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.

Pathophysiology of bone disease in chronic kidney disease: from basics to renal osteodystrophy and osteoporosis.

Chronic kidney disease (CKD) is a highly prevalent disease that has become a public health problem. Progression of CKD is associated with serious complications, including the systemic CKD-mineral and bone disorder (CKD-MBD). Laboratory, bone and vascular abnormalities define this condition, and all have been independently related to cardiovascular disease and high mortality rates. The "old" cross-talk between kidney and bone (classically known as "renal osteodystrophies") has been recently expanded to the cardiovascular system, emphasizing the importance of the bone component of CKD-MBD. Moreover, a recently recognized higher susceptibility of patients with CKD to falls and bone fractures led to important paradigm changes in the new CKD-MBD guidelines. Evaluation of bone mineral density and the diagnosis of "osteoporosis" emerges in nephrology as a new possibility "if results will impact clinical decisions". Obviously, it is still reasonable to perform a bone biopsy if knowledge of the type of renal osteodystrophy will be clinically useful (low versus high turnover-bone disease). However, it is now considered that the inability to perform a bone biopsy may not justify withholding antiresorptive therapies to patients with high risk of fracture. This view adds to the effects of parathyroid hormone in CKD patients and the classical treatment of secondary hyperparathyroidism. The availability of new antiosteoporotic treatments bring the opportunity to come back to the basics, and the knowledge of new pathophysiological pathways [OPG/RANKL (LGR4); Wnt-ß-catenin pathway], also affected in CKD, offers great opportunities to further unravel the complex physiopathology of CKD-MBD and to improve outcomes.

The receptor activator of nuclear factor κΒ ligand receptor leucine-rich repeat-containing G-protein-coupled receptor 4 contributes to parathyroid hormone-induced vascular calcification.

BACKGROUND: In chronic kidney disease, serum phosphorus (P) elevations stimulate parathyroid hormone (PTH) production, causing severe alterations in the bone-vasculature axis. PTH is the main regulator of the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, which is essential for bone maintenance and also plays an important role in vascular smooth muscle cell (VSMC) calcification. The discovery of a new RANKL receptor, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), which is important for osteoblast differentiation but with an unknown role in vascular calcification (VC), led us to examine the contribution of LGR4 in high P/high PTH-driven VC. METHODS: In vivo studies were conducted in subtotally nephrectomized rats fed a normal or high P diet, with and without parathyroidectomy (PTX). PTX rats were supplemented with PTH(1-34) to achieve physiological serum PTH levels. In vitro studies were performed in rat aortic VSMCs cultured in control medium, calcifying medium (CM) or CM plus 10-7 versus 10-9 M PTH. RESULTS: Rats fed a high P diet had a significantly increased aortic calcium (Ca) content. Similarly, Ca deposition was higher in VSMCs exposed to CM. Both conditions were associated with increased RANKL and LGR4 and decreased OPG aorta expression and were exacerbated by high PTH. Silencing of LGR4 or parathyroid hormone receptor 1 (PTH1R) attenuated the high PTH-driven increases in Ca deposition. Furthermore, PTH1R silencing and pharmacological inhibition of protein kinase A (PKA), but not protein kinase C, prevented the increases in RANKL and LGR4 and decreased OPG. Treatment with PKA agonist corroborated that LGR4 regulation is a PTH/PKA-driven process. CONCLUSIONS: High PTH increases LGR4 and RANKL and decreases OPG expression in the aorta, thereby favouring VC. The hormone's direct pro-calcifying actions involve PTH1R binding and PKA activation.

Osteoporosis, bone mineral density and CKD-MBD (II): Therapeutic implications. / Osteoporosis, densidad mineral ósea y complejo CKD-MBD (II): implicaciones terapéuticas.

Osteoporosis (OP) and chronic kidney disease (CKD) both independently affect bone health. A significant number of patients with CKD have decreased bone mineral density (BMD), are at high risk of fragility fractures and have an increased morbidity and mortality risk. With an ageing population, these observations are not only dependent on "renal osteodystrophy" but also on the associated OP. As BMD predicts incident fractures in CKD patients (partI), we now aim to analyse the potential therapeutic consequences. Post-hoc analyses of randomised studies have shown that the efficacy of drugs such as alendronate, risedronate, raloxifene, teriparatide and denosumab is similar to that of the general population in patients with a mild/moderate decline in their glomerular filtration rate (especially CKD-3). These studies have some flaws however, as they included mostly "healthy" women with no known diagnosis of CKD and generally with normal lab test results. Nevertheless, there are also some positive preliminary data in more advanced stages (CKD-4), even though in CKD-5D they are more limited. Therefore, at least in the absence of significant mineral metabolism disorders (i.e. severe hyperparathyroidism), the potential benefit of these drugs should be considered in patients with a high or very high fracture risk. It is an important change that the new guidelines do not make it a requirement to first perform a bone biopsy and that the risk/benefit ratio of these drugs may be justified. However, we must also be aware that most studies are not consistent and the level of evidence is low. Consequently, any pharmacological intervention (risk/benefit) should be prudent and individualised.

Osteoporosis and adynamic bone in chronic kidney disease.

Among the chronic kidney disease-mineral bone disease (CKD-MBD) disorders, osteoporosis and adynamic bone are highly prevalent, and they have been consistently associated with low bone mass, bone fractures, vascular calcifications and greater mortality in general and CKD populations. Despite the fact that osteoporosis and adynamic bone have similar clinical outcomes, they have different pathogeneses and clinical management. In osteoporosis, there is a lack of balance between bone formation and bone resorption, and less new bone is formed to replace bone losses. Osteoporosis is defined by the World Health Organization as "a disease characterized by low bone mineral density and micro architectural deterioration leading to low bone strength and increased risk of fractures." In the general population, there is a good correlation between dual-energy X-ray absorptiometry measurements and bone fractures, but this is not the case with CKD patients. Despite the fact that we have a great number of active antiosteoporotic drugs, the experience in CKD patients is limited. Adynamic bone is suspected based on biochemical parameters, mainly parathyroid hormone (PTH) and bone alkaline phosphatase, but it needs to be proven using a bone biopsy, where a low or zero bone formation rate and a reduction or absence of osteoblasts and osteoclasts should be found. The clinical management of adynamic bone has important limitations and currently does not allow taking many active measures. Treatment is mainly based on the prevention of risk factors known to induce PTH oversuppression, such as aluminium and calcium load and very high doses of vitamin D receptor activators. Due to the limitations in the treatment of both conditions, prevention plays a key role in the management of these disorders.

Vascular calcification in patients with chronic kidney disease: types, clinical impact and pathogenesis.

Vascular calcification plays a major role in cardiovascular disease, which is one of the main causes of mortality in chronic kidney disease patients. Vascular calcification is determined by prevalent traditional and uraemia-related (non-traditional) risk factors. It occurs mainly in the arteries, which are classified into three types according to their size and structural characteristics. In addition, vascular calcification has been associated with bone loss and fractures in chronic kidney disease patients and the general population, stressing the fact that both disorders can share pathogenetic pathways. The strategies to control vascular calcification involve several measures, chief among them the control of hyperphosphataemia. Furthermore, it has been recently described that strategies that reduce bone resorption and increase bone mineralization may decrease the risk of vascular calcifications; however, this approach still remains controversial. The mechanisms involved in vascular calcification are complex and not yet fully understood. Phosphorus plays a major role, while other factors related to bone formation have been recently identified.

Vascular calcifications, vertebral fractures and mortality in haemodialysis patients.

BACKGROUND: Vascular calcifications and the bone fractures caused by abnormal bone fragility, also called osteoporotic fractures, are frequent complications associated with chronic kidney diseases (CKD). The aim of this study was to investigate the association between vascular calcifications, osteoporotic bone fractures and survival in haemodialysis (HD) patients. METHODS: A total of 193 HD patients were followed up to 2 years. Vascular calcifications and osteoporotic vertebral fractures (quoted just as vertebral fractures in the text) were assessed by thoracic, lumbar spine, pelvic and hand X-rays and graded according to their severity. Clinical, biochemical and therapeutic data gathered during the total time spent on HD were collected. RESULTS: The prevalence of aortic calcifications was higher in HD patients than in a random-based general population (79% versus 37.5%, P < 0.001). Total time on any renal replacement therapy (RRT) and diabetes were positively associated with a higher prevalence of vascular calcifications. In addition to these factors, time on HD was also positively associated with the severity of vascular calcifications, and higher haemoglobin levels were associated with a lower prevalence of severe vascular calcifications in large and medium calibre arteries. The prevalence of vertebral fractures in HD patients was similar to that of the general population (26.5% versus 24.1%). Age and time on HD showed a positive and statistically significant association with the prevalence of vertebral fractures. Vascular calcifications in the medium calibre arteries were associated with a higher rate of prevalent vertebral fractures. In women, severe vascular calcifications and vertebral fractures were positively associated with mortality [RR = 3.2 (1.0-10.0) and RR = 4.8 (1.7-13.4), respectively]. CONCLUSIONS: Positive associations between vascular calcifications, vertebral fractures and mortality have been found in patients on HD.

Vascular calcifications: pathogenesis, management, and impact on clinical outcomes.

The predisposition to vascular calcifications in patients with chronic kidney disease (CKD) has gained great interest in recent years as many studies have described its likely impact on morbidity and mortality. The mechanism by which the process of vascular calcification is produced is complex, and it does not consist in a simple precipitation of calcium and phosphate but is instead an active and modifiable process. Several "modifiable and nonmodifiable" factors that are able to promote vascular calcification are extremely frequent in patients with CKD. Most of the present strategies to decrease vascular calcifications are based in the control of the more prevalent modifiable risk factors. Unfortunately, the extremely important nonmodifiable risk factors, which are highly prevalent, such as older age, time on dialysis, and diabetes, are not under one's control. Recent studies also have shown that vascular calcifications in some localizations were associated with increased osteoporotic fractures not only in dialysis patients but also in the general population, and interestingly, mortality also was associated significantly and positively with vascular calcifications and nontraumatic bone fractures. Despite that new strategies may improve the management of vascular diseases and specifically have a positive impact on the high prevalence of vascular calcifications, still the best possible control of the bone metabolic and inflammatory parameters are in the primary line. The horizon of the coming decade looks promising, but solid clinical and epidemiologic data are needed to manage better the bone- and cardiovascular-related disorders in patients with CKD.

Hyperphosphataemia as a cardiovascular risk factor -- how to manage the problem.

Hyperphosphataemia is a frequent and important cardiovascular risk factor in patients with chronic kidney disease (CKD). High phosphate levels may influence vascular calcifications by two separate mechanisms: by worsening secondary hyperparathyroidism, which in turn facilitates calcification, and by promoting calcium phosphate deposition in pre-formed endothelial plaques and in the arterial wall. Recent studies have shown that hyperphosphataemia induces the proliferation and differentiation of endothelial vascular cells into osteoblast-like cells, promoting vascular calcification. High phosphate levels also increase the risk of mortality in patients with CKD. To reduce the negative impact of high phosphate, serum phosphate levels should be <5 mg/dl and serum calcium <10 mg/dl. This allows the calcium x phosphate product to be maintained at < or =50 mg(2)/dl(2), reducing the risk of vascular, valvular, and extraskeletal calcification. A multiple-factor approach can be used to reduce serum phosphate: (i). decrease bone resorption by maintaining adequate serum parathyroid hormone levels; (ii). reduce phosphorous intake in the diet, (iii). use phosphate binders efficiently; and (iv). avoid under-dialysis. The patient's diet should be high in nutrition but with the lowest possible phosphorous content. Doses of phosphate binders should be tailored to individual dietary habits and must be taken during meals in a dose proportional to the phosphorous content of the meal. Because of the risk of increased extraskeletal calcification, calcium-containing phosphate-binder intake should not exceed 2-3 g/day. Sevelamer hydrochloride, a non-calcium and non-aluminium phosphate binder with a potency similar to that of calcium salts has shown beneficial effects on lipid profiles. Better control of serum phosphate is achieved in patients on continuous ambulatory peritoneal dialysis than in those on haemodialysis. Removal of phosphate is directly correlated with duration and frequency of dialysis sessions. Thus, it is advisable not to reduce the duration of dialysis sessions to <4 h three times per week.