Biomedical subjects

D R Abrahamson

Publications and source records attributed to D R Abrahamson.

At least 19 recordsLinked to original sources

PubMed1997

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.

Animals↗

PubMed1997

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.

Basement Membrane↗

PubMed1997

Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules.

We have characterized the Na+/H+ exchanger (NHE) isoforms expressed in rat renal cortical tubule fragments. Amiloride sensitivity of the Na(+)-dependent intracellular pH (pHi) recovery in suspended tubules that had been acid loaded by an NH4+ prepulse was determined in nominally CO2/HCO3(-)-free solution, using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In the presence of 140 mM extracellular Na+, 800 microM amiloride inhibited the rate of Na(+)-dependent pHi recovery by only 65%, demonstrating the presence of a Na(+)-dependent amiloride-insensitive H+ extrusion system. This system was not affected by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Lowering extracellular Na+ concentration permitted 300 microM amiloride to completely inhibit Na(+)-dependent pHi recovery. These results can be explained by the expression of a Na+/H+ exchange with the pharmacological properties of NHE4. Using reverse transcriptase-polymerase chain reaction, we found specific mRNA for NHE1, NHE2, NHE3, and NHE4 isoforms in the renal cortex. Immunohistochemical studies using polyclonal antibodies against rat NHE4 peptide demonstrated that NHE4 is heterogeneously expressed on basolateral membrane domains of cortical tubules. These results strongly suggest that amiloride-insensitive Na+/H+ exchange expressed in renal cortical tubule suspensions is mediated by NHE4.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Diabetes induces changes in glomerular development and laminin-beta 2 (s-laminin) expression.

Offspring of diabetic mothers have developmental renal abnormalities; thus, we investigated the effects of the diabetic milieu on kidney development. Four groups of host rats, including insulin-deficient and insulin-treated streptozotocin-induced diabetic rats, normal controls, and insulin-treated nondiabetic rats, were prepared. After 38 days, rats received ocular implants of E14 fetal rat kidneys. Nine days later the fetal kidney grafts were harvested for analysis of glomerular development and expression of fibronectin, laminin, laminin-beta 2, and alpha-smooth muscle actin and m170, two additional markers of mesangial maturation. The rate of glomerular maturation was delayed in grafts placed in hyperglycemic, insulin-deficient diabetic rats. These glomeruli contained few mesangial cells or matrix, and laminin-beta 2 expression was reduced as compared with controls. Mesangial expression of alpha-smooth muscle actin and m170 was not detected. In contrast, grafts placed in insulin-treated diabetic animals had increased numbers of mesangial cells and expanded mesangial matrix. The content of laminin-beta 2 and expression of m170 and alpha-smooth muscle actin were also increased in these grafts. These data show that hyperglycemia and insulin status influence laminin isoform expression and play important roles in mesangial development.

Actins↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys.

To address origins of glomerular endothelial and mesangial cells in embryonic mammalian kidneys, we established interspecies grafts between rats and mice, in which fetal kidneys were implanted into the anterior eye chamber of adult hosts. After 5-7 days, hosts bearing grafts received intravenous injections with species-specific monoclonal antibodies (MAbs) to matrix components. In all cases, glomerular basement membranes and mesangial matrices labeled solely for donor-derived matrix. Additionally, microvessel extracellular matrices within grafts were usually of donor origin. To examine directly the origin of glomerular endothelial and mesangial cells, we grafted embryonic gestational days 11-12 (E11-12) kidneys from normal mice into anterior eye chambers of host reverse-orientation splice acceptor 26 mice, which are transgenic animals that express beta-galactosidase in every cell. When grafts were developed for beta-galactosidase activity, host cells were seen in peripheral vessels, but the majority of glomerular endothelial cells were of donor, not host, origin. Where host-derived-endothelial cells were found in glomeruli, donor endothelial cells were present as well. Mesangial cells were always of donor origin. When E11 mouse kidneys were labeled with the endothelial cell-specific Bandeiraea simplicifolia isolectin B4, we determined that endothelial cells are present from the inception of metanephrogenesis. Together, the evidence shows that cells of endogenous kidney origin were almost entirely responsible for development of the glomerular microvasculature in oculo. External vessels from the host, although important for graft maintenance, were not major contributors to the glomerulus.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Effect of ethanol on the synthesis and secretion of apoA-I- and apoB-containing lipoproteins in HepG2 cells.

The short- and long-term effects of ethanol on the production of lipids and apolipoproteins (apo) in HepG2 cells were studied. Short-term incubation with 1% ethanol caused a significant 32% increase in the cellular content of both triglycerides and cholesteryl esters. Under these conditions, the net accumulation in the medium of triglycerides, unesterified cholesterol, cholesteryl esters, apoA-I, and apoE was stimulated by 75%, 41%, 43%, 19%, and 39%, respectively. ApoA-I and apoE mRNA levels increased by 15%. The major short-term effect of ethanol was on the net accumulation of apoB in the medium which was stimulated by 56-100% in the presence of 0.1-1.0% ethanol. Under these conditions, apoB mRNA abundance was elevated by 17-26% and LDL receptor activity was unchanged. The increase in apoB accumulation in the medium was predominantly due to augmented secretion of newly synthesized apoB-100 which was evident at 0.05% ethanol. The secretion of newly synthesized apoA-I was not altered by short-term incubation with < or = 0.5% ethanol. The rate of apoB production was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. Addition of Pfizer CP-113,818, an inhibitor of acyl-CoA:cholesterol acyltransferase, caused a 69% reduction in the secretion of cholesteryl esters and a 24% decrease in that of apoB-100. In contrast to the short-term effect of ethanol, long-term incubation with ethanol resulted in a dose-dependent increase in the secretion of newly synthesized apoA-I without significantly affecting that of apoB-100. The increase in apoA-I secretion was evident at 0.05% ethanol and reached a maximum of 77% at 0.5% ethanol. These observations indicate that in HepG2 cells the effect of ethanol on the production of apoA-I- and apoB- containing lipoproteins is both time- and dose-dependent and is different in these two apolipoproteins.

Apolipoprotein A-I↗

PubMed1996

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.

Animals↗

PubMed1995

Qualitative alterations in laminin expression in experimental lupus nephritis.

Previous studies have revealed quantitative alterations in laminin-1 expression at the mRNA and protein levels during the development of glomerulonephritis and glomerulosclerosis in chronic graft-versus-host disease in mice, a model for lupus nephritis. We have now studied the qualitative alterations in laminin expression with two monoclonal antibodies that recognize epitopes on either the E8 or the P1 fragment of laminin-1. Both of these fragments are involved in cell-matrix and matrix-matrix interactions. In normal glomeruli these laminin epitopes are present only in the mesangial matrix; during embryogenesis, however, they are also present in the glomerular basement membrane. The distribution of laminin epitopes was first studied by using immunofluorescence in kidneys of mice with graft-versus-host disease at different points in time after disease induction. Reflection contrast and immunoelectron microscopy were performed after in vivo injection of the horseradish peroxidase-coupled monoclonal antibodies. In glomeruli of mice 8 weeks after disease induction, both injected antibodies bound specifically in electron-dense immune deposits in the mesangium and subepithelially along the glomerular basement membrane as well as in the expanded mesangial matrix. At 11 and 12 weeks after disease induction, when focal and segmental glomerulosclerosis had developed, the antibodies additionally bound in the matrix subendothelially along the glomerular basement membrane and at the periphery of end-stage sclerotic lesions. To study changes in the distribution of laminin epitopes over time, mice were injected with either monoclonal antibody before induction of graft-versus-host disease. The antibodies were detected 8 and 12 weeks later in the mesangial matrix of mice with lupus nephritis. Once segmental glomerulosclerosis had developed, the antibodies were additionally detected within the thickened glomerular capillary wall. The specific binding of anti-laminin monoclonal antibodies in electron-dense immune deposits further substantiates the hypothesis that anti-laminin autoantibodies participate in glomerular immune complex formation in this model, as suggested by earlier studies. Furthermore, our results show that the distribution of glomerular laminin epitopes in the matrix is altered during the development of glomerular disease. These changes in the structure of the glomerular basement membrane may contribute to the abnormal cell-matrix and matrix-matrix interactions during the development of glomerular disease in this model for lupus nephritis.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

Developmental expression of the nephritogenic antigen of monoclonal antibody 5-1-6.

The biogenesis of p51, the target of nephritogenic monoclonal antibody 5-1-6, was studied in the developing glomerulus by immunolocalization and metabolic labeling. The localization of p51 was compared with that of ZO-1, a component of the cytoplasmic face of the epithelial slit diaphragm, and with that of podocalyxin, and apical marker of the podocyte. p51 first became faintly, but clearly, detectable on the basal and lateral sides of the developing podocytes at the S-shaped body stage. Staining intensity increased with further maturation and was restricted to the visceral epithelial cells. On immunoelectron microscopy, the antigen was seen along the basal and lateral surfaces below occluding junction at the early capillary loop stage and later, with the interdigitation of foot processes, became concentrated in the slit pores. At no stage was p51 seen on the apical surface. p51 and ZO-1 were closely localized in the mature glomerulus but arrived at their final positions from opposite directions. p51 was on basal and podocalyxin was on apical sides of the glomerular epithelium from the S-shaped body stage onwards. Metabolic labeling studies showed that p51 is actively synthesized during initial glomerular development and that the rate of synthesis declines substantially with maturation. We conclude that p51 is primarily synthesized during the initial glomerular development, becomes concentrated in the slit pores of mature podocytes, and serves as a basal differentiation marker for podocytes.

Aging↗

PubMed1994

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.

Animals↗

PubMed1994

Effects of laminin monoclonal antibodies on the development of cultured rat embryos.

Whole rat embryos (9.5 days of gestation) were exposed to six different monoclonal antibodies to laminin during 48 hr of culture. Four (LAM I, LAM V, 5A2, 9D2) of the six were teratogenic or lethal and two (LAM II, 5D3) were not toxic at comparable levels. Teratogenicity and lethality were not related to antibody level, subclass or affinity for whole laminin. Indirect immunofluorescence studies using mouse embryo sections revealed that the toxic antibodies bound in a diffuse manner, while the nontoxic antibodies showed distinct labeling of tissues. These observations suggest that previous varied responses seen in cultured rat embryos exposed to laminin antibodies obtained from humans, monkeys, and rats were the result of differences in the epitope specificity of those antibodies.

Amino Acid Sequence↗

PubMed1994

Cloning and biological function of laminin in Hydra vulgaris.

The Cnidarian, hydra, lends itself to studies related to the role of extracellular matrix (ECM) components in development because of its high regenerative capacity and its simple structure, which is organized as an epithelial bilayer with an intervening ECM termed the mesoglea. Previous immunocytochemical and biochemical studies have established that hydra mesoglea contains many of the major matrix components (e.g., fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan) associated with the ECM of vertebrate and more complex invertebrate species. Additional studies have also established that ECM components have a critical role in hydra development as monitored during head regeneration and morphogenesis of hydra cell aggregates. In the present study a monoclonal antibody (mAb52) raised to isolated hydra mesoglea was used as a probe in additional functional studies and to screen a cDNA expression library made from poly(A)+ RNA isolated from Hydra vulgaris. Immunofluorescent analysis indicated that mAb52 was localized along the entire longitudinal axis of adult polyps in what is termed the subepithelial zones of hydra mesoglea. Cytochemical studies found these subepithelial zones to be rich in anionic sites. Previous studies have shown that mAb52 blocks hydra cell aggregate development and experiments in the current study have shown that mAb52 also blocks in vivo interstitial cell (I-cell) migration in hydra grafts. Sequence analysis of cDNA clones isolated using mAb52 indicated that the protein encoded by these clones had structural homology with mammalian and Drosophila laminin B1 chain and hybridized to a single 6.75-kb band on Northern blots of total hydra RNA. One interesting difference in hydra laminin B1 was the presence of a FTGTQ amino acid sequence in place of the vertebrate YIGSR cell binding domain. Under nonreducing conditions, polyclonal antibodies against FTGTQ bound to the same > 200-kDa band on Western blots of mesoglea as mAb52 and also immunolocalized to the subepithelial zones. Under reducing conditions, anti-FTGTQ antibodies bound to a single band with a mass of approximately 200 kDa. In addition, FTGTQ peptide inhibited adhesion of dissociated hydra cells to mesoglea and anti-FTGTQ antibodies inhibited hydra cell binding to substrates coated with mesoglea or FTGTQ peptide. Anti-FTGTQ antibodies also inhibited in vivo I-cell migration in hydra grafts. Given the early divergence of Cnidarians during evolution, these studies indicate the highly conserved nature of laminin and provide additional information regarding the critical role of ECM components during hydra development.

Amino Acid Sequence↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1993

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.

Adult↗