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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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Ultrastructural analysis of glomeruli. Low and high magnification electron micrographs show E17.5 glomeruli from control, Lama5 −/−, and Lama5 −/−; Mr51 kidneys, as indicated. The normally continuous basement membrane underlying the podocytes in control (A and G, arrowheads) was disrupted in the Lama5 −/− mutant (B and E, arrows). The chimeric laminin Mr51 rescued assembly of the basement membrane and formation of podocyte foot processes on the Lama5 −/− genetic background (C and H, arrowheads). In the control (D and I), mesangial cells (mc) bound the GBM (I, white line) to maintain the capillary loop (cl) structure. In Lama5 −/−; Mr51 glomeruli (F and J), the ballooning of the capillaries, which were commonly filled with red blood cells (F, rbc), was associated with detachment of mesangial cells from the GBM (F, arrowheads; J, white line). However, podocytes (p) and endothelial cells (e) maintained their normal positions on either side of the GBM. To better present the structure of glomeruli, red blood cells were removed digitally in I and J.
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fig5: Ultrastructural analysis of glomeruli. Low and high magnification electron micrographs show E17.5 glomeruli from control, Lama5 −/−, and Lama5 −/−; Mr51 kidneys, as indicated. The normally continuous basement membrane underlying the podocytes in control (A and G, arrowheads) was disrupted in the Lama5 −/− mutant (B and E, arrows). The chimeric laminin Mr51 rescued assembly of the basement membrane and formation of podocyte foot processes on the Lama5 −/− genetic background (C and H, arrowheads). In the control (D and I), mesangial cells (mc) bound the GBM (I, white line) to maintain the capillary loop (cl) structure. In Lama5 −/−; Mr51 glomeruli (F and J), the ballooning of the capillaries, which were commonly filled with red blood cells (F, rbc), was associated with detachment of mesangial cells from the GBM (F, arrowheads; J, white line). However, podocytes (p) and endothelial cells (e) maintained their normal positions on either side of the GBM. To better present the structure of glomeruli, red blood cells were removed digitally in I and J.

Mentions: To further investigate the fine structure of the GBM and adjacent cells, we used transmission electron microscopy to visualize E17.5 control, Lama5 −/− and Lama5 −/−; Mr51 glomeruli (Fig. 5) . In the control, the GBM was clearly visible between podocytes and endothelial cells (Fig. 5, A and G). Mesangial cells were attached to the GBM at the bases of the capillary loops (Fig. 5 D); this allows them to maintain the glomerular capillary loop structure. In the Lama5 −/− glomerulus, the absence of laminin α5, coupled with the programmed disappearance of laminin α1, resulted in breakdown of the GBM by the late capillary loop stage (Fig. 5, B and E). In the Lama5 −/−; Mr51 glomerulus, the ultrastructure of the GBM was similar to that observed in control (Fig. 5, C and H), confirming that the transgene-derived chimeric laminin α chain must have been assembled into the GBM, as previously demonstrated by immunofluorescence (Fig. 3). This suggests that the short and long arm regions of laminin α5 are sufficient for directing incorporation of the chimera into the GBM. Although podocytes extended foot processes in both control and Lama5 −/−; Mr51 glomeruli (Fig. 5, A, C, G, and H), there appeared to be fewer processes in the latter. This may suggest that the α5 G domain has some role in inducing process extension. However, slit diaphragms, the delicate tight and adherens junctions–related structures that are required for glomerular filtration (Kerjaschki, 2001; Miner, 2002), were observed between many of those foot processes that did form (Fig. 5 H), indicating that this crucial aspect of podocyte differentiation was progressing appropriately. To investigate this in more detail, we used antibodies to slit diaphragm– and foot process–associated proteins. The results were essentially similar for the three genetic backgrounds; all podocytes expressed nephrin, CD2AP, podocin, and synaptopodin (unpublished data). The straightforward interpretation is that neither GBM composition nor its integrity is relevant for expression of these proteins. However, it is important to note that initiation of expression of these proteins has been shown to occur as early as the S-shape stage of glomerular development, when the GBM is structurally sound even in the Lama5 −/− background. Thus, we cannot rule out the possibility that podocyte interactions with the basement membrane at early stages are required for their proper differentiation and gene expression in vivo.


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)

Ultrastructural analysis of glomeruli. Low and high magnification electron micrographs show E17.5 glomeruli from control, Lama5 −/−, and Lama5 −/−; Mr51 kidneys, as indicated. The normally continuous basement membrane underlying the podocytes in control (A and G, arrowheads) was disrupted in the Lama5 −/− mutant (B and E, arrows). The chimeric laminin Mr51 rescued assembly of the basement membrane and formation of podocyte foot processes on the Lama5 −/− genetic background (C and H, arrowheads). In the control (D and I), mesangial cells (mc) bound the GBM (I, white line) to maintain the capillary loop (cl) structure. In Lama5 −/−; Mr51 glomeruli (F and J), the ballooning of the capillaries, which were commonly filled with red blood cells (F, rbc), was associated with detachment of mesangial cells from the GBM (F, arrowheads; J, white line). However, podocytes (p) and endothelial cells (e) maintained their normal positions on either side of the GBM. To better present the structure of glomeruli, red blood cells were removed digitally in I and J.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172883&req=5

fig5: Ultrastructural analysis of glomeruli. Low and high magnification electron micrographs show E17.5 glomeruli from control, Lama5 −/−, and Lama5 −/−; Mr51 kidneys, as indicated. The normally continuous basement membrane underlying the podocytes in control (A and G, arrowheads) was disrupted in the Lama5 −/− mutant (B and E, arrows). The chimeric laminin Mr51 rescued assembly of the basement membrane and formation of podocyte foot processes on the Lama5 −/− genetic background (C and H, arrowheads). In the control (D and I), mesangial cells (mc) bound the GBM (I, white line) to maintain the capillary loop (cl) structure. In Lama5 −/−; Mr51 glomeruli (F and J), the ballooning of the capillaries, which were commonly filled with red blood cells (F, rbc), was associated with detachment of mesangial cells from the GBM (F, arrowheads; J, white line). However, podocytes (p) and endothelial cells (e) maintained their normal positions on either side of the GBM. To better present the structure of glomeruli, red blood cells were removed digitally in I and J.
Mentions: To further investigate the fine structure of the GBM and adjacent cells, we used transmission electron microscopy to visualize E17.5 control, Lama5 −/− and Lama5 −/−; Mr51 glomeruli (Fig. 5) . In the control, the GBM was clearly visible between podocytes and endothelial cells (Fig. 5, A and G). Mesangial cells were attached to the GBM at the bases of the capillary loops (Fig. 5 D); this allows them to maintain the glomerular capillary loop structure. In the Lama5 −/− glomerulus, the absence of laminin α5, coupled with the programmed disappearance of laminin α1, resulted in breakdown of the GBM by the late capillary loop stage (Fig. 5, B and E). In the Lama5 −/−; Mr51 glomerulus, the ultrastructure of the GBM was similar to that observed in control (Fig. 5, C and H), confirming that the transgene-derived chimeric laminin α chain must have been assembled into the GBM, as previously demonstrated by immunofluorescence (Fig. 3). This suggests that the short and long arm regions of laminin α5 are sufficient for directing incorporation of the chimera into the GBM. Although podocytes extended foot processes in both control and Lama5 −/−; Mr51 glomeruli (Fig. 5, A, C, G, and H), there appeared to be fewer processes in the latter. This may suggest that the α5 G domain has some role in inducing process extension. However, slit diaphragms, the delicate tight and adherens junctions–related structures that are required for glomerular filtration (Kerjaschki, 2001; Miner, 2002), were observed between many of those foot processes that did form (Fig. 5 H), indicating that this crucial aspect of podocyte differentiation was progressing appropriately. To investigate this in more detail, we used antibodies to slit diaphragm– and foot process–associated proteins. The results were essentially similar for the three genetic backgrounds; all podocytes expressed nephrin, CD2AP, podocin, and synaptopodin (unpublished data). The straightforward interpretation is that neither GBM composition nor its integrity is relevant for expression of these proteins. However, it is important to note that initiation of expression of these proteins has been shown to occur as early as the S-shape stage of glomerular development, when the GBM is structurally sound even in the Lama5 −/− background. Thus, we cannot rule out the possibility that podocyte interactions with the basement membrane at early stages are required for their proper differentiation and gene expression in vivo.

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