Increased expression of H+-ATPase in inner medullary collecting duct of aquaporin-1-deficient mice

2003 ◽  
Vol 285 (3) ◽  
pp. F550-F557 ◽  
Author(s):  
Young-Hee Kim ◽  
Jin Kim ◽  
A. S. Verkman ◽  
Kirsten M. Madsen
Keyword(s):  
Kidney Development ◽  
De Novo ◽  
Collecting Duct ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Wild Type ◽  
Urinary Ph ◽  
Aquaporin 1 ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

Phenotype analysis has demonstrated that aquaporin-1 (AQP1) null mice are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. We report here that deletion of AQP1 is also associated with a decrease in urinary pH from 6.15 ± (SE) 0.1 to 5.63 ± 0.07. To explore the mechanism of the decrease in urinary pH, we examined the expression of H+-ATPase in kidneys of AQP1 null mice. There was strong labeling for H+-ATPase in intercalated cells and proximal tubule cells in both AQP1 null and wild-type mice. Strong H+-ATPase immunostaining was also present in the apical plasma membrane of inner medullary collecting duct (IMCD) cells in AQP1 null mice, whereas no H+-ATPase labeling was observed in IMCD cells in wild-type mice. In addition, there was an increase in the prevalence of type A intercalated cells in the IMCD of AQP1 null mice, suggesting that the deletion of intercalated cells from the IMCD, which normally occurs during postnatal kidney development, was impaired. Western blot analysis of H+-ATPase expression in the different regions of the kidney demonstrated a significant increase in H+-ATPase protein in the inner medulla of AQP1 null mice compared with wild-type mice. There were no changes in H+-ATPase expression in the cortex or outer medulla. These results represent the first demonstration of apical H+-ATPase immunoreactivity in IMCD cells in vivo and suggest that the decrease in urinary pH observed in AQP1 null mice is due to upregulation of H+-ATPase in the IMCD. The induction of H+-ATPase expression in IMCD cells of AQP1 null mice may be related to the chronically low interstitial osmolality in these animals. The challenge will be to identify the molecular signal(s) responsible for the de novo H+-ATPase expression.

scholarly journals Characterization of anion exchangers in an inner medullary collecting duct cell line.

1998 ◽  
Vol 9 (5) ◽  
pp. 746-754
Author(s):  
G Obrador ◽  
H Yuan ◽  
T M Shih ◽  
Y H Wang ◽  
M A Shia ◽  
...  
Keyword(s):  
Cell Line ◽  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Rat Stomach ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells, rat renal medulla, and freshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney homogenate and therefore probably represents AE1. The mobility of the 180-kD band is similar to that for rat stomach and kidney AE2 and therefore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that both AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these cells was found to be virtually identical to that reported for kidney AE1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, AE1 and AE2, and both of these exchangers probably mediate the electroneutral Cl--dependent HCO3-transport observed in this cell line.

scholarly journals Syntaxin specificity of aquaporins in the inner medullary collecting duct

2009 ◽  
Vol 297 (2) ◽  
pp. F292-F300 ◽  
Author(s):  
Abinash C. Mistry ◽  
Rickta Mallick ◽  
Janet D. Klein ◽  
Thomas Weimbs ◽  
Jeff M. Sands ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Snare Complex ◽  
Apical Plasma Membrane ◽  
Transport Activity ◽  
Inner Medullary Collecting Duct ◽  
Syntaxin 4 ◽  
Medullary Collecting Duct ◽  
Syntaxin 3

Proper targeting of the aquaporin-2 (AQP2) water channel to the collecting duct apical plasma membrane is critical for the urine concentrating mechanism and body water homeostasis. However, the trafficking mechanisms that recruit AQP2 to the plasma membrane are still unclear. Snapin is emerging as an important mediator in the initial interaction of trafficked proteins with target soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (t-SNARE) proteins, and this interaction is functionally important for AQP2 regulation. We show that in AQP2-Madin-Darby canine kidney cells subjected to adenoviral-mediated expression of both snapin and syntaxins, the association of AQP2 with both syntaxin-3 and syntaxin-4 is highly enhanced by the presence of snapin. In pull-down studies, snapin detected AQP2, syntaxin-3, syntaxin-4, and SNAP23 from the inner medullary collecting duct. AQP2 transport activity, as probed by AQP2's urea permeability, was greatly enhanced in oocytes that were coinjected with cRNAs of SNARE components (snapin+syntaxin-3+SNAP23) over those injected with AQP2 cRNA alone. It was not enhanced when syntaxin-3 was replaced by syntaxin-4 (snapin+syntaxin-4+SNAP23). On the other hand, the latter combination significantly enhanced the transport activity of the related AQP3 water channel while the presence of syntaxin-3 did not. This AQP-syntaxin interaction agrees with the polarity of these proteins' expression in the inner medullary collecting duct epithelium. Thus our findings suggest a selectivity of interactions between different aquaporin and syntaxin isoforms, and thus in the regulation of AQP2 and AQP3 activities in the plasma membrane. Snapin plays an important role as a linker between the water channel and the t-SNARE complex, leading to the fusion event, and the pairing with specific t-SNAREs is essential for the specificity of membrane recognition and fusion.

scholarly journals Histochemical carbonic anhydrase in rat inner medullary collecting duct.

1992 ◽  
Vol 40 (10) ◽  
pp. 1535-1545 ◽  
Author(s):  
J G Kleinman ◽  
J L Bain ◽  
C Fritsche ◽  
D A Riley
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cell Type ◽  
Variable Intensity ◽  
Inner Medullary Collecting Duct ◽  
Majority Population ◽  
Medullary Collecting Duct

Rat inner medullary collecting duct (IMCD) secretes substantial amounts of H+. However, carbonic anhydrase (CA), a concomitant of H+ secretion, has been generally reported absent in this segment. To reexamine this problem, we investigated CA and the morphological phenotypes of cells comprising the IMCD by CA histochemistry, using a modified Hansson technique with light and electron microscopy. Throughout the medulla, tubule cells exhibit histochemical CA activity. In the initial third of the inner medulla, a small proportion have features of intercalated cells and demonstrate some degree of CA activity. However, the majority population in the early portions of the IMCD appears to consist of principal cells. These also show CA staining of widely variable intensity, both among and within cells. A third cell type, previously called "IMCD cells", appears in the middle portion of the IMCD and is the only cell type present near the papilla tip. In contrast to previous reports, these "IMCD cells" have histochemical CA staining, also of highly variable intensity. These results demonstrate that stainable carbonic anhydrase to support acidification is present throughout the rat IMCD, both in intercalated cells and in some cells clearly not of this type. Therefore, the presence of CA is not specific for the intercalated cell type and suggests that other cell types may participate in acid secretion in IMCD.

scholarly journals Intercalated cells of the rat inner medullary collecting duct

1987 ◽  
Vol 31 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
William L. Clapp ◽  
Kirsten M. Madsen ◽  
Jill W. Verlander ◽  
C.Craig Tisher
Keyword(s):  
Collecting Duct ◽  
Intercalated Cells ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

Cellular response to acute respiratory acidosis in rat medullary collecting duct

1983 ◽  
Vol 245 (6) ◽  
pp. F670-F679 ◽  
Author(s):  
K. M. Madsen ◽  
C. C. Tisher
Keyword(s):  
Plasma Membrane ◽  
Surface Density ◽  
Collecting Duct ◽  
Respiratory Acidosis ◽  
Hydrogen Ion ◽  
Apical Plasma Membrane ◽  
Apical Region ◽  
Intercalated Cells ◽  
Ion Secretion ◽  
Medullary Collecting Duct

The collecting duct of the mammalian kidney is involved in urine acidification. Recent studies in the turtle bladder suggest that hydrogen ion secretion in response to elevated CO2 is regulated by insertion of hydrogen pumps into the luminal membrane of the mitochondria-rich cells. Because intercalated cells of the collecting duct are structurally similar to mitochondria-rich cells of the amphibian bladder, we studied the rat outer medullary collecting duct (OMCD) during respiratory acidosis to determine whether changes compatible with hydrogen ion secretion occur in the intercalated cells. Rats were studied during normal acid-base conditions and after 4-5 h of respiratory acidosis. After collection of physiologic data, the kidneys were fixed by in vivo perfusion and processed for electron microscopy. No changes were observed in the principal cells of the OMCD. Morphometric analysis revealed a significant increase in the surface density of the apical plasma membrane and a decrease in the number of tubulovesicular profiles in the apical region of the intercalated cells throughout the OMCD with respiratory acidosis. There were no changes in surface density of the basolateral membrane. These findings suggest that in response to respiratory acidosis there is transport of membrane from the tubulovesicular membrane compartment to the apical plasma membrane of the intercalated cells.

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

1996 ◽  
Vol 7 (12) ◽  
pp. 2533-2542 ◽  
Author(s):  
S M Ginns ◽  
M A Knepper ◽  
C A Ecelbarger ◽  
J Terris ◽  
X He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Specific Affinity ◽  
Basolateral Plasma Membrane ◽  
Medullary Collecting Duct ◽  
Antipeptide Antibody

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.

Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice

2005 ◽  
Vol 289 (1) ◽  
pp. F194-F199 ◽  
Author(s):  
Ryan G. Morris ◽  
Shinichi Uchida ◽  
Heddwen Brooks ◽  
Mark A. Knepper ◽  
Chung-Lin Chou
Keyword(s):  
Expression Profile ◽  
Knockout Mice ◽  
Collecting Duct ◽  
Water Channel ◽  
Major Protein ◽  
Altered Expression ◽  
Aquaporin 1 ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Concentrating Defect

Aquaporin-1 is the major protein responsible for transport of water across the epithelia of the proximal tubule and thin descending limbs. Rapid water efflux across the thin descending limb is required for the normal function of the countercurrent multiplier mechanism. Therefore, urinary concentrating capacity is severely impaired in aquaporin-1 knockout (AQP1 −/−) mice. Here, we have investigated the long-term consequences of deletion of the AQP1 gene product by profiling abundance changes in transporters expressed in the inner medullas of AQP1 (−/−) mice vs. heterozygotes [AQP1 (+/−)], which have a normal concentrating capacity. Semiquantitative immunoblotting demonstrated marked suppression of two proteins strongly expressed in the inner medullary collecting duct (IMCD): UT-A1 (a urea transporter) and AQP4 (a basolateral water channel). Furthermore, the urea permeability of the IMCD was significantly reduced in AQP1 (−/−) mice. In contrast, there was increased expression of three proteins normally expressed at higher levels in the cortical collecting duct (CCD) than in IMCD: AQP3 (another basolateral water channel) and the epithelial sodium channel subunits β-ENaC and γ-ENaC. Changes in expression of these proteins were confirmed by immunocytochemistry. Messenger RNA profiling (real-time RT-PCR) revealed changes in UT-A1, β-ENaC, γ-ENaC, and AQP3 transcript abundance that paralleled the changes in protein abundance. Thus, from the perspective of transport proteins, the IMCDs of AQP1 (−/−) mice have a significantly altered phenotype. To address whether these changes are specific to AQP1 (−/−) mice, we profiled IMCD transporter expression in a second knockout model manifesting a concentrating defect, that of ClC-nK1, a chloride channel in the ascending thin limb important for urinary concentration. As in the AQP1 knockout mice, ClC-nK1 (−/−) mice showed decreased expression of UT-A1 and increased expression of β-ENaC and γ-ENaC vs. WT controls. In conclusion, the expression profile of IMCD transporters is markedly altered in AQP1 −/− mice and this manifestation is related to the associated concentrating defect.

Fourfold reduction of water permeability in inner medullary collecting duct of aquaporin-4 knockout mice

1998 ◽  
Vol 274 (2) ◽  
pp. C549-C554 ◽  
Author(s):  
C. L. Chou ◽  
Tonghui Ma ◽  
Baoxue Yang ◽  
Mark A. Knepper ◽  
A. S. Verkman
Keyword(s):  
Water Permeability ◽  
Knockout Mice ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Inner Medullary Collecting Duct ◽  
Kidney Morphology ◽  
Medullary Collecting Duct ◽  
Mild Defect

Aquaporin (AQP)-3 and AQP4 water channels are expressed at the basolateral membrane of mammalian collecting duct epithelium. To determine the contribution of AQP4 to water permeability in the initial inner medullary collecting duct (IMCD), osmotic water permeability ( P f) was compared in isolated perfused IMCD segments from wild-type and AQP4 knockout mice. The AQP4 knockout mice were previously found to have normal gross appearance, survival, growth, and kidney morphology and a mild urinary concentrating defect (T. Ma, B. Yang, A. Gillespie, E. J. Carlson, C. J. Epstein, and A. S. Verkman. J. Clin. Invest. 100: 957–962, 1997). Transepithelial P f was measured in microdissected IMCDs after 18–48 h of water deprivation and in the presence of 0.1 nM arginine vasopressin (to make basolateral P f rate limiting). P fvalues (37°C; means ± SE in cm/s × 10−3) were 56.0 ± 8.5 for wild-type mice ( n = 5) and 13.1 ± 3.7 for knockout mice ( n = 6) ( P < 0.001). Northern blot analysis of kidney showed that transcript expression of AQP1, AQP2, AQP3, and AQP6 were not affected by AQP4 deletion. Immunoblot analysis indicated no differences in protein expression of AQP1, AQP2, or AQP3, and immunoperoxidase showed no differences in staining patterns. Coexpression of AQP3 and AQP4 in Xenopus laevis oocytes showed additive water permeabilities, suggesting that AQP4 deletion does not affect AQP3 function. These results indicate that AQP4 is responsible for the majority of basolateral membrane water movement in IMCD but that its deletion is associated with a very mild defect in urinary concentrating ability.

Water permeability of apical and basolateral cell membranes of rat inner medullary collecting duct

1990 ◽  
Vol 259 (6) ◽  
pp. F986-F999 ◽  
Author(s):  
B. Flamion ◽  
K. R. Spring
Keyword(s):  
Water Flow ◽  
Water Permeability ◽  
Cell Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Volume Regulatory Decrease ◽  
Water Permeation ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

To quantify the pathways for water permeation through the kidney medulla, knowledge of the water permeability (Posmol) of individual cell membranes in inner medullary collecting duct (IMCD) is required. Therefore IMCD segments from the inner two thirds of inner medulla of Sprague-Dawley rats were perfused in vitro using a setup devised for rapid bath and luminal fluid exchanges (half time, t1/2, of 55 and 41 ms). Differential interference contrast microscopy, coupled to video recording, was used to measure volume and approximate surface areas of single cells. Volume and volume-to-surface area ratio of IMCD cells were strongly correlated with their position along the inner medullary axis. Transmembrane water flow (Jv) was measured in response to a variety of osmotic gradients (delta II) presented on either basolateral or luminal side of the cells. The linear relation between Jv and delta II yielded the cell membrane Posmol, which was then corrected for membrane infoldings. Basolateral membrane Posmol was 126 +/- 3 microns/s. Apical membrane Posmol rose from a basal value of 26 +/- 3 microns/s to 99 +/- 5 microns/s in presence of antidiuretic hormone (ADH). Because of amplification of basolateral membrane, the ADH-stimulated apical membrane remained rate-limiting for transcellular osmotic water flow, and the IMCD cell did not swell significantly. Calculated transcellular Posmol, expressed in terms of smooth luminal surface, was 64 microns/s without ADH and 207 microns/s with ADH. IMCD cells in anisosmotic media displayed almost complete volume regulatory decrease but only partial volume regulatory increase.

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