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.

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 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.

K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct

1997 ◽  
Vol 272 (1) ◽  
pp. F124-F131 ◽  
Author(s):  
B. Buffin-Meyer ◽  
M. Younes-Ibrahim ◽  
C. Barlet-Bas ◽  
L. Cheval ◽  
S. Marsy ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Lower Sensitivity ◽  
Kinetic Properties ◽  
Type I ◽  
Type Ii ◽  
Distribution Profile ◽  
Adenosine Triphosphatases ◽  
Collecting Ducts ◽  
K Depletion

Two distinct Sch-28080-sensitive K-adenosine triphosphatases (K-ATPases) were previously described in the rat nephron: a ouabain-resistant K-ATPase (type I) present in collecting ducts (CD) and a ouabain-sensitive from (type II) located in proximal tubules (PT) and thick ascending limbs (TAL). In K-depleted rats, K-ATPase activity is increased in CD, whereas it is reduced in PT and TAL. Because expression of colonic H-K-ATPase is restricted to the CD of K-depleted rats, we hypothesized that K-ATPase from the CD of K-depleted rats might be different from types I and II. Indeed, type III K-ATPase displays higher sensitivities to ouabain and to Sch-28080 than type II, a lower sensitivity to Sch-28080 than type I, and, conversely to types I and II, it can be stimulated by Na+. Pharmacological differences between types II and III K-ATPases were confirmed by [3H]ouabain binding experiments. Thus the rat kidney expresses three K-ATPases that differ by their pharmacological and kinetic properties, their distribution profile along the nephron and their behavior during K depletion.

Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney

1994 ◽  
Vol 266 (5) ◽  
pp. F823-F828 ◽  
Author(s):  
Y. Sugimoto ◽  
T. Namba ◽  
R. Shigemoto ◽  
M. Negishi ◽  
A. Ichikawa ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Cellular Localization ◽  
Mouse Kidney ◽  
Prostaglandin E ◽  
Outer Medulla ◽  
Multiple Functions ◽  
Collecting Ducts ◽  
Distal Tubules ◽  
Stimulation Of

Distribution of the mRNAs for three subtypes of prostaglandin E (PGE) receptors in the mouse kidney was investigated by in situ hybridization. The mRNA for EP1 subtype, which is coupled to Ca2+ mobilization, was specifically localized to the collecting ducts from the cortex to the papilla. The mRNA for EP2 subtype, which is linked to stimulation of adenylate cyclase, was localized to the glomeruli. The mRNA for EP3 subtype, which is coupled to inhibition of adenylate cyclase, was located densely in the tubules in the outer medulla and in the distal tubules in the cortex. These results exhibit distinct cellular localization of three subtypes of PGE receptor in the kidney and suggest that PGE2 exerts multiple functions via these subtypes expressed in different segments of the nephron.

Increased apical targeting of renal ENaC subunits and decreased expression of 11βHSD2 in HgCl2-induced nephrotic syndrome in rats

2006 ◽  
Vol 290 (3) ◽  
pp. F674-F687 ◽  
Author(s):  
Soo Wan Kim ◽  
Sophie de Seigneux ◽  
Martin C. Sassen ◽  
JongUn Lee ◽  
Jin Kim ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Sodium Excretion ◽  
Collecting Duct ◽  
Urinary Sodium Excretion ◽  
Brown Norway ◽  
Apical Plasma Membrane ◽  
Control Group ◽  
Sodium Retention ◽  
Outer Medulla ◽  
Outer Stripe

Nephrotic syndrome is often accompanied by sodium retention and generalized edema. We hypothesize that dysregulation of the epithelial sodium channel (ENaC) and/or of sodium (co)transporters may be responsible for the increased sodium retention associated with HgCl2-induced nephropathy. In addition, we examined the hypothesis that the expression of type 2 11β-hydroxysteroid dehydrogenase (11βHSD2) is reduced, contributing to the enhanced mineralocorticoid activity. Membranous nephropathy was induced in Brown Norway rats by repeated injections of HgCl2 (1 mg/kg sc), whereas the control group received only vehicle. After 13 days of treatment, the abundance of ENaC subunits, sodium (co)transporters, and 11βHSD2 in the kidney was examined by immunoblotting and immunohistochemistry. HgCl2 treatment induced marked proteinuria, hypoalbuminemia, decreased urinary sodium excretion, and ascites. The protein abundance of α-ENaC was increased in the cortex/outer stripe of outer medulla (OSOM) and inner stripe of the outer medulla (ISOM). The protein abundances of β-ENaC and γ-ENaC were decreased in the cortex/OSOM while increased in the ISOM. Immunoperoxidase microscopy demonstrated increased targeting of ENaC subunits to the apical plasma membrane in the distal convoluted tubule, connecting tubule, and cortical and medullary collecting duct segments. Moreover, 11βHSD2 abundance was decreased in cortex/OSOM and ISOM. The protein abundances of type 3 Na/H exchanger (NHE3), Na-K-2Cl cotransporter (NKCC2), and thiazide-sensitive Na-Cl cotransporter (NCC) were decreased. Moreover, the abundance of the α-1 subunit of the Na-K-ATPase was decreased in the cortex/OSOM and ISOM but remained unchanged in the inner medulla. These results suggest that increased apical targeting of ENaC subunits combined with diminished abundance of 11βHSD2 may contribute to sodium retention associated with HgCl2-induced nephrotic syndrome. The decreased abundance of NHE3, NKCC2, NCC, and Na-K-ATPase may play a compensatory role in promoting sodium excretion.

scholarly journals Developmental expression of the epithelial Na+ channel in kidney and uroepithelia

1999 ◽  
Vol 276 (2) ◽  
pp. F304-F314 ◽  
Author(s):  
Shigeru Watanabe ◽  
Kazumichi Matsushita ◽  
Paul B. McCray ◽  
John B. Stokes
Keyword(s):  
Urinary Bladder ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Rnase Protection Assay ◽  
Adult Life ◽  
Developmental Expression ◽  
Na Channel ◽  
Rnase Protection ◽  
Collecting Ducts ◽  
Uroepithelial Cell

The epithelial Na+ channel (ENaC) plays an important role in regulating Na+ balance in neonatal and adult life. Using in situ hybridization, we localized α-, β-, and γ-rat ENaC (rENaC) mRNA in developing rat kidney and uroepithelia. rENaC mRNA was first detectable on fetal day 16, and by fetal day 17, mRNA was abundant in the terminal collecting duct and uroepithelia. After birth, the intensity of the signals for all three subunits increased in the cortical collecting ducts and by 9 days after birth had diminished in the inner medullary collecting ducts. Expression in uroepithelial cells was different. mRNA for β- and γ-rENaC, but not α-rENaC, was detected in pelvis, ureters, and bladder at all stages of development beyond fetal day 16. By RNase protection assay (RPA), the greatest increase in subunit abundance in the kidney occurred before birth. Between postnatal days 9 and 30, the abundance of β- and γ-rENaC decreased relative to α-rENaC in outer and inner medulla. The urinary bladder, in contrast, demonstrated the greatest increase in β- and γ-rENaC mRNA abundance after birth. We were generally unable to detect α-rENaC by RPA in urinary bladder. Feeding weaned rats a diet of high or low NaCl did not change the abundance of any of the subunit mRNAs in bladder. These results demonstrate additional heterogeneity of developmental expression and regulation of ENaC. The differences between the collecting duct and uroepithelial cell rENaC mRNA regulation raise the possibility of significant differences in function.

Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment

2000 ◽  
Vol 278 (1) ◽  
pp. F29-F42 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Complete Disappearance ◽  
Vasopressin Secretion ◽  
V2 Receptor ◽  
Intracellular Vesicles

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.

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.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

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.

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