Regulation of intracellular pH in rabbit cortical connecting tubule and cortical collecting duct

Abstract. Inward rectifier potassium channels (Kir) play an important role in the K+ secretion from the kidney. Recently, a new subfamily of Kir, Kir7.1, has been cloned and shown to be present in the kidney as well as in the brain, choroid plexus, thyroid, and intestine. Its cellular and subcellular localization was examined along the renal tubule. Western blot from the kidney cortex showed a single band for Kir7.1 at 52 kD, which was also observed in microdissected segments from the thick ascending limb of Henle, distal convoluted tubule (DCT), connecting tubule, and cortical and medullary collecting ducts. Kir7.1 immunoreactivity was detected predominantly in the DCT, connecting tubule, and cortical collecting duct, with lesser expression in the thick ascending limb of Henle and in the medullary collecting duct. Kir7.1 was detected by electron microscopic immunocytochemistry on the basolateral membrane of the DCT and the principal cells of cortical collecting duct, but neither type A nor type B intercalated cells were stained. The message levels and immunoreactivity were decreased under low-K diet and reversed by low-K diet supplemented with 4% KCl. By the double-labeling immunogold method, both Kir7.1 and Na+, K+-ATPase were independently located on the basolateral membrane. In conclusion, the novel Kir7.1 potassium channel is located predominantly in the basolateral membrane of the distal nephron and collecting duct where it could function together with Na+, K+-ATPase and contribute to cell ion homeostasis and tubular K+ secretion.

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Stimulation of Cl- self exchange by intracellular HCO3- in rabbit cortical collecting duct

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.1.c94 ◽

1989 ◽

Vol 257 (1) ◽

pp. C94-C101 ◽

Cited By ~ 16

Author(s):

K. Matsuzaki ◽

J. B. Stokes ◽

V. L. Schuster

Keyword(s):

Anion Exchange ◽

Intracellular Ph ◽

Rate Coefficient ◽

Collecting Duct ◽

Cortical Collecting Duct ◽

High K ◽

Shift Experiment ◽

Ethanesulfonic Acid ◽

The Individual ◽

Stimulation Of

In rabbit cortical collecting duct, Cl- self exchange accounts for most of the transepithelial Cl- tracer rate coefficient, KCl (nm/s); a small fraction is effected by Cl--HCO3- exchange and Cl- diffusion. We previously reported that changing from a CO2-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) bath to a 5% CO2-25 mM HCO3- bath stimulates Cl- self exchange. Here, we examine in further detail the individual components of the CO2-HCO3- system that stimulate KCl. Addition of 0.5% CO2 to a HEPES bath (final pH = 7.24) stimulated KCl by 70 +/- 19 nm/s, a delta KCl comparable to that induced by 1% CO2 (pH 7.12), 6% CO2 (pH 6.6), or 6% CO2-25 mM HCO3- (pH 7.4). The roles of intracellular pH (pHi) and HCO3- concentration were examined by clamping pHi using high K+ and nigericin. Increasing pHi from 6.9 to 7.6 in solutions without exogenous CO2 or HCO3- increased KCl by 71 +/- 17 nm/s. These results suggest that pHi might regulate anion exchange. However, during such a pHi-shift experiment, metabolically derived CO2 produces a concomitant change in intracellular HCO3- concentration [( HCO3-]i). To determine whether an increase in [HCO3-]i could stimulate Cl- self exchange, we replaced HEPES with 6% CO2-5 mM HCO3- isohydrically (pHi clamped at 6.9). With this increase in [HCO3-]i at constant pHi, KCl increased by 51 +/- 10 nm/s. These maneuvers had negligible effects on Cl- diffusion and Cl--HCO3- exchange. These experiments demonstrate that increases in cell [HCO3-] (or perhaps CO2) can stimulate transepithelial anion exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

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Effects of prostaglandin E2 on membrane voltage of the connecting tubule and cortical collecting duct from rabbits.

The Journal of Physiology ◽

10.1113/jphysiol.1993.sp019555 ◽

1993 ◽

Vol 462 (1) ◽

pp. 275-289 ◽

Cited By ~ 4

Author(s):

T Shimizu ◽

M Nakamura ◽

K Yoshitomi ◽

M Imai

Keyword(s):

Prostaglandin E2 ◽

Collecting Duct ◽

Membrane Voltage ◽

Cortical Collecting Duct ◽

Connecting Tubule

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Intracellular pH regulation in the rabbit cortical collecting duct A-type intercalated cell

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.3.f340 ◽

1997 ◽

Vol 273 (3) ◽

pp. F340-F347 ◽

Cited By ~ 10

Author(s):

A. E. Milton ◽

I. D. Weiner

Keyword(s):

Intracellular Ph ◽

Ph Regulation ◽

Collecting Duct ◽

Cortical Collecting Duct ◽

Bafilomycin A1 ◽

Proton Secretion ◽

Acid Load ◽

A Cell ◽

Phi Regulation ◽

Intracellular Acid

The A cell may possess multiple H+ transporters, including H(+)-adenosinetriphosphatase (H(+)-ATPase) and H(+)-K(+)-ATPase. The current study examines the relative roles of proton transporters in the A cell by observing their contribution to both basal intracellular pH (pHi) regulation and pHi recovery from an intracellular acid load. CCD were studied using in vitro microperfusion, and pHi was measured in the individual A cell using the fluorescent, pH-sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). Inhibiting H(+)-ATPase with luminal bafilomycin A1 decreased basal pHi, whereas inhibiting apical H(+)-K(+)-ATPase with either luminal Sch-28080 or luminal potassium removal did not. The predominant mechanism of pHi, recovery from an intracellular acid load was peritubular sodium dependent and peritubular ethylisopropylamiloride (EIPA) sensitive, identifying basolateral Na+/H+ exchange activity. In the absence of peritubular sodium, pHi recovery was inhibited by luminal bafilomycin A1 but not by luminal Sch-28080 addition or by luminal potassium removal. However, when Na+/H+ exchange was inhibited with EIPA, both bafilomycin A1 sensitive and potassium dependent, Sch-28080-sensitive components of pHi recovery were present. Quantitatively, the rate of H(+)-ATPase proton secretion was greater than the rate of H(+)-K(+)-ATPase proton secretion. We conclude that basolateral Na+/H+ exchange is the predominant mechanism of A cell pHi recovery from an intracellular acid load. An apical H(+)-ATPase is the primary apical transporter contributing to A cell pHi regulation. An apical H(+)-K(+)-ATPase, while present, plays a more limited role under the conditions tested.

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Expression of isoforms of the Na+/H+exchanger in M-1 mouse cortical collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00291.2000 ◽

2002 ◽

Vol 282 (4) ◽

pp. F649-F654 ◽

Cited By ~ 7

Author(s):

C. Hill ◽

A. N. Giesberts ◽

S. J. White

Keyword(s):

Intracellular Ph ◽

Apical Membrane ◽

Collecting Duct ◽

Extracellular Nucleotides ◽

Cortical Collecting Duct ◽

Intracellular Acidification ◽

Basolateral Membranes ◽

Nephron Segments ◽

Collecting Duct Cells ◽

Nhe Isoforms

Na+/H+ exchanger (NHE) proteins perform a variety of functions in the kidney and are differentially distributed among nephron segments. The purpose of this study was to identify NHE isoforms in murine M-1 cells as a model of cortical collecting duct principal cells. It was found that mRNAs corresponding to NHE1, NHE2, and NHE4 are expressed in M-1 cells. NHE-dependent regulation of intracellular pH (pHi) was investigated in the absence of extracellular HCO[Formula: see text]. Application of a 20 mM NH4Cl pulse resulted in a reversible intracellular acidification from which recovery was partially inhibited by application of 1 mM amiloride to either the apical or the basolateral membranes and was abolished when amiloride was applied to both sides of the monolayers, which suggests that NHEs are expressed in both the apical and the basolateral cell membranes of M-1 cells. The purinergic agonists ATP and benzoylbenzoyl-ATP caused a reduction of pHi when applied to the apical membrane, which suggests pHi may be influenced by extracellular nucleotides in the luminal fluid of the cortical collecting duct.

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ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. II. Bafilomycin-sensitive H+ secretion

AJP Renal Physiology ◽

10.1152/ajprenal.00120.2015 ◽

2015 ◽

Vol 309 (3) ◽

pp. F259-F268 ◽

Cited By ~ 7

Author(s):

Masayoshi Nanami ◽

Vladimir Pech ◽

Yoskaly Lazo-Fernandez ◽

Alan M. Weinstein ◽

Susan M. Wall

Keyword(s):

Intracellular Ph ◽

Collecting Duct ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Transport Pathway ◽

Anion Secretion ◽

Intercalated Cell ◽

Atpase Inhibitors ◽

Hcl Secretion ◽

Transepithelial Voltage

Epithelial Na+ channel (ENaC) blockade stimulates stilbene-sensitive conductive Cl− secretion in the mouse cortical collecting duct (CCD). This study's purpose was to determine the co-ion that accompanies benzamil- and DIDS-sensitive Cl− flux. Thus transepithelial voltage, VT, as well as total CO2 (tCO2) and Cl− flux were measured in CCDs from aldosterone-treated mice consuming a NaCl-replete diet. We reasoned that if stilbene inhibitors (DIDS) reduce conductive anion secretion they should reduce the lumen-negative VT. However, during ENaC blockade (benzamil, 3 μM), DIDS (100 μM) application to the perfusate reduced net H+ secretion, which increased the lumen-negative VT. Conversely, ENaC blockade alone stimulated H+ secretion, which reduced the lumen-negative VT. Application of an ENaC inhibitor to the perfusate reduced the lumen-negative VT, increased intercalated cell intracellular pH, and reduced net tCO2 secretion. However, benzamil did not change tCO2 flux during apical H+-ATPase blockade (bafilomycin, 5 nM). The increment in H+ secretion observed with benzamil application contributes to the fall in VT observed with application of this diuretic. As such, ENaC blockade reduces the lumen-negative VT by inhibiting conductive Na+ absorption and by stimulating H+ secretion by type A intercalated cells. In conclusion, 1) in CCDs from aldosterone-treated mice, benzamil application stimulates HCl secretion mediated by the apical H+-ATPase and a yet to be identified conductive Cl− transport pathway; 2) benzamil-induced HCl secretion is reversed with the application of stilbene inhibitors or H+-ATPase inhibitors to the perfusate; and 3) benzamil reduces VT not only by inhibiting conductive Na+ absorption, but also by stimulating H+ secretion.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00274.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. F1421-F1429 ◽

Cited By ~ 37

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.

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Regulation of the apical Cl−/HCO 3 − exchanger pendrin in rat cortical collecting duct in metabolic acidosis

AJP Renal Physiology ◽

10.1152/ajprenal.00205.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. F103-F112 ◽

Cited By ~ 42

Author(s):

Snezana Petrovic ◽

Zhaohui Wang ◽

Liyun Ma ◽

Manoocher Soleimani

Keyword(s):

Metabolic Acidosis ◽

Mrna Expression ◽

Intracellular Ph ◽

Northern Blot ◽

Collecting Duct ◽

Kidney Cortex ◽

Cortical Collecting Duct ◽

Acid Base ◽

Ph Change ◽

Immunofluorescence Labeling

Pendrin is an apical Cl−/OH−/HCO[Formula: see text] exchanger in β-intercalated cells (β-ICs) of rat and mouse cortical collecting duct (CCD). However, little is known about its regulation in acid-base disorders. Here, we examined the regulation of pendrin in metabolic acidosis, a condition known to decrease HCO[Formula: see text]secretion in CCD. Rats were subjected to NH4Cl loading for 4 days, which resulted in metabolic acidosis. Apical Cl−/HCO[Formula: see text] exchanger activity in β-ICs was determined as amplitude and rate of intracellular pH change when Cl was removed in isolated, microperfused CCDs. Intracellular pH was measured by single-cell digital ratiometric imaging using fluorescent pH-sensitive dye 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein-AM. Pendrin mRNA expression in kidney cortex was examined by Northern blot hybridizations. Expression of pendrin protein was assessed by indirect immunofluorescence. Microperfused CCDs isolated from acidotic rats demonstrated ∼60% reduction in apical Cl−/HCO[Formula: see text] exchanger activity in β-ICs ( P < 0.001 vs. control). Northern blot hybridizations indicated that the mRNA expression of pendrin in kidney cortex decreased by 68% in acidotic animals ( P < 0.02 vs. control). Immunofluorescence labeling demonstrated significant reduction in pendrin expression in CCDs of acidotic rats. We conclude that metabolic acidosis decreases the activity of the apical Cl−/HCO[Formula: see text] exchanger in β-ICs of the rat CCD by reducing the expression of pendrin. Adaptive downregulation of pendrin in metabolic acidosis indicates the important role of this exchanger in acid-base regulation in the CCD.

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High baseline ROMK activity in mouse late distal convoluted and early connecting tubule (DCT2/CNT) probably contributes to aldosterone-independent K+ secretion

AJP Renal Physiology ◽

10.1152/ajprenal.00252.2021 ◽

2021 ◽

Author(s):

Viatcheslav Nesterov ◽

Marko Bertog ◽

Christoph Korbmacher

Keyword(s):

Single Channel ◽

Apical Membrane ◽

Collecting Duct ◽

Standard Diet ◽

Cortical Collecting Duct ◽

Channel Activity ◽

Connecting Tubule ◽

Low Potassium ◽

K Secretion ◽

Baseline Conditions

The renal outer medullary K+ channel (ROMK) is co-localized with the epithelial Na+ channel (ENaC) in late distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD). ENaC-mediated Na+ absorption generates the electrical driving force for ROMK-mediated tubular K+ secretion which is critically important for maintaining renal K+ homeostasis. ENaC activity is aldosterone-dependent in late CNT and early CCD (CNT/CCD) but aldosterone-independent in DCT2 and early CNT (DCT2/CNT). This suggests that under baseline conditions with low plasma aldosterone ROMK-mediated K+ secretion mainly occurs in DCT2/CNT. Therefore, we hypothesized that baseline ROMK activity is higher in DCT2/CNT than in CNT/CCD. To test this hypothesis, patch-clamp experiments were performed in DCT2/CNT and CNT/CCD microdissected from mice maintained on standard diet. In single-channel recordings from outside-out patches we detected typical ROMK channel activity in both DCT2/CNT and CNT/CCD and confirmed that ROMK is the predominant K+ channel in the apical membrane. Amiloride-sensitive (ΔIami) and tertiapin-sensitive (ΔITPNQ) whole-cell currents were determined to assess ENaC and ROMK activity, respectively. As expected, baseline ΔIami was high in DCT2/CNT (~370 pA) but low in CNT/CCD (~60 pA). Importantly, ΔITPNQ was significantly higher in DCT2/CNT than in CNT/CCD (~810 pA versus ~350 pA). We conclude that high ROMK activity in DCT2/CNT is critical for aldosterone-independent renal K+ secretion under baseline conditions. A low potassium diet significantly reduced ENaC but not ROMK activity in DCT2/CNT. This suggests that modifying ENaC activity in DCT2/CNT plays a key regulatory role in adjusting renal K+ excretion to dietary K+ intake.

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