The formation of quiescent glomerular endothelial cell monolayer in vitro is strongly dependent on the choice of extracellular matrix coating

Pajęcka K., Nygaard Nielsen M., Krarup Hansen T., Williams J.M.
Experimental Cell Research, 2017

Primary glomerular endothelial cells (GEnCs) is an important tool for studying glomerulosclerotic mechanisms and in drug development. Primary GEnCs are commonly cultured either on gelatin or plasma-derived fibronectin coated surfaces, yet neither of the substrates are a major component of the healthy mature GBM. Here, the authors set out to establish a simple, reproducible model of quiescent hGEnC monolayer in vitro by determining the best commercially available ECM substrate- recombinant human laminin (rhLN)- 521, -511, -111, fibronectin (FN), collagen type IV and collagen I - based Attachment Factor. All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Laminin-521, laminin-511 and FN were best at supporting hGEnC attachment and spreading. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70 kDa dextrans which was 82 % tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions. The authors recommendations based on the results are that hGEnCs is best cultured on the mature glomerular basement membrane laminin - rhLN521 – which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined adherens junctions and physiological perm-selectivity.

 

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains

St John P.L. and Abrahamson D.R.
Kidney International, 2001

During the glomerular basement membrane assembly, laminin-111 is initially expressed in vascular clefts of comma-a dn S-shaped bodies and is eventually replaced by laminin-521 which persists into maturation. Post-fixation immunoelectron microscopy of developing mouse and 2-3 days old mice kidney was performed and showed Intracellular labeling for laminin-111 and laminin-521 in developing glomerular endothelial cells and podocytes. In early capillary loop stage GBMs laminin-111 was absent but the expression of a5 was strong (developmental switch). The results show that both endothelial cells and podocytes synthesize laminin but highest level of laminin synthesis by endothelial cells.

 

Roles for Laminin in Embryogenesis: Exencephaly, Syndactyly, and Placentopathy in Mice Lacking the Laminin α5 Chain

Miner J.H., Cunningham J., Sanes J.R.
J Cell Biol., 1998

Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure. In addition to exencephaly, syndactyly, and placentopathy, several defects were seen in internal organs of Lama5 −/− embryos, including small or absent kidneys. Some of these defects were variable in severity and penetrance, but all can be associated with BLs that normally contain laminin α5.

 

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 J.H.
J Cell Biol., 2003

In developing glomeruli, laminin alpha-5 (laminin-521 and -511) replaces laminin alpha-1 (laminin-111) in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. By the capillary loop stage, laminin-111 is eliminated and at maturity, only laminin-521 is detected in the GBM where it plays a crucial role in maintaining glomerular capillary loop structures. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, the authors produced transgenic mice that express a chimeric laminin composed of laminin alpha-5 domains fused to the human laminin alpha-1 globular (G) domain (Mr51). 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, suggesting that the G domain of laminin alpha-5 is essential for mesanglial adhesion. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha-5. These results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to laminin alpha-5 in the GBM.

 

ECM modulated early kidney development in embryonic organ culture

Sebinger D., Ofenbauer A., Gruber P., Malik S., Werner C.
Biomaterials, 2013

Here, the authors, explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. By culture of microdissected E11.5 kidney rudiments, the show that specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. Culture on laminin-111 promoted development as shown by more bud tips and more nephrons.

 

The Laminin a Chains: Expression, Developmental Transitions, and Chromosomal Locations of a 1-5, Identification of Heterotrimeric Laminins 8–11, and Cloning of a Novel a 3 Isoform

Miner J.H., Patton B.L., Lentz S.I., Gilbert D.J., Snider W.D., Jenkins N.A., Copeland N.G., Sane J.R.
The Journal of Cell Biology, 1997

Using a panel of nucleotide and antibody probes, we surveyed the expression of a 1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, a 4 and a 5 exhibited the broadest patterns of expression, while expression of a1 was the most restricted. Detailed analysis of developing kidney revealed that some individual basal laminae, including those of the tubule and glomerulus, changed in laminin chain composition as they matured. During glomerulogenesis there are developmental transitions in laminin trimer deposition. Initially the GBM contains laminin-111, laminin-411 and laminin-511. By the capillary loop stage during GMB maturation, laminin-111 is eliminated from the GBM and the beta 2 chain begins to accumulate while the beta 1 chain is down regulated.

 

Endothelin A receptor activation on mesangial cells initiates Alport glomerular disease

Dufek B. Meehan D.T., Delimont D., Cheung L., Gratton M.A., Phillips G., Song W., Liu S., Cosgrove D.
International Society of Nephrology, 2016

Normally, α5 laminins are highly expressed in the glomerular basement membranes (GBM).  In this article, the authors show that the mesangial filopodia in the GBM are depositing mesangial matrix proteins, including laminin 211, which activates focal adhesion kinase in glomerular podocytes, resulting in the activation of genes encoding proinflammatory cytokines and metalloproteinases through an nuclear factor kB–dependent signaling pathway, which drive the progression of glomerulonephritis. The authors test whether endothelial cell-derived endothelin-1 is up-regulated in Alport glomeruli and further elevated by hypertension. Endothelin A receptor activation on mesangial cells is a key event in initiation of Alport glomerular disease in this model.

 

Laminin a4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

C.K. Abrass, Hansen K.M., Patton B.L.
Matrix Pathobiology, 2010

Laminin-8/9 is synthesized by rat glomerular mesangial cells and is required for PDGF-induced mesangial cell migration. Here the authors show that Lama4-/- mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor beta in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to alpha4-containing laminins results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. These data suggest that failure of laminin alpha4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. 

 

The Glomerular Basement Membrane

Miner J.H.
Exp Cell Res. 2012

Like all basement membranes, glomerular basement membrane (GBM) consists mainly of laminin, type IV collagen, nidogen, and heparan sulfate proteoglycan. However, the GBM is unusually thick and contains particular members of these general protein families, including laminin-521, collagen α3α4α5(IV), and agrin. Knockout studies in mice and genetic findings in humans shows that the laminin and type IV collagen components are particularly important for GBM structure and function, as laminin or collagen IV gene mutations cause filtration defects and renal disease of varying severities, depending on the nature of the mutations.

 

Laminin isoforms in endothelial and perivascular basement membranes

Yuosif L., Russo J.D., Sorokin L.
Cell Adh Migr., 2013

α4 and α5 chain laminins are the predominant isoforms in the basal lamina of vascular endothelial cells. While the α4 chain is expressed ubiquitously throughout different developmental stages, the prominent expression of α5 chain appears postnatally and its distribution varies with vessel type.