The goal of the proposed study is to identify renal microRNAs and their target genes that could potentially contribute to the development of salt-induced hypertension and renal injury in the Dahl salt-sensitive (SS) rat. The SS rat is a widely used animal model for human salt-sensitive forms of hypertension and renal injury, the molecular mechanisms of which are poorly understood. MicroRNAs are a class of small regulatory RNA, the discovery of which has been hailed as one of the most important breakthroughs in biology in recent years. MicroRNAs are encoded by specific genes in plant and animal genomes, and have been predicted to regulate the protein expression of thousands of mammalian genes, primarily through translational repression. Intensive research in the last two years has assigned important and diverse functions, ranging from the regulation of cell differentiation to insulin secretion, to several dozen mammalian microRNAs. The relevance of microRNA to complex mammalian physiology such as blood pressure regulation is unknown. We hypothesize that specific microRNAs, through the suppression of their target genes, are involved in the development of Dahl salt- sensitive hypertension and renal injury. The current proposal outlines the exploratory/developmental phase of the study that will achieve two goals: discovering renal microRNAs potentially relevant to the SS model, and identifying their target genes.
In Aim 1, a custom-made microRNA microarray platform combined with a modified real-time PCR assay will be used to examine renal microRNA expression profiles in the SS rat. SS- 13BN, a consomic strain of rat with substantially attenuated hypertension and renal injury, will be used as the control.
In Aim 2, target genes for differentially expressed microRNAs found in Aim 1 will be identified according to four criteria including 1) the presence of certain sequence and thermodynamic characteristics, 2) an inverse correlation between the levels of the microRNA and the target protein in SS and SS-13BN rats, 3) differences in the level of the target protein unexplainable by mRNA, and 4) up- and down-regulation of the target protein occurring when the microRNA is inhibited and overexpressed, respectively, in cultured rat cells. Preliminary studies indicate that 1) microRNA expression can be reliably analyzed using custom-made microarrays;2) differential expression of microRNA exists between SS and SS-13BN rats;3) many proteins differentially expressed between SS and SS-13BN are not explained by mRNA levels and are predicted targets for microRNAs;and 4) several genomic regions associated with SS phenotypes contain microRNA genes. The results of the proposed, exploratory study will become the foundation for future studies determining the in vivo pathophysiological significance of the identified microRNAs. Project Narrative Relevance Identification of specific microRNAs that are functionally important in the SS model will open new directions of research exploring the significance of non-protein-coding genes in complex mammalian physiology, and improve our understanding of the pathophysiology of hypertension and renal injury.
