Endothelin‐1 increases Na+‐K+‐2Cl− cotransporter‐1 expression in cultured astrocytes and in traumatic brain injury model: An involvement of HIF1α activation

Na+‐K+‐2Cl− cotransporter‐1 (NKCC1) is present in brain cells, including astrocytes. The expression of astrocytic NKCC1 increases in the acute phase of traumatic brain injury (TBI), which induces brain edema. Endothelin‐1 (ET‐1) is a factor that induces brain edema and regulates the expression of several pathology‐related genes in astrocytes. In the present study, we investigated the effect of ET‐1 on NKCC1 expression in astrocytes. ET‐1 (100 nM)‐treated cultured astrocytes showed increased NKCC1 mRNA and protein levels. The effect of ET‐1 on NKCC1 expression in cultured astrocytes was reduced by BQ788 (1 μM), an ETB antagonist, but not by FR139317 (1 μM), an ETA antagonist. The involvement of ET‐1 in NKCC1 expression in TBI was examined using a fluid percussion injury (FPI) mouse model that replicates the pathology of TBI with high reproducibility. Administration of BQ788 (15 nmol/day) decreased FPI‐induced expressions of NKCC1 mRNA and protein, accompanied with a reduction of astrocytic activation. FPI‐induced brain edema was attenuated by BQ788 and NKCC1 inhibitors (azosemide and bumetanide). ET‐1‐treated cultured astrocytes showed increased mRNA and protein expression of hypoxia‐inducible factor‐1α (HIF1α). Immunohistochemical observations of mouse cerebrum after FPI showed co‐localization of HIF1α with GFAP‐positive astrocytes. Increased HIF1α expression in the TBI model was reversed by BQ788. FM19G11 (an HIF inhibitor, 1 μM) and HIF1α siRNA suppressed ET‐induced increase in NKCC1 expression in cultured astrocytes. These results indicate that ET‐1 increases NKCC1 expression in astrocytes through the activation of HIF1α. Main Points Endothelin‐1 (ET‐1) increases NKCC1 expression in cultured astrocytes and a TBI model via ETB receptor. Increased expression of astrocytic NKCC1 by ET‐1 is mediated by HIF1α. The ET‐induced NKCC1 expression may be a mechanism of brain edema.