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1.  Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys 
The Journal of Cell Biology  1985;100(6):1988-2000.
To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S- shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin- rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.
PMCID: PMC2113600  PMID: 3889015
2.  Transgenic Expression of Human LAMA5 Suppresses Murine Lama5 mRNA and Laminin α5 Protein Deposition 
PLoS ONE  2011;6(9):e23926.
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.
doi:10.1371/journal.pone.0023926
PMCID: PMC3168496  PMID: 21915268
3.  Organogenesis of the kidney glomerulus 
Organogenesis  2011;7(2):75-82.
The glomerular basement membrane (GBM) is a crucial component of the kidney's filtration barrier that separates the vasculature from the urinary space. During glomerulogenesis, the GBM is formed from fusion of two distinct basement membranes, one synthesized by the glomerular epithelial cell (podocyte) and the other by the glomerular endothelial cell. The main components of the GBM are laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogen and the heparan sulfate proteoglycan, agrin. By studying mice lacking specific GBM components, we have shown that during glomerulogenesis, laminin is the only one that is required for GBM integrity and in turn, the GBM is required for completion of glomerulogenesis and glomerular vascularization. In addition, our results from laminin β2-null mice suggest that laminin-521, and thus the GBM, contribute to the establishment and maintenance of the glomerular filtration barrier to plasma albumin. In contrast, mutations that affect GBM collagen IV or agrin do not impair glomerular development or cause immediate leakage of plasma proteins. However, collagen IV mutation, which causes Alport syndrome and ESRD in humans, leads to gradual damage to the GBM that eventually leads to albuminuria and renal failure. These results highlight the importance of the GBM for establishing and maintaining a perfectly functioning, highly selective glomerular filter.
doi:10.4161/org.7.2.15275
PMCID: PMC3142441  PMID: 21519194
laminin; collagen IV; nephrotic syndrome; alport syndrome; podocyte; mesangial cell; glomerulogenesis
4.  Glomerular basement membrane composition and the filtration barrier 
The glomerular basement membrane (GBM) is an especially thick basement membrane that contributes importantly to the kidney’s filtration barrier. The GBM derives from the fusion of separate podocyte and endothelial cell basement membranes during glomerulogenesis and consists primarily of laminin-521 (α5β2γ1), collagen α3α4α5(IV), nidogens-1 and -2, and agrin. Of these nine proteins, mutations in the genes encoding four of them (LAMB2, COL4A3, COL4A4, and COL4A5) cause glomerular disease in humans as well as in mice. Furthermore, mutation of a fifth (Lama5) gene in podocytes in mice causes proteinuria, nephrotic syndrome, and progression to renal failure. These results highlight the importance of the GBM for establishing and maintaining a properly functioning glomerular filtration barrier.
doi:10.1007/s00467-011-1785-1
PMCID: PMC3108006  PMID: 21327778
Laminin; Collagen IV; Podocyte; Basement membrane; Nephrotic syndrome
5.  Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys 
The Journal of Cell Biology  1986;103(6):2489-2498.
Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti- rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti- laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.
PMCID: PMC2114591  PMID: 3782306
6.  Glomerular basement membrane and related glomerular disease 
The glomerular basement membrane (GBM) is lined by fenestrated endothelium from the capillary-lumen side and by interdigitating foot processes of the podocytes from the urinary-space side. These three layers of the glomerular capillary wall constitute the functional unit of the glomerular filtration barrier. The GBM is assembled through an interweaving of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations in genes encoding LAMB2, COL4A3, COL4A4, and COL4A5 cause glomerular disease in humans as well as in mice. In addition, laminin α5 mutation in podocytes leads to proteinuria and renal failure in mice. Moreover, more neoepitopes in Goodpasture’s disease and for the first time alloepitopes in Alport post-transplantation nephritis have been located in the collagen α5(IV) NC1 domain. These discoveries underscore the importance of the GBM in establishing and maintaining the integrity of the glomerular filtration barrier.
doi:10.1016/j.trsl.2012.03.004
PMCID: PMC3477400  PMID: 22683419
7.  Role of the Podocyte (and Glomerular Endothelium) in Building the GBM 
Seminars in nephrology  2012;32(4):342-349.
This article summarizes the basic cellular and extracellular events in development of the glomerulus and assembly of the glomerular basement membrane (GBM), paying special attention to laminin and type IV collagen. Cellular receptors for GBM proteins, including the integrins, dystroglycan, and discoidin domain receptor 1 (DDR1) are also discussed. Evidence is reviewed showing that the laminin isoform present in the earliest GBM, LM-111, and final isoform found in the mature GBM, LM-521, are each derived from both endothelial cells and podocytes. Although the early collagen α1α2α1(IV) similarly derives from endothelial cells and podocytes, collagen α3α4α5(IV) found in fully mature GBM is a product solely of podocytes. Genetic diseases affecting laminin and type IV collagen synthesis are also presented, with an emphasis on mutations to LAMB2 (Pierson syndrome) and COL4A3, COL4A4, and COL4A5 (Alport syndrome) and their experimental mouse models. Stress is placed on the assembly of a compositionally correct GBM for the acquisition and maintenance of glomerular barrier properties.
doi:10.1016/j.semnephrol.2012.06.005
PMCID: PMC3438517  PMID: 22958488
Laminin; Type IV collagen; Alport syndrome; fibrosis
8.  Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains 
The Journal of Cell Biology  1989;109(6):3477-3491.
To examine the ultrastructural distribution of laminin within kidney basement membranes, we prepared rat anti-mouse laminin mAbs to use in immunolocalization experiments. Epitope domains for these mAbs were established by immunoprecipitation, immunoblotting, affinity chromatography, and rotary shadow EM. One mAb bound to the laminin A and B chains on blots and was located to a site approximately 15 nm from the long arm-terminal globular domain as shown by rotary shadowing. Conjugates of this long arm-specific mAb were coupled to horseradish peroxidase (HRP) and intravenously injected into mice. Kidney cortices were fixed for microscopy 3 h after injection. HRP reaction product was localized irregularly within the renal glomerular basement membrane (GBM) and throughout mesangial matrices. In addition, this mAb bound in linear patterns specifically to the laminae rarae of basement membranes of Bowman's capsule and proximal tubule. This indicates the presence of the long arm immediately beneath epithelial cells in these sites. The laminae densae of these basement membranes were negative by this protocol. In contrast, the lamina rara and densa of distal tubular basement membranes (TBM) were both heavily labeled with this mAb. A different ultrastructural binding pattern was seen with eight other mAbs, including two that mapped to different sites on the short arms by rotary shadowing and five that blotted to a large pepsin-resistant laminin fragment (P1). These latter mAbs bound weakly or not at all to GBM but all bound throughout mesangial matrices. In contrast, discrete spots of HRP reaction product were seen across all layers of Bowman's capsule BM and proximal TBM. These same mAbs, however, bound densely across the full width of distal TBM. Our findings therefore show that separate strata of different basement membranes are variably immunoreactive to these laminin mAbs. The molecular orientation or integration of laminin into the three dimensional BM meshwork therefore varies with location. Alternatively, there may be a family of distinct laminin-like molecules distributed within basement membranes.
PMCID: PMC2115970  PMID: 2480964
9.  Proteinuria precedes podocyte abnormalities inLamb2–/– mice, implicating the glomerular basement membrane as an albumin barrier  
The Journal of Clinical Investigation  2006;116(8):2272-2279.
Primary defects in either podocytes or the glomerular basement membrane (GBM) cause proteinuria, a fact that complicates defining the barrier to albumin. Laminin β2 (LAMB2) is a GBM component required for proper functioning of the glomerular filtration barrier. To investigate the GBM’s role in glomerular filtration, we characterized GBM and overlying podocyte architecture in relation to development and progression of proteinuria in Lamb2–/– mice, which model Pierson syndrome, a rare congenital nephrotic syndrome. We found ectopic deposition of several laminins and mislocalization of anionic sites in the GBM, which together suggest that the Lamb2–/– GBM is severely disorganized, although it is ultrastructurally intact. Importantly, albuminuria was detectable shortly after birth and preceded podocyte foot process effacement and loss of slit diaphragms by at least 7 days. Expression and localization of slit diaphragm and foot process–associated proteins appeared normal at early stages. GBM permeability to the electron-dense tracer ferritin was dramatically elevated in Lamb2–/– mice, even before widespread foot process effacement. Increased ferritin permeability was not observed in nephrotic CD2-associated protein–null (Cd2ap–/–) mice, which have a primary podocyte defect. Together these data show that the GBM serves as a barrier to protein in vivo and that the glomerular slit diaphragm alone is not sufficient to prevent the passage of albumin into the urinary space.
doi:10.1172/JCI28414
PMCID: PMC1523402  PMID: 16886065
10.  The Glomerular Basement Membrane 
Experimental Cell Research  2012;318(9):973-978.
The kidney’s glomerular filtration barrier consists of two cells—podocytes and endothelial cells—and the glomerular basement membrane (GBM), a specialized extracellular matrix that lies between them. Like all basement membranes, the 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. These studies suggest that the GBM plays a crucial role in establishing and maintaining the glomerular filtration barrier.
doi:10.1016/j.yexcr.2012.02.031
PMCID: PMC3334451  PMID: 22410250
laminin; collagen IV; Alport syndrome; Pierson syndrome
11.  Proteinuria and structural alterations in rat glomerular basement membranes induced by intravenously injected anti-laminin immunoglobulin G 
Antibodies against laminin, which is a defined glycoprotein of basement membranes, were produced in sheep and affinity purified by immunoadsorption on laminin-Sepharose (S alpha L). When injected intravenously into rats, S alpha L rapidly bound in a linear pattern to the glomerular basement membrane (GBM) in the peripheral and mesangial regions of all glomeruli, and, when greater than 0.5 mg S alpha L was injected, to some tubular BM as well. 1-2 h after the injection of conjugates of horseradish peroxidase (HRP) and S alpha L, HRP reaction product was present throughout the full thickness of the GBM and mesangial matrix. [125I]S alpha L binding to the kidney in vivo increased linearly over the dose range of 40-950 micrograms of IgG and accounted for approximately 2% of the injected dose/g kidney. When 4 mg of [125I]S alpha L was injected, 1.5% or 62 micrograms/g kidney was bound. Proteinuria did not develop within 7 wk of injection in rats that received 0.5-1.6 mg of S alpha L. In contrast, all animals that received injections of 4 mg of S alpha L gradually became proteinuric within 3-6 wk. Thickening, reduplication, and flocculent subendothelial deposits were observed in the GBM of these animals. In addition, mononuclear cells adhered to the GBM and infiltrated beneath the endothelium. However, the deposition of rat C3 was infrequently observed, and rat IgG was not seen in the glomeruli of any rat that received S alpha L. 10 wk after injection, much greater amounts of S alpha L appeared within the mesangium than the peripheral GBM. These results demonstrate that the interaction of S alpha L with the GBM, possibly in concert with infiltrating mononuclear cells, gradually altered the structure and permeability characteristics of the glomerulus independent of a host anti-S alpha L humoral response.
PMCID: PMC2186722  PMID: 7086353
12.  Scanning electron microscopy of cellular and acellular glomeruli of male dogs affected with Samoyed hereditary glomerulopathy and a carrier female. 
Glomeruli isolated from three male dogs affected with Samoyed hereditary glomerulopathy were compared by scanning electron microscopy with glomeruli of one carrier female and six unaffected dogs. Scanning electron microscopy was performed before and after removal of podocytes and endothelial cells with enzyme and detergent, producing cellular and acellular glomeruli respectively. Cellular glomeruli of unaffected dogs showed podocytes with normally arranged foot processes, while in acellular glomeruli, the subepithelial surface of glomerular capillary basement membranes appeared smooth to finely granular. In contrast, cellular glomeruli of affected males showed microvilli, globular cytoplasmic projections from podocytes, and effacement of foot processes; acellular glomeruli demonstrated ridges and plaque-like irregularities on the subepithelial surface of glomerular capillary basement membranes. Changes in the glomeruli of the carrier female were intermediate between those of unaffected and affected male dogs. The appearance of the subepithelial surface of glomerular capillary basement membranes of acellular glomeruli seen by scanning electron microscopy correlated with the extent of multilaminar splitting of glomerular capillary basement membranes seen by transmission electron microscopy.
Images
PMCID: PMC1255368  PMID: 3453267
13.  A Hypomorphic Mutation in the Mouse Laminin α5 Gene (Lama5) Causes Polycystic Kidney Disease 
Extracellular matrix abnormalities have been found in both human and animal models of polycystic kidney disease (PKD). We have produced a new mouse PKD model through insertion of a PGKneo cassette in an intron of the gene encoding laminin α5, a major tubular and glomerular basement membrane component important for glomerulogenesis and ureteric bud branching. Lama5neo represents a hypomorphic allele due to aberrant splicing. Lama5neo/neo mice exhibit PKD, proteinuria, and death from renal failure by 4 weeks of age. This contrasts with mice totally lacking laminin α5, which die in utero with multiple developmental defects. At 2 days of age, Lama5neo/neo mice exhibited mild proteinuria and microscopic cystic transformation. By 2 weeks, cysts were grossly apparent in cortex and medulla, involving both nephron and collecting duct segments. Tubular basement membranes appeared to form normally, and early cyst basement membranes showed normal ultrastructure but developed marked thickening as cysts enlarged. Overall, laminin α5 protein levels were severely reduced due to mRNA frameshift caused by exon skipping. This was accompanied by aberrant accumulation of laminin-332 (α3β3γ2; formerly called laminin-5) in some cysts, as also observed in human PKD. This constitutes the first evidence that a primary defect in an extracellular matrix component can cause PKD.
doi:10.1681/ASN.2005121298
PMCID: PMC1482806  PMID: 16790509
BM, basement membrane; E, embryonic day; GBM, glomerular BM; P, postnatal day; PKD, polycystic kidney disease; PC1, polycystin-1
14.  Blood pressure influences end-stage renal disease of Cd151 knockout mice  
Podocytes of the kidney adhere tightly to the underlying glomerular basement membrane (GBM) in order to maintain a functional filtration barrier. The clinical importance of podocyte binding to the GBM via an integrin-laminin-actin axis has been illustrated in models with altered function of α3β1 integrin, integrin-linked kinase, laminin-521, and α-actinin 4. Here we expanded on the podocyte-GBM binding model by showing that the main podocyte adhesion receptor, integrin α3β1, interacts with the tetraspanin CD151 in situ in humans. Deletion of Cd151 in mouse glomerular epithelial cells led to reduced adhesive strength to laminin by redistributing α3β1 at the cell-matrix interface. Moreover, in vivo podocyte-specific deletion of Cd151 led to glomerular nephropathy. Although global Cd151-null B6 mice were not susceptible to renal disease, as has been shown previously, increasing blood and transcapillary filtration pressure induced nephropathy in these mice. Importantly, blocking the angiotensin-converting enzyme in renal disease–susceptible global Cd151-null FVB mice prolonged their median life span. Together, these results establish CD151 as a crucial modifier of integrin-mediated adhesion of podocytes to the GBM and show that blood pressure is an important factor in the initiation and progression of Cd151 knockout–induced nephropathy.
doi:10.1172/JCI58878
PMCID: PMC3248294  PMID: 22201679
15.  Drosophila laminin: characterization and localization 
The Journal of Cell Biology  1987;105(5):2383-2391.
Drosophila laminin was isolated from the medium of Drosophila Kc cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given. Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.
PMCID: PMC2114870  PMID: 3119602
16.  The Glomerular Basement Membrane as a Barrier to Albumin 
Nature reviews. Nephrology  2013;9(8):10.1038/nrneph.2013.109.
The glomerular basement membrane (GBM) is the central, non-cellular layer of the glomerular filtration barrier that is situated between the two cellular components – fenestrated endothelial cells and interdigitated podocyte foot processes. The GBM is composed primarily of four extracellular matrix macromolecules – laminin-521, type IV collagen α3α4α5, the heparan sulfate proteoglycan agrin, and nidogen – that produce an interwoven meshwork thought to impart both size- and charge-selective properties. Although the composition and biochemical nature of the GBM have been known for a long time, the functional importance of the GBM vs. podocytes and endothelial cells for establishing the glomerular filtration barrier to albumin is still debated. Together with mouse genetics studies, the discoveries of four human mutations in GBM components in two inherited kidney disorders, Alport syndrome and Pierson syndrome, support essential roles for the GBM in glomerular permselectivity. Here we explain in detail the proposed mechanisms whereby the GBM can serve as the major albumin barrier and discuss possible approaches to circumvent GBM defects associated with loss of permselectivity.
doi:10.1038/nrneph.2013.109
PMCID: PMC3839671  PMID: 23774818
17.  The glomerular basement membrane: not gone, just forgotten 
The Journal of Clinical Investigation  2006;116(8):2090-2093.
The glomerular capillaries function as the filtration barrier that retains albumin and other plasma proteins in the circulation. The unresolved question that has been asked for more than 50 years is, Which structural component of these capillaries constitutes the main molecular sieve that normally retains albumin and allows its passage in diseases associated with proteinuria? There is considerable evidence implicating both the glomerular basement membrane (GBM) and the epithelial filtration slits as the barrier. However, the prevailing point of view at present is that the slit diaphragms bridging the filtration slits are responsible for this important function, and evidence implicating the GBM is largely ignored or forgotten. In this issue of the JCI, Jarad et al. show that in laminin β2–deficient (Lamb2–/–) mice, proteinuria can be directly attributed to the altered composition of the GBM (see the related article beginning on page 2272). Changes in the permeability of the GBM and its organization were primary to changes in the epithelium, as they preceded foot process effacement and loss of slit diaphragms.
doi:10.1172/JCI29488
PMCID: PMC1523392  PMID: 16886057
18.  Development of kidney glomerular endothelial cells and their role in basement membrane assembly 
Organogenesis  2009;5(1):275-287.
Data showing that the embryonic day 12 (E12) mouse kidney contains its own pool of endothelial progenitor cells is presented. Mechanisms that regulate metanephric endothelial recruitment and differentiation, including the hypoxia-inducible transcription factors and vascular endothelial growth factor/vascular endothelial growth factor receptor signaling system, are also discussed. Finally, evidence that glomerular endothelial cells contribute importantly to assembly of the glomerular basement membrane (GBM), especially the laminin component, is reviewed. Together, this forum offers insights on blood vessel development in general, and formation of the glomerular capillary in particular, which inarguably is among the most unique vascular structures in the body.
PMCID: PMC2659369  PMID: 19568349
glomerular basement membrane; laminin; podocytes; type IV collagen; vascular endothelial growth factor
19.  Preservation of mesangium and immunohistochemically defined antigens in glomerular basement membrane isolated by detergent extraction. 
Journal of Clinical Investigation  1982;69(5):1169-1175.
To define the characteristics of isolated glomerular basement membrane (GBM), immunohistochemical and morphometric analyses have been carried out on rat and human tissues. Site-specific arrays of antigens were identified in detergent-isolated GBM in a distribution similar to that observed in intact kidney. In the human, fibronectin, procollagen IV, and collagen V were observed along the internal aspect of GBM continuous with antigenic sites in the mesangium. Another array of antigens was identified in the GBM but not within the mesangium--Goodpasture's antigen, bovine lens capsule type IV collagen, and amyloid P component. In addition, sites reactive with rabbit antiserum to laminin were present on both sides of the lamina densa as well as within the mesangial region. Actomyosin, a presumed mesangial cell antigen persisted in the mesangium of isolated GBM. Mesangial matrix was identified in detergent-isolated GBM in an amount equivalent to that present in intact glomeruli. Sonicated GBM contained the same antigens but it was not possible to quantitate the amount of mesangial material by immunofluorescence or morphometric analysis. The thickness of the lamina densa was greater in sonicated and detergent-treated rat GBM preparations than in native rat kidney. These studies demonstrated that isolated GBM is heterogeneous with respect to its antigenic constituents and in addition contains mesangial matrix, which is morphologically and immunohistochemically distinct from peripheral GBM.
Images
PMCID: PMC370182  PMID: 7040475
20.  Transcriptional induction of slit diaphragm genes by Lmx1b is required in podocyte differentiation 
The Journal of Clinical Investigation  2002;109(8):1065-1072.
LMX1B encodes a LIM-homeodomain transcription factor. Mutations in LMX1B cause nail-patella syndrome (NPS), an autosomal dominant disease with skeletal abnormalities, nail hypoplasia, and nephropathy. Expression of glomerular basement membrane (GBM) collagens is reduced in Lmx1b–/– mice, suggesting one basis for NPS nephropathy. Here, we show that Lmx1b–/– podocytes have reduced numbers of foot processes, are dysplastic, and lack typical slit diaphragms, indicating an arrest in development. Using antibodies to podocyte proteins important for podocyte function, we found that Lmx1b–/– podocytes express near-normal levels of nephrin, synaptopodin, ZO-1, α3 integrin, and GBM laminins. However, mRNA and protein levels for CD2AP and podocin were greatly reduced, suggesting a cooperative role for these molecules in foot process and slit diaphragm formation. We identified several LMX1B binding sites in the putative regulatory regions of both CD2AP and NPHS2 (podocin) and demonstrated that LMX1B binds to these sequences in vitro and can activate transcription through them in cotransfection assays. Thus, LMX1B regulates the expression of multiple podocyte genes critical for podocyte differentiation and function. Our results indicate that reduced levels of proteins associated with foot processes and the glomerular slit diaphragm likely contribute, along with reduced levels of GBM collagens, to the nephropathy associated with NPS.
doi:10.1172/JCI13954
PMCID: PMC150942  PMID: 11956244
21.  Fibronectin localization in the rat glomerulus 
The Journal of Cell Biology  1980;87(3):691-696.
Fibronectin (FN) has been localized in the rat glomerulus using indirect immunolabeling. It was demonstrated in frozen sections by immunofluorescence, in sections of fixed kidneys by both peroxidase and ferritin-labeled antibodies, and in isolated glomerular basement membranes (GBM) with ferritin-labeled antibodies. Complementary and convergent results were obtained with these approaches. FN was most abundant in the mesangial matrix where it was especially concentrated at the interface between the endothelial and mesangial cells. In the peripheral capillary loop, FN was also detected in the laminae rarae (interna and externa) of the GBM--i.e., between the endothelial and epithelial cells, respectively, and the GBM. These findings indicate that FN is an important constituent of the glomerulus, and they are compatible with the assumption that, in the glomerulus, as in cultured cells, FN is involved in cell-to-cell (mesangial-mesangial, mesangial- endothelial) and cell-to-substrate (mesangial cell-mesangial matrix, epithelium-GBM, endothelium-GBM) attachment.
PMCID: PMC2110796  PMID: 7007395
22.  Laminin Network Formation Studied by Reconstitution of Ternary Nodes in Solution* 
The Journal of Biological Chemistry  2012;287(53):44270-44277.
Background: Laminin self-assembly into a cell-associated network is essential for basement membrane formation.
Results: The isolated tips of the laminin short arms form ternary complexes in solution.
Conclusion: The nodes in the laminin network are formed by the N-terminal domains of one α, one β, and one γ chain.
Significance: The reconstitution of laminin network nodes enables structure-function studies.
The polymerization of laminins into a cell-associated network is a key process in basement membrane assembly. Network formation is mediated by the homologous short arm tips of the laminin heterotrimer, each consisting of a globular laminin N-terminal (LN) domain followed by a tandem of laminin-type epidermal growth factor-like (LEa) domains. How the short arms interact in the laminin network is unclear. Here, we have addressed this question by reconstituting laminin network nodes in solution and analyzing them by size exclusion chromatography and light scattering. Recombinant LN-LEa1–4 fragments of the laminin α1, α2, α5, β1, and γ1 chains were monomeric in solution. The β1 and γ1 fragments formed the only detectable binary complex and ternary complexes of 1:1:1 stoichiometry with all α chain fragments. Ternary complex formation required calcium and did not occur at 4 °C, like the polymerization of full-length laminins. Experiments with chimeric short arm fragments demonstrated that the LEa2–4 regions of the β1 and γ1 fragments are dispensable for ternary complex formation, and an engineered glycan in the β1 LEa1 domain was also tolerated. In contrast, mutation of Ser-68 in the β1 LN domain (corresponding to a Pierson syndrome mutation in the closely related β2 chain) abolished ternary complex formation. We conclude that authentic ternary nodes of the laminin network can be reconstituted for structure-function studies.
doi:10.1074/jbc.M112.418426
PMCID: PMC3531742  PMID: 23166322
Basal Lamina; Chromatography; Laminin; Protein Complexes; Protein Self-assembly; Recombinant Protein Expression; Surface Plasmon Resonance (SPR)
23.  Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis. 
Journal of Clinical Investigation  1997;99(10):2470-2478.
Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of alpha1(IV) and alpha2(IV) isoforms because they fail to developmentally switch their alpha-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich alpha3(IV), alpha4(IV), and alpha5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate.
PMCID: PMC508088  PMID: 9153291
24.  Nanoscale protein architecture of the kidney glomerular basement membrane 
eLife  2013;2:e01149.
In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM.
DOI: http://dx.doi.org/10.7554/eLife.01149.001
eLife Digest
The blood that flows through the body must be continually filtered to remove waste products and to ensure that it contains optimal levels of water and salts. Filtration is performed inside the kidneys by tufts of small blood vessels called glomeruli. These glomerular capillaries allow water and waste products to pass from the blood into the urine, while holding back proteins and blood cells. The wall of a glomerular capillary consists of two layers of cells flanking a third layer called the glomerular basement membrane. If any of these layers malfunctions, it becomes possible for proteins to pass into the urine. This is a clear sign of kidney disease.
The basement membrane is composed of proteins secreted by the two layers of cells, but little was known about how these proteins are organized. Now, Suleiman et al. have adapted a new form of high-resolution optical microscopy called STORM to study the structure of the glomerular basement membrane in both mouse and human kidney tissue. By combining data from STORM and electron microscopy, Suleiman et al. showed that the proteins in the glomerular basement membranes of both species are arranged similarly to form a distinctive layered structure. This suggests that the organization of the basement membrane plays a critical role in its function.
The technique was used to demonstrate that proteins were not organized in the glomerular basement membrane in tissue samples taken from mice suffering from Alport syndrome, a genetic disorder of the kidneys. In addition to suggesting that the disorganization of basement membranes may play an important role in disease, this work also provides a method for investigating the structure of the basement membrane in diverse types of tissue.
DOI: http://dx.doi.org/10.7554/eLife.01149.002
doi:10.7554/eLife.01149
PMCID: PMC3790497  PMID: 24137544
super-resolution microscopy; extracellular matrix; kidney; basement membrane; Human; Mouse
25.  Functional role of laminin α1 chain during cerebellum development 
Cell Adhesion & Migration  2011;5(6):480-489.
We had developed a conditional Laminin α1 knockout-mouse model (Lama1cko) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1cko mice. Our study of the postnatal cerebellum of Lama1cko animals revealed a disrupted basement membrane correlated with an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.
doi:10.4161/cam.5.6.19191
PMCID: PMC3277781  PMID: 22274713
cerebellum; development; laminin; cell migration; laminin-111

Results 1-25 (470572)