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Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane.

Kikkawa Y, Virtanen I, Miner JH - J. Cell Biol. (2003)

Bottom Line: In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur.Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5.Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.

ABSTRACT
In developing glomeruli, laminin alpha5 replaces laminin alpha1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin alpha5 domains VI through I fused to the human laminin alpha1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin alpha5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the alpha5 G domain essential for mesangial cell adhesion to alpha5LG3-5. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

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Identification and localization of cell types in developing glomeruli. E17.5 control, Lama5 −/−, Lama5 −/−; Mr51, and Lama5 −/−; Mr5G2 frozen kidney sections (as indicated) were stained with antibodies to WT1 to label podocytes (A–H, green), PECAM to label endothelial cells (E–H, red), and desmin to label mesangial cells (I–L, green). Basement membranes were stained with anti–laminin γ 1 monoclonal antibody (A–D, red) or anti–laminin-1 polyclonal antibody (I–L, red). In Lama5 −/−; Mr51 or Mr5G2 glomeruli, the podocytes were arranged properly in a single cell layer at the periphery (C and D), and the glomerular endothelial cells were properly localized (G and H). However, defective capillary loop formation was observed, despite the presence of mesangial cells (K and L). Bar, 50 μm.
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fig4: Identification and localization of cell types in developing glomeruli. E17.5 control, Lama5 −/−, Lama5 −/−; Mr51, and Lama5 −/−; Mr5G2 frozen kidney sections (as indicated) were stained with antibodies to WT1 to label podocytes (A–H, green), PECAM to label endothelial cells (E–H, red), and desmin to label mesangial cells (I–L, green). Basement membranes were stained with anti–laminin γ 1 monoclonal antibody (A–D, red) or anti–laminin-1 polyclonal antibody (I–L, red). In Lama5 −/−; Mr51 or Mr5G2 glomeruli, the podocytes were arranged properly in a single cell layer at the periphery (C and D), and the glomerular endothelial cells were properly localized (G and H). However, defective capillary loop formation was observed, despite the presence of mesangial cells (K and L). Bar, 50 μm.

Mentions: To further investigate the effects of Mr51 and Mr5G2 on glomerulogenesis, we used cell type–specific antibodies to identify the three cell types found in glomeruli. Frozen sections of E17.5 control, Lama5 −/−, Lama5 −/−; Mr51, and Lama5 −/−; Mr5G2 kidneys were stained with antibodies to WT1, platelet endothelial cell adhesion molecule (PECAM), and desmin to label podocytes, endothelial cells, and mesangial cells, respectively (Fig. 4 , green), and doubly labeled with an anti–basement membrane antibody (Fig. 4, red). In the control, podocytes were observed in a single cell layer epithelium adjacent to the glomerular capillaries (Fig. 4 A), and mesangial cells, which provide tension to maintain the glomerular capillary loop structure, were found associated with endothelial cells in the interior of the glomerulus (Fig. 4 I). In the Lama5 −/− mutant, the podocytes were in disarray, and the endothelial cells and mesangial cells were extruded from glomerulus, as we showed previously (Miner and Li, 2000; Fig. 4, B, F, and J). In Lama5 −/−; Mr51 glomeruli, the transgene-derived chimeric laminin chain partially rescued the defects observed in the mutant. The podocytes were arranged in a single cell layer (Fig. 4 C), and the endothelial cells and mesangial cells were localized in the interior of the glomerulus, similar to the control (Fig. 4, G and K). These results suggest that the COOH-terminal portion of laminin α5 is dispensable for the assembly of the GBM and arrangement of podocytes. However, the great reduction in capillary looping (Fig. 4, C and K) is indicative of a mesangial cell defect because a similar phenotype has been observed in the total absence of mesangial cells in mice lacking either PDGF B or PDGF receptor β (Lindahl et al., 1998). Lama5 −/−; Mr5G2 glomeruli exhibited a very similar aberrant glomerular phenotype (Fig. 4, D, H, and L).


Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane.

Kikkawa Y, Virtanen I, Miner JH - J. Cell Biol. (2003)

Identification and localization of cell types in developing glomeruli. E17.5 control, Lama5 −/−, Lama5 −/−; Mr51, and Lama5 −/−; Mr5G2 frozen kidney sections (as indicated) were stained with antibodies to WT1 to label podocytes (A–H, green), PECAM to label endothelial cells (E–H, red), and desmin to label mesangial cells (I–L, green). Basement membranes were stained with anti–laminin γ 1 monoclonal antibody (A–D, red) or anti–laminin-1 polyclonal antibody (I–L, red). In Lama5 −/−; Mr51 or Mr5G2 glomeruli, the podocytes were arranged properly in a single cell layer at the periphery (C and D), and the glomerular endothelial cells were properly localized (G and H). However, defective capillary loop formation was observed, despite the presence of mesangial cells (K and L). Bar, 50 μm.
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Related In: Results  -  Collection

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fig4: Identification and localization of cell types in developing glomeruli. E17.5 control, Lama5 −/−, Lama5 −/−; Mr51, and Lama5 −/−; Mr5G2 frozen kidney sections (as indicated) were stained with antibodies to WT1 to label podocytes (A–H, green), PECAM to label endothelial cells (E–H, red), and desmin to label mesangial cells (I–L, green). Basement membranes were stained with anti–laminin γ 1 monoclonal antibody (A–D, red) or anti–laminin-1 polyclonal antibody (I–L, red). In Lama5 −/−; Mr51 or Mr5G2 glomeruli, the podocytes were arranged properly in a single cell layer at the periphery (C and D), and the glomerular endothelial cells were properly localized (G and H). However, defective capillary loop formation was observed, despite the presence of mesangial cells (K and L). Bar, 50 μm.
Mentions: To further investigate the effects of Mr51 and Mr5G2 on glomerulogenesis, we used cell type–specific antibodies to identify the three cell types found in glomeruli. Frozen sections of E17.5 control, Lama5 −/−, Lama5 −/−; Mr51, and Lama5 −/−; Mr5G2 kidneys were stained with antibodies to WT1, platelet endothelial cell adhesion molecule (PECAM), and desmin to label podocytes, endothelial cells, and mesangial cells, respectively (Fig. 4 , green), and doubly labeled with an anti–basement membrane antibody (Fig. 4, red). In the control, podocytes were observed in a single cell layer epithelium adjacent to the glomerular capillaries (Fig. 4 A), and mesangial cells, which provide tension to maintain the glomerular capillary loop structure, were found associated with endothelial cells in the interior of the glomerulus (Fig. 4 I). In the Lama5 −/− mutant, the podocytes were in disarray, and the endothelial cells and mesangial cells were extruded from glomerulus, as we showed previously (Miner and Li, 2000; Fig. 4, B, F, and J). In Lama5 −/−; Mr51 glomeruli, the transgene-derived chimeric laminin chain partially rescued the defects observed in the mutant. The podocytes were arranged in a single cell layer (Fig. 4 C), and the endothelial cells and mesangial cells were localized in the interior of the glomerulus, similar to the control (Fig. 4, G and K). These results suggest that the COOH-terminal portion of laminin α5 is dispensable for the assembly of the GBM and arrangement of podocytes. However, the great reduction in capillary looping (Fig. 4, C and K) is indicative of a mesangial cell defect because a similar phenotype has been observed in the total absence of mesangial cells in mice lacking either PDGF B or PDGF receptor β (Lindahl et al., 1998). Lama5 −/−; Mr5G2 glomeruli exhibited a very similar aberrant glomerular phenotype (Fig. 4, D, H, and L).

Bottom Line: In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur.Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5.Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.

ABSTRACT
In developing glomeruli, laminin alpha5 replaces laminin alpha1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin alpha5 domains VI through I fused to the human laminin alpha1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin alpha5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the alpha5 G domain essential for mesangial cell adhesion to alpha5LG3-5. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

Show MeSH
Related in: MedlinePlus