Fig 1.
Clinical parameters (average, range) at each milestone in AS (n = 8) vs WT (n = 4) dogs.
(A) Serum creatinine (sCr); (B) Symmetric dimethylarginine (SDMA); (C) Urine protein: urine creatinine (UPC); (D) Iohexol clearance; *p<0.05.
Fig 2.
Pathologic parameters (average, range) at each milestone in AS (n = 8) vs WT (n = 4) dogs.
(A) Glomerulosclerosis score; (B) Tubulointerstitial damage score; *p<0.05.
Fig 3.
Immunofluorescence staining for fibronectin of kidney from WT and AS dogs at milestone 1.
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.
Fig 4.
Identification of laminin 211 in the GBM of AS but not WT dogs.
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.
Fig 5.
Integrin α8 co-localizes with laminin 211 in the GBM of AS but not WT dogs.
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.
Fig 6.
Mesangial cell process extension into the GBM of AS but not WT dogs.
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).