*3.2. Inhibition of RAAS Signaling Normalizes Blood Pressure in Hyp Mice*

To test this hypothesis, we administered the aldosterone antagonist canrenone and the angiotensin receptor blocker losartan over five days to 12- to 14-month-old male *Hyp* and WT mice. Intraarterial catheterization confirmed hypertension in vehicle-treated *Hyp* mice compared to WT controls. Interestingly, daily subcutaneous injections of 30 mg/kg canrenone led to a distinct decrease in systolic, diastolic, and mean arterial pressure in *Hyp* mice relative to vehicle-treated *Hyp* mice (Figure 3A–C). In contrast, WT mice injected with canrenone showed only minor reductions in mean, systolic, and diastolic blood pressure (Figure 3A–C). Losartan was administered via oral gavage at the dose of 30 mg/kg twice daily, and the effects of losartan were examined one hour after the last administration, due to a shorter half-life of this drug compared with canrenone. Similar to canrenone, losartan caused a pronounced decrease in systolic, diastolic, and mean arterial pressure in *Hyp* mice, relative to vehicle-treated *Hyp* mice, but had only negligible effects in WT animals (Figure 3D–F).

**Figure 3. Inhibition of RAAS signaling normalizes blood pressure in** *Hyp* **mice.** (**A**,**D**) Systolic (SP), (**B**,**E**) diastolic (DP), and (**C**,**F**) mean arterial blood pressure (MAP), as well as (**G**,**H**) augmentation index (AI) in 12–14-month-old male WT and *Hyp* mice treated over 5 days with 30 mg/kg canrenone (s.c. once daily) or 30 mg/kg losartan (gavage twice daily) (n = 4–8). Bars represent mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 vs. vehicle by one-way ANOVA followed by Student–Newman–Keuls post-hoc test. Insets show results of two-way ANOVA.

To determine whether the beneficial effect of RAAS inhibition on hypertension was associated with a decrease in peripheral vascular resistance, we analyzed the augmentation index in WT and *Hyp* mice post-treatment. Interestingly, both treatments reduced the elevated augmentation index in *Hyp* mice. Canrenone did not alter the augmentation index in WT mice, but significantly lowered the augmentation index in *Hyp* mice, whereas losartan had a similar effect in both genotypes (Figure 3G,H). Two-way ANOVA revealed a significant two-way interaction between genotype and treatment for systolic pressure (*p* < 0.001), mean arterial pressure (*p* < 0.01), and augmentation index (*p* < 0.05) in animals treated with canrenone, whereas this interaction was only found significant for the reduction in systolic pressure (*p* < 0.01) in *Hyp* mice treated with losartan (Figure 3A–H). Collectively, our data suggest that the blood pressure-lowering response of *Hyp* mice to both canrenone and losartan is exaggerated compared with WT mice, supporting our hypothesis that activation of RAAS signaling contributes to hypertension in aged *Hyp* mice. Because both canrenone and losartan reduced blood pressure and augmentation index in aged *Hyp* mice, it is likely that the contributing effect of the RAAS to the development of hypertension is mediated through a combination of increased peripheral resistance together with increased blood volume.

#### **4. Discussion**

The central aim of this study was to characterize the pathophysiological role of long term endogenous FGF23 hypersecretion in the cardiovascular system of 14-month-old male *Hyp* mice. We found that aged *Hyp* mice were mildly hypertensive but did not develop LV hypertrophy. Hypertension in aged *Hyp* mice was associated with increases in serum aldosterone levels, in vascular peripheral resistance, and in renal pNCC abundance. Administration of the angiotensin II receptor blocker losartan and the mineralocorticoid receptor blocker canrenone rescued the cardiovascular phenotype by lowering blood pressure and vascular resistance in *Hyp* mice.

Elevated circulating FGF23 levels are associated with accelerated disease progression, morbidity, and/or mortality in several clinical disorders, including CKD but also cardiac failure [8–10,21]. It was proposed that excessive FGF23 causes LV hypertrophy by Klotho-independent binding to FGFR4 and subsequent activation of the calcineurin/NFAT pathway in cardiomyocytes [11,12]. However, it is still unclear whether this disease mechanism is relevant in the setting of normal kidney function, because evidence from clinical studies in XLH patients and mouse models of XLH suggests that chronic FGF23 excess does not invariably cause LV hypertrophy [15–18]. In our study, an increase in circulating intact FGF23 levels of ~10-fold was not sufficient to induce an increase in LV size or deterioration of LV function in 14-month-old *Hyp* mice when compared to WT littermates. Fractional shortening, ejection fraction, and molecular markers of hypertrophy (*Anp*, *Bnp*) all remained unchanged in these animals. This is in line with previous studies in animal models of XLH that did not find any association between increased levels of FGF23 and LV hypertrophy [17,19].

The current study has shown that aged *Hyp* mice are characterized by a small increase in systolic, diastolic, and mean arterial blood pressure, corroborating earlier studies in younger *Hyp* mice [13,22]. Although hypertension is not a universal complication in XLH patients [14], early-onset hypertension is frequently found in adult XLH patients [23]. Based on the sodium-conserving function of FGF23, elevated circulating FGF23 may predispose subjects to the development of hypertension through volume expansion [13]. However, an increase in serum intact FGF23 levels of about 6-fold did not increase blood pressure in mice with a loss-of-function point mutation in the *Phex* gene that are characterized by normal kidney function [19]. Rather, the latter study reported that systolic pressure was actually slightly reduced in aged *PhexC733R* male mice relative to WT controls. A possible explanation for these discrepant findings may be the greater increase in FGF23 serum levels in *Hyp* mice. However, elevated circulating FGF23 levels per se may not be sufficient to cause hypertensive changes in the absence of other contributing factors.

Our data have revealed for the first time that serum aldosterone as well as augmentation index were increased in aged *Hyp* mice. Although we did not measure renin activity and angiotensin II levels, these findings suggest a general stimulation of the RAAS in aged *Hyp* mice. In contrast, we found lower aldosterone levels in 3-month-old *Hyp* mice [13]. We do not have a conclusive explanation for the discrepancy between young and aged *Hyp* mice regarding aldosterone secretion. There may be age-related changes in aldosterone secretion in *Hyp* mice or differences in the interaction between FGF23 signaling and the RAAS in young, growing vs. aged, non-growing *Hyp* mice. Nevertheless, the findings in the current study led us to hypothesize that hypertension in aged *Hyp* mice may be caused by increased RAAS signaling alone or in combination with FGF23-mediated increased sodium absorption, leading to a combination of volume overload and increased peripheral vascular resistance. Aldosterone, similar to FGF23, enhances sodium and water reabsorption in the distal nephron, indirectly increasing blood pressure [24–26]. Furthermore, it was shown previously that Ang II administration to *Hyp* mice resulted in additive effects on blood pressure [21]. We used short term (5-day) treatment with the mineralocorticoid receptor blocker canrenone and the angiotensin II receptor type 1 antagonist losartan as tools to dissect the renal and cardiovascular effects of aldosterone and angiotensin II. However, both drugs lowered blood pressure and decreased peripheral resistance in aged *Hyp* mice. Therefore, it is likely that a combination of volume effects and increased peripheral vascular resistance is involved in the hypertension-promoting effects of RAAS stimulation in aged *Hyp* mice. Interestingly, these drugs had little effect in WT mice, further corroborating that augmented RAAS signaling contributes to the development of hypertension in *Hyp* mice.

Although our study has provided novel insights into the pathogenesis of hypertension in *Hyp* mice, a key question in this context remains unanswered: what is driving the stimulation of RAAS in aged *Hyp* mice? It has been suggested that FGF23 hypersecretion may stimulate the RAAS through the suppression of vitamin D hormone production in CKD [27,28]. However, whether RAAS is regulated by vitamin D remains a controversial issue [29], and it is currently not known if this proposed mechanism may also be relevant for *Hyp* mice. It is clear that further experimentation is required to define the molecular basis of the crosstalk between FGF23 and the RAAS. This is not only relevant for XLH patients, but also has much broader implications for diseases characterized by a combination of chronic FGF23 hypersecretion and increased RAAS signaling, such as CKD [30].

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomedicines10071691/s1, Table S1. Mouse primer sequences for quantitative real-time PCR analysis

**Author Contributions:** N.L. and R.G.E. conceived and designed the studies; N.L., A.Z. and D.F. performed experiments and analyzed the data; N.L. and R.G.E. wrote the manuscript; all authors discussed and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Open Access Funding by the University of Veterinary Medicine Vienna.

**Institutional Review Board Statement:** All animal procedures were approved by the Ethics and Animal Welfare Committee of the University of Veterinary Medicine Vienna, Austria, and by the Austrian Federal Ministry of Education, Science and Research (permit number BMWFW-68.205/0054- II/3b/2013 and 2021-0.331.140).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed in this study are included in this article.

**Acknowledgments:** We thank C. Schüler, C. Bergow, A. Petric, and N. Ginner for excellent technical assistance, and Ute Zeitz for help with animal breeding.

**Conflicts of Interest:** The authors have declared that no conflict of interest exists.
