Validation of the array expression pattern by quantitative RT-PCR.

<p>Expression of proinflammatory chemokines and adhesion molecules in glomeruli isolated from control mice (black bars), <i>Tnfr1</i>−/− (dark grey bars), <i>Tnfr2</i>−/− (bright grey bars), and <i>Tnfr1,2</i>−/− mice (white bars) was anlayzed after challenge with indicated concentrations of TNF in vitro for 12 or 24 hours. The mRNA levels of CCL2/MCP-1 (<b>A</b>), CCL5/RANTES (<b>B</b>), ICAM-1 (<b>C</b>), VCAM-1 (<b>D</b>), E-selectin (<b>E</b>), and P-selectin (<b>F</b>) were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Data shown are means and SE of n = 3 to 4 per group; ∗ p<0.05 versus wildtype.</p

Gene expression profiles in wildtype and <i>Tnfr1,2-</i>deficient glomeruli after TNF stimulation ex vivo.

<p>Expression profiling by microarrays was performed on isolated glomeruli from wildtype and <i>Tnfr1,2</i>−/− mice 12 after stimulation with 50 ng/ml TNF for 12 hours ex vivo. A negativ “fold change” (black to green) indicates decreased expression and a postive “fold change” (black to red) indicates increased expression in a glomerular sample compared with the average of all analyzed samples. The 290 differentially regulated genes identified in <i>Tnfr1,2</i>−/− glomeruli are listed according to fold-change values versus wildtype, starting with the most down-regulated gene (CCL2; -50.0-fold). Note that almost all genes found significantly regulated in <i>Tnfr1,2</i>−/− glomeruli had a similar trend of expression change in <i>Tnfr1</i>−/− glomeruli, although not reaching statistical significance in <i>Tnfr1</i>−/− glomeruli for several genes. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068167#pone.0068167.s003" target="_blank">Table S2</a> lists all differentially expressed genes with respective probe set IDs, gene bank accession numbers, gene names and fold-changes according to fold-change values compared to wildtype.</p

Glomerular expression of the small GTPase Rab6B.

<p>Rab6B expression was analyzed in glomeruli isolated from control mice (black bars), <i>Tnfr1</i>−/− (dark grey bars), <i>Tnfr2</i>−/− (bright grey bars), and <i>Tnfr1,2</i>−/− mice (white bars) after challenge with indicated concentrations of TNF in vitro for 12 or 24 hours. The mRNA levels of Rab6B were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Data shown are means and SE of n = 3 per group; ∗ p<0.05, ∗∗ p<0.01 versus wildtype.</p

Secretion of proinflammatory chemokines by TNF-stimulated glomeruli ex vivo.

<p>Concentrations of CCL2/MCP-1, CCL5/RANTES, and CXCL10/IP-10 were measured by ELISA in culture supernatants of wildtype, <i>Tnfr1</i>−/−, <i>Tnfr2</i>−/−, and <i>Tnfr1,2</i>−/− glomeruli 12 hours after stimulation with 50 ng/ml of TNF. Data shown are means and SD of n = 3 per group; ∗ p<0.05 versus wildtype.</p

TNF-induced expression of glomerular adhesion molecules and chemokines correlates with leukocyte infiltration in vivo.

<p><b>A:</b> Glomerular expression of adhesion molecules and proinflammatory chemokines in glomeruli isolated from control mice (white bars) and from mice 8 hours after intraperitoneal injection of 5 µg TNF (grey bars). mRNA levels were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Results are expressed as fold change compared to control glomeruli. Data shown are means and SE of n = 3 to 6 per group; ∗ p<0.05 versus control. <b>B:</b> Glomerular leukocyte infiltration 8 hours after intraperitoneal injection of TNF (5 µg) was analysed by compartment-specific flow cytometry. Leukocyte subpopulations were identified as follows: leukocytes: CD45⁺, neutrophils: CD45⁺ Ly6G⁺ F4/80⁻, F4/80 positive mononuclear phagocytes: CD45⁺ F4/80⁺ Ly6G⁻, and T cells: CD45⁺ CD3ε⁺. Leukocyte numbers are expressed as percentage of all glomerular cells. ∗ p<0.05 versus control.</p

Differentially expressed genes in <i>Tnfr</i>-deficient glomeruli compared to wildtype after TNF stimulation.

<p>Expression profiling by microarrays was performed on isolated glomeruli from wildtype, <i>Tnfr1,2</i>−/−, <i>Tnfr1</i>−/− and <i>Tnfr2</i>−/− mice after stimulation with 50 ng/ml TNF for 12 hours ex vivo. <b>A:</b> Significance analysis using SAM detected differential regulation of 290 unique genes in <i>Tnfr1,2</i>−/− glomeruli with at least 1.5-fold difference compared to wildtype (black data points). 219 genes were down-regulated (upper left), and 71 genes were up-regulated (lower right). <b>B:</b> In <i>Tnfr1</i>−/− mice 219 differentially regulated genes were identified, with lower expression of 159 genes and increased expression of 60 genes. <b>C:</b> In <i>Tnfr2</i>-deficient glomeruli only 5 genes were differentially expressed, all of them with decreased expression compared to wildtype. In all three genotypes, expression of the majority of genes with fluorescence signals above background level did not significantly differ from wildtype (grey data points). Data represent the mean log2 value of fluorescence signals from three independent experiments per genotype.</p

Overrepresented gene ontology terms within differentially expressed genes in TNF-stimulated <i>Tnfr1,2</i>−/− gomeruli ¹.

1<p>The 10 most overrepresented gene ontology (GO) terms are listed according to their enrichment scores. Terms with an EASE score of p ≤0.05, fold enrichment ≥1.5, and at least 4 genes per group were considered significant for overrepresentation.</p

Overrepresented gene ontology terms within differentially expressed genes in TNF-stimulated <i>Tnfr1</i>−/− glomeruli ¹.

1<p>The 10 most overrepresented gene ontology (GO) terms are listed according to their enrichment scores. Terms with an EASE score of p ≤0.05, fold enrichment ≥1.5, and at least 4 genes per group were considered significant for overrepresentation.</p

Contribution of TNFR1 and TNFR2 signaling to TNF-mediated infiltration of glomerular leukocytes.

<p>Compartment-specific flow cytometry was performed on glomerular tissue prepared from wildtype, <i>Tnfr1</i>−/−, <i>Tnfr2</i>−/−, and <i>Tnfr1,2</i>−/− mice 8 hours after intraperitoneal injection of 5 µg TNF. Leukocyte subpopulations were identified as follows: <b>A:</b> CD45⁺ leukocytes, <b>B:</b> CD45⁺ Ly6G⁺ F4/80⁻ neutrophils, <b>C:</b> CD45⁺ F4/80⁺ Ly6G⁻ mononuclear phagocytes, and <b>D:</b> CD45⁺ CD3ε⁺ T cells. Leukocyte numbers are expressed as percentage of all glomerular cells. ∗ p<0.05, ∗∗ p<0.01 versus wildtype.</p

Expression of proinflammatory chemokines and adhesion molecules in TNF-stimulated primary mesangial cells (pMCs).

<p>pMCs were isolated from control mice (black bars), <i>Tnfr1</i>−/− (dark grey bars), <i>Tnfr2</i>−/− (bright grey bars), and <i>Tnfr1,2</i>−/− mice (white bars) after stimulation with indicated concentrations of TNF in vitro for 12 hours. The mRNA levels of CCL2/MCP-1 (<b>A</b>), CCL5/RANTES (<b>B</b>), ICAM-1 (<b>C</b>), VCAM-1 (<b>D</b>), E-selectin (<b>E</b>), and P-selectin (<b>F</b>) were determined by quantitative real-time PCR, and values were normalized to 18S rRNA as reference gene. Data shown are means and SE of two independently performed experiments.</p