Vascular calcification has a global health impact that is closely linked to bone loss. The Receptor Activator of Nuclear Factor Kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, fundamental for bone metabolism, also plays an important role in vascular calcification. The Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), a novel receptor for RANKL, regulates bone remodeling, and it appears to be involved in vascular calcification. Besides RANKL, LGR4 interacts with R-spondins (RSPOs), which are known for their roles in bone but are less understood in vascular calcification. Studies were conducted in rats with chronic renal failure fed normal or high phosphorus diets for 18 weeks, with and without control of circulating parathormone (PTH) levels, resulting in different degrees of aortic calcification. Additionally, vascular smooth muscle cells (VSMCs) were cultured under non-calcifying (1 mM phosphate) and calcifying (3 mM phosphate) media with different concentrations of PTH. To explore the role of RANKL in VSMC calcification, increasing concentrations of soluble RANKL were added to non-calcifying and calcifying media. The effects mediated by RANKL binding to its receptor LGR4 were investigated by silencing the LGR4 receptor in VSMCs. Furthermore, the gene expression of the RANK/RANKL/OPG system and the ligands of LGR4 was assessed in human epigastric arteries obtained from kidney transplant recipients with calcification scores (Kauppila Index). Increased aortic calcium in rats coincided with elevated systolic blood pressure, upregulated Lgr4 and Rankl gene expression, downregulated Opg gene expression, and higher serum RANKL/OPG ratio without changes in Rspos gene expression. Elevated phosphate in vitro increased calcium content and expression of Rankl and Lgr4 while reducing Opg. Elevated PTH in the presence of high phosphate exacerbated the increase in calcium content. No changes in Rspos were observed under the conditions employed. The addition of soluble RANKL to VSMCs induced genotypic differentiation and calcification, partly prevented by LGR4 silencing. In the epigastric arteries of individuals presenting vascular calcification, the gene expression of RANKL was higher. While RSPOs show minimal impact on VSMC calcification, RANKL, interacting with LGR4, drives osteogenic differentiation in VSMCs, unveiling a novel mechanism beyond RANKL-RANK binding.

Phosphorus is an essential mineral involved in bone mineralization, energy metabolism, and cellular signaling, whose serum concentration is tightly regulated by an endocrine network including fibroblast growth factor 23 (FGF23), parathyroid hormone (PTH), vitamin D and Klotho. Disruption of this balance, particularly in chronic kidney disease (CKD), leads to hyperphosphatemia, which is strongly associated with bone fragility, vascular calcification, and increased mortality. In CKD, impaired phosphorus homeostasis triggers endocrine dysregulation characterized by elevated PTH and FGF23 levels, Klotho deficiency, and altered vitamin D metabolism, resulting in major skeletal and vascular consequences. Experimental and clinical evidence indicates that phosphorus overload contributes directly to skeletal deterioration and early vascular remodeling, even prior to clinically detectable renal impairment. Moreover, high dietary phosphorus intake under conditions of normal renal function reproduces several molecular and structural alterations typically observed in CKD, supporting a pathogenic role for chronic phosphorus excess. The dietary source of phosphorus has gained increasing relevance, as inorganic phosphate additives exhibit high intestinal bioavailability and impose a greater systemic phosphorus burden. Current management strategies rely on dietary restriction, phosphate binders, modulation of intestinal phosphorus transport and optimization of mineral-regulating hormones, although evidence for improved clinical outcomes remains limited. A deeper understanding of the molecular mechanisms linking phosphorus overload to bone and vascular pathology may facilitate the development of more effective preventive and therapeutic strategies.

Background: The increasing prevalence of processed foods has significantly elevated dietary phosphorus intake globally, posing a risk to skeletal health. Elevated serum phosphate promotes parathyroid hormone (PTH) release, leading to bone resorption and decreased bone formation. Objective: This study investigated the influence of chronically elevated phosphorus intake on bone structure in rats with normal renal function, focusing on the Receptor Activator of Nuclear factor Kappa-B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway and its related components, leucine rich repeat containing G protein-coupled receptor 4 (LGR4), and R-spondins (RSPOs). Methods: Rats were fed a high-phosphorus diet, followed by assessment of the bone microstructure and of the expression of key signalling molecules. Results: Elevated phosphorus intake induced significant bone deterioration, particularly in the trabecular bone compartment, associated with alterations in the RANK/RANKL/OPG pathway and in the LGR4 and RSPO1 and RSPO4 signalling components in bone. Moreover, we also observed changes in RANKL, RSPO1 and RSPO4 serum levels in the rats that had received a high-phosphorus diet. Conclusions: These findings highlight the detrimental impact of excessive dietary phosphorus on skeletal health, even without renal impairment, and suggest that components of this pathway, particularly RSPO1 and RSPO4, could serve as potential biomarkers of bone deterioration. The widespread consumption of phosphorus-rich processed foods underscores the importance of nutritional education to mitigate these skeletal risks in industrialized populations.

This study examined the expression of the renin-angiotensin system (RAS) and inflammatory markers in cardiovascular complications associated with long-term type 1 diabetes (T1D) using a rat model. After 24 weeks of streptozotocin-induced T1D, the animals exhibited metabolic alterations indicative of both cardiac and renal dysfunction. Tissue-specific dysregulation of RAS components and pro-inflammatory markers were observed in the heart, aorta, and perivascular adipose tissue (PVAT). In the heart, there was a significant upregulation of both classical (AT1R, 1.00 (0.22) vs. 1.70 (0.45) R.U.) and counter-regulatory RAS components (ACE2, 1.00 (0.43) vs. 1.96 (0.67) R.U.; p < 0.001) and MasR (1.00 (0.56) vs. 1.33 (0.29) R.U.; p = 0.004). The aorta displayed increased expression of classical RAS components alongside a significant reduction in ACE2 expression (1.00 (0.74) vs. 0.51 (0.48) R.U.; p < 0.032). Notably, PVAT showed a significant overexpression of classical RAS components (ACE 1.00 (0.22) vs. 4.08 (1.32) R.U.; p < 0.001, AT1R 1.00 (0.59) vs. 7.22 (4.14) R.U.; p < 0.001) and MasR (1.00 (0.70) vs. 4.52 (1.91) R.U.; p < 0.001), accompanied by increased expression of TNFα and ADAM17. These findings suggest that long-term T1D induces tissue-specific activation patterns of the RAS and inflammatory pathways within the cardiovascular system, which may contribute to the progression of diabetic cardiovascular complications. Therapeutic targeting of RAS components may represent a viable strategy for mitigating cardiovascular damage in T1D.

The European COSMOS study, which included 6,797 haemodialysis patients from 20 countries, demonstrated that elevated PTH levels were associated with a higher risk of mortality in diabetic patients compared to non-diabetic ones. Elevated PTH levels have been linked to inflammatory processes. This study aimed to analyse, in epigastric arteries of dialysis patients, whether diabetes and elevated PTH levels might synergistically modulate the expression of inflammatory and vascular calcification (VC) markers through ADAM17 and/or its inhibitor, TIMP3. Epigastric artery samples were examined from 62 chronic kidney disease patients, 31 non-diabetic and 31 diabetic with similar characteristics, obtained during kidney transplantation. The gene expression of ADAM17, TIMP3, inflammatory markers (TNFα, VCAM, and ICAM), α-actin, DKK1, and RUNX2, as well as protein expression of RUNX2 and vascular calcification, were evaluated. Analyses were performed based on the presence/absence of diabetes and serum PTH levels (using the median value as the cut-off). Differences between groups were assessed using Chi-squared tests for qualitative variables or Wilcoxon tests for quantitative variables. In the epigastric arteries of diabetic patients, there was lower gene expression of TIMP3 (1.0 (0.8) U.R. vs 0.6 (0.5) U.R.; p = 0.028) and α-actin (1.0 (0.6) U.R. vs 0.7 (0.5) U.R.; p = 0.006), higher gene expression of VCAM (1.0 (0.9) U.R. vs 2.1 (2.8) U.R.; p = 0.011), TNFα (1.0 (1.1) U.R. vs 2.0 (1.9) U.R.; p = 0.008), and RUNX2 (1.0 (0.8) U.R. vs 2.4 (2.8) U.R.; p = 0.009), and a higher percentage of RUNX2-positive nuclei (14.9 (15.8) vs 31.1 (27.1); p = 0.038) compared to non-diabetic patients. Arteries from patients with PTH levels above the median exhibited higher gene expression of ADAM17 (1.0 (0.6) U.R. vs 1.8 (1.6) U.R.; p = 0.026). The increase in ADAM17 expression persisted when only diabetic patients were analysed (1.0 (0.5) U.R. vs 2.9 (2.9) U.R.; p = 0.038), but not in non-diabetic patients. The combination of elevated PTH and diabetes was associated with higher gene expression of RUNX2 (1.0 (0.8) U.R. vs 2.7 (3.3) U.R.; p = 0.019), a higher percentage of RUNX2-positive nuclei (13.3 (17.0) vs 32.9 (27.2); p = 0.039), and a higher Kauppila index (3.9 (5.0) vs 9.7 (7.3); p = 0.045). Although changes in ADAM17 and TIMP3 gene expression were observed in diabetic patients, potentially related to increased synthesis and/or release of pro-inflammatory factors, these do not appear to explain the difference in mortality associated with elevated PTH levels between diabetic and non-diabetic patients observed in the COSMOS study. However, the increased mortality in diabetic patients with elevated PTH may be linked to greater VC progression mediated by RUNX2.