Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

Abstract

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl -and HCO -. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl -current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl-channel is involved in this process. (J. Clin. Invest. 1995.95:644-650.)

rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.

“…In addition to the steroids, 1 mM 4-methylumbelliferyl-D-xyloside and 2.5 mM D(+)-galactosamine were added. We have found that these compounds greatly enhance the rate of seal formation (36), probably due to their ability to inhibit proteoglycan synthesis (37,38). Pilot studies have shown that they do not alter the qualitative response of the IMCD monolayers to steroids and that they retain their sensitivity to benzamil (data not shown).…”

“…The transmonolayer Isc and transepithelial resistance measurements were made in a modified Ussing chamber as previously described (1,2,36) on the day the cells were to be used. For each isolation used for mRNA analysis, parallel cultures were made in the large and the small Millicell filters.…”

“…For each isolation used for mRNA analysis, parallel cultures were made in the large and the small Millicell filters. The large filters were used for RNA isolation and the small filters used for the electrical measurements to be sure that the values were typical of those we have previously reported (1,2,35,36 (40). Cell-attached patches were used to detect channel activity in intact cells.…”

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

“…Because CFTR functions not only as a Cl Ϫ channel, but also as a regulator of other cAMP-responsive apical membrane ion channels including the epithelial sodium channel (64,65), an outwardly rectifying chloride channel (28), and a renal potassium channel (66), we speculate that apical membrane resident CFTR Cl Ϫ channels serve at least two functions in distal nephron: first, mediation of transepithelial Cl Ϫ transport (14,15), and second, regulation of sodium chloride reabsorption/secretion as well as potassium secretion (64). Given that cAMP did not acutely stimulate CFTR trafficking in this study, it is unlikely that CFTR regulates these other ion channels by regulating their trafficking to the apical membrane.…”

“…In contrast, little is known about the intracellular trafficking of CFTR or the mechanism(s) by which cAMP stimulates CFTRmediated Cl Ϫ secretion in kidney epithelia. Because CFTR Cl Ϫ channels are expressed in all nephron segments of the kidney (3) and are important for transepithelial Cl Ϫ transport (14,15) and enlargement of renal cysts in polycystic kidney disease (16), it is important to elucidate the role of cAMP in the regulation of CFTR in normal and pathophysiological renal states.…”