scholarly journals Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase

2010 ◽  
Vol 298 (5) ◽  
pp. F1162-F1169 ◽  
Author(s):  
Fan Gong ◽  
Rodrigo Alzamora ◽  
Christy Smolak ◽  
Hui Li ◽  
Sajid Naveed ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Apical Membrane ◽  
Ex Vivo ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Kidney Tissue ◽  
Intercalated Cells ◽  
Clear Cells ◽  
Kidney Slices ◽  
Immunofluorescence Labeling

The vacuolar H+-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N6-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.

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

2004 ◽  
Vol 286 (1) ◽  
pp. F161-F169 ◽  
Author(s):  
Snezana Petrovic ◽  
Sharon Barone ◽  
Jie Xu ◽  
Laura Conforti ◽  
Liyun Ma ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Immunocytochemical Staining ◽  
Intercalated Cells ◽  
Outer Medulla ◽  
Anion Exchanger 1 ◽  
Bicarbonate Reabsorption ◽  
Medullary Collecting Duct ◽  
Immunofluorescence Labeling

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 [Formula: see text] exchanger, located in intercalated cells of OMCD, mediate the bicarbonate reabsorption. Here we report the identification of a new basolateral [Formula: see text] 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 ∼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 α-intercalated cells. Functional studies in oocytes demonstrated that increasing the osmolality of the media (to simulate the physiological milieu in the medulla) increased the [Formula: see text] exchanger activity mediated via SLC26A7 by about threefold ( P < 0.02 vs. normal condition). We propose that SLC26A7 is a basolateral [Formula: see text] exchanger in intercalated cells of the OMCD and may play an important role in bicarbonate reabsorption in medullary collecting duct.

scholarly journals Cellular localization of adenine receptors in the rat kidney and their functional significance in the inner medullary collecting duct

2013 ◽  
Vol 305 (9) ◽  
pp. F1298-F1305 ◽  
Author(s):  
Bellamkonda K. Kishore ◽  
Yue Zhang ◽  
Haykanush Gevorgyan ◽  
Donald E. Kohan ◽  
Anke C. Schiedel ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Ex Vivo ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Renal Vasculature ◽  
Outer Medulla ◽  
Collecting Ducts ◽  
Rabbit Polyclonal Antibody ◽  
Immunofluorescence Labeling

The Gi-coupled adenine receptor (AdeR) binds adenine with high affinity and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in the renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex and outer medulla compared with the inner medulla. Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid COOH-terminal sequence of rat AdeR, which we generated, detected two bands between ∼30 and 40 kDa (molecular mass of native protein: 37 kDa) in the cortex, outer medulla, and inner medulla. These bands were ablated by preadsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature, including the glomerular arterioles, and less intense labeling in the cells of the collecting duct system. Confocal immunofluorescence imaging colocalized AdeR with aquaporin-2 protein to the apical plasma membrane in the collecting duct. Functionally, adenine (10 μM) significantly decreased ( P < 0.01) 1-deamino-8-d-arginine vasopressin (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 μM, P < 0.01), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in the renal vasculature and collecting ducts and its functional relevance. This study may open a new avenue for the exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease.

scholarly journals Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

2014 ◽  
Vol 306 (9) ◽  
pp. F981-F995 ◽  
Author(s):  
Mohammad M. Al-bataineh ◽  
Fan Gong ◽  
Allison L. Marciszyn ◽  
Michael M. Myerburg ◽  
Núria M. Pastor-Soler
Keyword(s):  
Protein Kinase ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Phosphodiesterase Inhibitor ◽  
Cell Monolayers ◽  
Apical Pole ◽  
Kidney Slices ◽  
Immunofluorescence Labeling ◽  
Amp Activated Protein Kinase

The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress.

Three distinct cell populations in rat kidney collecting duct

1987 ◽  
Vol 253 (2) ◽  
pp. C323-C328 ◽  
Author(s):  
H. Holthofer ◽  
B. A. Schulte ◽  
G. Pasternack ◽  
G. J. Siegel ◽  
S. S. Spicer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Morphological Characteristics ◽  
Anion Channel ◽  
Band 3 ◽  
Cell Populations ◽  
Intercalated Cells ◽  
Distinct Cell ◽  
Collecting Ducts ◽  
Principal And Intercalated Cells

The morphologically heterogeneous cell populations in the collecting ducts of the rat kidney were studied using immunocytochemical detection of Na+-K+-ATPase and the anion channel (band 3) glycoprotein. Both enzymes were localized to the basal aspect of separate and morphologically distinct subpopulations of cells in various segments of the collecting duct. Na+-K+-ATPase appeared to be present exclusively in principal cells as identified by their morphology, whereas band 3 antibodies reacted only with intercalated cells. However, 5-20% of cells with the morphological characteristics of intercalated cells failed to react with either antisera in various segments of collecting ducts. As band 3 glycoprotein serves in exchanging intracellular bicarbonate for chloride, it is highly likely that the cells positive for this antigen secrete protons. The method introduced here appears thus useful for distinguishing between principal and intercalated cells by differences in their enzyme content and further for revealing two subpopulations of intercalated cells. This method promises to provide a useful approach for studying the principal and intercalated cell populations in various metabolic states.

Lectin-gold cytochemistry reveals intercalated cell heterogeneity along rat kidney collecting ducts

1985 ◽  
Vol 248 (3) ◽  
pp. C348-C356 ◽  
Author(s):  
D. Brown ◽  
J. Roth ◽  
L. Orci
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Intercalated Cells ◽  
Double Labeling ◽  
Thin Sections ◽  
Intercalated Cell ◽  
Collecting Ducts

The lectin-gold technique was used to detect Helix pomatia and Dolichos biflorus lectin binding sites directly on semithin and thin sections of rat kidney collecting ducts. Intercalated cell apical plasma membranes and the membranes of apical cytoplasmic vesicles were heavily labeled in the cortex and outer stripe of the outer medulla but were negative or very weakly labeled in the inner stripe and inner medulla. In contrast, clear cell apical membranes were labeled along the entire length of the collecting duct. Double labeling of semithin cryostat sections with a specific antibody and lectin-gold complexes was used to demonstrate that the intercalated cells in all regions studied contained carbonic anhydrase, even though the lectin binding differed. These results indicate that, in terms of their glycocalyx composition, intercalated cells represent a heterogeneous population in different regions of the collecting duct.

Differential localization of two glucose transporter isoforms in rat kidney

1990 ◽  
Vol 259 (2) ◽  
pp. C286-C294 ◽  
Author(s):  
B. Thorens ◽  
H. F. Lodish ◽  
D. Brown
Keyword(s):  
Proximal Tubule ◽  
Glucose Transporter ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Staining Intensity ◽  
Intercalated Cells ◽  
Brain Glucose ◽  
Nephron Segments ◽  
Glucose Transporter Isoforms

The localization of two glucose transporter isoforms was mapped in the rat kidney: the high-Michaelis constant (Km; 15-20 mM) low-affinity "liver" transporter and the low-Km (1-2 mM) high-affinity "erythroid/brain" transporter. Both are basolateral membrane proteins, but the liver transporter was present exclusively in the S1 part of the proximal tubule, whereas the erythroid/brain transporter was expressed at variable levels in different nephron segments. Staining intensity was low in the straight proximal tubule (S3), intermediate in the medullary thin and thick ascending limbs, and highest in connecting segments and collecting ducts. In the collecting duct, the erythroid/brain glucose transporter was expressed at the highest level in intercalated cells; less was present in principal cells. In the papilla, only intercalated cells expressed this transporter isoform. These results suggest specific involvements of each transporter isoform in transepithelial glucose reabsorption by different segments of the proximal tubule. They also indicate that while the liver glucose transporter is present in gluconeogenic cells, there is a good correlation between the level of expression of the erythroid/brain glucose transporter and the glycolytic activity of the different nephron segments.

Immunolocalization of electroneutral Na-HCO3 − cotransporter in rat kidney

2000 ◽  
Vol 279 (5) ◽  
pp. F901-F909 ◽  
Author(s):  
Henrik Vorum ◽  
Tae-Hwan Kwon ◽  
Christiaan Fulton ◽  
Brian Simonsen ◽  
Inyeong Choi ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Electron Microscopic Level ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Electron Microscopic ◽  
Outer Medulla ◽  
Outer Stripe ◽  
Medullary Collecting Duct

An electroneutral Na-HCO3 − cotransporter (NBCN1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBCN1. The affinity-purified antibody specifically recognized one band, ∼180 kDa, in rat kidney membranes. Peptide- N-glycosidase F deglycosylation reduced the band to ∼140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBCN1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBCN1 immunolabeling. Immunoelectron microscopy demonstrated that NBCN1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO3 −-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBCN1. The localization of NBCN1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBCN1 may be important for electroneutral basolateral HCO3 − transport in these cells.

Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney

2001 ◽  
Vol 280 (6) ◽  
pp. F1093-F1106 ◽  
Author(s):  
Henrik Hager ◽  
Tae-Hwan Kwon ◽  
Anna K. Vinnikova ◽  
Shyama Masilamani ◽  
Heddwen L. Brooks ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Subcellular Localization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Medullary Collecting Duct ◽  
Epithelial Sodium Channel Enac

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. α-ENaC was localized mainly in a zone in the apical domains, whereas β- and γ-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, α-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, α-ENaC was present in a narrow zone near the apical plasma membrane, whereas β- and γ-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na+ reabsorption.

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

2005 ◽  
Vol 289 (3) ◽  
pp. C665-C672 ◽  
Author(s):  
Guangmu Li ◽  
Qiongqiong Yang ◽  
Edward A. Alexander ◽  
John H. Schwartz
Keyword(s):  
Transmembrane Domain ◽  
Apical Membrane ◽  
Specific Binding ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Full Length ◽  
Snare Proteins ◽  
Glutathione S Transferase ◽  
Syntaxin 1A ◽  
Carboxy Terminal

H+ transport in the collecting duct is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H+-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H+-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H+-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1AΔC [amino acids (aa) 1–264] formed complexes with H+-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H+-ATPase (aa 235–264) and another that bound SNAP23 and VAMP (aa 190–234) to an equivalent degree as full-length syntaxin. However, the aa 235–264 cassette alone without the SNARE N (aa 1–160) does not bind but requires ligation to the SNARE N to bind H+-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H+-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H+-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H+-ATPase exocytosis.

Autoradiographic localization of beta-adrenergic receptors in rat kidney slices using [125I]iodocyanopindolol

1984 ◽  
Vol 246 (2) ◽  
pp. F240-F245
Author(s):  
P. A. Munzel ◽  
D. P. Healy ◽  
P. A. Insel
Keyword(s):  
Adrenergic Receptors ◽  
Rank Order ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Distal Tubule ◽  
Cortical Collecting Duct ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Kidney Slices ◽  
Adrenergic Antagonist

We have used autoradiography of [125I]iodocyanopindolol [( 125I]-ICYP) to define the distribution and localization of beta-adrenergic receptors in the rat kidney. [125I]ICYP, a radiolabeled beta-adrenergic antagonist, proved to be an excellent probe to identify these receptors in kidney slices in that specific [125I]ICYP binding was 1) saturable, 2) competed stereoselectively by the agonist isoproterenol and the antagonist propranolol, and 3) competed by agonists in a classical rank order of potency for beta 1-adrenergic receptors (isoproterenol greater than epinephrine congruent to norepinephrine). Autoradiographic studies demonstrated that specific [125I]ICYP sites were present exclusively in the cortex and outer medulla and were localized in glomeruli, ascending limb, and distal tubule-cortical collecting duct. Autoradiography of probes like [125I]ICYP appears to offer a simple and rapid method to assess distribution of adrenergic receptors in the kidney and presumably in other tissues as well.

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