INTRODUCTION: Genetic defects in COL4A3, COL4A4 or COL4A5 cause a broad spectrum of progressive glomerulopathies and associated hearing loss categorised as Alport syndrome. Despite being one of the best characterised genetic kidney diseases, patients rely on blockade of the renin-angiotensin system to delay the onset of renal failure and requirement for renal replacement therapy. There are currently no targeted therapies for Alport syndrome. We aimed to build our basic understanding about the glomerular extracellular matrix (ECM) in Alport syndrome by employing global analysis of composition and ultrastructural imaging in the Col4a3-/- mouse.

METHODS: Cellular and extracellular matrix (ECM) fractions from wild type and Col4a3-/- glomeruli at 6 and 16 weeks of age were analysed by mass spectrometry (MS) and imaged at 6, 16 and 28 weeks of age using serial-block face scanning electron microscopy (SBFSEM).

RESULTS: At 6-weeks of age glomerular basement membrane (GBM) and podocyte structure remained within normal ranges in Col4a3-/- mice, however ECM composition, even prior to the onset of barrier dysfunction was already significantly altered compared to wild type mice. These changes included complete absence of type IV collagen α3, α4, α5 and upregulation of type IV collagen α1, α2, α6 and type VI collagen chains. In contrast, at 16-weeks of age we found dramatic changes in the structure (GBM, podocytes) and composition (glomerular ECM) including: increased type IV collagen α1 and α2, fibronectin, type I collagen, laminin α2 and fibrinogen chains. Global and pathway analysis of glomerular cellular fractions indicated changes in actin regulating proteins at 6-weeks and mitochondrial dysfunction at 16-weeks. With SBFSEM we discovered podocyte foot process invaginations into the GBM, a novel feature of Alport nephropathy. The frequency and length of these invaginations increased with age and correlated with GBM disruption.

CONCLUSIONS: Our data demonstrate that Alport syndrome progresses with distinct early changes in ECM composition but not ultrastructure, followed by more profound ECM accumulation, podocyte invagination into the GBM, disruption of the GBM and latterly mitochondrial dysfunction. Enhanced understanding about the pathways that control podocyte cell-matrix adhesion may ultimately inform therapeutic strategies to correct or repair glomerular barrier function in Alport syndrome.