Laminin-521 Protein Therapy for Glomerular Basement Membrane and Podocyte Abnormalities in a Model of Pierson Syndrome

Abstract

Background Laminin α5β2γ1 (LM-521) is a major component of the GBM. Mutations in LAMB2 that prevent LM-521 synthesis and/or secretion cause Pierson syndrome, a rare congenital nephrotic syndrome with diffuse mesangial sclerosis and ocular and neurologic defects. Because the GBM is uniquely accessible to plasma, which permeates endothelial cell fenestrae, we hypothesized that intravenous delivery of LM-521 could replace the missing LM-521 in the GBM of Lamb2 mutant mice and restore glomerular permselectivity.Methods We injected human LM-521 (hLM-521), a macromolecule of approximately 800 kD, into the retro-orbital sinus of Lamb2-/- pups daily. Deposition of hLM-521 into the GBM was investigated by fluorescence microscopy. We assayed the effects of hLM-521 on glomerular permselectivity by urinalysis and the effects on podocytes by desmin immunostaining and ultrastructural analysis of podocyte architecture.Results Injected hLM-521 rapidly and stably accumulated in the GBM of all glomeruli. Super-resolution imaging showed that hLM-521 accumulated in the correct orientation in the GBM, primarily on the endothelial aspect. Treatment with hLM-521 greatly reduced the expression of the podocyte injury marker desmin and attenuated the foot process effacement observed in untreated pups. Moreover, treatment with hLM-521 delayed the onset of proteinuria but did not prevent nephrotic syndrome, perhaps due to its absence from the podocyte aspect of the GBM.Conclusions These studies show that GBM composition and function can be altered in vivovia vascular delivery of even very large proteins, which may advance therapeutic options for patients with abnormal GBM composition, whether genetic or acquired.

Keywords: glomerular basement membrane; glomerular filtration barrier; laminin; podocyte.

Graphical abstract

Figure 1.

Recombinant hLM-521 is trimeric and adhesive in vitro. (A) Five nanograms of recombinant hLM-521 trimers were subjected to 4%–15% SDS-PAGE and detected by silver staining. The trimers (approximately 800 kD) were too large to migrate out of the well without reduction (lane 1) but separated into approximately 400-kD α5 chains and β2 and γ1 chains that comigrated at approximately 180–200 kD under reducing conditions (lane 2). (B) With a sandwich ELISA using anti-FLAG as a capturing antibody and anti-hLAMC1 as a detection antibody, hLM-521 showed a concentration-dependent (from 0.08 to 20 µg/ml) increase in OD at 450 nm. (C) K562 cells expressing integrin α3β1 were plated onto hLM-521–coated dishes and assayed for the number of cells adhered. Adhesion was specifically blocked by antibodies to integrin α3 and β1 but not by antibodies to integrin α6 or to Lu or by nonimmune mouse and rat IgG.

Figure 2.

Injected hLM-521 is deposited into the GBM. Kidneys from (A) P13 mice either uninjected or at 15 minutes after one injection of hLM-521 (1.2 mg/kg), (B) P17 mice either uninjected or injected with PBS or hLM-521 once daily starting at P12 for 5 days, and (C) P37 mice injected with hLM-521 once daily starting at P12 for 6 days were subjected to confocal immunofluorescence assays. Antibodies to hLAMA5 (green) and agrin (red) were used with nuclear counterstaining (blue). (A) Lamb2+/+ mice showed mostly mesangial staining for hLAMA5 (magenta arrowheads in middle column) that did not overlap with the GBM marker agrin, and weak GBM staining of hLAMA5 (white arrowheads in middle column); Lamb2−/− mice showed hLAMA5 signals that were more distinct in the GBM than in the mesangial matrix (compare white arrowheads to magenta arrowheads in right column). (B) After five doses of hLM-521, Lamb2−/− mice displayed more robust accumulation of hLM-521 in the GBM compared with Lamb2+/− mice. Uninjected or PBS-injected mice showed no specific LAMA5 signal. Dashed lines demarcate the surface of the kidney. (C) After 20 days of chase, Lamb2−/− mice retained hLM-521 in the GBM, even when the mesangial matrix had expanded (asterisks).

Figure 3.

Injected hLM-521 is localized to the endothelial aspect of the GBM and properly oriented. Lamb2−/− and +/− mice also null for Rag1 either uninjected (A and E) or injected with hLM-521 once daily starting at P12 for 6 days and dissected at P18 or P25 were analyzed by STORM using antibodies to hLAMA5 (red) and agrin (blue). (B and C) hLM-521–injected Lamb2−/− mice showed discrete linear signals for human LAMA5 confined to the endothelial aspect of the GBM versus (D) the interrupted linear pattern in hLM-521–injected Lamb2+/− mice. (F and G) hLM-521–injected Lamb2−/− mice also showed linear staining of human laminin γ1 at the center of the GBM between the agrin layers compared with (H) the scattered staining in hLM-521–injected Lamb2+/− mice.

Figure 4.

hLM-521 treatment delays albuminuria in Lamb2−/− mice. Mice were untreated or injected with hLM-521 once daily starting at P11 or P12 for 5–7 days. Urine was collected before injection and at various time points thereafter for assessing ACRs (milligrams per gram). In Lamb2−/− mice with albuminuria<9000 before injection, treatment with hLM-521 kept proteinuria low through P18, compared with uninjected Lamb2−/− mice that showed high proteinuria at P18 (4651±6770, n=12 versus 20,826±11,580, n=8; ***P<0.001). At P20 albuminuria in treated mice began to rise but was still significantly lower than that in untreated Lamb2−/− mice (11,696±12,406, n=5 versus 32,711±13,184, n=5; *P<0.05).

Figure 5.

hLM-521–treated Lamb2−/− mice show attenuated podocyte injury. (A) Kidneys collected from P18 mice either untreated or injected with PBS or hLM-521 once daily for 5 days were sectioned and immunostained for desmin (green) and WT1, a podocyte nucleus marker (red), and examined by confocal microscopy after nuclear counterstaining (blue). Desmin was normally detected in mesangial cells (Lamb2+/−; left column). In PBS-injected Lamb2−/− mice (middle column), there was occasional ectopic expression of desmin in WT1-positive podocytes (arrowheads). With hLM-521 treatment (right column), there was a dramatic reduction in desmin-positive podocytes. (B) Quantification of the ratio of desmin-WT1 double-positive cells to total WT1-positive cells shows the significant reduction in podocyte injury with hLM-521 treatment (3.2%±2.3%, n=3 versus 14.9%±4.3%, n=4; **P<0.01).

Figure 6.

Podocyte FPE is reduced in hLM-521–treated Lamb2−/− mice. Kidneys collected from P18 mice uninjected or injected with PBS or hLM-521 once daily for 5 days were analyzed by transmission electron microscopy. (A) The GBMs (asterisks in d–f) appeared normal, detected between the podocyte foot processes (arrows) and the endothelium (fenestrae marked with white arrowheads). Villous transformation in podocytes of Lamb2−/− mice (black arrowheads in e) was reduced (f) in Lamb2−/− mice treated with hLM-521. (B) When quantified by number of foot processes per micrometer of GBM, uninjected or PBS-injected Lamb2−/− mice showed significant reduction in the density of foot processes compared with Lamb2+/+ and Lamb2+/− controls (1.22±0.14, n=4 versus 2.18±0.07, n=3 glomeruli; ***P<0.001). hLM-521 treatment significantly increased the density of podocyte foot processes in Lamb2−/− mice (1.63±0.17, n=3; *P<0.05). FP, foot processes.

Figure 7.

The Lamb2−/− glomerular endothelium appears normal with or without hLM-521 treatment. Kidneys collected from P18 Lamb2−/− mice, either uninjected or injected with PBS or hLM-521 once daily starting at P12 for 5 days, and from uninjected Lamb2+/− mice were analyzed by (A) immunofluorescence staining for PECAM and (B) transmission electron microscopy. (A) Localization of PECAM in the glomerular capillary wall of both untreated and hLM-521–treated Lamb2−/− mice was similar to that of Lamb2+/− mice. (B) In all mice the glomerular endothelium was thin and well fenestrated (arrows in d–f), and the capillary loops were patent (black asterisks in a–c). The boxed regions in (a–c) are magnified in (d–f), respectively. White asterisks, red blood cells.