Aldosterone-dependent miRNA regulation of endothelin-1

The kidney is the organ principally responsible for maintaining acid-base homeostasis and ion balance in the blood stream. Aldosterone is the major known hormone involved in regulation of sodium retention in the kidney collecting duct. The hormone has non-genomic effects in renal cells, but its primary mode of action is regulation of transcription of many genes. Some of those genes encode proteins that function in sodium retention. The endothelin-1 (Edn1) gene is among those induced by aldosterone in the renal collecting duct. Endothelin favors sodium secretion in the urine, so aldosterone and endothelin-1 participate in a feedback loop that adjusts sodium levels in the body. MicroRNAs (miRNAs) are a family of small noncoding RNAs that have been shown to regulate gene expression by inhibiting translation or targeting specific mRNAs for degradation. miRNAs play important roles in the regulation of metabolism in the healthy cell and in the pathogenesis of many diseases including diabetic nephropathy and hypertension-related renal disease. My working hypothesis is that aldosterone action alters the miRNA content in the inner medullary collecting duct resulting in increased translation and stabilization of mRNAs involved in sodium reabsorption. The primary focus is on miRNA action on Edn1 mRNA. In this dissertation, a reporter gene strategy was employed to conduct a systematic functional analysis of the Edn1 mRNA 3' untranslated region. The work confirmed that the mRNA contains many elements important for its stability and translation scattered along the length of the 3' untranslated region. The miRNA landscape in cells derived from the murine renal inner medullary collecting duct (mIMCD-3) was determined using Toray 3D-GeneTM miRNA microarray analysis to evaluate 1080 miRNAs. Microarray analysis identified 34 highly expressed miRNAs. Other miRNAs were present in lesser amounts, and many were not found in mIMCD-3 cells. The levels of a number of miRNAs appeared to be affected by the presence of aldosterone. Aldosterone responsive miRNAs affected levels of serum/glucocorticoid regulated kinase 1 mRNA, sodium/potassium-transporting ATPase subunit beta-1 mRNA, and endothelin-1 mRNA. Finally, argonaute immunoprecipitation experiments demonstrated that endothelin-1 (Edn1) mRNA is a direct target of the RNA-induced silencing complex indicating miRNA action on this transcript. The RISC-Edn1 mRNA interaction was specifically inhibited by anti-miR-709 implicating this particular miRNA in the regulation of Edn1 expression in the renal collecting duct.