Aging impairs vascular function, but the mechanisms involved are unknown. The aim of this study was to analyze whether aging-related hyperphosphatemia is implied in this effect by elucidating the role of oxidative stress. C57BL6 mice that were aged 5 months (young) and 24 months (old), receiving a standard (0.6%) or low-phosphate (0.2%) diet, were used. Isolated mesenteric arteries from old mice showed diminished endothelium-dependent vascular relaxation by the down-regulation of NOS3 expression, increased inflammation and increased fibrosis in isolated aortas, compared to those isolated from young mice. In parallel, increased Nox4 expression and reduced Nrf2, Sod2-Mn and Gpx1 were found in the aortas from old mice, resulting in oxidant/antioxidant imbalance. The low-phosphate diet improved vascular function and oxidant/antioxidant balance in old mice. Mechanisms were analyzed in endothelial (EC) and vascular smooth muscle cells (SMCs) treated with the phosphate donor ß-glycerophosphate (BGP). In EC, BGP increased Nox4 expression and ROS production, which reduced NOS3 expression via NFκB. BGP also increased inflammation in EC. In SMC, BGP increased Collagen I and fibronectin expression by priming ROS production and NFκB activity. In conclusion, hyperphosphatemia reduced endothelium-dependent vascular relaxation and increased inflammation and vascular fibrosis through an impairment of oxidant/antioxidant balance in old mice. A low-phosphate diet achieved improvements in the vascular function in old mice.

Integrin-linked kinase (ILK) is a key scaffolding protein between extracellular matrix protein and the cytoskeleton and has been implicated previously in the pathogenesis of renal damage. However, its involvement in renal mitochondrial dysfunction remains to be elucidated. We studied the role of ILK and its downstream regulations in renal damage and mitochondria function both in vivo and vitro, using a folic acid (FA)-induced kidney disease model. Wild type (WT) and ILK conditional-knockdown (cKD-ILK) mice were injected with a single intraperitoneal dose of FA and studied after 15 days of chronic renal damage progression. Human Kidney tubular epithelial cells (HK2) were transfected with specific siRNAs targeting ILK, glycogen synthase kinase 3-β (GSK3β), or CCAAT/enhancer binding protein-β (C/EBPβ). The expressions and activities of renal ILK, GSK3β, C/EBPβ, mitochondrial oxidative phosphorylation enzymes, and mitochondrial membrane potential were assessed. Additionally, the expression of markers for fibrosis fibronectin (FN) and collagen 1 (COL1A1), for autophagy p62 and cytosolic light chain 3 (LC3B) isoforms II and I, and mitochondrial homeostasis marker carnitine palmitoyl-transferase 1A (CPT1A) were evaluated using immunoblotting, RT-qPCR, immunofluorescence, or colorimetric assays. FA upregulated ILK expression, leading to the decrease of GSK3β activity, increased tubular fibrosis, and produced mitochondrial dysfunction, both in vivo and vitro. These alterations were fully or partially reversed upon ILK depletion, mitigating FA-induced renal damage. The signaling axis composed by ILK, GSK3β, and C/EBPβ regulated CPT1A transcription as the limiting factor in the FA-based impaired mitochondrial activity. We highlight ILK as a potential therapeutical target for preserving mitochondrial function in kidney injury.

Hyperlipidemia-associated atherosclerosis promotes plaque formation and extracellular matrix accumulation in the vascular media, increasing also the production of reactive oxygen species (ROS), inflammation, and the oxidation of LDL to oxidized-LDL, through mechanisms that remain unknown. Our objective was to determine whether oxidizedLDL-induced oxidative stress drives fibrosis in the arterial media during atherosclerosis and whether antioxidants can prevent or reduce this effect. Experiments were conducted in vascular smooth muscle cells (VSMCs) and in apolipoproteinE-knockout (KO-apoE) mice. ROS production was measured by confocal microscopy and fibrosis by immunofluorescence, Western and RT-qPCR in VSMCs and by Sirius red staining and RT-qPCR in mice. In VSMCs, oxidized-LDL treatment significantly increased the expression of TGF-beta, fibronectin and collagen-I, indicating fibrosis. Additionally, oxidized-LDL enhanced ROS production and NF-κB activation. The fibrotic response was prevented by treatment with the antioxidant N-acetylcysteine (NAC) and an NF-κB inhibitor, suggesting that the ROS-NF-κB axis mediates these effects in VSMCs. In vivo, KO-apoE mice exhibited higher cholesterol levels and increased ROS production compared with wild-type controls. These mice also showed enhanced aortic fibrosis. Importantly, NAC administration reduced both ROS production and fibrosis in KO-apoE mice. Overall, the findings suggest that both oxidized-LDL-induced vascular fibrosis and hyperlipidemia-induced fibrosis are mediated by ROS.