Autophagy is a dynamic and highly regulated process of self-digestion responsible for cell survival and reaction to oxidative stress. As oxidative stress is increased in uremia and is associated with vascular calcification, we studied the role of autophagy in vascular calcification induced by phosphate. In an in vitro phosphate-induced calcification model of vascular smooth muscle cells (VSMCs) and in an in vivo model of chronic renal failure, autophagy was inhibited by the superoxide dismutase mimic MnTMPyP, superoxide dismutase-2 overexpression, and by knockdown of the sodium-dependent phosphate cotransporter Pit1. Although phosphate-induced VSMC apoptosis was reduced by an inhibitor of autophagy (3-methyladenine) and knockdown of autophagy protein 5, calcium deposition in VSMCs was increased during inhibition of autophagy, even with the apoptosis inhibitor Z-VAD-FMK. An inducer of autophagy, valproic acid, decreased calcification. Furthermore, 3-methyladenine significantly promoted phosphate-induced matrix vesicle release with increased alkaline phosphatase activity. Thus, autophagy may be an endogenous protective mechanism counteracting phosphate-induced vascular calcification by reducing matrix vesicle release. Therapeutic agents influencing the autophagic response may be of benefit to treat aging or disease-related vascular calcification and osteoporosis.

Hyperphosphatemia is the major risk factor associated with vascular calcification (VC) in end-stage renal disease. As oxidative stress is increased in uremia, we studied the role of mitochondrial reactive oxygen species (ROS) and nuclear factor-κB signaling in phosphate-induced VC. In an in vitro calcification model (β-glycerophosphate (BGP) induction) using bovine aortic smooth muscle cells, the production of intracellular and mitochondrial ROS, or superoxide anion, was stimulated by increased mitochondrial membrane potential. This effect was blocked by the superoxide dismutase (SOD) mimic MnTMPyP, a respiratory chain inhibitor rotenone, or a protonophore. Calcium deposition and the switch of smooth muscle cells from a contractile to an osteogenic phenotype were decreased when mitochondrial ROS generation was inhibited by the respiratory chain inhibitor, MnTMPyP, or the overexpression of SOD1 and SOD2 and uncoupling protein 2. The phosphorylation of IkKβ, IκBα degradation, and p65 nuclear translocation were increased by BGP but reversed when mitochondrial ROS production was blocked by protonophore or MnTMPyP. Knockdown of endogenous p65 or overexpression of IκBα reduced calcium deposition in the cultured cells. Furthermore, in a rat model of dietary adenine-induced chronic renal failure, MnTMPyP reduced aortic ROS levels, p65 activation, and calcium deposition. Thus, mitochondrial ROS-mediated p65 nuclear translocation is involved in phosphate-induced VC.

Peroxisome proliferator-activated receptors (PPARs, α, β/δ, and γ) are members of the nuclear receptor superfamily of ligand-activated transcription factors. PPARs regulate the expression of genes involved in lipid metabolism. 8( S)-hydroxyeicosatetraenoic acid (8- S-HETE), leukotriene B4(LTB4), and hypolipidemic fibrates activate PPARα, whereas PPARγ is activated by prostaglandin metabolites. The present studies examined the intrarenal and tissue distribution of rabbit and human PPARα, -β/δ, and -γ mRNAs. Nuclease protection showed PPARα predominated in liver, heart, and kidney, whereas PPARγ, a putative adipose-specific transcription factor, was in white adipose tissue, bladder, and ileum, followed by kidney and spleen. Lower expression levels of PPARβ/δ were observed in several tissues. In situ hybridization of kidney showed PPARα mRNA predominated in proximal tubules and medullary thick ascending limbs of both rabbit and human. PPARγ was exclusively expressed in medullary collecting duct and papillary urothelium. Immunoblot confirmed the expression of PPARγ protein in freshly isolated inner medullary collecting ducts. mRNAs for all the PPARs were expressed in the ureter and bladder in both rabbit and human, but PPARγ expression was greatest. This distinct distribution of PPAR isoforms has important implications for lipid-activated gene transcription in urinary epithelia.

Periostin neutralizing Ab. The hybridoma cell lines secreting mouse mAb against mouse periostin were generated by Abmart Co., according to the standard hybridoma technique (72). The mouse mAbs were purified by Protein A affinity chromatograph. mAb purity was confirmed by HPLC. The concentrations of the obtained mAbs were measured by Mouse IgG ELISA Quantitation kit (Bethyl Laboratories), following the manufacturer's instructions. The kinetic parameters of the mAbs were determined using a Biacore T100 instrument (Biacore AB). Purified Ab was diluted in saline and injected at a dose of 5 mg/kg into db/db mice for 2 weeks.Statistics. All values are shown as mean ± SEM. Statistical differences were determined by 2-way ANOVA with Bonferroni-adjusted post-test or by 2-tailed Student's t test. A P value less than 0.05 was considered significant.

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