Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells.

Previous studies indicate that the colonic H-K-ATPase mRNA is expressed as the distal nephron. However, the exact intrarenal localization of the colonic H-K-ATPase protein is still unclear. The goal of the present study was to determine the cellular and subcellular localization of the colonic H-K-ATPase protein in the rabbit kidney. We used three monoclonal antibodies (MAbs) directed against different epitopes of the rabbit colonic H-K-ATPase alpha-subunit (HKalpha(2)) to localize HKalpha(2) protein by immunofluorescence labeling of kidney sections and laser-scanning confocal microscopy. The specificity of the MAbs was confirmed by reaction with a single ~100-kDa band on Western blots of distal colon. Specific immunohistochemical reaction with the apical membrane of surface epithelial cells was observed with all three MAbs on distal colon sections. In rabbit kidney, immunofluorescence was detected only on the apical membrane of connecting tubule cells. Immunofluorescence was not detected in the cortical-, outer-, and inner-medullary collecting ducts. Furthermore, costaining with principal- and intercalated cell-specific MAbs and a MAb against the thick ascending limb suggests that these cell types express HKalpha(2) protein at levels that are below the detection limit with this method. We conclude that in the rabbit kidney, under normal dietary conditions, the HKalpha(2) protein is expressed in the apical membrane of connecting tubule cells.

The sgk, an aldosterone-induced gene in mineralocorticoid target cells, regulates the epithelial sodium channel.

The sgk, an aldosterone-induced gene in mineralocorticoid target cells, regulates the epithelial sodium channel. Aldosterone increases sodium reabsorption in tight epithelia. The early phase of this stimulatory effect is thought to involve activation of apical sodium channels. To identify immediate-early genes that initiate this effect, we used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques. This review summarizes our recent findings. Aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum- and glucocorticoid-regulated kinase), in the native mineralocorticoid target cells, that is, in cortical collecting duct (CCD) cells. The induction of sgk mRNA occurs within 30 minutes of the addition of aldosterone and does not require de novo protein synthesis, indicating that sgk is an immediate/early aldosterone-induced gene. Induction of sgk by aldosterone is mediated through mineralocorticoid receptors (MRs), since it is prevented by ZK91857, an MR antagonist, but not by RU486, a glucocorticoid antagonist. In addition to aldosterone, RU28362, a pure glucocorticoid receptor agonist, also induced sgk mRNA, both in primary cultures of rabbit CCD cells and in the M-1 mouse CCD cell line. Sgk mRNA levels are also influenced by changes in the osmolality of the medium. In M-1 cells, incubation of cells for one hour in a mildly hypotonic medium decreased sgk mRNA levels, whereas incubation in hypertonic medium brought about opposite changes. To determine whether sgk is involved in the regulation of the epithelial sodium channel (ENaC), we coexpressed the full-length sgk cRNA in Xenopus oocytes with the three ENaC subunits. Expression of sgk resulted in a significant increase in the amiloride-sensitive Na current, suggesting that this protein kinase plays an important role in the early phase of aldosterone-stimulated Na transport. These results indicate that sgk is an aldosterone-induced immediate/early gene in native MR target cells, and is involved in the regulation of ion transport and possibly cell volume.

SGK is a primary glucocorticoid-induced gene in the human.

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes.

The serum- and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloride-sensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of beta and gamma subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Intrarenal distribution of the colonic H,K-ATPase mRNA in rabbit.

BACKGROUND: Evidence suggests that the colonic H,K-ATPase isoform is expressed in the kidney and that a mRNA species highly homologous to the rat and guinea pig HKalpha2 is expressed in the cortical collecting duct (CCD) of the rabbit. The goals of this study were to determine if this mRNA is the rabbit homologue of HKalpha2 or a novel isoform and to determine intrarenal distribution of the HKalpha2 mRNA in rabbit. METHODS: 5'-RACE and Dye Deoxy Terminator chemistry were used to determine the full-length sequence of the rabbit HKalpha2 mRNA. The intrarenal distribution of HKalpha2 mRNA was determined in microdissected nephron segments, connecting tubule (CNT), and CCD cells isolated by immunodissection, as well as in the three cell types of the CCD. Principal cells and alpha- and beta-intercalated cells were isolated by fluorescence-activated cell sorting. HKalpha2 mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) or single-nephron RT-PCR (SN-RTPCR). RESULTS: The full-length sequence of the rabbit kidney HKalpha2 mRNA was determined. This transcript is identical to the one expressed in rabbit distal colon. In microdissected nephron segments, strong HKalpha2 amplicons were present in the CNT, CCD, and outer medullary collecting duct (OMCD), whereas no signal was detected in the proximal tubule, distal convoluted tubule, think ascending limb, and inner medullary collecting duct. Roughly comparable levels of HKalpha2 mRNA were present in all three CCD cell types, and the highest levels were observed in a subpopulation most likely corresponding to CNT cells. CONCLUSIONS: These results suggest that the HKalpha2 mRNA is expressed in rabbit collecting duct is identical in size and sequence to the one expressed in rabbit distal colon. HKalpha2 mRNA in the rabbit kidney is selectively expressed in the CNT, CCD, and OMCD, and all three collecting duct subtypes express its mRNA.

sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels.

The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum- and glucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The full-length sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88-94% at the nucleotide level, and 96-99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na+ channel results in a significantly enhanced Na+ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na+ transport.

Rabbit distal convoluted tubule coexpresses NaCl cotransporter and 11 beta-hydroxysteroid dehydrogenase II mRNA.

BACKGROUND: Although the renal cortical collecting duct (CCD) is a principal target for aldosterone, recent evidence suggests that salt transport by other nephron segments may also be regulated by aldosterone. Electroneutral and thiazide-sensitive NaCl cotransport by the distal convoluted tubule (DCT) of the rat is increased in animals deprived of dietary NaCl. We tested the hypothesis that the DCT of the rabbit is an aldosterone target tissue. METHODS: The single-nephron reverse-transcriptase/polymerase chain reaction (RT-PCR) technique was used to determine mRNA expression of NaCl cotransporter and 11 beta-HSD 2 in dissected nephron segments. The rabbit NaCl cotransporter was first cloned and rabbit-specific primers selected. A micro-assay was developed to assess 11 beta-HSD 2 enzyme activity in 0.5 mm samples of the same nephron segments. RESULTS: NaCl cotransporter was expressed in 0 of 6 proximal tubule (PT), 6 of 6 DCT and 3 of 6 CCD samples, while 11 beta-HSD was found in 0 of 7 PT, 7 of 7 DCT and 9 of 9 CCD samples. Corticosterone was converted to 11-dehydrocorticosterone at a high rate and to a similar extent by both the DCT and CCD, but not the PT. CONCLUSIONS: We conclude that the DCT is a target tissue for the action of aldosterone. Axial heterogeneity of electroneutral (in DCT) and electrogenic (in CCD) Na transporters along the distal nephron may improve sodium recovery in low salt and volume states.

The role of 11beta-hydroxysteroid dehydrogenase in steroid hormone specificity.

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11beta-HSD is present, which, in contrast to the type 1 11beta-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11beta-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11Beta-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11beta-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11beta-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11beta-HSD2 using a chimera encoding 11beta-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11beta-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11Beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11beta-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11beta-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11beta-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11beta-HSD activity.

Extranuclear localization of endogenous 11beta-hydroxysteroid dehydrogenase-2 in aldosterone target cells.

Type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2) plays a key role in conferring aldosterone selectivity on the mineralocorticoid receptor (MR) by inactivating intracellular glucocorticoids before they can occupy the MR. 11betaHSD2 is a microsomal enzyme expressed in aldosterone target cells, although its subcellular distribution is still unclear. The goal of this study was to determine the subcellular localization of the endogenous 11betaHSD2 in renal aldosterone target cells. We generated an antibody against rabbit 11betaHSD2 and used it in combination with a nuclear marker and confocal laser scanning microscopy. On Western blots the antibody recognized a single band of approximately 41 kDa in the renal cortical collecting duct, outer medullary collecting duct, submandibular gland and adrenal cortex, whereas the colon, liver, renal medulla, and heart were negative. Immunohistochemistry showed specific reaction in the known aldosterone target cells of the kidney (connecting tubule, cortical collecting duct, and outer medullary collecting duct) with no signals over glomeruli, proximal nephron segments, and blood vessels. Staining for 11betaHSD2 was very weak in rabbit colon, and no immunoreactivity could be detected in the heart and brain. Confocal microscopy of kidney sections costained with the 11betaHSD2 antibody and the nuclear marker propidium iodide demonstrated that 11betaHSD2 is in the cytoplasmic compartment with no evidence for nuclear localization. Subcellular localization of 11betaHSD2 to a cytoplasmic compartment seems ideal for fulfilling its biological function, i.e. the efficient inactivation of intracellular glucocorticoids before they occupy MRs, which are predominantly cytoplasmic in the absence of hormone.

Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists.

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green fluorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by fluorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclear-to-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were retained in nuclear matrix preparations after in vivo crosslinking. These data strongly suggest that hormone-activated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids.

Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance.

AE2 mRNA and protein is expressed in several nephron segments, one of which is the cortical collecting duct (CCD). However, the distribution of AE2 among the different cell types of the CCD and the function of AE2 in the kidney are not known. The purpose of this study was to determine the distribution of AE2 mRNA among the three CCD cell types and to examine the effects of changes in acid/base balance on its expression. Following NH4Cl (acid) or NaHCO3 (base) loading of rabbits for approximately 18 h, CCD cells were isolated by immunodissection. AE2 mRNA levels were determined by RT-PCR and were normalized for beta-actin levels. We found that CCD cells express high levels of AE2 mRNA (approximately 500 copies/cell). AE2 mRNA levels were significantly higher in CCD cells originating from base-loaded than acid-loaded rabbits, with an average increase of 3.7 +/- 1.07-fold. The effect of pH on AE2 mRNA levels was also tested directly using primary cultures of CCD cells. CCD cells incubated in acidic media expressed significantly lower levels of AE2 mRNA than those in normal or alkaline media. Experiments with isolated principal cells, alpha-intercalated cells, and beta-intercalated cells (separated by fluorescence-activated cell sorting) demonstrated that AE2 mRNA levels are comparable in the three collecting duct cell subtypes and are similarly regulated by changes in acid/base balance. Based on these results, we conclude that adaptation to changes in extracellular H+ concentration is accompanied by opposite changes in AE2 mRNA expression. The observations that AE2 mRNA is not expressed in a cell-type-specific manner and that changes in acid/base balance have similar effects on each CCD cell subtype suggest that AE2 might serve a housekeeping function rather than being the apical anion exchanger of beta-intercalated cells.

11 beta-hydroxysteroid dehydrogenase-2 is a high affinity corticosterone-binding protein.

In addition to mineralocorticoid and glucocorticoid receptors, a third category of corticosteroid binding sites has been described in the kidney, the Type III binding protein. This intracellular binder has high affinity for corticosterone, but binds neither aldosterone nor synthetic glucocorticoids. Based on similarities in steroid specificity and kinetic parameters, we hypothesized that these corticosterone binding sites belong to the type 2 isoform of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2). The goal of this study was to express the recombinant rabbit 11 beta-HSD2 in mammalian cells and test if such cells acquire both NAD-dependent 11 beta-HSD2 activity as well as high affinity corticosterone binding sites. Stably transfected CHO cell lines expressed high, NAD-dependent, unidirectional 11 beta-HSD2 activity. At the same time, the transfected cells also acquired a large number of corticosterone-specific binding sites (1.21 +/- 0.3 x 10[6]), whereas non-transfected cells had no corticosterone binding above background. The Kd for corticosterone was 25 +/- 8 nM. Neither the glucocorticoid receptor (GR) agonists dexamethasone and RU 28362 nor the mineralocorticoid receptor (MR) agonist aldosterone bound to these sites. The steroid specificity of the binding sites, as determined by competing [3H]corticosterone with unlabeled steroids, is identical to that of 11 beta-HSD2: corticosterone >> 11-hydroxyprogesterone > carbenoxolone > 11 dehydrocorticosterone > cortisol > progesterone approximately DOC >>> DEX > RU 28362 - aldosterone. These results strongly suggest that the previously described high affinity corticosterone binding sites are 11 beta-HSD2. Thus, though Type III binding sites are not corticosteroid receptors as originally thought, they play an important role in regulating the activity of both mineralocorticoid- and glucocorticoid receptors.

Aberrant 11beta-hydroxysteroid dehydrogenase-1 activity in the cpk mouse: implications for regulation by the Ke 6 gene.

Glucocorticoids have been used to create experimental polycystic kidney disease in rodents and to induce cysts in embryonic kidneys cultures. In addition, the plasma corticosterone levels are higher in a heritable murine model of polycystic kidney disease, cpk mice, in the first postnatal week. Previously, we had shown that the 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) gene is down-regulated in the cpk mice in a coordinated pattern with the Ke 6 gene. In this study, we measured the level of 11betaHSD-1 activity in kidney and liver tissues of cpk homozygote mice and found a reduction in its activity only in the kidney, not in the liver. The activity of the 11betaHSD-1 enzyme appears to be tightly correlated to the level of Ke 6 protein in these tissues. We discuss the possibility that the activity of the 11betaHSD-1 enzyme may be regulated by the Ke 6 enzyme. Ke 6 gene expression has been located to the outer stripe region of rodent kidneys, which is the same region of expression as that for the 11betaHSD-1 gene. These results suggest that down-regulation of the Ke 6 gene may lead to elevated corticosterone levels, mediated through an inhibition of 11betaHSD-1 activity.

Aldosterone regulation of sodium channel gamma-subunit mRNA in cortical collecting duct cells.

Specific regulatory mechanisms of aldosterone-stimulated Na+ reabsorption through the apical amiloride-sensitive channel are unknown. In this study, we examined the effects of aldosterone on Na+ channel gamma-subunit mRNA levels in cultured rabbit cortical collecting duct cells. With the use of reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from aldosterone-treated cells and degenerate primers, a 446-base pair (bp) PCR product was amplified and further characterized by nested PCR and sequencing. The nested PCR yielded a predicted 164-bp product. Sequencing of the 446-bp PCR product revealed 83% nucleotide and 91% amino acid identity to the rat colonic Na+ channel gamma-subunit. The relative abundance of Na+ channel mRNA was determined by quantitative PCR after a 24-h aldosterone treatment. The results demonstrate that Na+ channel gamma-subunit mRNA levels were significantly higher (2.6 +/- 0.42) in aldosterone-treated cultures vs. the controls. This increase, however, is less than the aldosterone-induced increase (3.2 +/- 2.0) in the amiloride-sensitive short-circuit current. These results indicate that Na+ channel gamma-subunit mRNA levels are increased by aldosterone and that this increase is likely to be responsible, at least in part, for the aldosterone-induced Na+ current in the kidney.

Subcellular localization of the type 2 11beta-hydroxysteroid dehydrogenase. A green fluorescent protein study.

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11beta-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11beta-HSD2. We generated a chimeric gene encoding the full-length rabbit 11beta-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8-10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11beta-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11beta-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11beta-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11beta-HSD2 is localized exclusively to the ER. Since 11beta-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.

CFTR expression in cortical collecting duct cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a adenosine 3',5'-cyclic monophosphate-activated chloride channel located in the apical membrane of many epithelial cells, and it may play a significant role in the kidney. Recent functional evidence from our laboratory suggests that CFTR may be expressed by the cortical collecting duct (CCD). Therefore, in the present study, the reverse transcription-polymerase chain reaction (RT-PCR) technique was utilized to detect CFTR mRNA in the M-1 mouse CCD cell line and in immunoselected rabbit CCD cells. Primers were constructed to amplify the cDNA sequence encoding the first nucleotide binding domain of CFTR. CFTR PCR products were obtained from both M-1 and rabbit CCD cDNA preparations. The identify of the product amplified from M-1 cell cDNA was confirmed by restriction digestion analysis. The rabbit CCD PCR product was sequenced, and its deduced amino acid sequence was found to be 97% homologous to the corresponding regions of human CFTR. The level of CFTR cDNA detected after 30 cycles of amplification of CCD cDNA was only 49 +/- 8 (n = 9) times lower than the level of beta-actin PCR product obtained from the same sample, suggesting that the levels of CFTR mRNA present in the CCD are physiologically relevant. Northern analysis, using a cRNA probe corresponding to the amplified region on the mRNA from CCD cells, revealed a single hybridizing species with a size of approximately 6.5 kb. Finally, CFTR PCR was performed with cDNA preparations originating from principal cells (PC), beta-intercalated cells (beta-ICC), and alpha-ICC obtained by fluorescence-activated cell sorting of rabbit CCD. CFTR PCR products were obtained from all three cell types, with the most abundant levels found in beta-ICC. beta-ICC expressed 25-fold (n = 4, P < 0.001) and 4.5-fold (n = 7, P < 0.001) higher levels than PC and alpha-ICC, respectively. This distribution pattern suggests that, within the CCD, CFTR plays a role primarily in beta-ICC function.

Effect of acid/base balance on H-ATPase 31 kD subunit mRNA levels in collecting duct cells.

The cortical collecting duct (CCD) adapts to disturbances of acid/base balance by adjusting the direction and magnitude of its HCO3 transport. The molecular events involved in this adaptation are incompletely understood, but it seems that adaptation is accompanied by changes in the activity and intracellular distribution of the vacuolar H-ATPase. The goal of this study was to examine the effects of metabolic acidosis and alkali load on the expression of the mRNA encoding the 31 kD subunit of the vacuolar H-ATPase in rabbit CCD cells. Pairs of rabbits received either a NH4Cl load or a NaHCO3 load for 16 hours, resulting in a urinary pH of 5.53 +/- 0.38 and 8.42 +/- 0.10, respectively. CCD cells were isolated by immunodissection and mRNA levels of the H-ATPase 31 kD subunit and of beta-actin were determined from the same cDNA samples by quantitative RT-PCR. H-ATPase mRNA levels were significantly higher in CCD cells from acidotic than alkali-loaded rabbits (2.51 +/- 1.3 vs. 0.65 +/- 0.2; P < 0.05). Similar differences in the H-ATPase 31 kD subunit mRNA levels were observed by Northern blotting. beta-actin mRNA levels were comparable in CCD cells of the two groups. The distribution of the H-ATPase 31 kD subunit mRNA was determined among the three cell types of the CCD, that is in alpha- and beta-intercalated cells (alpha-ICC and beta-ICC) and principal cells (PC) isolated by fluorescence-activated cell sorting. The level of expression was comparable in alpha-ICCs and beta-ICCs, whereas PCs contained very low levels of H-ATPase mRNA. In both alpha-ICC and beta-ICC the levels of the 31 kD H-ATPase mRNA were significantly higher in acidotic than in alkali-loaded rabbits. These results indicate that in the rabbit CCD changes in acid/base balance not only regulate the subcellular distribution of the vacuolar H-ATPase but also alter its expression, at least at the mRNA level.

Expression of colonic H-K-ATPase mRNA in cortical collecting duct: regulation by acid/base balance.

In addition to the gastric isoform of H-K-ATPase, the colonic isoform is also expressed in the kidney, but its intrarenal localization and exact function are not known. The goal of this study was to determine whether the colonic H-K-ATPase is expressed in the rabbit cortical collecting duct (CCD) and whether it is regulated by changes in acid/base balance. With quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from immunodissected rabbit CCD cells and degenerate oligonucleotide primers, a PCR product of the predicted size (approximately 430 bp) was amplified. The amplified DNA was further characterized by nested PCR and sequencing. Direct sequencing of the 434-bp PCR product revealed 83% identity at the nucleotide level and an 80.4% identity at the deduced amino acid level to the rat colonic H-K-ATPase. With the same primers and cDNA originating from rabbit distal colon, a DNA fragment with a size and nucleotide sequence identical to that originating from CCD cells was amplified. Furthermore, using PCR screening, we isolated and sequenced a 1.5-kb cDNA clone from a rabbit CCD library. The predicted amino acid sequence of the protein encoded by this cDNA is 85 and 82% identical to the corresponding regions of the guinea pig and rat colonic H-K-ATPase, respectively, and 70% identical to the H-K-ATPase recently cloned from Bufo marinus, whereas it shows only 45 and 42% homology to the rat Na-K-ATPase alpha 1-subunit and the rat gastric H-K-ATPase, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression cloning of the aldosterone target cell-specific 11 beta-hydroxysteroid dehydrogenase from rabbit collecting duct cells.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor from occupancy by endogenous glucocorticoids. We have recently described a novel isoform of 11-OHSD in the renal aldosterone target cells (11 beta-OHSD/CD) that differs from the previously characterized isoform (11 beta-OHSD-1). Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation, has a very high affinity for its substrate, and is tissue-specific. We report here the isolation, sequence, and characterization of a complementary DNA (cDNA) encoding the rabbit collecting duct 11 beta-OHSD/CD or 11 beta-OHSD type 2. The cDNA, isolated using expression screening in Xenopus oocytes, is 1.9 kilobases in length and encodes a protein of 406 amino acids with a predicted molecular mass of 44,130 daltons. The cloned enzyme has a Michaelis constant (Km) for corticosterone of 6.6 +/- 3 nM, catalyzes exclusively dehydrogenation, and uses only NAD as cofactor. The cloned enzyme shows 85% and 75% amino acid identity to the recently cloned human type 2 11 beta-OHSD and sheep kidney 11 beta-OHSD, respectively, whereas the overall homology to rat liver 11 beta-OHSD-1 is less than 20% The messenger RNA for this 11 beta-OHSD is expressed at very high levels in the renal collecting duct and at much lower levels in the colon. The intrarenal distribution was determined by reverse-transcription polymerase chain reaction in isolated nephron segments or cell types. The messenger RNA is present only in aldosterone target cells within the kidney, at highest levels in principal cells, at lower levels in intercalated cells, and in inner medullary cells. These data suggest that the 11 beta-OHSD cDNA from rabbit collecting duct cells encodes the enzyme that confers aldosterone selectivity to mineralocorticoid target cells.

Vasopressin activates a chloride conductance in cultured cortical collecting duct cells.

In rabbit cortical collecting duct (CCD) cells, arginine vasopressin (AVP) causes a transient increase followed by a sustained depression of transepithelial potential difference (PDte). Mechanisms underlying the decrease in PDte are not well understood. In this study, we used primary cultures of rabbit CCD cells to study effects of AVP. Basolateral addition of AVP caused a dose-dependent increase in transepithelial conductance (Gte) and a corresponding decrease in PDte. A significant effect was observed at 1 pM AVP, and half-maximal response occurred at 30 pM AVP; 1 nM AVP increased Gte and decreased PDte. Replacement of apical Na+ with N-methyl-D-glucamine did not prevent the effect of AVP on Gte, suggesting that it is not mediated by an increase in apical Na+ conductance. Similarly, apical Ba2+ (1 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.1 mM) failed to prevent the effect of AVP. On the other hand, 5-nitro-2(3-phenylpropylamino)benzoic acid (0.1 mM) caused partial inhibition, whereas substitution of apical Cl- with gluconate or cyclamate almost completely prevented the AVP-induced increase in Gte. In unidirectional ion-flux studies, 1 nM AVP caused only a modest increase in apical-to-basolateral (A-->BL) flux of 22Na and had no effect on transepithelial flux of 86Rb in either direction. On the other hand, AVP caused a pronounced increase in A-->BL flux and BL-->A flux of 36Cl, resulting in an increased net Cl- absorption. The effect of AVP on Gte could be mimicked by 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) and isoproterenol, and effects of AVP and isoproterenol were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)