal collecting duct cells.     

Regulation of cGMP production by intracellular alkalinization in cultured rat inner medullary collecting duct cells

We evaluated the relationship between cell pH and cGMP production in cultured rat renal inner medullary collecting duct cells. The cGMP level, 21 +/- 6, was not different in control vs. alkalinized cells, 49 +/- 17 fmol/mg protein (p greater than 0.5). 10(-11) M atrial natriuretic peptide (ANF) enhanced cGMP production in alkalinized cells, 426 +/- 34 vs. 141 +/- 9*. Conversely, alkalinization inhibited 10(-4)M nitroprusside (SNP) induced cGMP formation, 29 +/- 9 vs. 332 +/- 67*. Phosphodiesterase inhibition abolished the difference in cGMP production by ANF but did not reverse the inhibitory effect of alkalinization on SNP induced cGMP production. In rat renal inner medullary collecting duct cells, cellular alkalinization plays a significant role in the regulation of guanylate cyclase mediated cGMP production. * = p less than 0.05).     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immunohistochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a 'leaky' to a 'tight' epithelium is evident from the acquisition of the alpha-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins, PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, PCD2 and PCD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PCD1, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is, however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells

In the renal collecting duct (CD) the major physiological role of aldosterone is to promote Na+ reabsorption. In addition, aldosterone may also influence CD water permeability elicited by vasopressin (AVP). We have previously shown that endogenous expression of the aquaporin-2 (AQP2) water channel in immortalized mouse cortical CD principal cells (mpkCCDC14) grown on filters is dramatically increased by administration of physiological concentrations of AVP. In the present study, we investigated the influence of aldosterone on AQP2 expression in mpkCCDC14 cells by RNase protection assay and Western blot analysis. Aldosterone reduced AQP2 mRNA and protein expression when administered together with AVP for short periods of time (< or =24 h). For longer periods of time, however, aldosterone increased AQP2 protein expression despite sustained low expression levels of AQP2 mRNA. Both events were dependent on mineralocorticoid receptor occupancy because they were both induced by a low concentration of aldosterone (10-9 m) and were abolished by the mineralocorticoid receptor antagonist canrenoate. Inhibition of lysosomal AQP2 protein degradation increased AQP2 protein expression in AVP-treated cells, an effect that was potentiated by aldosterone. Finally, both aldosterone and actinomycin D delayed AQP2 protein decay following AVP washout, but in a non-cumulative manner. Taken together, our data suggest that aldosterone tightly modulates AQP2 protein expression in cultured mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.     

Effects of glycoprotein and basement membrane synthesis inhibitors on the growth of cultured renal collecting duct epithelium

The biochemical and morphological extent of glycoprotein synthesis inhibition of cellular and extracellular proteins was studied on cultured renal collecting duct (CD) epithelium. We found that tunicamycin (4 micrograms/ml) inhibits the glycosylation of a 150,000 d glycoprotein (gpCDI). A 85,000 d glycoprotein (gpCDII) was not affected. The inhibition by tunicamycin demonstrates that gpCDI has characteristics of a N-glycan, whereas gpCDII seems to be an O-glycan. 6-diazo-5-oxo-norleucine (4 X 10(-5) M) which was used as glutamine analogue, did not show a comparable inhibitory effect as seen with tunicamycin. The lack of effect of norleucine demonstrates that glutamine is not the locus of glycosylation in both proteins. However, because of the tunicamycin inhibition it points to asparagine as the site of glycosylation in the gpCDI. Long term cultures of the tissue up to 15 days in the presence of tunicamycin and norleucine and of substances usually used as basement membrane inhibitors, such as hydroxy-D-proline (1 mM), L-azetidine-2-carboxylic acid (1 mM) and o- and p-nitrophenyl-xylopyranoside 1 mM), revealed that it is possible to eliminate completely the fibroblasts beneath the cultured epithelium and within the degenerating corematerial. Experiments with hydroxy-D-proline showed the most striking effect. Experiments with L-azetidine-2-carboxylic acid and nitrophenyl-xylopyranoside resulted in the elimination of fibroblasts and dedifferentiation of the collecting duct epithelium.     

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.     

Aldosterone and tight junctions: modulation of claudin-4 phosphorylation in renal collecting duct cells

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immuno-histochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a ‘leaky’ to a ‘tight’ epithelium is evident from the acquisition of the α-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins. PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, P^CD2 and P^CD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PcDl, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is. however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Thyroid hormone. Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T3) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have examined the effect of T3 on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5 X 10(-8) M T3 to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T3 completely inhibits the maximal increase in short-circuit current in response to 1 X 10(-7) M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T3 and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5 X 10(-9) M T3. T3 has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T3 on nuclear binding of [3H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K'd1 = (0.82 +/- 0.36) X 10(-10) M and K'd2 = (3.2 +/- 0.60) X 10(-8) M. The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1 X 10(-8) M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5 X 10(-8) M T3 showed a K'd1 = (0.15 +/- 0.10) X 10(-10) M and K'd2 = (3.5 +/- 0.10) X 10(-8) M. We conclude that T3 inhibits the action of aldosterone on sodium transport at a site after receptor binding in the nucleus.