Outer Medullary Collecting Duct Cells Research Articles

Comparison of Isotonic Activation of Cell Volume Regulation in Rat Peritoneal Mesothelial Cells and in Kidney Outer Medullary Collecting Duct Principal Cells.

In disease states, mesothelial cells are exposed to variable osmotic conditions, with high osmotic stress exerted by peritoneal dialysis (PD) fluids. They contain unphysiologically high concentrations of glucose and result in major peritoneal membrane transformation and PD function loss. The effects of isotonic entry of urea and myo-inositol in hypertonic (380 mOsm/kg) medium on the cell volume of primary cultures of rat peritoneal mesothelial cells and rat kidney outer medullary collecting duct (OMCD) principal cells were studied. In hypertonic medium, rat peritoneal mesothelial cells activated a different mechanism of cell volume regulation in the presence of isotonic urea (100 mM) in comparison to rat kidney OMCD principal cells. In kidney OMCD cells inflow of urea into the shrunken cell results in restoration of cell volume. In the shrunken peritoneal mesothelial cells, isotonic urea inflow caused a small volume increase and activated regulatory volume decrease (RVD). Isotonic myo-inositol activated RVD in hypertonic medium in both cell types. Isotonic application of both osmolytes caused a sharp increase of intracellular calcium both in peritoneal mesothelial cells and in kidney OMCD principal cells. In conclusion, peritoneal mesothelial cells exhibit RVD mechanisms when challenged with myo-inositol and urea under hyperosmolar isotonic switch from mannitol through involvement of calcium-dependent control. Myo-inositol effects were identical with the ones in OMCD principal cells whereas urea effects in OMCD principal cells led to no RVD induction.

Expression of SLC4A11 protein in mouse and rat medulla: a candidate transporter involved in outer medullary ammonia recycling.

SLC4A11 is a multifunctional membrane transporter involved with H+ transport, NH 3 and alkaline pH stimulated H+ transport, and water transport. The role of SLC4A11 in the kidney is not well understood. A prior study has shown that in murine kidney, SLC4A11/LacZ staining is primarily in the long‐looped descending thin limb (DTL) as determined by colocalization with aquaporin 1 (AQP1), a protein that is expressed in some, but not all, descending thin limb segments. Using a previously characterized polyclonal antibody, we demonstrate the selective expression of SLC4A11 in the upper DTLs (which are AQP1‐positive) in the outer medulla and inner medulla with little or no expression in the lower DTLs (which are AQP‐1‐null). SLC4A11 also colocalized with AQP1 and the urea transporter UT‐B in the mouse descending vasa recta, but was absent in mouse and rat ascending vasa recta. Mouse, but not rat, outer medullary collecting duct cells also labeled for SLC4A11. Our results are compatible with the hypothesis that in the inner stripe of the outer medulla, SLC4A11 plays a role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb and secreted into the long‐looped DTLs. SLC4A11 can potentially modulate the rate of ammonia transport in the mouse outer medullary collecting duct. Our data suggest functionally unique SLC4A11 pathways in mouse and rat and complement previous studies of DTL Na+, urea and water permeability indicating that the upper and lower DTLs of long‐looped nephrons are functionally distinct.

Pyk2 regulates H+-ATPase-mediated proton secretion in the outer medullary collecting duct via an ERK1/2 signaling pathway

Acid-secreting intercalated cells respond to changes in systemic pH through regulation of apical H(+) transporters. Little is known about the mechanism by which these cells sense changes in extracellular pH (pH(o)). Pyk2 is a nonreceptor tyrosine kinase activated by autophosphorylation at Tyr402 by cell-specific stimuli, including decreased pH, and is involved in the regulation of MAPK signaling pathways and transporter activity. We examined whether the Pyk2 and MAPK signaling pathway mediates the response of transport proteins to decreased pH in outer medullary collecting duct cells. Immunoblot analysis of phosphorylated Pyk2 (Tyr402), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) was used to assay protein activation. To examine specificity of kinase activation and its effects, we used Pyk2 small interfering RNA to knockdown Pyk2 expression levels, the Src kinase inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP 1) to inhibit Pyk2 phosphorylation, and the MEK inhibitor U0126 to inhibit ERK1/2 phosphorylation. The pH-sensitive fluorescent probe 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was used to assay H(+) transporter activity. The activity of H(+) transporters was measured as the rate of intracellular pH (pH(i)) recovery after an NH(4)Cl prepulse. We show that Pyk2 is endogenously expressed and activated by acid pH in mouse-derived outer medullary collecting duct (mOMCD1) cells. Incubation of mOMCD1 cells in acid media [extracellular pH (pH(o)) 6.7] increased the phosphorylation of Pyk2, ERK1/2, and p38. Reduction in pH(i) induced by an NH(4)Cl prepulse also increased the phosphorylation of Pyk2, ERK1/2, and p38. Consistent with our previous studies, we found that mOMCD1 cells exhibit H(+)-ATPase and H(+),K(+)-ATPase activity. Pyk2 inhibition by Pyk2 siRNA and PP 1 prevented Pyk2 phosphorylation as well as H(+)-ATPase-mediated recovery in mOMCD1 cells. In addition, ERK1/2 inhibition by U0126 prevented acid-induced ERK1/2 phosphorylation and H(+)-ATPase-mediated pH(i) recovery but not phosphorylation of p38. We conclude that Pyk2 and ERK1/2 are required for increasing H(+)-ATPase, but not H(+),K(+)-ATPase, activity at decreased pH(i) in mOMCD1 cells.

PH sensor in the kidney

Proton receptors are G‐protein‐coupled receptors, which can sense pH in the physiological pH range. One of the receptors, GPR4, is expressed at high levels in kidneys, overexpressed in kidney tumors, and mediates pH‐dependent increase of intracellular cAMP. We used cultured outer medullary collecting duct cells, which express GPR4, and GPR4 knockout mice (KOs) (Yang et al., Mol Cell Biol, 2006) to examine if GPR4 is involved in acid‐base regulation. Acid secretion in OMCD cells was measured with NH4Cl−prepulse and pH sensitive dye BCECF‐AM as pH recovery from intracellular acidification. Control cells increase acid secretion if incubated in media of low pH (7.0), whereas cells transfected with anti‐GPR4 synthetic siRNA do not. Cells transfected with control siRNA were not different from the control, non‐transfected cells. GPR4 KOs have mild metabolic acidosis (arterial blood HCO3−of 21.2 ± 1.5 vs. 24.9 ± 0.62 mM in WTs; pCO2 of 34 ± 2.6 vs. 38 ± 1.1 mmHg in WTs, and slightly higher Cl− concentration). After acid loading (280 mM NH4Cl for 5 days), both WTs and GPR4 KOs were acidotic, but WTs decreased urine pH from 6.0 ± 0.08 to 5.5 ± 0.05 whereas urine pH of GPR4 KOs did not change (6.1 ± 0.05 vs. 6.0 ± 0.1). mRNA expression of other pH sensing receptors, OGR1 and TDAG8, examined with real‐time RT‐PCR, was not increased in the kidneys of GPR4 KOs. We also examined the expression of unrelated but exquisitely pH sensitive calcium receptor (CaR). CaR expression was increased in the kidneys of GPR4 KOs at baseline (60%) and after acid loading (100%) compared to WTs. Our in vitro and in vivo data suggest that GPR4 mediates adaptive response to acidosis and that it may act in concert with CaR, a possible explanation for only mild acidosis in GPR4 KOs.

Immunolocalization of anion exchanger 1 (Band 3) in the renal collecting duct of the common marmoset

The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl-/HCO3- exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.

H +-ATPase activity in selective disruption of H +-K +-ATPase alpha 1 gene of mice under normal and K-depleted conditions

The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by two luminal proton ATPases, H(+)-ATPase and H(+)-K(+)-ATPase (HKA), both of which are in the intercalated cells (ICs) of OMCD. We showed previously that HKAalpha1 (gastric H(+)-K(+)-ATPase) activity is the essential H(+)-K(+)-ATPase activity under normal conditions, and that HKAalpha2 (colonic H(+)-K(+)-ATPase) is induced and mediates increased proton-secretion under K-depleted conditions. To better understand the role of H(+)-ATPase (potassium-independent) in acid secretion and the relationship between H(+)-ATPase and a specific HKA isoform, we examined H(+)-ATPase activity in the H(+)-K(+)-ATPasealpha1 knockout (KO) mice under normal and K-depleted conditions. Mice were fed a potassium-free diet and studied after 7 days. Segments of the OMCD were perfused in vitro, and intracellular pH (pH(i)) was measured by ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. The isolated OMCD tubules obtained from mice fed a potassium-free diet were examined by fluorescent immunocytochemistry with an antibody to the 31-kDa subunit of H(+)-ATPase (E-11) and were compared with those obtained from a normal diet. In the absence of Na(+) and K(+), the H(+)-ATPase-mediate pH(i) recovery rates were 6.7 +/- 1.1 x 10(-4) units/s (n = 7 ICs) in wild-type (WT) mice and increased to 8.7 +/- 1.8 x 10(-4) (P < 0.05; n = 6) in HKAalpha1 KO mice. K-independent proton transport activity was significantly inhibited by the H(+)-ATPase inhibitor bafilomycin A(1) (BAF, 10 nM) with luminal applied in both WT and KO mice. Comparison of the results indicated upregulation of BAF-sensitive H(+)-ATPase activity in KO mice. To determine the intracellular localization of H(+)-ATPase in the intercalated cells of OMCD, we dissected the OMCD and performed fluorescent immunocytochemistry with the H(+)-ATPase antibody in the WT and KO mice. In the WT mice, on normal diet, H(+)-ATPase staining distributed diffusely throughout the intercalated cells and was slightly polarized to the apical plasma membrane in the KO mice, consistent with increase in the H(+)-ATPase-mediate pH(i) recovery in the KO mice. One week of a potassium-free diet resulted in a significant increase in the degree of H(+)-ATPase polarization at the apical plasma membrane in both WT and KO mice. Hypokalemia stimulates H(+)-ATPase in the intercalated cells of OMCD of both WT and KO mice. The enhanced activity of H(+)-ATPase plays an important role in compensatory proton secretion in the HKAalpha1 KO mice under normal conditions.

Vasopressin induces expression of the Cl−/HCO3− exchanger SLC26A7 in kidney medullary collecting ducts of Brattleboro rats

SLC26A7 is a newly identified basolateral Cl(-)/HCO(3)(-) exchanger specific to alpha-intercalated cells of the outer medullary collecting duct (OMCD). The purpose of the present experiments was to examine the expression of SLC26A7 in kidneys of vasopressin-deficient Brattleboro rats before and after treatment with desamino-Cys(1),d-Arg(8)-vasopressin (dDAVP). Brattleboro rats were treated with dDAVP, a vasopressin analog, for 8 days, and their kidneys were examined for the expression of SLC26A7. The expression of SLC26A7 protein, as examined by immunofluorescence, was undetectable in kidneys of Brattleboro rats. However, treatment with dDAVP induced expression of SLC26A7 protein, restoring it to levels observed in normal rats. These results were verified by Western blot analysis. The mRNA expression of SLC26A7 remained unchanged in response to dDAVP. Immunofluorescent labeling demonstrated abundant levels of anion exchanger type 1 in the OMCD of Brattleboro rats and a mild reduction in response to dDAVP. The abundance of H(+)-ATPase was not affected by dDAVP. The increased SLC26A7 expression directly correlated with enhanced aquaporin-2 expression, which is proportional to increased interstitial osmolarity in the medulla. In conclusion, vasopressin increases the expression of SLC26A7 protein through posttranscriptional mechanisms in the OMCD. The induction of SLC26A7 by vasopressin in OMCD cells of Brattleboro rats is likely an attempt by cells to regulate their cell volume and maintain HCO(3)(-) absorption in a state associated with increased interstitial medullary tonicity.

Regulation of Potassium Excretion in the Kidney

The maintenance of potassium balance depends primarily on excretion by the kidney. The regulated secretion of potassium normally accounts for most of urinary potassium excretion. Potassium transport along the nephron has two main features : the ubiquitous Na,K-ATPase defines basolateral membranes, whereas site-specific potassium transporters are responsible for the apical transport. Two different cell types mediate secretion and reabsorption of potassium. Principal cells secrete potassium, whereas intercalated cells, especially those belonging to the subfamily of β-intercalated cells, reabsorb potassium. The factors that stimulate potassium secretion by the principal cells include (1) increased extracellular fluid potassium concentration (2) increased aldosterone and (3) increased tubular flow rate. One factor that decreases potassium secretion is increased hydrogen concentration (acidosis). In situations associated with severe potassium depletion, there is a cessation of potassium secretion and net reabsorption of potassium. It is believed that H,K-ATPase transport mechanism located in the luminal membrane of the cortical and outer medullary collecting duct cells reabsorb potassium in exchange for hydrogen secreted into the tubular lumen.

Differential regulation of basolateral Cl-/HCO3- exchangers SLC26A7 and AE1 in kidney outer medullary collecting duct.

SLC26A7 is a recently identified Cl(-)/HCO(3)(-) exchanger that co-localizes with AE1 on the basolateral membrane of Alpha intercalated cells (A-IC) in outer medullary collecting duct (OMCD). The purpose of these studies was to determine whether AE1 and SLC26A7 are differentially regulated in OMCD in pathophysiologic states. Toward this end, the expression and regulation of AE1 and SLC26A7 was examined in water deprivation, a condition known to increase the osmolality of the medulla. Rats were subjected to 3 d of water deprivation while having free access to food. Northern hybridizations demonstrated that in the outer medulla, the mRNA expression of SLC26A7 increased by approximately 300% (P < 0.01 versus control; n = 3), whereas the expression of AE1 decreased by approximately 50% (P < 0.05 versus control, n = 3) in water-deprived rats. Immunoblot analysis studies demonstrated that in the outer medulla, SLC26A7 abundance increased by approximately 3.5-fold (P < 0.02 versus control; n = 3), whereas the AE1 abundance decreased by approximately 55% (P < 0.05 versus control) in water deprivation. The expression of SLC26A7 remained unchanged in the kidney cortex and stomach in water deprivation, indicating the specificity of SLC26A7 upregulation in outer medulla. In situ hybridization indicated the exclusive expression of SLC26A7 in the outer medulla and double immunofluorescence labeling confirmed the co-localization of AE1 and SLC26A7 on the basolateral membrane of A-IC cells in OMCD. It is concluded that AE1 and SLC26A7 are differentially regulated in OMCD in water deprivation. On the basis of these results and previous functional studies indicating the activation of SLC26A7 activity by high osmolality, it is proposed that SLC26A7 may play an important role in bicarbonate reabsorption and or cell volume regulation in OMCD (specifically under hypertonic conditions).

Renal and hepatic expression of the ammonium transporter proteins, Rh B Glycoprotein and Rh C Glycoprotein.

A family of ammonium transporter proteins was recently identified. Members of this family, Rh B Glycoprotein (RhBG) and Rh C Glycoprotein (RhCG) are expressed in the kidney and the liver, important tissues for ammonium metabolism. Immunohistochemical studies demonstrate basolateral RhBG immunoreactivity in the connecting segment (CNT) and collecting ducts, but not in the proximal tubule or the loop of Henle. Colocalization with thiazide sensitive cotransporter and carbonic anhydrase II confirms expression in the CNT, initial collecting tubule (ICT), and throughout the collecting duct. Colocalization with AE1 and pendrin demonstrates expression is greatest in A-type intercalated cells in the cortical collecting duct (CCD), outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD), present in the CCD principal cell, and not detectable in either pendrin-positive CCD intercalated cells or in non-intercalated cells in the OMCD and IMCD. RhCG immunoreactivity has a similar axial distribution as RhBG. However, RhCG immunoreactivity is apical, and is detectable in all CCD and outer stripe of OMCD cells. The liver, a second organ involved in ammonia metabolism, also expresses both RhBG and RhCG. Basolateral RhBG immunoreactivity is present in the perivenous hepatocyte, but is not present in either the periportal or mid-zonal hepatocyte. Hepatic RhCG mRNA is expressed at lower levels than RhBG, and RhCG protein is detected in bile duct epithelium. These findings indicate that RhBG and RhCG are involved in at least two organs that transport ammonia, and that they are located in sites where they are likely to mediate important roles in ammonia transport.

SLC26A7: a basolateral Cl-/HCO3-exchanger specific to intercalated cells of the outer medullary collecting duct

The outer medullary collecting duct (OMCD) plays an important role in bicarbonate reabsorption and acid-base regulation. An apical V-type H+-ATPase and a basolateral Cl-/HCO3- exchanger, located in intercalated cells of OMCD, mediate the bicarbonate reabsorption. Here we report the identification of a new basolateral Cl-/HCO3- exchanger in OMCD intercalated cells in rat kidney. Northern hybridizations demonstrated the predominant expression of this transporter, also known as SLC26A7, in the outer medulla, with lower expression levels in the inner medulla. SLC26A7 was recognized as a approximately 90-kDa band in the outer medulla by immunoblot analysis and was localized on the basolateral membrane of a subset of OMCD cells by immunocytochemical staining. No labeling was detected in the cortex. Double-immunofluorescence labeling with the aquaporin-2 and SLC26A7 antibodies or anion exchanger-1 and SLC26A7 antibodies identified the SLC26A7-expressing cells as alpha-intercalated cells. Functional studies in oocytes demonstrated that increasing the osmolality of the media (to simulate the physiological milieu in the medulla) increased the Cl-/HCO3- exchanger activity mediated via SLC26A7 by about threefold (P < 0.02 vs. normal condition). We propose that SLC26A7 is a basolateral Cl-/HCO3- exchanger in intercalated cells of the OMCD and may play an important role in bicarbonate reabsorption in medullary collecting duct.

Carbonic anhydrase IV is expressed in H(+)-secreting cells of rabbit kidney.

Carbonic anhydrase (CA) IV is a membrane-bound enzyme that catalyzes the dehydration of carbonic acid to CO(2) and water. Using peptides from each end of the deduced rabbit CA IV amino acid sequence, we generated a goat anti-rabbit CA IV antibody, which was used for immunoblotting and immunohistochemical analysis. CA IV was expressed in a variety of organs including spleen, heart, lung, skeletal muscle, colon, and kidney. Rabbit kidney CA IV had two N-glycosylation sites and was sialated, the apparent molecular mass increasing by at least 11 to approximately 45 kDa in the cortex. Medullary CA IV was much more heavily glycosylated than CA IV from cortex or any other organ, such modifications increasing the molecular mass by at least 20 kDa. CA IV was expressed on the apical and basolateral membranes of proximal tubules with expression levels on the order of S2 > S1 > S3 = 0. Because CA IV is believed to be anchored to the apical membrane by glycosylphosphatidylinositol, the presence of basolateral CA IV suggests an alternative mechanism. CA IV was localized on the apical membranes of outer medullary collecting duct cells of the inner stripe and inner medullary collecting duct cells, as well as on alpha-intercalated cells. However, CA IV was not expressed by beta-intercalated cells, glomeruli, distal tubule, or Henle's loop cells. Thus CA IV was expressed by H(+)-secreting cells of the rabbit kidney, suggesting an important role for CA IV in urinary acidification.

Establishment of a mouse clonal early proximal tubule cell line and outer medullary collecting duct cells expressing P2 purinoceptors.

The purpose of this study was to establish tubule cells expressing P2 purinoceptors. Use of the polymerase chain reaction coupled with reverse transcription showed that mouse clonal early proximal tubule (S1) cell line (NF-5) and outer medullary collecting tubule (OMCT) cells expressed mRNA for P2X4, P2Y1 and P2Y2 purinoceptors. ATP and its analogues induced a dose-dependent increase in cytosolic free calcium concentration ([Ca2+]i) in both NF-5 and OMCT cells. ATP enhanced [3H]-thymidine uptake by both NF-5 and OMCT cells in a dose-dependent manner. In conclusion, NF-5 and OMCT cells can be used in the further analysis of the function and regulation of activity and expression of P2 purinoceptors in the renal tubule.

Involvement of macromolecule synthesis, endonuclease activation and c-fos expression in cisplatin-induced apoptosis of mouse proximal tubule cells

We have previously demonstrated that cisplatin-induced nephrotoxicity is associated with the induction of apoptosis using mouse renal cells derived from the terminal proximal tubule (S 3) which is the major target site of cisplatin-induced injury. The purpose of this study was to elucidate the intracellular mechanisms leading to the cisplatin-induced apoptosis of S 3 cells. Actinomycin D (an inhibitor of RNA synthesis), cycloheximide (an inhibitor of protein synthesis) and aurintricarboxylic acid (an endonuclease inhibitor) reduced the extent of DNA fragmentation, a biochemical parameter of apoptosis, in cisplatin-treated S 3 cells. Furthermore, cisplatin-induced apoptosis of S 3 cells was accompanied by an increase in the level of c-fos mRNA expression, which is inhibited by pretreatment of the cells with actinomycin D, but not with cycloheximide or aurintricarboxylic acid. In contrast, outer medullary collecting duct cells treated with cisplatin exhibited morphological changes characteristic of apoptosis and an increase in the level of c- fos mRNA expression, but no increase in the extent of DNA fragmentation. In conclusion, the synthesis of macromolecules such as RNA and protein, endonuclease activation and c-fos expression appear to be involved in the intracellular pathways leading to the induction of apoptosis in cisplatin-treated S 3 cells. In addition, the response to cisplatin may be different in different cells.

Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells.

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

Repressive regulation of the aquaporin-2 gene.

Expression of aquaporin-2 (AQP2) is exclusively limited to kidney collecting duct cells, and this strictly limited expression could be mediated by transcription of the gene. We first examined AQP2 mRNA expression in many cultured epithelial cells derived from kidney. Northern blot using OK, LLC-PK1, Madin-Darby canine kidney, and outer medullary collecting duct (OMCD) cells and primary culture of inner medullary collecting duct (IMCD) cells did not reveal any significant signal. A more sensitive method, ribonuclease protection assay, could detect a faint signal in OMCD cells when they were bathed in a hypertonic medium. Reverse-transcribed polymerase chain reaction applied to primary culture of IMCD cells showed a rapid dissipation of AQP2 mRNA within 4 days after culture. A reporter gene assay performed in the 1st day of primary culture of IMCD cells showed that the 5' region up to -2.9 kb worked as a promoter. Deletion experiments showed that at least two regions, from -434 to -364 and from -153 to -84, contain negatively acting cis-elements. When connected to a heterologous promoter, these regions repressed the activity in an orientation-dependent manner. These results suggest that transcription of AQP2 gene is strictly regulated and its ability is rapidly depressed in culture condition. This cell differentiation-specific expression of the gene may be, at least in part, mediated by the repressors present in its 5'-flanking region.

Beta-adrenergic regulation of H+ secretion by cultured outer medullary collecting duct cells.

The effects of the beta-adrenergic agonist isoproterenol (Iso) on cells of the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture were examined using whole cell patch-clamp techniques and measurements of intracellular pH (pHi) and Ca2+. Iso (10(-6) M) increased the cellular Cl- conductance, and this effect was mimicked by treatment of the cells with dibutyryladenosine 3',5'-cyclic monophosphate (cAMP, 10(-5) M) or protein kinase A (PKA, 0.4 U/ml). Iso did not alter the baseline pHi, but it did increase the activity of both the Cl-/HCO3- antiporter and the H(+)-adenosinetriphosphatase (H(+)-ATPase). The increase in Cl-/HCO3- antiporter rate was mimicked by dibutyryl-cAMP plus 3-isobutyl-1-methylxanthine (cAMP + IBMX, 10(-4) M + 10(-5) M). However, the Iso-induced stimulation of the H(+)-ATPase activity was not mimicked by cAMP + IBMX. Measurements of intracellular Ca2+ showed that Iso also increased intracellular Ca2+ levels. This response was not dependent on extracellular Ca2+, nor did cAMP + IBMX appreciably alter intracellular Ca2+. Consequently, we postulate that beta-adrenergic agonists are potential stimulators of OMCDi H+ secretion. These agonists stimulate cellular HCO3- efflux through a signal transduction pathway involving cAMP and PKA. However, a different signal transduction pathway appears to mediate the stimulation of cellular H+ efflux. This second pathway may involve an elevation of intracellular Ca2+.

Electrophysiological properties of cultured outer medullary collecting duct cells.

Whole cell patch-clamp techniques were used to characterize the electrophysiological properties of cells from the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture. With pipette and bathing solutions mimicking intracellular and extracellular fluid, the resting membrane voltage was -30 to -40 mV. The whole cell conductance exhibited slight outward rectification, and at the resting membrane voltage the cell conductance averaged 2.58 +/- 0.49 nS (n = 17). The major conductive ion species was Cl-. The Cl- conductance was also found to have a significant permeability to HCO3- and was inhibited by the Cl(-)-channel blockers diphenylamine carboxylic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. A small K+ conductance was also present, but no Na+ conductance was detected. Current generated by the H(+)-adenosinetriphosphatase (H(+)-ATPase) was quantitated. This current was dependent on the presence of ATP in the pipette. Dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin A1, inhibitors of the vacuolar H(+)-ATPase, also reduced this outward current in an ATP-dependent manner. The inhibitor-sensitive component of the outward current, a measure of the current generated by the H(+)-ATPase, was in the range of 35-100 pA/cell.

Characterization of acid-base transporters in cultured outer medullary collecting duct cells.

Cells from the inner stripe of the rabbit outer medullary collecting duct (OMCDi) were grown in primary culture, and their acid-base transport properties were characterized using intracellular pH (pHi) measurements with the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal pHi in HCO(3-)-buffered solutions was 7.28 +/- 0.04 (n = 20). The presence of a Cl-/HCO(3-)-antiporter was demonstrated by reversible alkalinization on bath Cl- removal. The mean alkalinization seen on Cl- removal was 0.16 +/- 0.02 pH units (n = 20) and was inhibited 92% by 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Studies were also performed to determine the presence of an Na+/H+ antiporter and an H(+)-adenosinetriphosphatase (H(+)-ATPase). After an NH4Cl acid load the cells exhibited both Na(+)-dependent and Na(+)-independent pHi recovery mechanisms. The Na(+)-dependent mechanism was inhibited by amiloride. The Na(+)-independent mechanism was completely inhibited by 10(-3) M N-ethylmaleimide or 2.5 x 10(-9) M bafilomycin A1, but was not significantly altered by removal of bathing solution K+. Thus, the Na(+)-dependent recovery mechanism exhibited characteristics of an Na+/H+ antiporter, whereas the Na(+)-independent recovery mechanism was consistent with the presence of an H(+)-ATPase.

Cl-HCO3 exchange and Na-HCO3 symport in rabbit outer medullary collecting duct cells

The mechanisms of basolateral HCO3- (or related base) transport were examined in the outer stripe of the outer medullary collecting duct (OMCDo). Rabbit OMCDo were perfused in vitro and cell pH (pHi) of principal cells (PC) and intercalated cells (IC) was monitored with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using fluorescence ratio imaging microscopy. Basolateral HCO3- exit was stimulated either by bath HCO3- reduction or by NH3/NH4(+)-induced cell alkalinization. Rates of pHi decrease were twice as high in IC than in PC. In both cell types HCO3- exit was dependent on Cl(-)-coupled and Na(+)-coupled mechanisms; the former was dominant in IC, whereas the latter was dominant in PC. pHi of PC and IC significantly increased by bath Cl- removal in the absence of Na+ and decreased by bath Na+ removal in the absence of Cl- but with amiloride. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited these pHi responses to a reduction of bath HCO3-, Cl-, or Na+, and an alkali loading. Our results suggest that 1) the capacity for basolateral HCO3- transport is twice as high in IC than in PC, 2) both PC and IC possess basolateral Na(+)-independent Cl(-)-HCO3- exchange and Na(+)-HCO3- cotransport, and 3) basolateral HCO3- transport is mediated mainly by Na(+)-HCO3- cotransport in PC and by Na(+)-independent Cl(-)-HCO3- exchange in IC.