Increased astrocytic marker expression in the brainstem in rats with a mutation in the inwardly rectifying potassium channel 5.1 (Kir5.1)
Authors: Alvarez‐Argote Santiago , Hodges Mathew R. 
Published: June 2021

Inwardly rectifying potassium (Kir) channels modulate membrane potentials and contribute to K+ recycling in the kidney and brain. We recently showed in rats with mutations in Kcnj16 (SSKcnj16−/− rats), the gene that encodes Kir5.1 channels, lead to dysfunction in renal salt handling, prevents salt‐sensitive hypertension, but also leads to sudden unexpected death during salt‐loading (Palygin et al., JCI Insight 2017). In addition, SSKcnj16−/− rats show renal and ventilatory pH regulation dysfunction, and have audiogenic seizures. Given that Kir4.1 and Kir5.1 channels are thought to be co‐expressed in CNS astrocytes and drive K+ recycling in the CNS, we sought to test the hypothesis that SSKcnj16−/− rats will have an astrocytic expansion within brainstem nuclei that contribute to ventilatory control mechanisms. To test this hypothesis, we used an immunohistochemical approach to assess neuronal cell counts (NeuN) and the density of expression of an astrocytic marker (GFAP) within multiple nuclei in the brainstem of adult (>8 weeks of age) naïve control SS rats (n=2), in addition to SSKcnj16−/− rats with and without (naïve; n=3) audiogenic seizure stimulation once/day for 3 consecutive days. Rats were anesthetized, transcardially perfused and fixed (4% PFA), cryoprotected in sucrose before frozen sectioning (20μm thickness) and placement on coated slides. Slides were incubated sequentially with anti‐GFAP and anti‐NeuN antibodies which were detected with 594 anti‐rabbit and 488 anti‐mouse as secondary antibodies, respectively, and images obtained using standard epifluorescence microscopy and analyzed using image J within which an algorithm was developed to count NeuN positive neurons and to determine the percent of background positive for GFAP. The nuclei analyzed were: Raphe Pallidus (RPa), Obscurus (Rob) and Magnus (Rm), hypoglossal motor nuclei (XII), dorsal motor nucleus of the vagus (DMV), nucleus tractus solitarus (NTS), nucleus ambiguous (AMB), the ventral respiratory column (VRC) and cochlear (Co) nuclei. In control SS rats, we noted that the relative percentage of each area stained positive for GFAP was 17.2% (RPa), 7.8 (Rob), 6.4 (Rm), 5.0 (XII), 5.6 (DMV), 6.0 (NTS), 4.6 (AMB), 4.8 (VRC) and 15.6 (Co) compared to the naïve SSKcnj16−/− rats 30.1, 14.1, 16, 17, 11.5, 13.8, 13.1, 15 and 24 respectively. An unpaired t‐test analysis was performed, and statistically significant differences were found for the AMB nucleus (p=0.035). Ongoing experiments will determine if additional animals in each group will resolve other potential differences in neurons and/or astrocytes among naïve control and SSKcnj16−/− rats, or if there are additional alterations in brainstem neurons/glia after multiple audiogenic seizures, which may induce reactive gliosis. We conclude that mutations in Kir5.1 channels may alter astrocytes but not neuron counts within respiratory nuclei.Support or Funding InformationSupported by NIH HL122358This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.