Dopamine D4 receptor isoform mRNA and protein  are expressed in the rat cortical collecting duct

We reported previously [ Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F391–F400, 1996] that dopamine inhibits vasopressin (AVP)-dependent water permeability and Na+ transport in the rat cortical collecting duct (CCD) apparently through a D4 dopamine receptor. The present experiments used RT-PCR of total RNA extracted from microdissected rat CCD to determine whether the D4and D1A dopamine receptor isoforms are expressed. Specific primers were used to amplify three regions of the D4 cDNA. All three gave products with 98–100% nucleotide identity to the known rat D4 sequence; however, there was an extra 6-bp insert at the 3′ end of the second transmembrane region that was identical to the human and mouse sequences but which had not been documented in the rat sequence. D4 receptor protein was also localized exclusively to the CCD and medullary collecting ducts by immunohistochemistry. Two regions of the D1A dopamine receptor message were also amplified by RT-PCR of RNA from rat CCD and were verified by sequencing and immunohistochemistry. We conclude that both D4 and D1A dopamine receptors are expressed in the rat CCD, but the physiological effects are attributable to a D4 receptor.

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Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.1.f195 ◽

2000 ◽

Vol 279 (1) ◽

pp. F195-F202 ◽

Cited By ~ 21

Author(s):

Randi B. Silver ◽

Sylvie Breton ◽

Dennis Brown

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Collecting Duct ◽

Proton Pumps ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Collecting Ducts ◽

Acid Load ◽

Collecting Tubules ◽

Atpase Staining

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

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Automated method for the isolation of collecting ducts

AJP Renal Physiology ◽

10.1152/ajprenal.00273.2005 ◽

2006 ◽

Vol 291 (1) ◽

pp. F236-F245 ◽

Cited By ~ 18

Author(s):

R. Lance Miller ◽

Ping Zhang ◽

Tong Chen ◽

Andreas Rohrwasser ◽

Raoul D. Nelson

Keyword(s):

Flow Cytometry ◽

Large Particle ◽

Fluorescent Protein ◽

Collecting Duct ◽

Complex Object ◽

Cortical Collecting Duct ◽

Fluorescent Intensity ◽

Automated Method ◽

Collecting Ducts ◽

Time Required

The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68 ± 0.44–14.5 ± 0.66 cm or 9.2 ± 0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.

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RenalAT1Receptor Protein Expression During the Early Stage of Diabetes Mellitus

International Journal of Experimental Diabetes Research ◽

10.1080/15604280214483 ◽

2002 ◽

Vol 3 (2) ◽

pp. 97-108 ◽

Cited By ~ 30

Author(s):

Lisa M. Harrison-Bernard ◽

John D. Imig ◽

Pamela K. Carmines

Keyword(s):

Diabetes Mellitus ◽

Protein Expression ◽

Early Stage ◽

Collecting Duct ◽

Receptor Protein ◽

Cortical Collecting Duct ◽

Distal Nephron ◽

Protein Levels ◽

Nephron Segments

Experiments were performed to evaluate the hypothesis that the early stage of Type 1 diabetes mellitus (DM) increases renal angiotensin II (AngII) concentration and angiotensin type 1 (AT1) receptor protein levels. Nineteen or twenty days after vehicle (Sham rats) or streptozotocin (STZ rats) treatment, plasma [AngII] was higher in STZ rats (152±23 fmol/ml) than in Sham rats (101±7 fmol/ml); however, kidney [AngII] did not differ between groups.AT1receptor protein expression was greater in STZ kidneys than in Sham kidneys. This increase was restricted to the cortex, whereAT1protein levels were elevated by 77±26% (42 kDa) and 101±16% (58 kDa) in STZ kidneys. Immunohistochemistry revealed this effect to be most evident in distal nephron segments including the connecting tubule/cortical collecting duct. Increased renal corticalAT1receptor protein and circulating AngII levels are consistent with an exaggerated AngII-dependent influence on renal function during the early stage of DM in the rat.

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Evidence of corticosteroid action along the nephron

AJP Renal Physiology ◽

10.1152/ajprenal.1984.246.2.f111 ◽

1984 ◽

Vol 246 (2) ◽

pp. F111-F123 ◽

Cited By ~ 3

Author(s):

D. Marver

Keyword(s):

Collecting Duct ◽

Type I ◽

Type Ii ◽

Cortical Collecting Duct ◽

Distal Nephron ◽

Type Iii ◽

Collecting Ducts ◽

Collecting Tubule ◽

Cortical Collecting Tubule

The kidney contains three classes of corticosteroid-binding proteins receptors. They include a mineralocorticoid-specific (Type I), a glucocorticoid-specific (Type II), and a corticosterone-specific (Type III) site. The Type I and Type III sites roughly parallel each other along the nephron, with maximal binding occurring in the late distal convoluted or connecting segment and the cortical and medullary collecting ducts. Type II sites occur throughout the nephron, with maximal concentrations appearing in the proximal tubule and the late distal convoluted-cortical collecting duct region. The function of the Type I sites in the connecting segment is unclear since chronic mineralocorticoid therapy does not influence the potential difference in this segment as it does in the cortical collecting tubule. Furthermore, the specific role of Type II versus Type III sites in the distal nephron is unknown. Finally, the possible influence of sodium on both latent and steroid-induced renal cortical and medullary Na-K-ATPase is discussed.

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ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. I. Stilbene-sensitive Cl− secretion

AJP Renal Physiology ◽

10.1152/ajprenal.00471.2013 ◽

2015 ◽

Vol 309 (3) ◽

pp. F251-F258 ◽

Cited By ~ 6

Author(s):

Masayoshi Nanami ◽

Yoskaly Lazo-Fernandez ◽

Vladimir Pech ◽

Jill W. Verlander ◽

Diana Agazatian ◽

...

Keyword(s):

Channel Blocker ◽

Single Channel ◽

Collecting Duct ◽

Cortical Collecting Duct ◽

Collecting Ducts ◽

Hcl Secretion ◽

Transepithelial Voltage ◽

Apical Membranes ◽

Rats And Mice

Inhibition of the epithelial Na+ channel (ENaC) reduces Cl− absorption in cortical collecting ducts (CCDs) from aldosterone-treated rats and mice. Since ENaC does not transport Cl−, the purpose of the present study was to explore how ENaC modulates Cl− absorption in mouse CCDs perfused in vitro. Therefore, we measured transepithelial Cl− flux and transepithelial voltage in CCDs perfused in vitro taken from mice that consumed a NaCl-replete diet alone or the diet with aldosterone administered by minipump. We observed that application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid unmasks conductive Cl− secretion. During ENaC blockade, this Cl− secretion fell with the application of a nonselective Cl− channel blocker [DIDS (100 μM)] to the perfusate. While single channel recordings of intercalated cell apical membranes in split-open CCDs demonstrated a Cl− channel with properties that resemble the ClC family of Cl− channels, ClC-5 is not the primary pathway for benzamil-sensitive Cl− flux. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl− absorption is benzamil sensitive, and, second, benzamil application stimulates stilbene-sensitive conductive Cl− secretion, which occurs through a ClC-5-independent pathway.

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Deoxycorticosterone-stimulated bicarbonate secretion in rabbit cortical collecting ducts: effects of luminal chloride removal and in vivo acid loading

AJP Renal Physiology ◽

10.1152/ajprenal.1985.249.2.f205 ◽

1985 ◽

Vol 249 (2) ◽

pp. F205-F212 ◽

Cited By ~ 9

Author(s):

J. Garcia-Austt ◽

D. W. Good ◽

M. B. Burg ◽

M. A. Knepper

Keyword(s):

Collecting Duct ◽

High Rate ◽

Bicarbonate Secretion ◽

Cortical Collecting Duct ◽

Acid Base ◽

Urinary Ph ◽

Collecting Ducts ◽

Acid Loading

To assess the role of cortical collecting duct bicarbonate secretion in the regulation of net acid excretion, we have sought to identify what factors influence the secretion rate. Net and unidirectional bicarbonate fluxes were measured in isolated perfused cortical collecting ducts from deoxycorticosterone-treated rabbits. The collecting ducts secreted bicarbonate at 11-24 pmol X mm-1 X min-1, confirming the high rate seen in earlier studies. Oral acid loading (50 mM NH4Cl drinking water) completely inhibited the net bicarbonate secretion. The bath-to-lumen flux was markedly reduced with acid loading, but the lumen-to-bath flux changed very little. In tubules from rabbits treated with deoxycorticosterone (but not NH4Cl), luminal chloride replacement with either sulfate or gluconate completely and reversibly inhibited the net bicarbonate secretion. The bath-to-lumen flux was greatly inhibited, but there was little change in the lumen-to-bath flux. We conclude: 1) High rates of bicarbonate secretion can be induced in rabbit cortical collecting ducts by chronic treatment of the animals with deoxycorticosterone. 2) When deoxycorticosterone-treated rabbits were made acidotic by oral administration of NH4Cl, the bicarbonate secretion was prevented, indicating that the systemic acid-base state of the animal may be an important factor regulating bicarbonate secretion. 3) Replacement of chloride in the lumen with sulfate inhibits bicarbonate secretion in the cortical collecting duct, an effect which may explain in part the decrease in urinary pH in response to sulfate infusions in mineralocorticoid-stimulated animals.

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Rabbit cortical collecting ducts express a novel prostacyclin receptor

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.1.f145 ◽

1995 ◽

Vol 268 (1) ◽

pp. F145-F154 ◽

Cited By ~ 4

Author(s):

R. L. Hebert ◽

L. Regnier ◽

L. N. Peterson

Keyword(s):

Prostaglandin E2 ◽

Calcium Ion ◽

Collecting Duct ◽

Ion Concentration ◽

Cortical Collecting Duct ◽

Water Conductivity ◽

Prostacyclin Receptor ◽

Collecting Ducts ◽

Intracellular Calcium Ion ◽

Calcium Ion Concentration

Prostaglandin E2 (PGE2) inhibits vasopressin-stimulated water conductivity (AVP-Lp) and inhibits Na+ reabsorption in the rabbit cortical collecting duct (CCD). Inhibition of Na+ reabsorption is mediated by increased intracellular calcium ion concentration ([Ca2+]i). Prostacyclin (PGI2) has also been shown to inhibit Na+ reabsorption in the CCD. The present studies were designed to examine the effect of the PGI2 agonist, Iloprost (ILP), on AVP-Lp and [Ca2+ in the isolated perfused rabbit CCD and to determine whether ILP activates different receptors than PGE2. ILP and PGE2 each maximally inhibited AVP-Lp equipotently at 10(-7) M. When CCDs were exposed to PGE2 and ILP simultaneously, or if PGE2 was added in the presence of ILP, inhibition of AVP-Lp was additive. Additivity was not observed if the PGI2 agonist, carbaprostacyclin (c-PGI2), was added with ILP, or if the PGE2 agonist, sulprostone, was added with PGE2, or if ILP was added to CCDs preexposed to PGE2. In fura 2-loaded CCD, ILP and PGE2 added separately increased [Ca2+]i. The response to c-PGI2 could be desensitized by prior exposure to ILP. ILP did not cause desensitization to PGE2, but PGE2 could desensitize the CCD to ILP. We conclude that PGI2 inhibits AVP-Lp by activation of a novel IP3 prostacyclin receptor and increases [Ca2+]i by activation of an IP1 prostacyclin receptor in the rabbit CCD. Functional evidence is presented that PGI2 cannot occupy PGE2 receptors and that PGE2 can occupy but cannot activate PGI2 receptors linked to inhibition of AVP-Lp.

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Increased collecting duct urea transporter expression in Dahl salt-sensitive rats

AJP Renal Physiology ◽

10.1152/ajprenal.00073.2003 ◽

2003 ◽

Vol 285 (1) ◽

pp. F143-F151 ◽

Cited By ~ 16

Author(s):

Robert A. Fenton ◽

Chung-Lin Chou ◽

Shana Ageloff ◽

William Brandt ◽

John B. Stokes ◽

...

Keyword(s):

Collecting Duct ◽

Hydroxysteroid Dehydrogenase ◽

Plasma Corticosterone ◽

Urea Transporter ◽

Rt Pcr ◽

Collecting Ducts ◽

Medullary Collecting Duct ◽

Transporter Expression ◽

Solute Transporter

Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11β-HSD2 abundance in DS rats. Consistent with the view that an elevated 11β-HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11β-HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.

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Functional characterization of alpha- and beta-intercalated cell types in rabbit cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.1991.261.3.f377 ◽

1991 ◽

Vol 261 (3) ◽

pp. F377-F385 ◽

Cited By ~ 6

Author(s):

H. Furuya ◽

M. D. Breyer ◽

H. R. Jacobson

Keyword(s):

Collecting Duct ◽

Basolateral Membrane ◽

Functional Characterization ◽

Cell Types ◽

Disulfonic Acid ◽

Cortical Collecting Duct ◽

Electrical Measurements ◽

Collecting Ducts

Single-cell electrical measurements and spectrophotometric determinations of intracellular pH were used to determine unique features of alpha- and beta-intercalated cells (alpha-IC, beta-IC) in in vitro perfused rabbit cortical collecting ducts (CCD). pHi rose in alpha-IC and fell in beta-IC after bath Cl- removal. Luminal Cl- removal did not change pHi of alpha-IC, but pHi of beta-IC rose by 0.36 +/- 0.01 pH units. Cl- concentration-dependent recovery of beta-IC pHi revealed a Cl- Km of 18.7 mM for the luminal Cl(-) -HCO3- exchanger. Measurements of basolateral membrane voltage (Vbl) also showed two IC cell types. Removal of luminal Cl- did not change Vbl in alpha-IC, whereas Vbl hyperpolarized by a mean of 73.2 +/- 3.5 mV in beta-IC. Reducing bath Cl- depolarized both alpha- and beta-IC Vbl. In alpha-IC a large repolarization of 39.8 +/- 5.2 mV followed acute depolarization after bath Cl- removal. Reducing bath HCO3- (constant CO2) had little effect on beta-IC Vbl, whereas alpha-IC Vbl depolarized by 5.2 +/- 0.7 mV. Reducing luminal HCO3- in the absence of luminal Cl- produced a 17.6 +/- 1.8 mV depolarization in beta-IC. This change was independent of luminal Na+ and was not blocked by luminal 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In beta-IC, Vbl was not altered by either bath or lumen DIDS in the presence of luminal Cl-. However, when luminal Cl- was removed, luminal DIDS reversibly depolarized Vbl by 9.6 +/- 2.9 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

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Vitamin D receptor gene expression in mammalian kidney.

Journal of the American Society of Nephrology ◽

10.1681/asn.v551251 ◽

1994 ◽

Vol 5 (5) ◽

pp. 1251-1258

Author(s):

L Liu ◽

M Ng ◽

A M Iacopino ◽

S T Dunn ◽

M R Hughes ◽

...

Keyword(s):

Gene Expression ◽

Vitamin D ◽

Vitamin D Receptor ◽

Collecting Duct ◽

Receptor Gene ◽

Receptor Protein ◽

Vitamin D Receptor Gene ◽

Cortical Collecting Duct ◽

Distal Nephron ◽

Receptor Gene Expression

The vitamin D-receptor protein and its mRNA were localized in microscope sections of paraffin-embedded mammalian kidneys by means of immunocytochemistry and in situ hybridization, respectively. A monoclonal antibody against chicken intestinal vitamin D receptor immunostained the nucleus and cytoplasm of cells within the distal convoluted tubule, connecting segment, and initial cortical collecting duct of both rats and pigs. Although fainter, immunostaining also was present over proximal tubular cells. (35S)UTP-labeled cRNA probes were detected over both the proximal and distal portions of the mouse nephron, but silver grain densities were 5.8-fold greater over the latter. In conclusion, localization of both the vitamin D-receptor protein and its mRNA in both the proximal and distal nephron of adult mammals suggests that the gene for this protein is expressed in cells at both of these sites. The intensity of immunostaining and the density of cRNA-associated silver grains suggest that vitamin D-receptor gene expression is greatest in the distal nephron.

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