Endogenous ligands expression in kidney in animals subjected to CLP.

<p>mRNA expression of HMGB1 (a), HSP70 (b) in the kidney of WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals per group. Data presented as mean ± standard deviation (SD), * p<0.05 vs WT; ** p<0.01 vs WT.</p

NF-κB activity in the kidney of TLR2, TLR4 and MyD88 deficient mice subjected to CLP.

<p>(<b>a</b>) Histological analysis of NF-κB p65 by immunohistochemistry of control (a), WT (b), TLR2 ^−/− (c), TLR4 ^−/− (d) and MyD88 ^−/− (e) mice respectively, 24 hours after CLP . Results of a representative experiment with five animals per group. (<b>b</b>) Score of NF-κB p65 in kidney represented in figure A. Data shown as mean ± standard deviation (SD), *** p<0.01 vs WT.</p

Neutrophil infiltration in renal tissue 24 hours after CLP.

<p>Gene expression of KC (a), IL-17 (b) , iNOS (c) in kidney 24 hours after CLP in WT, TLR2^−/− , TLR4^−/− and MyD88−/− mice. The mRNA was normalized to HPRT expression and compared to normal group. (d) Assay of myeloperoxidase activity in kidney of control, WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice 24 hours after CLP. (e) GR1 expression in the kidney of mice 24 hours after CLP. (f) Results of a representative experiment with five animals/group. Data shown as mean ± standard deviation (SD).* p<0.05 vs WT and ** p<0.01 vs WT. ND: not detected.</p

Apoptosis in the kidney of TLR2, TLR4 and MyD88 deficient mice subjected to CLP.

<p>(<b>a</b>) Histological analysis of apoptosis by immunohistochemistry of cleaved caspase 3 of control (a), WT (b), TLR2 ^−/− (c), TLR4 ^−/− (d) and MyD88 ^−/− (e) mice respectively, 24 hours after CLP . Results of a representative experiment with 5 animals per group. (<b>b</b>) Histological analysis of apoptosis by immunofluorescence to TUNEL of control (a), WT (b), TLR2 ^−/− (c), TLR4 ^−/− (d) and MyD88 ^−/− mice (e) respectively, 24 hours after sepsis. (<b>c</b>) Score of cleaved caspase-3 in kidney represented in figure A. (<b>d</b>) Score of TUNEL in kidney represented in figure B. (<b>e</b>) Gene expression of BCL-2 in the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals/group. Data shown as mean ± standard deviation (SD), * p<0.05 vs WT and ** p<0.01 vs WT.</p

Expression of pro-inflammatory cytokines after CLP.

<p>mRNA expression of IL-1β (a), TNF-α (b), IL-6 (c), KC (d), IL-17 (e) and iNOS (f) in the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals per group. (g) Analysis of expression of α-IKK in kidney of WT, TLR2^−/− , TLR4^−/− and MyD88^−/− mice 24 hours after CLP. Results representative of a experiment with two animals/group Data shown as mean ± standard deviation (SD), * p<0.05, ** p<0.01 and *** p<0.001 vs WT.</p

Acute tubular necrosis in TLR2, TLR4 and MyD88 deficient mice subjected to CLP.

<p>Histology and quantification of ATN of control(a) and WT(b), TLR2^−/− (c), TLR4 ^−/− (d) and MyD88^−/− mice (e) 24 hours after CLP. Results of a representative experiment with 5 animals per group. Data shown as mean ± standard deviation (SD), *** p<0.0001 vs WT.</p

Migration GR1⁺ neutrophils into the peritoneal cavity after CLP.

<p>(a) Flow cytometry of the peritoneal cavity 24 hours after CLP in control, WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice. (b, c) Frequency of GR1^low and GR1^high cells population in control, WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice 24 hours after CLP. Data shown as mean ± standard deviation (SD).* p<0.05 vs. WT and ** p<0.01 vs. WT.</p

Partially neutrophils depletion in WT mice subjected to CLP.

<p>(a) Flow cytometry of spleen to determine the success of depletion in control mice without depletion in mice only depleted and depleted mice subjected to sepsis (*p<0.05 vs control). (b, c) Kidney function of WT mice and WT depleted mice subjected to CLP at time 24 hours, assessed by levels of serum creatinine and blood urea (*p<0.05 vs WT sepsis; **p<0.01 vs WT sepsis). (d, e, f, g, h, i) Gene expression of TNF-α, IL-6, IL1-β, IL-17, KC and iNOS in the kidney of WT mice and WT depleted mice 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with three animals/group. Data shown as mean ± standard deviation (SD). *p<0.05 vs WT sepsis and **p<0.01 vs WT sepsis.</p

Renal hypoxia in TLR2, TLR4 and MyD88 deficient mice subjected to CLP.

<p>(a) Histological analysis of hypoxia by immunohistochemistry (IHC) of control (a), WT (b), TLR2^−/− (c) , TLR4^−/− (d) and MyD88^−/− mice (e) respectively, 24 hours after CLP. Results of a representative experiment with 5 animals per group. (b) Score of hypoxia level represented by iminohistochemistry in figure A. (c) Gene expression of HIF-1α in the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals per group. Data shown as mean ± standard deviation (SD), * p<0.05 vs WT and ** p<0.01 vs WT.</p