Molecular and functional evidence for a role of a K,Cl cotransporter in the transepithelial secretion of fluid by mosquito renal tubules

Abstract

In the mammalian renal collecting tubule, a K,Cl cotransporter (KCC4) recycles Cl− across the basolateral membrane of type A intercalated cells to permit the function of a basolateral Cl/HCO3 exchanger (AE1). Thus, it contributes to the transepithelial, V-type H+-ATPase-driven secretion of acid. In the renal (Malpighian) tubules of the mosquito Aedes aegypti, we have recently demonstrated that a basolateral, DIDS-sensitive AE contributes to the transepithelial, V-type H+-ATPase-driven secretion of urine during diuretic conditions. The presence of a KCC that regulates the AE is unknown. In the present study, we used RT-PCR on Malpighian tubules to clone a full-length cDNA (AeKCC) that encodes 1096 amino acids with ~55% identity to human KCCs. When expressed heterologously in Xenopus oocytes, the AeKCC mediates 86Rb+ uptake that is 1) activated by cell swelling and 2) inhibited by lowering external Cl− or adding DIOA. In isolated Malpighian tubules, adding peritubular DIOA reduced the spontaneous rates of fluid secretion by ~85%. In tubules secreting fluid at diuretic rates, adding peritubular DIOA returned secretion rates to unstimulated levels. The latter effect of DIOA resembles that of DIDS, which indicates that the KCC and AE may be functionally coupled. The former effect of DIOA indicates that the KCC also plays a role in tubule function when the AE is less active. Supported by NIH Grant K01-DK080194-01 to PMP.