Biochemical requirements for singlet oxygen production by purified human myeloperoxidase.

The myeloperoxidase (MPO)-hydrogen peroxide (H2O2)-halide systems were found to produce chemiluminescence at 1,268 nm, a characteristic emission band for singlet oxygen (1O2). The emission was enhanced by a factor of 29 +/- 5 in deuterium oxide and was inhibited by the 1O2 quenchers, histidine and azide ion. Inactivation of MPO with heat or with cyanide ion prevented light production. The combined weight of all data strongly supported the production of 1O2 by these enzyme systems. The amount of 1O2 produced was sensitive to the conditions employed. Under optimal conditions at pH 5, the MPO-H2O2-bromide (Br-) system produced 0.42 +/- 0.03 mol 1O2/mol H2O2 consumed, close to the theoretical value of 0.5 that was predicted by the reaction stoichiometry. In contrast, the MPO-H2O2-chloride (Cl-) system was much less efficient. The maximum yield of 1O2 was 0.09 +/- 0.02 mol/mol H2O2 consumed and required pH 4 and 5 mM H2O2. At higher pH, the 1O2 production rapidly decreased. The yield at pH 7 was 0.0004 +/- 0.0002 mol/mol H2O2 consumed. Enzyme inactivation was a major factor limiting the yield of 1O2 with both Cl- and Br-. While the MPO-H2O2-halide systems can efficiently produce 1O2, the conditions required are not physiologic, which suggests that the chemiluminescence of the stimulated neutrophil does not derive from 1O2 generated by a MPO mechanism.     

Platelets mediate neutrophil-dependent immune complex nephritis in the rat.

Neutrophils and platelets are frequently present in glomeruli in immune glomerulonephritis (GN). No role for the platelet in acute neutrophil-mediated renal injury has been defined. We investigated a neutrophil-mediated model of subendothelial immune complex GN in the rat. Rats were platelet-depleted (mean platelet less than 10,000/microliter) with goat anti-platelet IgG before induction of GN by the renal artery perfusion of concanavalin A followed by anti-concanavalin A IgG. Platelet-depletion resulted in a significant reduction in albuminuria (7 +/- 2 vs. 55 +/- 10 mg/24 h) and fractional albumin excretion (0.045 +/- 0.01 vs. 0.410 +/- 0.09) compared with controls. The decrease in albuminuria was not due to differences in blood or glomerular neutrophil counts, complement, renal function, or glomerular antibody binding. Platelet-depleted rats had equivalent subendothelial deposits and glomerular endothelial cell injury but had minimal platelet infiltrates and fibrin deposition compared with controls. These studies demonstrate a role for platelets in mediating acute neutrophil-induced glomerular injury and proteinuria in this model of GN.     

Antimyeloperoxidase-associated proliferative glomerulonephritis: an animal model

To develop an animal model for antimyeloperoxidase (MPO)-associated necrotizing crescentic glomerulonephritis (NCGN), we immunized Brown Norway rats with MPO and localized a neutrophil lysosomal enzyme extract, primarily consisting of MPO and elastinolytic enzymes, plus H2O2, the substrate of MPO, to the glomerular basement membrane (GBM). Upon immunization rats developed antibodies and positive skin tests to MPO. After unilateral perfusion of the left kidney with the lysosomal enzyme extract and H2O2, MPO and immunoglobulin (Ig)G localized transiently along the GMB. At the time of maximal inflammation, at 4 and 10 d after perfusion, MPO, IgG, and C3 could not be detected anymore. MPO-immunized rats perfused with the lysosomal enzyme extract and H2O2, in contrast to control-immunized and/or control-perfused rats, developed a proliferative GN characterized by intra- and extracapillary cell proliferation, ruptured Bowman's capsule, periglomerular granulomatous inflammation, and formation of giant cells. Monocytes, polymorphonuclear leukocytes (PMN), and to a far lesser extent T cells were found in the glomeruli. Interstitial infiltrates consisted of monocytes, PMN, and T cells. Granulomatous vasculitis of small vessels was found at 10 d after perfusion. The proliferative NCGN in this rat model closely resembles human anti-MPO- associated pauci-immune NCGN, and enables the study of the pathophysiology of anti-MPO-associated NCGN.     

Myeloperoxidase modulates the phagocytic activity of polymorphonuclear neutrophil leukocytes. Studies with cells from a myeloperoxidase-deficient patient

Patients lacking the primary granulae enzyme, myeloperoxidase (MPO), do not usually show any increased susceptibility to infection or altered inflammatory response, in contrast to several other biochemical defects in polymorphonuclear neutrophils. We have now evaluated the role of MPO on phagocyte function in a patient with complete MPO deficiency suffering from generalized pustular psoriasis. We found that the MPO-deficient neutrophils showed enhanced phagocytosis (greater than 200% of normal) of IgG- and C3b-opsonized yeast particles and prolonged N-formylmethionyl-leucyl-phenylaline-mediated stimulation of superoxide production. When purified human MPO was added to normal neutrophils during cell adhesion, their Fc- and C3b-mediated phagocytosis was reduced without affecting cell viability. 1 microgram/ml of MPO reduced the Fc and C3b phagocytosis to 47 and 65%, respectively, whereas 10 micrograms/ml reduced the activity to 20 and 54%. Both attachment and ingestion were reduced to a similar extent, indicating that MPO affected the receptor function per se. When MPO was added to the hyperactive MPO-deficient cells, phagocytosis was reduced more rapidly. Catalase, azide, and methionine eliminated the inhibitory effect, and catalase and methionine, in fact, enhanced the phagocytic activity of adherent neutrophils. These data indicate that, apart from being a potent antimicrobial system, the oxidizing activity of the MPO-H2O2-halide system may modulate the inflammatory response by impairing certain receptor-mediated recognition mechanisms of phagocytic cells, which otherwise could elicit inflammatory reactions and tissue injury.     

Degradation and oxidation of methionine enkephalin by human neutrophils

Met5-enkephalin, tyr-gly-phe-met, is an endogenous pentapeptide, with morphine agonist activity. In this study, we demonstrated that met5-enkephalin was degraded with the release of tyrosine by resting human PMN, whereas it was degraded as well as oxidized to its sulfoxide derivative, met5-(O)-enkephalin, by phagocytosing PMN. PMN also degraded met5-(O)-enkephalin but to a lesser extent. Bacitracin at 1 gm/L inhibited the degradation and oxidation of met5-enkephalin without affecting the production of superoxide and viability of PMN. The oxidation of met5-enkephalin by phagocytosing PMN was inhibited by catalase or NaN3 but not by SOD. This suggests that the oxidation of met5-enkephalin by phagocytosing PMN was, at least in part, dependent on the MPO system (MPO-H2O2-halide). Using purified canine MPO, we further demonstrated that MPO-H2O2-CI- oxidized met5-enkephalin to met5-(O)-enkephalin. The MPO-mediated oxidation of met5-enkephalin was inhibited by methionine but not by methionine sulfoxide, tyrosine, glycine, or phenylalanine, confirming that it was the methionine moiety of met5-enkephalin which was oxidized. Since both the sulfoxide derivative and the degradation products met5-enkephalin have reduced opiate agonist activity, oxidation and degradation of met5-enkephalin by PMN may contribute to the pain at the site of inflammation. (J Lab Clin Med 99:418, 1982.)     

The glomerular mesangium: I. Kinetic studies of macromolecular uptake in normal and nephrotic rats

This study was designed to define quantitatively the function of the rat glomerular mesangium in the uptake and processing of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, AHIgG-(125)I), to relate this function to that of the general reticuloendothelial system, and to examine the effects of increased glomerular permeability to protein on the mesangial cell system.Mesangial localization of human IgG as demonstrated by immunofluorescent microscopy showed good correlation with concentrations of AHIgG-(125)I in preparations of isolated glomeruli. In normal rats the concentrations of AHIgG-(125)I in glomeruli were similar to those of lung, liver, and spleen and demonstrated a rapid decrease with increasing time intervals after aggregate administration.In rats given aminonucleoside of puromycin a marked increase in mesangial uptake of aggregates was found while studies of nephrotic lungs, liver, spleen, and blood showed no such differences. Glomerular levels of AHIgG-(125)I in aminonucleoside animals could not be correlated with the quantity of proteinuria.Nephrotic and control animals given unaggregated human IgG showed little glomerular localization by immunofluorescent microscopy; no difference in the concentration of this protein in nephrotic as compared to control glomerular isolates was found.Thus, the mesangium in normal animals functions in a manner analogous to that of the general reticuloendothelial system. In nephrotic rats the mesangial uptake of macromolecules is makedly increased, a finding not observed in other tissues.     

Glomerular arachidonate lipoxygenation in rat nephrotoxic serum nephritis.

Arachidonate lipoxygenation to monohydroxylated eicosatetraenoic acids (HETE) was studied in rat nephrotoxic serum nephritis (NSN). A single infusion of nephrotoxic serum enhanced conversion of [3H]arachidonic acid ([3H]C20:4) to [3H]12-HETE in glomeruli isolated from nephritic rats compared with controls. The percent conversion of [3H]arachidonic acid was 1.95 +/- 0.2% in control glomeruli and 14.2 +/- 2% in nephritic glomeruli 2 d after induction of disease. No significant changes in the conversion of [3H]C20:4 to [3H]5-, 8-, and 9-HETE were noted. Extraction of glomerular HETE by alkaline hydrolysis, to evaluate possible reacylation of HETE after their production, confirmed the presence of 12-HETE and did not provide evidence of 5-HETE synthesis. Increased glomerular 12-HETE synthesis in nephritic rats was also demonstrated by high pressure liquid chromatography-UV detection and by 12-HETE radioimmunoassay. The enhanced glomerular 12-HETE synthesis commenced as early as 3-5 h after administration of nephrotoxic serum and peaked at day 2 with 10-fold enhancement of 12-HETE production. Increments of glomerular 12-HETE persisted on day 7 and returned toward control levels by day 14. Platelet depletion, induced by antiplatelet antisera, did not decrease glomerular 12-HETE synthesis in NSN, thereby eliminating platelets as the cellular origin of 12-HETE. Glomerular epithelial and mesangial cells are the most likely sources of enhanced 12-lipoxygenase activity. The enhanced arachidonate 12-lipoxygenation in glomerular immune injury could have important proinflammatory effects in the evolution of glomerulonephritis since 12-HETE has important effects on leukocyte function.     

The glomerular mesangium. II. Studies of macromolecular uptake in nephrotoxic nephritis in rats

These studies were designed to explore the effects of nephrotoxic serum (NTS) in rats on the uptake and processing by the glomerular mesangium of intravenously administered protein macromolecules (radiolabeled aggregated human IgG, [125I]AHIgG). Measurements of [125I]AHIgG levels in preparations of isolated glomeruli correlated well with qualitative estimates of glomerular IgG deposition seen by immunofluorescent microscopy. Rats given 2 ml NTS received 25 mg/100 g body wt [125I]AHIgG 48 h later and were sacrificed at varying time intervals thereafter. NTS-treated animals demonstrated a marked increase in glomerular uptake of [125I]AHIgG as compared with concurrent controls but a normal ability to clear [125I]AHIgG from the mesangium over 72 hr. Animals given 1.0, 0.5, and 0.25 ml NTS had neither proteinuria nor significant light microscopic changes, yet had increased uptake of [125I]AHIgG of 4.4, 3.0, and 2.1 times control values, respectively at 8 h after the infusion of aggregates. Rats given 1 ml NTS and 12.5, 6.25, and 3.125 mg [125I]AHIgG/100 g body wt had increased glomerular uptake at 8 h, but there was, within both NTS and control groups, a disproportionate decrease in uptake at lower [125I]AHIgG doses. Rats given cobra venom factor (CoF) followed by a NTS shown to be complement dependent (proteinuria reduced by prior complement depletion with CoF) had less mesangial [125I]AHIgG uptake than NTS controls but greater uptake compared with normal controls. CoF itself had no effect on mesangial or reticuloendothelial system [125I]AHIgG uptake. These studies demonstrate a striking change in glomerular mesangial activity after fixation of nephrotoxic antibodies to the glomerular basement membrane, even in the absence of proteinuria and suggest that subtle alterations of the filtration surface can influence mesangial function. The effect of NTS on the mesangium is due, in large part, to the glomerular injury and proteinuria which nephrotoxic antibodies produce.     

Macrophage-induced glomerular fibrin deposition in experimental glomerulonephritis in the rabbit.

Glomerular fibrin deposition is important in the pathogenesis of renal failure and crescent formation in glomerulonephritis. The mechanisms of glomerular fibrin deposition are unknown. The current studies explored the role of macrophages in this process. Methods were developed for measuring glomerular fibrin deposition and glomerular procoagulant activity in a passive model of the autologous phase of antiglomerular basement membrane antibody-induced glomerulonephritis in rabbits. Significant fibrin deposition was observed to be associated with glomerular macrophage accumulation. Leukocyte ablation with mustine hydrochloride prevented both glomerular macrophage accumulation and fibrin deposition without affecting the coagulation system or the deposition of disease-inducing antibodies and complement. Repletion with mononuclear inflammatory cells produced significant fibrin deposition. To examine the role of tissue injury per se in glomerular fibrin deposition, a macrophage-independent model of glomerular injury (heterologous phase glomerulonephritis) was also studied. Although a similar degree of glomerular injury occurred, there was no significant fibrin deposition. This suggests that macrophages, rather than injury alone, are responsible for fibrin deposition. Lysates of isolated glomeruli containing macrophages demonstrated greatly enhanced procoagulant activity compared with lysates of glomeruli without macrophages. Thus macrophages appear to be directly responsible for glomerular fibrin deposition in antiglomerular basement membrane antibody-induced glomerulonephritis, and this appears to be due to their ability to express procoagulant activity rather than their propensity to cause glomerular injury.     

Superoxide dismutase mimetic preserves the glomerular capillary permeability barrier to protein

Overproduction of superoxide (O2*) occurs in glomerular disease and may overwhelm the capacity of superoxide dismutase (SOD), thereby intensifying oxidant injury by O2* and related radical species that disrupt the glomerular capillary permeability barrier to protein. We examined the efficacy of the SOD mimetic tempol in preserving glomerular permeability to protein using 1) a rat model of glomerular immune injury induced by an antiglomerular basement membrane antibody (anti-GBM), and 2) isolated rat glomeruli in which injury was induced by the cytokine tumor necrosis factor-alpha (TNFalpha). To induce glomerular immune injury, rats received anti-GBM using a protocol that results in prominent infiltration of glomeruli by macrophages and in which macrophage-derived TNFalpha has been shown to mediate albuminuria. To increase glomerular capillary permeability to albumin (P(alb)) ex vivo, isolated glomeruli were incubated with TNFalpha at concentrations (0.5-4.0 microg/ml) known to stimulate O2* production. Increments in P(alb) were detected by measuring changes in glomerular volume in response to an applied oncotic gradient. Significant increases in the urine excretion of albumin and F(2alpha)-isoprostane were observed in rats with glomerular immune injury without a significant change in systolic blood pressure. Tempol treatment significantly reduced urine isoprostane and albumin excretion. In isolated glomeruli, TNFalpha increased P(alb) and tempol abrogated this effect, both in a dose-dependent manner. These observations indicate that SOD mimetics can preserve the glomerular permeability barrier to protein under conditions of oxidative stress from O2* production.     

The Inhibition of Polymorphonuclear Leukocyte Cytotoxicity by Dapsone: A POSSIBLE MECHANISM IN THE TREATMENT OF DERMATITIS HERPETIFORMIS

The effect of the sulfone compound 4,4'-diaminodiphenyl sulfone (dapsone) on normal human polymorphonuclear leukocytes (PMNL) has been investigated in vitro. The drug has a dramatically beneficial effect in dermatitis herpetiformis in which the PMNL and immune complexes has been stressed to be of importance for the development of the skin lesions. Pruritus disappears and the inflammatory eruptions clear within a few days of starting therapy. The effect of dapsone has been evaluated on the different stages of phagocytosis. Using dapsone concentrations (1-30 mug/ml) comparable with those found after therapeutic doses, we have found that the drug interferes primarily with the myeloperoxidase (MPO)-H(2)O(2)-halide-mediated cytotoxic system in the PMNL. No effect was observed on random locomotion, chemotaxis, phagocytic ingestion, oxidative metabolism, or the release of lysosomal enzymes. Kinetic studies in a cell-free system with purified MPO revealed a competitive type of inhibition using varying concentrations of NaI. Furthermore, the inhibition resulted in reduced candidicidal activity during phagocytosis of Candida albicans, and reduced cytotoxicity to adjacent mammalian cells measured as the (51)Cr release from virus-induced lymphoma cells. Because the MPO-H(2)O(2)-halide system not only fulfills the antimicrobial activity but is suggested to be a modulator of the inflammatory reaction as well, the action of dapsone in dermatitis herpetiformis may in part be explained by its effect on this system.     

Clindamycin, erythromycin, and roxithromycin inhibit the proinflammatory interactions of Pseudomonas aeruginosa pigments with human neutrophils in vitro.

The Pseudomonas aeruginosa-derived phenazine pigments pyocyanin and 1-hydroxyphenazine (1-hp) prime human neutrophils for enhanced, stimulus-activated release of superoxide and myeloperoxidase (MPO), respectively. In the present study, the modulatory potentials of the antimicrobial agents clindamycin, erythromycin, and roxithromycin (10 and 20 micrograms/ml) on the prooxidative interactions of pyocyanin and 1-hp (12.5 microM) with human neutrophils have been investigated. Clindamycin, erythromycin, and especially roxithromycin caused dose-related inhibition of the generation of superoxide by both untreated and pyocyanin-treated neutrophils during activation with either the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or the calcium ionophore A23187. The antimicrobial agents also inhibited the generation of reactive oxidants by the MPO-H2O2-halide system during activation of both untreated and 1-hp-treated neutrophils by FMLP. These effects appeared to be due to drug-related interference with membrane-associated oxidative metabolism, since none of the antimicrobial agents inhibited the release of MPO by activated neutrophils, nor did they possess oxidant-scavenging properties. These data demonstrate that clindamycin, erythromycin, and especially roxithromycin antagonize the proinflammatory interactions of pyocyanin and 1-hp with neutrophils and indicate a possible therapeutic role for these antimicrobial agents in the prevention of tissue damage in diseases characterized by P. aeruginosa infection.     

Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase.

The neutrophil has been implicated as an important mediator of vascular injury, especially after endotoxemia. This study examines neutrophil-mediated injury to human microvascular endothelial cells in vitro. We found that neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, or lipopolysaccharide (LPS) (1-1,000 ng/ml) alone produced minimal endothelial injury over a 4-h assay. In contrast, neutrophils incubated with endothelial cells in the presence of low concentrations of LPS (1-10 ng/ml) could then be stimulated by FMLP or C5a to produce marked endothelial injury. Injury was maximal at concentrations of 100 ng/ml LPS and 10(-7) M FMLP. Pretreatment of neutrophils with LPS resulted in a similar degree of injury, suggesting that LPS effects were largely on the neutrophil. Endothelial cell injury produced by LPS-exposed, FMLP-stimulated neutrophils had a time course similar to that induced by the addition of purified human neutrophil elastase, and different from that induced by hydrogen peroxide (H2O2). Further, neutrophil-mediated injury was not inhibited by scavengers of a variety of oxygen radical species, and occurred with neutrophils from a patient with chronic granulomatous disease, which produced no H2O2. In contrast, the specific serine elastase inhibitor methoxy-succinyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone inhibited 63% of the neutrophil-mediated injury and 64% of the neutrophil elastase-induced injury. However, neutrophil-mediated injury was not inhibited significantly by 50% serum, 50% plasma, or purified alpha 1 proteinase inhibitor. These results suggest that, in this system, chemotactic factor-stimulated human neutrophil injury of microvascular endothelial cells is enhanced by small amounts of LPS and may be mediated in large part by the action of neutrophil elastase.     

Increased leukotriene B4 synthesis in immune injured rat glomeruli

We examined glomerular synthesis of the 5-lipoxygenase metabolite, LTB4, in normal and immune-injured rat glomeruli. Glomeruli isolated from normal rats and from rats with nephrotoxic serum nephritis (NSN), passive Heymann nephritis (PHN) and cationic bovine gamma globulin (CBGG)-induced glomerulonephritis were incubated with the calcium ionophore A23187 (3 microM). Lipids in the glomeruli and media were extracted with ethyl acetate, and were purified and fractionated by HPLC. Immunoreactive-LTB4 (i-LTB4) was determined by radioimmunoassay on HPLC fractions with a detection limit of 50 pg of i-LTB4. A large peak of i-LTB4 that comigrated with authentic LTB4 was found exclusively in glomeruli isolated from the CBGG-injected rats. Addition of the lipoxygenase inhibitor BW755C (50 micrograms/ml) to glomerular incubation resulted in greater than 90% inhibition of i-LTB4. Synthesis of i-LTB4 by glomeruli from normal, NSN and PHN rats was undetectable. Glomerular LTB4 synthesis by CBGG-injected rats was confirmed by radiometric HPLC and by gas chromatography mass-spectroscopy (GC-MS) analysis. In order to rule out synthesis of LTB4 by neutrophils entrapped in the glomeruli, a group of rats received 1,000 rad total body x irradiation, with shielding of the kidneys before induction of CBGG glomerulonephritis. Despite greater than 95% reduction in total leukocyte count, glomerular synthesis of LTB4 remained enhanced. Augmented glomerular synthesis of the proinflammatory lipid, LTB4, in the CBGG model of glomerular disease could have an important role in the development of glomerular injury and proteinuria.     

Receptor antagonist of platelet activating factor inhibits inflammatory injury induced by in situ formation of immune complexes in renal glomeruli and in the skin

Experiments were designed to test the effect of a receptor antagonist of platelet activating factor (PAF), SRI63072, on inflammatory injury induced by in situ formation of immune complexes in two vascular districts of the rat that have different structural and hemodynamic characteristics: unilateral glomerulonephritis induced by perfusion of the left kidney of preimmunized animals with cationic human IgG, and passive reversed Arthus reaction in the skin. Three days after perfusion the left kidneys of rats not treated with SRI63072 (group I) were greatly enlarged, and pale and severe exudative and proliferative lesions were present in glomeruli. Granular deposits of human IgG, rat IgG, and rat C3 were seen by immunofluorescence microscopy, and subepithelial electron-dense deposits were visualized by electron microscopy. The right kidneys were consistently normal. The animals were severely proteinuric and had increased levels of PAF in the circulation. In contrast, rats treated with SRI63072 (group II) and studied at the same interval of time developed only mild, focal glomerulonephritis in the perfused kidneys. By immunofluorescence microscopy the glomerular deposits of human IgG and rat IgG were similar in quantity and distribution to those observed in rats of group I. Despite the fact that SRI63072 did not influence the level or the activity of the rat serum complement system, the deposits of C3 were less abundant and more focal. As in animals of group I, electron-dense deposits were present at the epithelial side of the glomerular basement membrane. Proteinuria was slight, and levels of circulating PAF were not significantly increased. These effects cannot be ascribed to interference of SRI63072 with antigen "implantation," antibody binding, or local hemodynamic conditions, because the amounts of glomerular human and rat IgG were the same in treated and untreated rats; SRI63072 did not decrease the glomerular filtration rate; and SRI63072 prevented the increase in vascular permeability and the exudative lesions in passive Arthus reaction in the skin, a model less affected by hemodynamic changes than glomerulonephritis. The beneficial effect of SRI63072 indicates that PAF is an important mediator of the inflammatory process generated either in glomeruli or in the skin by in situ immune complex formation.     

Hydroxyl radical scavengers ameliorate proteinuria in rat immune complex glomerulonephritis

We examined the effect of the administration of different oxygen radical scavengers on the development of glomerulonephritis induced by cationic bovine gamma-globulin (cBGG). Treatment with the H2O2 scavenger catalase or the superoxide anion (O2-) scavenger superoxide dismutase (SOD) did not significantly reduce proteinuria. In contrast, treatment with the hydroxyl radical (OH.) scavengers dimethyl sulfoxide (DMSO) or dimethylthiourea resulted in significant decrements in proteinuria, from 156 +/- 20 mg/24 hours in saline solution--treated control rats to 70 +/- 17 mg/24 hours (p less than 0.05) and 37 +/- 10 mg/24 hours (p less than 0.01) in DMSO- and dimethylthiourea-treated rats, respectively. Therapy with DMSO for 5 days after induction of glomerular disease also resulted in amelioration of proteinuria, 10.0 +/- 5.0 mg/24 hours versus saline solution-treated rats, 67.6 +/- 16.2 mg/24 hours (p less than 0.005). OH. scavenger therapy did not influence glomerular morphology, glomerular immunoglobulin G (IgG), or complement deposition, or creatinine clearances of rats with glomerulonephritis. Furthermore, there were no significant differences in serum levels of C3 and C5 or anti-BGG antibody production between DMSO-treated rats and control rats. None of the radical scavengers administered altered the enhanced glomerular thromboxane synthesis characteristic of this model. Our results suggest that OH. generation mediates in part glomerular injury in cBGG-induced glomerulonephritis.     

Rat glomerular mesangial cells synthesize basic fibroblast growth factor. Release, upregulated synthesis, and mitogenicity in mesangial proliferative glomerulonephritis.

Mesangial injury and cell proliferation are frequent findings in various glomerular diseases in man. Previous studies have demonstrated that basic fibroblast growth factor (bFGF) is a potent mesangial cell mitogen in vitro. To further elucidate the role of bFGF in rat mesangial cell (RMC) proliferation, we examined whether RMC synthesize bFGF in vitro and whether bFGF is involved in mesangial proliferation in vivo. Cultured RMC expressed bFGF protein (23, 21.5, and 18 kD forms) and bFGF mRNA, and released biologically active bFGF into the culture medium after antibody- and complement-mediated injury. Normal rat glomeruli in vivo contained no detectable bFGF mRNA, but bFGF protein (23 and 21.5 kD) could be demonstrated, which immunolocalized to the mesangium. Glomerular bFGF decreased markedly during the acute phase of glomerulonephritis induced by anti-Thy 1.1 antibody, compatible with mesangial bFGF release after complement-mediated mesangiolysis. During the subsequent mesangial proliferative phase, glomerular bFGF protein and mRNA increased above normal. Intrarenal infusion of heparin did not affect the bFGF immunostaining of glomeruli at this stage, indicating a predominantly intracellular localization of the bFGF. The capability of bFGF to mediate proliferation in the anti-Thy 1.1 model was further supported by experiments in which intravenous bFGF given 24 h after a subnephritogenic dose of anti-Thy 1.1 antibody led to a 4.9- to 5.1-fold increase in glomerular cell proliferation (with > 60% of the cells identified as mesangial cells by double immunolabeling). No such increase was observed in normal rats injected with bFGF. These data show that mesangial cells produce and release bFGF after injury and that bFGF is mitogenic for injured mesangial cells in vivo. Release of mesangial cell bFGF thus may be an important mechanism involved in the initiation of mesangial cell proliferation in vivo.     

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus.     

Essential fatty acid deficiency depletes rat glomeruli of resident macrophages and inhibits angiotensin II-induced eicosanoid synthesis.

Essential fatty acid (EFA) deficiency exerts a beneficial effect on immune-mediated glomerulonephritis, preventing both the tissue injury and consequent mortality. Because both macrophages and eicosanoids are thought to play pathogenic roles in glomerulonephritis, and because macrophages play an important role in modulating arachidonate metabolism at sites of renal injury, the effects of EFA deficiency on the population of resident glomerular macrophages and on glomerular eicosanoid generation were examined. EFA deficiency led to a striking reduction in the number of resident glomerular macrophages and a corresponding reduction in the number of resident glomerular Ia+ cells. This phenomenon was not strain-specific, was not due to a decrease in circulating monocytes, was not a function of changes in cell surface labeling characteristics, and was not restricted to a specific subset of glomeruli. In addition, EFA deficiency affected other areas of the renal cortex: a comparable depletion of interstitial macrophages and Ia+ cells was also observed. In conjunction with the decrease in glomerular macrophages seen with the deficiency state, a marked decrease in both basal and angiotensin II-stimulated glomerular eicosanoid production was noted. In contrast to angiotensin II, platelet-activating factor-induced eicosanoid production was not significantly affected by the deficiency state. These changes in glomerular eicosanoid production could not be attributed to changes in glomerular cyclooxygenase or reacylation capacity. Dietary (n-6) fatty acid supplementation, but not (n-3) fatty acid supplementation, reversed both the decrease in glomerular macrophages and the diminished eicosanoid metabolism seen with the deficiency state. Understanding the mechanisms behind the changes in the glomerular microenvironment induced by EFA deficiency may provide a basis for elucidating the protective effect of dietary fatty acid manipulation on immune-mediated glomerulonephritis.     

EXPERIMENTAL NEPHRITIS IN RATS INDUCED BY INJECTION OF ANTIKIDNEY SERUM : V. CHRONIC NEPHRITIS OF INSIDIOUS DEVELOPMENT FOLLOWING APPARENT RECOVERY FROM ACUTE NEPHROTOXIC NEPHRITIS

1. Three different inbred lines of rats were found to vary in their response to antikidney serum: rats of the Whelan strain were most susceptible to nephrotoxin and most prone to develop chronic glomerulonephritis immediately following the acute injury induced by this agent; animals of the Evans strain were almost as vulnerable to the acute effects of nephrotoxin; Wistar rats were the least affected. 2. Both Evans and Wistar rats usually recovered quickly from the acute injury, and between the 2nd and 5th months after injection they excreted normal or only slightly abnormal urines. During this period of absence of clinical signs of disease, histopathological examination of their kidneys revealed only minor scarring in the glomerular tufts. 3. Most of these apparently recovered rats subsequently developed a slowly progressing chronic glomerulonephritis irrespective of whether they were fed a basal or high protein diet. 4. Histopathologically similar renal lesions were present in all three strains of rats with active chronic nephritis regardless of whether the chronic disease followed immediately the acute nephrotoxic injury or was separated from it by an interval of months. These lesions were somewhat more severe, however, in Whelan rats. 5. Some intraglomerular scarring was present in the kidneys of all rats which survived acute nephrotoxic nephritis. It was especially prominent in those animals that remained clinically cured for as long as a year. 6. The permanent clinical recovery of certain animals, which were found to have moderate glomerular fibrosis on postmortem examination, suggests that factors other than this residual scarring contributed to the development of the recurrent nephritis observed in most of the Evans and Wistar rats. These unknown factors may produce varying degrees of renal functional trauma affecting both glomeruli and tubules.