RANKL, but Not R-Spondins, Is Involved in Vascular Smooth Muscle Cell Calcification through LGR4 Interaction.

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.

Novel Biomarkers of Bone Metabolism.

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.

Soluble Klotho, a Potential Biomarker of Chronic Kidney Disease-Mineral Bone Disorders Involved in Healthy Ageing: Lights and Shadows.

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, ßKlotho 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.

Redox Metabolism and Vascular Calcification in Chronic Kidney Disease.

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.

Phosphorus May Induce Phenotypic Transdifferentiation of Vascular Smooth Muscle Cells through the Reduction of microRNA-145.

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.

Experimental Models to Study Diabetes Mellitus and Its Complications: Limitations and New Opportunities.

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.

Effects of a Losartan-Antioxidant Hybrid (GGN1231) on Vascular and Cardiac Health in an Experimental Model of Chronic Renal Failure.

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-ß1 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.

Serum and Urinary Soluble α-Klotho as Markers of Kidney and Vascular Impairment.

This study was designed to investigate the controversy on the potential role of sKlotho as an early biomarker in Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD), to assess whether sKlotho is a reliable marker of kidney α-Klotho, to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and to evaluate the role of autophagy in this process. Experimental studies were conducted in CKD mice fed a normal phosphorus (CKD+NP) or high phosphorus (CKD+HP) diet for 14 weeks. The patients' study was performed in CKD stages 2-5 and in vitro studies which used VSMCs exposed to non-calcifying medium or calcifying medium with or without sKlotho. The CKD experimental model showed that the CKD+HP group reached the highest serum PTH, P and FGF23 levels, but the lowest serum and urinary sKlotho levels. In addition, a positive correlation between serum sKlotho and kidney α-Klotho was found. CKD mice showed aortic osteogenic differentiation, together with increased autophagy. The human CKD study showed that the decline in serum sKlotho is previous to the rise in FGF23. In addition, both serum sKlotho and FGF23 levels correlated with kidney function. Finally, in VSMCs, the addition of sKlotho prevented osteogenic differentiation and induced autophagy. It can be concluded that serum sKlotho was the earliest CKD-MBD biomarker, a reliable indicator of kidney α-Klotho and that might protect against osteogenic differentiation by increasing autophagy. Nevertheless, further studies are needed to investigate the mechanisms of this possible protective effect.

Role of Klotho and AGE/RAGE-Wnt/ß-Catenin Signalling Pathway on the Development of Cardiac and Renal Fibrosis in Diabetes.

Fibrosis plays an important role in the pathogenesis of long-term diabetic complications and contributes to the development of cardiac and renal dysfunction. The aim of this experimental study, performed in a long-term rat model, which resembles type 1 diabetes mellitus, was to investigate the role of soluble Klotho (sKlotho), advanced glycation end products (AGEs)/receptor for AGEs (RAGE), fibrotic Wnt/ß-catenin pathway, and pro-fibrotic pathways in kidney and heart. Diabetes was induced by streptozotocin. Glycaemia was maintained by insulin administration for 24 weeks. Serum and urine sKlotho, AGEs, soluble RAGE (sRAGE) and biochemical markers were studied. The levels of Klotho, RAGEs, ADAM10, markers of fibrosis (collagen deposition, fibronectin, TGF-ß1, and Wnt/ß-catenin pathway), hypertrophy of the kidney and/or heart were analysed. At the end of study, diabetic rats showed higher levels of urinary sKlotho, AGEs and sRAGE and lower serum sKlotho compared with controls without differences in the renal Klotho expression. A significant positive correlation was found between urinary sKlotho and AGEs and urinary albumin/creatinine ratio (uACR). Fibrosis and RAGE levels were significantly higher in the heart without differences in the kidney of diabetic rats compared to controls. The results also suggest the increase in sKlotho and sRAGE excretion may be due to polyuria in the diabetic rats.

MicroRNA-145 and microRNA-486 are potential serum biomarkers for vascular calcification.

INTRODUCTION: MicroRNAs (miRs) regulate vascular calcification (VC), and their quantification may contribute to suspicion of the presence of VC. METHODS: The study was performed in four phases. Phase 1: miRs sequencing of rat calcified and non-calcified aortas. Phase 2: miRs with the highest rate of change, plus miR-145 [the most abundant miR in vascular smooth muscle cells (VSMCs)], were validated in aortas and serum from rats with and without VC. Phase 3: the selected miRs were analyzed in epigastric arteries from kidney donors and recipients, and serum samples from general population. Phase 4: VSMCs were exposed to different phosphorus concentrations, and miR-145 and miR-486 were overexpressed to investigate their role in VC. RESULTS: miR-145, miR-122-5p, miR-486 and miR-598-3p decreased in the rat calcified aortas, but only miR-145 and miR-486 were detected in serum. In human epigastric arteries, miR-145 and miR-486 were lower in kidney transplant recipients compared with donors. Both miRs inversely correlated with arterial calcium content and with VC (Kauppila index). In the general population, the severe VC was associated with the lowest serum levels of both miRs. The receiver operating characteristic curve showed that serum miR-145 was a good biomarker of VC. In VSMCs exposed to high phosphorus, calcium content, osteogenic markers (Runx2 and Osterix) increased, and the contractile marker (α-actin), miR-145 and miR-486 decreased. Overexpression of miR-145, and to a lesser extent miR-486, prevented the increase in calcium content induced by high phosphorus, the osteogenic differentiation and the loss of the contractile phenotype. CONCLUSION: miR-145 and miR-486 regulate the osteogenic differentiation of VSMCs, and their quantification in serum could serve as a marker of VC.

Contribución de fósforo y PTH al desarrollo de hipertrofia y fibrosis cardíaca en un modelo experimental de insuficiencia renal crónica / Contribution of phosphorus and PTH to the development of cardiac hypertrophy and fibrosis in an experimental model of chronic renal failure

Antecedentes y objetivo: Mantener niveles adecuados de fósforo sérico en el paciente con enfermedad renal crónica es fundamental para su correcto manejo clínico. Sin embargo resulta difícil su control de forma aislada porque normalmente se asocian con aumentos séricos de hormona paratiroidea (PTH). En el presente estudio se analizaron los efectos de la hiperfosfatemia aislada, en presencia de PTH elevada y normal, sobre la inflamación, hipertrofia y fibrosis cardíaca en un modelo de insuficiencia renal experimental. Materiales y métodos: Se formaron cuatro grupos de ratas. A dos grupos se les realizó paratiroidectomía total (PTx). A las ratas con Ca < 7,5 mg/dL y PTH < 50 pg/mL se les realizó nefrectomía 7/8 (IRC) y se les colocó un pellet subcutáneo que libera PTH 1-34 (5 μg/kg/día). Un grupo recibió dieta con P normal (PN) (grupo IRC + PTx + rPTH + PN) y otro dieta con P alto (0,9% PA) (grupo IRC + PTx + rPTH + PA). Otros dos grupos que solo tenían IRC recibieron dieta PN (IRC + PN) y PA (IRC + PA). Se añadió también un grupo SHAM para nefrectomía y paratiroidectomía. Tras 14 semanas las ratas fueron sacrificadas. Resultados: Los grupos con dieta alta en fósforo (IRC + PA e IRC + PTx + rPTH + PA) tuvieron una reducción significativa del aclaramiento de creatinina y también del peso corporal, con un aumento del fósforo sérico independientemente de la paratiroidectomía, pero no así con los niveles séricos de calcio, FGF23 y de calcitriol que fueron 2-3 veces superiores en el grupo con hiperparatiroidismo secundario (IRC + PA). El diámetro de los cardiomiocitos fue superior en el grupo IRC + PA, mientras la paratiroidectomía (IRC + PTx + rPTH + PA) los redujo significativamente, a pesar de los elevados y similares valores de fósforo sérico. El TNF-α, Adam17 y la fibrosis cardíaca a nivel histológico y molecular mostraron un patrón similar con aumentos en el grupo con hiperparatiroidismo secundario severo (IRC + PA). (AU)

Background and objective: Adequate serum phosphorus levels in patients with chronic kidney disease is essential for their clinical management. However, the control of hyperphosphatemia is difficult because is normally associated with increases in serum PTH. In the present study, the effects of hyperphosphatemia, in the presence of elevated and normal PTH, on cardiac inflammation, hypertrophy and fibrosis in an experimental renal failure model were analyzed. Materials and methods: Four groups of rats were formed. Two groups underwent total parathyroidectomy (PTx). Rats with Ca < 7.5 mg/dL and PTH < 50 pg/mL underwent 7/8 nephrectomy (CRF) and a subcutaneous pellet was placed that releases PTH 1-34 (5 μg/kg/day). One group received a diet with normal P (NP) (CRF + PTx + rPTH + NP group) and another with a high P diet (0.9% HP) (CRF + PTx + rPTH + HP group). Other two groups that only had CRF received NP (CRF + NP) and HP (CRF + HP) diet. A SHAM group for nephrectomy and parathyroidectomy was also added. After 14 weeks the rats were sacrificed. Results: The groups with a diet high in phosphorus (CRF + H A and CRF + PTx + rPTH + HP) had a significant reduction in creatinine clearance and also in body weight with an increase in serum phosphorus regardless of parathyroidectomy, but not serum levels of calcium, FGF23 and calcitriol that were 2-3 times higher in the group with secondary hyperparathyroidism (CRF + HP). The diameter of the cardiomyocytes was greater in the CRF + HP group, while parathyroidectomy (CRF + PTx + rPTH + HP) significantly reduced them, despite the high and similar serum phosphorus values. TNF-α, Adam17 and cardiac fibrosis at the histological and molecular level showed a similar pattern with increases in the group with severe secondary hyperparathyroidism (CRF + HP). (AU)

Contribution of phosphorus and PTH to the development of cardiac hypertrophy and fibrosis in an experimental model of chronic renal failure. / Contribución de fósforo y PTH al desarrollo de hipertrofia y fibrosis cardíaca en un modelo experimental de insuficiencia renal crónica.

BACKGROUND AND OBJECTIVE: Adequate serum phosphorus levels in patients with chronic kidney disease is essential for their clinical management. However, the control of hyperphosphatemia is difficult because is normally associated with increases in serum PTH. In the present study, the effects of hyperphosphatemia, in the presence of elevated and normal PTH, on cardiac inflammation, hypertrophy and fibrosis in an experimental renal failure model were analyzed. MATERIALS AND METHODS: Four groups of rats were formed. Two groups underwent total parathyroidectomy (PTx). Rats with Ca < 7.5 mg/dL and PTH < 50 pg/mL underwent 7/8 nephrectomy (CRF) and a subcutaneous pellet was placed that releases PTH 1-34 (5 µg/kg/day). One group received a diet with normal P (NP) (CRF + PTx + rPTH + NP group) and another with a high P diet (0.9% HP) (CRF + PTx + rPTH + HP group). Other two groups that only had CRF received NP (CRF + NP) and HP (CRF + HP) diet. A SHAM group for nephrectomy and parathyroidectomy was also added. After 14 weeks the rats were sacrificed. RESULTS: The groups with a diet high in phosphorus (CRF + H A and CRF + PTx + rPTH + HP) had a significant reduction in creatinine clearance and also in body weight with an increase in serum phosphorus regardless of parathyroidectomy, but not serum levels of calcium, FGF23 and calcitriol that were 2-3 times higher in the group with secondary hyperparathyroidism (CRF + HP). The diameter of the cardiomyocytes was greater in the CRF + HP group, while parathyroidectomy (CRF + PTx + rPTH + HP) significantly reduced them, despite the high and similar serum phosphorus values. TNF-α, Adam17 and cardiac fibrosis at the histological and molecular level showed a similar pattern with increases in the group with severe secondary hyperparathyroidism (CRF + HP). CONCLUSIONS: Hyperphosphatemia confirmed its importance in the genesis of secondary hyperparathyroidism, but also of kidney damage that was independent of PTH levels. However, inflammation, fibrosis, and cardiomyocyte growth were more closely related to PTH levels, since in the presence of similar severe hyperphosphatemia, parathyroidectomy reduced the values of inflammatory parameters, cardiac hypertrophy, and fibrosis.

Role of the RANK/RANKL/OPG and Wnt/ß-Catenin Systems in CKD Bone and Cardiovascular Disorders.

In the course of chronic kidney disease (CKD), alterations in the bone-vascular axis augment the risk of bone loss, fractures, vascular and soft tissue calcification, left ventricular hypertrophy, renal and myocardial fibrosis, which markedly increase morbidity and mortality rates. A major challenge to improve skeletal and cardiovascular outcomes in CKD patients requires a better understanding of the increasing complex interactions among the main modulators of the bone-vascular axis. Serum parathyroid hormone (PTH), phosphorus (P), calcium (Ca), fibroblast growth factor 23 (FGF23), calcidiol, calcitriol and Klotho are involved in this axis interact with RANK/RANKL/OPG system and the Wnt/ß-catenin pathway. The RANK/RANKL/OPG system controls bone remodeling by inducing osteoblast synthesis of RANKL and downregulating OPG production and it is also implicated in vascular calcification. The complexity of this system has recently increased due the discovery of LGR4, a novel RANKL receptor involved in bone formation, but possibly also in vascular calcification. The Wnt/ß-catenin pathway plays a key role in bone formation: when this pathway is activated, bone is formed, but when it is inhibited, bone formation is stopped. In the progression of CKD, a downregulation of the Wnt/ß-catenin pathway has been described which occurs mainly through the not coincident elevations of sclerostin, Dickkopf1 (Dkk1) and the secreted Frizzled Related Proteins (sFRPs). This review analyzes the interactions of PTH, P, Ca, FGF23, calcidiol, calcitriol and Klotho with the RANKL/RANKL/OPG system and the Wnt/ß-catenin, pathway and their implications in bone and cardiovascular disorders in CKD.

Effects of calcitriol and paricalcitol on renal fibrosis in CKD.

BACKGROUND: In chronic kidney disease, the activation of the renin-angiotensin-aldosterone system (RAAS) and renal inflammation stimulates renal fibrosis and the progression to end-stage renal disease. The low levels of vitamin D receptor (VDR) and its activators (VDRAs) contribute to worsen secondary hyperparathyroidism and renal fibrosis. METHODS: The 7/8 nephrectomy model of experimental chronic renal failure (CRF) was used to examine the anti-fibrotic effects of treatment with two VDRAs, paricalcitol and calcitriol, at equivalent doses (3/1 dose ratio) during 4 weeks. RESULTS: CRF increased the activation of the RAAS, renal inflammation and interstitial fibrosis. Paricalcitol treatment reduced renal collagen I and renal interstitial fibrosis by decreasing the activation of the RAAS through renal changes in renin, angiotensin receptor 1 (ATR1) and ATR2 mRNAs levels and renal inflammation by decreasing renal inflammatory leucocytes (CD45), a desintegrin and metaloproteinase mRNA, transforming growth factor beta mRNA and protein, and maintaining E-cadherin mRNA levels. Calcitriol showed similar trends without significant changes in most of these biomarkers. CONCLUSIONS: Paricalcitol effectively attenuated the renal interstitial fibrosis induced by CRF through a combination of inhibitory actions on the RAAS, inflammation and epithelial/mesenchymal transition.

Influence of Kv11.1 (hERG1) K+ channel expression on DNA damage induced by the genotoxic agent methyl methanesulfonate.

Besides their crucial role in cell electrogenesis and maintenance of basal membrane potential, the voltage-dependent K+ channel Kv11.1/hERG1 shows an essential impact in cell proliferation and other processes linked to the maintenance of tumour phenotype. To check the possible influence of channel expression on DNA damage responses, HEK293 cells, treated with the genotoxic agent methyl methanesulfonate (MMS), were compared with those of a HEK-derived cell line (H36), permanently transfected with the Kv11.1-encoding gene, and with a third cell line (T2) obtained under identical conditions as H36, by permanent transfection of another unrelated plasma membrane protein encoding gene. In addition, to gain some insights about the canonical/conduction-dependent channel mechanisms that might be involved, the specific erg channel inhibitor E4031 was used as a tool. Our results indicate that the expression of Kv11.1 does not influence MMS-induced changes in cell cycle progression, because no differences were found between H36 and T2 cells. However, the canonical ion conduction function of the channel appeared to be associated with decreased cell viability at low/medium MMS concentrations. Moreover, direct DNA damage measurements, using the comet assay, demonstrated for the first time that Kv11.1 conduction activity was able to modify MMS-induced DNA damage, decreasing it particularly at high MMS concentration, in a way related to PARP1 gene expression. Finally, our data suggest that the canonical Kv11.1 effects may be relevant for tumour cell responses to anti-tumour therapies.

Contribution of phosphorus and PTH to the development of cardiac hypertrophy and fibrosis in an experimental model of chronic renal failure.

BACKGROUND AND OBJECTIVE: Adequate serum phosphorus levels in patients with chronic kidney disease is essential for their clinical management. However, the control of hyperphosphatemia is difficult because is normally associated with increases in serum PTH. In the present study, the effects of hyperphosphatemia, in the presence of elevated and normal PTH, on cardiac inflammation, hypertrophy and fibrosis in an experimental renal failure model were analyzed. MATERIALS AND METHODS: 4 groups of rats were formed. Two groups underwent total parathyroidectomy (PTx). Rats with Ca <7.5 mg/dL and PTH < 50 pg/mL underwent 7/8 nephrectomy (CRF) and a subcutaneous pellet was placed that releases PTH 1-34 (5 µg/kg/day). One group received a diet with normal P (NP) (CRF + PTx + rPTH + NP group) and another with a high P diet (0.9% - HP) (CRF + PTx + rPTH + HP group). Other 2 groups that only had CRF received NP (CRF + NP) and HP (CRF + HP) diet. A SHAM group for nephrectomy and parathyroidectomy was also added. After 14 weeks the rats were sacrificed. RESULTS: The groups with a diet high in phosphorus (CRF + H A and CRF + PTx + rPTH + HP) had a significant reduction in creatinine clearance and also in body weight with an increase in serum phosphorus regardless of parathyroidectomy, but not serum levels of calcium, FGF23 and calcitriol that were 2-3 times higher in the group with secondary hyperparathyroidism (CRF + HP). The diameter of the cardiomyocytes was greater in the CRF + HP group, while parathyroidectomy (CRF + PTx + rPTH + HP) significantly reduced them, despite the high and similar serum phosphorus values. TNF-α, Adam17 and cardiac fibrosis at the histological and molecular level showed a similar pattern with increases in the group with severe secondary hyperparathyroidism (CRF + HP). CONCLUSIONS: Hyperphosphatemia confirmed its importance in the genesis of secondary hyperparathyroidism, but also of kidney damage that was independent of PTH levels. However, inflammation, fibrosis, and cardiomyocyte growth were more closely related to PTH levels, since in the presence of similar severe hyperphosphatemia, parathyroidectomy reduced the values ​​of inflammatory parameters, cardiac hypertrophy, and fibrosis.