Forced expression of laminin beta1 in podocytes prevents nephrotic syndrome in mice lacking laminin beta2, a model for Pierson syndrome

Abstract

Pierson syndrome is a congenital nephrotic syndrome with ocular and neurological defects caused by mutations in LAMB2, the gene encoding the basement membrane protein laminin β2 (Lamβ2). It is the kidney glomerular basement membrane (GBM) that is defective in Pierson syndrome, as Lamβ2 is a component of laminin-521 (LM-521; α5β2γ1), the major laminin in the mature GBM. In both Pierson syndrome and the Lamb2(-/-) mouse model for this disease, laminin β1 (Lamβ1), a structurally similar homolog of Lamβ2, is marginally increased in the GBM, but it fails to fully compensate for the loss of Lamβ2, leading to the filtration barrier defects and nephrotic syndrome. Here we generated several lines of Lamβ1 transgenic mice and used them to show that podocyte-specific Lamβ1 expression in Lamb2(-/-) mice abrogates the development of nephrotic syndrome, correlating with a greatly extended lifespan. In addition, the more Lamβ1 was expressed, the less urinary albumin was excreted. Transgenic Lamβ1 expression increased the level of Lamα5 in the GBM of rescued mice, consistent with the desired increased deposition of laminin-511 (α5β1γ1) trimers. Ultrastructural analysis revealed occasional knob-like subepithelial GBM thickening but intact podocyte foot processes in aged rescued mice. These results suggest the possibility that up-regulation of LAMB1 in podocytes, should it become achievable, would likely lessen the severity of nephrotic syndrome in patients carrying LAMB2 mutations.

Fig. 1.

Generation and characterization of transgenic mice expressing a chimeric Lamβ1 chain. (A) Shared structure of Lamβ1 and -β2, and percentage of similarities between mouse Lamβ1 and -β2 and between human Lamβ1 (hβ1) and mouse Lamβ1 (mβ1). LN, laminin NH2-terminal domain; LE, laminin EGF-like domain; LF, laminin four domain; Lβ, laminin β-knob. (B) Schematic diagram of the Neph-B1 transgene. The 4.1-kb nephrin (Nphs1) promoter drives expression of the mouse/human chimeric Lamβ1 cDNA with a SV40 polyadenylylation signal sequence (pA). Asterisks indicate the epitopes that the antimouse and antihuman antibodies recognize, respectively. (C–F) Mouse laminin β1 confocal immunofluorescence micrographs. The antibody used recognizes both endogenous Lamβ1 and transgenic Lamβ1. Although wild-type GBM lacks deposition of Lamβ1 and Lamb2^−/− GBM shows only a low level of Lamβ1, transgenic mice show linear and higher level deposition of Lamβ1. Arrows indicate GBM, and M indicates mesangial matrix. (G) Quantification of Lamβ1 fluorescence intensity in the GBM. *P < 0.001. (Scale bars, 20 μm.)

Fig. 2.

Increased Lamβ1 in the GBM in human Pierson syndrome. Immunofluorescence analysis of Lamβ1 in human kidney sections. A 3-mo-old Pierson syndrome patient's specimen (A) shows linear staining for Lamβ1 in the GBM (arrows), whereas a normal adult control (B) shows weak mesangial staining and the absence of staining in the GBM. (Scale bars, 50 μm.)

Fig. 3.

Forced Lamβ1 expression in podocytes of Lamb2^−/− mice prevents nephrotic syndrome. (A) Reduced albuminuria in Lamb2^−/−; Neph-B1 mice. SDS/PAGE/Coomassie blue analysis of 1 μL of urine from mice of the following genotypes: lane 3, Lamb2^−/−; Neph-B1H; lane 4, Lamb2^−/−; lane 5, Lamb2^+/−; Neph-B1H (lanes 3–5 were from 3-wk-old mice); lane 7, Lamb2^+/−; Neph-B1L; lane 8, Lamb2^−/−; Neph-B1L (lanes 7 and 8 were from 10-mo-old mice). Lane 1, markers. Arrow indicates the size of albumin. (B) Graph showing albumin-to-creatinine ratios (g/g) in urine of Lamb2^−/−; Neph-B1 and control mice. Lamb2^−/−; Neph-B1 mice survived more than 1 y and had greatly reduced albuminuria compared with Lamb2^−/− mice, which survive 1 mo with ∼100 g urinary albumin/g creatinine. Note that the low Lamβ1-expressing mice (Neph-B1L) showed heavier albuminuria than the high expressing mice (Neph-B1H).

Fig. 4.

Histological analysis reveals maintenance of podocyte phenotype in Lamb2^−/−; Neph-B1 kidneys. (A–C and G–I) PAS staining of kidneys from 3-wk-old (A–C) and 1 y-old (G–I) mice. Asterisks in B indicate protein casts in tubules of a nephrotic Lamb2^−/− mouse, which were not observed in Lamb2^−/−; Neph-B1 mice (A and C). Arrows in G and I indicate thickening of the GBM at 1 y in Lamb2^−/−; Neph-B1 mice. (D–F and J–L) Immunofluorescence analysis of podocin (green) and desmin (red) in kidney sections from 3-wk-old (D–F) and 1-y-old (J–L) mice. Arrow in E indicates an injured podocyte expressing both podocin and desmin; desmin is confined to mesangial cells in normal glomeruli, as observed in the Lamb2^−/−; Neph-B1 and Lamb2^+/− panels. (Scale bars, 50 μm.)

Fig. 5.

Normalization of laminin composition in the GBM of Lamb2^−/−; Neph-B1 mice. Immunofluorescence analysis of deposition of (A–C) Lamα1 (green) and Lamα5 (red); (D–F) laminin-332; and (G–I) Lamα2 in the GBMs of 3-wk-old mice. Ectopic deposition of Lamα1, LM-332, and Lamα2 in the Lamb2^−/− GBM (B, E, and H) did not occur in the Lamb2^−/−; Neph-B1 GBM (A, D, and G). Arrows indicate GBM; Lamα2 is normally present in the mesangium (M) (G and I). Asterisk in H indicates a sclerotic glomerulus. (J) Lamα5 fluorescence intensity in the GBM was quantified using images in Fig. S3. Transgenic Lamβ1 expression increased Lamα5 in the GBM to a level comparable to that of the control. (J) *P < 0.001. (Scale bars, 50 μm.)

Fig. 6.

Ultrastructural analysis of glomerular capillary walls. (A–E) Transmission electron micrographs of glomerular capillary loops from 3-wk-old (A–C) and from 1-y-old (D and E) mice. Note that the severe podocyte foot process effacement observed in Lamb2^−/− mice (B) was not observed in young or old Lamb2^−/−; Neph-B1 mice (A and D). Arrow in D indicates segmental thickening of the GBM. Asterisk indicates electron lucent areas in the expanded lamina densa. (F and G) Scanning electron micrographs of glomeruli from 1-y-old mice. Podocyte foot processes in Lamb2^−/−; Neph-B1 mice were intact. (Scale bar, 2 μm.) Po, podocyte; CL, capillary lumen; En, endothelial cell; R, red blood cell.