Regulation of intracellular pH in rat renal inner medullary thin limbs of Henle’s loop

Regulation of intracellular pH (pH i ) was studied in isolated rat renal inner medullary thin limbs of Henle’s loop in bicarbonate/phosphate-buffered medium with high p CO 2 , high osmolality (≅670 mosmol/kg H 2 O; 270 mM urea; 180 mM NaCl), organic osmolytes, and a pH of 6.8 to approximate the physiological in vivo environment. The pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pH i . Resting pH i was always acid and significantly more acid in descending thin limb (DTL) cells than in ascending thin limb (ATL) cells from pure or mixed-type thin limbs. Resting pH i was slightly but significantly higher in both DTLs and ATLs in high osmolality (≅670 mosmol/kg H 2 O) than in low osmolality (≅290 mosmol/kg H 2 O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pH i following additional acidification with an NH 4 Cl pulse was reduced by Na + removal from the medium and by the addition of 60 µM HOE642 (an inhibitor of the Na + /H + exchanger, NHE1), 55 µM S1611 (inhibitor of Na + /H + exchanger, NHE3), 1 µM bafilomycin Aff1 (an inhibitor of vacuolar H + -ATPase), or 20 µM Schering 28080 (an inhibitor of H + -K + -ATPase) to the medium. Resting pH i was also reduced by 60 µM HOE642, 55 µM S1611, and 20 µM Schering 28080. In both DTLs and ATLs, RT-PCR revealed message for NHE1, NHE3, and vacuolar H + -ATPase; immunocytochemistry demonstrated the expression of the protein for NHE1 (basolateral membrane), NHE3 (luminal membrane), and H + -K + -ATPase (luminal membrane). These data suggest that pH i in rat inner medullary thin limbs is regulated by urea and by basolateral and luminal H + extrusion via NHE1, NHE3, vacuolar H + -ATPase, and H + -K + -ATPase.