The effect of sodium/hydrogen exchanger inhibition on chemotherapy-induced intestinal epithelial damage
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The small intestinal epithelium is one of the most rapidly dividing tissues in the mammalian body, making it an inadvertent target of non-specific chemotherapy. Proliferation rates of intestinal stem cells (ISCs) are largely dependent on the Wnt/ß-catenin pathway, which is controlled in part by intracellular pH (pHi) with acidic pHi reducing proliferation rates. An important regulator of pHi in the intestinal epithelium is the sodium/hydrogen exchanger (NHE), which removes cytosolic hydrogen in exchange for extracellular sodium. Commercially available inhibitors of NHE, including the amiloride analog EIPA, have been shown to decrease pHi as well as proliferation rates in certain cell types. Since many chemotherapy drugs indiscriminately target rapidly replicating cells, decreasing ISC proliferation rate by inhibiting NHE function may prevent chemotherapy induced ISC damage. We hypothesize that enteroids treated with EIPA before treatment with doxorubicin (dox) will acidify ISCs, decreasing rates of proliferation, and thereby decreasing percentages of apoptotic cells compared with the enteroids that were not pre-treated. The number of viable enteroids and average number of bud per enteroid (a representation of intestinal stem cell number) was determined three days post-treatment using light microscopy. In 16 addition, markers of apoptosis (?-H2Ax and cleaved caspase 3) were quantified using western blot. Pre-treating cells with EIPA showed no benefit to enteroid survival or bud number post-dox treatment. The quantity of ?-H2Ax, a marker of double stranded DNA breakage, showed no change between EIPA pre-treated cells and groups not pre-treated with EIPA. Cleaved caspase-3 was significantly elevated in EIPA pre-treated cells compared to groups not pre-treated with EIPA. This study did not determine the mechanism by which EIPA pre-treatment increased measurements of apoptosis although it is possible that since NHE inhibition is a component of some apoptotic pathways, that pre-inhibiting it increased the proportion of cells entering apoptosis. These data show that there is no cell-survival advantage to acidifying the intestinal epithelium prior to treatment with chemotherapeutic agents. In fact, inhibiting NHE prior to chemotherapy challenge may increase the risk of mucositis in patients undergoing chemotherapy treatment.