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D J Salant

Publications and source records attributed to D J Salant.

At least 19 recordsLinked to original sources
PubMed1992

Quantification of glomerular epithelial cell adhesion by using anti-DNA antibodies in ELISA.

A sensitive and reproducible microassay is described for quantification of adhesion of cells to matrix-coated 96-wells plates under different experimental conditions. For this purpose glomerular visceral epithelial cells (GVEC) were used. Attached GVEC were fixed with methanol and incubated with a monoclonal anti-DNA antibody. Following standard procedures, the amount of bound antibody was quantified by ELISA. A positive linear relationship in the range of 800-5000 cells per well was found between OD values and cell numbers obtained by hand-counting (r = 0.94, p less than 0.001). The assay is 10 to 100 times more sensitive than most other adhesion assays. The applicability of the ELISA assay was demonstrated by manipulation of the temperature during adhesion and by using different concentrations of the matrix-molecules fibronectin, EHS-laminin and collagen type I. The ELISA assay was found to be unaffected by non-specific interaction of anti-DNA antibodies with the matrix molecules used for coating. The assay was neither affected by potential release of DNA from the GVEC under these different experimental conditions. In conclusion, this cell adhesion microassay is simple, reliable, sensitive, and cost-effective, since it requires small amounts of GVEC and reagents.

Animals↗
PubMed1992

Management of glomerular diseases of primary and secondary origin.

Most recent information on the management of glomerular diseases is clustered in three areas. In nephrotic syndrome, interest has focused on the use of cyclosporine in steroid-resistant patients, treatment of progressive membranous nephropathy with alkylating agents, and symptomatic management of unresponsive cases with angiotensin-converting enzyme inhibitors. The most recent data on lupus nephritis establish the efficacy of intravenous cyclophosphamide in the long-term preservation of renal function and the lack of benefit of plasmapheresis in patients with severe disease. In rapidly progressive glomerulonephritis, the discovery of circulating antibodies to neutrophil cytoplasmic antigens has proved valuable in diagnosing certain forms of renal vasculitis. A rational approach to treating such patients is beginning to crystallize.

Adjuvants, Immunologic↗
PubMed1992

Production and polarized secretion of basement membrane components by glomerular epithelial cells.

To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme-linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side.

Animals↗
PubMed1992

Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells.

A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.

Animals↗
PubMed1991

Effect of nephritogenic antibody on complement regulation in cultured rat glomerular epithelial cells.

In passive Heymann nephritis, a rat model of membranous nephropathy, antibody (anti-Fx1A) activates C on the surface of the glomerular epithelial cell (GEC), leading to GEC injury and proteinuria. In this study, we examined C activation by anti-Fx1A in cultured rat GEC. In addition to anti-Fx1A IgG, anti-Fx1A F(ab')2 and Fab' led to GEC injury in the presence of rat or human sera as sources of C. Cytotoxicity was Mg2+ and factor B dependent, but Ca2+ independent, indicating that anti-Fx1A activated the C alternative pathway (AP). Furthermore, in the presence of Mg2+ and factor B, anti-Fx1A enhanced 125I-C3b deposition on GEC in the absence of classical pathway activation. AP C3 and C5 convertases formed on GEC (GEC-C3bBbP) were inactivated over time, probably due to binding of GEC C regulatory proteins. This inactivation was prevented when GEC-C3bBbP were incubated with anti-Fx1A IgG. An antibody raised against cultured GEC that binds to GEC in vitro and in vivo had no effect on C3 and C5 convertases, suggesting that stabilization of C3bBbP is unique to anti-Fx1A. Anti-Fx1A Fab' also stabilized GEC-C3bBbP, indicating that cross-linking of membrane Ag was not required. C3bBbP on E were not affected by anti-Fx1A, excluding direct stabilization of convertases by anti-Fx1A. Therefore, anti-Fx1A inhibits C regulation on GEC, which can account for its ability to activate the AP. This represents a potentially powerful mechanism of producing disease in vivo.

Animals↗
PubMed1991

Glomerular basement membrane expansion in passive Heymann nephritis. Absence of increased synthesis of type IV collagen, laminin, or fibronectin.

The distribution and synthetic rate of glomerular basement membrane components was examined in the Passive Heymann Nephritis model of experimental membranous nephropathy. The extensive tissue injury that developed included subepithelial electron-dense deposits, podocyte foot process effacement, and expansion of the glomerular basement membrane. Levels of mRNA for type IV collagen, laminin, and fibronectin from isolated glomeruli was quantitated by slot-blot analysis and showed no change in experimental animals as compared to controls at either 1 week, 3 weeks, or 3 months after disease induction. Immunoelectron microscopy with gold-labeled anti-laminin IgG revealed no difference in the number of particles bound to the glomerular basement membrane of experimental animals and controls. Immunofluorescence with both type IV collagen antisera and anti-laminin antibody showed no difference in the intensity or pattern of staining. Despite extensive glomerular damage and glomerular basement membrane thickening, no evidence was found for either an increase in the synthetic rate of type IV collagen, laminin, or fibronectin or for an accumulation of basement membrane laminin within the damaged glomeruli. Alternate processes, such as diminished density of matrix components or accumulation of other unmeasured matrix constituents, presumably account for the expansion of the glomerular basement membrane seen in experimental membranous nephropathy.

Animals↗
PubMed1990

Cytosolic calcium and protein kinase C reduce complement-mediated glomerular epithelial injury.

In rat membranous nephropathy, protein-uria is due to formation of the C5b-9 membrane attack complex of complement (C), and is associated with morphological evidence of glomerular epithelial cell (GEC) injury. Analogous morphological changes are induced by C5b-9 in cultured GEC. In addition, in cultured GEC C5b-9 induces Ca2+ influx, as well as Ca2+ mobilization and increased 1,2-diacylglycerol due to the activation of phospholipase C. In this study we investigated how this GEC activation pattern might influence C-mediated GEC injury. We demonstrate that the C5b-9-induced increase in cytosolic Ca2+ concentration ([Ca2+]i) did not impair ATP generation by mitochondria, suggesting that it does not contribute to cytotoxicity. Moreover, this increase in [Ca2+]i protected GEC from C-mediated cytolysis. However, a large increase in [Ca2+]i (produced by the Ca2+ ionophore A23187) impaired ATP generation and aggravated C-mediated cytotoxicity, suggesting that intact mitochondrial activity is necessary for GEC to withstand C attack. Activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) also decreased C-mediated cytolysis. Conversely, C lysis was enhanced in GEC that had been pretreated for 18 hours with a high dose of PMA to deplete PKC, and following PKC inhibition with H-7. Therefore, PKC activation, possibly resulting from C5b-9-induced increase in 1,2-diacylglycerol, triggered mechanisms that protected GEC from C-mediated injury. Thus, as a consequence of C5b-9-induced phospholipase activation, the amount of C-induced GEC injury is diminished.

Adenosine Triphosphate↗
PubMed1990

Extracellular matrix regulates proliferation and phospholipid turnover in glomerular epithelial cells.

To understand how glomerular epithelial cell (GEC) growth might be regulated in health and disease, we studied the effects of growth factors and extracellular matrix on proliferation and membrane phospholipid turnover in cultured rat GECs. In GECs adherent to type I collagen matrix, epidermal growth factor (EGF), insulin, and serum stimulated DNA synthesis and increased cell number. In addition, GECs proliferated when adherent to type IV collagen, but not to laminin or plastic substrata. Attachment of GECs to the substrata that facilitated proliferation (types I or IV collagen) produced increases in 1,2-diacylglycerol (DAG), an activator of protein kinase C (PKC). Increased DAG was associated with hydrolysis of inositol phospholipids and an increase in inositol trisphosphate and was not dependent on the presence of growth factors. After PKC downregulation (by preincubation with a high dose of phorbol myristate acetate), DNA synthesis was enhanced in GECs adherent to collagen. Thus contact of GECs with collagen matrices is required for serum, EGF, or insulin to induce proliferation. Collagen matrix also activates phospholipase C. As a result, the DAG-PKC signaling pathway desensitizes GECs to the mitogenic effects of growth factors and might promote cell differentiation. Understanding the interaction between GECs, growth factors, and extracellular matrix may elucidate the mechanisms of proliferation during glomerular injury.

Animals↗
PubMed1990

Loss and rearrangement of glomerular basement membrane laminin during acute nephrotoxic nephritis in the rat.

Many earlier studies have shown that the intravenous injection into rats of sheep antibodies against rat glomerular basement membrane (GBM) induces a rapid influx of neutrophils and proteinuria (nephrotoxic nephritis or NTN). The GBM antigens recognized by nephrotoxic antibodies (NTAbs) have not been identified conclusively. Our experiments presented here, however, showed that NTAbs did not significantly reduce binding of anti-laminin IgGs to laminin-coated enzyme-linked immunosorbent assay (ELISA) plates or to the GBM in vivo, indicating little cross-reactivity between the NTAbs and laminin. To evaluate possible changes in GBM architecture during acute stages of NTN, the ultrastructural distribution of laminin was determined by postfixation, postembedding immunogold labeling, and compared between normal and nephritic rats. The density of immunoreactive GBM laminin was significantly reduced in rats with acute NTN. In addition, conjugates of anti-laminin IgG and horseradish peroxidase were intravenously injected into rats that then received injections of NTAbs. Anti-laminin peroxidase conjugates were also injected after administering NTAbs. In both cases, an overall decrease in anti-laminin peroxidase reaction product was observed as compared to normal controls. The densest labeling was seen in the lamina rara interna, especially in areas of endothelial cell detachment. Some immunoperoxidase reaction product was also bound to basal surfaces of detaching endothelial cells, demonstrating the removal of at least some laminin from the GBM. A decrease in GBM binding of intravenously injected anti-laminin IgG, both before and after injection of rats with NTAbs, was also confirmed by postembedding immunogold labeling. Furthermore, morphometry showed that the GBM was significantly wider in nephritic rats than in controls, indicating a redistribution of laminin over a greatly increased area. These immunoultrastructural findings show, therefore, that GBM architecture is altered in the early phase of NTN.

Acute Disease↗
PubMed1989

Decay accelerating factor regulates complement activation on glomerular epithelial cells.

Epithelial cells of the glomerular capillary are the site of C5b-9 mediated injury in rat membranous nephropathy. We investigated the regulation of C activation by cultured glomerular epithelial cells (GEC). Rat and human GEC were more resistant to C injury by homologous C than heterologous C. In human GEC homologous C cytotoxicity was enhanced by antiserum to decay accelerating factor (DAF) indicating that homologous C activation was, at least in part, restricted by membrane DAF. Anti-DAF immunoprecipitated a 67-kDa protein from human glomeruli. In rat GEC, pronase and phosphatidylinositol-specific phospholipase C (which are known to inactivate human DAF) enhanced cytotoxicity by homologous C. Thus, DAF is present on human GEC in culture and in human kidney glomeruli, and a DAF-like protein is present on cultured rat GEC. These proteins regulate C activation in vitro and may play a role in controlling C activation on GEC in vivo.

Animals↗
PubMed1989

Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.

In rat membranous nephropathy, formation of the C5b-9 membrane attack complex (MAC) leads to proteinuria in association with glomerular visceral epithelial cell (GEC) injury. These alterations in GEC function and morphology might result from changes in intracellular free Ca2+ concentration [( Ca2+]i) and activation of phospholipases. We demonstrate that in cultured rat GEC, antibody-directed formation of noncytolytic amounts of the MAC induced a rapid and sustained increase in [Ca2+]i that was partly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The MAC elevated levels of inositol bis- (IP2) and trisphosphate (IP3), as well as 1,2-diacylglycerol (DAG) and phosphatidic acid (PA). In permeabilized GEC, IP3 released Ca2+ from intracellular stores. Cellular 45Ca2+ uptake was also increased by the MAC. Thus, in GEC, the MAC induced Ca2+ mobilization from intracellular stores secondary to activation of phospholipase C and production of IP3, as well as enhanced Ca2+ influx. In addition, C5b-9 stimulated release of arachidonic acid (AA), prostaglandin F2 alpha, and thromboxane A2. Indomethacin partially inhibited the increase in DAG levels observed with the MAC, whereas the prostaglandin H2/thromboxane A2 analogue U46619 elevated DAG, suggesting that an eicosanoid product of MAC-induced AA release may enhance the activation of phospholipase C. Activation of phospholipases by the MAC may lead to altered GEC function and thereby contribute to the pathophysiological changes that characterize complement-dependent rat membranous nephropathy.

Animals↗
PubMed1989

Studies with antibodies to cultured rat glomerular epithelial cells. Subepithelial immune deposit formation after in vivo injection.

To investigate the role of glomerular epithelial cell (GEC) membrane proteins in the in situ formation of subepithelial immune deposits, the authors raised a rabbit antiserum against GEC that had been grown in culture (anti-GEC). By indirect immunofluorescence (IF) on normal rat kidney, anti-GEC stained proximal tubular brush border (BB). After intravenous injection into animals, granular glomerular capillary wall staining for IgG was present by IE and subepithelial immune deposits were identified by standard transmission and immunoelectron microscopy. Using the latter technique, injected anti-GEC IgG was identified beneath slit diaphragms and in endocytic-coated pits and intracellular vesicles of podocytes. Anti-GEC immunoprecipitated gp330 and two other proteins from radiolabeled BB. These proteins also were identified by sheep anti-rat Fx1A, the antiserum responsible for passive Heymann nephritis. Anti-GEC and anti-Fx1A also immunoprecipitated five identical proteins from surface-labeled GEC. Biosynthetically-labeled but not surface-labeled GEC contained immunoprecipitable gp330. Thus, injection into rats of antibodies raised against cultured GEC can produce subepithelial immune deposits, a disease process classically induced by antibodies to BB or its purified components. In addition to gp330, GEC and BB share other antigenic determinants that may contribute to the formation of these immune deposits.

Animals↗
PubMed1988

Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.

Glomerular injury in passive Heymann nephritis (PHN) in rats is mediated by the C5b-9 membrane attack complex (MAC) and is associated with morphologic changes in glomerular visceral epithelial cells (GEC). We determined if the nephritogenic antibody of PHN (gamma 1 sheep anti-Fx1A IgG) directs insertion of the MAC into GEC plasma membranes with consequent cytotoxicity. Antibody-sensitized GEC were exposed to various sera serving as sources of complement. Loss of cell viability was determined by trypan blue uptake and/or by release of cellular lactate dehydrogenase (LDH). Incubation of antibody-sensitized primary and passaged GEC in fresh human serum (FHS) resulted in sigmoidal relationships between cytotoxicity and complement dose (r = 0.97 and 0.94, respectively) such that cytolysis approached 100% with FHS (10% vol/vol). Cytotoxicity was not evident if C8-deficient (C8D) plasma was substituted for FHS, but was restored in a dose-dependent manner by reconstitution with purified rat C8. Sublytic injury was demonstrated by wide separation between simultaneous release curves of cell-incorporated biscarboxyethyl carboxyfluorescein (BCECF; mol wt approximately equal to 520) and LDH at limiting doses of complement (at 2% FHS, BCECF release was 51.1 +/- 0.6% of maximum vs. 3.2 +/- 1.3% for LDH; N = 3) and by blebbing of the plasma membrane on electron microscopy. Thus, the pathogenic antibody of PHN produces complement-mediated sublytic as well as lytic cytotoxicity of GEC.

Animals↗
PubMed1988

Effect of diet, age and sex on the renal response to immune injury in the rat.

We investigated the effect of three factors, namely dietary protein intake, age and sex, on the susceptibility of the renal glomerulus to the binding of antiglomerular basement membrane antibody (anti-GBM) in the early (heterologous) phase of anti-GBM nephritis, and the consequent reduction in glomerular filtration rate (GFR) as measured by inulin clearance (CIn). The effect of diet was examined in approximately equal to 8 week-old female Munich-Wistar rats fed a 40% high (HP) or a 6% low (LP) protein diet, and that of sex and age in male and female rats, 6 week or 10 month old. Following an intravenous dose (3 to 20 micrograms/g body wt) of radiolabeled nephritogenic anti-GBM, assessment of glomerular function was followed by quantitation of anti-GBM binding (values corrected for GBM surface area) in isolated glomeruli. At a given plasma level of antibody, the degree of binding of anti-GBM was slightly but significantly higher in HP than LP-fed rats; the decrease in GFR was significantly more pronounced in HP than LP-fed animals. The amount of anti-GBM binding was significantly greater in adult than young animals; however, the consequent decrease in GFR was more pronounced in the young than adult animals. Sex dependency was not discernible in anti-GBM binding or reduction in GFR. In all of the above experimental groups, the degree of anti-GBM binding was closely correlated with the plasma level of anti-GBM, but not with effective renal plasma flow rate, measured by PAH clearance. Separate groups of rats were subjected to experimental manipulation of single nephron GFR, glomerular capillary hydraulic pressure and glomerular plasma flow rate, by partial aortic constriction and saralasin administration. This set of experiments, using a tracer amount of non-nephritogenic anti-GBM, revealed that glomerular anti-GBM binding is independent of any of the above parameters. The studies indicate that dietary protein intake and age, but not sex, are among the factors determining the susceptibility of the glomerulus to acute immune injury. Since the binding of anti-GBM is determined by the affinity property of the glomerulus per se, and not by the prevailing hemodynamic pattern, the observed dependence of susceptibility to functional impairment on age and protein intake appears to also reflect a property of the glomerulus, which is influenced by age and the degree of dietary protein intake.

Aging↗