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Madin-Darby canine kidney cells. III. Aldosterone stimulates an apical H+/K+ pump
H Oberleithner1, W Steigner, S Silbernagl
1Department of Physiology, University of Würzburg, Federal Republic of Germany.
Insights
Madin-Darby canine kidney cells exhibit apical H+ secretion via Na+/H+ exchange and an aldosterone-stimulated H(+)-K+ pump. This pump is inhibited by omeprazole, suggesting a role in urinary acid-base balance.
Area of Science:
- Cell Biology
- Renal Physiology
- Epithelial Transport
Background:
- Madin-Darby canine kidney (MDCK) cells serve as a model for intercalated cells in urinary epithelia.
- Understanding the mechanisms of proton (H+) secretion in these cells is crucial for comprehending renal acid-base regulation.
Purpose of the Study:
- To investigate the apical H+ secretion mechanisms in MDCK cell domes.
- To identify the specific transporters and regulatory factors involved in this process.
Main Methods:
- Experiments utilized MDCK cell monolayers grown as domes to measure pH gradients (delta pHd).
- Short-circuit current (SCC) measurements assessed transepithelial ion transport.
- Histochemical analysis evaluated carbonic anhydrase (CA) activity.
- Pharmacological agents (amiloride, barium, omeprazole) and ion substitutions (Na+, K+) were employed to probe transport pathways.
Main Results:
- Apical Na+/H+ exchange was confirmed by inhibition with amiloride and Na+-free solutions.
- An aldosterone-stimulated, acetazolamide-sensitive SCC indicated a basolateral to apical current.
- Apical K+ substitution, barium, and omeprazole significantly reduced delta pHd, suggesting an apical H(+)-K+ pump.
- Carbonic anhydrase activity was localized in dome-forming cells.
Conclusions:
- MDCK cells possess both apical Na+/H+ exchange and an apical H(+)-K+ pump.
- The H(+)-K+ pump is stimulated by aldosterone and inhibited by omeprazole and barium.
- Carbonic anhydrase plays a role in H+ production for apical secretion.
- These findings elucidate key mechanisms in renal acid-base transport.
Abstract:
Functionally and morphologically, Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of urinary epithelia. Experiments were performed on domes of confluent MDCK monolayers to test for apical H+ secretion. Apical application of 10(-3) mol/l amiloride or of Na(+)-free solution significantly reduced the limiting pH gradient across the dome epithelium (delta pHd) consistent with inhibition of apical Na+/H+ exchange. Short-circuit current (SCC) measurements disclosed an acetazolamide-sensitive, (basolateral to apical) positive transepithelial current stimulated by 10(-7) mol/l aldosterone and inhibited by acidification of apical medium to pH = 4.5. Histochemical evaluation of carbonic anhydrase (CA) activity revealed cytoplasmic and apical-membrane-bound CA particularly in dome-forming cells. Apical substitution of Na+ by K+ increased delta pHd, whereas a reduction of K+ concentration to 0.5 mmol/l or addition of barium or omeprazole (10(-5) mol/l) to the apical superfusate reduced delta pHd by at least 75%. Aldosterone-stimulated SCC was completely abolished by the apical application of barium. We conclude that besides Na+/H+ exchange MDCK cells can express an apically located H(+)-K+ pump stimulated by aldosterone and inhibited directly by the anti-ulcer agent omeprazole or indirectly, either by blocking apical K+ recycling or by interfering with the CA-dependent intracellular formation of H+ ions.