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 pCO(2) high osmolality (congruent to670 mosmol/kg H2O, 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 (congruent to670 mosmol/kg H2O) than in low osmolality (congruent to290 mosmol/kg H2O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pH, following additional acidification with an NH4Cl pulse was reduced by Na+ removal from the medium and by the addition of 60 muM HOE642 (an inhibitor of the Na+/H+ exchanger, NHE1), 55 muM S1611 (inhibitor of Na+/H+ exchanger, NHE3), 1 muM bafilomycin A1 (an inhibitor of vacuolar H+-ATPase), or 20 muM Schering 28080 (an inhibitor of H+-K+-ATPase) to the medium. Resting pH(i) was also reduced by 60 muM HOE642, 55 muM S1611, and 20 muM 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.