Cisplatin Decreases ENaC Activity Contributing to Renal Salt Wasting Syndrome

Abstract

Cisplatin (CDDP) is an important anticancer drug. A common side effect of CDDP is renal salt and water-wasting syndrome (RSWS). The origin of RSWS is obscure. Emerging evidence, though, suggests that broad inhibition of sodium transport proteins by CDDP may result in decreases in tubular reabsorption, causing increases in sodium and water excretion. In this sense, CDDP would be acting like a diuretic. The effect of CDDP on the epithelial Na⁺ channel (ENaC), which is the final arbiter fine-tuning renal Na⁺ excretion, is unknown. We test here whether CDDP affects ENaC to promote renal salt and water excretion. The effects of CDDP and benzamil (BZM), a blocker of ENaC, on excretion of a sodium load were quantified. Similar to BZM, CDDP facilitated renal Na⁺ excretion. To directly quantify the effects on ENaC, principal cells in split-open tubules were patch clamped. CDDP, at doses comparable to those used for chemotherapy (1.5 µM), significantly decreased ENaC activity in native tubules. To further elaborate on this mechanism, the dose-dependent effects of CDDP on mouse ENaC (mENaC) heterologously expressed in Chinese Hamster Ovary (CHO) cells were tested using patch clamping. As in native tubules, CDDP significantly decreased the activity of mENaC expressed in CHO cells. Dose-response curves and competition with amiloride identified CDDP as a weak inhibitor of ENaC (apparent IC₅₀ = 1 µM) that competes with amiloride for inhibition of the channel, weakening the inhibitory actions of the latter. Such observations are consistent with CDDP being a partial modulator of ENaC, which possibly has a binding site that overlaps with that of amiloride. These findings are consistent with inhibition of ENaC by CDDP contributing to the RSWS caused by this important chemotherapy drug.

Keywords: Deg/ENaC channels; chemotherapy; diuretic; hypertension; pseduohypoaldosteronism; renal sodium excretion.

Figure 1

Cisplatin (CDDP) is natriuretic. Summary graph showing cumulative Na⁺ excretion in mice that received an Na⁺ load and treatment with cisplatin (CDDP, the black circles, where n = 5 independent trials with three mice per trial), benzamil (BZM, the white squares, where n = 5 independent trials with three mice per trial) or the vehicle (VEH, the white circles, where n = 5 independent trials with three mice per trial) over a 4 h period. Treatment with BZM significantly increased Na⁺ excretion at both the 2 h and 4 h time points. Treatment with CDDP significantly increased excretion at the 4 h time point, where BZM and CDDP were not different. * p < 0.05 vs. VEH group.

Figure 2

CDDP decreased the activity of the epithelial Na⁺ channel (ENaC) in native murine Principal cells. (A) Representative current traces of ENaC in cell-attached patches made on the apical plasma membrane of Principal cells in freshly isolated tubules from naïve mice before (top) and after (bottom) treatment with 1.5 µM CDDP. Negative pipette potential −60 mV and inward Na⁺ current downwards. (B) Summary graph of (n = 7) paired experiments showing ENaC activity (NP_o) before and after treatment with CDDP. Summary data from experiments were identical to that shown in (A) * p < 0.05 vs. Before.

Figure 3

CDDP is a partial inhibitor of ENaC. Summary graph of the dose-dependent inhibitory effects of amiloride (black circles) and cisplatin (white boxes) on mENaC expressed in Chinese Hamster Ovary (CHO) cells (n = 3–5). CDDP significantly reduced ENaC activity with a reduced Emax compared with amiloride.

Figure 4

CDDP competes with amiloride, lessening the inhibitory effects of amiloride on ENaC. (A) Overlay of typical macroscopic current traces from a representative CHO cell expressing mENaC before and after treatment, with (0.2–10 µM) amiloride in the presence (dashed lines) and absence (full lines) of 10 µM of CDDP. Currents elicited by voltage ramps were stepped from a holding potential of 40 mV to 60 mV and ramped to −100 mV. (B) Summary graph of (n = 3–5 for each amiloride concentration) experiments showing concentration-dependent effects of amiloride on mENaC in the absence (black circles, full line) and presence (white boxes, dashed line) of 10 µM of CDDP. Exposure to CDDP reduced the maximal inhibition percentage of amiloride and caused a dextral displacement of the amiloride inhibition curve.