Effects of the EGFR Inhibitor Erlotinib on Magnesium Handling

Abstract

A mutation in pro-EGF causes isolated hypomagnesemia, and monoclonal antibodies targeting the extracellular domain of the EGF receptor (EGFR) affect epithelial Mg(2+) transport. The effect of the EGFR tyrosine kinase inhibitor erlotinib on Mg(2+) homeostasis, however, remains unknown. Here, we injected C57BL/6 mice with erlotinib for 23 days and observed a small but significant decrease in serum Mg(2+) concentrations at days 16 and 23, but the fractional excretion of Mg(2+) remained unchanged after 23 days. Semiquantitative immunohistochemical evaluation did not reveal detectable changes in renal expression of transient receptor potential melastatin 6 (TRPM6) protein, the channel that mediates Mg(2+) reabsorption. Patch clamp analysis in TRPM6-expressing cells demonstrated that 30 muM erlotinib inhibited EGF-induced changes in TRPM6 current density and tyrosine phosphorylation of EGFR; 0.3 muM erlotinib did not have these effects. Furthermore, 30 muM erlotinib inhibited EGF-stimulated increases in the mobile fraction of endomembrane TRPM6 channels. In summary, erlotinib can influence Mg(2+) handling but its effect on the systemic Mg(2+) concentration seems less potent than that observed with antibody-based EGFR inhibitors. These data suggest that typical human dosages of erlotinib are unlikely to severely affect serum Mg(2+) concentrations.

Figure 1.

Effect of erlotinib on serum Mg²⁺ and Ca²⁺ concentrations. (A, B) Changes in serum Mg²⁺ and Ca²⁺ concentrations after 16 days, in mice receiving daily injections with erlotinib or vehicle. (C, D) Effect of erlotinib or vehicle on serum Mg²⁺ and Ca²⁺ concentrations after 23 days. Values are presented as means ± SEM (n = 9). *P < 0.05 is considered statistically significant.

Figure 2.

Functional data after 23 days of erlotinib injections. (A, B) Daily urinary excretion of Mg²⁺ and Ca²⁺ in mice injected with erlotinib for 23 days. (C) Creatinine clearance (estimated GFR) in erlotinib- and vehicle-injected mice and also the (D, E) corresponding fractional excretions of Mg²⁺ (FEMg) and Ca²⁺ (FECa). Values are presented as means ± SEM (n = 9). *P < 0.05 is considered statistically significant. FECa, corresponding fractional excretion of Ca²⁺; FEMg, corresponding fractional excretion of Mg²⁺.

Figure 3.

Effect of erlotinib on mRNA and protein abundance of TRPM6. (A) Semiquantitative real-time PCR was used to determine the abundance of TRPM6 mRNA extracted from kidney. (B) Histogram depicting TRPM6 protein abundance determined by computerized analysis of immunohistochemical images. Representative immunohistochemical pictures of TRPM6 in vehicle- and erlotinib-injected mice. (C) Semiquantitative real-time PCR determination of TRPM6 mRNA expression in the colon. Data are presented as means ± SEM. *P < 0.05 is considered statistically significant.

Figure 4.

Erlotinib modulates EGFR mRNA expression. (A, B) Semiquantitative determination of the mRNA abundance of EGFR and EGF in the kidney of vehicle- and erlotinib-injected mice. (C, D) Measurements of urinary EGF excretion and also the urinary EGF/creatinine ratio. (E, F) Colonic mRNA abundance of EGFR and EGF. Data are presented as means ± SEM. *P < 0.05 is considered statistically significant.

Figure 5.

EGFR blockade by erlotinib can prevent EGF-induced changes in TRPM6 current density. (A) Time course of the current development (pA/pF) at +80 mV of TRPM6-transfected (■) HEK293 cells, pretreated with EGF (●) and erlotinib 30 μM (▲) or 0.3 μM (▼). (B) Current recorded after 200-second stimulation by a voltage ramp between −100 and +100 mV of TRPM6-transfected HEK293 cells (1), pretreated with EGF (2) or erlotinib (3) alone, or pretreated with EGF and erlotinib 30 μM (4)/0.3 μM (5). (C) Histogram summarizing the current density (pA/pF) at +80 of TRPM6-transfected HEK293 cells pretreated with EGF and/or erlotinib as indicated. # indicates P < 0.01 compared with TRPM6 current (n = 12 to 26 cells). * indicates P < 0.05 compared with TRPM6 pretreated with EGF (n = 12 to 26 cells). (D) The immunoprecipitated EGFR was placed on Western blots for the detection of pTyr and the EGFR itself. In addition, α-tubulin was detected in whole-cell lysates as a control for total expression. pTyr, tyrosine phosphorylation.

Figure 6.

Erlotinib inhibits EGF-stimulated changes in the mobile fraction of TRPM6. (A) Fluorescence recovery kinetics as a function of time, measured in HEK293 cells transiently transfected with GFP-TRPM6. Cells were preincubated with erlotinib (30 μM, 30 minutes) alone (▲) or before EGF application (10 nM, 30 to 60 minutes, △), and compared with control (■) or EGF-treated cells (□). (B) Histogram representing the maximal recovery of fluorescence (estimated mobile fraction) in HEK293 cells expressing GFP-TRPM6 with or without application of erlotinib, EGF, or both. Data are presented as means ± SEM (n = 9). *P < 0.05 is considered statistically significant from control. †P < 0.05 statistically significant from EGF-treated.