Laminin Compensation in Collagen α3(IV) Knockout (Alport) Glomeruli Contributes to Permeability Defects

Abstract

Alport disease is caused by mutations in genes encoding the ␣3, ␣4, or ␣5 chains of type IV collagen, which form the collagenous network of mature glomerular basement membrane (GBM). In the absence of ␣3, ␣4, ␣5 (IV) collagen, ␣1, ␣2 (IV) collagen persists, which ordinarily is found only in GBM of developing kidney. In addition to dysregulation of collagen IV, Alport GBM contains aberrant laminins, which may contribute to the progressive GBM thickening and splitting, proteinuria, and renal failure seen in this disorder. This study sought to characterize further the laminin dysregulation in collagen ␣3(IV) knockout mice, a model of Alport disease. With the use of confocal microscopy, laminin ␣1 and ␣5 abundance was quantified, and it was found that they co-distributed in significantly large amounts in areas of GBM thickening. In addition, labeling of entire glomeruli for laminin ␣5 was significantly greater in Alport mice than in wild-type siblings. Reverse transcriptase-PCR from isolated glomeruli demonstrated significantly more laminin ␣5 mRNA in Alport mice than in wild-type controls, indicating upregulated transcription of Lama5. For testing glomerular barrier function, ferritin was injected into 2-wk-old Alport and control mice, and GBM was examined by electron microscopy. Highest ferritin levels were seen in Alport GBM thickenings beneath effaced podocyte foot processes, but morphologically normal GBM was significantly permeable as well. We concluded that (1) ultrastructurally normal Alport GBM residing beneath differentiated podocyte foot processes is inherently and abnormally permeable, and (2) upregulation of Lama5 transcription and concentration of laminin ␣1 and ␣5 within Alport GBM thickenings contribute to abnormal permeabilities.

Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes

“…Transgene expression was assayed by both quantitative confocal immunofluorescence microscopy 19,32 and in situ hybridization on the Lamb2 2/2 background. As shown in Figure 1A, dual immunostaining of kidney sections for rat laminin b2 and the GBM marker agrin identified three independent lines of transgenic mice with differing levels of rat b2 in the GBM (Tg Lo , Tg Med , and Tg Hi represent the low, medium, and high transgene expressors, respectively).…”

The Renal Lesions of Alport Syndrome

“…In young Alport mice, the ultrastructurally normal GBM is known to already be abnormally permeable. 13 The concomitant accumulation of mRNAs encoding TGF␤1 and extracellular matrix components in human and mouse Alport podocytes are thought to reflect key events in renal disease progression. 14 Blocking the TGF␤1 pathway prevents GBM thickening in Alport mice.…”

Basement membranes and human disease

“…This hypothesis is supported by the presence of structural abnormalities in mature GBM in X-linked Alport's syndrome that contain α1.α1.α2(IV), and filtration defects in Col4a3 -/-mice in areas of the GBM that are structurally normal (Abrahamson et al 2007). …”

“…Moreover, although the actual contribution to the phenotype of altered laminin α5 and α1 expression in the GBM of Col4a3 -/-mice (Abrahamson et al 2007) remains to be determined, the above data do suggest that clinical severity and disease progression in Alport's syndrome is subject to multiple modifiers. This knowledge may shed light on potential treatment options.…”