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

Abstract

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.

Keywords: AQP9; human placenta; lactic acid transport; mitochondria; preeclampsia.

Figure 1

Effect of the availability of glucose and lactate on the expression of Aquaporin-9 (AQP9) and the viability of explants. Explants were cultured in the different conditions: Control (25 mM glucose medium), Low Glucose medium (Low Glu, 5 mM glucose), Low Glucose medium with D-Lactic acid, and Low Glucose medium with L-Lactic acid. (A) Expression of AQP9. Representative Western blot for AQP9 protein expression in different culture conditions. Densitometry of immunoblot, the values were plotted as the relative abundance of AQP9 expression. (n = 6 placentas; NS = Non-significant). (B) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) incorporation. The viability was evaluated by MTT assay in placental explants cultured in the different conditions. CHC was used to block MTCs. The effect of 0.5 mM Phloretin or 0.3 mM HgCl₂ to block AQP9 was evaluated in all the treatments. All the experiments were independently conducted in triplicate. Data are expressed as means ± SEM. (n = 6 placentas; ^*p < 0.05 and ^**p < 0.001 compared to control without blockers, NS = Non-significant; and ^#p < 0.05 compared to low glucose medium with L-lactic acid without blockers). (C) Lactate dehydrogenase (LDH) release. LDH release assay was performed for determining the rate of cell death by necrosis in placental explants cultured in different conditions. CHC was used to block MTCs. The effect of 0.5 mM Phloretin or 0.3 mM HgCl₂ to block AQP9 was evaluated in all the treatments. The experiments were independently conducted in triplicate at least three times. Data are expressed as means ± SEM. (n = 6 placentas; NS = Non-significant). All the experiments were independently conducted in triplicates at least three times.

Figure 2

Bax and Bcl-2 expression. (A) Representative Western Blot images and (B) densitometry for Bax/Bcl-2 ratio in placental explants cultured in the different conditions: Control (25 mM glucose medium), Low Glucose medium (Low Glu, 5 mM glucose), Low Glucose medium with D-Lactic acid, and Low Glucose medium with L-Lactic acid. The effect of 0.5 mM Phloretin or 0.3 mM HgCl₂ to block AQP9 was evaluated in all the treatments. Data are expressed as means ± SEM. n = 6 placentas. (^*p < 0.05, ^***p < 0.001 compared to control without AQP9 blockers, NS = Non-significant; and ^##p < 0.01 compared to low glucose medium with L-lactic acid without AQP9 blockers).

Figure 3

TUNEL assay. (A) Representative images and (B) % of apoptotic nuclei for each treatment were shown. TUNEL positive cells (red); stroma nuclei stained with DAPI (blue). Image magnification: × 1,000. Data are expressed as means ± SEM. (n = 6 placentas; ^***p < 0.001 compared to control without AQP9 blockers, NS = Non-significant; and ^###p < 0.001 compared to low glucose medium with L-lactic acid without blockers).

Figure 4

Expression of AQP9 in trophoblast mitochondria. (A) Representative transmission electron microscope images of heavy/large and light/small mitochondria found in the isolated subpopulations. Individual mitochondria were falsely colored to aid in identification. The scale bar represents 100 nm and the magnification is 50,000 × (n = 4 placentas). (B) AQP9 expression in microsomal and mitochondrial fractions. Representative immunoblot revealed that AQP9 is expressed in both mitochondrial and microsomal fractions isolated from villous trophoblast cells. (C) AQP9 expression in heavy/large mitochondria subpopulation. Representative immunoblot showed the expression of AQP9 only in the heavy/large fraction (n = 12 placentas).

Figure 5

Schematic representation of lactate use by trophoblast cells. In normal pregnancies, trophoblast cells use glucose as a fuel to support the placenta’s cellular growth (A). The excess of lactate due to fetal metabolism may be driven into the maternal circulation by monocarboxylate transports system (MCTs) and AQP9. When the availability of glucose is reduced (B), trophoblast cells can use lactate as a carbon source substitute (dot lines). The uptake of lactate into the cytosol may be facilitated by AQP9 and MCTs localized in the plasma membrane. In the cytosol, lactate can be metabolized into pyruvate or it can pass across the external and inner mitochondria membranes by MCTs or AQP9. In the mitochondria matrix, lactate may be oxidated to pyruvate by a mitochondrial LDH while NAD⁺ is reduced to NADH. The produced NADH may act as a reactive oxygen species scavenger. In preeclamptic placentas, AQP9 is not functional altering the use of lactate by the trophoblast cells. This may affect the mitochondria function, leading to the activation of the mitochondrial pathway of apoptosis. OMM, Outer Mitochondria membrane; IMM, Inner Mitochondria membrane; ECT, Electron transport chain; TCA cycle, tricarboxylic acid cycle; and MPC 1/2, Mitochondrial pyruvate carrier 1 and 2.