Kcnj10 (Kir 4.1) Knockout in Dahl SS Rats Determines the Expression of Kcnj10 and Kcnj16 Proteins in Brain and Kidney

Abstract

Loss‐of‐function mutation in Kcnj10 (Kir4.1) results in a number of neurological and renal pathologies in humans commonly known as EAST/SeSAME syndrome. The neurological complications, including the ataxia, associated with genetic mutations in Kcnj10 seems to be the most debilitating features of the syndrome. On the cell membrane, Kir4.1 forms a potassium inwardly rectifying channel either as homomer or heteromer together with other Kir channels such as Kir5.1 (Kir4.1/Kir5.1). Our recently published (Palygin et al., JCI Insight, 2017) and novel data revealed that the knockout of Kcnj16 (Kir5.1) in Dahl salt‐sensitive (SS) rats (SSKcnj16−/−) did not recapitulate Kcnj10 neurological phenotype, but has similar renal pathologies including severe hypokalemia and salt wasting. The goal of this study was to define the expression, localization, and functional mechanisms of Kir4.1 and Kir5.1 in the brain and kidney. To address this, we have used previously described SSKcnj16−/− rats and newly generated Kcnj10 knockout (SSKcnj10−/−) on the same SS rat background. SSKcnj10−/− was created using CRISPR/Cas9 mutagenesis, resulting in a one base pair insertion in the Kcnj10 gene exon 2 and predicted frameshift in Kir4.1 protein translation after just 26 amino acids.Similar to previously reported Kcnj10 KO mice, SSKcnj10−/− rats exhibit early mortality. Supplementation of diet chow with high potassium (2% KCl) expanded survival rate for both SSKcnj10−/− rats (up to 3–4 weeks) and SSKcnj10+/− (up to 40 weeks). Despite the potassium dietary supplement, SSKcnj10−/− rats revealed severe hypokalemia (2.9±0.3 compared to 4.3±0.1 and 4.7±0.4 mM of K+ for SSKcnj10+/− and WT rats, correspondingly). Significant reduction in Ca2+ and Na+ blood concentrations were also observed (0.85±0.13; 1.4±0.02 vs 1.36±0.03 mM of Ca2+ and 125.5±7.3; 135.1±0.6 vs 133.5±0.5 mM of Na+ for SSKcnj10−/−, SSKcnj10+/− and WT, correspondingly). The Kcnj10 deletion resulted in changes in growth and development (16.3±0.8; 32.1±2.6 vs 40.5±4.5 g of body weight for SSKcnj10−/−, SSKcnj10+/− and WT rats, respectively). An immunohistochemical (IHC) analysis of the rat kidney tissues in combination with genetically modified animals allowed us to precisely detect expression and localization of Kir4.1 and Kir5.1 in the kidney and brain. Our results indicate strong Kir4.1 localization in astroglial cells with the complete absence in neurons and reverse neuronal localization for Kir5.1 in the brain stem region. Importantly, the knockout of Kir4.1 did not remove Kir5.1 neuronal staining. However, Kir4.1 and Kir5.1 in the kidney co‐localized in distal renal tubules and knockout of Kir4.1 resulted in the absence of both proteins.Therefore, our results indicate that Kir4.1 and Kir5.1 are co‐localized in the kidney cortex, but not in the nuclei of the brain stem. As it was described previously, Kir4.1/Kir5.1 is a predominant channel in the distal tubules, and its specific renal localization makes it potentially a highly valuable pharmacological target for the regulation of potassium homeostasis and blood pressure.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.