INSIGHTS INTO SALT-SENSITIVE HYPERTENSION USING RESPONSE-RELEVANT CLUSTERING: PP.21.317

Objective: The stroke prone spontaneously hypertensive rat (SHRSP) is an excellent model of human essential hypertension and exhibits salt sensitivity. The aims of this study were to utilise a combination of microarray profiling, construction of congenic strains and novel statistical methods to identify genes and pathophysiological pathways involved in salt-sensitive hypertension. Design and Methods: Renal microarray gene expression profiling was undertaken in 21 week old SHRSP, WKY and SP.WKYGla2a (2a) congenic strains to identify differentially expressed genes in non-salt (n = 3 males/strain) and salt loaded animals (n = 3 males/strain) using the Affymetrix Rat230–2 Gene Chips. The meta-covariate analysis procedure combines model based clustering and binary classification. It exploits the underlying covariance structure in microarray data by identifying meta-covariates that are lower dimensional representations of probe clusters. These clusters are based on similar patterns of gene expression and the ability of the gene cluster to predict the response (e.g., disease vs control). Therefore clusters were obtained of co-varying, response-relevant genes which aided in the biological interpretation of the gene expression data. qRT-PCR was used to validate the microarray data and clusters explored using Ingenuity Pathway Analysis (IPA) software. Results: The most significant cluster in all comparisons was cluster 13 identified in the Salt-No Salt comparison with a regression coefficient (w) of -46.76. Functional and Canonical pathway analysis of cluster 13 in IPA implicated transcriptional activation and circadian rhythm signalling, respectively. Salt-NoSalt renal expression profiles validated by qRT-PCR from each of the strain comparisons identified significant genes in the 2a and WKY strains that were not changing in the SHRSP including 4 transcription factors (TF) (Arntl, Bhlhe41, Nfil3 and NPAS2). Potential upstream regulatory factors of this cluster 13, mapping to the congenic interval on chromosome 2, were identified including Arnt, mir9–1 and mir9–2. Conclusion: Meta-covariate analysis identifies genes involved in transcriptional activation and circadian rhythm which may contribute to the enhanced salt sensitivity in the SHRSP compared to the 2a and WKY strains.