Sara Panizo's research while affiliated with University of Alcalá and other places

Background: 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. Methods: 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). Results: 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. Conclusions: 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.

Bone represents a metabolically active tissue subject to continuous remodeling orchestrated by the dynamic interplay between osteoblasts and osteoclasts. These cellular processes are modulated by a complex interplay of biochemical and mechanical factors, which are instrumental in assessing bone remodeling. This comprehensive evaluation aids in detecting disorders arising from imbalances between bone formation and reabsorption. Osteoporosis, characterized by a reduction in bone mass and strength leading to heightened bone fragility and susceptibility to fractures, is one of the more prevalent chronic diseases. Some epidemiological studies, especially in patients with chronic kidney disease (CKD), have identified an association between osteoporosis and vascular calcification. Notably, low bone mineral density has been linked to an increased incidence of aortic calcification, with shared molecules, mechanisms, and pathways between the two processes. Certain molecules emerging from these shared pathways can serve as biomarkers for bone and mineral metabolism. Detecting and evaluating these alterations early is crucial, requiring the identification of biomarkers that are reliable for early intervention. While traditional biomarkers for bone remodeling and vascular calcification exist, they suffer from limitations such as low specificity, low sensitivity, and conflicting results across studies. In response, efforts are underway to explore new, more specific biomarkers that can detect alterations at earlier stages. The aim of this review is to comprehensively examine some of the emerging biomarkers in mineral metabolism and their correlation with bone mineral density, fracture risk, and vascular calcification as well as their potential use in clinical practice.

Shortly after the discovery of Klotho, interest grew in its potential role in chronic kidney disease (CKD). There are three isoforms of the Klotho protein: αKlotho, βKlotho and γKlotho. This review will focus on αKlotho due to its relevance as a biomarker in CKD. αKlotho is synthesized mainly in the kidneys, but it can be released into the bloodstream and urine as soluble Klotho (sKlotho), which undertakes systemic actions, independently or in combination with FGF23. It is usually accepted that sKlotho levels are reduced early in CKD and that lower levels of sKlotho might be associated with the main chronic kidney disease–mineral bone disorders (CKD-MBDs): cardiovascular and bone disease. However, as results are inconsistent, the applicability of sKlotho as a CKD-MBD biomarker is still a matter of controversy. Much of the inconsistency can be explained due to low sample numbers, the low quality of clinical studies, the lack of standardized assays to assess sKlotho and a lack of consensus on sample processing, especially in urine. In recent decades, because of our longer life expectancies, the prevalence of accelerated-ageing diseases, such as CKD, has increased. Exercise, social interaction and caloric restriction are considered key factors for healthy ageing. While exercise and social interaction seem to be related to higher serum sKlotho levels, it is not clear whether serum sKlotho might be influenced by caloric restriction. This review focuses on the possible role of sKlotho as a biomarker in CKD-MBD, highlighting the difference between solid knowledge and areas requiring further research, including the role of sKlotho in healthy ageing.

Vascular calcification (VC) is a common complication in patients with chronic kidney disease which increases their mortality. Although oxidative stress is involved in the onset and progression of this disorder, the specific role of some of the main redox regulators, such as catalase, the main scavenger of H2O2, remains unclear. In the present study, epigastric arteries of kidney transplant recipients, a rat model of VC, and an in vitro model of VC exhibiting catalase (Cts) overexpression were analysed. Pericalcified areas of human epigastric arteries had increased levels of catalase and cytoplasmic, rather than nuclear runt-related transcription factor 2 (RUNX2). In the rat model, advanced aortic VC concurred with lower levels of the H2O2-scavenger glutathione peroxidase 3 compared to controls. In an early model of calcification using vascular smooth muscle cells (VSMCs), Cts VSMCs showed the expected increase in total levels of RUNX2. However, Cts VMSCs also exhibited a lower percentage of the nucleus stained for RUNX2 in response to calcifying media. In this early model of VC, we did not observe a dysregulation of the mitochondrial redox state; instead, an increase in the general redox state was observed in the cytoplasm. These results highlight the complex role of antioxidant enzymes as catalase by regulation of RUNX2 subcellular location delaying the onset of VC.

Vascular calcification (VC), is a common complication in patients with chronic kidney disease and increases mortality. Although oxidative stress is involved in the onset and progression of this disorder, the specific role of some main redox regulators such as catalase, the main scavenger of H2O2, remains unclear. In the present study, epigastric arteries of kidney transplant recipients, an in vivo model of VC and an in vitro model of VC exhibiting catalase overexpression (Cts) were analyzed. Peri-calcification areas of human epigastric arteries have increased levels of catalase and cytoplasmic rather than nuclear RUNX2. In the in vivo model, advanced aortic VC concurs with lower levels of the H2O2 scavenger, glutathione peroxidase 3 compared to controls. In an early model of calcification using vascular smooth muscle cells (VSMCs), Cts VSMCs showed the expected increase in RUNX2 total levels. However, Cts VMSC also exhibited lower percentage of the nucleus stained for RUNX2 in response to calcifying media. At this early model of VC we did not observe dysregulation of mitochondrial redox state, an increase in general redox state was observed in the cytoplasm. These in vivo and in vitro results highlight the complex role of antioxidant enzymes as catalase in the process of VC by regulation of RUNX2 location.

Phosphorus is a vital element for life found in most foods as a natural component, but it is also one of the most used preservatives added during food processing. High serum phosphorus contributes to develop vascular calcification in chronic kidney disease; however, it is not clear its effect in a population without kidney damage. The objective of this in vivo and in vitro study was to investigate the effect of high phosphorus exposure on the aortic and serum levels of miR-145 and its effect on vascular smooth muscle cell (VSMCs) changes towards less contractile phenotypes. The study was performed in aortas and serum from rats fed standard and high-phosphorus diets, and in VSMCs exposed to different concentrations of phosphorus. In addition, miR-145 silencing and overexpression experiments were carried out. In vivo results showed that in rats with normal renal function fed a high P diet, a significant increase in serum phosphorus was observed which was associated to a significant decrease in the aortic α-actin expression which paralleled the decrease in aortic and serum miR-145 levels, with no changes in the osteogenic markers. In vitro results using VSMCs corroborated the in vivo findings. High phosphorus first reduced miR-145, and afterwards α-actin expression. The miR-145 overexpression significantly increased α-actin expression and partially prevented the increase in calcium content. These results suggest that miR-145 could be an early biomarker of vascular calcification, which could give information about the initiation of the transdifferentiation process in VSMCs.

Background: Diabetic patients on haemodialysis have a higher risk of mortality than non-diabetic patients. The aim of this COSMOS analysis was to assess whether bone and mineral laboratory values (calcium, phosphorus, and PTH) contribute to such risk. Methods: COSMOS is a multicentre, open-cohort, 3-year prospective study, which includes 6797 patients from 227 randomly selected dialysis centres from 20 European countries. The association between mortality and calcium, phosphate or PTH was assessed using Cox proportional hazard regression models using both penalized splines smoothing and categorization according to KDIGO guidelines. The effect modification of the association between the relative risk of mortality and serum calcium, phosphate or PTH by diabetes was assessed. Results: There was a statistically significant effect modification of the association between the relative risk of mortality and serum PTH by diabetes (p = 0.011). The slope of the curve of the association between increasing values of PTH and relative risk of mortality was steeper for diabetic compared with non-diabetic patients, mainly for high levels of PTH. In addition, high serum PTH (> 9 times the normal values) was significantly associated with a higher relative risk of mortality in diabetic patients but not in non-diabetic patients (1.53[95%CI:1.07-2.19] and 1.17[95%CI:0.91-1.52]). No significant effect modification of the association between the relative risk of mortality and serum calcium or phosphate by diabetes was found (p = 0.2 and p = 0.059, respectively). Conclusion: The results show a different association of PTH with the relative risk of mortality in diabetic and non-diabetic patients. These findings could have relevant implications for the diagnosis and treatment of CKD-MBD.

Preclinical biomedical models are a fundamental tool to improve the knowledge and management of diseases, particularly in diabetes mellitus (DM) since, currently, the pathophysiological and molecular mechanisms involved in its development are not fully clarified, and there is no treatment to cure DM. This review will focus on the features, advantages and limitations of some of the most used DM models in rats, such as the spontaneous models: Bio-Breeding Diabetes-Prone (BB-DP) and LEW.1AR1-iddm, as representative models of type 1 DM (DM-1); the Zucker diabetic fatty (ZDF) and Goto-kakizaki (GK) rats, as representative models of type 2 DM (DM-2); and other models induced by surgical, dietary and pharmacological—alloxan and streptozotocin—procedures. Given the variety of DM models in rats, as well as the non-uniformity in the protocols and the absence of all the manifestation of the long-term multifactorial complications of DM in humans, the researchers must choose the one that best suits the final objectives of the study. These circumstances, added to the fact that most of the experimental research in the literature is focused on the study of the early phase of DM, makes it necessary to develop long-term studies closer to DM in humans. In this review, a recently published rat DM model induced by streptozotocin injection with chronic exogenous administration of insulin to reduce hyperglycaemia has also been included in an attempt to mimic the chronic phase of DM in humans.

Background and Aims Phosphorus (P) is found in most foods as a natural component, but nowadays, with the rise in the consumption of processed foods, P intake has increased in the developed world. Elevated serum P in patients with chronic kidney disease (CKD) contributes to development of vascular calcification. However, it is poorly understood the relationship between high P dietary intake and vascular calcification in healthy population with normal renal function, and the molecular pathways responsible for the transdifferentiation of vascular smooth muscle cells (VSMCs) that precedes it. Recent studies show that miR-145 is the most abundant microRNA (miR) in VSMCs, and its decrease is associated with loss of contractile phenotype -lower α-actin levels- and increased osteogenic differentiation. In addition, low serum levels of miR-145 has been associated with the presence of vascular calcification. Thus, the aim of the study was to evaluate the effect of high P dietary intake on aortic and serum miR-145 levels, phenotypic alterations of VSMCs and vascular calcification. Method The study was performed: a) in vivo in aortas and serums from rats with normal renal function fed normal P (NP, 0,6% P -n = 10-) and high P diets (HP, 0,9% P -n = 10-), and b) in vitro exposing the VSMCs to different concentrations of P (up to 3 mM), and overexpressing and silencing miR-145 to investigate its potential role in the VSMCs phenotypic differentiation. Results a) In vivo study: The HP group showed higher serum P (NP: 1.29 [1.15-1.35] vs HP:1.40 [1.33-1.48] mmol/L, p = 0.02) and PTH levels (NP: 235.58 ± 75.58 vs HP: 498.67 ± 142.07 pg/ml, p = 0.0001), without significant differences in serum Ca and FGF23 levels. There were not differences in aortic Ca content, although there was a significant reduction in α-actin expression in the HP group (NP: 2.49 [2.10-3.25] vs HP:1.23 [1.04-1.50] R.U., p = 0.002). Moreover, the aortic and serum levels of miR-145 were lower in the HP group: aortic miR-145 (NP: 1.26 [0.97-1.49] vs HP: 0.56 [0.38-0.69] R.U., p = 0.004) and serum miR-145 (NP: 0.84 [0.70-0.99] vs HP: 0.37 [0.31-0.52] R.U., p = 0.001). b) In vitro study: A7r5 cells exposure to high P reduced miR-145 (1mM P: 1.00 [0.88-1.28] vs 3mM P: 0.41 [0.32-0.52], p = 0.001), and α -actin (1mM P: 1.03 [0.96-1.15] vs 3mM P: 0.73 [0.61-0.74], p = 0.0008) levels increasing Ca content in a P concentration-dependent manner (1 mM P: 0.24 [0.00-1.17]; 1.5 mM P: 0.66 [0.47-2.61]; 2 mM P: 2.72 [1.06-4.01]; 2.5 mM P: 4.60 [3.02-4.91]; 3 mM P: 5.81 [5.58-6.34], p = 0.000). the silencing of miR-145 under standard P content (1mM), decreased α -actin (control: 1.02 [0.99-1.04] vs miR-145 antagomir: 0.75 [0.69-0.90] R.U., p = 0.0007), meanwhile its overexpression increased it (control: 1.02 [0.99-1.04] vs miR-145 mimic: 2.70 [2.68-3.07] R.U., p = 0.001). The α -actin levels and Ca content were not modified after silencing miR-145 under high P conditions C (3 mM), meanwhile miR-145 overexpression under high P conditions increased α -actin levels (control: 0.54 [0.41-0.61] vs miR-145 mimic: 1.82 [1.68-2.04] R.U., p = 0.0005), and partially prevented the increases in Ca content (control: 182.77 [176.78-194.84] vs miR-145 mimic: 117.16 [112.27-122.69] R.U., p = 0.0005). Conclusion The results indicate the high P exposure could lead to an impairment of vascular health by causing decrease in miR-145 levels with the consequent reduction in α -actin expression in VSMC cells.

Drugs providing antihypertensive and protective cardiovascular actions are of clinical interest in controlling cardiovascular events and slowing the progression of kidney disease. We studied the effect of a hybrid compound, GGN1231 (derived from losartan in which a powerful antioxidant was attached), on the prevention of cardiovascular damage, cardiac hypertrophy, and fibrosis in a rat model of severe chronic renal failure (CRF). CRF by a 7/8 nephrectomy was carried out in male Wistar rats fed with a diet rich in phosphorous (0.9%) and normal calcium (0.6%) for a period of 12 weeks until sacrifice. In week 8, rats were randomized in five groups receiving different drugs including dihydrocaffeic acid as antioxidant (Aox), losartan (Los), dihydrocaffeic acid+losartan (Aox+Los) and GGN1231 as follows: Group 1 (CRF+vehicle group), Group 2 (CRF+Aox group), Group 3 (CRF+Los group), Group 4 (CRF+Aox+Los group), and Group 5 (CRF+GGN1231 group). Group 5, the CRF+GGN1231 group, displayed reduced proteinuria, aortic TNF-α, blood pressure, LV wall thickness, diameter of the cardiomyocytes, ATR1, cardiac TNF-α and fibrosis, cardiac collagen I, and TGF-β1 expression. A non-significant 20% reduction in the mortality was also observed. This study showed the possible advantages of GGN1231, which could help in the management of cardiovascular and inflammatory processes. Further research is needed to confirm and even expand the positive aspects of this compound.