Gene expression profiles linked to AT1 angiotensin receptors in the kidney

Abstract

To characterize gene expression networks linked to AT(1) angiotensin receptors in the kidney, we carried out genome-wide transcriptional analysis of RNA from kidneys of wild-type (WT) and AT(1A) receptor-deficient mice (KOs) at baseline and after 2 days of angiotensin II infusion (1,000 ng·kg(-1)·min(-1)). At baseline, 405 genes were differentially expressed (>1.5×) between WT and KO kidneys. Of these, >80% were upregulated in the KO group including genes involved in inflammation, oxidative stress, and cell proliferation. After 2 days of angiotensin II infusion in WT mice, expression of ≈805 genes was altered (18% upregulated, 82% repressed). Genes in metabolism and ion transport pathways were upregulated while there was attenuated expression of genes protective against oxidative stress including glutathione synthetase and mitochondrial superoxide dismutase 2. Angiotensin II infusion had little effect on blood pressure in KOs. Nonetheless, expression of >250 genes was altered in kidneys from KO mice during angiotensin II infusion; 14% were upregulated, while 86% were repressed including genes involved in immune responses, angiogenesis, and glutathione metabolism. Between WT and KO kidneys during angiotensin II infusion, 728 genes were differentially expressed; 10% were increased and 90% were decreased in the WT group. Differentially regulated pathways included those involved in ion transport, immune responses, metabolism, apoptosis, cell proliferation, and oxidative stress. This genome-wide assessment should facilitate identification of critical distal pathways linked to blood pressure regulation.

Fig. 1.

Hierarchical clustering of differentially expressed genes between kidneys from AT_1A receptor-deficient [knockout (KO)] and wild-type (WT) control mice. More than 80% of genes were upregulated in the kidneys of KO compared with WT mice including genes involved in inflammation, oxidative stress, and cell proliferation. Yellow depicts no change, whereas red represents upregulation, and green represents downregulation.

Fig. 2.

Network 1 created by Ingenuity Pathway Analysis from differentially expressed genes between kidneys from AT_1A receptor-deficient and WT control mice. Blue indicates downregulation and red indicates upregulation. Square, cytokine; vertical oval, transmembrane receptor; rectangle, nuclear receptor; diamond, enzyme; rhomboid, transporter; hexagon, translation factor; horizontal oval, transcription factor; circle, others. A line indicates gene products that bind one another. A line with arrow indicates that a gene products is acting on another.

Fig. 3.

Expression of serum/glucocorticoid-regulated kinase (Sgk1) in kidneys of KO and WT mice at baseline and after angiotensin (Ang) II infusion. A: Sgk1 mRNA levels (relative to WT control mice as 100%). Data were normalized to 18S RNA from the same samples (n = 3 for each group). Open column, control mice; gray column, Ang II infusion. B: comparison of the levels of Sgk1 protein using Western immunoblotting analysis. The values above the bands are the means of relative changes in WT and KO mice after Ang II infusion compared with WT control mice. ***P < 0.01 compared with WT control mice; ***P < 0.001 compared with WT control mice; ††P < 0.01 compared with WT mice infused with Ang II.

Fig. 4.

Cluster analysis of genes that showed interaction of genotype and Ang II treatment. The mean appears yellow, whereas red represents upregulation, and green represents downregulation.

Fig. 5.

Differential gene expression confirmed by qRT-PCR. Data were normalized to 18S RNA from the same samples (n = 3 for each group). Open column, control mice; gray column, Ang II infusion. Mean of expression levels in WT control mice were assigned a value of unity. *P < 0.05, **P < 0.01 compared with WT control mice; †P < 0.05, ††P < 0.01 compared with WT mice infused with Ang II. Agt, angiotensinogen; Cyp4a14, cytochrome P450, family 4, subfamily a, polypeptide 14; Scnn1a, sodium channel, nonvoltage-gated 1α (ENaC-α); Slc13a1, solute carrier family 13 (sodium/sulfate symporters), member 1; Camk2b, calcium/calmodulin-dependent protein kinase IIβ; Gpx6, glutathione peroxidase 6 (olfactory).