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

Wideband dual-polarized 1-Bit unit-cell design for mmWave reconfigurable intelligent surface

This study presents a novel reflective unit-cell with wideband characteristics at millimeter-wave (mmWave) bands for application in Reconfigurable Intelligent Surfaces (RIS). The proposed unit-cell design demonstrates through full-wave simulations a superior bandwidth performance over the 26.50-29.45 GHz, targeting the n257 band of mmWave 5G. The design was created for dual-polarization operation, each controlled by a p-i-n diode to realise a 1-bit RIS. The design achieves impressive performance, maintaining a phase difference error within ±20 degrees across most of the 3 GHz bandwidth while reflecting over 80% of the energy. The increased reflectivity minimizes losses, while precise phase control improves beam pointing accuracy, crucial in low-complexity 1-bit systems. Numerical simulations also indicate that this unit-cell performs effectively in a 6-layer PCB stack-up. A comparison with state-of-the-art unit-cells for RIS design is also presented, demonstrating the advantages of the proposed design in terms of bandwidth, dual-polarization operation, and phase accuracy.</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

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

Proteins altered in Alport glomeruli identified by 2D DIGE and MALDI-TOF.

<p>n/c = no change, n/a = not applicable.</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

There is no change in expression of integrin β1 in Alport glomeruli.

<p>A–C: Fresh frozen kidney sections from 4 week old wild-type (wt) mice, immunolabeled with anti-integrin β1 (A), anti collagen α3α4α5(IV) (B) and overlap of labeling is shown in C (merge). D–F: Representative fluorescence micrographs of wildtype (D) and Alport glomeruli immunolabled with anti-integrin β1. Glomerular fluorescence intensities were averaged for n = 3 mice of each genotype, wild-type (wt, blue) or Alport (red), and there is no statistical difference.</p

Integrin α1 protein is upregulated in the mesangium of Alport glomeruli.

<p>A–C: Fresh frozen kidney sections from 4 week old Alport mice were labeled with a combination of hamster anti-integrin α1 and rat anti-laminin β1 IgGs, followed by the appropriate species-specific Alexa Fluor secondary antibody. Anti-integrin α1 labeling (A, Itgα1) is restricted to the mesangial layer, marked by anti-laminin β1 staining (B, Lamβ1), and overlap of staining is shown in C (merge). D–F: Representative fluorescence micrographs are shown of anti-integrin α1 labeling of wild-type (D, wt), or Alport (E) mouse glomeruli. Glomerular fluorescence intensities were averaged for n = 3 mice of each genotype, wildtype (wt, blue) or Alport (red), and integrin α1 signals were significantly greater in Alport. * p = 0.01.</p