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A V Cybulsky

Publications and source records attributed to A V Cybulsky.

47 records · Page 3Linked to original sources
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↗
PubMed1987

A role for thromboxane in complement-mediated glomerular injury.

The membrane attack complex (MAC) of complement (C) has been shown to stimulate prostaglandin (PG) and thromboxane (Tx) synthesis in nucleated cells. Because glomerular epithelial cell injury and altered permeability in rat membranous nephropathy are mediated by the MAC, the authors examined whether MAC-induced proteinuria is linked to glomerular prostanoid synthesis. In kidneys containing non-nephritogenic, non-C-fixing gamma 2 sheep anti-Fx1A (planted antigen) that were perfused in vitro with C-fixing guinea pig anti-sheep IgG and a C source (fresh human plasma, 50% vol/vol in buffered bovine albumin), heavy proteinuria developed, reaching 4.27 +/- 1.20 mg/min/g at 100-120 minutes (n = 8). Cyclooxygenase blockade with 10(-4) M indomethacin (n = 6) inhibited urinary PGE2 excretion (569 +/- 47 to 124 +/- 18 pg/min/g, P less than 0.001) and lowered proteinuria (1.06 +/- 0.42 mg/min/g, P less than 0.001). Reduced protein excretion (0.88 +/- 0.12 mg/min/g, n = 6, P less than 0.001) also occurred with inhibition of Tx synthetase by OKY-046, 10(-4) M, a dose that was shown in separate perfusions to inhibit urinary TxB2 excretion by greater than 85%. Control kidneys, without planted antigen and perfused with anti-sheep IgG and plasma, excreted 0.30 +/- 0.05 mg protein/min/g (n = 6). Because inulin clearance was reduced by indomethacin, renal hemodynamic factors may have contributed to the reduction in proteinuria observed with this drug. However, insulin clearance was not significantly affected by OKY-046, implying that inhibition of Tx synthetase reduced proteinuria independently of changes in renal hemodynamics. Thus, proteinuria in rat membranous nephropathy is due to MAC-dependent glomerular epithelial injury and is mediated, in part, by Tx.

Animals↗
PubMed1987

Anti-Fx1A induces association of Heymann nephritis antigens with microfilaments of cultured glomerular visceral epithelial cells.

Binding of anti-Fx1A to Heymann nephritis antigens (HA) on rat glomerular epithelial cells (GECs) in culture leads to capping and disappearance of antigens from the cell surface. This process may contribute to the formation of glomerular subepithelial immune deposits in vivo. The authors differentially extracted GECs to determine whether HA redistribution is mediated by cytoskeletal components. Observations were made by phase-contrast and immunofluorescence microscopy on primary and passaged GECs in monolayer culture and by spectrofluorimetry on GECs in suspension. GEC-bound sheep anti-Fx1A IgG was detected by fluoresceinated anti-sheep IgG. Microfilaments were identified by rhodamine-phalloidin staining of F-actin. After cross-linking HA on GECs by anti-Fx1A IgG at 0 C, GECs remained polygonal in shape and had diffuse granular IgG staining of their plasma membranes. Treatment of GECs at 0 C with hypotonic buffer containing 0.5% Triton X-100 produced microfilament-rich cytoskeletons that retained the shape of unextracted GECs. Further incubation with DNase I at 37 C removed microfilaments (mean fluorescence declined by 90%) and resulted in the rounding of cytoskeletons. After Triton X-100 treatment, 85% of initial GEC-bound anti-Fx1A IgG remained, but only 29% remained after DNase I. In contrast to intact IgG, detergent-extraction resulted in the complete loss of GEC-bound anti-Fx1A Fab'. Anti-Fx1A IgG did not bind to GECs pretreated with Triton X-100. Thus, cross-linking of HA by anti-Fx1A converts HA from a detergent-soluble, membrane-associated form to an insoluble, cytoskeleton-bound form. Attachment of cross-linked HA to the cytoskeleton is mediated by microfilaments.

Actin Cytoskeleton↗
PubMed1986

The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy.

Using a model of rat membranous nephropathy (MN), we examined the relationship between the development of glomerular epithelial cell injury and the formation and stability of the membrane attack complex (MAC) of complement. Isolated rat kidneys were perfused with buffered bovine albumin (BSA) or various plasmas (complement source). Kidneys containing nephritogenic amounts of complement-fixing sheep antibody to glomerular epithelial antigens (aFx1A) perfused with BSA (n = 5), and normal kidneys perfused with normal human plasma in BSA (50% v/v, n = 6) excreted 0.30 +/- 0.02 mg protein/min/g during 90 min perfusion (control groups). When normal plasma was added to the perfusate of aFx1A kidneys at concentrations of 12.5, 25, and 50% v/v, protein excretion rose in a time- and concentration-dependent manner. Perfusions with 25% plasma resulted in baseline proteinuria from 0 to 20 min that increased to 2.8 +/- 0.9 mg/min/g at 20 to 40 min and 8.6 +/- 2.1 at 40 to 60 min (n = 4, p less than 0.01 vs control groups). Removal of plasma at 20 min did not prevent this rise in protein excretion (3.9 +/- 2.4 and 5.8 +/- 2.6 mg/min/g at 30 to 40 and 55 to 65 min respectively, p less than 0.01, n = 4). Perfusion of aFx1A kidneys with C8-deficient (C8D) human plasma (25% v/v, n = 4) or C6D rabbit serum (25% v/v, n = 2) independently produced low levels of proteinuria comparable with BSA, but in combination, the two reagents restored enhanced protein excretion (n = 2). In aFx1A kidneys containing C5b-7, addition of C8 and C9 (C6D serum) after intervals of 20, 60, or 90 min immediately reconstituted heavy proteinuria. Thus, the magnitude of MAC-induced glomerular epithelial injury in rat MN is related to the complement dose. Altered glomerular permeability is delayed with respect to the onset of complement activation. Once sufficient C5b-9 is formed, proteinuria can develop despite cessation of new MAC assembly, implying that C5b-9 persists after formation. Moreover, the C5b-7 MAC intermediate is not eliminated rapidly in this model.

Animals↗
PubMed1986

Antibody, antigen, and glomerular capillary wall charge interactions: influence of antigen location on in situ immune complex formation.

These studies examined the charge interactions between the glomerular capillary wall, antibody and antigen at different sites in the glomerulus. Sheep IgG was separated into differently charged subclasses and immunologically placed in one of two glomerular locations (subepithelial or subendothelial) to serve as planted antigen. Single kidneys with planted antigen were transplanted into uninephrectomized recipients that received affinity-purified, cationic and anionic rat anti-sheep IgG labelled with 125I and 131I, respectively. Glomerular bound antibody was determined and corrected for antibody delivery. Specificity of antibody binding was confirmed by comparison of kidneys with or without planted antigen. The results indicate that the influence of charge on glomerular antibody binding depends on the site of the antigen. When antigen was planted in the subepithelial space, significantly more (15 to 25%) cationic than anionic antibody bound despite the fact that the antigen was cationic. Conversely, when the antigen was planted subendothelially, significantly more anionic (13 to 22%) antibody bound when the antigen was cationic, and significantly more cationic (7 to 16%) antibody bound when the antigen was anionic. Thus, the negatively-charged glomerular filtration barrier retards the permeation of anionic antibodies that complex with antigens located in the subepithelial space, but antigen-antibody charge interactions appear to predominate when the antigen is more proximally located.

Animals↗
PubMed1986

Complement-induced glomerular epithelial cell injury. Role of the membrane attack complex in rat membranous nephropathy.

In passive Heymann nephritis (PHN) in rats, antibody (anti-Fx1A) reacts in situ with a glomerular epithelial antigen and induces complement (C)-mediated cell-independent proteinuria. To assess the role of the membrane attack complex (MAC), we determined the need for C8 in the pathogenesis of proteinuria in an autologous-phase model of PHN. Isolated rat kidneys, containing nonnephritogenic, non-C-fixing gamma 2 sheep anti-Fx1A (planted antigen), when perfused in vitro with C-fixing guinea pig anti-sheep IgG and a source of C (fresh human plasma 50% vol/vol in buffer containing bovine serum albumin), developed marked proteinuria after 20 min (0.58 +/- 0.08 mg/min X g, n = 8) that increased further to 3.20 +/- 0.93 mg/min X g after 80 min. In contrast, identical kidneys perfused with antibody and heat-inactivated or C8-deficient human plasma and normal kidneys perfused with antibody and fresh plasma excreted only 0.27 +/- 0.03 (n = 6), 0.27 +/- 0.04 (n = 5), and 0.40 +/- 0.05 mg/min X g (n = 6) after 20 min, and 0.13 +/- 0.02, 0.22 +/- 0.03, and 0.32 +/- 0.05 mg/min X g after 80 min, respectively. When C8-deficient plasma was reconstituted with sources of C8 (n = 3), proteinuria was restored to the level observed with fresh normal plasma. Differences in protein excretion could not be explained by quantitative differences in glomerular antigen or antibody content. Extensive ultrastructural damage to glomerular visceral epithelial cells was exclusively seen in antigen-containing kidneys perfused with antibody and C8-replete plasma. Thus, glomerular injury in this model results from an antigen-specific, antibody-directed, C8-dependent reaction involving assembly of the MAC. The ultrastructural findings argue in favor of MAC-induced cytotoxicity of the glomerular visceral epithelial cells.

Animals↗
PubMed1986

Nephritogenic potential of sheep antibodies against glomerular basement membrane laminin in the rat.

Antilaminin antibodies have been shown to bind to laminin within the glomerular basement membrane (GBM) and mesangium of experimental animals but have induced little or no glomerular injury. We used a sheep antiserum to murine Englebreth-Holm-Swarm sarcoma laminin (sheep antilaminin) to further study the potential of antilaminin antibodies to cause glomerular injury. Intravenous injections of sheep antilaminin into rats produced intense linear GBM deposits of sheep IgG but consistently failed to induce heterologous phase proteinuria as previously shown. In addition, no autologous phase injury appeared even after preimmunization with sheep IgG (N = 4) or passive administration of rat anti-sheep IgG (N = 3) (mean urine protein less than 4 mg/24 hours up to 16 days). GBM deposits of rat C3 in vivo were absent despite the ability of both sheep antilaminin and rat anti-sheep IgG to fix human and rat C3 in vitro as determined by an indirect immunofluorescent assay. In contrast, when kidneys containing sheep antilaminin were transplanted into naive recipients that were passively immunized with rat anti-sheep IgG, severe proteinuria occurred (range 7 to 109 mg/24 hours on day 2; 49 to 350 mg/24 hours on day 5 posttransplantation) in association with glomerular deposition of C3. Histological evaluation at day 5 showed a severe proliferative glomerulonephritis with infiltrating polymorphonuclear and mononuclear leukocytes. Electron microscopy showed endothelial and epithelial cell detachment from the GBM and inflammatory cell adherence to denuded GBM. Epithelial foot process effacement and cytoplasmic absorption droplets were also noted. Identical kidneys transplanted into nonimmunized recipients or immunized recipients depleted of complement had significantly less (p less than 0.05) proteinuria (nonimmunized: 5 to 18 mg/24 hours on day 2, 4 to 9 mg/24 hours on day 5; complement-depleted: 6 to 13 mg/24 hours on day 2, 4 to 27 mg/24 hours on day 5) and no glomerular complement fixation was seen in these animals. Thus, severe glomerular injury can be induced by a focused, amplified, complement-dependent immune attack on glomerular laminin. In contrast, the widespread distribution of laminin and antilaminin probably dilutes the total glomerular immune reaction and precludes effective complement fixation and glomerular injury during the autologous phase in nontransplanted kidneys. A similar explanation might account for the lack of glomerular injury in previous studies that utilized antisera to known GBM constituents.

Animals↗
PubMed1985

Effects of high sodium dialysate during maintenance hemodialysis.

The effects of high sodium 144 mmol/l (mEq/l) dialysate were studied in normotensive, hypertensive and anephric chronic hemodialysis patients. Comparisons of blood pressures, weights and side effects associated with the hemodialysis procedure were made between two 6-month periods using dialysate sodium concentration of 133 mmol/l (mEq/l), followed by a high dialysate sodium of 144 mmol/l (mEq/l), each patient acting as his own control. No difference was found in the frequency of cramps or 'disequilibrium' side effects (nausea, vomiting, headache, restlessness). High sodium dialysate is beneficial for normotensive and anephric patients in reducing dialysis-induced hypotension and was not associated with any deleterious effects on long-term blood pressure control. In hypertensive patients, the benefit is less clear, and hypertension may increase.

Adult↗
PubMed1984

Late development of purpura in a patient with glomerulonephritis.

A 20-year-old man with a 10-year history of glomerulonephritis presented with a purpuric rash on his legs. A renal biopsy specimen obtained when he was 11 years old had shown mesangial glomerulonephritis; staining 9 years later for IgA had negative results. A second renal biopsy, performed when the rash was present, revealed mesangial glomerulonephritis and mesangial deposits of IgA; biopsies of the involved skin showed leukocytoclastic vasculitis. In this case isolated glomerulonephritis appeared to change to a multisystem illness, with a different immunologic character, through one of several possible pathogenetic mechanisms.

Adult↗
PubMed1980

Activation of prorenin by proteases from polymorphonuclear leukocytes.

Inactive renin (prorenin) can be activated by certain proteases in human blood, of which a possible source in vivo is polymorphonuclear leukocytes (PMNs). We extracted enzyme from PMNs using methods established for the recovery of neutral and acid protease fractions, and tested their effectiveness on plasma prorenin in vitro. Neutral protease preparations, possessing mainly chymotrypsin and elastase activity, produce no activation of prorenin. Exogenous pancreatic alpha-chymotrypsin does activate plasma prorenin, but less effectively than trypsin. From the quantity of PMNs extracted for neutral protease, and its failure to activate protenin, we deduce that this enzyme preparation, like exogenous chymotrypsin, is qualitatively unimportant. In contrast, the extracted PMN acid protease fraction, believed to be rich in cathepsin D, exhibited high prorenin activating ability, suggesting both quantitative and qualitative importance. The low pH requirement of this acid protease (near pH 4.0), together with its inactivity at neutral pH, argues against an important systemic role in the conversion of prorenin. However, it may contribute to systemic activation in partnership with other enzymes, or else play a specialized local role in situations where PMN concentration and activity increase, and the pH is on the acid side.

Chymotrypsin↗