Aggravated renal dysfunction in Sirt1 knockout mouse after LPS challenge.

<p>Sirt1^−/− mice and Sirt1^+/+ littermates were divided into four groups (Sirt1^+/+/PBS, Sirt1^−/−/PBS, Sirt1^+/+/LPS, Sirt1^−/−/LPS). Serum BUN (A) and KIM-1(B) Levels were measured 24 h after LPS challenge n≥4 mice/group. * P<0.05 versus LPS/Sirt1^+/+ group.</p

Sirt1 deletion causes significant increase of pro-inflammatory cytokine production after LPS challenge.

<p>Serum IL-6 and TNF-alpha levels were measured by ELISA 6 h (A, B) or 24 h (C, D) after LPS challenge (n≥4 mice/group). *P<0.05 versus Sirt1^+/+/LPS group.</p

Sirt1 deletion enhances LPS-induced Stat3 and ERK1/2 activation in the kidney.

<p>Kidney tissues were harvested 6(n≥7 mice/group). Samples were subjected to immunoblotting assay. STAT3 (A) and ERK1/2 (B) phosphorylation were examined in the kidney tissues by immunoblotting assay. (C, D) Densitometry analysis. *P<0.05 versus Sirt1^+/+/LPS group.</p

Increased neutrophil infiltration in Sirt1 knockout mice after LPS challenge.

<p>Twenty four hours after LPS challenge, kidney tissues from Sirt1^−/− mice and sirt1^+/+ littermates were collected (n = 5 mice/group). Cryosections were prepared. (<b>A</b>) Gr-1 was used as a specific marker for neutrophil staining. Neutrophil (arrow) infiltration was detected. (B) The number of neutrophils was counted. *P<0.05 versus Sirt1^+/+/LPS group.</p

Aggravated renal tubular injury in Sirt1 knockout mice after LPS challenge.

<p>Kidney tissues were harvested 24(n = 5 mice/group). The collected kidneys were stained with H&E staining. (A) Histological examination shows increased tubular injury in kidney cortex of Sirt1^−/− mice after LPS challenge, including tubular dilatation, flattening (*) and vacuolization (arrows). (B) Quantitative evaluation of morphological tubular damage 24 h after LPS challenge. *P<0.05 versus Sirt1^+/+/LPS group.</p

Sirt1 deletion enhances LPS-induced ICAM-1/VCAM-1 expression in the kidney.

<p>ICAM-1 and VCAM-1expression were assayed 24 h after LPS challenge (n≥4 mice/group). (A, B) Representative blots showing ICAM-1 and VCAM-1 expression in the kidney. (C, D) Densitometry analysis. *P<0.05 versus Sirt1^+/+/LPS group.</p

Sirt1 deletion enhances LPS-induced NF-κB activation.

<p>Kidney tissues were harvested 6(n≥7 mice/group). Kidney samples were then subjected to immunoblotting assay. IκBα phosphorylation and IκBα level were examined. (A) Representative blots of IκBα phosphorylation and total IκBα. (B, C) Densitometry analysis. *P<0.05 versus Sirt1^+/+/LPS group.</p

p53-mediated induction of PAI-1 expression contributes to increased pulmonary MPO levels in mice with PCSE.

<p>(<b>A</b>) WT mice were exposed to ambient air (control) or PCS (n = 5/group) for 20 weeks. LL collected from these mice was subjected to total cell counting. (<b>B</b>) Total protein in the lavage was quantified from the above exposed mice. (<b>C</b>) Paraffin embedded lung sections from WT, p53- and PAI-1-deficient mice (n = 5mice/group) exposed to ambient air (control) or PCS for 20 weeks were subjected to H&E staining. Representative fields from 1 of 3 sections per subject are shown at X 400 magnification. Lung sections were subjected to IHC analysis for neutrophils using anti-MPO antibody and for macrophages using anti-F4/80 antibody. Neutrophils (<b>D</b>) and macrophages (<b>E</b>) were counted in 10 high-power fields (hpf) are shown as bar graph. (<b>F</b>) Lung homogenate from WT, p53- and PAI-1-deficient mice exposed to ambient air or PCS for 20 weeks were immunoblotted for changes in the levels of MPO using anti-MPO antibody. These membranes were later stripped and analyzed for β-actin to assess loading. Data shown in bar graphs are mean ± SD of two independent experiments (n = 5 mice/group). Differences between treatments are statistically significant *(P<0.05).</p

Mice lacking PAI-1 expression resist type II AEC apoptosis.

<p>Mice in ambient AIR or exposed to PCS were treated with 50 μl saline or purified IAV in saline via intranasal instillation. One week after IAV infection, the mice were sacrificed. (<b>A</b>) Lung sections were subjected to H & E and IHC staining for M2 antigen. Representative fields from 1 of 3 sections per subject are shown at X 400 magnification (n = 5 mice/group). (<b>B</b>) Lung homogenates were immunoblotted for changes in IAV M2 and active caspase-3antigens to assess severity of IAV infection and apoptosis respectively. The plot represents ratios in the densities of bands normalized against β-actin levels in the same sample (n = 5 mice/group). (<b>C</b>) Lung homogenates were tested for MPO activity by colorimetric assay and represented as a bar graph of two independent experiments (n = 5 mice/group). (<b>D</b>) Lung homogenates from the mice in ambient AIR or exposed to PCS for 20 weeks were immunoblotted for changes in EAR1 with β-actin antibody as a loading control. (<b>E</b>) Total RNA from the mice exposed to AIR or PCS was analyzed for EAR and β-actin mRNA (n = 5 mice/group).</p

Increased PAI-1 expression sensitizes mice to IAV infection and alveolar injury.

<p>(<b>A</b>) Mice (n = 3) treated with saline or 0.5 LD₅₀ of purified mouse-adapted IAV (strain A/PR/8/34) in 50 μl by intranasal instillation. LL fluids were analyzed for PAI-1, and isolated type II AECs lysates were immunoblotted for p53, activation of caspase-3 and β-actin. Data shown in bar graphs are means ± SD of two independent experiments. Differences between treatments are statistically significant *(P<0.05) (n = 3 mice/group). (<b>B</b>) Mice exposed to saline or IAV as described above were analyzed for SP-C and β-actin. Data shown in bar graphs are means ± SD of two independent experiments. Differences between treatments are statistically significant *(P<0.05) (n = 3 mice/group). (<b>C</b>) Lung sections from the mice treated as described above were subjected to IHC analysis for MPO and macrophage antigens, and TUNEL staining to assess inflammation and apoptosis. Neutrophils, macrophages and apoptotic (TUNEL-positive) cells were quantified by counting positive cells in 10 high-powered fields (hpf) are shown as bar graph. (<b>D</b>) WT mice or transgenic mice that over express PAI-1 (PAI-1^+/+) or PAI-1-deficient mice (PAI-1^-/-) were exposed to 50 μl saline or IAV in saline. Lung homogenates were immunoblotted for changes in IAV M2, MPO and active caspase-3 antigen levels to assess severity of IAV infection, inflammation and lung injury. β-actin was tested to gauge similar loading. Bar represents fold changes in the densities of bands (IAV M2) normalized against β-actin levels in the same sample (n = 3 mice/group). (<b>E</b>) Lung homogenates from WT mice or transgenic mice that overexpress PAI-1 (PAI-1^+/+) or PAI-1-deficient mice (PAI-1^-/-) were also tested for MPO activity by colorimetric assay. Data shown in bar graphs are means ± SD of two independent experiments (n = 3 mice/group).</p