To establish and evaluate a novel approach for quantification of the thiazide sensitive sodium chloride cotransporter (NCC) mRNA in human urinary exosomes, and to investigate the relationship between NCC mRNA expression and the increased NCC protein abundance previously reported by us to be induced by fludrocortisone administration in primary aldosteronism (PA) patients.Design and Method:
Morning spot urine samples from healthy volunteers were collected and stored at −80oC for method optimization. Urinary exosomes were harvested by progressive ultracentrifugation, and NCC abundance quantified by Western blot. Exosomal total RNA was isolated by spin column-based isolation and combined phenol and column-based isolation. NCC expression was determined and quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).Results:
Among healthy volunteers, exosomal total RNA isolated by combined phenol and column-based isolation yielded 2 times as much as the column-based method, and NCC expression (Ct value∼35) could be observed only with the combine phenol and column-based approach. The minimum urine volume required for RNA isolation was 200 ml with the average concentration of total RNA being 18.4 ng/ul eluted in 16 ul RNase free water. In ongoing studies, the optimised method of quantitative analysis is being applied to hypertensive patients with raised plasma aldosterone/renin ratios who are undergoing fludrocortisone suppression testing (100 mcg q6 h) in order to diagnose or exclude PA, and NCC expression and NCC abundance will be compared.Conclusions:
NCC mRNA in human urinary exosomes has been successfully isolated by a combined phenol and column-based isolation method. This will help in the exploration of molecular mechanisms that govern NCC expression and abundance, including that induced by mineralocorticoids, and may ultimately aid in the clinical diagnosis and treatment of PA.