**2. Materials and Methods**

#### *2.1. Animal Study Design of Ang II-Dependent Hypertension*

Experiments were performed in male Sprague–Dawley rats (body weight 150–175 g) in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Animal husbandry was in conformity with the institutional guidelines in compliance with national laws and policies (D.L.n. 116, Gazzetta Ufficiale della Repubblica Italiana, suppl.40, 18 February 1992). Rats were individually housed in cages (or metabolic cages as necessary) in a temperaturecontrolled room with a 12:12 light:dark cycle, with free access to a standard rat chow and tap water. One week before the beginning of the protocol, rats were accustomed to metabolic cages and experimental procedures. Body weight (BW, g) was measured once a week. Systolic blood pressure was measured by plethysmography technique (tail cuff method, average of six recordings using a BP Recorder (Ugo Basile Instruments, Varese, Italy)) at the beginning and at the end of the experimental protocols [29,30]. Ang II was administrated through osmotic minipumps (Alzet 2004, Palo Alto, CA, USA), subcutaneously implanted under sodium pentobarbital anesthesia (40 mg/kg/i.p.), in order to administer Ang II

(200 ng/Kg/min for 4 weeks, *n* = 8, Sigma) or physiological saline in the control group (*n* = 9). Losartan (Los, 50 mg/kg/day), dissolved in drinking water, was administered in control + Los (*n* = 6) and Ang II + Los treated rats (*n* = 6). Glomerular filtration rate (mL/min) was evaluated as creatinine clearance using 24 h diuresis (ml/24 h).

Non-fasting plasma glucose (mg/dL), creatinine (mg/dL), sodium (mEq/L), potassium (mEq/L), calcium (mg/dL), phosphate (mg/dL), alkaline phosphatase (U/L), uric acid (mg/dL), total cholesterol (mg/dL), triglycerides (mg/dL) and 24 h urinary calcium (mg/24 h), phosphate (mg/24 h), and sodium (mEq/24 h) were measured by colorimetric technique on Cobas Roche (Mannheim, Germany) [15].

At the end of the experimental periods, rats were euthanized by an overdose of anesthesia. Hearts and kidneys were excised and weighted. The femurs were excised and fixed with 10% formalin, embedded in paraffin and used for light microscopic examination and morphometric analysis. Contralateral femur with tibia was also dissected, fixed with 10% formalin solution, neutral buffered, and used for peripheral quantitative computed tomography.
