The aim of this study was to determine the effect of RNA interference inhibition of mineralocorticoid receptor (MR) on cold-induced hypertension (CIH) and renal damage. Recombinant adeno-associated virus (AAV) carrying short hairpin small interference (si)RNA for MR (AAV.MR-shRNA) was constructed and tested for the ability to inhibit renal MR and to control CIH. Three groups of rats with CIH received AAV.MR-shRNA (1.25 Â 10 9 particles/rat, intravenous), AAV carrying scrambled shRNA (AAV.Control-shRNA) (1.25 Â 10 9 particles/rat, intravenous) and phosphate buffer solution (PBS), respectively. All rats were kept in a cold chamber (6.71C) throughout the experiment. Adeno-associated virus delivery of MR-shRNA prevented progression of CIH. Blood pressure (BP) of the AAV.MR-shRNA-treated group did not increase and remained at 14573 mm Hg, whereas BP of the AAV.Control-shRNA-treated and PBStreated group increased to 16774 and 16173 mm Hg, respectively, at 3 weeks after gene delivery. Thus, the antihypertensive effect of a single injection of AAV.MRshRNA lasted for at least 3 weeks (length of the study). Adeno-associated virus carrying short hairpin siRNA for MR significantly increased urinary sodium excretion and decreased proteinuria. It also decreased serum creatinine and blood urea nitrogen, suggesting enhanced renal function. Both Western blot and immunohistochemical analysis showed that MR expression was decreased significantly in the kidney in the AAV.MR-shRNA-treated rats, confirming that renal MR is effectively inhibited by AAV.MR-shRNA. Adeno-associated virus carrying short hairpin siRNA for MR also significantly attenuated renal hypertrophy. In addition, AAV delivery of MR-shRNA prevented atrophy and dilation of renal tubules and abolished tubular deposition of proteinaceous material seen in CIH rats. Conclusions: (1) AAV delivery of MR-shRNA effectively silenced MR in vivo. (2) RNA interference inhibition of MR may open a new avenue for the long-term control of hypertension and renal damage.

Clinical observations and epidemiological surveys indicated that the prevalence of hypertension and heart diseases is increased in cold regions or during winter. Cold exposure increased NADPH oxidase gp91 phox protein expression in heart, kidneys, and aorta in rats. The aim of this study was to investigate if RNA interference (RNAi) silencing of gp91 phox would attenuate cold-induced hypertension and cardiovascular and renal damage. The recombinant adeno-associated virus serotype 2 (AAV-2) vector carrying gp91 phox -shRNA (gp91-shRNA) was constructed for inhibiting gp91 phox protein expression in cold-exposed rats. Blood pressure (BP) was monitored using a telemetry system. BP was increased in the Control-shRNA and PBS groups within 1 week of exposure to moderate cold (5°C) and reached a plateau after 7 weeks. The cold-induced increase in BP was attenuated significantly by intravenous delivery of gp91-shRNA (1.25 · 10 10 particles/rat, 0.5 mL). One single dose of gp91-shRNA controlled hypertension for up to 10 weeks. In addition, gp91-shRNA reversed coldinduced vascular dysfunction. gp91-shRNA abolished the cold-induced up-regulation of gp91 phox protein expression in heart, kidneys, and aorta, confirming effective silencing of gp91phox . The cold-induced increases in NADPH oxidase activity and superoxide production were eliminated by silencing of gp91 phox , suggesting that the cold-induced up-regulation of NADPH oxidase activity may be attributed to the increased gp91 phox protein expression. RNAi silencing of gp91 phox abolished cold-induced cardiac and renal hypertrophy and attenuated aortic, coronary, and renal remodeling. The up-regulation of gp91 phox may play a critical role in cold-induced cardiovascular dysfunction and organ damage. AAV delivery of gp91-shRNA may be a new and effective therapeutic approach for cold-related cardiovascular disorders.

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