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.
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.
Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.
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.
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.
laminin; collagen IV; nephrotic syndrome; alport syndrome; podocyte; mesangial cell; glomerulogenesis
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.
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.
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.
super-resolution microscopy; extracellular matrix; kidney; basement membrane; Human; Mouse
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.
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.
It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.
Podocytes are specialized epithelial cells that cover the outer surfaces of glomerular capillaries. Unique cell junctions, known as slit diaphragms, which feature nephrin and Neph family proteins in addition to components of adherens, tight, and gap junctions, connect adjacent podocyte foot processes. Single gene disorders affecting the slit diaphragm result in nephrotic syndrome in humans, characterized by massive loss of protein across the capillary wall. In addition to specialized cell junctions, interconnecting podocytes also adhere to the glomerular basement membrane (GBM) of the capillary wall. The GBM is a dense network of secreted, extracellular matrix (ECM) components and contains tissue-restricted isoforms of collagen IV and laminin in addition to other structural proteins and ECM regulators such as proteases and growth factors. The specialized niche of the GBM provides a scaffold for endothelial cells and podocytes to support their unique functions and human genetic mutations in GBM components lead to renal failure, thus highlighting the importance of cell–matrix interactions in the glomerulus. Cells adhere to ECM via adhesion receptors, including integrins, syndecans, and dystroglycan and in particular the integrin heterodimer α3β1 is required to maintain barrier integrity. Therefore, the sophisticated function of glomerular filtration relies on podocyte adhesion both at cell junctions and at the interface with the ECM. In health, the podocyte coordinates signals from cell junctions and cell–matrix interactions, in response to environmental cues in order to regulate filtration and as our understanding of mechanisms that control cell adhesion in the glomerulus develops, then insight into the effects of disease will improve. The ultimate goal will be to develop targeted therapies to prevent or repair defects in the filtration barrier and to restore glomerular function.
podocyte; adhesion and signaling molecules; cell junction; extracellular matrix; nephrotic syndrome
Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1β1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5β2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newborn mouse GBMs. Our results show loss of laminin α1 from GBMs in early capillary loop stages and continuous linear deposition of laminin bearing the α5 chain thereafter. In contrast, collagen α1α2α1(IV) persisted in linear patterns into late capillary loop stages, when collagen α3α4α5(IV) first appeared in discontinuous, non-linear patterns. This patchy pattern for collagen α3α4α5(IV) continued into maturing glomeruli where there were lengths of linear, laminin α5-positive GBM entirely lacking either isoform of collagen IV. Relative abundance of laminin and collagen IV mRNAs in newborn and 5-week-old mouse kidneys also differed, with those encoding laminin α1, α5, β1, β2, and γ1, and collagen α1(IV) and α2(IV) chains all significantly declining at 5 weeks, but α3(IV) and α4(IV) were significantly upregulated. We conclude that different biosynthetic mechanisms control laminin and type IV collagen expression in developing glomeruli.
glomerular filtration barrier; glomerular endothelial cells; podocytes
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.
Laminin; Collagen IV; Podocyte; Basement membrane; Nephrotic syndrome
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.
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.
Basal Lamina; Chromatography; Laminin; Protein Complexes; Protein Self-assembly; Recombinant Protein Expression; Surface Plasmon Resonance (SPR)
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.
Laminin; Type IV collagen; Alport syndrome; fibrosis
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.
Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here.
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.
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.
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.
BM, basement membrane; E, embryonic day; GBM, glomerular BM; P, postnatal day; PKD, polycystic kidney disease; PC1, polycystin-1
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.
laminin; collagen IV; Alport syndrome; Pierson syndrome
Antibodies specific for the core protein of basement membrane HSPG (Mr = 130,000) were administered to rats by intravenous injection, and the pathologic consequences on the kidney were determined at 3 min to 2 mo postinjection. Controls were given antibodies against gp330 (the pathogenic antigen of Heymann nephritis) or normal rabbit IgG. The injected anti-HSPG(GBM) IgG disappeared rapidly (by 1 d) from the circulation. The urinary excretion of albumin increased in a dose- dependent manner during the first 4 d, was increased 10-fold at 1-2 mo, but remained moderate (mean = 12 mg/24 h). By immunofluorescence the anti-HSPG(GBM) was seen to bind rapidly (by 3 min) to all glomerular capillaries, and by immunoperoxidase staining the anti-HSPG was seen to bind exclusively to the laminae rarae of the GBM where it remained during the entire 2-mo observation period. C3 was detected in glomeruli immediately after the injection (3 min), where it bound exclusively to the lamina rara interna; the amount of C3 bound increased up to 2 h but decreased rapidly thereafter, and was not detectable after 4 d. Mononuclear and PMN leukocytes accumulated in glomerular capillaries, adhered to the capillary wall, and extended pseudopodia through the endothelial fenestrae to contact in the LRI of the GBM where the immune deposits and C3 were located. At 1 wk postinjection, staining for C3 reappeared in the glomeruli of some of the rats, and by this time most of the rats, including controls injected with normal rabbit IgG, had circulating anti-rabbit IgG (by ELISA) and linear deposits of rat IgG along the GBM (by immunofluorescence). Beginning at 9 d, there was progressive subepithelial thickening of the GBM which in some places was two to three times its normal width. This thickening was due to the laying down of a new layer of basement membrane-like material on the epithelial side of the GBM, which gradually displaced the old basement membrane layers toward the endothelium. The results show that the core proteins of this population of basement membrane HSPG (Mr = 130,000), which are ubiquitous components of basement membranes, are exposed to the circulation and can bind anti-HSPG(GBM) IgG in the laminae rarae of the GBM. Binding of these antibodies to the GBM leads to changes (C3 deposition, leukocyte adherence, moderate proteinuria, GBM thickening) considered typical of the acute phase of anti-GBM glomerulonephritis. Antibody binding interferes with the normal turnover of the GBM, presumably by affecting the biosynthesis and/or degradation of basement membrane components.
Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.
The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM.
In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg175, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5.
This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.