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.

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.

Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties.

We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain-specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three-armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophosphate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.

Human neutrophils do not degrade major basement membrane components during chemotactic migration.

At sites of inflammation, circulating neutrophils (PMNs) migrate through microvessel walls into the subendothelial interstitium. While endothelial passage is mediated by adhesion proteins, including those of the integrin, selectin and immunoglobulin superfamily classes, the mechanisms used to cross the subendothelial basement membrane (BM) are unclear. Studies examining tumour cell invasion and lymphocyte extravasation suggest several possible mechanisms, including proteolysis. Different cells, however, may use different mechanisms to effect passage. To examine neutrophil-basement membrane interactions in more detail, human PMNs were embedded within reconstituted BM (Matrigel) and used in migration assays. The integrity of the gel following migration was assessed by assaying for the release of incorporated radiolabelled products and by-immunoblotting for specific matrix molecule epitopes. PMNs migrated through Matrigel in response to the chemotactic peptide FMLP. Degradation products of laminin, heparan sulphate proteoglycan or of gelatin, however, were not detected. In contrast, phorbol ester, which triggers activation without migration, released approximately 40% of incorporated HSPG, 30% of gelatin and 20% of laminin as intact molecules or degraded fragments. Electron microscopy of migrating cells demonstrated pseudopodia associated with channels within the Matrigel. Although the serine proteinase inhibitor DFP, plasma and a specific anti-neutrophil elastase IgG blocked degradation, these agents failed to inhibit migration. Migration was inhibited, however, when the Matrigel concentration was increased to 10 mg/ml. Thus, although PMNs will degrade matrix components they do not do so during migration, and proteolytic remodelling of the BM is not a pre-requisite for neutrophil passage.

Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys.

Kidney glomerular basement membranes (GMBs) originate in development from fusion of a dual basement membrane between endothelial cells and primitive epithelial podocytes. After fusion, segments of newly synthesized matrix, derived primarily from podocytes, appear as subepithelial outpockets and are spliced into GBMs during glomerular capillary loop expansion. To investigate GBM assembly further, we examined newborn mouse kidneys with monoclonal rat anti-mouse laminin IgGs (MAb) conjugated to horseradish peroxidase (HRP). In adults, these MAb strongly label glomerular mesangial matrices but bind only weakly or not at all to mature GBMs. In contrast, anti-laminin MAb intensely bound newborn mouse GBMs undergoing initial assembly. After intraperitoneal injection of MAb-HRP into neonates, dense binding occurred across both subendothelial and subepithelial pre-fusion GMBs as well as forming mesangial matrices. Considerably less MAb binding was seen, however, in post-fusion GBMs from more mature glomeruli in the same section, although mesangial matrices remained positive. In addition, new subepithelial segments in areas of splicing were negative. These results conflict with those obtained previously with injections of polyclonal anti-laminin IgGs into newborns or adults, which result in complete labeling of all GBMs. Although epitope masking cannot be completely excluded, we believe that decreased MAb binding to developing GBM reflects actual epitope loss. This loss could occur by laminin isoform substitution, conformational change, and/or proteolytic processing during GBM assembly.

Basement membrane proteoglycans in glomerular morphogenesis: chondroitin sulfate proteoglycan is temporally and spatially restricted during development.

We previously reported the presence of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG) in basement membranes of almost all adult tissues. However, an exception to this ubiquitous distribution was found in the kidney, where BM-CSPG was absent from the glomerular capillary basement membrane (GBM) but present in other basement membranes of the nephron, including collecting ducts, tubules, Bowman's capsule, and the glomerular mesangium. In light of this unique pattern of distribution and of the complex histoarchitectural reorganization occurring during nephrogenesis, the present study used light and electron microscopic immunohistochemistry to examine the distribution of BM-CSPG and basement membrane heparan sulfate proteoglycan (BM-HSPG) during prenatal and postnatal renal development in the rat. Our results show that the temporal and spatial pattern of expression of BM-CSPG during nephrogenesis is unlike that reported for other basement membrane components such as laminin, fibronectin, and BM-HSPG, all of which can be found in the earliest formed basement membranes of the vesicle-stage nephron. Although BM-CSPG is present in the basement membranes of the invading vasculature and ureteric buds, its first appearance in nephron basement membrane occurs during the late comma stage. In capillary loop-stage glomeruli of prenatal animals, BM-CSPG is present in the presumptive mesangial matrix but undetectable in the GBM. However, as postnatal glomerular maturation progresses BM-CSPG is also found in both the lamina rara interna and lamina densa of the GBM in progressively increasing amounts, being most evident in the GBM of 21-day-old animals. Micrographs of glomeruli from 42-day-old animals show that BM-CSPG gradually disappears from the GBM and, by 56 days after birth, appears to be completely absent from the GBM, its pattern of distribution resembling that of the adult animal. Our results show that BM-CSPG is not required for the initial assembly of basement membranes but may in fact serve to stabilize basement membrane structure after histoarchitectural reorganization is completed.

Binding of injected laminin to developing kidney glomerular mesangial matrices and basement membranes in vivo.

During glomerular development, subendothelial and -epithelial basement membrane layers fuse to produce the glomerular basement membrane (GBM) shared by endothelial cells and epithelial podocytes. As glomeruli mature, additional basement membrane derived from podocytes is spliced into the fused GBM and loose mesangial matrices condense. The mechanisms for GBM fusion, splicing, and mesangial matrix condensation are not known but might involve intermolecular bond formation between matrix molecules. To test for laminin binding sites, we intravenously injected mouse laminin containing alpha1-, beta1-, and gamma1-chains into 2-day-old rats. Kidneys were immunolabeled for fluorescence and electron microscopy with domain-specific rat anti-mouse laminin monoclonal antibodies (MAbs), which recognized only mouse and not endogenous rat laminin. Intense labeling for injected laminin was found in mesangial matrices and weaker labeling was seen in GBMs of maturing glomeruli. These patterns persisted for at least 2 weeks after injection. In control newborns receiving sheep IgG, no binding of injected protein was observed and laminin did not bind adult rat glomeruli. To assess which molecular domains might mediate binding to immature glomeruli, three proteolytic laminin fragments were affinity-isolated by MAbs and injected into newborns. These failed to bind glomeruli, presumably owing to enzymatic digestion of binding domains. Alternatively, stable incorporation may require multivalent laminin binding. We conclude that laminin binding sites are transiently present in developing glomeruli and may be functionally important for GBM assembly and mesangial matrix condensation.

Basement membrane-specific chondroitin sulfate proteoglycan is abnormally associated with the glomerular capillary basement membrane of diabetic rats.

We have previously reported the production of monoclonal antibodies (MAb) recognizing the core protein of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG). Using immunohistochemical techniques, we have shown that BM-CSPG is present in almost every basement membrane, one exception being the normal glomerular capillary basement membrane (GBM), where it is absent. In the present study of mature kidneys we examined the distribution of BM-CSPG in streptozocin-induced diabetes mellitus in rats. We found BM-CSPG atypically associated with the GBM of diabetic animals as early as 1 month after induction of diabetes mellitus. Immunoelectron microscopy (IEM) of affected capillary loops showed BM-CSPG present in the subendothelial matrix in areas of GBM thickening and absent in areas where the GBM appears to be of normal thickness. Moreover, the association of BM-CSPG with regions of the pericapillary GBM affects the morphology of the capillary endothelial cells within these areas, directly displacing the cell body from the GBM proper and causing loss of fenestrae. These new data on BM-CSPG distribution reflect abnormal glomerular extracellular matrix protein biosynthesis/turnover in diabetes and suggest that BM-CSPG in the GBM might in turn affect normal capillary structure and/or function.

Ultrastructure of developing kidney glomerular basement membranes: temporal changes in binding of anti-laminin IgG and cationized ferritin.

In vivo labeling of infant rat and mouse glomerular basement membranes (GBMs) with polyclonal anti-laminin IgGs results in binding across the full widths of GBMs at all stages of development. These stages include the pre-fusion, double basement membranes found beneath endothelial cells and podocytes in early glomeruli, and the subepithelial matrix outpockets where newly synthesized GBM is spliced into fused basement membrane during glomerular maturation. Identical binding results are obtained either with peroxidase or post-embedding immunogold techniques. Although injected cationized ferritin also binds abundantly to all developing GBMs, it quickly disappears and, 24 hours after injection, is generally absent from GBMs but remains within mesangial matrices. Injection of newborn mice with monoclonal anti-laminin IgGs results in dense labeling of pre-fusion GBMs but post-fusion GBMs and subepithelial outpockets are weak-negative. Although masking can not be excluded, these results indicate that laminin epitopes are removed during GBM fusion and splicing, either by isoform substitution or proteolytic processing. The loss of bound cationized ferritin is believed to occur mainly through rapid turnover of GBM proteoglycans.

Origins and formation of microvasculature in the developing kidney.

Regulation of microvessel assembly in the developing kidney is not known and may occur through vasculogenic, angiogenic, or both processes. To examine this question, we grafted rat and mice embryonic (E) day 12 (E12) kidneys, which have only a rudimentary vasculature, into anterior eye chambers of mouse and rat hosts. Species-specific, monoclonal anti-basement membrane antibodies showed that glomerular basement membranes, mesangial matrices, and microvessel basement membranes were always derived from the graft. When wild-type E12 mouse kidneys were grafted into anterior chambers of ROSA26 mice, in which the beta-galactosidase transgene is expressed ubiquitously, glomerular and microvascular endothelial cells stemmed from the graft, even after maintenance of kidneys in organ culture for 6 days before grafting. Immunolabeling with antibodies against the vascular endothelial growth factor (VEGF) receptor, Flk1, the EphB1 receptor, and its ligand, ephrin-B1, labeled discrete mesenchymal cells in embryonic and newborn kidney cortex, as well as developing microvessel and glomerular endothelium. In adult kidneys, Flk1 labeled glomeruli weakly, other vascular structures were unlabeled. When wild-type E12 kidneys were grafted under renal capsules of adult ROSA26 hosts, endothelium developing within the graft again came from the implanted kidney. In contrast, when E12 kidneys were grafted into renal cortices of newborns, glomeruli within grafts now contained host-derived endothelium. Similarly, when ROSA26 E12 kidneys were implanted into newborn wild-type hosts, chimeric vessels containing graft- and host-derived endothelium were seen in nearby host tissue. Our results indicate that cells capable of forming the entire microvascular tree of grafted metanephroi are already present in the E12 kidney. We hypothesize that Flk1/VEGF and EphB1/ephrin-B1 mediate renal endothelial mitosis-motility and cell guidance-aggregation behavior, respectively.

ELK and LERK-2 in developing kidney and microvascular endothelial assembly.

Eph family receptor tyrosine kinases direct neuronal cell targeting, bundling and intercellular aggregation activity, yet their role in mammalian kidney development has been unexplored to date. We recently identified expression of ELK (Eph-like kinase) receptors in cultured human renal microvascular endothelial cells (HRMEC), and showed that ELK mediates their in vitro assembly into capillary-like structures in response to the exogenous ligand, LERK-2. Here we identify expression of the ELK ligand, LERK-2, in HRMEC and in primitive vascular structures of developing murine kidney. ELK and LERK-2 are expressed on endothelial progenitor cells of primitive microvasculature in a pattern similar to that of the VEGF receptor, flk-1. ELK LERK-2 and flk-1 antigens are also displayed on the branching ureteric bud epithelium; ELK and LERK-2 expression persists in mature collecting ducts, glomeruli and arterioles. To explore whether renal-derived endothelial cells may distinguish LERK-2 from the angiogenic Eck ligand, LERK-1 (B61), and whether endothelial cells from different sources may distinguish among Eph receptor ligands, we compared HRMEC and human umbilical vein endothelial cell (HUVEC) responses in an in vitro capillary-like assembly assay. HRMEC endothelial cells assembled capillary-like structures in response to LERK-2, but not LERK-1, under conditions that promoted HUVEC to assemble in response to LERK-1, but not LERK-2. Therefore, responses mediated through specific Eph family receptors (ELK and Eck) are discriminated by endothelial cells from different vascular bed sources. ELK and its ligand, LERK-2, are spatially and temporally coordinated in expression and may function in morphogenesis of the renal microvasculature.

Laminin distribution in developing glomerular basement membranes.

The renal glomerular basement membrane (GMB) separates two distinctly different cell layers: the vascular endothelium, and visceral epithelial podocytes. When initial vascularization of the forming glomerulus takes place during nephrogenesis, the early GBM forms by fusion of a dual basement membrane between endothelial cells and podocytes. As glomerular capillary loops blossom, newly synthesized basement membrane segments derived from podocytes are then inserted or spliced into the fused GBM. The molecular processes accounting for either basement membrane fusion or splicing are unresolved. Using monoclonal anti-mouse laminin antibodies (mAbs) against the end of the laminin long arm (5D3), we have shown in adult mice that peripheral loop GBM is only weakly immunoreactive but the mesangial matrix and tubular basement membrane (TBM) is intensely positive. In contrast, mAbs against domains in the center of the laminin cross only label TBMs and mesangial matrices of mature mice and GBMs are negative. Immunofluorescence microscopy of neonatal mouse kidneys showed, however, that anti-laminin mAbs brightly labeled developing GBMs of glomeruli undergoing initial vascularization and capillary loop formation. Post-fusion GBMs of maturing stage glomeruli became unreactive for most anti-laminin mAbs but remained positive for 5D3. Our results therefore show that some GBM laminin epitopes are transiently expressed during glomerular development. These changes in GBM immunoreactivities may reflect proteolytic processing during basement membrane fusion and splicing, or temporally controlled synthesis of different laminin isoforms.

Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mutant mice.

Flk1, a receptor tyrosine kinase for vascular endothelial growth factor (VEGF), is the earliest known marker for endothelial precursors (angioblasts). We examined heterozygous mice in which the Flk1 gene was partially replaced by a promoter-less LacZ insert and used beta-galactosidase histochemistry to view cells transcribing Flk1. In day 10 (E10) embryos, a Flk1-positive network surrounded the metanephric blastema, and, at E11, a vessel entered the metanephros from its ventral aspect alongside the ingrowing ureteric bud. However, aortic branches did not engage embryonic kidneys at these time points. In newborns, beta-galactosidase was localized exclusively and intensely to endothelial cells of all vessels and glomeruli. In contrast, when E12 kidneys grown in organ culture for 6 days were examined, only scattered Flk1-positive cells were seen, glomeruli were unlabeled, and vessels were absent. When organ-cultured kidneys were then grafted into wild-type anterior eye chambers, numerous Flk1-positive endothelial cells in vessels and glomeruli were found, all stemming from the graft. Image analysis showed that grafts with the most abundant glomerulo- and tubulogenesis were also those with the richest expression of Flk1. We conclude that 1) kidney microvessels precede renal artery development, 2) angioblast differentiation is arrested in organ culture but released on grafting when vasculogenesis resumes, and 3) nephrogenesis and microvessel assembly are tightly coupled in vivo.

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

BACKGROUND: The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. METHODS: To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. RESULTS: Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. CONCLUSIONS: Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.

Cyclosporine-induced nephrotoxicity in deoxycorticosterone-NaCl treated rats.

This study tested the hypothesis that the adverse effects of cyclosporine (Cy) are accelerated in animals with induced hypertension. Four groups of rats were unilaterally nephrectomized at 5 weeks of age. Two weeks later, two of these groups received implantations of Silastic strips impregnated with deoxycorticosterone acetate (DOCA) and were maintained on 1% saline (DOCA-NaCl); the other two groups served as sham controls. Daily injections of Cy (20 mg/kg) were given to one DOCA-NaCl and one control group. During the initial 3 days, the Cy/DOCA-NaCl treated rats displayed a significant increase in systolic arterial pressure (SAP), but their SAP did not increase significantly thereafter. Cy/DOCA-NaCl treatment caused severe nephrotoxicity 18 days after initiation of treatment, but neither cyclosporine nor DOCA-NaCl treatment alone resulted in morphological renal damage. In Cy/DOCA-NaCl rats, the interstitial spaces between renal tubules were dramatically increased in size and contained abundant bundles of collagenous fibres, deposits of immunoreactive laminin, and infiltrates of mononuclear cells. In a second experiment, bilateral renal denervation prior to treatment did not lessen the Cy-induced renal damage. These results indicate that in the DOCA-NaCl rat, Cy treatment damages the renal cortex in a pattern similar to that observed in humans treated chronically with Cy. Further, the results indicate that an absence of renal innervation does not decrease the nephrotoxic effects of Cy in this rapid onset model.

Glomerular development in intraocular and intrarenal grafts of fetal kidneys.

To examine nephrogenesis in allogeneic grafts, we implanted fetal (day 14 postconception) rat kidneys, which contained only immature tubules, and day 15 to 17 postconception kidneys (with immature tubules and developing glomeruli) into the anterior eye chamber or under the renal capsule of mature rat hosts. Within 9 to 10 days, every graft became richly vascularized, new nephrons were induced to form, and extensive glomerular and tubular cytodifferentiation occurred in all implants. Electron microscopy showed well-formed maturing-stage glomeruli containing erythrocytes, fenestrated endothelial cells, mesangial cells, and fully developed podocytes with foot processes and slit diaphragms. Tubules morphologically corresponding to proximal tubules and containing epithelial cells with tall apical microvilli, as well as distal tubular segments, were also observed. Intravenous injections of antilaminin IgG into hosts resulted in labeling of glomerular basement membranes in grafted kidneys, confirming perfusion of the grafts by host vasculature. Some ultrastructural abnormalities were observed in the grafted kidneys, including the persistence of unfused basement membranes in capillary walls of some maturing-stage glomeruli, and signs of graft rejection were obvious in most samples by 16 days after implantation. Despite these irregularities, however, the ability to establish allogeneic grafts of developing kidneys presents an opportunity to explore glomerular development in a variety of genetic and physiological backgrounds.

Permeability of the macula densa basement membrane area to high molecular weight molecules.

To investigate the permeability properties of the basement membrane beneath macula densa cells and between extraglomerular mesangial cells, thick ascending limbs with attached glomeruli were dissected from rabbit kidney and incubated in a Ringers solution containing either horseradish peroxidase (HRP) (1 mg/ml; molecular weight approximately 40,000) or native or cationic ferritin (both at 5 mg/ml molecular weight approximately 450,000) for 5 and 20 min at room temperature. Tubules were processed for electron microscopy. At both time points, HRP reaction product fully permeated the matrix material between extraglomerular mesangial cells, the basement membrane underneath the macula densa, and also was occasionally located in intercellular spaces and intracellular vesicles within macula densa cells. Similar results were obtained with native and cationic ferritin. In separate experiments, thick ascending limbs with attached glomeruli were perfused for 20 min at room temperature with a Ringer solution containing HRP (1 mg/ml). HRP was found in tubulovesicular bodies within the apical cytoplasm of macula densa cells again indicating that these cells exhibit endocytotic activity. However, HRP did not gain access to intercellular spaces indicating that the apical junctional complex was impermeable to HRP. These results demonstrate that the macula densa basement membrane and matrix material between extraglomerular mesangial cells is permeable to high molecular weight molecules and suggest unhindered diffusion of water and solutes within this area.

Evidence that embryonic kidney cells expressing flk-1 are intrinsic, vasculogenic angioblasts.

Renal glomerular capillary tufts have been believed to arise from angiogenic ingrowth of extrinsic vessels. We found, however, that when embryonic day 12 (E12) mouse kidneys were maintained in culture for 6 days and then grafted into anterior eye chambers of adult transgenic ROSA26 host mice (which carry the beta-galactosidase transgene), glomerular endothelial cells within the grafts were predominantly of intrinsic, kidney origin. To identify potential endothelial precursors, we immunolabled kidneys with antibodies against the vascular endothelial growth factor receptor, flk-1. Numerous discrete cells expressing flk-1 were scattered throughout the nephrogenic mesenchyme of both E12 and newborn kidneys, and with development these cells became concentrated in microvessels, glomerular vascular clefts, and glomerular tufts. In adults, flk-1 was weakly expressed in glomeruli but absent elsewhere. To examine abilities of flk-1-positive cells to establish glomeruli, E12 kidneys were grafted into kidney cortices of adult and newborn ROSA26 hosts. Grafts into adults resulted in few glomeruli containing host-derived endothelium, whereas a majority of glomeruli grafted into newborns contained host cells. Cells of graft origin were found in vessels forming in renal cortices of newborn hosts, but not in adults. Our findings indicate that embryonic kidney cells expressing flk-1 are angioblasts that create microvessels and glomeruli by vasculogenesis.

Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney.

BACKGROUND: Glomerular epithelial protein 1 (GLEPP1) is a receptor-like membrane protein tyrosine phosphatase (RPTP) with a large ectodomain consisting of multiple fibronectin type III repeats, a single transmembrane segment, and a single cytoplasmic phosphatase active site sequence. In adult human and rabbit kidneys, GLEPP1 is found exclusively on apical membranes of podocytes and especially on surfaces of foot processes. Although neither ligand nor function for this protein is known, other RPTPs with similar topologies have been implicated in mediating adherence behavior of cells. METHODS: To evaluate potential roles of GLEPP1 further, we cloned the full-length mouse GLEPP1 cDNA and examined its expression patterns in developing kidney by Northern blot analysis, in situ hybridization, and immunofluorescence microscopy. RESULTS: Nucleotide sequencing showed that mouse GLEPP1 was approximately 80% identical to rabbit and human GLEPP1 and approximately 91% identical at the amino acid level. The membrane-spanning and phosphatase domains of mouse GLEPP1 shared> 99% homology with PTPphi, a murine macrophage cytoplasmic phosphatase. Northern analysis identified a single GLEPP1 transcript of approximately 5.5 kb in fetal kidney that became approximately threefold more abundant in adults. In situ hybridization of newborn mouse kidney revealed GLEPP1 mRNA in visceral epithelial cells (developing podocytes) of comma- and S-shaped nephric figures, and expression increased in capillary loop and maturing stage glomeruli. Beginning on embryonic day 14, GLEPP1 protein was first observed on cuboidal podocytes of capillary loop stage glomeruli, but nascent podocytes of earlier comma- and S-shaped nephric figures were negative. At later stages of glomerular maturation, where foot process elongation and interdigitation occurs, GLEPP1 immunolabeling intensified on podocytes and then persisted at high levels in fully developed glomeruli. CONCLUSION: Our findings are consistent with a role for GLEPP1 in mediating and maintaining podocyte differentiation specifically.

Carboxy terminal sequence and synthesis of rat kidney laminin gamma 1 chain.

We used antibodies against mouse Englebreth-Holm-Swarm (EHS) tumor laminin to screen a newborn rat kidney lambda gt11 expression library and isolated three overlapping cDNA clones, termed 2b-11 (401 bp), 10-b7 (779 bp), and 2a (2,095 bp). DNA sequence analysis identified these cDNAs as encoding much of the carboxy terminal domain I/II of laminin gamma 1 chain (formerly referred to as B2e), and 1436 bp of the 3' untranslated region. In situ hybridization of fetal (E15) rat sections localized laminin gamma 1 chain mRNA primarily to meninges of the brain, auditory and peripheral nerve fibers, gastrointestinal system, and developing lung airway epithelium. Intense hybridization was also found in early nephric structures and glomeruli of fetal kidneys. In kidneys of three-day-old rats, hybridization persisted over early nephric figures, developing glomeruli, and collecting ducts, but considerably less hybridization was seen over tubules. On Northern blots of neonatal kidney RNA, the three cDNA clones hybridized to two species of 7.5 and 5.5 kb, suggesting that developing rat kidney laminin gamma 1 mRNAs are processed using two different polyadenylation signals.

Extracellular matrix (mesoglea) of Hydra vulgaris III. Formation and function during morphogenesis of hydra cell aggregates.

Hydra, as a member of the phylum Cnidaria, is characterized by a body lining organized as an epithelial bilayer with an intervening extracellular matrix (ECM) termed the mesoglea. Previous studies have established that the mesoglea has components indicative of mammalian ECM such as type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan, and these components appear to play a critical role in hydra head regeneration. A remarkable feature of hydra is its ability to reorganize into its adult structure within 96 hr to 7 days from pellets formed from dissociated hydra cells. This regenerative model has been termed the hydra cell aggregate system. The present study has been designed to characterize the biogenesis of mesoglea in hydra cell aggregates and to determine its role in morphogenesis of aggregates. We find that hydra cell aggregates first form an epithelial bilayer by 12 hr of development and then subsequently develop a mesoglea. Morphogenesis of hydra structure then follows formation of the mesoglea. Immunofluorescence studies indicate that mesoglea components are first deposited between the epithelial bilayer by about 12-17 hr of pellet formation, and pulse-labeling studies indicate that the translation rate of matrix components peaks by 48-72 hr of development. Ultrastructural studies indicate that a mature mesoglea is formed by 48-96 hr of pellet formation. Drugs such as beta-aminoproprionitrile and 2,2'-dipydridyl, which interfere with the cross-linking of collagens, and p-nitrophenyl-beta-D-xylopyranoside, which interferes with the addition of GAG moieties to proteoglycan core molecules, were found to reversibly block development of hydra cell aggregates. Transmission electron microscopy studies indicate that these drugs affect the ultrastructure of the mesoglea. In addition, both polyclonal and monoclonal antibodies raised to isolated mesoglea were found to block development of hydra cell aggregates. These studies indicate that (1) mesoglea formation is rapid and precedes morphogenetic processes during aggregate development, and (2) formation of mesoglea is essential for normal morphogenesis of hydra cell aggregates.