Abstract
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 in vitro and in vivo. 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.
Highlights
The pathologic glomerular basement membrane in Alport syndrome is irregularly thickened and thinned, with a multilaminar or ‘‘basketweave’’ appearance that is unique to the disease and a definitive diagnostic test for Alport syndrome [1]
We demonstrate that CD151 knockout mouse, which has a specific defect in a3b1 integrin binding affinity, a characteristic likely to impact the structural integrity of the capillary tuft [12], shows mesangial filopodial invasion of the glomerular capillaries, deposition of a2 containing laminins in the glomerular basement membranes (GBM), podocyte focal adhesion kinase (FAK) activation and MMP dysregulation
We provide evidence that laminin a2 activates FAK on glomerular podocytes resulting in downstream activation of MMPs and pro-inflammatory cytokines that contribute to the progressive glomerular pathogenesis
Summary
The pathologic glomerular basement membrane in Alport syndrome is irregularly thickened and thinned, with a multilaminar or ‘‘basketweave’’ appearance that is unique to the disease and a definitive diagnostic test for Alport syndrome [1]. It has been shown that the thickened regions are more permeable to injected ferritin than the non-thickened regions of the GBM [2]. This property is consistent with a partially degraded matrix network, suggesting proteolytic damage may contribute to focal thickening of the Alport GBM. The work includes roles for pro-inflammatory cytokines, such as TGF-b1 [5,6], CTGF [7], and the adhesion molecule a1b1 integrin [8,9], all of which contribute to the glomerular pathology in Alport syndrome. MMPs are induced as a function of disease progression, and several MMPs, including MMP-2, MMP-9, and MMP-12 have been functionally linked to progressive destruction of the GBM [4,5]
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