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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.

Controlling the excessive fracture burden in patients with chronic kidney disease (CKD) Stages G4–G5D remains an impressive challenge. The reasons are 2-fold. First, the pathophysiology of bone fragility in patients with CKD G4–G5D is complex and multifaceted, comprising a mixture of age-related (primary male/postmenopausal), drug-induced and CKD-related bone abnormalities. Second, our current armamentarium of osteoporosis medications has not been developed for, or adequately studied in patients with CKD G4–G5D, partly related to difficulties in diagnosing osteoporosis in this specific setting and fear of complications. Doubts about the optimal diagnostic and therapeutic approach fuel inertia in daily clinical practice. The scope of the present consensus paper is to review and update the assessment and diagnosis of osteoporosis in patients with CKD G4-G5D and to discuss the therapeutic interventions available and the manner in which these can be used to develop management strategies for the prevention of fragility fracture. As such, it aims to stimulate a cohesive approach to the management of osteoporosis in patients with CKD G4–G5D to replace current variations in care and treatment nihilism.

Background and Aims Bone fragility fractures (bone fractures) are extremely frequent in haemodialysis (HD) patients. Serum phosphate (P) has been suggested as a risk factor for bone fracture, nonetheless, evidence is poor. The aim of this study was to assess the association between incidence of bone fractures and serum phosphate (P), calcium (Ca) and parathyroid hormone (PTH) in patients from the COSMOS study. Method COSMOS is an observational, prospective, open cohort study with 3 years of follow-up including 6797 haemodialysis patients from 227 centres randomly selected from 20 European countries. At baseline, demographics, comorbidities, treatments, serum biochemical parameters of the previous six months and bone fractures of the previous 12 months were collected. Every 6 months, all these variables, outcomes, and incident bone fractures, were collected. Patients who had at least one bone fracture during follow-up were compared with those who had not. Multivariate binary logistic regression and Poisson regression were used to assess the association between incidence of bone fractures and serum P, Ca and PTH. Time to fracture (and possible re-fracture) was also evaluated using Cox regression and Cox regression for recurrent events. All the results were adjusted by 23 variables including Ca, P, PTH, albumin and haemoglobin (full adjusted model). Results Analysis included 6274 patients who had follow-up data and non-missing information regarding bone fractures at baseline and during follow-up; 252 patients (4.0%), suffered at least one incident bone fracture. The fractured patients were older (68.1±12.9 vs. 63.8±14.5 years, p Multivariate binary logistic regression showed that “well known risk factors for bone fracture”, such as a previous bone fracture (OR: 7.78[95%CI:4.83-12.55], p 800 pg/mL (OR: 1.60[95%CI: 1.01-2.55], p=0.047), were associated with a higher incidence of follow-up bone fractures. In addition to the “well known bone risk factors for fracture”, serum P > 6.1 mg/dL (OR: 1.50[95% CI: 1.07-2.11], p=0.02) and serum Ca > 9.7 mg/dL (OR: 1.42[95%CI: 1.01-2.03], p=0.043), were also associated with a higher incidence of bone fractures in the full-adjusted model. These findings were partly consistent with Poisson regression for serum P >6.1 mg/dL (IRR: 1.46[95% CI: 1.00-2.13], p=0.0085) and serum Ca > 9.7 mg/dL (IRR: 1.49[95%CI: 1.06-2.08], p=0.0047), but not for PTH. In addition, Cox regression analysis showed association between bone fractures and serum P >6.1 mg/dl (HR: 1.61 [95%CI: 1.16-2.24], p=0.0049) and 1.60 [95%CI: 1.14-2.24] (p=0.0066) for simple and recurrent events respectively, but no association was found with serum Ca and PTH in the 2 full-adjusted models for the Cox regression analyses. Conclusion In COSMOS, a large haemodialysis patient’s cohort with 3 years follow up, serum P was the only biochemical parameter that remained as a significant risk factor for bone fractures after 4 statistic approaches. Thus, for the first time, in a large scale well controlled study, high serum P has been identified as a new independent potential risk factor for incident bone fractures in haemodialysis patients.

BackgroundMutations in Melanoma Antigen-encoding Gene D2 (MAGED2) promote tubular dysfunction, suggesting that MAGE proteins may play a role in kidney pathophysiology. We have characterized the expression and regulation of MAGE genes in normal kidneys and during kidney disease.MethodsThe expression of MAGE genes and their encoded proteins was explored by systems biology multi-omics (kidney transcriptomics and proteomics) in healthy adult murine kidneys and following induction of experimental acute kidney injury (AKI) by a folic acid overdose. Changes in kidney expression during nephrotoxic AKI were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry. Factors regulating gene expression were studied in cultured tubular cells.ResultsFive MAGE genes (MAGED1, MAGED2, MAGED3, MAGEH1, MAGEE1) were expressed at the mRNA level in healthy adult mouse kidneys, as assessed by RNA-Seq. Additionally, MAGED2 was significantly upregulated during experimental AKI as assessed by array transcriptomics. Kidney proteomics also identified MAGED2 as upregulated during AKI. The increased kidney expression of MAGED2 mRNA and protein was confirmed by qRT-PCR and western blot, respectively, in murine folic acid- and cisplatin-induced AKI. Immunohistochemistry located MAGED2 to tubular cells in experimental and human kidney injury. Tubular cell stressors [serum deprivation and the inflammatory cytokine tumour necrosis factor-like weak inducer of apoptosis (TWEAK)] upregulated MAGED2 in cultured tubular cells.ConclusionsMAGED2 is upregulated in tubular cells in experimental and human kidney injury and is increased by stressors in cultured tubular cells. This points to a role of MAGED2 in tubular cell injury during kidney disease that should be dissected by carefully designed functional approaches.

Background and Aims Massive intravascular hemolysis is a common condition of several pathologies. It is associated with acute kidney injury (AKI) and progressive impairment of renal function. In this context, free hemoglobin (Hb) can exert harmful effects by accumulating in the kidney, where induces oxidative stress and it becomes cytotoxic. NADPH oxidase 4 (Nox4) is the principal source of reactive oxygen species (ROS) in the kidney. Nox4 is mostly expressed in proximal tubular cells with lower levels in glomerulus. The role of Nox4 in renal damage is not clear, with studies reporting beneficial or deleterious actions depending of the environmental conditions. For that reason we aimed to investigate the role of Nox4 in massive intravascular hemolysis-associated AKI. Method To study the role of Nox4 in AKI caused by massive intravascular hemolysis, we performed an experimental model of intravascular hemolysis by intraperitoneal injection of phenylhydrazine (200 mg/kg) in wild type (Nox4+/+) and Nox4 knockout mice (Nox4-/-). Mice were sacrificed 24 and 72 hours after intravascular hemolysis induction. We collected serum, urine and tissues sample. We analyzed renal function, oxidative stress, cell death and inflammation in these samples. In other experiments, wild type mice were treated with GKT137831 (10mg/kg/day), a potent Nox4 and Nox1 inhibitor, and mice were sacrificed 72h after induction of hemolysis. We also performed in vitro experiments in murine tubular epithelial cells (MCT) and murine podocytes cells to investigate the regulation of Nox4 in Hb-stimulated cells treated or not with GKT137831. Results Induction of intravascular hemolysis in Nox4+/+ mice increased creatinine and BUN levels and enhanced the expression of tubular injury markers, such as NGAL. These pathological effects were reduced in Nox4 knockout mice. Then, we analyzed oxidative stress in our experimental model thought determination of HO-1, ferritin, GSH and lipid peroxidation levels. All of these oxidative markers were reduced in Nox4-/- mice with intravascular hemolysis as compared with Nox4+/+ mice. We also observed that inflammatory markers such as IL-6, cell death and podocytes injury markers were reduced in Nox4-/- mice than in wild type mice, specially 72 hours after phenylhydrazine injection. In line with these results, GKT137831 administration ameliorated intravascular hemolysis-associated renal function impairment. Moreover, oxidative stress, tubular injury markers and podocyte injury were reduced in hemolytic mice treated with GKT137831. GKT137831 also reduced Hb- and heme-mediated oxidative stress in MCT and podocytes. Conclusion Our results show the important role of Nox4 in renal injury associated to massive intravascular hemolysis. Moreover, the inhibition of Nox4 may be a potential therapeutic target to prevent renal damage associated to Hb accumulation. These findings provide new insights into novel aspects of Hb-toxicity and may have important pathogenic and therapeutic implications for intravascular hemolysis related diseases

Background and Aims Rhabdomyolysis is characterized by the breakdown of the skeletal muscle and the subsequent myoglobin (Mb) release into the bloodstream. A common complication of this syndrome is acute renal injury (AKI). Once filtered by the kidney, Mb causes oxidative stress, inflammation and tubular cell death. There is no specific treatment for rhabdomyolysis-AKI, so it is crucial a better understanding of this syndrome to identify new therapeutic targets. Klotho is an anti-aging protein mostly expressed by the kidney. In addition to its functions in the regulation of mineral metabolism, Klotho protects from AKI-harmful effects. However, no previous studies analyzed the role of Klotho in rhabdomyolysis. Method We performed a pre-clinical model of rhabdomyolysis in C57BL/6J mice (male, 12 weeks old, n=30) by intramuscular injection of 10 ml/kg of 50% glycerol (≥99.5% m/v). Mice were sacrificed 3 and 6 hours or 1, 3, 7 and 30 days after glycerol administration. To evaluate to beneficial effect of Klotho in rhabdomyolysis, C57BL/6J mice were injected intraperitoneally with 0.1 mg/kg recombinant mouse Klotho (1819-KL, R&D Systems), or vehicle (PBS) 30 minutes before and 1, 3 and 5 days after glycerol injection. Blood, urine and renal samples were collected to analyze renal function, Klotho/FGF23 levels, oxidative stress, inflammation, fibrosis and cell death, all of them pathological processes affecting Klotho expression. In addition, we carried out studies in murine tubular cells (MCTs) to study the molecular mechanisms involved in Klotho regulation. Results Our results indicate that rhabdomyolysis induces an early decrease in Klotho renal mRNA and protein expression as well as Klotho serum levels. Klotho levels decreased in line with augmentation of creatinine concentration, kidney inflammation (CCL2 and IL-6 mRNA expression) and tubular injury marker NGAL. Moreover, patients with rhabdomyolysis-AKI also showed lower plasma Klotho levels and increased FGF23 plasma concentration than age-matched healthy individuals. Renal klotho protein expression remained reduced one month after rhabdomyolysis-induction, in line with long term renal fibrosis and pro-inflammatory macrophage accumulation (F4/80+ cells). Exogenous recombinant Klotho administration ameliorated renal function and reduced rhabdomyolysis-mediated tubular cell death oxidative stress (4-HNE staining) and tubular injury 24h after glycerol injection. In the same line, Klotho administration during AKI development reduced long term renal fibrosis and macrophage infiltration one month later. Antioxidant therapies with N-acetylcysteine (NAC) and sulforaphane, a potent Nuclear factor erythroid-2-related factor 2 (Nrf2) inducer, reduced Mb-mediated Klotho decrease in cultured tubular cells. Inhibition of TNF-α and IL-6 with infliximab and tocilizumab, respectively, also reverted Mb-mediated Klotho decrease. Inhibition of the inflammatory NFkB and p38 pathways also prevented Mb-mediated Klotho reduction. Conclusion Our findings are the first to demonstrate decreased renal and soluble Klotho levels not only in the early phases of rhabdomyolysis-induced AKI, but also when renal function was recovered, indicating that long-term consequences of AKI, such as inflammation and fibrosis, are also involved in Klotho downregulation. In addition, our results also indicate that Klotho administration may be a potential strategy to decrease rhabdomyolysis- long term negative effects.

Background and Aims The stratification of early cardiovascular (CV) risk is a major unmet need in chronic kidney disease (CKD). Since angiogenic T-cells are emerging mediators of vascular homeostasis and have been linked to CV disease (CVD), this study evaluated the frequency of circulating angiogenic T-cells in CKD patients undergoing peritoneal dialysis (CKD-5D) and their potential as predictive biomarkers of CVD. Method In 30 peritoneal dialysis patients and 16 matched healthy controls (HC), we quantified: Vascular calcification (by Kauppila Index); left carotid intima media thickness (lc-IMT by ultrasound), carotid adventitial vasa vasorum density (an early marker of atherosclerosis stage, by Superb Microvascular Ultrasound Image), pulse wave velocity (a marker of vascular stiffness and dysfunction, using the Complior Analyzer), angiogenic T cell frequency in blood (by Flow cytometry) and the traditional 10-year CVD risk (using the SCORE chart). Results As expected, CKD-5D patients exhibited higher Kauppila index (p0.05) and did not correlate with Kauppila (r=-0.350, p=0.080) or lcIMT (r=-0.154, p=0.600), but strongly associated negatively with both pulse wave velocity (r=-0.734, p Conclusion Angiogenic-T cell frequency was decreased in CKD-5D patients and could be considered as biomarkers of vascular dysfunction and early atherosclerosis stage.

Background and Aims Diabetes is the most common cause of chronic kidney disease (CKD) stage 5 and a relevant risk factor for mortality. The available CKD-MBD guidelines do not make differences between diabetics and non-diabetics regarding the optimal targets of the serum bone and mineral biochemical parameters. Thus, the objective of this analysis of COSMOS was to compare the association between the main serum bone and mineral biochemical parameters (calcium-Ca, phosphorus-P and parathyroid hormone-PTH) and mortality in diabetic and non-diabetic patients in haemodialysis. Method COSMOS is a 3-year, observational, prospective, open-cohort study that includes 6797 patients from 227 dialysis centres that were randomly selected from 20 European countries. Cox proportional hazard regression models with penalized splines smoothing were used to analyse the association between relative risk of mortality vs. Ca, P or PTH. Hazard ratios were adjusted by demographics, comorbidities, treatments and biochemical parameters. Interaction analysis was used to analyse if diabetes can influence the association between relative risk of mortality and Ca, P and PTH. Results The present analysis includes 6306 patients with follow-up data and accurate information on diabetes status. The mortality rate in COSMOS was 13.3 deaths per 100 patient-years (17.8 in diabetics and 11.4 in non-diabetics). At baseline, diabetic patients were older (66.7 ± 11.7 vs. 62.8 ± 15.3 years, p Conclusion These results suggest for the first time a differential effect of diabetes on the association between relative risk of mortality and serum PTH. High serum PTH predicts death only in diabetics. These findings could have relevant implications for the diagnosis and treatment of CKD-MBD in diabetic and non-diabetic patients in haemodialysis.

Background Uraemic cardiomyopathy, a process mainly associated with increased myocardial fibrosis, is the leading cause of death in chronic kidney disease patients and can be prevented by vitamin D receptor activators (VDRAs). Since some microRNAs (miRNAs) have emerged as regulators of the fibrotic process, we aimed to analyse the role of specific miRNAs in VDRA prevention of myocardial fibrosis as well as their potential use as biomarkers. Methods Wistar rats were nephrectomized and treated intraperitoneally with equivalent doses of two VDRAs: calcitriol and paricalcitol. Biochemical parameters, cardiac fibrosis, miRNA (miR-29b, miR-30c and miR-133b) levels in the heart and serum and expression of their target genes collagen I (COL1A1), matrix metalloproteinase 2 (MMP-2) and connective tissue growth factor (CTGF) in the heart were evaluated. Results Both VDRAs attenuated cardiac fibrosis, achieving a statistically significant difference in the paricalcitol-treated group. Increases in RNA and protein levels of COL1A1, MMP-2 and CTGF and reduced expression of miR-29b and miR-30c, known regulators of these pro-fibrotic genes, were observed in the heart of chronic renal failure (CRF) rats and were attenuated by both VDRAs. In serum, significant increases in miR-29b, miR-30c and miR-133b levels were observed in CRF rats, which were prevented by VDRA use. Moreover, vitamin D response elements were identified in the three miRNA promoters. Conclusions VDRAs, particularly paricalcitol, attenuated cardiac fibrosis acting on COL1A1, MMP-2 and CTGF expression, partly through regulation of miR-29b and miR-30c. These miRNAs and miR-133b could be useful serum biomarkers for cardiac fibrosis and also potential new therapeutic targets.