Increased expression of WNK3 during the perinatal period in newborn rats with hypoxic-ischemic encephalopathy.

Abstract

Objectives: Neonatal hypoxic-ischemic encephalopathy (HIE) is brain damage caused by reduced blood/oxygen supply during the perinatal period. There is no adequate treatment currently. The kinase WNK3 is associated with cerebral edema and stroke prognosis, so we assessed its expression in a neonatal rat model of HIE.

Methods: The Rice method was used to induce HIE in 7-day-old rat pups by ligating the left carotid artery followed by hypoxia exposure. Rats were divided into sham, 6 h, 12 h, 24 h, and 48 h groups (n = 5 each). Neurological function was evaluated by negative geotaxis, righting reflex, and Morris water maze tests. WNK3 expression was measured by Western blotting, RT-PCR, immunohistochemistry, and immunofluorescence in brain samples.

Results: HIE rats showed significant neurological impairments in short and long-term tests compared to shams. Negative geotaxis and righting reflex times were prolonged in HIE rats (all p < 0.01), and Morris water maze performance was impaired at 4 weeks (p < 0.05). Western blotting revealed an approximate three-fold increase in cortical WNK3 protein expression by 48 h post-HIE (p < 0.001), while RT-PCR showed reduced WNK3 mRNA expression with a nadir at 6 h, a partial rebound at 24 h, and a decline again at 48 h. Histological staining confirmed increased proportions of WNK3-positive cells in peri-infarct cortex after HIE (p < 0.001).

Conclusion: Our study demonstrated a dissociation between WNK3 protein (upregulated ~3-fold) and mRNA (downregulated except for a transient 24 h rebound) in neonatal HIE, suggesting post-transcriptional regulation. The WNK3 upregulation may contribute to cerebral edema formation and neurological deficits. These findings are correlative; larger, sex-balanced studies incorporating WNK3 inhibition, direct brain water measurements, and integration with hypothermia therapy are warranted to test WNK3 as a therapeutic target in neonatal HIE.