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

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

doi:10.1371/journal.pone.0023926

Abstract

Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ∼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1–α5 isoform switch, or that for mouse laminin β1–β2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.

Highlights

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
Expression of a human LAMA5 transgene under control of a doxycyclin inducible, podocytespecific expression system rescues glomerular and tubular defects caused by a hypomorphic Lama5 mutation [29]. These findings demonstrate that the timely expression of LN-521 is needed for glomerular endothelial cell and podocyte differentiation, and the appearance of collagen a3a4a5(IV) is required for long term glomerular basement membrane (GBM) stability
Identification of human LAMA5 bacterial artificial chromosome (BAC) transgenics Transgenic mice were established from 25 progeny born from zygotes injected with BAC DNA RP11-1023E23

Summary

Introduction

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. To confirm transcription and translation of the human LAMA5 gene in the BAC transgenic mice, frozen sections of kidney from transgenic and wildtype siblings were incubated with monoclonal mouse anti-human laminin a5 IgG and fluorochrome-conjugated secondary antibody.

Results

Conclusion