Glucocorticoids are reported to have stimulatory effects on renal proximal tubule Na⁺/H⁺ exchanger activity and on the expression of the NHE3 isoform. Here we have examined using spectrofluorescence microscopy, the effects of both acute and chronic exposure to hydrocortisone on luminal Na⁺/H⁺ exchanger activity in monolayers of primary cultured human proximal tubule (HPT) cells loaded with the pH-sensitive dye BCECF.

The cells were acid-loaded using the NH₄Cl pulse method under sodium-free conditions and Na⁺/H⁺ exchanger activity was measured as the initial slope of pH_i recovery (ΔpH_i units per min, means ± S.E.M., Student’s paired t test) following the introduction of Na⁺ to the luminal bath. Chronic exposure of HPT cells to hydrocortisone (10 nM for 90 min) increased the rate of Na⁺-dependent pH_i recovery twofold when compared to untreated controls (control ΔpH_i = 0.10 ± 0.02, chronic hydrocortisone ΔpH_i = 0.23 ± 0.03, n = 6, P ≤ 0.001). Acute (seconds) exposure to hydrocortisone produced an inhibition of NHE activity. Under these conditions, when hydrocortisone was introduced into the luminal bath with sodium, the pH_i recovery rate was reduced to 13 % of control (control ΔpH_i = 0.11 ± 0.02, acute hydrocortisone ΔpH_i = 0.013 ± 0.012, n = 6, P ≤ 0.005). Pre-incubation of HPT cells for 10 min with the PKC inhibitor chelerythrine chloride (1 µM) had no effect on the stimulation of NHE after chronic exposure to hydrocortisone but did block the acute inhibition of NHE. On the contrary, preincubation of the cells for 20 min with the PKA inhibitor (RP)-cAMP (200 µM) inhibited the chronic stimulation of NHE but had no effect on the acute inhibition of NHE.

We conclude that chronic activation of NHE by hydrocortisone occurs via a PKA-dependent signalling pathway whereas acute inhibition of NHE is via PKC.

This work was funded by the Health Research Board of Ireland