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.

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.

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.

Mishra R, Rao V, Ta R, Shobeiri N, Hill CE. Mg 2ϩ -and MgATP-inhibited and Ca 2ϩ /calmodulin-sensitive TRPM7-like current in hepatoma and hepatocytes. Am J Physiol Gastrointest Liver Physiol 297: G687-G694, 2009. First published August 6, 2009 doi:10.1152/ajpgi.90683.2008.-Although understood to be ubiquitously expressed, the functional identification and significance of Mg 2ϩ -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 Nagluconate solutions, the latter Ca 2ϩ and Mg 2ϩ free, current reversed close to 0 mV, was time independent, and was greater than 10 times higher at ϩ120 mV compared with Ϫ120 mV. Outward current at Ϫ120 mV developed slowly, from 17.7 Ϯ 10.3 pA/pF at patch rupture to 106.6 Ϯ 15.6 pA/pF at 12 min in WIF-B cells, and 4.9 Ϯ 2.7 to 20.6 Ϯ 5.6 pA/pF in rat hepatocytes. The nonspecific TRP channel inhibitor, 2-aminoethoxyphenylborate (2-APB), inhibited current (IC 50 ϭ 72 Ϯ 13 M) 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 2ϩ 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 2ϩ (IC50 ϭ 125 Ϯ 35 nM) also inhibited current development, and this could be partially relieved by the calmodulin and Ca 2ϩ /calmodulin-dependent kinase inhibitors W-7, staurosporine, or (2,3,13,17,26,35,39), and pathophysiological situations such as anoxic cell death and ischemia-reperfusion injury (1, 41). Although a role for TRPC1 in stimulus-induced Ca 2ϩ influx in the major liver epithelial cell, the hepatocyte, has been suggested (5), and both TRPM6 and TRPM7 are expressed in relatively high amounts in liver tissue (7, 9, 22), Mg 2ϩ -sensitive nonselective cation currents have not been demonstrated in these cells. Nevertheless, 2-aminoethoxyphenylborate (2-APB), albeit a relatively nonspecific inhibitor of TRPM and TRPC channels and other transporters, inhibited bile generation in perfused rat livers (10), and reduced hepatic store-operated Ca 2ϩ current and ischemia-reperfusion injury (15,27).TRPM7 currents are outwardly rectifying and exhibit very small inward currents under divalent-free intracellular recording conditions due to block of inward current by external Mg 2ϩ (26, 34). These currents develop over the course of minutes following the initiation of intracellular dialysis due to the dilution of cytosolic Mg 2ϩ . Dialysis with Mg 2ϩ -containing solutions also induces a slow inactivation, or "rundown," of TRPM7 current [IC 50 0.1-0.7 mM (26,31,40)]. T...