Effect of signaling pathway inhibitors on ox-LDL-induced Klotho downregulation in proximal tubular cells.

<p>Pretreatment of MCT with parthenolide (1 µM) or PD098059 (50 µM) for 1 hour attenuates the decrease in Klotho mRNA (<b>A</b>) and protein (<b>B</b>) induced by ox-LDL (25 µg/mL). (<b>C</b>) Treatment with ox-LDL (25 µg/mL) promotes IκB degradation (left panel) and ERK1/2 phosphorylation (right panel) in MCT cells. Cell lysates were analyzed by Western blot for IκB and phospho-ERK 1/2. Each blot was stripped and reprobed with beta-actin and anti-ERK antibody, respectively. Mean±SD of 3 independent experiments. *p<0.05 vs control, # p<0.05 vs ox-LDL.</p

Oxidized-LDL stimulates expression and secretion of pro-inflammatory chemokines and ROS production by proximal tubular MCT cells.

<p>(<b>A</b>) Quantitative RT-PCR analyses of IL-6, TNF-α, RANTES and MCP-1 in MCT stimulated with ox-LDL (25 µg/mL) at different time points. (<b>B</b>) Quantitative RT-PCR analyses of IL-6, TNF-α, RANTES and MCP-1 in MCT stimulated with ox-LDL at different doses (0–25 µg/mL). Values for chemokines were normalized to GAPDH expression and results are expressed as fold-change vs control. (<b>C–E</b>) Secretion of RANTES, MCP-1 and IL-6 (at 18 and 24 h) in MCT treated with 25 µg/mL ox-LDL. Supernatants were tested by ELISA. (<b>F</b>) NADPH-dependent ROS production in MCT cells treated with ox-LDL (25 µg/ml) for 24 h. (<b>G</b>) Representative DHE staining of MCT cells under basal conditions and stimulated with ox-LDL (25 µg/ml) for 24 h. Mean±SD of 3 independent experiments. *p<0.05 vs control.</p

Inflammatory response and oxidative stress in murine kidneys.

<p>Representative inmunohistochemistry (<b>A</b>) and semiquantitative assessment (<b>B–C</b>) of renal macrophage infiltration (F4/80) and inflammatory cytokines (RANTES and MCP-1) in kidneys from C57BL/6 (WT) and ApoE knockout (ApoE) mice fed normal chow-(ND) or hyperlipidemic-diet (HC) for 5 or 10 weeks. Original magnification x 100. (<b>D</b>) Expression of MCP-1 and RANTES, as determined by real time RT-PCR, in kidneys from all the studied groups. (<b>E</b>) Representative DHE staining and semiquantitative DHE assessment in kidneys from all mice groups (magnification x 200). *p<0.05 vs WT with ND in each time-period, † p<0.05 vs WT with HC in each time-period, ‡ p<0.05 vs ApoE knockout mice with ND in each time-period. Values shown are mean±SD, n = 8 per group.</p

Murine model of renal damage induced by hyperlipidemia.

<p>(<b>A</b>) Serum cholesterol levels from C57BL/6 (WT) and ApoE knockout (ApoE) mice fed a normal chow diet (ND) or a hyperlipidemic diet (HC) for 5 or 10 weeks. (<b>B</b>) Semiquantitative assessment and representative secondary ion images under the irradiation of bismuth cluster showing lipid content in kidneys from mice at 10 weeks of dietary intervention. In order to normalize tissue size variation, metabolite measurements were expressed as % relative intensity = ratio between the metabolite area and total tissue area (phosphate area) (<b>C</b>) Representative Masson trichrome staining in mouse kidneys and semiquantitative assessment of lesions in glomerular, tubular and interstitial compartments. Original magnification x 100. *p<0.05 vs WT with ND in each time-period, † p<0.05 vs WT with HC in each time-period, ‡ p<0.05 vs ApoE knockout mice with ND in each time-period. Values shown are mean±SD, n = 8 per group.</p

Oxidized LDL decrease Klotho expression in cultured tubular cells.

<p>(<b>A</b>) Oil-Red-O staining in murine proximal tubular cells (MCT) showing increased lipid accumulation after 24 h incubation with ox-LDL (25 µg/mL). Ox-LDL decreases Klotho mRNA expression, as determined by quantitative RT-PCR, in a time (<b>B</b>) and dose-dependent manner (<b>C</b>) in proximal tubular cells (MCT). Mean±SD of three independent experiments. *p<0.05 vs control. Klotho protein expression, as determined by Western blot (<b>D</b>) and confocal microscopy (<b>E</b>), in MCT treated with ox-LDL (25 µg/mL) for 24 h. Indirect immunofluorescence using anti-Klotho with secondary Alexa Fluor 488–conjugated antibody (green). Nuclei were stained with propidium iodide (PI, red). Images are representative of three independent experiments.</p

Decreased Klotho expression in kidneys from hyperlipidemic mice.

<p>(A) Localization of renal Klotho using anti-Klotho with secondary Alexa Fluor 488–conjugated antibody (green) in a normolipidemic WT mice. (<b>B</b>) Klotho-expressing tubular cells in kidney from WT mice were detected using anti-Klotho with secondary Alexa Fluor 633–conjugated antibody (red) and then stained with proximal tubule marker FITC-Tetranogolobus lotus (green). Nuclei were stained with DAPI (blue). Arrows and arrowheads identify Klotho expression in proximal and distal nephron tubules, respectively. (<b>C</b>) Total Klotho kidney protein expression, as determined by Western-blot in kidneys from C57BL/6 (WT) and ApoE knockout (ApoE) mice fed normal chow-(ND) or hyperlipidemic-diet (HC) for 5 or 10 weeks. (<b>D</b>) Klotho mRNA expression in kidneys as determined by real time RT-PCR. *p<0.05 vs WT with ND in each time-period, † p<0.05 vs WT with HC in each time-period, ‡ p<0.05 vs ApoE knockout mice with ND in each time-period. Values shown are mean±SD, n = 8 per group.</p