Abstract-CYP11B2 is the enzyme responsible for aldosterone synthesis mainly in the adrenal gland. In this study, we hypothesized that CYP11B2 gene, protein, and aldosterone are produced locally in kidney and regulated by low salt intake, angiotensin II type 1 (AT 1 ) receptor and insulin-deficient diabetes hyperglycemia. We used real-time RT-PCR, immunohistochemistry staining, and microdialysis techniques to monitor changes in renal CYP11B2 mRNA and protein and aldosterone production in normal, adrenalectomized, or streptozotocin-induced insulin-deficient diabetic hyperglycemic rats. In normal kidney, CYP11B2 mRNA and protein were localized mainly in the renal cortex and upregulated by angiotensin II and low salt intake. The key enzyme for aldosterone synthesis is CYP11B2, a member of cytochrome P450 family with molecular weight 48.5 kDa. The CYP11B2 gene is Ϸ45 kb, contains 9 exons and is located on chromosome 8. 5 Aldosterone stimulates cellular hypertrophy, matrix formation, 6,7 and cell death. 8 Although aldosterone can be produced in tissues other than the adrenal gland, 9 its production in the kidney has never been reported. The demonstrated benefits of aldosterone receptor blockers in diabetic nephropathy, 6,10 -12 despite reported normal  or low 16 -19 levels of plasma aldosterone, suggest that this hormone may be produced locally within the kidney. Thus, we hypothesized that there is a local renal aldosterone system. Identification of different components of an aldosterone system in the kidney would boost this hypothesis. In this study, we confirmed that CYP11B2 gene, protein, and aldosterone production are locally present in kidney and regulated by low salt intake, Ang II type 1 (AT 1 ) receptor, and insulin-deficient diabetes hyperglycemia.
Animal PreparationStudy protocols were approved by the University of Virginia animal research committee. Sprague-Dawley rats (Harlan Teklad; Madison, Wis) weighing 245 to 255 g were used in this study. Rats were housed in a well-ventilated room (21Ϯ1°C; 12-hour light/dark cycle). Animals were weighed at the end of each study week.
Adrenalectomy and Microdialysis ProceduresBilateral adrenalectomy was performed according to previously published methods. 20 A single microdialysis probe was inserted into left renal cortex as described previously. 21 Each animal in the control group was instrumented by microdialysis probe but did not have adrenalectomy (sham). After adrenalectomy, rats received dexamethasone (Sigma; 250 mg/kg per day SC). On the fifth day after surgery, renal interstitial fluid (RIF) samples 21 were collected from normal (nϭ6) and adrenalectomized (nϭ6) rats.
Salt Intake and Renal Aldosterone Synthase ExpressionThe effects of different levels of salt intake on the renal expression of aldosterone synthase were studied by placing animals for 1 week on low-salt (0.05% NaCl), normal-salt (0.5% NaCl), or high-salt (4% NaCl) diet (nϭ8 for each group).