A deletion in the N-terminal polymerizing domain of laminin ß2 is a new mouse model of chronic nephrotic syndrome.

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin ß2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.

Endothelial cell-specific collagen type IV-α3 expression does not rescue Alport syndrome in Col4a3-/- mice.

The glomerular basement membrane (GBM) is a critical component of the kidney's blood filtration barrier. Alport syndrome, a hereditary disease leading to kidney failure, is caused by the loss or dysfunction of the GBM's major collagen type IV (COL4) isoform α3α4α5. The constituent COL4 α-chains assemble into heterotrimers in the endoplasmic reticulum before secretion into the extracellular space. If any one of the α3-, α4-, or α5-chains is lost due to mutation of one of the genes, then the entire heterotrimer is lost. Patients with Alport syndrome typically have mutations in the X-linked COL4A5 gene or uncommonly have the autosomal recessive form of the disease due to COL4A3 or COL4A4 mutations. Treatment for Alport syndrome is currently limited to angiotensin-converting enzyme inhibition or angiotensin receptor blockers. Experimental approaches in Alport mice have demonstrated that induced expression of COL4A3, either widely or specifically in podocytes of Col4a3-/- mice, can abrogate disease progression even after establishment of the abnormal GBM. While targeting podocytes in vivo for gene therapy is a significant challenge, the more accessible glomerular endothelium could be amenable for mutant gene repair. In the present study, we expressed COL4A3 in Col4a3-/- Alport mice using an endothelial cell-specific inducible transgenic system, but collagen-α3α4α5(IV) was not detected in the GBM or elsewhere, and the Alport phenotype was not rescued. Our results suggest that endothelial cells do not express the Col4a3/a4/a5 genes and should not be viewed as a target for gene therapy.

Alport syndrome and Pierson syndrome: Diseases of the glomerular basement membrane.

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5ß2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases.

Pathogenicity of a Human Laminin ß2 Mutation Revealed in Models of Alport Syndrome.

Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin ß2 (LAMB2), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3-/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5+/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α3α4α5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.

EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis.

BACKGROUND: Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. METHODS: Here, we assessed the role of EphA2 in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe-/-) and by assessing EphA2 function in multiple vascular cell culture models. After 8 to 16 weeks on a Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. RESULTS: Despite enhanced weight gain and plasma lipid levels compared with Apoe-/- controls, EphA2-/-Apoe-/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. Although plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest that hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2-/-Apoe-/- mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression after smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. CONCLUSIONS: Together, these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by promoting monocyte firm adhesion, whereas smooth muscle EphA2 expression may regulate the progression to advanced atherosclerosis by regulating smooth muscle proliferation and extracellular matrix deposition.

Muscular dystrophy meets protein biochemistry, the mother of invention.

Muscular dystrophies result from a defect in the linkage between the muscle fiber cytoskeleton and the basement membrane (BM). Congenital muscular dystrophy type MDC1A is caused by mutations in laminin α2 that either reduce its expression or impair its ability to polymerize within the muscle fiber BM. Defects in this BM lead to muscle fiber damage from the force of contraction. In this issue of the JCI, McKee and colleagues use a laminin polymerization-competent, designer chimeric BM protein in vivo to restore function of a polymerization-defective laminin, leading to normalized muscle structure and strength in a mouse model of MDC1A. Delivery of such a protein to patients could ameliorate many aspects of their disease.

Functional assessment of a novel COL4A5 splice region variant and immunostaining of plucked hair follicles as an alternative method of diagnosis in X-linked Alport syndrome.

BACKGROUND: Many COL4A5 splice region variants have been described in patients with X-linked Alport syndrome, but few have been confirmed by functional analysis to actually cause defective splicing. We sought to demonstrate that a novel COL4A5 splice region variant in a family with Alport syndrome is pathogenic using functional studies. We also describe an alternative method of diagnosis. METHODS: Targeted next-generation sequencing results of an individual with Alport syndrome were analyzed and the results confirmed by Sanger sequencing in family members. A splicing reporter minigene assay was used to examine the variant's effect on splicing in transfected cells. Plucked hair follicles from patients and controls were examined for collagen IV proteins using immunofluorescence microscopy. RESULTS: A novel splice region mutation in COL4A5, c.1780-6T>G, was identified and segregated with disease in this family. This variant caused frequent skipping of exon 25, resulting in a frameshift and truncation of collagen α5(IV) protein. We also developed and validated a new approach to characterize the expression of collagen α5(IV) protein in the basement membranes of plucked hair follicles. Using this approach we demonstrated reduced collagen α5(IV) protein in affected male and female individuals in this family, supporting frequent failure of normal splicing. CONCLUSIONS: Differing normal to abnormal transcript ratios in affected individuals carrying splice region variants may contribute to variable disease severity observed in Alport families. Examination of plucked hair follicles in suspected X-linked Alport syndrome patients may offer a less invasive alternative method of diagnosis and serve as a pathogenicity test for COL4A5 variants of uncertain significance.