INTRACELLULAR PH REGULATION IN CULTURED RENAL PROXIMAL TUBULE CELLS IN DIFFERENT STAGES OF MATURATION

This study examines the ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8- to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pH(i)) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis and acidosis using 15 mM NH4+ salt. The net recovery mte (dpH(i)/dt) from intracellular acidosis increases significantly between 10 and 12 days of age from 0.39 +/- 0.04 to 0.54 +/- 0.06 pH units/min (P < 0.05, n = 10 vs. 6). This increase can be completely accounted for by an increase in the rate of amiloride (100 muM)-inhibitable Na+-H+ exchange (0.29 +/- 0.04 vs. 0.42 +/- 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na+-H+ exchange increases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increased by Na+ depleting the cells (48 and 49%, respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS, 500 muM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosis is significantly lower in RPTC-10 than in RPTC-40 (0.16 +/0.02 vs. 0.28 +/- 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500 muM) inhibits the recovery by 27 +/- 8 and 26 +/- 9%, respectively, whereas amiloride has no effect. In RPTC-40 the SITS-sensitive recovery is largely Cl- dependent, and a developmental increase in the activity of Cl--HCO3- exchange between 12 and 14 days of age is demonstrated. No developmental change is seen in either steady-state pH(i) (7.27-7.35) or in cytoplasmic buffer capacity (37.6-44.4 mM/pH(i) unit). In summary, the capacity to protect RPTC from derangements in pH(i) is lower in infancy than in adulthood, which might have important clinical implications in the neonate.