Lactic Acid Permeability of Aquaporin-9 Enables Cytoplasmic Lactate Accumulation via an Ion Trap

(1) Background: Human aquaporin-9 (AQP9) conducts several small uncharged metabolites, such as glycerol, urea, and lactic acid. Certain brain tumors were shown to upregulate AQP9 expression, and the putative increase in lactic acid permeability was assigned to severity. (2) Methods: We expressed AQP9 and human monocarboxylate transporter 1 (MCT1) in yeast to determine the uptake rates and accumulation of radiolabeled l-lactate/l-lactic acid in different external pH conditions. (3) Results: The AQP9-mediated uptake of l-lactic acid was slow compared to MCT1 at neutral and slightly acidic pH, due to low concentrations of the neutral substrate species. At a pH corresponding to the pKa of l-lactic acid, uptake via AQP9 was faster than via MCT1. Substrate accumulation was fundamentally different between AQP9 and MCT1. With MCT1, an equilibrium was reached, at which the intracellular and extracellular l-lactate/H+ concentrations were balanced. Uptake via AQP9 was linear, theoretically yielding orders of magnitude of higher substrate accumulation than MCT1. (4) Conclusions: The selectivity of AQP9 for neutral l-lactic acid establishes an ion trap for l-lactate after dissociation. This may be physiologically relevant if the transmembrane proton gradient is steep, and AQP9 acts as the sole uptake path on at least one side of a polarized cell.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

G-CSF Upregulates the Expression of Aquaporin-9 through C/EBP-Beta to Enhance the Cytotoxic Activity of Arsenic Trioxide to Acute Myeloid Leukemia Cells

Arsenic trioxide (ATO) is the first-line drug for the treatment of acute promyelocytic leukemia (APL). Aquaporin-9 (AQP9), a transmembrane transporter, is required for leukemia cells to uptake ATO. APL cells express high levels of AQP9, while other types of acute myeloid leukemia (AML) cells express much lower levels of AQP9, which limits the transportation and the cytotoxic activity of ATO in those types of leukemia. Recently, we found that granulocyte colony stimulating factor (G-CSF) can significantly upregulate the expression of AQP9 in AML cell lines (THP-1 and HL-60), and can significantly enhance the intracellular concentrations of ATO, when compared with the treatment with ATO alone. We also found that the combination of ATO and G-CSF inhibited the cell proliferation and induced cell apoptosis more significantly than the treatment with ATO or G-CSF alone. The overexpression of AQP9 with lentivirus in HL-60 or THP-1 cells significantly enhanced the cytotoxic activity of ATO, while the knock-down of AQP9 gene with small interfering RNA (siRNA) significantly attenuated the combination effect of G-CSF and ATO. Moreover, we found that the upregulation of AQP9 by G-CSF depends on the upregulation of C/EBP-beta, the transcription factor downstream of G-CSF. In conclusion, our study suggests that the pretreatment of G-CSF could significantly enhance the cytotoxic activity of ATO in AML cells through the upregulation of AQP9, and that the combination of G-CSF and ATO would be a potential therapeutic strategy for AML patients. Disclosures No relevant conflicts of interest to declare.

Aquaporin 9 Represents a Novel Target of Chronic Liver Injury That May Antagonize Its Progression by Reducing Lipotoxicity

In recent years, chronic liver injury has become a common disease that harms human health. Its clinical manifestations are hepatic steatosis and secondary chronic steatohepatitis, which can quickly transform into liver fibrosis and cirrhosis if not treated in time. Therefore, this study is aimed at searching for new therapeutic targets of chronic liver injury and clarifying the molecular mechanisms of the new targets involved in chronic liver injury. After aquaporin 9 was identified as a target by proteomics, Aqp9-/- mice were constructed using the CRISPR/Cas9 system. Biochemical and morphological tests were used to verify the effect of Aqp9 knockout on early chronic liver injury. Proteomics, molecular biology, and morphology experiments were used to screen and verify the effects of Aqp9 knockout on its downstream pathway. Through the above experiments, we demonstrated that aquaporin 9 could be used as an intervention target for antagonizing the development of early chronic liver injury and its gene knockout affected downstream inflammation, oxidative stress, apoptosis, and pyroptosis by alleviating hepatic lipotoxicity.