Identification of laminin 211 in the GBM of AS but not WT dogs.

<p>Dual immunofluorescence immunostaining of kidney from a WT dog (A-C) and an AS dog at milestone 2 (D-F); 63x1.4 n.a. oil. Laminin 521 of the GBM was labeled with anti-laminin β2 (LAMB2), and laminin 211 produced by mesangial cells, was labeled with anti-laminin α2 (LAMA2), demonstrating co-localization of laminin 211 with the GBM of several capillary loops in the AS dog.</p

Mesangial cell process extension into the GBM of AS but not WT dogs.

<p>A-B: Dual immunofluorescence immunostaining of kidney from a WT dog and an AS dog, 63x1.4 n.a. oil with 3X zoom. Anti-laminin β2 and anti-integrin α8 antibodies were used to stain the GBM and mesangial cells, respectively. Staining reveals distinct delineation of mesangium absent from the GBM of the normal dog (A) but extension of mesangium within the GBM of the AS dog (B). C: Transmission electron microscopy of kidney tissue from an AS dog at milestone 2. Cytoplasmic extensions, also described as cellular interpositioning, are observed at the base of the capillary loops, consistent with invasion of mesangial cell processes (arrow) corresponding with extension of the mesangium (B).</p

Clinical parameters (average, range) at each milestone in AS (n = 8) vs WT (n = 4) dogs.

<p>(A) Serum creatinine (sCr); (B) Symmetric dimethylarginine (SDMA); (C) Urine protein: urine creatinine (UPC); (D) Iohexol clearance; *p<0.05.</p

Pathologic parameters (average, range) at each milestone in AS (n = 8) vs WT (n = 4) dogs.

<p>(A) Glomerulosclerosis score; (B) Tubulointerstitial damage score; *p<0.05.</p

Immunofluorescence staining for fibronectin of kidney from WT and AS dogs at milestone 1.

<p>Staining for fibronectin reveals fibrosis in AS dogs (B) as early as milestone 1 on confocal microscopy when compared to WT littermates (A) at the same milestone, 10x0.3 n.a. dry.</p

Integrin α8 co-localizes with laminin 211 in the GBM of AS but not WT dogs.

<p>A-C: Dual immunofluorescence immunostaining of kidney from a WT dog; 63x 1.4 n.a. oil. The GBM was localized with anti-collagen α5 (α5(IV)) and the mesangium was localized with anti-integrin α8 (INTα8). D-I: Dual immunofluorescence immunostaining of kidney tissue from an AS dog at milestone 2. Laminin 211, produced by mesangial cells, was labeled with anti-laminin α2 (LAMA2) and mesangial cells were localized with anti-integrin α8 (INTα8), demonstrating co-localization of laminin 211 with mesangial cell extension in capillary loops. Images D-F were taken with 40x1.3 n.a. oil; images G-I were taken with 63x1.4 n.a. oil with 2X zoom.</p

Endothelial alterations in a canine model of immune thrombocytopenia

<p>Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (<i>P</i> = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (<i>R</i> = 0.99103; <i>P</i> < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.</p