SUMMARY Dietary potassium deficiency, common in Western diets, raises blood pressure and enhances salt sensitivity. Potassium homeostasis requires a molecular switch in the distal convoluted tubule (DCT), which fails in familial hyperkalemic hypertension (pseudohypoaldosteronism type 2), activating the thiazide-sensitive NaCl cotransporter, NCC. Here, we show that dietary potassium deficiency activates NCC, even in the setting of high salt intake, thereby causing sodium retention and a rise in blood pressure. The effect is dependent on plasma potassium, which modulates DCT cell membrane voltage and, in turn, intracellular chloride. Low intracellular chloride stimulates WNK kinases to activate NCC, limiting potassium losses, even at the expense of increased blood pressure. These data show that DCT cells, like adrenal cells, sense potassium via membrane voltage. In the DCT, hyperpolarization activates NCC via WNK kinases, whereas in the adrenal gland, it inhibits aldosterone secretion. These effects work in concert to maintain potassium homeostasis.

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Excess aldosterone is an important contributor to hypertension and cardiovascular disease. Conversely, low circulating aldosterone causes salt wasting and hypotension. Aldosterone activates mineralocorticoid receptors (MRs) to increase epithelial sodium channel (ENaC) activity. However, aldosterone may also stimulate the thiazide-sensitive Na + -Cl 2 cotransporter (NCC). Here, we generated mice in which MRs could be deleted along the nephron to test this hypothesis. These kidney-specific MR-knockout mice exhibited salt wasting, low BP, and hyperkalemia. Notably, we found evidence of deficient apical orientation and cleavage of ENaC, despite the salt wasting. Although these mice also exhibited deficient NCC activity, NCC could be stimulated by restricting dietary potassium, which also returned BP to control levels. Together, these results indicate that MRs regulate ENaC directly, but modulation of NCC is mediated by secondary changes in plasma potassium concentration. Electrolyte balance and BP seem to be determined, therefore, by a delicate interplay between direct and indirect mineralocorticoid actions in the distal nephron.

Purpose: Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically.Experimental Design: AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized.Results: We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) signifi-cantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redoxtargeted combination therapies are effective in eradicating ALL cells in vitro and in vivo.Conclusions: Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional genotoxic chemotherapy and a diagnosis of AML is usually fatal.More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. Here, we show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating acute lymphoblastic leukemia cells in vitro and in vivo. Thus, targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia. Key Points• Glutaminase inhibition commonly impairs glutathione metabolism and induces mitochondrial oxidative stress in acute myeloid leukemia cells • A glutaminase inhibitor synergizes with pro-oxidant drugs in inducing apoptosis and eliminating leukemia cells in vitro and in vivo Homoharringtonine (HHT; omacetaxine mepesuccinate), arsenic trioxide (ATO), cell-permeable glutathione reduced ethyl ester (GSH-MEE) and dimethyl 2oxoglutarate (α-ketoglutarate) were purchased from Millipore Sigma. Metabolic tracing experimentsCells were seeded at 3 x 10 5 /ml (replicates of 3) and treated with vehicle (DMSO) or CB-839 at 500 nM for 8 h, followed by incubation in glutamine-free RPMI 1640 supplemented with 13 C 5 , 15 N 2 -labeled L-glutamine (Cambridge Isotope Laboratories) for up to 12 h, in the presence of vehicle or drug. Flash frozen cell pellets (~ 1 x 10 6 cells) or supernatants (50 µl) were extracted and subjected to analysis by ultra-high pressure liquid chromatography and mass spectrometry (UHPLC/MS) as was previously described 20 . Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of 13 C and 15 N isotopes were performed using MAVEN (Princeton University, Princeton, NJ) and manually validated. Cell viability assaysCells were seeded at 0.5-1.0 x10 5 /ml in triplicate wells of 48-well tissue culture plates. Where indicated, the cells were treated with drug for a period of 48-72 h.After treatment, a sample of cells from each well was stained with PI (10 µg/ml) and viable cells (PI -) were counted with a flow cytometer (Millipore Guava