**1. Introduction**

The most frequent inherited renal phosphate-wasting disease in humans is X-linked hypophosphatemia (XLH). The murine homolog is *Hyp* (hypophosphatemia). XLH patients and *Hyp* mice lose phosphate via the urine and show impaired bone mineralization as a consequence of hypophosphatemia and alterations in the organic bone matrix. The disease is caused by loss-of-function mutations in *PHEX* (Phosphate-regulating endopeptidase homolog, X-linked) in mice and men. By mechanisms that are poorly understood, *PHEX* mutations lead to excessive secretion of the bone-derived, phosphotropic hormone fibroblast growth factor-23 (FGF23) [1–3].

FGF23 is a 32 kDa glycoprotein circulating in the bloodstream. Only the intact molecule is biologically active. FGF23 signals through FGF receptors (FGFRs), with FGFR1c being the most important receptor under physiological conditions. High affinity binding of FGF23 to FGFRs in the cell membrane requires the presence of the co-receptor protein αKlotho [4,5]. The major site of action of FGF23 is the kidney, where it inhibits tubular reabsorption of phosphate and suppresses vitamin D hormone (1,25(OH)2D) production in proximal renal tubules [6]. Excessive concentrations of intact FGF23 lead to renal phosphate wasting,

**Citation:** Latic, N.; Zupcic, A.; Frauenstein, D.; Erben, R.G. Activation of RAAS Signaling Contributes to Hypertension in Aged *Hyp* Mice. *Biomedicines* **2022**, *10*, 1691. https://doi.org/10.3390/ biomedicines10071691

Academic Editor: Ramón C. Hermida

Received: 7 June 2022 Accepted: 9 July 2022 Published: 13 July 2022

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hypophosphatemia, and impaired bone mineralization in patients with normal kidney function [7].

Although FGF23 mainly acts on epithelial cells in the kidney under physiological conditions, other organs such as the cardiovascular system may become targets of FGF23 signaling in pathological settings. The cardiovascular sequelae of chronically elevated FGF23 are still controversial. Studies in chronic kidney disease (CKD) patients have shown that elevated FGF23 levels are independently associated with CKD progression, left ventricular (LV) hypertrophy, cardiovascular risk, and all-cause mortality [8–10]. A key question in this context is whether FGF23 is only a biomarker of impaired phosphate metabolism or chronic inflammation or whether there is a causal link between FGF23 and increased cardiovascular risk. The myocardium does not express αKlotho [10,11], so the heart is likely not a target of FGF23 action under normal conditions. However, at high circulating concentrations such as those found in CKD patients, FGF23 may promote LV hypertrophy by a direct, αKlotho-independent, FGFR4-mediated action on cardiomyocytes [10–12]. Alternatively, FGF23 excess may cause LV hypertrophy by contributing to volume overload through its sodium-conserving effect [13].

Whether LV hypertrophy is typically associated with other diseases (than CKD) characterized by FGF23 excess, such as XLH, is unclear. Some studies find a high incidence of LV hypertrophy in XLH patients [14,15], but others report no such association [16]. In contrast, the data from experimental studies are more clear-cut: several independent studies failed to find LV hypertrophy in *Hyp* mice [17,18] or in mice with a loss-of-function point mutation in the *Phex* gene [19]. Although LV hypertrophy appears to be absent at least in younger *Hyp* mice up to 9 months of age, we and others found mild hypertension in these mice [6,13]. The pathophysiological mechanisms underlying hypertension in *Hyp* mice are not entirely clear, but they may be related to an FGF23-mediated upregulation in renal sodium-chloride cotransporter (NCC) abundance [13]. Injection of recombinant FGF23 for five days into wild-type mice led to an increase in systolic, diastolic, and mean arterial pressure [13]. *Hyp* mice are characterized by chronic elevation of FGF23 and may, therefore, serve as a model to investigate the pathophysiology of hypertension in diseases associated with excessive FGF23 secretion.

An improved understanding of the mechanistic link between chronically elevated FGF23 and LV hypertrophy or other untoward cardiovascular side effects such as hypertension is of major importance, not only for XLH patients but for all diseases characterized by chronic elevations of circulating intact FGF23, such as CKD. Here, we sought to further elucidate the long term cardiovascular sequelae of elevated circulating levels of FGF23 in 14-month-old *Hyp* mice as a model of FGF23 excess. We found that aged *Hyp* mice lacked LV hypertrophy but were characterized by increased serum aldosterone and hypertension that could be rescued by inhibitors of the renin–angiotensin–aldosterone system (RAAS).

#### **2. Materials and Methods**

#### *2.1. Animals*

The study was undertaken in accordance with prevailing EU and national guidelines for animal care and welfare and in compliance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. 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).

Male wild-type (WT) controls and *Hyp* mice were bred by mating WT females with *Hyp* males on C57BL/6 background in our animal facility. Tail length at the time of weaning was used for genotyping. Animals were kept at 24 ◦C with a 12/12-h light/dark cycle, and were housed in stable groups of 2–5 mice from the time of weaning. They were fed a normal mouse diet (V1124-000, Sniff, Soest, Germany) containing 1.0% calcium, 0.7% phosphorus, and 1000 IU vitamin D/kg, and had access to food and tap water ad libitum. At necropsy, the mice were exsanguinated from the abdominal vena cava under general

anesthesia with ketamine/medetomidine (100/0.25 mg/kg i.p.) for serum collection. No mice were excluded from the study.

#### *2.2. Losartan and Canrenone Treatment*

Some *Hyp* and WT mice were randomized to treatment with losartan (Sandoz, Austria, 50 mg) or vehicle (0.9% NaCl) via oral gavage twice a day or with subcutaneous injections of canrenone (Aldactone, Reimser, Germany, 200 mg/10 mL) or vehicle (0.9% NaCl) daily. Both treatments were given for 5 days.
