ERRgamma regulates cardiac, gastric, and renal potassium homeostasis

Abstract

Energy production by oxidative metabolism in kidney, stomach, and heart, is primarily expended in establishing ion gradients to drive renal electrolyte homeostasis, gastric acid secretion, and cardiac muscle contraction, respectively. In addition to orchestrating transcriptional control of oxidative metabolism, the orphan nuclear receptor, estrogen-related receptor gamma (ERRgamma), coordinates expression of genes central to ion homeostasis in oxidative tissues. Renal, gastric, and cardiac tissues subjected to genomic analysis of expression in perinatal ERRgamma null mice revealed a characteristic dysregulation of genes involved in transport processes, exemplified by the voltage-gated potassium channel, Kcne2. Consistently, ERRgamma null animals die during the first 72 h of life with elevated serum potassium, reductions in key gastric acid production markers, and cardiac arrhythmia with prolonged QT intervals. In addition, we find altered expression of several genes associated with hypertension in ERRgamma null mice. These findings suggest a potential role for genetic polymorphisms at the ERRgamma locus and ERRgamma modulators in the etiology and treatment of renal, gastric, and cardiac dysfunction.

Figure 1

Genomic analysis of ERRγ function. Stomach (S) and kidney (K) genome-wide analyses of perinatal gene expression were compared with our previous heart (H) perinatal expression analysis to uncover processes that are regulated by ERRγ across tissues. A, Expression analysis detected altered expression of 1788 genes in the heart, kidney, and stomach tissues of ERRγ null mice. B, Functionally, 7–29% of these genes are associated with the GO classifications of Transport (GO:0006810) and Localization (GO:0051179), in addition to the 8–15% associated with the previously reported role of ERRγ in metabolic processes (GO:0008152). C, At the intersection of heart, stomach, and kidney expression changes, 150 genes associated with metabolic, and ion-handing processes were detected, as well as hypertension. Heart is Salk strain; stomach and kidney are GlaxoSmithKline strain (Materials and Methods).

Figure 2

ERRγ loss results in prolonged QT interval and down-regulated potassium channels. A, Representative averaged ECG demonstrates fractionated QRS complex in ERRγ heterozygous (HET) and KO and prolonged QT intervals in ERRγ KO mice at E18.5 (29). B, Whole-mount X-gal staining reveals the ubiquitous expression of ERRγ throughout the heart at E18.5. C, P0 ERRγ null mice have normal cardiomyocyte organization by hematoxylin and eosin (H&E) histology. D, Loss of ERRγ results in the altered expression of potassium channels of the heart. E, HL-1 cardiomyocyte cell line stably expressing small interfering RNA against ERRγ (siERRγ) has down-regulated expression of potassium channels, as seen in vivo. Values ± sd; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student's t test. All figures from Salk mice. GFP, Green fluorescent protein.

Figure 3

Loss of ERRγ alters parietal cell markers. Panel A, Immunohistochemistry against β-galactosidase reveals parietal cells express ERRγ. Panel B, Whole-mount X-gal staining demonstrates ERRγ expression is limited to the secretory portion of the gastric fundus. Panel C, A striking reduction in the expression of the H⁺/K⁺ATPase is seen in ERRγ null mice. Panel D, Although DBA-lectin staining is reduced in ERRγ null mice, parietal cells are present. Panels E–G, Anti-ERRγ staining colocalizes with Atp4b expression. Panel H, Loss of ERRγ in stomach results in profound down-regulation of parietal cell markers by triplicate quantitative RT-PCR (n = 3 WT; 9 HET; 6 KO). Values ± sd; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student's t test. S, Salk mice; G, GlaxoSmithKline mice; HET, heterozygous.

Figure 4

ERRγ binds and directly regulates Kcne2 and Atp4a promoters. A,) The mouse Atp4a and Kcne2 promoters contain putative ERREs (Materials and Methods and supplemental Fig. 1). B–D, ERRγ binds the putative ERREs found in Atp4a and Kcne2 genes. Two picomoles of biotinylated gene-specific or a consensus ERRE were preimmobilized on a strepavidin-coated 96-well plate. Nuclear extracts containing V5-tagged ERRγ were added to the wells in the presence of increasing amounts of either a WT or a mutated ERRE as indicated. Unbound proteins were washed away, and the bound ERRγ was detected using an anti-V5 antibody. E and F, Luciferase induction was observed in CV-1 cell transfection of reporter constructs containing WT Atp4a and Kcne2 promoter fragments, by nonmutant variants, when ERRγ and PGC-1α are cotransfected. *, P < 0.05; **, P < 0.01, by two-tailed Student's t test.

Figure 5

Loss of ERRγ alters renal markers. A, Hematoxylin and eosin staining of kidney does not reveal significant differences in ERRγ null mice. B, Anti-β-galactosidase staining indicates ERRγ expression in the distal tubules. C, X-gal staining of frozen sections shows that ERRγ is expressed in the distal tubules and collecting ducts of the kidney. D, Whole-mount X-gal staining reveals the high expression of ERRγ throughout the cortex of the kidney. E, In kidney, loss of ERRγ produces a down-regulation of potassium channels and components of the KSS as detected by triplicate quantitative PCR (n = 3 WT; 9 HET; 6 KO). F, Serum sodium levels did not increase to statistically significant degree in ERRγ null mice at E18.5. G, Serum potassium levels were increased by more than 70% in ERRγ null animals at E18.5. Values ± sd; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student's t test. S, Salk mice; G, GlaxoSmithKline mice; H&E, hematoxylin and eosin; HET, heterozygous.