*2.2. Peripheral Quantitative Computed Tomography (pQCT) Analysis*

The measurements were obtained by using a Stratec Research SA+ pQCT scanner (Stratec Medizintechnik GmbH, Pforzheim, Germany) with a voxel size of 70 mm and a scan speed of 3 mm/s. The excised tibiae were hold in place with manufacturer-made plastic holders in order to position the long axes of the specimen parallel to the image planes. The correct longitudinal positioning was resolved by means of a "scout scan". The scans were performed at the proximal metaphysis and at the diaphysis of the tibiae. The scans were analyzed with pQCT software 6.00B using contour mode 2 and peel mode 2. The threshold for the calculation of trabecular and total bone parameters was 500 mg/cm<sup>3</sup> and for cortical bone parameters 710 mg/cm3.

#### *2.3. Bone Histology and Histomorphometry*

For each rat the formalin fixed femur was decalcified in 10% EDTA for 30–45 days at 4 ◦C with gentle shaking and processed for paraffin embedding. Three-micron-thick paraffin embedded sections were used for standard histology after staining with Hematoxylin-Eosin (H&E) or with Sirius red to visualize collagen fibers with morphometric analysis.

Quantitative bone histomorphometry was conducted on distal femora. Experiments were performed in a blinded fashion. The region of interest (ROI) was identified in the secondary spongiosa of distal femora, starting 500 μm below the growth plate and for a length of 5 mm. Sections were stained with H&E and Sirius red and used to measure trabecular bone volume per tissue volume (BV/TV), osteoblast number per bone surface (N.Ob/BS) and osteoblast surface per bone surface (Ob.S/BS). Ten pictures at 20X were acquired with an optical microscope (Leica DMRB, Leica Biosystems, Muttenz, Switzerland) through a digital camera (Leica LAS EZ, Leica Biosystems, Switzerland) and all histomorphometric analyses were performed using Image J [31].

#### *2.4. Statistical Analysis*

Data are reported as means ± standard error of the mean (SEM). Differences among the groups of rats (control, control+losartan, Ang II, Ang II+losartan) for systolic blood pressure, body weight, glomerular filtration rate, heart/body weight, kidney/body weight, blood glucose, alkaline phosphatase, sodium, potassium, calcium, phosphate, uric acid, cholesterol and triglycerides, 24 h diuresis, 24 h urinary calcium, phosphate and sodium excretion, pQCT parameters, trabecular bone volume per tissue volume (BV/TV), osteoblast number per bone surface (N.Ob/BS) and osteoblast surface per bone surface (Ob.S/BS) were assessed using ANOVA followed by Fisher's protected least-significant test for post hoc comparisons. Differences between means were considered significant at *p* < 0.05.

#### **3. Results**

*3.1. Effects of Ang II and Losartan Administration on Systolic Blood Pressure, Body Weight, Glomerular Filtration Rate, Heart/Body Weight Ratio, Kidney/Body Weight Ratio and Serological Parameters*

Ang II administration caused a marked increase in blood pressure, which was prevented by losartan treatment in Ang II treated rats (Table 1). Losartan administration did not significantly modify blood pressure in control rats (Table 1).

**Table 1.** Systolic blood pressure (SBP, mmHg), body weight (BW, g.), glomerular filtration rate (GFR, ml/min), heart/body weight ratio (mg/g), kidney/body weight ratio (mg/g), non-fasting plasma glucose (mg/dL), alkaline phosphatase (U/L), sodium (mEq/L), potassium (mEq/L), calcium (mg/dL), phosphate (mg/dL), uric acid (mg/dL), total cholesterol (mg/dL), triglycerides (mg/dL) in control, control+los, Ang II, Ang II+los-treated rats at the end of the experimental period (four weeks). Data are means ± SEM. \* = *p* < 0.05 vs. control; † = *p* < 0.01 vs. control; ‡ = *p* < 0.0001 vs. control; ◦ = *p* < 0.05 vs. control+los; δ = *p* < 0.01 vs. control+los; § = *p* < 0.0001 vs. control+los; ˆ = *p* < 0.05 vs. Ang II+los; & = *p* < 0.01 vs. Ang II+los; ζ = *p* < 0.0001 vs. Ang II+los.


As compared to control rats, Ang II administration caused a significant reduction in BW, which was blunted by losartan treatment (Table 1). Ang II administration caused a slight decrease in glomerular filtration rate as compared to control rats. Losartan treatment prevented the decrease of GFR in Ang II treated rats (Table 1).

Ang II administration caused a significant increase in the heart/BW and kidney/BW ratios as compared to control rats, in line with the possible development of myocardial and kidney hypertrophy/fibrosis [15–17]. Losartan administration prevented the increase of heart and kidney/BW ratio in Ang II treated rats (Table 1).

As shown in Table 1, Ang II administration caused a slight decrease in plasma sodium, potassium and phosphate values as compared to control rats, which was prevented by losartan treatment in Ang II treated rats. No significant changes in plasma alkaline phosphatase, calcium, uric acid and in glyco-metabolic parameters (non-fasting glucose, cholesterol, triglycerides) were observed among the different groups (Table 1).

#### *3.2. Effects of Ang II and Losartan Administration on 24 h Diuresis, Urinary Sodium, Calcium, and Phosphate Excretion*

Before starting the treatments, diuresis and urinary sodium, calcium, and phosphate excretion were similar in all the groups (Figures 1 and 2).

**Figure 1.** Effects of Ang II and losartan administration on 24 h diuresis and urinary sodium excretion before and after two and four weeks of different treatments. Data are means ± SEM. \* = *p* < 0.05; § = *p* < 0.01.

**Figure 2.** Effects of Ang II and losartan administration on 24 h urinary calcium and phosphate excretion before and after two and four weeks of different treatments. Data are means ± SEM. \* = *p* < 0.05; § = *p* < 0.01.

Ang II administration increased diuresis just after two weeks of administration and until the end of the experimental period as compared to other groups (Figure 1). Losartan treatment blocked the increase in diuresis in Ang II treated rats during the entire experimental period (Figure 1).

After two weeks of Ang II administration and until the end of the experimental protocol Ang II administration caused a significant increase in urinary sodium excretion as compared to control rats (Figure 1). Losartan treatment blocked the increase in urinary sodium excretion in Ang II-treated rats (Figure 1).

After two weeks of Ang II administration no significant changes in urinary calcium excretion were observed in Ang II treated rats, as compared to other groups (Figure 2).

At the end of the experimental period, treatment with Ang II, alone or in combination with losartan, induced an increase of urinary calcium excretion as compared to control rats (Figure 2). Ang II administration increased phosphaturia just after two weeks of administration and until the end of the experimental period as compared to control rats (Figure 2). Otherwise, losartan treatment blocked the increase in urinary phosphate excretion in Ang II treated rats just after two weeks of administration until the end of experimental protocol (Figure 2).

#### *3.3. pQCT Analysis of Bone Parameters after Four Weeks of Treatment with Ang II and Losartan*

After four weeks of treatment, Ang II did not modify any bone parameters compared to control measured at the proximal metaphysis and at the diaphysis midshaft of the tibiae. Losartan induced an increase in Tb.BMD in rats chronically treated with Ang II and in control rats, although not reaching statistical significance (Table 2, Figure 3).


**Table 2.** Bone parameters measured by pQCT.

Tb.BMD: trabecular bone mineral density, Tb.Area: trabecular area, Ct.BMD: cortical bone mineral density, Ct.Area: cortical area, Tot.Area: total area. Tb.BMD and Tb.Area were measured at the proximal metaphysis of the tibia, Ct.BMD, Ct.Area and Tot.Area were measured at the diaphyseal midshaft. Data are means ± SEM.

**Figure 3.** Representative image of the metaphyseal site of the rat tibiae analyzed by pQCT. The bar on the left is the colour scale bar of the density (mg/cm3): white > 700 mg/cm3; grey <sup>≤</sup> 100 mg/cm3.
