293 - Neonatal Sustained Furosemide Exposure Alters KCC2/NKCC1 Expression and Causes Behavioral Abnormalities during Immature Brain Development

Publication Number: 4288.293

Yue Xu, Nagoya-University Graduate School of Medicine, Nagoya, Aichi, Japan; Atsuto Onoda, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi, Japan; Takahiro Kanzawa, Division of Neonatology, Center for Maternal-Neonatal Care, Nagoya University Hospital, Nagoya, Aichi, Japan; Ryoko Shimode, Division of Neonatology, Center for Maternal-Neonatal Care, Nagoya University Hospital, Gifu, Gifu, Japan; Yukina Takamoto, Division of Neonatology, Center for Maternal-Neonatal Care, Nagoya University Hospital, Nagoya, Aichi, Japan; Shintaro Ichimura, Nagoya University hospital, Nagoya, Aichi, Japan; Norika Mamiya, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; Shota Fujito, nagoya university hospital, nagoya, Aichi, Japan; Sakiko Suzuki, Nagoya University, Nagoya, Aichi, Japan; Zhiyue Deng, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; Kazuto Ueda, Nagoya University, Nagoya, Aichi, Japan; Ryosuke Miura, Nagoya University Hospital, Nagoya, Aichi, Japan; Toshihiko Suzuki, Nagoya University Hospital,, Nagoya, Aichi, Japan; Yukako Muramatsu, Nagoya university hospital, Nagoya, Aichi, Japan; Yoshiyuki Takahashi, Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; Yoshiaki Sato, Nagoya University Hospital, Nagoya, Aichi, Japan

Background: The potassium-chloride cotransporter (KCC2) and sodium-potassium-2-chloride cotransporter (NKCC1) regulate neuronal chloride homeostasis and brain maturation. In early postnatal life, a shift from NKCC1 to KCC2 dominance is crucial for proper neural circuit development. Furosemide, a loop diuretic and non-selective antagonist of NKCC1/KCC2, is widely used in neonatal intensive care units, sometimes long-term. Its effects on the immature brain and later neurobehavioral outcomes remain unclear.

Objective: To determine whether prolonged exposure to furosemide during the neonatal period induces behavioral and cognitive abnormalities, and to elucidate the underlying mechanisms related to the expression of cation-chloride cotransporters.

Design/Methods: Neonatal rats received daily intraperitoneal furosemide (2 or 20 mg/kg) or saline from postnatal day (P)1-14. Behavioral assessments included negative geotaxis (sensorimotor coordination) and shuttle avoidance (learning/memory) on P1-14 and P23-24, respectively. Hippocampal KCC2 expression was analyzed by immunohistochemistry on P7, P11, and P14. Furosemide (1 or 10 µg/mL) was applied to primary neurons from E17 embryos; NKCC1/KCC2 expression was evaluated using immunocytochemistry and qPCR.

Results: Behavioral analyses revealed that neonatal furosemide exposure (2-20 mg/kg, P1-P14) impaired both sensorimotor coordination and learning ability. In the negative geotaxis test, treated pups showed delayed righting responses, while the shuttle avoidance task demonstrated significantly reduced learning acquisition(Figure1). These findings indicate disrupted neurodevelopment affecting both reflexive and cognitive domains acquisition.
In both treatment groups, KCC2 expression in the hippocampal CA3 region was significantly reduced from P7 to P14(Figure2). In vitro, 1 µg/mL furosemide transiently decreased NKCC1/KCC2 at P7, with KCC2 recovery observed at P11. Conversely, 10 µg/mL persistently inhibited KCC2, reduced NKCC1 at an early stage, and exhibited dose-dependent and developmentally stage-dependent effects (Figure3). These molecular-level alterations correlated with behavioral and cognitive impairments, suggesting a disruption in the transition from NKCC1 to KCC2 during brain maturation.

Conclusion(s): Sustained neonatal furosemide exposure alters cation-chloride cotransporter expression, impairs neurodevelopment, and causes behavioral and cognitive abnormalities, underscoring the need for caution with prolonged neonatal use.

Behavioral effects of neonatal furosemide exposure
Negative geotaxis and shuttle avoidance tests show delayed sensory-motor development and learning progression in furosemide-treated rats.

Reduced hippocampal KCC2 expression during neonatal furosemide exposure
Representative immunofluorescence images of hippocampal CA3 neurons showing reduced KCC2 (green)
expression in furosemide-treated rats compared with controls at P7-14.

. Developmental changes in KCC2/NKCC1 expression during neonatal furosemide exposure
Developmental changes in KCC2/NKCC1 expression during neonatal furosemide exposure. Relative mRNA levels measured by qPCR in primary cortical neurons at DIV3, 7, and 11.