benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Kidney-Failure--Chronic

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.

Topics: Adenine; Alkaline Phosphatase; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Autophagy; Autophagy-Related Protein 5; Caspase Inhibitors; Cattle; Cells, Cultured; Disease Models, Animal; Kidney Failure, Chronic; Metalloporphyrins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphates; Proteins; Rats; RNA Interference; Secretory Vesicles; Sodium-Phosphate Cotransporter Proteins, Type III; Superoxide Dismutase; Time Factors; Transfection; Valproic Acid; Vascular Calcification

Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (>2.5 mM) induced apoptosis in two endothelial cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at approximately 2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Atherosclerosis; Calcium; Caspase Inhibitors; Cells, Cultured; Cysteine Proteinase Inhibitors; Endothelial Cells; Gene Expression; Humans; Hyperphosphatemia; In Vitro Techniques; Kidney Failure, Chronic; Membrane Potential, Mitochondrial; Phosphates; Reactive Oxygen Species; Sodium-Phosphate Cotransporter Proteins; Umbilical Veins

Although heat shock protein 72 kDa (HSP72) protects tubular epithelium from a variety of acute insults, its role in chronic renal injury and fibrosis is poorly characterized. In this study, we tested the hypothesis that HSP72 reduces apoptosis and epithelial-to-mesenchymal transition (EMT), important contributors to tubular cell injury in vitro and in vivo. In rats, orally administered geranylgeranylacetone (GGA), an agent that selectively induces HSP72, markedly reduced both apoptosis and cell proliferation in tubular epithelium and decreased both interstitial fibroblast accumulation and collagen I deposition after unilateral ureteric obstruction, a model of chronic renal tubulointerstitial fibrosis and dysfunction. In cultured renal NRK52E cells, exposure to TGF-beta1 induced EMT and apoptosis, major causes of renal fibrosis and tubular atrophy, respectively. Exposure to a pan-caspase inhibitor (ZVAD-FMK) prevented TGF-beta1-induced apoptosis but did not reduce EMT. In contrast, selective HSP72 expression in vitro inhibited EMT caused by TGF-beta1 as indicated by preserving the E-cadherin expression level and alpha-smooth muscle actin induction. Small interfering RNA directed against HSP72 blocked the cytoprotective effects of HSP72 overexpression on EMT in TGF-beta1-exposed cells. Taken together, our data indicate that HSP72 ameliorates renal tubulointerstitial fibrosis in obstructive nephropathy by inhibiting both renal tubular epithelial cell apoptosis and EMT.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Line; Cell Proliferation; Cell Transdifferentiation; Diterpenes; Epithelial Cells; Fibrosis; HSP72 Heat-Shock Proteins; Kidney Failure, Chronic; Kidney Tubules, Proximal; Male; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1