Effects of conditional p38α deletion on anti-GBM nephritis.

<p>(A) p38MAPKα deletion is depending on tissue and reaches from 50–100%. (B) Western blot experiments reveal significant reduction of p38 phosphorylation in kidneys of <i>MxCre-p38α^Δ/Δ</i> transgenic mice at day 14 after induction of anti-GBM nephritis. Downstream activation of MK2 is also strongly reduced. Three representative animals are shown in each group. (C) Survival of wild type and <i>MxCre-p38α^Δ/Δ</i> mice during anti-GBM nephritis shows no difference (controls n = 10, continuous line; wild type n = 16, spotted line; <i>MxCre-p38α^Δ/Δ</i> mice n = 18, dashed line). (D) Serum urea levels in wild-type (day 14: mean 66.61±18.33 mg/dl; n = 9) and <i>MxCre-p38α^Δ/Δ</i> mice (day 14: mean 58.43±10.46 mg/dl, p = ns vs. wild-type; n = 9). (E) Creatinine clearance decreases in both wild type (134.4±29.70; n = 8) and <i>MxCre-p38α^Δ/Δ</i> (97.79±16.15; n = 8) mice.</p

Functionality of the anti- GBM nephritis model and differential cytokine activation.

<p>Periodic acid-Schiff reagent (PAS) stain of a control kidney (A) and kidneys affected by crescentic GN over time are shown. BC = Bowmańs capsule, CL = capillary loop, BM = basement membrane, Pod = podocyte, U = urinary space. Development of full crescents occurs within 14 days (B–D). Mice were sacrificed at indicated days (original magnification 20×). (E) Expression analyses of pro- (TNFα, IL-1, IL-8) and anti-inflammatory (IL-10, TGFβ1) cytokines by qPCR in kidneys of control mice (white bars) and anti GBM-IgG treated mice (black bars). RNA was isolated from whole kidney lysates. Data are the mean value ± SEM (n = 4 for each time point).</p

Inflammatory gene expression during anti-GBM nephritis is partially p38α dependent.

<p>qPCRs from renal tissue of wild-type and <i>MxCre-p38α^Δ/Δ</i> mice 14 days after injection with anti-GBM antibodies. There is no difference in the mRNA expression of pro-inflammatory cytokines like TNF, IL-1β and IL-6. Furthermore there is a significant regulation of IL-8, IL-12 and IL-18. There are no significant changes in anti-inflammatory cytokines like IL-10, IL-13 and TGFβ1. MCP-1 is down-regulated in <i>MxCre-p38α^Δ/Δ</i> mice. n = 4/group measured in duplicates (A). <b>Leukocyte infiltration is regulated by corresponding chemokines:</b> Chemokines participating in (B) macrophage and (C) neutrophil recruitment are remarkably downregulated in <i>MxCre-p38α^Δ/Δ</i> mice. (D) Chemotactic regulation of T cells is unaffected in <i>MxCre-p38α^Δ/Δ</i> mice. In each group the RNA of five mice was pooled and then analyzed by RT-PCR with a RT² Profiler™ PCR Array kit. mRNA expression was normalized to beta-actin of wildtype mice.</p

Deletion of p38α ameliorates tubular but not glomerular damage during anti-GBM nephritis.

<p>(A) Semi-quantitative scoring of tubular damage. p38α deletion significantly diminishes tubular damage (mean score 0.6±0.1, p<0.05 vs. control; n = 7) as compared to wild type mice (mean score 0.9±0.01; n = 7). Tubules of wild type mice (B) are dilated, whereas tubules of <i>MxCre-p38α^Δ/Δ</i> (C) mice are still tightly packed. (D) KIM-1 mRNA level is dramatically increased in wild type mice (wild type 84.23±32.57; n = 4 vs. <i>p38α^Δ/Δ</i> 2.675±1.248; n = 4) indicating high tubular damage. (E) Vimentin shows clear upregulation of its mRNA in wild type mice (2.900±0.1732; n = 4) whereas it is reduced nearly to the baseline level in <i>MxCre-p38α^Δ/Δ</i> mice (1.375±0.3497; n = 4). Dashed line indicates RNA base level of control mice. (F–H) Analysis of crescent formation during anti GBM nephritis. Similar amounts of glomeruli revealed crescent formation in wild type (n = 6) and <i>MxCre-p38α^Δ/Δ</i> mice (n = 6) (wild type: mean 3.3±1.2% vs. <i>MxCre-p38α^Δ/Δ</i>: 4.2±1.5, p = ns). (I–K) Fibrotic tissue remodelling occurred in both wild type (n = 7) and <i>MxCre-p38α^Δ/Δ</i> mice (n = 7) (wild-type: mean 1.409±0.1132 vs <i>MxCre-p38α^Δ/Δ</i>: 1.615±0.1617, p = ns). Sirius red staining was performed 14 days after induction of anti-GBM nephritis. (L–M) p38α deletion affects the immune response to sheep IgG by decreased murine IgG depositions in the glomeruli.</p

Recruitment of leukocytes to kidneys in anti-GBM nephritis is p38α-dependent.

<p>(A–C) F4/80 staining to detect macrophages clearly reveals a prominent infiltration in the kidneys of wild-type mice (mean 155.4±58.6 cells/mm²), whereas <i>MxCre-p38α^Δ/Δ</i> mice show dramatically reduced macrophage numbers (mean 25.7±3.3 cells/mm², p<0.05 vs. wild type). (D–F) Wild type mice show prominent neutrophil infiltration (mean 43.8±5.5 cells/mm²). There is a similar infiltration but less severe in <i>MxCre-p38α^Δ/Δ</i> mice (mean 26.2±6.6 cells/mm², p<0.05 vs. wild type). (G–I) In contrast to the other lymphocytes the number of T cells is not different among wild type (mean 5.0±1.5 cells/mm²) and <i>MxCre-p38α^Δ/Δ</i> mice (mean 9.1±2.1 cells/mm², p = ns vs. wild-type). Staining was performed 14 days after induction of anti-GBM nephritis (n = 7/group).</p