Calcification In Chronic Kidney Disease Research Articles

#289 Lipocalin-2 promotes cardiovascular calcification in chronic kidney disease by upregulating STAT3/NCOA4-mediated ferritinophagy and ferroptosis

Abstract Background and Aims Cardiovascular calcification (CVC) is a common complication of chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality without any effective therapies. To date, the underlying mechanism of CKD-CVC has not been elucidated. Recent studies suggest that ferroptosis is a novel programmed cell death that may play a role in the process of CKD-CVC, but the mechanism is largely unclear. Ferritinophagy is an important inducer of ferroptosis that is regulated by nuclear receptor coactivator 4 (NCOA4). However, the relationship between ferritinophagy and CKD-CVC has not been explored. Lipocalin-2 (LCN2) is an iron-trafficking protein that has been associated with both osteogenesis and ferroptosis, but the exact role and molecular mechanism of LCN2 in CKD-CVC needs to be explored. Method A cross-sectional clinical study was utilized to examine the association between LCN2 expression and CKD-CVC in serum and calcified radial arteries of CKD patients. LCN2 knockout mice, their wild type littermates and mice infected with VSMCs-targeted adeno-associated virus encoding LCN2 gene or negative control gene were all fed by the high adenine and phosphate diet to induce CKD-CVC.VSMCs transfected with LCN2-overexpressing lentivirus, LCN2-shRNA lentivirus or LCN2 siRNA and recombinant LCN2 stimulated VSMCs were treated under the condition of calcifying medium to investigate the role of LCN2 in CKD-CVC in vitro. Cardiovascular calcification was evaluated by Von-kossa staining and tissue calcium content assay. VSMCs calcification were checked by the Alizarin Red Staining and cellular calcium content assay. Cellular reactive oxygen species (ROS) were measured by the DCFDA assay. Lipid peroxidation was measured by the fluorescence of the fatty acid analog C11-BODIPY581/591 probe. Intracellular labile ferrous iron levels were measured by fluorescence of Phen Green SK probe and ferro-orange probe. Transmission electron microscopy was also used to examine mitochondrial change of ferroptosis. Results The cross-sectional study identified that LCN2 levels in serum and radial arteries were significantly increased as the severity of CKD-CVC increased. High adenine and phosphate diet intake provokes renal fibrosis and cardiovascular calcification in vivo. In vitro, five-days high phosphate sodium stimulation induced calcium deposition and osteoblastic trans differentiation of VSMCs. Meanwhile, high phosphate also increased the levels of ferroptosis in VSMCs as evidenced by decreased cell viability, iron and ROS accumulation, upregulated PTGS2 and MDA levels, reduced glutathione peroxidase 4 (GPX4) levels and significantly mitochondrial damage in VSMCs. Intriguingly, calcium deposition in VSMCs could be reversed by the ferroptosis inhibitor ferrostatin-1. LCN2 expression was significantly upregulated both in the aorta of mice and calcified VSMCs. Compared with WT mice, deletion of LCN2 inhibited cardiovascular calcification whereas VSMC-targeted LCN2 overexpression aggravated CKD-CVC. Consistently, LCN2 knockdown also alleviated calcium deposition of VSMCs. Meanwhile, inhibition of ferroptosis were observed after LCN2 silencing as evidenced by decreased ferrous and lipid oxidation while increased levels of GPX4. Mechanically, we demonstrated high phosphate increased the protein level of NCOA4 and downregulated FTH1, which was reversed by depletion of LCN2. Knockdown of NCOA4 partly alleviated the ferritin reduction, ferroptosis, and calcification in VSMCs, indicating that NCOA4-mediated ferritinophagy was required for VSMCs calcification. Furthermore, we found that the recombinant LCN2 treatment significantly upregulated phosphorylated STAT3. STAT3 signaling inhibitor abolished the recombinant LCN2 induced NCOA4 upregulation and calcification in VSMCs. Conclusion Our findings indicate that the pro-calcific effect of high phosphate is due to VSMCs ferroptosis mediated by LCN2 upregulation. LCN2 silencing alleviates CKD-CVC by suppressing the STAT3/ NCOA4/FTH1 signaling mediated ferritinophagy. LCN2 could serve as a candidate therapeutic strategy for CKD-CVC.

FHL2 in arterial medial calcification in chronic kidney disease.

Arterial medial calcification (AMC) is a common complication in individuals with chronic kidney disease (CKD), which can lead to cardiovascular morbidity and mortality. The progression of AMC is controlled by a key transcription factor called runt-related transcription factor 2 (RUNX2), which induces vascular smooth muscle cells (VSMCs) transdifferentiation into a osteogenic phenotype. However, RUNX2 has not been targeted for therapy due to its essential role in bone development. The objective of our study was to discover a RUNX2 coactivator that is highly expressed in arterial VSMCs as a potential therapy for AMC. We employed transcriptomic analysis of human data and an animal reporter system to pinpoint FHL2 as a potential target. Subsequently, we investigated the mRNA and protein expression patterns of FHL2 in the aortas of both human and animal subjects with CKD. To examine the role of FHL2 in the RUNX2 transcription machinery, we conducted coimmunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) experiments. Next, we manipulated FHL2 expression in cultured VSMCs to examine its impact on high phosphate-induced transdifferentiation. Finally, we employed FHL2 null mice to confirm the role of FHL2 in the development of AMC in vivo. Among all the potential RUNX2 cofactor, FHL2 displays selective expression within the cardiovascular system. In the context of CKD subjects, FHL2 undergoes upregulation and translocation from the cytosol to the nucleus of arterial VSMCs. Once in the nucleus, FHL2 interacts structurally and functionally with RUNX2, acting as a coactivator of RUNX2. Notably, the inhibition of FHL2 expression averts transdifferentiation of VSMCs into an osteogenic phenotype and mitigates aortic calcification in uremic animals, without causing any detrimental effects on the skeletal system. These observations provide evidence that FHL2 is a promising target for treating arterial calcification in patients with CKD.

New therapeutic perspectives for vascular and valvular calcifications in chronic kidney disease.

Vascular and valvular calcification are associated with cardiovascular morbidity and mortality in people with chronic kidney disease (CKD). Uncertainty exists regarding therapeutic strategies to attenuate calcification. This review outlines the pathophysiological mechanisms contributing to vascular and valvular calcification, considers the mechanisms of action of therapeutic interventions, and reports the latest outcomes from interventional studies. Conventional therapies targeted at CKD-mineral and bone disorder (MBD) modulation have yielded conflicting or inconclusive results. Magnesium and vitamin K supplementation appear to offer attenuation of coronary artery calcification but inconsistent findings justify the need for further studies. Strategies targeting hydroxyapatite formation such as sodium thiosulphate and hexasodium fytate show promise and are worthy of further evaluation. The serum calcification propensity assay (T50) correlates with severity and progression; it holds promise as a potential future clinical tool for screening monitoring calcification risk. Whilst knowledge of the pathophysiology of vascular calcification has grown and therapeutic approaches appear promising, as yet no medication has been approved to treat vascular or valvular calcification, or calciphylaxis.

Detection of medial vascular calcification in chronic kidney disease based on pulse wave analysis in the frequency domain

Medial vascular calcification (mVC), particularly prevalent in patients with chronic kidney disease (CKD), is a pathological deposition of minerals in the medial layer of the vessel wall and is associated with an increased risk of cardiovascular disease. Currently, there is no cost–effective and non-invasive procedure of mVC assessment in routine clinical practice. We explored whether in–depth analysis of non-invasively recorded peripheral pulse waves can serve as an effective mVC biomarker.The study included 97 CKD patients with histological assessment of mVC in the epigastric artery and pulse wave measurements in the brachial artery. Pulse waves were analysed in the frequency domain to obtain features, which, together with traditional mVC risk factors (i.e., age, sex, and body mass index), were used to build generalized linear models for mVC prediction. The classifiers with the best scores in terms of balanced accuracy were combined in an ensemble establishing the final model.The final, ensembled model, assessed using a leave-one-out cross-validation process, achieved a balanced accuracy equal to 0.87, an accuracy of 0.93, an AUC ROC of 0.91, and an F-score of 0.96. Apart from the features associated with pulse waves, the selected variables included age, sex, body mass index, heart rate and diastolic blood pressure.Analysis of non-invasively recorded peripheral pulse waveforms combined with traditional risk factors, can help to detect mVC in CKD patients and thus, potentially introduce risk-lowering therapeutic strategies at earlier disease stages in the future.

Associations of urinary fetuin-A with histopathology and kidney events in biopsy-proven kidney disease.

Fetuin-A is implicated in the pathogenesis of vascular calcification in chronic kidney disease (CKD); however, the relationship between fetuin-A, histopathologic lesions and long-term kidney outcomes in patients with various types of kidney disease remains unclear. We measured urinary fetuin-A levels in 335 individuals undergoing clinically indicated native kidney biopsy. The expressions of fetuin-A mRNA and protein in the kidney were assessed using RNA sequencing and immunohistochemistry. The association of urinary fetuin-A with histopathologic lesions and major adverse kidney events (MAKE), defined as a decline in estimated glomerular filtration rate (eGFR) of at least 40%, kidney failure or death, was analyzed. Urinary fetuin-A levels showed a positive correlation with albuminuria (rs=0.67, P<.001) and a negative correlation with eGFR (rs=-0.46, P<.001). After multivariate adjustment, higher urinary fetuin-A levels were associated with glomerular inflammation, mesangial expansion, interstitial fibrosis and tubular atrophy, and arteriolar sclerosis. Using a 1 transcript per million gene expression cutoff, we found kidney fetuin-A mRNA levels below the threshold in both individuals with normal kidney function and those with CKD. Additionally, immunohistochemistry revealed reduced fetuin-A staining in tubular cells of CKD patients compared with normal controls. During a median 21-month follow-up, 115 patients experienced MAKE, and Cox regression analysis confirmed a significant association between elevated urinary fetuin-A and MAKE. This association remained significant after adjusting for potential confounding factors. Urinary fetuin-A is associated with chronic histological damage and adverse clinical outcomes across a spectrum of biopsy-proven kidney diseases.

Aortic calcification is associated with the difference between invasive central and cuff-measured brachial blood pressure in chronic kidney disease.

Chronic kidney disease (CKD) is associated with accelerated vascular calcification and increased central systolic blood pressure when measured invasively (invCSBP) relative to cuff-based brachial systolic blood pressure (cuffSBP). The contribution of aortic wall calcification to this phenomenon has not been clarified. We therefore examined the effects of aortic calcification on cuffSBP and invCSBP in a cohort of patients representing all stages of CKD. During elective coronary angiography, invCSBP was measured in the ascending aorta with a fluid-filled catheter with simultaneous recording of cuffSBP using an oscillometric device. Furthermore, participants underwent a non-contrast computed tomography scan of the entire aorta with observer blinded calcification scoring of the aortic wall ad modum Agatston. We included 168 patients (mean age 67.0±10.5, 38 females) of whom 38 had normal kidney function, while 30, 40, 28, and 32 had CKD stage 3a, 3b, 4, and 5, respectively. Agatston scores adjusted for body surface area ranged from 48 to 40,165. We found that invCSBP increased 3.6 (95% confidence interval 1.4-5.7) mmHg relative to cuffSBP for every 10,000-increment in aortic Agatston score. This association remained significant after adjustment for age, diabetes, antihypertensive treatment, smoking, eGFR and BP level. No such association was found for diastolic BP. Patients with advanced aortic calcification have relatively higher invCSBP for the same cuffSBP as compared to patients with less calcification. Advanced aortic calcification in CKD may therefore result in hidden central hypertension despite apparently well-controlled cuffSBP.

WITHDRAWN: Potential mechanisms underlying?Taohong?Siwu?Decoction?ameliorates vascular calcification in chronic kidney disease based on network pharmacology and molecular docking

WITHDRAWN: Potential mechanisms underlying?Taohong?Siwu?Decoction?ameliorates vascular calcification in chronic kidney disease based on network pharmacology and molecular docking

Food to Prevent Vascular Calcification in Chronic Kidney Disease.

Vascular calcification (VC) is a consequence of chronic kidney disease (CKD) which is of paramount importance regarding the survival of CKD patients. VC is far from being controlled with actual medication; as a result, in recent years, diet modulation has become more compelling. The concept of medical nutritional therapy points out the idea that food may prevent or treat diseases. The aim of this review was to evaluate the influence of food habits and nutritional intervention in the occurrence and progression of VC in CKD. Evidence reports the harmfulness of ultra-processed food, food additives, and animal-based proteins due to the increased intake of high absorbable phosphorus, the scarcity of fibers, and the increased production of uremic toxins. Available data are more supportive of a plant-dominant diet, especially for the impact on gut microbiota composition, which varies significantly depending on VC presence. Magnesium has been shown to prevent VC but only in experimental and small clinical studies. Vitamin K has drawn considerable attention due to its activation of VC inhibitors. There are positive studies; unfortunately, recent trials failed to prove its efficacy in preventing VC. Future research is needed and should aim to transform food into a medical intervention to eliminate VC danger in CKD.

Extracellular Histones Promote Calcium Phosphate-Dependent Calcification in Mouse Vascular Smooth Muscle Cells.

Vascular calcification, a major risk factor for cardiovascular events, is associated with a poor prognosis in chronic kidney disease (CKD) patients. This process is often associated with the transformation of vascular smooth muscle cells (VSMCs) into cells with osteoblast-like characteristics. Damage-associated molecular patterns (DAMPs), such as extracellular histones released from damaged or dying cells, are suspected to accumulate at calcification sites. To investigate the potential involvement of DAMPs in vascular calcification, we assessed the impact of externally added histones (extracellular histones) on calcium and inorganic phosphate-induced calcification in mouse VSMCs. Our study found that extracellular histones intensified calcification. We also observed that the histones decreased the expression of VSMC marker genes, while simultaneously increasing the expression of osteoblast marker genes. Additionally, histones treated with DNase I, which degrades dsDNA, attenuated this calcification, compared with the non-treated histones, suggesting a potential involvement of dsDNA in this process. Elevated levels of dsDNA were also detected in the serum of CKD model mice, underlining its potential role in vascular calcification in CKD. Our findings suggest that extracellular histones could play a pivotal role in the vascular calcification observed in CKD.

The functional role of cellular senescence during vascular calcification in chronic kidney disease.

Vascular calcification (VC) has emerged as a key predictor of cardiovascular events in patients with chronic kidney disease (CKD). In recent years, an expanding body of research has put forth the concept of accelerated vascular aging among CKD patients, highlighting the significance of vascular cells senescence in the process of VC. Within the milieu of uremia, senescent vascular endothelial cells (VECs) release extracellular microvesicles (MV) that promote vascular smooth muscle cells (VSMCs) senescence, thereby triggering the subsequent osteogenic phenotypic switch and ultimately contributing to the VC process. In addition, senescent vascular progenitor or stem cells with diminished ability to differentiate into VECs and VSMCS, compromise the repair of vascular integrity, on the other hand, release a cascade of molecules associated with senescence, collectively known as the senescence-associated secretory phenotype (SASP), perpetuating the senescence phenomenon. Furthermore, SASP triggers the recruitment of monocytes and macrophages, as well as adjacent VECs and VSMCs into a pro-adhesive and pro-inflammatory senescent state. This pro-inflammatory microenvironment niche not only impacts the functionality of immune cells but also influences the differentiation of myeloid immune cells, thereby amplifying the reduced ability to effectively clear senescent cells of senescent macrophages, promoted calcification of VSMCs. The objective of this paper is to provide a comprehensive review of the contribution of vascular cell senescence to the emergence and advancement of VC. Gaining a comprehensive understanding of the involvement of cellular senescence within the vessel wall is pivotal, especially when it comes to its intersection with VC. This knowledge is essential for advancing groundbreaking anti-aging therapies, aiming to effectively mitigate cardiovascular diseases.

Magnesium and Vascular Calcification in Chronic Kidney Disease: Current Insights.

Magnesium (Mg) plays crucial roles in multiple essential biological processes. As the kidneys are the primary organ responsible for maintaining the blood concentration of Mg, people with chronic kidney disease (CKD) may develop disturbances in Mg. While both hyper- and hypomagnesemia may lead to adverse effects, the consequences associated with hypomagnesemia are often more severe and lasting. Importantly, observational studies have shown that CKD patients with hypomagnesemia have greater vascular calcification. Vascular calcification is accelerated and contributes to a high mortality rate in the CKD population. Both in vitro and animal studies have demonstrated that Mg protects against vascular calcification via several potential mechanisms, such as inhibiting the formation of both hydroxyapatite and pathogenic calciprotein particles as well as limiting osteogenic differentiation, a process in which vascular smooth muscle cells in the media layer of the arteries transform into bone-like cells. These preclinical findings have led to several important clinical trials that have investigated the effects of Mg supplementation on vascular calcification in people with CKD. Interestingly, two major clinical studies produced contradictory findings, resulting in a state of equipoise. This narrative review provides an overview of our current knowledge in the renal handling of Mg in health and CKD and the underlying mechanisms by which Mg may protect against vascular calcification. Lastly, we evaluate the strength of evidence from clinical studies on the efficacy of Mg supplementation and discuss future research directions.

An Update on Hypomagnesemia and Hypermagnesemia

Magnesium is an essential element with a pleiotropic role in human biology. Despite tight intestinal and renal regulation of its balance, insufficient intake can finally result in hypomagnesemia, which is a proxy of intracellular deficiency. Conditions such as diabetes, cancer, and infections are often associated with hypomagnesemia, which mostly predicts an unfavorable outcome. The effects of hypomagnesemia can either be direct and include neurological and cardiovascular symptoms or indirect, taking a mechanistic role in inflammation, endothelial dysfunction, and oxidative stress. The indication for intravenous magnesium as a treatment of torsades de pointes and pre-eclampsia is unrefuted, but new indications of peroral or intravenous supplementation, albeit with less supporting evidence, have emerged suggesting, respectively, an attenuation of vascular calcification in chronic kidney disease and improved rate control in atrial fibrillation. Other potential beneficial properties of magnesium, which were claimed by observational data, such as lipid lowering and renal protection, were not, or only partially, investigated in randomized controlled trials. Thus, the role of peroral supplementation of mild chronic asymptomatic hypomagnesemia should be separated from the more targeted prescription of magnesium in specific study populations. (Severe) hypermagnesemia is potentially life-threatening and occurs almost uniformly in subjects with severe renal failure exposed to either supplements or to magnesium-containing cathartics or antacids. Moderate hypermagnesemia is very common in pre-eclamptic women treated with intravenous magnesium. For most (but not all) studied endpoints, mild hypermagnesemia yields a survival benefit. Long-lasting concerns about the potential negative effects of mild hypermagnesemia on bone physiology and structure have so far not been unequivocally demonstrated to be troublesome.

Moderate Iron Deficiency Does Not Potentiate Renal or Cardiac Calcification and Subsequent Tissue Injury in Mouse Models of CKD

Chronic kidney disease (CKD) is a public health challenge that affects over 26 million Americans and many more worldwide. Many develop renal and cardiac calcification, which promotes arterial wall stiffness. These structural changes of the vasculature contribute to hypertension and heart failure, increasing the risk of premature death. Dysregulated iron homeostasis also occurs in many patients with CKD, and iron deficiency and anemia are common complications that are independently associated with CKD progression, cardiovascular disease and all-cause mortality. Furthermore, lower transferrin saturation (TSAT) levels are independently associated with higher coronary artery calcification scores in CKD patients receiving hemodialysis. However, whether iron deficiency and/or anemia directly contribute to the pathogenesis of calcification in CKD is unknown. We examined the effects of iron deficiency on renal and cardiac calcification, and tissue injury in mouse models of CKD. We used a dietary model of progressive CKD: 8-week-old male C57BL/6J mice were fed for 8 weeks a customized 0.2% adenine-rich diet that was either iron-replete (100 ppm iron; “adenine-IR” group) or iron-deficient (4 ppm iron, “adenine-ID” group). Control mice were fed a diet without adenine, but with matching iron content (iron-replete “CTRL-IR” and iron-deficient “CTRL-ID” groups). Compared to CTRL-IR mice, CTRL-ID mice developed widespread tissue iron deficiency and anemia as expected. Surprisingly, despite 8 weeks of low iron diet which led to lower serum iron and worse anemia, adenine-ID mice had similarly high liver iron as adenine-IR mice, similar expression of transferrin receptor 1 (marker of tissue iron deficiency) in kidney or heart, and similarly elevated liver hepcidin mRNA. This suggests that hepcidin induction in this CKD model likely protected tissues from iron depletion, at least on the time scale of the experiment. Calcification in adenine mice was evident but was similar between adenine-ID and adenine-IR mice, as observed by the comparable expression of calcification markers in kidney and heart ( Runx2, Sox9, Opn). Additionally, markers of kidney function (BUN and serum creatinine), kidney injury ( Kim1, Lcn2) and cardiac injury( Myh7) were also comparable between adenine-ID and adenine-IR mice. Inflammation and fibrosis were increased in kidney and heart of adenine mice in general, but once again similar between adenine-ID and adenine-IR mice (serum IL-6, Tnfa, Tgfb Col1a1). We then employed a genetic model of progressive CKD ‒ constitutive Col4a3 −/−mice (Alport syndrome, life expectancy in this model ~11 weeks of age), and fed male mice either an IR or ID diet for 7 weeks. Diets were started in juvenile mice (4-week-old), a strategy known to exacerbate the effect of ID diet on tissue iron deficiency and anemia. Despite this, Alport-ID and Alport-IR mice still displayed similar liver iron levels and liver hepcidin expression. Alport-IR and -ID groups also had comparable renal and cardiac calcification, injury, inflammation and fibrosis. Altogether, our findings in two different mouse models of CKD indicate that moderate iron deficiency does not potentiate renal or cardiac calcification and subsequent tissue injury. Our data suggest that despite causing hypoferremia and anemia, chronically elevated hepcidin in CKD might delay the development of tissue iron deficiency in the liver, kidney and heart, and potentially protect against deleterious vascular changes and subsequent tissue injury.

Regulatory effects of nutritional and metabolic disorders on vascular calcification in chronic kidney disease: a narrative review

Regulatory effects of nutritional and metabolic disorders on vascular calcification in chronic kidney disease: a narrative review

Phosphate Toxicity and Vascular Calcification in Chronic Kidney Disease: A Closer Look Utilizing Transmission Electron Microscopy.

Hyperphosphatemia is independently linked with vascular calcification, cardiovascular disease, bone-mineral disease, progression of renal insufficiency, and all-cause mortality in chronic kidney disease (CKD) and end-stage renal disease (ESRD). The emerging importance of fibroblast growth factor-23 (FGF-23) and its co-factor Klotho play very important roles as phosphaturic hormones; however, phosphate levels rise due to a loss of renal Klotho production and the phosphaturic effects of the FGF-23/Klotho axis. Hyperphosphatemia is also associated with calciphylaxis, acceleration of renal tubulointerstitial disease, renal osteodystrophy, and uremic cardiomyopathy. This review incorporates ultrastructural remodeling of the thoracic aorta to provide a different perspective on vascular calcification. Nine-week-old male heterozygous (mRen2) 27 (Ren2) rat models of hypertension, insulin resistance, vascular oxidative stress and albuminuria are utilized to demonstrate aortic remodeling associated with vascular calcification. Nine-week-old male Zucker obese (fa/fa) rat models are utilized to better understand nephrolith formation. Phosphate homeostasis, toxicity, multiple metabolic and uremic toxicities, renal osteodystrophy, and vascular calcification are also discussed. Additionally, the role of the endothelium, vascular smooth muscle cells, inflammatory monocytes/macrophages and mast cells, pericytes, oxidative stress, hydrogen sulfide, and extraosseous calcification in the kidney are discussed as they relate to CKD, ESRD and calciphylaxis.

Antihypertensive treatment with hydrochlorothiazide-hydralazine combination aggravates medial vascular calcification in CKD rats with mineral bone disorder.

Arterial stiffness and medial vascular calcification, leading to isolated systolic blood pressure (BP), are major cardiovascular risk factors in patients with chronic kidney disease (CKD) and mineral bone disorders (MBD). The impact of BP on MBD-induced medial vascular calcification in CKD remains uncertain. We investigated whether BP reduction improves arterial stiffness and medial vascular calcification in a rat model of CKD-MBD. CKD was induced in Wistar rats by subtotal nephrectomy. Then, MBD was generated by a Ca/P-rich diet with calcitriol supplementation to induce medial vascular calcification. Two antihypertensive treatments were evaluated: (1) the angiotensin AT1 receptor antagonist losartan, and (2) the combination of the thiazide diuretic hydrochlorothiazide and the direct vasodilator hydralazine (HCTZ/HY). After 5 weeks, mean BP (MBP), pulse pressure (PP), and pulse wave velocity (PWV) were determined. Vascular calcification was assessed in the thoracic aorta. While MBP was similar in CKD-MBD and control CKD rats, PP and PWV were increased in CKD-MBD rats. The heightened arterial stiffness in CKD-MBD rats was associated with diffused medial calcification along the thoracic aorta. Although both losartan and HCTZ/HY reduced MBP in CKD-MBD rats, losartan did not affect PP and PWV nor medial vascular calcification, whereas HCTZ/HY, unexpectedly, further increased arterial stiffness and medial vascular calcification. In the rat model of CKD-MBD, antihypertensive treatment with losartan did not affect arterial stiffness or medial vascular calcification. However, HCTZ/HY treatment aggravated arterial stiffness and vascular calcification despite a similar reduction of MBP, suggesting a blood pressure-independent mechanism for vascular calcification.

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.

Chronic kidney disease mineral bone disorder in childhood and young adulthood: a ‘growing’ understanding

Chronic kidney disease (CKD) mineral and bone disorder (MBD) comprises a triad of biochemical abnormalities (of calcium, phosphate, parathyroid hormone and vitamin D), bone abnormalities (turnover, mineralization and growth) and extra-skeletal calcification. Mineral dysregulation leads to bone demineralization causing bone pain and an increased fracture risk compared to healthy peers. Vascular calcification, with hydroxyapatite deposition in the vessel wall, is a part of the CKD-MBD spectrum and, in turn, leads to vascular stiffness, left ventricular hypertrophy and a very high cardiovascular mortality risk. While the growing bone requires calcium, excess calcium can deposit in the vessels, such that the intake of calcium, calcium- containing medications and high calcium dialysate need to be carefully regulated. Normal physiological bone mineralization continues into the third decade of life, many years beyond the rapid growth in childhood and adolescence, implying that skeletal calcium requirements are much higher in younger people compared to the elderly. Much of the research into the link between bone (de)mineralization and vascular calcification in CKD has been performed in older adults and these data must not be extrapolated to children or younger adults. In this article, we explore the physiological changes in bone turnover and mineralization in children and young adults, the pathophysiology of mineral bone disease in CKD and a potential link between bone demineralization and vascular calcification.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Lactobacillus rhamnosus GG aggravates vascular calcification in chronic kidney disease: A potential role for extracellular vesicles

AimsLactobacillus rhamnosus GG (LGG) is a probiotic with great promise in future clinical application, which can significantly promote bone formation. However, the effect of LGG on CKD-related vascular calcification is unclear. In this study, we aimed to investigate the effect of LGG on CKD-related vascular calcification. Materials and methodsAfter 2 weeks of 5/6 nephrectomy, CKD rats received a special diet (4 % calcium and 1.8 % phosphate) combined with 1,25-dihydroxyvitamin D3 to induce vascular calcification. Meanwhile, CKD rats in the LGG group were gavaged orally with LGG (1 × 109 CFU bacteria/day). 16S RNA amplicon sequencing was performed to analyze the effect of LGG treatment on gut microbiota composition. Furthermore, differential ultracentrifugation was utilized to extract EVs. The effects of EVs on vascular calcification were evaluated in rat VSMCs, rat aortic rings, and CKD rat calcification models. In this study, vascular calcification was assessed by microcomputed tomography analysis, alizarin red staining, calcium content determination, and the expression of osteogenic transcription factors RUNX2 and BMP2. Key findingsLGG remarkably aggravated vascular calcification. LGG supplementation significantly altered gut microbiota composition in CKD rats, particularly increasing Lactobacillus. Interestingly, EVs presented a significant promoting effect on the development of calcification. Finally, mechanistic analysis proved that EVs aggravated vascular calcification through PI3K/AKT signaling. SignificanceThese results do not support the supplementation of LGG in CKD-associated vascular calcification patients. Our study presented a fresh perspective on LGG with potential risks and adverse effects. CKD patients should use specific probiotic strains cautiously.

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.