*3.4. Histomorphometric Analysis of Bone Parameters after Four Weeks of Treatment with Ang II and Losartan*

Osteoblast parameters, quantified by morphology and histomorphometry and normalized for trabecular bone surfaces (N.Ob/BS and Ob.S/BS) did not differ between Ang II treated rats and control animals. Losartan treatment did not modify osteoblast number/bone surface and osteoblast surface/bone surface in control and Ang II treated rats (Figure 4).

**Figure 4.** Effects of Ang II and Losartan administration on bone tissue. (**a**) Representative images from H&E-stained sections showing osteoblasts (arrow) and (**b**) quantification of osteoblast parameters in the different groups of rats. Data are means ± SEM. N.Ob: Osteoblast number; BS: bone surface; Ob.S: osteoblast surface; BS: bone surface. Ang II: Angiotensin II; Los: losartan.

Trabecular bone volume of femora in Ang II treated rats did not show significant difference as compared with control groups and Ang II+losartan treated rats (Figure 5).

**Figure 5.** Effects of Ang II and losartan administration on bone tissue. (**a**) Representative histological pictures from Sirius red-stained bone tissue sections (trabecular bone stained in red) and (**b**) quantification of femoral trabecular bone in the different groups of rats. BV: Trabecular bone volume; TV: tissue volume. Data are means ± SEM. Ang II: Angiotensin II; Los: losartan.

#### **4. Discussion**

The results of this study demonstrate that chronic Ang II administration caused an early increase in urinary excretion of calcium and phosphate that lasted a long time. The increase was paralleled by a decrease in plasma phosphate levels, while plasma calcium levels did not change.

The blockade of AT1 receptors by losartan treatment prevented the increase of phosphaturia, but not the increase in urinary calcium excretion.

A link between hypertension and bone metabolism has been described in the literature, in particular a relationship between high blood pressure and hypercalciuria, which may promote bone mineral loss [32–34].

In our case the Ang II-induced increase of calcium and phosphate excretion is not associated with changes in bone mass.

The observed enhancement of renal phosphate loss in Ang II treated animals might be primarily related to a direct effect of Ang II on the type II sodium-phosphate co-transporter (NaPi-IIa) that is the critical player in renal Pi regulation. It has indeed been shown that phosphate excretion increased seven fold in rats chronically treated with Ang II with an associated enhancement, likely induced by post-transcriptional mechanisms, of NaPi-IIa protein level with Western blot analysis of the brush border membrane vesicles exposed to Ang II [35]. This effect is potentially mediated by the Ang II type 1 receptor, and, in line with our experimental model, losartan prevented the increase of phosphate excretion in Ang II treated rats.

Beyond the direct effect of Ang II on NaPi-IIa, it should be considered that the endocrine regulators of the renal tubular maximum phosphate reabsorption (TmP) might be affected by RAS activation and subsequently constitute an endocrine milieu favoring kidney phosphate loss. In fact, the increased (sodium-driven) calcium excretion induced by Ang II administration could give rise to a negative calcium balance with secondary activation of parathyroid hormone (PTH). This phenomenon would result, just as in our case, an increased urinary excretion of phosphate and a decrease in plasma phosphate. PTH is in fact a known regulator of TmP and could directly contribute to bone and heart damage. PTH secretion could also be directly enhanced by aldosterone [36] that can in turn be stimulated by Ang II. This assumption is supported by the evidence described in the literature that RAS inhibitors lower PTH [37,38].

Taking into account the extent of the effects on phosphate metabolism in relation to the minor effects on calcium, it is also possible, as an additional hypothesis, that the increased urinary excretion of phosphate and reduced plasma phosphate levels are related to the activation of Fibroblast Growth Factor23 (FGF23) secretion by Ang II and/or aldosterone [39]. It has indeed been shown that Ang II and aldosterone stimulate FGF23 transcription and secretion in cultured osteoblast-like cells [40] and cardiac myocytes [41]. FGF23 is a 251-amino-acid long, primarily bone derived, hormone that is critical to the maintenance of phosphate homeostasis [42]. In the proximal kidney tubule, FGF23 binds to the FGF receptor (FGFR) and its co-receptor klotho and downregulates the membrane availability of the phosphate type II sodium-phosphate co-transporter, NaPi-IIa. This effect, acting in a combined manner with PTH signaling [43], results in increased urinary excretion of phosphate and reduced plasma phosphate levels. The interactions among PTH, FGF23 and RAS signaling on NaPi-IIa might therefore constitute the endocrine milieu which drives the phosphate loss in a synergistic manner, although the specific contribution of each regulatory signal remains unclear. If this was the case, the activation of FGF23 could at the same time participate in cardiac damage. In fact, Ang II inhibition has been shown to reduce FGF23-induced changes in Ca2+ homeostasis and hypertrophy in cardiac cells [44]

Interestingly, in our experimental conditions, an increase in urinary calcium excretion, but not an increase in urinary phosphate excretion, was present in Ang II+losartan treated group, in the absence of both hypertension and increased urinary sodium excretion. Whether this increase is a transitory effect or may last a long time, mediated by other mechanisms not related to the blockade of AT1 receptors or by the increase in blood pressure caused by Ang II, remains to be clarified.

However, in our experimental model of Ang II dependent hypertension we did not find significant changes in bone mass measured by both pQCT and histomorphometric analysis in the different appendicular skeleton sites. In line with our results, no detectable changes in the trabecular and cortical bone parameters, as assessed by micro-CT, were shown to be at the bone sites far from the inflamed joints after Ang II administration for four weeks in male mice with Tumor Necrosis Factor-mediated arthritis [45]. Our findings differ from results previously described in experimental models that used chronic administration of Ang II at the same dosage and for the same time used in our experimental model, with subsequent enhancement of bone loss [20,23]. These differences could depend on the different pathophysiologic conditions of animal model applied. In the present study we administered Ang II to male adult Sprague–Dawley rats in physiological conditions. Conversely, in the osteoporosis animal model, Ang II was administered to female animals that had developed ovariectomy induced bone loss [20,23]. It is therefore likely that the gender and the pre-activation of bone remodeling with a negative bone balance might be both critical determinants of RAS activation in bone. Taking into account that in our experimental model we administered Ang II to healthy male Sprague–Dawley rats for four weeks, we can reasonably hypothesize that the duration of the treatment, which was shown to be sufficient to cause changes to other organs, such as the heart [15,17] and kidney [16], is probably too short to cause an alteration in the bone tissue. In fact, in the absence of a pre-activation of bone remodeling characterized by bone loss, it is likely that, to observe a significant modification of bone density, it should be necessary to prolong the duration of administration of Ang II, and possibly also to increase the dosage. Further experimental studies will be needed to verify these hypotheses.

In conclusion, in our experimental model of Ang II dependent hypertension we found changes in urinary calcium/phosphate excretion, which might be due to direct or endocrine mediated mechanisms. In our experimental model, chronic administration of Ang II and/or increased blood pressure values did not affect bone tissue in the absence of pre-activation of bone loss. However, we cannot exclude that, by further extending the experimental time, a detrimental effect of Ang II on bone could become apparent.

**Author Contributions:** Conceptualization, G.C., G.Z. (Gianpaolo Zerbini) and C.R.T.d.G.; methodology, G.C., R.C., S.I., M.M. and B.P.; software, R.C. and B.P.; validation, R.C. and S.I.; formal analysis, G.C., M.M., R.C. and S.I.; investigation G.C., R.C., G.C., S.I., I.V., S.B., A.R., M.M. and C.R.T.d.G.; resources, G.Z. (Giovanni Zatti) and C.R.T.d.G.; data curation, G.C., R.C., S.I., G.Z. (Gianpaolo Zerbini), I.V., S.B. and G.Z. (Giovanni Zatti); writing—original draft preparation, G.C., G.Z. (Gianpaolo Zerbini), G.Z. (Giovanni Zatti), S.I., I.V., S.B., A.R. and C.R.T.d.G. writing—reviewing, interpreting the results and editing, G.C., G.Z. (Gianpaolo Zerbini), S.I., A.R., G.Z. (Giovanni Zatti) and C.R.T.d.G.; visualization, R.C., M.M. and B.P.; supervision, G.C., G.Z. (Gianpaolo Zerbini) and C.R.T.d.G.; funding acquisition, G.Z. (Giovanni Zatti). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fondo Ateneo Ricerca (Giovanni Zatti).

**Institutional Review Board Statement:** The study was conducted according to the Guidelines of the Declaration of Helsinki and approved by the Ministero della Salute della Repubblica Italiana-Direzione Generale della Sanita' Animale e dei Farmaci Veterinari (n. 1123/2015, 22 October 2015).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
