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

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

Upregulated expression of integrin α1 in mesangial cells and integrin α3 and vimentin in podocytes of Col4a3-null (Alport) mice.

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM

The intermediate filament protein vimentin is upregulated in Alport glomeruli.

<p>A: A digital scan of a portion of the 2D gel showing the position of the 8 vimentin spots robotically picked for LC-MS/MS. B: Western blot of wild-type (wt) or Alport mouse glomerular lysates harvested at 4 weeks of age probed with goat anti-vimentin IgGs (Vim, upper blot), then stripped and re-probed with mouse anti-smooth muscle actin (α-SMA, lower blot) as a loading control. Asterisks (*) indicate lower molecular weight bands that are more prominent in the Alport glomerular lystates, possibly representing proteolytic fragments of vimentin.</p

Vimentin is upregulated in podocytes of Alport glomeruli.

<p>A–C: Fresh frozen kidney sections from Alport mice were labeled with a combination of goat anti-vimentin and rabbit anti-GLEPP1 IgGs, followed by the appropriate species-specific Alexa Fluor secondaries. Vimentin labeling (A) is restricted to the epithelial podocyte layer, marked by GLEPP1 staining (B), overlap of staining is shown in C (merge). D–F: Representative fluorescence micrographs are shown of anti-vimentin labeling (Vim) of wild-type (D, wt), or Alport (E) mouse glomeruli. The relative glomerular fluorescence intensities were measured and averaged for n = 3 mice of each genotype, wildtype (wt, blue) or Alport (red). * p = 0.04.</p

The mRNA levels encoding <i>Itga3</i> and <i>Itgb1</i> are upregulated in Alport glomeruli.

<p>Quantitative real time RT-PCR was performed on n = 3 wild-type (wt, blue) and n = 3 Alport (red) glomerular RNA isolated at 4 weeks of age. Both <i>Itga3</i> and <i>Itgb1</i> mRNAs are significantly increased in Alport glomerular RNA. * p = 0.02.</p

Integrin α3 protein is upregulated in podocytes of Alport glomeruli.

<p>A–C: Fresh frozen kidney sections from 4 week old Alport mice were labeled with a combination of rabbit anti-integrin α3 and mouse anti-synaptopodin IgGs, followed by the appropriate species-specific Alexa Fluor secondaries. Anti-integrin α3 immunolabeling (A) is restricted to the epithelial podocyte layer, marked by synaptopodin staining (B), and overlap of staining is shown in C (merge). D–F: Representative fluorescence micrographs are shown of anti-integrin α3 labeling of wild-type (D, wt), or Alport (E) mouse glomeruli. The glomerular fluorescence intensities were averaged for n = 3 mice of each genotype, wild-type (wt, blue) or Alport (red), and integrin α3 signals were significantly greater in Alport. * p = 0.006.</p