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.

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys.

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.

Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine.

Fc receptors on the luminal membranes of intestinal epithelial cells in the neonatal rat mediate the vesicular transfer of functionally intact IgG from the intestinal lumen to the circulation. In addition, there is a low level of nonselective protein uptake, but in this case transfer does not occur. To determine whether a specialized class of endocytic vesicles could account for the selective transfer of IgG, mixtures of IgG conjugated to ferritin (IgG-Ft) and unconjugated horseradish peroxidase (HRP) were injected together into the proximal intestine of 10-d-old rats, and the cellular distribution of these two different tracers was determined by electron microscopy. Virtually all apical endocytic vesicles contained both tracers, indicating simultaneous uptake of both proteins within the same vesicle. However, only IgG-Ft bound to the apical plasma membrane, appeared within coated vesicles at the lateral cell surface, and was released from cells. HRP did not bind to the luminal membrane and was not transferred across cells but was confined to apical lysosomes as identified by acid phosphatase and aryl sulfatase activities. To test the possibility that the binding of IgG to its receptor stimulated endocytosis, HRP was used as a fluid volume tracer, and the amount of HRP taken up by cells in the presence and absence of IgG was measured morphologically and biochemically. The results demonstrate that endocytosis in these cells is constitutive and occurs at the same level in the absence of IgG. The evidence presented indicates that the principal selective mechanism for IgG transfer is the binding of IgG to its receptor during endocytosis. Continued binding to vesicle membranes appears to be required for successful transfer because unbound proteins are removed from the transport pathway before exocytosis. These results favor the proposal that IgG is transferred across cells as an IgG-receptor complex.

Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys.

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.

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.

Expression and polarized targeting of a rab3 isoform in epithelial cells.

Pathways of polarized membrane traffic in epithelial tissues serve a variety of functions, including the generation of epithelial polarity and the regulation of vectorial transport. We have identified a candidate regulator of polarized membrane traffic in epithelial cells (i.e., rab3B), which is a member of the rab family of membrane traffic regulators. Rab3B is highly homologous to a brain-specific rab3 isoform (rab3A) that targets in a polarized fashion to the presynaptic nerve terminal, where it probably regulates exocytosis. The coding region for human rab3B was cloned from epithelial mRNA using a reverse-transcription polymerase chain reaction strategy. This cDNA clone hybridized to a single mRNA species in Northern blots of poly(A)+ RNA isolated from epithelial cell lines. A rab3B-specific antibody that was raised against recombinant fusion protein recognized a 25-kD band in immunoblots of cell lysates prepared from cultured epithelial cells (e.g., T84 and HT29-CL19A), but not from a variety of nonepithelial cells (e.g., PC12 neuroendocrine cells). Immunofluorescence analysis confirmed that rab3B protein is preferentially expressed in cultured epithelial cells as well as in a number of native epithelial tissues, including liver, small intestine, colon, and distal nephron. Rab3B localized to the apical pole very near the tight junctions between adjacent epithelial cells within all of these cell lines and native epithelial tissues, as determined by immunofluorescence and immunoelectron microscopic analysis. Moreover, this pattern of intracellular targeting was regulated by cell contact; namely, rab3B was reversibly retrieved from the cell periphery as epithelial cell contact was inhibited by reducing the extracellular Ca2+ concentration. Our results indicate that neurons and epithelial cells express homologous rab3 isoforms that target in a polarized fashion within their respective tissues. The pattern and regulation of rab3B targeting in epithelial cells implicates this monomeric GTPase as a candidate regulator of apical and/or junctional protein traffic in epithelial tissues.

Intestinal absorption of immune complexes by neonatal rats: a route of antigen transfer from mother to young.

Horseradish peroxidase (HRP) in the presence of specific immunoglobulin G antibody to HRP is selectively absorbed from the gut lumen and transferred by intestinal epithelial cells to the lamina propria in newborn rats. The HRP is not transferred in detectable amounts in the absence of the antibody. Transport of maternally derived antigen via antigen-antibody complexes may have important influences on the developing immune system in young mammals.

Epithelial basement membrane of mouse jejunum. Evidence for laminin turnover along the entire crypt-villus axis.

Little is known regarding turnover of the epithelial basement membrane in adult small intestine. Are components degraded and inserted along the length of the crypt-villus axis or selectively in the crypt region with subsequent migration of basement membrane from crypt to villus tip in concert with epithelium? We injected affinity-purified sheep anti-laminin IgG or sheep anti-laminin IgG complexed to horseradish peroxidase (HRP) into mice to label basement membrane laminin in vivo. Fluorescence microscopy revealed linear fluorescence along the length of the jejunal epithelial basement membrane 1 d after anti-laminin IgG injection. By 1 wk, small nonfluorescent domains were interposed between larger fluorescent domains. Over the next 5 wk the lengths of nonfluorescent domains increased progressively whereas those of fluorescent domains decreased. Additionally, electron microscopy revealed HRP reaction product along the length of the epithelial basement membrane after 1 d whereas unlabeled or lightly labeled domains that increased in length with time were observed interposed between heavily labeled domains by 2 and 4 wk along the entire crypt-villus axis. We conclude that laminin turnover occurs focally in the epithelial basement membrane of mouse jejunum along the crypt-villus axis over a period of weeks and that this basement membrane does not comigrate in concert with its overlying epithelium.

Adult tissue angiogenesis: evidence for negative regulation by estrogen in the uterus.

Increased uterine vascular permeability and angiogenesis are two major events of embryo implantation and placentation during pregnancy. These latter processes require coordinated, uterine-specific interactions between progesterone (P4) and estrogen (E) signaling. Although roles of these steroids have long been suspected, definitive functions of E and/or P4 in uterine angiogenesis still remain elusive. We have therefore exploited the availability of reporter and mutant mice to explore the regulation of angiogenesis in response to steroid hormonal changes in vivo. We present here molecular, genetic, physiological, and pharmacological evidence that E and P4 have different effects in vivo: E promotes uterine vascular permeability but profoundly inhibits angiogenesis, whereas P4 stimulates angiogenesis with little effect on vascular permeability. These effects of E and P4 are mediated by differential spatiotemporal expression of proangiogenic factors in the uterus.

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.

Epitope mapping of the laminin molecule in murine skin basement membrane zone: demonstration of spatial differences in ultrastructural localization.

Results of studies performed to date with polyclonal antilaminin antibodies have been conflicting as to the ultrastructural localization of this glycoprotein in skin basement membrane zone (BMZ). Whereas initial reports suggested its presence solely within the lamina lucida (LL), others have suggested that laminin is instead an exclusive component of the lamina densa (LD). In an attempt to more critically address this issue, we have examined both intact and partially separated (via 1 M NaCl) murine skin BMZ by indirect immunoelectron microscopy via a two-step immunoperoxidase technique on unfixed cryopreserved tissue, utilizing nine well-characterized monoclonal antibodies with binding specificity for laminin. Localization of the sites of the epitopes recognized by these antibodies on isolated laminin molecules was previously determined by rotary shadowing and by biochemical analyses on enzymatic fragments of laminin. Whereas at least faint immunoreactants were detected in both regions with eight of nine antibodies, predominant staining was noted within the LL with three of eight and within (and even sparsely below) the LD in three of eight. One antibody bound solely to the LL; another bound equally within both regions. Although some overlap was noted, it appears that the epitope on the distal portion of the long arm of the laminin molecule resides primarily within the skin LD, whereas epitopes on more central portions of the short arms are present within the LL or within both LL and LD. The findings of stratification of laminin epitopes within skin BMZ supports a similar recent observation in mouse kidney and suggests that portions of the laminin molecule span both LD and LL, and that there may be a non-random spatial orientation for the laminin molecule within murine skin BMZ.

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.

Post-embedding colloidal gold immunolocalization of laminin to the lamina rara interna, lamina densa, and lamina rara externa of renal glomerular basement membranes.

Ultrastructural distribution of laminin within renal glomerular (GBM) and tubular basement membranes (TBM) was investigated using post-embedding immunolocalization with colloidal gold. Rat kidneys were fixed with 4% formaldehyde and embedded at 4 degrees C in Lowicryl K4M medium. Thin sections were then sequentially treated with affinity-purified rabbit anti-laminin IgG and anti-rabbit IgG conjugated to 10 nm diameter colloidal gold. Gold bound specifically to the GBM and TBM with particle densities of 690/micron2 and 731/micron2, respectively. In the GBM, the number of gold particles bound/micron2 of lamina densa greater than lamina rara externa greater than lamina rara interna. Closely similar binding patterns were found when kidneys were fixed with 0.5% glutaraldehyde plus 3% formaldehyde and embedded at 60 degrees C in L.R. White resin, but slightly less gold bound to sections overall than that seen with formaldehyde alone and Lowicryl. Taken together, these results illustrate that anti-laminin IgG, whether applied to fixed sections in vitro or introduced in vivo, bound to the lamina rara interna, lamina densa, and lamina rara externa of the GBM and throughout the TBM.

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.

Origin of the glomerular vasculature in the developing kidney.

Mechanisms regulating the establishment of the glomerular endothelium and mesangium during glomerulogenesis are not understood. In this article, we discuss two different blood vessel growth processes: vasculogenesis and angiogenesis, with particular attention on how these processes might operate in the developing kidney. Results from metanephric organ cultures and interspecies grafts are also interpreted with an emphasis on generation of the renal microvasculature. Among the several growth factors identified in the metanephros, many (eg, fibroblast growth factor [FGF], vascular endothelial growth factor [VEGF], platelet-derived growth factor [PDGF], epidermal growth factor/transforming growth factor-alpha [EGF/TGF-alpha], hepatocyte growth factor/scatter factor [HGF/SF], and insulinlike growth factor [IGF]) have angiogenic properties, and these are discussed in relation to formation of the glomerulus. How extracellular matrices and proteases might be involved in vascularization are also considered.

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.

Diverse aspects of metanephric development.

Mammalian nephrogenesis constitutes a series of complex developmental processes in which there is a differentiation and rapid proliferation of pluripotent cells leading to the formation of a defined sculpted tissue mass, and this is followed by a continuum of cell replication and terminal differentiation. Metanephrogenesis ensues with the intercalation of epithelial ureteric bud into loosely organized metanephric mesenchyme. Such an interaction is reciprocal, such that the intercalating ureteric bud induces the conversion of metanephric mesenchyme into an epithelial phenotype, while the mesenchyme stimulates the iterations of the ureteric bud. The induced mesenchyme then undergoes a series of developmental stages to form a mature glomerulus and tubular segments of the kidney. Coincidental with the formation of these nephric elements, the developing kidney is vascularized by the process of vasculogenesis and angiogenesis. Thus, the process of metanephric development is quite complex, and it involves a diverse group of molecules who's biological activities are inter-linked with one another and they regulate, in a concerted manner, the differentiation and maturation of the mammalian kidney. This diverse group of molecules include extracellular matrix (ECM) proteins and their receptors, ECM-degrading enzymes and their inhibitors, growth factors and their receptors, proto-oncogenes and transcription factors. A large body of literature data are available, which suggest a critical role of these molecules in metanephric development, and this review summarizes the recent developments that relate to metanephrogenesis.