B‐methyl‐cyclodextrin (B‐methyl‐CD) blocks constitutive NKCC2 endocytosis, increases surface NKCC2 and stimulates net NaCl absorption in Thick Ascending Limbs.

Abstract

The Na/K/2Cl cotransporter (NKCC2) mediates NaCl absorption by the epithelial cells of the thick ascending limb (TAL). Trafficking into (exocytosis) and out (endocytosis) of the apical membrane regulates surface NKCC2 levels. It is not known whether NKCC2 undergoes constitutive endocytosis and whether this regulates NaCl absorption by the TAL. We hypothesized that constitutive endocytosis regulates steady‐state surface NKCC2 and its activity in TALs. We measured surface NKCC2 and the rate of NKCC2 endocytosis by biotinylation and Western blot. To block endocytosis we used B‐methyl‐CD, a compound that depletes membrane cholesterol. In control TALs, we observed constitutive NKCC2 endocytosis over 30 min that averaged 7.0 ± 0.8 a.u/min. However, in TALs incubated with B‐methyl‐CD (5 mM) NKCC2 endocytosis was blocked, averaging only 0.9 ± 0.3 a.u/min (n = 6, p<0.01 vs control). Acute (20 min) blockade of endocytosis with B‐methyl‐CD increased steady‐state surface NKCC2 by 60 ± 16% (p<0.05). The total pool of NKCC2 was not changed by B‐methyl‐CD. Finally, we tested whether the increase in surface NKCC2 caused by inhibiting endocytosis stimulates Cl absorption. Incubating isolated perfused TALs with B‐methyl‐CD for 20 min enhanced net Cl absorption by 46 ± 15 % (n = 5, p<0.05). We concluded that constitutive NKCC2 endocytosis maintains steady‐state surface NKCC2 and regulates NaCl absorption.