Electrophysiological Identification of Cell Types in Cortical Collecting Duct Monolayers

1991 ◽  
Vol 14 (1-2) ◽  
pp. 1-11 ◽  
Author(s):  
Elsa Bello-Reuss
Keyword(s):  
Collecting Duct ◽  
Cell Types ◽  
Cortical Collecting Duct

Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance

1998 ◽  
Vol 274 (3) ◽  
pp. F596-F601 ◽  
Author(s):  
Géza Fejes-Tóth ◽  
Erzsébet Rusvai ◽  
Emily S. Cleaveland ◽  
Anikó Náray-Fejes-Tóth
Keyword(s):  
Mrna Expression ◽  
Collecting Duct ◽  
Cell Types ◽  
Alkaline Media ◽  
Mrna Levels ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base Balance ◽  
Acid Base ◽  
Base Balance

AE2 mRNA and protein is expressed in several nephron segments, one of which is the cortical collecting duct (CCD). However, the distribution of AE2 among the different cell types of the CCD and the function of AE2 in the kidney are not known. The purpose of this study was to determine the distribution of AE2 mRNA among the three CCD cell types and to examine the effects of changes in acid/base balance on its expression. Following NH4Cl (acid) or NaHCO3 (base) loading of rabbits for ∼18 h, CCD cells were isolated by immunodissection. AE2 mRNA levels were determined by RT-PCR and were normalized for β-actin levels. We found that CCD cells express high levels of AE2 mRNA (∼500 copies/cell). AE2 mRNA levels were significantly higher in CCD cells originating from base-loaded than acid-loaded rabbits, with an average increase of 3.7 ± 1.07-fold. The effect of pH on AE2 mRNA levels was also tested directly using primary cultures of CCD cells. CCD cells incubated in acidic media expressed significantly lower levels of AE2 mRNA than those in normal or alkaline media. Experiments with isolated principal cells, α-intercalated cells, and β-intercalated cells (separated by fluorescence-activated cell sorting) demonstrated that AE2 mRNA levels are comparable in the three collecting duct cell subtypes and are similarly regulated by changes in acid/base balance. Based on these results, we conclude that adaptation to changes in extracellular H+ concentration is accompanied by opposite changes in AE2 mRNA expression. The observations that AE2 mRNA is not expressed in a cell-type-specific manner and that changes in acid/base balance have similar effects on each CCD cell subtype suggest that AE2 might serve a housekeeping function rather than being the apical anion exchanger of β-intercalated cells.

Minealocorticoid receptors and 11 beta-steroid dehydrogenase activity in renal principal and intercalated cells

1994 ◽  
Vol 266 (1) ◽  
pp. F76-F80 ◽  
Author(s):  
A. Naray-Fejes-Toth ◽  
E. Rusvai ◽  
G. Fejes-Toth
Keyword(s):  
Cell Sorting ◽  
Direct Action ◽  
Collecting Duct ◽  
Cell Types ◽  
Target Cells ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Specific Receptors ◽  
Principal And Intercalated Cells ◽  
Steroid Dehydrogenase

Aldosterone exerts complex effects on the cortical collecting duct (CCD): it increases Na+ and K+ transport, and it also influences H+ and HCO3 transport. Whether these latter effects represent direct action of aldosterone on intercalated cells (ICC) or are secondary to changes in the transport of other electrolytes is unclear. Because the presence of specific receptors is the prerequisite of a direct steroid action, and mineralocorticoid receptors (MR) have not yet been demonstrated in ICC, in this study we determined the density of MR directly in isolated principal cells (PC) and beta-ICC. Purified populations of these two cell types were obtained from rabbit renal cortex by immunodissection and fluorescence-activated cell sorting. We found that both PC and beta-ICC contained a significant number of MR, although receptor density was higher in PC than in beta-ICC (6,704 +/- 912 vs. 2,181 +/- 388 MR sites/cell; P < 0.001). 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD), an enzyme that is present predominantly in mineralocorticoid target cells, exhibited a distribution similar to that of MR in the two cell types. 11 beta-OHSD activity, determined by measuring the rate of conversion of [3H]corticosterone to 11-dehydrocorticosterone, was 1.08 +/- 0.14 and 0.34 +/- 0.08 fmol.min-1 x 1,000 cells-1 (P < 0.001) in intact PC and beta-ICC, respectively. 11 beta-OHSD in both cell types utilized NAD as cofactor. These results suggest that beta-ICC are potential direct targets of aldosterone and that MR in both PC and beta-ICC are protected by 11 beta-OHSD.

A mathematical model of rat distal convoluted tubule. II. Potassium secretion along the connecting segment

2005 ◽  
Vol 289 (4) ◽  
pp. F721-F741 ◽  
Author(s):  
Alan M. Weinstein
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Cell Types ◽  
Distal Convoluted Tubule ◽  
Cortical Collecting Duct ◽  
Transport Activity ◽  
Luminal Membrane ◽  
In Series ◽  
Potassium Secretion ◽  
Osmotic Equilibration

A simulation of the rat distal convoluted tubule (DCT) is completed with a model of the late portion, or connecting tubule (CNT). This CNT model is developed by relying on a prior cortical collecting duct (CCD) model (Weinstein AM. Am J Physiol Renal Physiol 280: F1072–F1092, 2001), and scaling up transport activity of the three cell types to a level appropriate for DCT. The major difference between the two tubule segments is the lower CNT water permeability. In early CNT the luminal solution is hypotonic, with a K+ concentration less than that of plasma, and it is predicted that osmotic equilibration requires the whole length of CNT, to end with a nearly isotonic fluid, whose K+ concentration is severalfold greater than plasma. With respect to potassium secretion, early CNT conditions are conducive to maximal fluxes, whereas late conditions require the capacity to transport against a steep electrochemical gradient. The parameter dependence for K+ secretion under each condition is different: maximal secretion depends on luminal membrane K+ permeability, but the limiting luminal K+ concentration does not. However, maximal secretion and the limiting gradient are both enhanced by greater Na+ reabsorption. While higher CNT water permeability depresses K+ secretion, it favors Na+ reabsorption. Thus in antidiuresis there is a trade-off between enhanced Na+-dependent K+ secretion and the attenuation of K+ secretion by slow flow. When the CNT model is configured in series with the early DCT, thiazide diuretics promote renal K+ wasting by shifting Na+ reabsorption from early DCT to CNT; they promote alkalosis by shifting the remaining early DCT Na+ reabsorption to Na+/H+ exchange. This full DCT is suitable for simulating the defects of hyperkalemic hypertension, but the model offers no suggestion of a tight junction abnormality that might contribute to the phenotype.

Axial heterogeneity of rabbit cortical collecting duct

1991 ◽  
Vol 260 (4) ◽  
pp. F498-F505
Author(s):  
C. L. Emmons ◽  
K. Matsuzaki ◽  
J. B. Stokes ◽  
V. L. Schuster
Keyword(s):  
Cross Sections ◽  
Cell Function ◽  
Rate Coefficient ◽  
Collecting Duct ◽  
Lectin Binding ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Inner Cortex

The rabbit cortical collecting duct (CCD) consists of three major cell types: principal cells transport K+, beta-intercalated cells absorb Cl-, and alpha-intercalated cells secrete H+. We used functional and histological methods to assess axial distribution of these cell types along rabbit CCD. In perfused CCDs, lumen-to-bath Rb+ rate coefficient (an index of principal cell K+ transport) was not different in tubules from outer cortex (1 mm from renal surface) compared with those from inner cortex (2 mm from renal surface), suggesting that principal cell function is homogeneous along the CCD. In contrast, Cl- rate coefficient (a measure of beta-intercalated cell function) was twice as high in CCDs from outer compared with inner cortex, suggesting heterogeneity of beta-intercalated cells along the CCD. To further investigate these regional differences, we fixed and embedded kidneys and identified three cell types in CCD cross sections using carbonic anhydrase staining and peanut lectin binding. Comparing tubule cross sections from outer with those from inner cortex, we found no axial difference in the fraction of cells that were either principal cells (64%) or total (lectin binding and nonlectin binding) intercalated cells (36%). However, the lectin-binding intercalated cell subset was significantly increased in outer compared with inner cortex. We conclude that there is not heterogeneity of principal cells along the rabbit CCD; however, beta-cell number and function are increased in outer CCD. Collecting duct heterogeneity begins within the cortical segment.

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

1991 ◽  
Vol 261 (3) ◽  
pp. F377-F385 ◽  
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)

Interaction of Cl- and other halogens with Cl- transport systems in rabbit cortical collecting duct

1992 ◽  
Vol 263 (5) ◽  
pp. F870-F877 ◽  
Author(s):  
S. Muto ◽  
M. Imai ◽  
Y. Asano
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cell Types ◽  
Transport Systems ◽  
Cortical Collecting Duct ◽  
Cl Transport ◽  
Distinct Cell ◽  
Cl Conductance ◽  
Basolateral Membrane Potential

We have reported that in the rabbit cortical collecting duct (CCD) we can identify electrophysiologically three distinct cell types; the collecting duct (CD) cell and the alpha- and beta-intercalated (IC) cell. To further characterize the Cl- transport properties of each cell type, we examined the interaction between Cl- and other halogens or SCN- in the isolated and perfused CCD by intracellular microelectrode impalement. The rapid depolarization of the basolateral membrane potential (VB) caused by replacement of bath Cl- with each anion revealed that the sequences of apparent halogen selectivity for the basolateral Cl- conductance were similar in all three cell types. The ranking of Cl- > Br- > F- > I- corresponds to the sequence 5 of Eisenman's series, indicating “strong” interaction of the anions with the selectivity site. The basolateral Cl- conductance of these three cell types may share common characteristics, although I- permeability is less in IC cells than in CD cells. Hyperpolarization of the basolateral membrane of the beta-IC cell upon reduction of luminal Cl- reflects alterations in either Cl- entry across the apical membrane, or Cl- exit across the basolateral membrane, or both. Luminal Cl- replacement with each anion showed that the sequence of the hyperpolarization of the basolateral membrane was I- >> cyclamate = SCN- > F- > Br-, suggesting that I-inhibits either apical Cl- entry or basolateral Cl- exit. On the other hand, in the CD cell reduction of the perfusate Cl- by replacement with each anion caused the basolateral membrane to hyperpolarize with a different ranking: cyclamate = F- > I- = SCN- > Br-.(ABSTRACT TRUNCATED AT 250 WORDS)

Similar chloride channels in the connecting tubule and cortical collecting duct of the mouse kidney

2006 ◽  
Vol 290 (6) ◽  
pp. F1421-F1429 ◽  
Author(s):  
Antoine Nissant ◽  
Marc Paulais ◽  
Sahran Lachheb ◽  
Stéphane Lourdel ◽  
Jacques Teulon
Keyword(s):  
Chloride Channels ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Reversal Potential ◽  
Cell Types ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Connecting Tubule ◽  
Recording Conditions ◽  
Rich Solution

Using the patch-clamp technique, we investigated Cl− channels on the basolateral membrane of the connecting tubule (CNT) and cortical collecting duct (CCD). We found a ∼10-pS channel in CNT cell-attached patches. Substitution of sodium gluconate for NaCl in the pipette shifted the reversal potential by +25 mV, whereas N-methyl-d-gluconate chloride had no effect, indicating anion selectivity. On inside-out patches, we determined a selectivity sequence of Cl− > Br− ∼ NO3− > F−, which is compatible with that of ClC-K2, a Cl− channel in the distal nephron. In addition, the number of open channels ( NPo) measured in cell-attached patches was significantly increased when Ca2+ concentration or pH in the pipette was increased, which is another characteristic of ClC-K. These findings suggest that the basis for this channel is ClC-K2. A similar Cl− channel was found in CCD patches. Because CNT and CCD are heterogeneous tissues, we studied the cellular distribution of the Cl− channel using recording conditions (KCl-rich solution in the pipette) that allowed us to detect simultaneously Cl− channels and inwardly rectifying K+ channels. We detected Cl− channels alone in 45% and 42% and K+ channels alone in 51% and 58% of CNT and CCD patches, respectively. Cl− and K+ channels were recorded simultaneously from two patches (4% of patches) in the CNT and from none of the patches in the CCD. This indicates that Cl− and K+ channels are located in different cell types, which we suggest may be the intercalated cells and principal cells, respectively.

scholarly journals Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells

2016 ◽  
Vol 311 (6) ◽  
pp. F1360-F1368 ◽  
Author(s):  
Ming-Ming Wu ◽  
Yu-Jia Zhai ◽  
Yu-Xia Li ◽  
Qing-Qing Hu ◽  
Zhi-Rui Wang ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Apical Membrane ◽  
Intact Cell ◽  
Collecting Duct ◽  
Physiological Role ◽  
Cell Types ◽  
Cortical Collecting Duct ◽  
Principal Cells

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

NH3 permeability of principal cells and intercalated cells measured by confocal fluorescence imaging

1995 ◽  
Vol 269 (4) ◽  
pp. F545-F550 ◽  
Author(s):  
K. P. Yip ◽  
I. Kurtz
Keyword(s):  
Time Course ◽  
Collecting Duct ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Apical Surface ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Image Pairs ◽  
Confocal Fluorescence Imaging

The cortical collecting duct (CCD) is an important site for NH3 secretion in mammalian nephron. However, given the cellular heterogeneity of this epithelium, the transcellular sites for NH3 secretion are unknown. In the present study, a dual-excitation confocal microscope was designed and optimized to have sufficient temporal resolution to measure the permeability of ammonia (PNH3) across the basolateral and apical membrane of principal cells (PCs) and intercalated cells (ICs) in perfused rabbit CCDs. The rate of cellular NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein after 20 mM NH4Cl was added to the bath or luminal perfusate. The time course of increase in pHi was calculated from 488/442 image pairs stored at a rate of 4 Hz. The apparent basolateral and apical PNH3 values of PCs were 36 +/- 5 and 113 +/- 11 microns/s, respectively. The values were 5.0 +/- 0.7 and 34 +/- 3 microns/s after membrane folding correction. The apparent basolateral and apical PNH3 values of ICs were 38 +/- 6 and 132 +/- 15 microns/s. Corrected for membrane folding, the values were 9.0 +/- 1.0 and 47 +/- 5 microns/s, respectively. The results demonstrate that the apical surface was more permeable than the basolateral surface in both cell types. In addition, ICs were more permeable to NH3 than PCs across both membranes. The transcellular PNH3 of PCs and ICs were 27.3 and 29.5 microns/s, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

CFTR expression in cortical collecting duct cells

1996 ◽  
Vol 270 (1) ◽  
pp. F237-F244 ◽  
Author(s):  
K. M. Todd-Turla ◽  
E. Rusvai ◽  
A. Naray-Fejes-Toth ◽  
G. Fejes-Toth
Keyword(s):  
Collecting Duct ◽  
Cell Types ◽  
Northern Analysis ◽  
Transmembrane Conductance Regulator ◽  
Cortical Collecting Duct ◽  
Transmembrane Conductance ◽  
Pcr Product ◽  
Pcr Products ◽  
Collecting Duct Cells ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a adenosine 3',5'-cyclic monophosphate-activated chloride channel located in the apical membrane of many epithelial cells, and it may play a significant role in the kidney. Recent functional evidence from our laboratory suggests that CFTR may be expressed by the cortical collecting duct (CCD). Therefore, in the present study, the reverse transcription-polymerase chain reaction (RT-PCR) technique was utilized to detect CFTR mRNA in the M-1 mouse CCD cell line and in immunoselected rabbit CCD cells. Primers were constructed to amplify the cDNA sequence encoding the first nucleotide binding domain of CFTR. CFTR PCR products were obtained from both M-1 and rabbit CCD cDNA preparations. The identify of the product amplified from M-1 cell cDNA was confirmed by restriction digestion analysis. The rabbit CCD PCR product was sequenced, and its deduced amino acid sequence was found to be 97% homologous to the corresponding regions of human CFTR. The level of CFTR cDNA detected after 30 cycles of amplification of CCD cDNA was only 49 +/- 8 (n = 9) times lower than the level of beta-actin PCR product obtained from the same sample, suggesting that the levels of CFTR mRNA present in the CCD are physiologically relevant. Northern analysis, using a cRNA probe corresponding to the amplified region on the mRNA from CCD cells, revealed a single hybridizing species with a size of approximately 6.5 kb. Finally, CFTR PCR was performed with cDNA preparations originating from principal cells (PC), beta-intercalated cells (beta-ICC), and alpha-ICC obtained by fluorescence-activated cell sorting of rabbit CCD. CFTR PCR products were obtained from all three cell types, with the most abundant levels found in beta-ICC. beta-ICC expressed 25-fold (n = 4, P < 0.001) and 4.5-fold (n = 7, P < 0.001) higher levels than PC and alpha-ICC, respectively. This distribution pattern suggests that, within the CCD, CFTR plays a role primarily in beta-ICC function.

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