Jose M. Valdivielso

RANKL Increases Vascular Smooth Muscle Cell Calcification Through a RANK-BMP4–Dependent Pathway

Vascular calcification commonly associated with several pathologies and it has been suggested to be similar to bone mineralization. The axis RANKL-OPG (receptor activator of nuclear factor &kgr;B ligand–osteoprotegerin) finely controls bone turnover. RANKL has been suggested to increase vascular calcification, but direct evidence is missing. Thus, in the present work, we assess the effect of RANKL in vascular smooth muscle cell (VSMC) calcification. VSMCs incubated with RANKL showed a dose-dependent increase in calcification, which was abolished by coincubation with OPG. To test whether the effect was mediated by signaling to its receptor, knockdown of RANK was accomplished by short hairpin (sh)RNA. Indeed, cells lacking RANK showed no increases in vascular calcification when incubated with RANKL. To further elucidate the mechanism by which RANK activation increases calcification, we blocked both nuclear factor (NF)-&kgr;B activation pathways. Only IKK&agr; inactivation inhibited calcification, pointing to an involvement of the alternative NF-&kgr;B activation pathway. Furthermore, RANKL addition increased bone morphogenetic protein (BMP)4 expression in VSMCs, and that increase disappeared in cells lacking RANK or IKK&agr;. The increase in calcification was also blunted by Noggin, pointing to a mediation of BMP4 in the calcification induced by RANKL. Furthermore, in an in vivo model, the increase in vascular calcium content was parallel to an increase in RANKL and BMP4 expression, which was localized in calcified areas. However, blood levels of the ratio RANKL/OPG did not change. We conclude that RANKL increases vascular smooth muscle cell calcification by binding to RANK and increasing BMP4 production through activation of the alternative NF-&kgr;B pathway.

Differential Effects of Vitamin D Analogs on Vascular Calcification

We tested the effects of calcitriol and its analog paricalcitol on VSMC calcification in vitro and in vivo. For that reason, cells and animals with five‐sixths nephrectomy were treated with both compounds. Calcitriol, but not paricalcitol, increased VSMC calcification in vitro and in vivo independently of calcium and phosphate levels. This increase in calcification was parallel to an increase in the RANKL/OPG ratio.

Globotriaosylsphingosine actions on human glomerular podocytes: implications for Fabry nephropathy

BACKGROUND Transforming growth factor-β1 (TGF-β1) and the macrophage inhibitory factor receptor CD74 link the metabolic disorder with tissue injury in diabetic nephropathy. Fabry disease is an X-linked lysosomal glycosphingolipid storage disorder resulting from a deficient activity of α-galactosidase A that leads to proteinuric renal injury. However, the link between the metabolic abnormality and renal injury is poorly characterized. Globotriaosylsphingosine (lyso-Gb3) was recently identified as a bioactive molecule accumulating in Fabry disease. We hypothesized that lyso-Gb3 could modulate the release of secondary mediators of injury in glomerular podocytes and that recently described nephroprotective actions of vitamin D receptor activation in diabetic nephropathy may apply to lyso-Gb3. METHODS Real time RT-PCR, ELISA and Western blot were used to study the biological activity of lyso-Gb3 in cultured human podocytes and potential modulation by vitamin D receptor activation. RESULTS In human podocytes, lyso-Gb3 dose and time dependently increased the expression of TGF-β1, extracellular matrix proteins (fibronectin and type IV collagen) and CD74. TGF-β1 mediated lyso-Gb3 effects on extracellular matrix production. Vitamin D receptor activation with paricalcitol or calcitriol prevented the increase in TGF-β1, CD74 and extracellular matrix induced by lyso-Gb3. CONCLUSIONS Lyso-Gb3 may have a role in glomerular injury in Fabry disease by promoting the release of secondary mediators of glomerular injury common to diabetic nephropathy. These effects are prevented by paricalcitol, raising the issue of vitamin D receptor activation as potential adjunctive therapy in Fabry nephropathy.

Simultaneous changes in the calcium-sensing receptor and the vitamin D receptor under the influence of calcium and calcitriol

BACKGROUND The regulatory mechanisms of parathyroid hormone (PTH) synthesis are complex, involving calcium, calcitriol, the calcium-sensing receptor (CaR) and the vitamin D receptor (VDR). In this study, the effects of calcium and calcitriol on the simultaneous expression of CaR and VDR mRNA and protein levels were assessed in parathyroid glands cultured in vitro. METHODS Parathyroid glands (N = 424) were removed and cultured for 24 h to study the effect of calcium on the CaR, VDR and PTH. In addition, the effect of calcitriol at low calcium concentrations (0.6 mM) on CaR and VDR levels was studied after 48 h of incubation. CaR, VDR and PTH mRNAs were measured by quantitative real-time PCR (qRT-PCR), and CaR and VDR protein levels were measured by immunohistochemistry. RESULTS PTH gene expression was reduced by high calcium concentration. No differences were found in the CaR mRNA levels among the different calcium concentrations tested (0.6 mM calcium: 100%; 1.2 mM calcium: 120%; 2.0 mM calcium: 112%; median values), but VDR gene expression rose when calcium increased (0.6 mM calcium: 100%; 1.2 mM calcium: 164%; 2.0 mM calcium: 195%; median values). Calcitriol increased both CaR (control: 100%; 10(-8) M calcitriol: 196%; median values) and VDR genes expression (control: 100%; 10(-8) M calcitriol: 176%; median values). The same findings were corroborated at protein levels for both CaR and VDR. CONCLUSIONS In parathyroid glands cultured in vitro, calcium up-regulates VDR but not CaR. Conversely, calcitriol up-regulates both VDR and CaR mRNAs and protein levels, even at low calcium concentrations.

Predicting cardiovascular disease morbidity and mortality in chronic kidney disease in Spain. The rationale and design of NEFRONA: a prospective, multicenter, observational cohort study

BackgroundCardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). Cardiovascular risk assessment in this population is hampered by the failure of traditional risk factors to fully account for the elevated CVD risk (reverse epidemiology effect) and the presence of emerging risk factors specifically related to kidney failure. Therefore, diagnostic tools capable of improving cardiovascular risk assessment beyond traditional risk factors are currently warranted. We present the protocol of a 4-year prospective study aimed to assess the predictive value of non-invasive imaging techniques and biomarkers for CVD events and mortality in patients with CKD.MethodsFrom November 2009 to October 2010, 4137 asymptomatic adult patients with stages 2 to 5 CKD will be recruited from nephrology services and dialysis units throughout Spain. During the same period, 843 participants without CKD (control group) will be recruited from lists of primary care physicians, only at baseline. During the follow-up, CVD events and mortality will be recorded from all CKD patients. Clinical and laboratory characteristics will be collected in a medical documentation sheet. Three trained itinerant teams will carry out a carotid ultrasound to assess intima-media thickness and presence of plaques. A composite atherosclerosis score will be constructed based on carotid ultrasound data and measurement of ankle-brachial index. In CKD patients, presence and type of calcifications will be assessed in the wall of carotid, femoral and brachial arteries, and in cardiac valves, by ultrasound. From all participants, blood samples will be collected and stored in a biobank to study novel biomarkers.ConclusionsThe NEFRONA study is the first large, prospective study to examine the predictive value of several non-invasive imaging techniques and novel biomarkers in CKD patients throughout Spain. Hereby, we present the protocol of this study aimed to explore the most effective way in which these tests can be integrated with traditional risk factors to maximize CVD detection in this population.

Cardiovascular risk factors underestimate atherosclerotic burden in chronic kidney disease: usefulness of non-invasive tests in cardiovascular assessment

BACKGROUND Cardiovascular risk scoring (Score) does not specifically address chronic kidney disease (CKD) patients. The aim of our study is to quantify atherosclerosis using carotid ultrasound and ankle-brachial index (ABI) and to assess its additional value in risk scoring. METHODS In this cross-sectional, observational study, patients were studied according to a standardized protocol including carotid ultrasound and ABI to determine the atherosclerosis score (AS), ranging from absence of to severe atherosclerosis (AS 0 to AS 3). RESULTS We included 409 CKD-affected patients (231 on dialysis, 99 in CKD Stages IV-V and 79 in CKD Stages I-III) and 851 subjects with normal renal function. The presence and severity of atherosclerosis was significantly higher in the CKD group than in the controls at every decade of age studied. Among the CKD-affected subjects, the prevalence of carotid plaques was significantly higher in the dialysis group (78.3%) than in the group in CKD Stages I-III (55.6%, P < 0.001). We identified 174 patients at low-intermediate risk. Among them, 110 (63.2%) presented either moderate (AS 2) or severe (AS 3) atherosclerosis. Variables significantly (P < 0.05) and positively related to atherosclerosis were being on dialysis [OR = 3.40, 95% CI (1.73, 6.78) vs CKD Stages I-III], age [OR = 1.08, 95% CI (1.06-1.11)] and C-reactive protein [OR = 1.04, 95% CI (1.01-1.08)]. Conversely, female sex was negatively related to atherosclerosis [OR = 0.40, 95% CI (0.23-0.71), P = 0.002]. CONCLUSION The use of carotid ultrasound and ABI identifies atherosclerosis in a population of CKD patients in which risk scoring underestimates atherosclerosis burden.

Vitamin D receptor activation, left ventricular hypertrophy and myocardial fibrosis

BACKGROUND Left ventricular hypertrophy (LVH), a common complication in chronic kidney disease (CKD), is associated with high cardiovascular mortality. The aim of this experimental study was to analyze the effect of different vitamin D receptor activators (VDRAs) on both LVH and myocardial fibrosis in chronic renal failure (CRF). METHODS Male Wistar rats with CRF, carried out by 7/8 nephrectomy, were treated intraperitoneally with equivalent doses of VDRAs (calcitriol, paricalcitol and alfacalcidol, 5 days per week) during 4 weeks. A placebo group (CRF + vehicle) and a Sham group with normal renal function served as controls. Biochemical, morphological, functional and molecular parameters associated with LVH were evaluated, as well as cardiac fibrosis, collagen I, transforming growth factor β1 (TGFβ1) and matrix metalloproteinase-1 (MMP1) expression. RESULTS All VDRAs treatment prevented LVH, with values of cardiomyocyte size, LV wall and septum thickness and heart-body weight ratio similar to those observed in the Sham group. At molecular levels, all VDRAs attenuated atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression compared with CRF + vehicle. The phosphorylation of ERK1/2, a signal for activating growth, was stimulated in the CRF + vehicle group; VDRAs use prevented this activation. Paricalcitol was the only VDRA used that maintained in the normal range all parameters associated with myocardial fibrosis (total collagen, collagen I, TGFβ1 and MMP1). CONCLUSIONS Our findings demonstrated that the three VDRAs used induced similar changes in bone metabolic parameters and LVH. In addition, paricalcitol was the only VDRA which showed a relevant beneficial effect in the reduction of myocardial fibrosis, a key factor in the myocardial dysfunction in CKD patients.

N-methyl-d-aspartate receptors are expressed in rat parathyroid gland and regulate PTH secretion

N-methyl-d-aspartate receptors (NMDAR) are tetrameric amino acid receptors which act as membrane calcium channels. The presence of the receptor has been detected in the principal organs responsible for calcium homeostasis (kidney and bone), pointing to a possible role in mineral metabolism. In the present work, the presence of the receptor was determined in normal parathyroid glands (PTG) by real-time PCR, immunoprecipitation, and immunohistrochemistry. Healthy animals showed a decrease in blood parathyroid hormone (PTH) levels 15 min after the treatment with NMDA. This effect was also observed in animals with high levels of PTH-induced EDTA injection, but not in uremic animals with secondary hyperparathyroidism (2HPT). Normal rat PTG incubated in media with low calcium concentration (0.8 mM CaCl2) showed a decrease in PTH release when NMDA was added to the media. This effect of NMDA was abolished when glands were coincubated with MK801 (a pharmacological blocker of the NMDA channel) or PD98059 (an inhibitor of the ERK-MAPK pathway). Glands obtained from animals with 2HPT showed no effect of NMDA in the in vitro release of PTH, together with a decrease in the expression of NMDAR1. In conclusion, NMDA receptor is present in PTG and is involved in the regulation of the PTH release. The mechanism by which NMDAR exerts its function is through the activation of the MAPK cascade. In uremic 2HPT animals the receptor expression is downregulated and the treatment with NMDA does not affect PTH secretion.

A Forgotten Method to Induce Experimental Chronic Renal Failure in the Rat by Ligation of the Renal Parenchyma

Background: Animal models of chronic renal failure have been widely used in the experimental nephrology laboratories. The most common technique used is the 5/6 reduction of renal mass, either by surgical resection or by infarction. Methods: In the present work, we describe a forgotten technique based in the ligation of the renal parenchyma in both renal poles. This technique combines the advantages of the resection model, like the reproducibility and homogeneity, with the ones of the infarction technique, like the absence of bleeding. Results: 8 weeks after the procedure, animals showed a decrease in creatinine clearance together with an increase in plasma creatinine. Furthermore, glomeruli of animals with 5/6 nephrectomy showed a marked hypertrophy, with a glomerular volume significantly higher than control animals. Serum levels of parathyroid hormone were also increased, consistent with the development of secondary hyperparathyroidism. Conclusions: We conclude that the present technique is a valid and improved tool for the study of chronic renal failure.

Serum levels of matrix metalloproteinase-10 are associated with the severity of atherosclerosis in patients with chronic kidney disease.

Cardiovascular disease is the leading cause of mortality in chronic kidney disease (CKD). As matrix metalloproteinases have a major role in atherosclerosis, we hypothesized that alterations in metalloproteinases-8, -10 and their tissue inhibitor-1 can be associated with the severity of atherosclerosis in patients with kidney disease. This was evaluated in a cross-sectional, observational study of 111 patients with stages I-V kidney disease, 217 patients on dialysis and 50 healthy controls. The severity of atherosclerosis was estimated with the atherosclerosis score (AS), combining the results of ankle-brachial index and carotid ultrasound. Serum levels of the two metalloproteinases and tissue inhibitor-1 were measured by enzyme-linked immunosorbent assay and were significantly increased in patients with kidney disease compared with the healthy controls, and higher in patients on dialysis than in earlier stages of CKD. The severity of the AS was also more prevalent in the dialysis group, in which serum levels of both metalloproteinases and tissue inhibitor-1 were significantly higher. After multivariate analysis, metalloproteinase-10, dialysis, C-reactive protein, age, and male gender were associated with increased risk of atherosclerosis. Thus, patients with CKD exhibit elevated levels of circulating metalloproteinase-10, and this was independently associated with the severity of atherosclerosis and may represent a new biomarker of atherosclerotic diseases.