Rukshana Shroff

Dialysis Accelerates Medial Vascular Calcification in Part by Triggering Smooth Muscle Cell Apoptosis

Background— Vascular calcification is associated with increased morbidity and mortality in stage V chronic kidney disease, yet its early pathogenesis and initiating mechanisms in vivo remain poorly understood. To address this, we quantified the calcium (Ca) load in arteries from children (10 predialysis, 24 dialysis) and correlated it with clinical, biochemical, and vascular measures. Methods and Results— Vessel Ca load was significantly elevated in both predialysis and dialysis and was correlated with the patients’ mean serum Ca×phosphate product. However, only dialysis patients showed increased carotid intima-media thickness and increased aortic stiffness, and calcification on computed tomography was present in only the 2 patients with the highest Ca loads. Importantly, predialysis vessels appeared histologically intact, whereas dialysis vessels exhibited evidence of extensive vascular smooth muscle cell (VSMC) loss owing to apoptosis. Dialysis vessels also showed increased alkaline phosphatase activity and Runx2 and osterix expression, indicative of VSMC osteogenic transformation. Deposition of the vesicle membrane marker annexin VI and vesicle component mineralization inhibitors fetuin-A and matrix Gla-protein increased in dialysis vessels and preceded von Kossa positive overt calcification. Electron microscopy showed hydroxyapatite nanocrystals within vesicles released from damaged/dead VSMCs, indicative of their role in initiating calcification. Conclusions— Taken together, this study shows that Ca accumulation begins predialysis, but it is the induction of VSMC apoptosis in dialysis that is the key event in disabling VSMC defense mechanisms and leading to overt calcification, eventually with clinically detectable vascular damage. Thus the identification of factors that lead to VSMC death in dialysis will be of prime importance in preventing vascular calcification.

Prelamin A Acts to Accelerate Smooth Muscle Cell Senescence and Is a Novel Biomarker of Human Vascular Aging

Background— Hutchinson-Gilford progeria syndrome is a rare inherited disorder of premature aging caused by mutations in LMNA or Zmpste24 that disrupt nuclear lamin A processing, leading to the accumulation of prelamin A. Patients develop severe premature arteriosclerosis characterized by vascular smooth muscle cell (VSMC) calcification and attrition. Methods and Results— To determine whether defective lamin A processing is associated with vascular aging in the normal population, we examined the profile of lamin A expression in normal and aged VSMCs. In vitro, aged VSMCs rapidly accumulated prelamin A coincidently with nuclear morphology defects, and these defects were reversible by treatment with farnesylation inhibitors and statins. In human arteries, prelamin A accumulation was not observed in young healthy vessels but was prevalent in medial VSMCs from aged individuals and in atherosclerotic lesions, where it often colocalized with senescent and degenerate VSMCs. Prelamin A accumulation correlated with downregulation of the lamin A processing enzyme Zmpste24/FACE1, and FACE1 mRNA and protein levels were reduced in response to oxidative stress. Small interfering RNA knockdown of FACE1 reiterated the prelamin A–induced nuclear morphology defects characteristic of aged VSMCs, and overexpression of prelamin A accelerated VSMC senescence. We show that prelamin A acts to disrupt mitosis and induce DNA damage in VSMCs, leading to mitotic failure, genomic instability, and premature senescence. Conclusions— This study shows that prelamin A is a novel biomarker of VSMC aging and disease that acts to accelerate senescence. It therefore represents a novel target to ameliorate the effects of age-induced vascular dysfunction.

Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.

Mechanistic Insights into Vascular Calcification in CKD

Cardiovascular disease begins early in the course of renal decline and is a life-limiting problem in patients with CKD. The increased burden of cardiovascular disease is due, at least in part, to calcification of the vessel wall. The uremic milieu provides a perfect storm of risk factors for accelerated calcification, but elevated calcium and phosphate levels remain key to the initiation and progression of vascular smooth muscle cell calcification in CKD. Vascular calcification is a highly regulated process that involves a complex interplay between promoters and inhibitors of calcification and has many similarities to bone ossification. Here, we discuss current understanding of the process of vascular calcification, focusing specifically on the discrete and synergistic effects of calcium and phosphate in mediating vascular smooth muscle cell apoptosis, osteochondrocytic differentiation, vesicle release, calcification inhibitor expression, senescence, and death. Using our model of intact human vessels, factors initiating vascular calcification in vivo and the role of calcium and phosphate in driving accelerated calcification ex vivo are described. This work allows us to link clinical and basic research into a working theoretical model to explain the pathway of development of vascular calcification in CKD.

Abnormal High-Density Lipoprotein Induces Endothelial Dysfunction via Activation of Toll-like Receptor-2

Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDL(CKD)), a population with high cardiovascular risk, we have demonstrated that HDL(CKD) in contrast to HDL(Healthy) promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDL(CKD) that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDL(CKD) reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.

A Bimodal Association of Vitamin D Levels and Vascular Disease in Children on Dialysis

In addition to its classical role in calcium-phosphate homeostasis, vitamin D has anti-inflammatory effects that may influence vascular disease. This study examined the impact of vitamin D levels on the vascular phenotype in 61 children who had been on dialysis for >or=3 mo and in 40 age-matched control subjects. All dialysis patients were prescribed daily oral 1-alpha hydroxyvitamin D(3). 92% of patients were deficient in 25-hydroxyvitamin D [25(OH)D]. 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels were low in 36% and high in 11% of patients. There was a weak correlation between 1 alpha-hydroxyvitamin D(3) dosage and 1,25(OH)(2)D levels. Both carotid intima-media thickness and calcification scores showed a U-shaped distribution across 1,25(OH)(2)D levels: patients with both low and high 1,25(OH)(2)D had significantly greater carotid intima-media thickness (P < 0.0001) and calcification (P = 0.0002) than those with normal levels. Low 1,25(OH)(2)D levels were associated with higher high-sensitivity C-reactive protein (P < 0.0001). Calcification was most frequently observed in patients with the lowest 1,25(OH)(2)D and the highest high-sensitivity C-reactive protein. In contrast, 25(OH)D levels did not correlate with any vascular measure. In conclusion, both low and high 1,25(OH)(2)D levels are associated with adverse morphologic changes in large arteries, and this may be mediated through the effects of 1,25(OH)(2)D on calcium-phosphate homeostasis and inflammation. For optimization of strategies to protect the vasculature of dialysis patients, careful monitoring of 1,25(OH)(2)D levels may be required.

Mineral Metabolism and Vascular Damage in Children on Dialysis

Cardiovascular disease is increasingly recognized as a life-limiting problem in young patients with chronic kidney disease, but there are few studies in children that describe its determinants. We studied the association of intact parathyroid hormone (iPTH) levels and their management on vascular structure and function in 85 children, ages 5-18 years, who had received dialysis for > or =6 months. Compared to controls, dialysis patients had increased carotid intima-media thickness and pulse-wave velocity. All vascular measures positively correlated with serum phosphorus levels, while carotid intima-media thickness and cardiac calcification score also correlated with iPTH levels. Patients with mean time-integrated iPTH levels less than twice the upper limit of normal (n = 41) had vascular measures that were comparable to age-matched controls, but those with iPTH levels greater than twice the upper limit of normal (n = 44) had greater carotid intima-media thickness, stiffer vessels, and increased cardiac calcification than controls. Patients with increased carotid intima-media thickness had stiffer vessels and a greater prevalence of cardiac calcification. There was a strong dose-dependent correlation between vitamin D and all vascular measures, and calcium intake from phosphate binders weakly correlated with carotid intima-media thickness. In conclusion, both iPTH level and dosage of vitamin D are associated with vascular damage and calcification in children on dialysis.

Vascular Smooth Muscle Cell Calcification Is Mediated by Regulated Exosome Secretion

RATIONALE Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

HNF1B Mutations Associate with Hypomagnesemia and Renal Magnesium Wasting

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (<1.58 mg/dl) compared with 2% (one of 48) of those without mutations (P < 0.0001). The median plasma magnesium was significantly lower among mutation carriers than those without mutations (1.68 versus 2.02 mg/dl; P < 0.0001). Because hypermagnesuria and hypocalciuria accompanied the hypomagnesemia, we analyzed genes associated with hypermagnesuria and detected highly conserved HNF1 recognition sites in FXYD2, a gene that can cause autosomal dominant hypomagnesemia and hypocalciuria when mutated. Using a luciferase reporter assay, we demonstrated HNF1B-mediated transactivation of FXYD2. These results extend the phenotype of HNF1B mutations to include hypomagnesemia. HNF1B regulates transcription of FXYD2, which participates in the tubular handling of Mg(2+), thus describing a role for HNF1B not only in nephrogenesis but also in the maintenance of tubular function.

Long-term outcome of chronic dialysis in children.

As the prevalence of children on renal replacement therapy (RRT) increases world wide and such therapy comprises at least 2% of any national dialysis or transplant programme, it is essential that paediatric nephrologists are able to advise families on the possible outcome for their child on dialysis. Most children start dialysis with the expectation that successful renal transplantation is an achievable goal and will provide the best survival and quality of life. However, some will require long-term dialysis or may return intermittently to dialysis during the course of their chronic kidney disease (CKD). This article reviews the available outcome data for children on chronic dialysis as well as extrapolating data from the larger adult dialysis experience to inform our paediatric practice. The multiple factors that may influence outcome, and, particularly, those that can potentially be modified, are discussed.