X-Linked Alport Dogs Demonstrate Mesangial Filopodial Invasion of the Capillary Tuft as an Early Event in Glomerular Damage

BACKGROUND: X-linked Alport syndrome (XLAS), caused by mutations in the type IV collagen COL4A5 gene, accounts for approximately 80% of human Alport syndrome. Dogs with XLAS have a similar clinical progression. Prior studies in autosomal recessive Alport mice demonstrated early mesangial cell invasion as the source of laminin 211 in the glomerular basement membrane (GBM), leading to proinflammatory signaling. The objective of this study was to verify this process in XLAS dogs. METHODS: XLAS dogs and WT littermates were monitored with serial clinicopathologic data and kidney biopsies. Biopsies were obtained at set milestones defined by the onset of microalbuminuria (MA), overt proteinuria, onset of azotemia, moderate azotemia, and euthanasia. Kidney biopsies were analyzed by histopathology, immunohistochemistry, and electron microscopy. RESULTS: XLAS dogs showed progressive decrease in renal function and progressive increase in interstitial fibrosis and glomerulosclerosis (based on light microscopy and immunostaining for fibronectin). The only identifiable structural abnormality at the time of microalbuminuria was ultrastructural evidence of mild segmental GBM multilamination, which was more extensive when overt proteinuria developed. Co-localization studies showed that mesangial laminin 211 and integrin α8β1 accumulated in the GBM at the onset of overt proteinuria and coincided with ultrastructural evidence of mild cellular interpositioning, consistent with invasion of the capillary loops by mesangial cell processes. CONCLUSION: In a large animal model, the induction of mesangial filopodial invasion of the glomerular capillary loop leading to the irregular deposition of laminin 211 is an early initiating event in Alport glomerular pathology

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

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

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

Laminin α2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis

<div><p>It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes <i>in vitro</i> and <i>in vivo</i>. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.</p></div

Laminin α2, but not laminin α1 activates FAK on podocytes <i>in vivo</i> and <i>in vitro</i>.

<p>Panels A–C; 7 week old wild type glomerulus stained with antibodies specific for laminin 111 and pFAK³⁹⁷ show absence of pFAK immunostaining. Panels D–F; 7 week Alport glomerulus stained with antibodies specific for laminin α1 and pFAK³⁹⁷ pFAK immunostaining in podocytes adjacent to laminin α1-immunopositive GBM. Panels G-I show the same immunostaining as for D–F using Alport mice that do not express laminin α2 (the dy/dy muscular dystrophy mutation). Note the absence of pFAK³⁹⁷ immunostaining even though GBM is immunopositive for laminin α1. Panel J. Wild type podocytes were differentiated for 2 weeks and then plated on placental laminin, EHS laminin, or merosin for 15 hours. Extracts were prepared and analyzed by western blot for expression of pFAK³⁹⁷ and total FAK. β-actin was used as a loading control). Panel K shows quantitative analysis of pFAK397 relative to total FAK for several western blots. Panel L shows real time qRT-PCR results for transcripts endocing the indicated MMPs, demonstrating significantly elevated expression of MMP-9 and MMP-10 for cells cultured on merosin (MERO) relative to cells cultured on placental laminin (PLAM). Scale bar = 10 µm.</p