**1. Introduction**

Phosphate is part of the fundamental chemical necessary for cellular structure, signaling and energy production, making it essential for various biological processes. Phosphate

**Citation:** Latic, N.; Peitzsch, M.; Zupcic, A.; Pietzsch, J.; Erben, R.G. Long-Term Excessive Dietary Phosphate Intake Increases Arterial Blood Pressure, Activates the Renin–Angiotensin–Aldosterone System, and Stimulates Sympathetic Tone in Mice. *Biomedicines* **2022**, *10*, 2510. https://doi.org/10.3390/ biomedicines10102510

Academic Editors: Josef Zicha and Ivana Vanˇeˇcková

Received: 22 July 2022 Accepted: 5 October 2022 Published: 7 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

metabolism is mainly regulated by the vitamin D hormone (1,25(OH)2D3) and by two major phosphaturic hormones, fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH). FGF23 and PTH act on proximal tubules in the kidney to increase renal phosphate excretion by inhibiting reabsorption of filtered phosphate. Therefore, FGF23 and PTH lower phosphate levels in the blood. 1,25(OH)2D3 on the other hand stimulates phosphate absorption from the intestine, thereby increasing serum phosphate levels [1].

Despite its importance for various processes in the body, the accumulation of phosphate in the blood, hyperphosphatemia, can have deleterious effects. Epidemiological and observational studies have reported an association between increased serum phosphate levels and dietary phosphate load with left ventricular hypertrophy (LVH), cardiac calcification, as well as morbidity and mortality in patients suffering from chronic kidney disease (CKD) [2–5]. Cardiovascular events are the most frequent cause of death in CKD patients [6]. As the glomerular filtration rate decreases in CKD, the kidney is unable to adequately excrete phosphate, leading to hyperphosphatemia. Hyperphosphatemia has been identified as an independent risk factor for accelerated cardiovascular disease (CVD) development in these patients, but the exact mechanisms have not yet been elucidated [7]. Interestingly, high serum phosphate levels are also associated with an increased risk for CVD in adults with no history of CVD or CKD [8–10].

High dietary phosphate may also be linked to hypertension. Hypertension is a major health burden affecting about 1.3 billion people worldwide [11]. The existing epidemiological data on the effect of dietary phosphate on hypertension are inconsistent. While some studies have found a clear association between phosphate intake and hemodynamic parameters, others have not observed changes in blood pressure that were attributable to a high dietary phosphate load [10,12,13]. A recent intervention study shed additional light on this issue. Mohammad and coworkers examined the effects of a high phosphate diet in healthy subjects with normal renal function, and found that a 6-week dietary phosphate load induced small, but significant increases in systolic and diastolic blood pressure that were accompanied by activation of the sympathetic tone, relative to subjects receiving the low phosphate diet [10].

It still remains an open question whether the untoward effects of excess dietary phosphate on the cardiovascular system are direct or indirect. Experimental studies have attempted to clarify the mechanisms involved. Studies supporting a direct role of phosphate postulate that hyperphosphatemia leads to arterial calcification by directly stimulating the differentiation of vascular smooth muscle cells into osteoblast-like cells [14–16]. Furthermore, a study in rat aortas demonstrated that an increase in extracellular phosphate is followed by an increase in oxidative stress, a decrease in nitric oxide production, and inhibitory phosphorylation of endothelial nitric oxide synthase (eNOS) [17]. On the other hand, phosphate may act indirectly by inducing changes in the endocrine regulators of phosphate homeostasis, FGF23, PTH and 1,25(OH)2D3. An increase in serum phosphate stimulates FGF23 and PTH secretion, while at the same time inhibiting production of 1,25(OH)2D3. In this context, it is established that FGF23 causes LVH via FGF receptor 4-dependent activation of the calcineurin-NFAT signaling pathway [17]. PTH may cause hypertension by its direct effects on arteries and myocytes to promote arterial stiffness and LVH [18]. Indeed, Bozic and coworkers found an increase in blood pressure and LVH in normotensive and spontaneously hypertensive rats with normal kidney function that was driven by an increase in PTH [19].

It is clear that elucidation of the mechanisms underlying the potential hypertensive and hypertrophy-promoting effects of a high phosphate intake may have major implications for the prevention of CVD and its sequelae in humans. Previous experimental studies in rodents mostly used a phosphate-enriched diet to investigate the effects of an increased dietary phosphate intake on the cardiovascular system or the kidney [19–22]. However, an isolated increase in dietary phosphate leads to secondary hyperparathyroidism, making it difficult to dissect the effects of phosphate from that of increased PTH on cardiovascular endpoints. In this study, we sought to further explore the cardiovascular sequelae of longterm elevated phosphate intake in aged mice, largely independent of changes in PTH. To this end, we maintained male C57BL/6 mice on a calcium, phosphate, and lactose-enriched diet (CPD) with a balanced calcium/phosphate ratio for 14 months and compared them with age-matched mice fed a normal mouse diet. We found that 14-month-old mice on a CPD were characterized by hypertension and increased arterial stiffness, and that these changes were associated with increased urinary aldosterone excretion and augmented sympathetic tone.
