Tweak/Fn14 system is involved in rhabdomyolysis-induced acute kidney injury.

BACKGROUND: Rhabdomyolysis is a severe clinical syndrome associated to acute kidney injury (AKI) and chronic kidney disease (CKD). TWEAK/Fn14 signaling axis regulates renal inflammation and tubular cell death. However, the functional role of TWEAK/Fn14 in rhabdomyolysis remains unknown. METHODS: Rhabdomyolysis was induced in wild-type, TWEAK- and Fn14-deficient mice or mice treated with TWEAK blocking antibody. Renal injury, inflammation, fibrosis and cell death were assessed. Additionally, we performed in vivo and in vitro studies to explore the possible signalling pathways involved in Fn14 regulation. FINDINGS: Fn14 renal expression was increased in mice with rhabdomyolysis, correlating with decline of renal function. Mechanistically, myoglobin (Mb) induced Fn14 expression via ERK and p38 pathway, whereas Nrf2 activation diminished Mb-mediated Fn14 upregulation in cultured renal cells. TWEAK or Fn14 genetic depletion ameliorated rhabdomyolysis-associated loss of renal function, histological damage, tubular cell death, inflammation, and expression of both tubular and endothelial injury markers. Deficiency of TWEAK or Fn14 also decreased long-term renal inflammation and fibrosis in mice with rhabdomyolysis. Finally, pharmacological treatment with a blocking TWEAK antibody diminished the expression of acute renal injury markers and cell death and lessened residual kidney fibrosis and chronic inflammation in rhabdomyolysis. INTERPRETATION: TWEAK/Fn14 axis participates in the pathogenesis of rhabdomyolysis-AKI and subsequent AKI-CKD transition. Blockade of this signaling pathway may represent a promising therapeutic strategy for reducing rhabdomyolysis-mediated renal injury. FUNDING: Spanish Ministry of Science and Innovation, ISCIII and Junta de Andalucía.

What happens under the flexor tendons of the fingers in dactylitis?

AIM: Tenosynovitis is one of the most frequently described inflammatory lesions in psoriatic dactylitis. The aim of the study was to assess by ultrasound the distribution of content within the synovial sheath of the finger flexor tendons in a cadaveric experimental model of tenosynovitis and to describe anatomically the elements of the space between the flexor tendons and the palmar aspect of the proximal phalanx of the fingers. MATERIAL AND METHOD: Silicone was injected under ultrasound guidance into the digital flexor sheath of the index finger of a hand specimen. Ultrasound images of the distribution of the filling of the flexor synovial space with the injected material were obtained. These images were compared with images from patients with psoriatic dactylitis. The palmar regions of the hand and fingers were dissected to check the distribution of the injected silicone in the synovial cavity. Additionally, we dissected the 2nd to 5th fingers of five cadaveric hands, including the one used for the experiment. RESULTS: During the injection of the substance, we observed an increasing homogeneous hypoechoic band around the flexor tendons that differed from the images of patients. Dissection of the specimen showed the injected silicone distributed throughout the digital flexor sheath to the distal interphalangeal joint. In addition, we provided an illustrated anatomical description of the elements located between the flexor tendons and the palmar aspect of the proximal phalanx, the inflammation of which could simulate flexor tenosynovitis. CONCLUSION: The observations of this study may contribute to a better understanding of the anatomical structures involved in PsA dactylitis.

The direct effect of fibroblast growth factor 23 on vascular smooth muscle cell phenotype and function.

BACKGROUND: In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype. METHODS: This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2-3 studying a relationship between FGF23 and vascular dysfunction. RESULTS: In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2-3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222. CONCLUSION: In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.

Role of Toll-like receptor 4 in intravascular hemolysis-mediated injury.

Massive intravascular hemolysis is a common characteristic of several pathologies. It is associated with the release of large quantities of heme into the circulation, promoting injury in vulnerable organs, mainly kidney, liver, and spleen. Heme activates Toll-like receptor 4 (TLR4), a key regulator of the inflammatory response; however, the role of TLR4 in hemolysis and whether inhibition of this receptor may protect from heme-mediated injury are unknown. We induced intravascular hemolysis by injection of phenylhydrazine in wildtype and Tlr4-knockout mice. In this model, we analyzed physiological parameters, histological damage, inflammation and cell death in kidney, liver, and spleen. We also evaluated whether heme-mediated-inflammatory effects were prevented by TLR4 inhibition with the compound TAK-242, both in vivo and in vitro. Induction of massive hemolysis elicited acute kidney injury characterized by loss of renal function, morphological alterations of the tubular epithelium, cell death, and inflammation. These pathological effects were significantly ameliorated in the TLR4-deficient mice and in wildtype mice treated with TAK-242. In vitro studies showed that TAK-242 pretreatment reduced heme-mediated inflammation by inhibiting the TLR4/NF-κB (nuclear factor kappa B) axis. However, analysis in liver and spleen indicated that TLR4 deficiency did not protect against the toxic accumulation of heme in these organs. In conclusion, TLR4 is a key molecule involved in the renal inflammatory response triggered by massive intravascular hemolysis. TLR4 inhibition may be a potential therapeutic approach to prevent renal damage in patients suffering from hemolysis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

6-Shogaol (enexasogoal) treatment improves experimental knee osteoarthritis exerting a pleiotropic effect over immune innate signalling responses in chondrocytes.

BACKGROUND AND PURPOSE: The pathogenesis of osteoarthritis implicates a low-grade inflammation associated to the innate immune system activation. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate osteoarthritis. We studied the preventive effect of 6-shogaol, a potential TLR4 inhibitor, on the treatment of experimental knee osteoarthritis. EXPERIMENTAL APPROACH: Osteoarthritis was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6-shogaol for eight weeks. Cartilage damage, inflammatory mediator presence and disease markers were assessed in joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6-shogaol into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we elucidated 6-shogaol action mechanisms. KEY RESULTS: 6-Shogaol treatment prevented articular cartilage lesions, synovitis and the presence of pro-inflammatory mediators, and disease markers in osteoarthritis animals. Molecular modelling studies predicted 6-shogaol interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6-shogaol reduced LPS-induced TLR4 inflammatory signalling pathways. Besides, MAPK assay demonstrated that 6-shogaol directly inhibits the ERK1/2 phosphorylation activity. CONCLUSION AND IMPLICATIONS: 6-Shogaol evoked a preventive action on cartilage and synovial inflammation in osteoarthritis mice. 6-shogaol effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during osteoarthritis, which proposes it as an attractive drug for osteoarthritis treatments.

Magnesium supplementation reduces inflammation in rats with induced chronic kidney disease.

BACKGROUND: Inflammation is a common feature in chronic kidney disease (CKD) that appears specifically associated with cardiovascular derangements in CKD patients. Observational studies have revealed a link between low Mg levels and inflammation. In this study, we hypothesize that Mg might have a modulatory effect on the inflammation induced under the uraemic milieu. METHODS: In vivo studies were performed in a 5/6 nephrectomized rat model of CKD. Furthermore, a possible direct effect of Mg was addressed through in vitro studies with vascular smooth muscle cells (VSMCs). RESULTS: Uraemic rats fed a normal (0.1%) Mg diet showed a systemic inflammatory response evidenced by the elevation in plasma of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6, and GPx activity, a marker of oxidative stress. Importantly, an increased expression of these cytokines in the aortic tissue was also observed. In contrast, a dietary Mg supplementation (0.6%) greatly prevented the oxidative stress and the pro-inflammatory response. In vitro, in VSMCs cultured in a pro-inflammatory high phosphate medium, incubation with Mg 1.6 mM inhibited the increase in the production of ROS, the rise in the expression of TNF-α, IL-1ß, IL-6 and IL-8 and the activation of NF-κB signalling that was observed in cells incubated with a normal (0.8 mM) Mg. CONCLUSION: Mg supplementation reduced inflammation associated with CKD, exerting a direct effect on vascular cells. These findings support a possible beneficial effect of Mg supplementation along the clinical management of CKD patients.

Pathogenic Pathways and Therapeutic Approaches Targeting Inflammation in Diabetic Nephropathy.

Diabetic nephropathy (DN) is associated with an increased morbidity and mortality, resulting in elevated cost for public health systems. DN is the main cause of chronic kidney disease (CKD) and its incidence increases the number of patients that develop the end-stage renal disease (ESRD). There are growing epidemiological and preclinical evidence about the close relationship between inflammatory response and the occurrence and progression of DN. Several anti-inflammatory strategies targeting specific inflammatory mediators (cell adhesion molecules, chemokines and cytokines) and intracellular signaling pathways have shown beneficial effects in experimental models of DN, decreasing proteinuria and renal lesions. A number of inflammatory molecules have been shown useful to identify diabetic patients at high risk of developing renal complications. In this review, we focus on the key role of inflammation in the genesis and progression of DN, with a special interest in effector molecules and activated intracellular pathways leading to renal damage, as well as a comprehensive update of new therapeutic strategies targeting inflammation to prevent and/or retard renal injury.

Protective Role of Nrf2 in Renal Disease.

Chronic kidney disease (CKD) is one of the fastest-growing causes of death and is predicted to become by 2040 the fifth global cause of death. CKD is characterized by increased oxidative stress and chronic inflammation. However, therapies to slow or prevent CKD progression remain an unmet need. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that plays a key role in protection against oxidative stress and regulation of the inflammatory response. Consequently, the use of compounds targeting Nrf2 has generated growing interest for nephrologists. Pre-clinical and clinical studies have demonstrated that Nrf2-inducing strategies prevent CKD progression and protect from acute kidney injury (AKI). In this article, we review current knowledge on the protective mechanisms mediated by Nrf2 against kidney injury, novel therapeutic strategies to induce Nrf2 activation, and the status of ongoing clinical trials targeting Nrf2 in renal diseases.

Inflammation both increases and causes resistance to FGF23 in normal and uremic rats.

Fibroblast growth factor 23 (FGF23) increases phosphorus excretion and decreases calcitriol (1,25(OH)2D) levels. FGF23 increases from early stages of renal failure. We evaluated whether strict control of phosphorus intake in renal failure prevents the increase in FGF23 and to what extent inflammation impairs regulation of FGF23. The study was performed in 5/6 nephrectomized (Nx) Wistar rats fed diets containing 0.2-1.2% phosphorus for 3 or 15 days. FGF23 levels significantly increased in all Nx groups in the short-term (3-day) experiment. However, at 15 days, FGF23 increased in all Nx rats except in those fed 0.2% phosphorus. In a second experiment, Nx rats fed low phosphorus diets (0.2 and 0.4%) for 15 days received daily intraperitoneal lipopolysaccharide (LPS) injections to induce inflammation. In these rats, FGF23 increased despite the low phosphorus diets. Thus, higher FGF23 levels were needed to maintain phosphaturia and normal serum phosphorus values. Renal Klotho expression was preserved in Nx rats on a 0.2% phosphorus diet, reduced on a 0.4% phosphorus diet, and markedly reduced in Nx rats receiving LPS. In ex vivo experiments, high phosphorus and LPS increased nuclear ß-catenin and p65-NFκB and decreased Klotho. Inhibition of inflammation and Wnt signaling activation resulted in decreased FGF23 levels and increased renal Klotho. In conclusion, strict control of phosphorus intake prevented the increase in FGF23 in renal failure, whereas inflammation independently increased FGF23 values. Decreased Klotho may explain the renal resistance to FGF23 in inflammation. These effects are likely mediated by the activation of NFkB and Wnt/ß-catenin signaling.

Nrf2 Plays a Protective Role Against Intravascular Hemolysis-Mediated Acute Kidney Injury.

Massive intravascular hemolysis is associated with acute kidney injury (AKI). Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a central role in the defense against oxidative stress by activating the expression of antioxidant proteins. We investigated the role of Nrf2 in intravascular hemolysis and whether Nrf2 activation protected against hemoglobin (Hb)/heme-mediated renal damage in vivo and in vitro. We observed renal Nrf2 activation in human hemolysis and in an experimental model of intravascular hemolysis promoted by phenylhydrazine intraperitoneal injection. In wild-type mice, Hb/heme released from intravascular hemolysis promoted AKI, resulting in decreased renal function, enhanced expression of tubular injury markers (KIM-1 and NGAL), oxidative and endoplasmic reticulum stress (ER), and cell death. These features were more severe in Nrf2-deficient mice, which showed decreased expression of Nrf2-related antioxidant enzymes, including heme oxygenase 1 (HO-1) and ferritin. Nrf2 activation with sulforaphane protected against Hb toxicity in mice and cultured tubular epithelial cells, ameliorating renal function and kidney injury and reducing cell stress and death. Nrf2 genotype or sulforaphane treatment did not influence the severity of hemolysis. In conclusion, our study identifies Nrf2 as a key molecule involved in protection against renal damage associated with hemolysis and opens novel therapeutic approaches to prevent renal damage in patients with severe hemolytic crisis. These findings provide new insights into novel aspects of Hb-mediated renal toxicity and may have important therapeutic implications for intravascular hemolysis-related diseases.

Bevacizumab Allows Preservation of Liver Function and its Regenerative Capacity after Major Hepatectomy.

BACKGROUND: Parallel to the safety of liver resections, new chemotherapy drugs have emerged for the control of liver metastases. However, there is unclear evidence about the combination of intensive BVZ-therapy and extended resections. The main aim was to analyse the impact of Bevacizumab (BVZ) in terms of liver safety and tolerability in two experimental models: a basal-toxicity situation and after major hepatectomy. METHODS: Eighty male-Wistar rats were grouped as toxicity analysis (sham-operated rats-OS-) and regeneration after- surgery analysis (hepatectomy rats-H-). Eight further subgroups were created according to sacrifice (6- hours-6h- or 24-hours-24h-) and dose (µg) of BVZ (none, 100, 200, 400). Several measurements were performed, including biochemical serum samples, histopathological analysis, cytokines (IL-6, TNF-α, TGF-ß), oxidative-stress (GSH/GSSG, ATP), lipid-peroxidation (TBARS) and epidermal and vascular endothelium growth-factors (EGF and VEGF). RESULTS: In the toxicity analysis, safe results with BVZ were observed, with no significant differences among the groups. A trend towards a lower oxidative status was observed in the OS 6 h-100, -200 and -400 versus the OS 6 h-none group. Similar results were observed in the hepatectomy model, with stable oxidative-stress-index and IL-6, TNF- α, and TGF- ß levels. Despite higher lipid peroxidation status, overall regeneration was preserved. As expected, VEGF was almost undetectable in BVZ-treated groups after resection, but not in the non-resection group. CONCLUSION: It was concluded that liver status was not impaired by BVZ even at the high-dose. Similarly, liver regeneration after extended hepatectomy in BVZ-treated animals was well-preserved. Extended liver resections may be encouraged in BVZ-treated patients due to its excellent tolerability and good liver regeneration status.

Serum Magnesium is associated with Carotid Atherosclerosis in patients with high cardiovascular risk (CORDIOPREV Study).

This study aimed to ascertain whether there is an independent association between serum magnesium (Mg) and the Carotid Intima-Media Thickness (IMT-CC), a well-accepted atherosclerotic-biomarker surrogate of cardiovascular disease (CVD), in a population with high cardiovascular risk. Serum Mg and traditional atherosclerotic risk factors were recorded in 939 patients (mean age, 59.6 ± 0.3 years, 83.2% men) with coronary heart disease (CHD) enrolled in the CORDIOPREV trial. Serum Mg strongly associated with IMT-CC. Before adjusting for potential confounding factors, IMT-CC decreased by 0.111 ± 0.011 mm per mg/dl increase in serum Mg (p < 0.001). After adjustment, the effect of Mg did not appear mediated through factors related to glucose metabolism, the lipid profile or the mineral metabolism and renal function. Multivariate models showed the lower Mg levels (quartile 1) as a strong independent factor contributing to IMT-CC along with age, sex, SBP, HDL-C, and diuretic use. Logistic regression analysis confirmed the predictive ability of serum Mg to differentiate patients at higher atherosclerotic risk as defined by an IMT-CC ≥ 1.0 mm, yielding a OR for the lower quartile of 10.623 (95%CI 2.311-48.845; P = 0.002) and a ROC-derived cutoff of 1.61 mg/dl. Therefore, our findings outline low serum magnesium as a possible independent risk factor for carotid atherosclerosis.

Glomerular Hematuria: Cause or Consequence of Renal Inflammation?

Glomerular hematuria is a cardinal symptom of renal disease. Glomerular hematuria may be classified as microhematuria or macrohematuria according to the number of red blood cells in urine. Recent evidence suggests a pathological role of persistent glomerular microhematuria in the progression of renal disease. Moreover, gross hematuria, or macrohematuria, promotes acute kidney injury (AKI), with subsequent impairment of renal function in a high proportion of patients. In this pathological context, hemoglobin, heme, or iron released from red blood cells in the urinary space may cause direct tubular cell injury, oxidative stress, pro-inflammatory cytokine production, and further monocyte/macrophage recruitment. The aim of this manuscript is to review the role of glomerular hematuria in kidney injury, the role of inflammation as cause and consequence of glomerular hematuria, and to discuss novel therapies to combat hematuria.

Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis-mediated cell death.

Acute kidney injury is a common complication of rhabdomyolysis. A better understanding of this syndrome may be useful to identify novel therapeutic targets because there is no specific treatment so far. Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death that is involved in renal injury. In this study, we investigated whether ferroptosis is associated with rhabdomyolysis-mediated renal damage, and we studied the therapeutic effect of curcumin, a powerful antioxidant with renoprotective properties. Induction of rhabdomyolysis in mice increased serum creatinine levels, endothelial damage, inflammatory chemokines, and cytokine expression, alteration of redox balance (increased lipid peroxidation and decreased antioxidant defenses), and tubular cell death. Treatment with curcumin initiated before or after rhabdomyolysis induction ameliorated all these pathologic and molecular alterations. Although apoptosis or receptor-interacting protein kinase (RIPK)3-mediated necroptosis were activated in rhabdomyolysis, our results suggest a key role of ferroptosis. Thus, treatment with ferrostatin 1, a ferroptosis inhibitor, improved renal function in glycerol-injected mice, whereas no beneficial effects were observed with the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone or in RIPK3-deficient mice. In cultured renal tubular cells, myoglobin (Mb) induced ferroptosis-sensitive cell death that was also inhibited by curcumin. Mechanistic in vitro studies showed that curcumin reduced Mb-mediated inflammation and oxidative stress by inhibiting the TLR4/NF-κB axis and activating the cytoprotective enzyme heme oxygenase 1. Our findings are the first to demonstrate the involvement of ferroptosis in rhabdomyolysis-associated renal damage and its sensitivity to curcumin treatment. Therefore, curcumin may be a potential therapeutic approach for patients with this syndrome.-Guerrero-Hue, M., García-Caballero, C., Palomino-Antolín, A., Rubio-Navarro, A., Vázquez-Carballo, C., Herencia, C., Martín-Sanchez, D., Farré-Alins, V., Egea, J., Cannata, P., Praga, M., Ortiz, A., Egido, J., Sanz, A. B., Moreno, J. A. Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis-mediated cell death.

Calcimimetics maintain bone turnover in uremic rats despite the concomitant decrease in parathyroid hormone concentration.

Calcimimetics decrease parathyroid hormone (PTH) secretion in patients with secondary hyperparathyroidism. The decrease in PTH should cause a reduction in bone turnover; however, the direct effect of calcimimetics on bone cells, which express the calcium-sensing receptor (CaSR), has not been defined. In this study, we evaluated the direct bone effects of CaSR activation by a calcimimetic (AMG 641) in vitro and in vivo. To create a PTH "clamp," total parathyroidectomy was performed in rats with and without uremia induced by 5/6 nephrectomy, followed by a continuous subcutaneous infusion of PTH. Animals were then treated with either the calcimimetic or vehicle. Calcimimetic administration increased osteoblast number and osteoid volume in normal rats under a PTH clamp. In uremic rats, the elevated PTH concentration led to reduced bone volume and increased bone turnover, and calcimimetic administration decreased plasma PTH. In uremic rats exposed to PTH at 6-fold the usual replacement dose, calcimimetic administration increased osteoblast number, osteoid surface, and bone formation. A 9-fold higher dose of PTH caused an increase in bone turnover that was not altered by the administration of calcimimetic. In an osteosarcoma cell line, the calcimimetic induced Erk1/2 phosphorylation and the expression of osteoblast genes. The addition of a calcilytic resulted in the opposite effect. Moreover, the calcimimetic promoted the osteogenic differentiation and mineralization of human bone marrow mesenchymal stem cells in vitro. Thus, calcimimetic administration has a direct anabolic effect on bone that counteracts the decrease in PTH levels.

Dietary magnesium supplementation prevents and reverses vascular and soft tissue calcifications in uremic rats.

Although magnesium has been shown to prevent vascular calcification in vitro, controlled in vivo studies in uremic animal models are limited. To determine whether dietary magnesium supplementation protects against the development of vascular calcification, 5/6 nephrectomized Wistar rats were fed diets with different magnesium content increasing from 0.1 to 1.1%. In one study we analyzed bone specimens from rats fed 0.1%, 0.3%, and 0.6% magnesium diets, and in another study we evaluated the effect of intraperitoneal magnesium on vascular calcification in 5/6 nephrectomized rats. The effects of magnesium on established vascular calcification were also evaluated in uremic rats fed on diets with either normal (0.1%) or moderately increased magnesium (0.6%) content. The increase in dietary magnesium resulted in a marked reduction in vascular calcification, together with improved mineral metabolism and renal function. Moderately elevated dietary magnesium (0.3%), but not high dietary magnesium (0.6%), improved bone homeostasis as compared to basal dietary magnesium (0.1%). Results of our study also suggested that the protective effect of magnesium on vascular calcification was not limited to its action as an intestinal phosphate binder since magnesium administered intraperitoneally also decreased vascular calcification. Oral magnesium supplementation also reduced blood pressure in uremic rats, and in vitro medium magnesium decreased BMP-2 and p65-NF-κB in TNF-α-treated human umbilical vein endothelial cells. Finally, in uremic rats with established vascular calcification, increasing dietary magnesium from 0.1% magnesium to 0.6% reduced the mortality rate from 52% to 28%, which was associated with reduced vascular calcification. Thus, increasing dietary magnesium reduced both vascular calcification and mortality in uremic rats.

Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are osteoblasts progenitors and a variety of studies suggest that they may play an important role for the health in the field of bone regeneration. Magnesium supplementation is gaining importance as adjuvant treatment to improve osteogenesis, although the mechanisms involving this process are not well understood. The objective of this study was to investigate the effects of magnesium on MSC differentiation. Here we show that in rat bone marrow MSC, magnesium chloride increases MSC proliferation in a dose-dependent manner promoting osteogenic differentiation and mineralization. These effects are reduced by 2-APB administration, an inhibitor of magnesium channel TRPM7. Of note, magnesium supplementation did not increase the canonical Wnt/ß-catenin pathway, although it promoted the activation of Notch1 signaling, which was also decreased by addition of 2-APB. Electron microscopy showed higher proliferation, organization and maturation of osteoblasts in bone decellularized scaffolds after magnesium addition. In summary, our results demonstrate that magnesium chloride enhances MSC proliferation by Notch1 signaling activation and induces osteogenic differentiation, shedding light on the understanding of the role of magnesium during bone regeneration.

Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

In renal failure, hyperphosphatemia occurs despite a marked elevation in serum fibroblast growth factor (FGF)-23. Abnormal regulation of the FGFR1-Klotho receptor complex may cause a resistance to the phosphaturic action of FGF23. The purpose of the present study was to investigate the regulation of renal Klotho and FGF receptor (FEFR)-1 in healthy and uremic rats induced by 5/6 nephrectomy. In normal rats, the infusion of rat recombinant FGF23 enhanced phosphaturia and increased renal FGFR1 expression; however, Klotho expression was reduced. Uremic rats on a high-phosphate (HP) diet presented hyperphosphatemia with marked elevation of FGF23 and an increased fractional excretion of phosphate (P) that was associated with a marked reduction of Klotho expression and an increase in FGFR1. After neutralization of FGF23 by anti-FGF23 administration, phosphaturia was still abundant, Klotho expression remained low, and the FGFR1 level was reduced. These results suggest that the expression of renal Klotho is modulated by phosphaturia, whereas the FGFR1 expression is regulated by FGF23. Calcitriol (CTR) administration prevented a decrease in renal Klotho expression. In HEK293 cells HP produced nuclear translocation of ß-catenin, together with a reduction in Klotho. Wnt/ß-catenin inhibition with Dkk-1 prevented the P-induced down-regulation of Klotho. The addition of CTR to HP medium was able to recover Klotho expression. In summary, high FGF23 levels increase FGFR1, whereas phosphaturia decreases Klotho expression through the activation of Wnt/ß-catenin pathway.-Muñoz-Castañeda, J. R., Herencia, C., Pendón-Ruiz de Mier, M. V., Rodriguez-Ortiz, M. E., Diaz-Tocados, J. M., Vergara, N., Martínez-Moreno, J. M., Salmerón, M. D., Richards, W. G., Felsenfeld, A., Kuro-O, M., Almadén, Y., Rodríguez, M. Differential regulation of renal Klotho and FGFR1 in normal and uremic rats.

High phosphate induces a pro-inflammatory response by vascular smooth muscle cells and modulation by vitamin D derivatives.

In chronic kidney disease patients, high phosphate (HP) levels are associated with cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently correlated with inflammation, the present study aimed to investigate an independent direct effect of HP as a pro-inflammatory factor in VSMCs. A possible modulatory effect of vitamin D (VitD) was also investigated. The study was performed in an in vitro model of human aortic smooth muscle cells (HASMCs). Incubation of cells in an HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators intercellular adhesion molecule 1 (ICAM-1), interleukins (ILs) IL-1ß, IL-6, IL-8 and tumour necrosis factor α (TNF-α) (not corroborated at the protein levels for ICAM-1), as well as an increase in reactive oxygen/nitrogen species (ROS/RNS) production. This was accompanied by the activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signalling as demonstrated by the increase in the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κΒ) assessed by Western blotting and confocal microscopy. Since all these events were attenuated by an antioxidant pre-incubation with the radical scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), it is suggested that the inflammatory response is upstream mediated by the ROS/RNS-induced activation of NF-κΒ. Addition of paricalcitol (PC) 3·10-8 M to cells in HP prevented the phosphate induced ROS/RNS increase, the activation of NF-κΒ and the cytokine up-regulation. A bimodal effect was observed, however, for different calcitriol (CTR) concentrations, 10-10 and 10-12 M attenuated but 10-8 M stimulated this phosphate induced pro-oxidative and pro-inflammatory response. Therefore, these findings provide novel mechanisms whereby HP may directly favour vascular dysfunctions and new insights into the protective effects exerted by VitD derivatives.

Procaine Inhibits Osteo/Odontogenesis through Wnt/ß-Catenin Inactivation.

INTRODUCTION: Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. METHODS: In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. RESULTS: Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/ß-catenin pathway, observing a loss of nuclear ß-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3ß and an increase of phosphorylated ß-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3ß, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3ß and ß-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. CONCLUSIONS: Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/ß-catenin pathway through the increase of Gsk3ß expression and ß-catenin phosphorylation.