Effect of separate and additive inhibition of TLR2 and TLR4 signalling pathways on NF-ĸB activation.

<p>(A) Reduction in NF-ĸB p65 subunit expression when TLR2 and 4 inhibitors were used separately and further abrogation of NF-ĸB p65 subunit expression when the inhibitors were used additively in control media (B) An attenuation in NF-ĸB-DNA binding was observed when anti-TLR2-IgA and TAK-242 were used separately and further downregulation in NF-ĸB-DNA binding when both inhibitors were used additively in control media. Normalized results are expressed as mean ± SEM, n = 4. *P<0.05 versus HMEC-1 cells treated with DMSO + control IgA (control). **P<0.01 versus HMEC-1 cells treated with DMSO + control IgA (control). ^††P<0.01 versus HMEC-1 cells treated with Anti-TLR2-IgA. ^‡‡P<0.01 versus HMEC-1 cells treated with TAK-242.</p

The effect of recombinant HMGB1 on NF-ĸB p65 subunit expression and NF-ĸB-DNA binding.

<p>(A) Exposure to recombinant HMGB1 (500 ng/ml) in control media for 2 hours induced nuclear NF-ĸB p65 subunit expression and, (B) NF-ĸB-DNA binding was also induced with exposure to recombinant HMGB1 (500 ng/ml) in HMEC-1 cells. Normalized results are expressed as mean ± SEM, n = 5. *P<0.05 versus HMEC-1 cells cultured in the absence of recombinant HMGB1. **P<0.01 versus HMEC-1 cells cultured in the absence of recombinant HMGB1.</p

Expression of cytokines and cell adhesion molecules with exposure to defined conditions for 72 hours.

<p>(A) Reduction in MCP-1 transcription was detected in the fluctuating glucose limb whereas an increase in IL-8 transcription was detected in the 30 mM glucose and fluctuating glucose limbs with maximal increase in cells exposed to fluctuating glucose concentrations for 72 hours. (B) ICAM-1 protein expression increased in both the fluctuating and 11.2 mM glucose limbs however; (C) VCAM-1 expression was not induced by any experimental condition. Normalized results are expressed as mean ± SEM, n = 4. *P<0.05 versus HMEC-1 cells cultured with 5 mM glucose. **P<0.01 versus HMEC-1 cells cultured with 5 mM glucose.</p

Expression of secreted HMGB1 with exposure to high and moderate glucose concentrations for 72 hours.

<p>(A)An increase in secreted levels of HMGB1 was detected in the supernatant of cells exposed to high glucose levels of 30 mM and 11.2 mM glucose for 72 hours. Western blot analysis was performed on equal volumes of supernatant and normalized to total protein in corresponding cell lysates. (B) HMGB1 protein expression was increased in the fluctuating glucose limb. No significant increase in HMGB1 expression was observed with exposure to high and moderate levels of glucose. Normalized results are expressed as mean ± SEM, n = 3. *P<0.05 versus HMEC-1 cells cultured with 5 mM glucose. **P<0.01 versus HMEC-1 cells cultured with 5 mM glucose.</p

ICAM-1 expression in wildtype, TLR2^-/- and TLR4^-/- murine models.

<p>(A) In the wildtype, TLR2^-/- and TLR4^-/- murine models, ICAM-1 was expressed in the glomeruli (black arrows), peritubular capillaries (asterisks), epithelial cells (green arrows) and tubular brush border (arrowheads) (B) There was a significant upregulation of ICAM-1 in the glomeruli of wildtype diabetic mice compared to wildtype mice without diabetes. This upregulation was attenuated in TLR2^-/- and TLR4^-/- diabetic mice compared to wildtype diabetic mice. *P<0.01 versus wildtype mice without diabetes. ^†P<0.05 versus wildtype mice induced with diabetes. ^††P<0.01 versus wildtype mice induced with diabetes.</p

The effect of recombinant HMGB1 on stimulating inflammatory cytokines and cell adhesion molecules.

<p>(A) Stimulating HMEC-1 cells with recombinant HMGB1 in control media for 2 hours induced the secretion of MCP-1 and IL-8 into the media (B) Exposure to recombinant HMGB1 also induced a moderate increase in ICAM-1. Normalized results are expressed as mean ± SEM, n = 3. *P<0.05 versus HMEC-1 cells cultured in control media. **P<0.01 versus HMEC-1 cells cultured in control media.</p

TLR2 and 4 expression in cells exposed to the defined experimental conditions for 72 hours.

<p>(A) Reduction in TLR2 expression with 11.2 mM glucose and (B) maximal TLR4 expression with fluctuating glucose conditions. Normalized results are expressed as mean ± SEM, n = 4. *P<0.05 versus HMEC-1 cells cultured with 5 mM glucose. **P<0.01 versus HMEC-1 cells cultured with 5 mM glucose.</p

Expression of NF-ĸB p65 and NF-ĸB-DNA binding from cells exposed to experimental conditions for 72 hours.

<p>(A) Maximal increase in nuclear NF-ĸB p65 subunit expression was observed in fluctuating glucose limb and, (B) concomitant maximal increase in NF-ĸB-DNA binding was detected in cells with exposure to fluctuating glucose conditions for 72 hours. Normalized results are expressed as mean ± SEM, n = 3. *P<0.05 versus HMEC-1 cells cultured with 5 mM glucose. **P<0.01 versus HMEC-1 cells cultured with 5 mM glucose.</p

Effect of anti-TLR2-IgA or TAK-242 on inflammatory cytokines and cell adhesion molecules.

<p>(A) With exposure to TLR2 neutralizing antibody in control media, there was no reduction in MCP-1 and IL-8 expression however with exposure to TAK-242 in control media, there was a suppression in MCP-1 and IL-8 expression (B) With exposure to TLR2 neutralizing antibody in control media, there was no reduction in ICAM-1 expression but TAK-242 suppressed ICAM-1 expression. Normalized results are expressed as mean ± SEM, n = 3. **P<0.01 versus control.</p

The cumulative incremental benefits of listing compared with non-waitlisting among individuals with ESKD and varying age and co-morbidities.

<p>The cumulative incremental benefits of listing compared with non-waitlisting among individuals with ESKD and varying age and co-morbidities.</p