A Missense LAMB2 Mutation Causes Congenital Nephrotic Syndrome by Impairing Laminin Secretion

Abstract

Laminin ␤2 is a component of laminin-521, which is an important constituent of the glomerular basement membrane (GBM). Null mutations in laminin ␤2 (LAMB2) cause Pierson syndrome, a severe congenital nephrotic syndrome with ocular and neurologic defects. In contrast, patients with LAMB2 missense mutations, such as R246Q, can have less severe extrarenal defects but still exhibit congenital nephrotic syndrome. To investigate how such missense mutations in LAMB2 cause proteinuria, we generated three transgenic lines of mice in which R246Q-mutant rat laminin ␤2 replaced the wild-type mouse laminin ␤2 in the GBM. These transgenic mice developed much less severe proteinuria than their nontransgenic Lamb2-deficient littermates; the level of proteinuria correlated inversely with R246Q-LAMB2 expression. At the onset of proteinuria, expression and localization of proteins associated with the slit diaphragm and foot processes were normal, and there were no obvious ultrastructural abnormalities. Low transgene expressors developed heavy proteinuria, foot process effacement, GBM thickening, and renal failure by 3 months, but high expressors developed only mild proteinuria by 9 months. In vitro studies demonstrated that the R246Q mutation results in impaired secretion of laminin. Taken together, these results suggest that the R246Q mutation causes nephrotic syndrome by impairing secretion of laminin-521 from podocytes into the GBM; however, increased expression of the mutant protein is able to overcome this secretion defect and improve glomerular permselectivity.

Laminin β2 Gene Missense Mutation Produces Endoplasmic Reticulum Stress in Podocytes

“…It was also noted that the highest C321R expressor (C321R-Tg Hi ) exhibited a lower level of b2 in the GBM than the lowest R246Q expressor (R246Q-Tg Lo ) studied previously ( Figure 1A). Moreover, as we previously showed, 19 rat b2 protein levels in the GBM were much higher in Lamb2 2/2 mice expressing WT rat b2 (Lamb2 2/2 ; Tg-WT) than in R246Q-Tg Lo mice ( Figure 1B). Surprisingly, transgene-derived mRNA was easily detected by in situ hybridization in C321R-Tg Med and -Tg Hi and the Tg-WT kidneys in a podocyte-specific pattern, although not in C321R-Tg Lo or R246Q-Tg Lo kidneys ( Figure 1C).…”

“…Surprisingly, transgene-derived mRNA was easily detected by in situ hybridization in C321R-Tg Med and -Tg Hi and the Tg-WT kidneys in a podocyte-specific pattern, although not in C321R-Tg Lo or R246Q-Tg Lo kidneys ( Figure 1C). Taken together, analysis of transgene-derived mRNA and protein levels suggests that, even with transcription levels comparable with those levels of the WT b2 transgene in podocytes (and higher than the endogenous mouse Lamb2 mRNA 19 ), C321R-LAMB2 protein levels in the GBM were comparatively very low.…”

“…Scale bar, 10 mm. (B) Panels from our previous paper 19 show that Tg-WT mice exhibited much higher rat b2 in the GBM compared with R246Q-Tg . These findings suggest that the increased transgene expression and the subsequent increased deposition of C321R-LAMB2 in the GBM partly ameliorate the severe GFB defect in Lamb2 2/2 mice in a dose-dependent manner.…”

“…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).…”

“…We previously showed that defective laminin secretion from podocytes to the GBM underlies the R246Q mutation-caused congenital nephrotic syndrome. 19 Missense mutations can lead to protein misfolding and disruption of protein trafficking. 20,21 Alterations in protein trafficking occur mainly in the ER, the central site for folding, post-translational modifications, and transport of secretory, luminal, and membrane proteins.…”