Transgenic Expression of Human LAMA5 Suppresses Murine Lama5 mRNA and Laminin α5 Protein Deposition

2011-09-02 19:13:15

PLoS One; 2011 Sept; 6(9):e23926

Brooke M. Steenhard, Adrian Zelenchuk, Larysa Stroganova, Kathryn Isom, Patricia L. St. John, Glen K. Andrews, Kenneth R. Peterson, Dale R. Abrahamson


The human kidney contains approximately one million individual nephrons, each beginning with a glomerulus, which is a unique capillary tuft that largely restricts the passage of serum albumin and larger proteins into the primary nephron filtrate. All three layers of the glomerular capillary wall, namely the glomerular endothelial cells, glomerular epithelial cells (podocytes), and an intervening glomerular basement membrane (GBM), are required for maintenance of normal filtration barrier properties. For example, enzymatic degradation of glycosaminoglycans within the glomerular endothelial surface glycocalyx results in an increased fractional clearance for albumin. Additionally, blockage of podocyte-derived VEGF signaling causes glomerular endothelial cell abnormalities in developing or mature kidneys and proteinuria.

A host of defects that affect the podocyte and its specialized intercellular junction, the epithelial slit diaphragm, also cause abnormal glomerular permeabilities. These include mutations in the NPHS1 gene encoding the slit diaphragm component, nephrin, which causes congenital nephrotic syndrome of the Finnish type and results in massive proteinuria at birth. Mutations to NPHS2, which encodes another slit diaphragm protein, podocin, also causes proteinuria in autosomal recessive steroid-resistant nephrotic syndrome, a disease often diagnosed in childhood. Intracellularly, podocin and nephrin are both linked indirectly to the actin cytoskeleton through interaction with CD2-associated protein (CD2ap). Slit diaphragms are absent in mice that lack nephrin or podocin and these animals die perinatally with renal failure. Mice deficient in CD2ap also die from renal failure, but at 6–7 weeks of age.

Like all basement membranes, the GBM is composed of type IV collagen, laminin, nidogen, and proteoglycans. Unlike most other basement membranes, however, the GBM undergoes type IV collagen and laminin isoform substitution during glomerular development. Specifically, basement membranes within the earliest glomerular regions of comma- and S-shaped nephric figures contain collagen α1α2α1(IV) and laminin α1β1γ1 (LN-111). These isoforms are later replaced by collagen α3α4α5(IV), laminin α5β1γ1 (LN-511), and laminin α5β2γ1 (LN-521) as glomerular capillary loops expand. Subsequently, LN-521 is the only laminin isoform found in GBMs of fully mature glomeruli. Collagen α1α2α1(IV) and all of the GBM laminin chains originate from both glomerular endothelial cells and podocytes, but collagen α3α4α5(IV) derives solely from podocytes.

The reasons why the GBM collagen IV and laminin composition changes during development are not fully understood, but evidence indicates that this is necessary for final glomerular maturation and full acquisition and maintenance of filtration barrier properties. Alport disease, which is a familial nephropathy marked by focal splitting, thinning, and regional thickening of the GBM and leads to renal failure, is caused by mutations in either the COL4A3, COL4A4, or COL4A5 genes encoding the collagen α3(IV), α4(IV), and α5(IV) protein chains, respectively. Most Alport patients fail to assemble a stable network of collagen α3α4α5(IV) in the GBM, and there is retention of the infantile, collagen α1α2α1(IV) network. This isoform appears to be more susceptible to proteolysis, which may explain why the GBMs of Alport patients ultimately deteriorate. A model of Alport disease has been created in mice through the deletion of the Col4a3 gene, and these animals die of renal failure 2–4 months after birth with the same glomerular defects as those seen in Alport patients. The mouse Alport phenotype can be rescued when transgenic mice expressing human COL4A3-COL4A4 genes are crossed onto the mouse Col4a3 knockout background.

Failure to undergo laminin isoform transitioning from LN-111 to LN-521 also results in kidney malfunction in mice and in humans. Although normal glomerular development is seen in mice with laminin β2 deficiencies, they eventually exhibit podocyte foot process broadening, proteinuria, and die of renal failure. Humans with mutations in the LAMB2 gene suffer from Pierson syndrome, which usually presents at birth as congenital nephrotic syndrome with severe neuromuscular junction abnormalities (owing to the presence of laminin β2 in the neuromuscular junction basement membrane as well).

There are no human mutations described for LAMA5, but experiments in mice have shown its expression to be absolutely crucial for normal glomerular development and function. Mice with deletions of Lama5 die before birth with neural tube closure defects and placental dysmorphogenesis. In kidney, a stable GBM fails to assemble, and endothelial cells do not form vascularized glomerular tufts. This Lama5 knockout phenotype can be partially rescued when fetal kidneys from Lama5 mutants are grafted into newborn kidneys of normal, wildtype hosts. In this case, host endothelial cells, which express laminin α5, migrate into the engrafted Lama5 null kidneys and vascularized glomeruli form within grafts. The host endothelial cell-derived laminin α5 does not project across the full width of these GBMs, however. This results in an unusual situation where there is retention of the infantile laminin α1 on the outer, sub-podocyte layer of matrix and laminin α5 is present only on the inner, subendothelial layer. Additionally, these hybrid GBMs are abnormally wide and not as well condensed as normal GBM, and podocyte foot processes are absent. In other experiments, deletion of Lama5 only in podocytes results in mild to severe proteinuria, and variable defects in GBM and podocyte ultrastructure. In this same study, expression of a human LAMA5 transgene under control of a doxycyclin inducible, podocyte-specific expression system rescues glomerular and tubular defects caused by a hypomorphic Lama5 mutation.

Taken together, these findings demonstrate that the timely expression of LN-521 is needed for glomerular endothelial cell and podocyte differentiation, and the appearance of collagen α3α4α5(IV) is required for long term GBM stability. However, very little is known at the gene level regarding activation of any of the mature GBM protein isoforms, what silences transcription of infantile chain genes at the appropriate developmental stages, and how the infantile collagen α1α2α1(IV) and LN-111 networks are removed from developing GBM. In addition, we do not understand what causes upregulation of Lama5 in Col4a3 knockout (Alport) mice, which may be an important contributor to fibrosis in that model.

To begin addressing some of these questions, we have developed bacterial artificial chromosome (BAC) transgenic mice expressing human LAMA5. These transgenics deposited apparently large amounts of human laminin α5 protein in basement membranes widely, and, specifically in glomeruli, at the appropriate developmental stage. Expression of human LAMA5 did not appear harmful and kidney functional tests and morphology were normal. The results suggest that the BAC used for transgenic injections contained all of the necessary regulatory information for proper LAMA5 expression. Of great interest, in kidneys from lines with the highest levels of human LAMA5 expression, there were significant decreases in native mouse Lama5 mRNA and mouse laminin α5 protein deposition.

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