RVD in principal and intercalated cells of rabbit cortical collecting tubule

1988 ◽  
Vol 255 (5) ◽  
pp. C612-C621 ◽  
Author(s):  
K. Strange
Keyword(s):  
Mammalian Cells ◽  
Control Volume ◽  
Regulatory Volume Decrease ◽  
Isotonic Saline ◽  
Cell Types ◽  
Intercalated Cells ◽  
Intercalated Cell ◽  
Collecting Tubule ◽  
Principal And Intercalated Cells ◽  
Cortical Collecting Tubule

Cells of the rabbit renal cortical collecting tubule possess significant regulatory volume decrease (RVD) capabilities. After a 100-mosmol/kg reduction in peritubular osmolality, principal and intercalated cells swell 40-45 and 30-35%, respectively, and immediately activate RVD mechanisms. Both cell types downregulate their volume to within 5-6% of control volume at initial rates of 3-6%/min. Return to isotonic saline causes both cell types to shrink (isotonic shrinkage) 25-35% below control volume due to the loss of osmotically active intracellular solutes during RVD. In most mammalian cells studied to date, RVD is mediated largely by passive KCl efflux via KCl cotransport, parallel K+ and Cl- channels, or parallel K+-H+ and Cl- -HCO3- exchange mechanisms. Peritubular application of 0.1 mM ouabain (0 Na+ lumen), bilateral CO2-HCO3- removal, or bilateral application of 0.02 mM bumetanide, 2.0 mM Ba2+, 2.0 mM anthracene-9-carboxylic acid, or 0.5 mM SITS had no significant effect on rates or magnitudes of RVD and isotonic shrinkage in either cell type. Bilateral elevation of K+ from 5 to 52.5 mM reverses or reduces the electrochemical gradient for K+ movement, causing accumulation of this ion in the cytoplasm, but had no effect on the rates or magnitude of principal and intercalated cell RVD. Principal and intercalated cells from K+- or Cl- -depleted tubules (1 h bilateral perfusion with K+- or Cl- -free saline at 37 degrees C) showed normal rates and magnitudes of RVD in K+- or Cl- -free hypotonic saline. Taken together, these results argue against a significant role of passive KCl efflux pathways in mediating principal and intercalated cell RVD.

Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule

1993 ◽  
Vol 264 (2) ◽  
pp. F259-F266 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt
Keyword(s):  
Cell Types ◽  
Intercalated Cells ◽  
Functional Identification ◽  
Proton Extrusion ◽  
Collecting Tubule ◽  
Initial Ph ◽  
Acid Load ◽  
Extrusion Mechanism ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

A K-dependent proton extrusion mechanism was investigated by means of fluorescence techniques in rabbit cortical collecting tubules. These experiments were performed in split opened tubules from normal animals exposed to the intracellular pH (pH(i)) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This preparation permitted the separate study of the intercalated cells (IC) from the principal cells (PC). In IC pH(i) recovery in response to an acute acid load was observed under Na-free conditions on addition of 5 mM K. This K-dependent recovery of pH(i) in the IC was only partial, but was Sch 28080 inhibitable (10(-5) M) and ouabain insensitive. This suggests the process is mediated by an H-K-adenosinetriphosphatase similar to that of gastric cells. The PC were capable of recovering from the acid load, but this Na-independent response was not blocked by the Sch 28080, suggesting some other mechanism for this result. In both cell types reintroduction of Na into the superfusate resulted in full recovery back to the initial pH(i), presumably the result of Na/H exchange.

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.

CFTR-dependent and -independent swelling-activated K+ currents in primary cultures of mouse nephron

2003 ◽  
Vol 284 (4) ◽  
pp. F812-F828 ◽  
Author(s):  
Radia Belfodil ◽  
Hervé Barrière ◽  
Isabelle Rubera ◽  
Michel Tauc ◽  
Chantal Poujeol ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Regulatory Volume Decrease ◽  
Mouse Kidney ◽  
Wild Type ◽  
Collecting Tubule ◽  
K Currents ◽  
Volume Decrease ◽  
Autocrine Mechanism ◽  
Cortical Collecting Tubule

The role of CFTR in the control of K+ currents was studied in mouse kidney. Whole cell clamp was used to identify K+ currents on the basis of pharmacological sensitivities in primary cultures of proximal (PCT) and distal convoluted tubule (DCT) and cortical collecting tubule (CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT cells, forskolin activated a 293B-sensitive K+ current in WT, but not in KO, mice. In these cells, a hypotonic shock induced K+ currents blocked by charybdotoxin in WT, but not in KO, mice. In PCT cells from WT and KO mice, the hypotonicity-induced K+ currents were insensitive to these toxins and were activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting that the corresponding channel was TASK2. In conclusion, CFTR is implicated in the control of KCNQ1 and Ca2+-sensitive swelling-activated K+ conductances in DCT and CCT, but not in proximal convoluted tubule, cells. In KO mice, impairment of the regulatory volume decrease process in DCT and CCT could be due to the loss of an autocrine mechanism, implicating ATP and adenosine, which controls swelling-activated Cl− and K+channels.

The "coat" of kidney intercalated cell tubulovesicles does not contain clathrin

1986 ◽  
Vol 250 (4) ◽  
pp. C605-C608 ◽  
Author(s):  
D. Brown ◽  
L. Orci
Keyword(s):  
Coat Protein ◽  
Rat Kidney ◽  
Cell Types ◽  
Intercalated Cells ◽  
Coated Pits ◽  
Thin Sections ◽  
Intercalated Cell ◽  
Apical Cytoplasm ◽  
Collecting Ducts ◽  
Lowicryl K4m

Intercalated cells of kidney collecting ducts contain a population of tubulovesicles in their apical cytoplasm, whose limiting membranes are decorated by arrays of dense, club-shaped projections oriented toward the cytoplasm. These tubulovesicles have been implicated in endo-exocytotic events in these cells. To determine a possible relationship between this “coating” material and clathrin, the coat protein associated with endocytotic coated pits and coated vesicles in other cell types, we applied a monospecific, affinity-purified anti-clathrin antibody to thin sections of rat kidney embedded at low temperature in Lowicryl K4M. We found that no specific labeling was associated with the studlike material of intercalated cell tubulovesicles.

Immunological segmentation of the rabbit distal, connecting, and collecting tubules

1987 ◽  
Vol 252 (3) ◽  
pp. F412-F422
Author(s):  
P. Poujeol ◽  
P. Ronco ◽  
M. Tauc ◽  
M. Geniteau ◽  
F. Chatelet ◽  
...  
Keyword(s):  
Cell Types ◽  
Cellular Level ◽  
Cell Fractionation ◽  
Intercalated Cells ◽  
Cortical Cells ◽  
Double Labeling ◽  
Principal Cells ◽  
Group 2 ◽  
Principal And Intercalated Cells ◽  
Collecting Tubules

To obtain monoclonal antibodies (MAB) specific for the different cell types of distal and collecting tubules, BALB/c mice were immunized with cell suspensions highly enriched in cells from the distal segments of the rabbit nephron. Nine MAB were selected and cloned. Four groups could be identified on the basis of double-labeling immunofluorescence (IF) on frozen kidney sections and on microdissected tubules. In addition, binding specificity at the cellular level was studied by immunoelectronmicroscopy (IEM) for selected MAB. A single MAB (group 1) was specific for distal bright cells and a subpopulation of cortical ascending limb cells. Six MAB (group 2) reacted with connecting and collecting tubules. Five of these (group 2A) had similar binding patterns and reacted identically with the two tubular segments. The MAB studied by IEM was specific for connecting and principal cells. One antibody (group 2B) reacted with only a fraction of the cells associated with the connecting tubule (CNT), but with all cells of the cortical collecting tubule (CCT). By IEM, this antibody was found to be specific for intercalated cells in CNT and bound both principal and intercalated cells of the CCT. Two MAB (group 3) reacted with antigen(s) expressed by the various terminal segments of renal tubule. MAB of groups 1 and 2A, which define distal bright cells and connecting-principal cells from the CNT-CCT, respectively, were used for cell fractionation experiments. Heterogeneous rabbit cortical cells were first incubated with the selected MAB. MAB-bearing renal cells were separated on plastic dishes previously coated with an affinity-purified goat anti-mouse immunoglobulin. Using these procedures it was possible to obtain highly purified subpopulations of distal, bright, or connecting-principal cells.

Expression of acid-base-related proteins in mesonephric kidney of the rabbit

1994 ◽  
Vol 267 (6) ◽  
pp. F987-F997 ◽  
Author(s):  
T. Matsumoto ◽  
C. A. Winkler ◽  
L. P. Brion ◽  
G. J. Schwartz
Keyword(s):  
Collecting Duct ◽  
Cell Types ◽  
Intercalated Cells ◽  
Acid Base ◽  
Principal Cells ◽  
Collecting Tubule ◽  
Metanephric Kidney ◽  
Collecting Tubules ◽  
Related Proteins ◽  
Different Cell Types

The mesonephric kidney, precursor to the metanephric kidney, comprises 30-50 nephrons, each with a glomerulus and proximal, distal, and collecting tubules. Although two different cell types have been identified in the mesonephric collecting tubule, no relationship to cells of the metanephric collecting duct has been established. To characterize expression of some of the acid-base-related proteins, we assayed for carbonic anhydrase (CA) activity and performed immunocytochemistry in mesonephroi from 15- to 20-day-old fetal rabbits. From total RNA, we detected expression of CA II and CA IV mRNA. Microdissected proximal and collecting tubules abundantly expressed both CA II and CA IV, at least to the extent observed in mature metanephric proximal tubules and collecting ducts. Histochemistry confirmed the expression of CA activity in these segments; in the collecting tubule, 28% of the collecting tubule cells were CA rich. Most CA-rich cells showed apical H(+)-ATPase and basolateral band 3 anion exchanger staining consistent with the findings in mature H(+)-secreting (alpha) intercalated cells of the metanephric collecting duct. CA-negative cells could be labeled with an antibody that identifies mature metanephric principal cells. Thus the mesonephric collecting tubule has many cells resembling mature alpha-intercalated cells and a majority of cells resembling principal cells. The similarity to the metanephric collecting duct suggests that the lineages of metanephric alpha-intercalated and principal cells may be closely related to those of the mesonephros.

Isolated principal and intercalated cells: hormone responsiveness and Na+-K+-ATPase activity

1989 ◽  
Vol 256 (4) ◽  
pp. F742-F750 ◽  
Author(s):  
G. Fejes-Toth ◽  
A. Naray-Fejes-Toth
Keyword(s):  
Binding Sites ◽  
Single Cells ◽  
Functional Characterization ◽  
Cell Types ◽  
Rabbit Kidney ◽  
Intercalated Cells ◽  
Functional Differences ◽  
Maximal Stimulation ◽  
Principal And Intercalated Cells ◽  
Camp Levels

To explore biochemical and functional differences between principal (PC) and intercalated cells (ICC), we have developed a method for separating them from rabbit kidney. Fragments of cortical collecting ducts were isolated by immunodissection, and single cells obtained from these clusters were stained with fluorochrome-conjugated, cell-specific markers. PC and ICC were then separated by fluorescence-activated cell sorting. Identity of the sorted cells was confirmed by staining with other cell-specific monoclonal antibodies (MCABs) or peanut lectin. Purity was greater than 99% for ICC and greater than 96% for PC. Arginine vasopressin (AVP) increased adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in both cell types, but maximal stimulation was significantly greater with PC (approximately 20-fold) than with ICC (2.7-fold). Half-maximal stimulation was seen at approximately 2 x 10(-10) M AVP with both cell types. Isoproterenol increased cAMP levels only with ICC (from 1.23 +/- 0.16 to 12.06 +/- 1.25 fmol/cell; P less than 0.001). The number of ouabain binding sites and the activity of Na+-K+-ATPase was significantly higher in sorted PC than ICC (2.2 X 10(6) vs. 9.6 X 10(5) binding sites; 19.2 vs. 9.6 fmol.min-1.cell-1 ATP hydrolyzed in PC vs. ICC, respectively). These results demonstrate the feasibility of isolating homogeneous populations of PC and ICC, which is useful for further studies of their biochemical and functional characterization.

Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct

1998 ◽  
Vol 275 (1) ◽  
pp. F94-F102 ◽  
Author(s):  
Randi B. Silver ◽  
Han Choe ◽  
Gustavo Frindt
Keyword(s):  
Plasma Levels ◽  
Collecting Duct ◽  
Plasma Aldosterone ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Elevated Plasma ◽  
Ratiometric Fluorescence ◽  
Collecting Tubule ◽  
Acid Load ◽  
Cortical Collecting Tubule

Extracellular K+-dependent H+ extrusion after an acute acid load, an index of H/K exchange, was monitored in intercalated cells (ICs) from rat cortical collecting tubule (CCT) using ratiometric fluorescence imaging of the intracellular pH (pHi) indicator, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The hypothesis tested was that 12- to 14-day NaCl deprivation increases H-K-ATPase in rat ICs. The rate of H/K exchange in the low-NaCl ICs was double that of controls. In control ICs, H/K exchange was inhibited by Sch-28080 (10 μM). In the low-NaCl ICs, it was partially blocked by Sch-28080 or ouabain (1 mM). Simultaneous addition of both inhibitors abolished K-dependent pHirecovery. The induced H/K exchange observed with NaCl restriction was not due to elevated plasma aldosterone as evidenced by experiments on ICs from rats implanted with osmotic minipumps administering aldosterone such that plasma levels were comparable to those of NaCl-deficient rats. The results suggest that NaCl deficiency induces two isoforms of H-K-ATPase in ICs and that this effect is not mediated by elevated plasma aldosterone.

Volume regulatory Cl- loss after Na+ pump inhibition in CCT principal cells

1991 ◽  
Vol 260 (2) ◽  
pp. F225-F234 ◽  
Author(s):  
K. Strange
Keyword(s):  
Steady State ◽  
High Selectivity ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Principal Cells ◽  
Na Pump ◽  
Collecting Tubule ◽  
Volume Decrease ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

Ouabain caused rabbit cortical collecting tubule (CCT) principal cells to swell 53% and then undergo regulatory volume decrease (RVD) at a rate of 4%/min to a new steady-state volume 10% below control. Reduction of peritubular Cl- concentration transiently depolarized transepithelial potential (Vte) by 36 mV and stimulated the rate of RVD 30-fold. Peritubular application of 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited RVD 74%. In contrast, luminal Cl- reduction or application of DIDS had no effect on RVD. A 10-fold elevation of perfusate K+ caused volume-regulated cells to swell 23% at a rate of 60%/min. Removal of luminal Cl- had no effect on either the rate or magnitude of K+ swelling. Peritubular or bilateral Cl- removal, however, inhibited the rate of K+ swelling by 96 and 99%, respectively. Substitution of bath Cl- for Br-, SCN-, or I- inhibited the rate of K+ swelling by 40, 38, and 98%, respectively. Surprisingly, NO3- inhibited the rate of K+ swelling by 82%. All Cl- substitutes tested transiently depolarized Vte by 3–49 mV. These results suggest strongly that RVD is mediated by a basolateral Cl- channel with a high selectivity for Cl- over other anions.

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