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 pCO₂, high osmolality (≅670 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 (≅670 mosmol/kg H₂O) than in low osmolality (≅290 mosmol/kg H₂O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pH_i following additional acidification with an NH₄Cl 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 A1 (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.