Diabetic Vascular Calcification Mediated by the Collagen Receptor Discoidin Domain Receptor 1 via the Phosphoinositide 3-Kinase/Akt/Runt-Related Transcription Factor 2 Signaling Axis.

Objective- Vascular calcification is a common and severe complication in patients with atherosclerosis which is exacerbated by type 2 diabetes mellitus. Our laboratory recently reported that the collagen receptor discoidin domain receptor 1 (DDR1) mediates vascular calcification in atherosclerosis; however, the underlying mechanisms are unknown. During calcification, vascular smooth muscle cells transdifferentiate into osteoblast-like cells, in a process driven by the transcription factor RUNX2 (runt-related transcription factor 2). DDR1 signals via the phosphoinositide 3-kinase/Akt pathway, which is also central to insulin signaling, and upstream of RUNX2, and this led us to investigate whether DDR1 promotes vascular calcification in diabetes mellitus via this pathway. Approach and Results- Ddr1+/+ ; Ldlr-/- (single knock-out) and Ddr1-/- ; Ldlr-/- (double knock-out) mice were placed on high-fat diet for 12 weeks to induce atherosclerosis and type 2 diabetes mellitus. Von Kossa staining revealed reduced vascular calcification in the aortic arch of double knock-out compared with single knock-out mice. Immunofluorescent staining for RUNX2 was present in calcified plaques of single knock-out but not double knock-out mice. Primary vascular smooth muscle cells obtained from Ddr1+/+ and Ddr1-/- mice were cultured in calcifying media. DDR1 deletion resulted in reduced calcification, a 74% reduction in p-Akt levels, and an 88% reduction in RUNX2 activity. Subcellular fractionation revealed a 77% reduction in nuclear RUNX2 levels in Ddr1-/- vascular smooth muscle cells. DDR1 associated with phosphoinositide 3-kinase, and treatment with the inhibitor wortmannin attenuated calcification. Finally, we show that DDR1 is important to maintain the microtubule cytoskeleton which is required for the nuclear localization of RUNX2. Conclusions- These novel findings demonstrate that DDR1 promotes RUNX2 activity and atherosclerotic vascular calcification in diabetes mellitus via phosphoinositide 3-kinase/Akt signaling.

Phosphate excretion is decreased in older cardiac patients with normal kidney function: an emerging dietary risk factor?

Serum phosphate independently predicts cardiovascular events and mortality. Sixteen healthy adults and 9 adults with cardiovascular disease (CVD) ingested 500 mg of sodium phosphate after an over-night fast. In control subjects, the urine phosphate/creatinine ratio was significantly higher at 2 h (3.12 ± 1.02) than at baseline (1.98 ± 0.58, p < 0.001) but no change was observed in CVD patients. Decreased postprandial urinary excretion of phosphate could accelerate vascular calcification and may be an under-recognized risk factor for CVD.

Phosphate: an old bone molecule but new cardiovascular risk factor.

Phosphate handling in the body is complex and involves hormones produced by the bone, the parathyroid gland and the kidneys. Phosphate is mostly found in hydroxyapatite. however recent evidence suggests that phosphate is also a signalling molecule associated with bone formation. Phosphate balance requires careful regulation of gut and kidney phosphate transporters, SLC34 transporter family, but phosphate signalling in osteoblasts and vascular smooth muscle cells is likely mediated by the SLC20 transporter family (PiT1 and PiT2). If not properly regulated, phosphate imblanace could lead to mineral disorders as well as vascular calcification. In chronic kidney disease-mineral bone disorder, hyperphosphataemia has been consistently associated with extra-osseous calcification and cardiovascular disease. This review focuses on the physiological mechanisms involved in phosphate balance and cell signalling (i.e. osteoblasts and vascular smooth muscle cells) as well as pathological consequences of hyperphosphataemia. Finally, conventional as well as new and experimental therapeutics in the treatment of hyperphosphataemia are explored.

Dietary vitamin K and therapeutic warfarin alter the susceptibility to vascular calcification in experimental chronic kidney disease.

The leading cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease, with vascular calcification being a key modifier of disease progression. A local regulator of vascular calcification is vitamin K. This γ-glutamyl carboxylase substrate is an essential cofactor in the activation of several extracellular matrix proteins that inhibit calcification. Warfarin, a common therapy in dialysis patients, inhibits the recycling of vitamin K and thereby decreases the inhibitory activity of these proteins. In this study, we sought to determine whether modifying vitamin K status, either by increasing dietary vitamin K intake or by antagonism with therapeutic doses of warfarin, could alter the development of vascular calcification in male Sprague-Dawley rats with adenine-induced CKD. Treatment of CKD rats with warfarin markedly increased pulse pressure and pulse wave velocity, as well as significantly increased calcium concentrations in the thoracic aorta (3-fold), abdominal aorta (8-fold), renal artery (4-fold), and carotid artery (20-fold). In contrast, treatment with high dietary vitamin K1 increased vitamin K tissue concentrations (10-300-fold) and blunted the development of vascular calcification. Thus, vitamin K has an important role in modifying mechanisms linked to the susceptibility of arteries to calcify in an experimental model of CKD.

Cardiovascular disease in an adenine-induced model of chronic kidney disease: the temporal link between vascular calcification and haemodynamic consequences.

OBJECTIVES: Medial vascular calcification is highly prevalent in chronic kidney disease (CKD), and it is a risk factor for mortality. This study characterizes the time course and the link between calcification of major arteries, changes in blood pressure (BP) and cardiac growth in experimental CKD. METHODS: CKD (elevated serum creatinine and urea) was induced with a 0.25% adenine diet (5, 8 and 11 weeks). BP was measured by radiotelemetry in conscious rats or indwelling catheter under anaesthesia. At each time point, serum biochemistry and tissue calcification was quantified. RESULTS: CKD was present in all animals by 5 weeks and the ensuing 6 weeks (11 weeks total). CKD animals developed elevated serum phosphate (5-8 weeks) and fibroblast growth factor-23 (FGF-23; 5-11 weeks) levels. There was a 100% incidence of calcification at 11 weeks, 71% at 8 weeks and 33% at 5 weeks, and distal arteries appeared more susceptible than proximal arteries. Calcification was associated with widening of pulse pressure (PP), and a higher pulse wave. Continuous radiotelemetry revealed a significant increase in SBP variability and an accelerated (<24 h) elevation in PP of at least 10 mmHg following 8 weeks of CKD. This precipitous change was driven by a drop in mean DBP rather than elevated mean SBP. PP, duration of CKD and FGF-23 levels correlated with left ventricular hypertrophy. CONCLUSION: The unique haemodynamic consequences of medial calcification, combined with the hormonal consequences of hyperphosphatemia (i.e. FGF-23), seem to have an exacerbated risk for left ventricular hypertrophy.

Vascular calcification in animal models of CKD: A review.

Vascular calcification is a significant contributor to the cardiovascular mortality observed in chronic kidney disease (CKD). This review discusses the animal models (5/6 nephrectomy, mouse electrocautery model and dietary adenine) that have been employed in the study of vascular calcification outcomes in CKD. Rodent models of CKD generate a range of severity in the vascular calcification phenotype. Major limitations of the 5/6th nephrectomy model include the requirement for surgery and the need to use either excessive dietary phosphorus or vitamin D. Major limitations of the mouse electrocautery model include the requirement for surgery, the mortality rate when very advanced CKD develops, and resistance to vascular calcification without the use of transgenic animals. This is balanced against the major advantage of the ability to study transgenic animals to further understand the mechanisms associated with either the acceleration or inhibition of calcification. Dietary adenine generates severe CKD and does not require surgery. The major disadvantage is the weight loss that ensues when rats receive a diet containing 0.75% adenine. In summary, animal models are useful to study CKD-associated vascular calcification and the results obtained in these pre-clinical animal studies appear to translate to the evidence, however limited, which exists in humans with CKD.

Mg2+- and MgATP-inhibited and Ca2+/calmodulin-sensitive TRPM7-like current in hepatoma and hepatocytes.

Although understood to be ubiquitously expressed, the functional identification and significance of Mg(2+)-inhibited, nonspecific cation currents has been established in only a few cell types. Here we identified an outwardly rectifying nonspecific cation current in quiescent rat hepatocytes and the proliferating and polarized rat hepatoma, WIF-B. Under whole cell recording conditions in which cells were bathed and dialyzed with Na-gluconate solutions, the latter Ca(2+) and Mg(2+) free, current reversed close to 0 mV, was time independent, and was greater than 10 times higher at +120 mV compared with -120 mV. Outward current at -120 mV developed slowly, from 17.7 +/- 10.3 pA/pF at patch rupture to 106.6 +/- 15.6 pA/pF at 12 min in WIF-B cells, and 4.9 +/- 2.7 to 20.6 +/- 5.6 pA/pF in rat hepatocytes. The nonspecific TRP channel inhibitor, 2-aminoethoxyphenylborate (2-APB), inhibited current (IC(50) = 72 +/- 13 microM) and caused apoptotic cell death in WIF-B cells. Rat hepatocyte survival was more resistant to 2-APB. Dialysis of WIF-B cells with physiological concentrations of Mg(2+) and Mg-ATP, but not ATP alone, inhibited current development, suggesting that Trpm7 rather than Trpm6 underlies this current. RT-PCR demonstrated that both Trpm6 and Trpm7 are expressed at similar levels in both cell types, suggesting that the functional differences noted are not transcript dependent. Intracellular Ca(2+) (IC(50) = 125 +/- 35 nM) also inhibited current development, and this could be partially relieved by the calmodulin and Ca(2+)/calmodulin-dependent kinase inhibitors W-7, staurosporine, KN-93, or calmodulin kinase II (CaMKII) inhibitory peptide. To summarize, our results show that in addition to their established Mg(2+) sensitivity, Trpm7-like channels are inhibited by cytosolic Ca(2+) in a CaMKII-dependent manner and may support hepatocellular survival during proliferation.