Kcna3 Deficiency Promotes Renin-Associated Hypertension Through Ca2+-Dependent AKT–PKA–CREB Signaling

Figures

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  Figure 1. Kcna3 expression is not altered in hypertension. (A) Volcano plot revealed that the renal levels of Kcna3 were not significantly different between Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) (GSE41453). Dashed lines indicate the thresholds for statistical significance (p < 0.05, horizontal) and fold change (|log2 fold change| > 1, vertical). (B) Boxplot of Kcna3 expression levels in WKY rats and SHRs. (C) Western blotting analysis of Kv1.3 expression in the renal and cardiac tissues of wild-type (WT) and Kcna3 knockout (KO) mice. (D) Quantification of Kv1.3 protein levels in renal and cardiac tissues of WT and Kcna3 KO mice. (E) Quantitative real-time polymerase chain reaction analysis of Kcna3 mRNA levels in different tissues of WT and Kcna3 KO mice. (F) Representative immunofluorescence staining images of Kv1.3 in the kidneys of WT and Kcna3 KO mice. Scale bar = 100 μm. In all bar graphs, blue bars denote KO and red bars denote WT. Data are presented as mean ± standard error of the mean. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (unpaired Student’s t-test). Original Western blot images are provided in the Supplementary Materials.

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  Figure 2. Kcna3 deficiency promotes hypertension and renal injury. (A–C) Telemetric measurements of systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) in wild-type (WT) and Kcna3 knockout (KO) mice across a 3-week observation period. Data were assessed by two-way ANOVA (time × genotype), revealing a significant genotype effect for SBP, DBP, and MAP (p < 0.001 for all) without a significant time × genotype interaction, supporting a stable hypertensive phenotype across the observation period. (D) Carotid artery-based measurement of arterial blood pressure confirmed elevated blood pressure in Kcna3 KO mice. (E) The plasma levels of renin, angiotensin II, and aldosterone. (F) Representative images of renal sections subjected to hematoxylin and eosin (H&E) and Masson’s trichrome staining showing structural damage and increased collagen deposition. Scale bar = 100 μm. (G) Western blotting analysis of fibrosis-related proteins Mmp2 and Mmp9 in the kidney tissues. (H) Quantification of collagen-positive area. (I) Quantitative analysis of Mmp2 and Mmp9 levels. In all bar graphs, blue bars denote KO and red bars denote WT. Data are presented as mean ±…

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  Figure 3. Kcna3 deficiency activates renin–angiotensin–aldosterone system (RAAS)-related pathways and promotes renin expression. (A) Principal component analysis (PCA) of renal transcriptomic profiles of wild-type (WT) and Kcna3 knockout (KO) mice. (B) Volcano plot showing differentially expressed genes between WT and Kcna3 KO mice. (C) Heatmap of hierarchical clustering of differentially expressed genes. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes. (E,F) Gene set enrichment analysis (GSEA) showing the activation of cortisol synthesis and secretion and aldosterone synthesis and secretion pathways in Kcna3 KO mice. (G) Western blotting analysis of renin expression in the kidney tissues of WT and Kcna3 KO mice. (H) Western blotting analysis of renin expression after treatment with the Kv1.3 inhibitor 5-(4-phenoxybutoxy) psoralen (PAP-1). (I) Representative immunofluorescence staining images of renin (green) and Aqp2 (red) in the renal sections. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 100 μm. (J,K) Quantitative analysis of renin levels. (L) Quantification of renin fluorescence…

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  Figure 4. Kcna3 deficiency activates Ca2+-dependent PI3K-AKT-CREB signaling in the kidney. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes revealed the significant enrichment of PI3K-AKT, cAMP, and calcium signaling pathways. (B) Quantification of intracellular Ca2+ levels in wild-type (WT) and Kcna3 knockout (KO) mice. (C) Schematic diagram of the proposed mechanism (Proposed mechanism): Kcna3 deficiency impairs K+ efflux, leading to membrane depolarization and increased Ca2+ influx. (D) Western blotting analysis of Pik3ca, Akt1, p-Pik3ca, and p-Akt1 levels in the kidney tissues of WT and Kcna3 KO mice. (E) Quantification of Pik3ca, Akt1, p-Pik3ca, and p-Akt1 levels. (F) Western blotting analysis of Creb1 and p-Creb1 in the kidney tissues of WT and Kcna3 KO mice. (G) Quantification of Creb1 and p-Creb1 levels. (H) Representative immunofluorescence staining of p-Creb1 (green) in the kidney sections of WT and Kcna3 KO mice. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 100 μm. (I) Quantification of p-Creb1-positive area and fluorescence intensity. In all bar graphs, blue bars denote KO and…

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  Figure 5. Pharmacological Kv1.3 inhibition promotes renin expression via Ca2+-dependent and PKA-dependent activation of AKT-CREB signaling. (A) Western blotting analysis of Akt1, p-Akt1, Creb1, p-Creb1, and renin levels in cells treated with vehicle, 5-(4-phenoxybutoxy) psoralen (PAP-1), or PAP-1 + the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). (B) Quantification of expression levels shown in (A). (C) Western blotting analysis of Creb1, p-Creb1, and renin in cells treated with vehicle, PAP-1, or PAP-1 + the protein kinase A (PKA) inhibitor H89. (D) Immunofluorescence staining of p-Creb1 (green) in different groups. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar = 100 μm. (E) Quantification of expression levels shown in (C). (F) Quantification of renin expression and p-Creb1 fluorescence intensities. In the bar graphs, yellow represents Vehicle, green represents PAP-1, light purple represents PAP-1+BAPTA, and dark purple represents PAP-1+H89. Data are presented as mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. (one-way analysis of variance, followed by a…