Kidney immunolocalization of tubulointerstitial osteopontin, TNF-α and IL-6.

<p>Osteopontin (A–D), TNF-α (E–H) and IL-6 (I–L) after 7, 14 or 21 days of treatment with vehicle (VH) or 15 mg/kg cyclosporine (CsA). Black arrows indicate renal inflammation, p≤0.01 (B–D and H). Hematoxylin counterstain. Bars = 10 µm.</p

Macrophage infiltration.

<p>Immunohistochemistry by ED-1 immunostaining (arrows) after 7, 14 or 21 days of treatment with vehicle (VH) or 15 mg/kg cyclosporine (CsA). The data are presented as the mean ± SD. * p<0.05 (CsA versus VH). Hematoxylin counterstain. Bars = 10 µm.</p

Urinary biomarker levels.

<p>Urinary ELISA assays (ng/mL) after 7, 14 or 21 days of treatment with vehicle (VH) or 15 mg/kg cyclosporine (CsA). The data are presented as the mean ± SD. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 (CsA versus VH).</p

Immunohistochemical detection of injury markers in renal cortex tissue.

<p>Mean optical densitometry, MOD, arbitrary units after 7, 14 or 21 days of treatment with vehicle (VH) or 15 mg/kg cyclosporine (CsA). The data are presented as the mean ± SD. * p<0.05, ** p<0.01 and *** p<0.001 (CsA versus VH).</p

Weight gain, renal function and hemodynamics, hematocrit and CsA blood levels after 7, 14 or 21 days of daily subcutaneous injection of vehicle (control group, VH) or CsA 15 mg/kg/day (treatment group, CsA).

<p>The data are presented as the mean ± SD.</p><p>* p<0.05,</p><p>** p<0.01,</p><p>*** p<0.001 and</p><p>**** p<0.0001 (CsA versus VH).</p

Kidney injury induced by CsA.

<p>Collagen fibers stained green by Gomori's method (A–D). Red arrows indicate fibrosis (D). Immunolocalization of tubulointerstitial α-SMA (E–H) and vimentin (I–L) after 7, 14 or 21 days of treatment with vehicle (VH) or 15 mg/kg cyclosporine (CsA). Black arrows indicate renal injury, p≤0.001 (G–H and L). Hematoxylin counterstain. Bars = 10 µm.</p

Histopathological analysis of kidneys after CV- and MjTX-II-induced peritonitis.

<p>(A) Normal histological condition of proximal (PT) and distal convuluted tubules (DT) in the renal control. (B-E) CV- and MjTX-II PLA₂-induced peritonitis provoked renal damage characterized by the presence of pyknotic nuclei (arrowheads), karyolisis (white arrow) and hyaline casts (asterisks) in proximal tubules. (F) Ac2-26 peptide restores the normal morphological aspect of juxtamedullary structures. Stain: Hematoxylin-Eosin. Scale bars: 20 μm.</p

Effect of Ac2-26 treatment on the mesenteric inflammation induced by CV and MjTX-II.

<p>Intact mast cells (arrows) in the control mesentery (A). Inflamed mesentery of CV—(B) and MjTX-II-induced peritonitis (C-D) with extravasated neutrophils in the tissue (arrowheads) as observed at 4 and 24 h. Reduced neutrophil influx (arrowheads) after Ac2-26 post-treatment at 4 (E) and 24 h (F) of MjTX-II-induced peritonitis. Vessels (V). Stain: Toluidine blue. Scale bars: 10 μm. Quantitative analysis of extravasated neutrophils in the mesentery after CV- (G) and MjTX-II–induced peritonitis (H). The data represent the mean ± SEM of cell numbers/mm² (n = 5 animals/group). **P < 0.01 vs control; ^§P < 0.05 vs MjTX-II-4 h.</p

Analysis of AnxA1 expression in the juxtamedullary renal structures.

<p>AnxA1 immunostaining was detected in the epithelial cells under all of the experimental conditions (A, C, E, G, I), concentrated in the glomerular capsule (arrows) and distal convuluted tubules (asterisks). Ac2-26 post-treatment decreased the immunostaining for endogenous AnxA1 in the distal tubules and glomerular capsule (D, F, H, J) 4 and 24 h after CV- and MjTX-II-induced peritonitis. Negative control of the reaction (B). Counterstain: hematoxylin. Scale bars: 20 μm. Densitometric analysis of the epithelial cells from distal convuluted tubules (K-L) and glomerular capsule (M-N) immunostained for AnxA1. Values (arbitrary units) are expressed as the mean ± SEM of sections analyzed from 5 rats /group. **P < 0.01 and ***P < 0.001 vs control; ^###P < 0.001 vs CV groups; ^§§P < 0.01 and ^§§§P < 0.001 vs MjTX-II groups at the corresponding experimental time.</p

Effect of Ac2-26 treatment on proinflammatory cytokine secretion.

<p>Peritonitis was induced in rats by i.p. injection of CV (100 μg) or MjTX-II (100 μg) in 0.5 mL of PBS. Control group received i.p. only PBS. Another set of animals were treated i.p. with 1 mg/kg of Ac2-26 peptide after 15 minutes of CV or MjTX-II injection. Peritoneal exudate was collected 4 and 24 hours after peritonitis inductions and cytokine dosages were performed by ELISA, as described in Methods. Values are expressed as the mean ± SEM of cytokine levels (n = 5 rats/group). ND < 31.25 pg/mL (not detected).</p><p>* P < 0.05 and</p><p>*** P < 0.001 vs control</p><p>^##P < 0.01 vs CV 4 h.</p><p>Effect of Ac2-26 treatment on proinflammatory cytokine secretion.</p