Victoria Cattell

Expression of the gene for inducible nitric oxide synthase in experimental glomerulonephritis in the rat.

Nitrite, a stable product of nitric oxide (NO), is synthesized in vitro by glomeruli in experimental glomerulonephritis. We have now studied the expression of the gene for inducible NO synthase (iNOS) in accelerated nephrotoxic nephritis (NTN). The purpose of the study was to confirm in vivo induction of iNOS in this model of immune complex disease, and to relate the onset of induction and the level of expression to pathogenic events in the model. Glomeruli from rats with NTN were isolated at 6 h, 24 h and 2, 4 and 7 days and total RNA extracted. RNA (10/μg) was reverse transcribed and polymerase chain reaction (PCR) was performed with primers homologous to rat vascular smooth muscle iNOS and rat β actin. A 222‐base PCR product corresponding to iNOS mRNA was present in all experimental animals. iNOS expression was also found in activated macrophages, neutrophils and IL‐1‐stimulated but not unstimulated mesangial cells. Quantitative competitive PCR was carried out on glomerular samples using a 514‐bp mutant of a 735‐bp PCR product. iNOS expression was present at low levels in normal glomeruli and was markedly enhanced at 6 h after the induction of glomerulonephritis and peaked at 24 h. Increased iNOS expression persisted to day 7. β actin mRNA levels were similar in all glomerular specimens. This study demonstrates that there is in vivo induction of iNOS in immune complex glomerulonephritis, corresponding to the generation of nitrite we have previously reported. iNOS gene expression is detectable within 6 h of induction of NTN, indicating the onset of gene transcription is closely related lo the initial formation of immune complexes.

Cutting Edge: Amelioration of Kidney Disease in a Transgenic Mouse Model of Lupus Nephritis by Administration of the Caspase Inhibitor Carbobenzoxy-Valyl-Alanyl-Aspartyl-(β-o-methyl)-Fluoromethylketone

Systemic lupus erythematosus (SLE) is a common, potentially fatal, non-organ-specific autoimmune disorder. Immune complex-mediated kidney disease is the major cause of mortality. Apoptotic cells in the epidermis are a possible source of self Ags, and apoptosis of endothelial cells and lymphocytes is thought to contribute to end-organ damage. We have previously shown that female transgenic mice expressing IFN-γ in the epidermis develop inflammatory skin disease and features of SLE that have striking parallels with the human condition. We have now tested the effects of a pan-caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-(β-o-methyl)-fluoromethylketone, on disease progression. Daily s.c. administration of carbobenzoxy-valyl-alanyl-aspartyl-(β-o-methyl)-fluoromethylketone to female transgenic mice over a 3-wk period resulted in significant amelioration of both glomerular and interstitial renal damage, independent of the effects on autoantibody levels or skin inflammation. We propose that apoptosis inhibitors could be beneficial in the treatment of human SLE.

Inducible Nitric Oxide Synthase Induction in Thy 1 Glomerulonephritis Is Complement and Reactive Oxygen Species Dependent

Thy 1 glomerulonephritis (GN) is a rat model of complement-dependent immune mesangial injury with induced glomerular nitric oxide (NO) synthesis. To examine mechanisms of inducible nitric oxide synthase (iNOS) induction, we studied the effects of treatment with the antioxidant N-acetyl-cysteine (NAC) and soluble complement receptor 1 (sCR1). Thy 1 GN was induced by intravenous anti-Thy 1 antibody. Glomeruli were isolated and kidney tissue taken from 30 min to 24 h after induction. Nitrite (NO–2) synthesis, luminol chemiluminescence for reactive oxygen species (ROS), and iNOS and cytokine mRNA were assayed in isolated glomeruli. Mesangial injury (mesangiolysis) and leucocyte infiltration were quantitated on tissue sections. NAC (i.p. 1,000 mg/kg, 1 h prior to anti-Thy 1) reduced glomerular NO–2 synthesis (3.5 ± 0.66 vs. untreated 8.2 ± 1.1, p = 0.02), and iNOS mRNA expression, and abolished enhanced chemiluminescence. In vitro incubation of nephritic glomeruli with 20 mM NAC also suppressed nitrite production (4.7 ± 0.8 vs. untreated 12.2 ± 0.7 nmol NO–2/2,000 glomeruli/48 h, p = 0.003), and chemiluminescence. In NAC-treated animals, neutrophil infiltration (0.5 ± 0 vs. untreated 9.6 ± 1.6 glomerulus, p = 0.0005), and macrophage infiltration (1.7 ± 0.4 vs. untreated 12.0 ± 0.1, p = 0.006) were abolished, and mesangiolysis was significantly reduced (45.9 ± 1.3 vs. untreated 34.4 ± 2.1 cells/glomerulus, p = 0.009). NAC did not inhibit anti-Thy 1 antibody deposition. C1q was unaffected, but C3 was reduced. sCR1 treatment prevented iNOS mRNA induction, the enhanced chemiluminescence, and the neutrophil infiltration at 1 h. IL-1β and TNFα mRNAs were not affected by either NAC or sCR1. These results show that NAC inhibits iNOS induction and NO synthesis in this model, and suppresses ROS synthesis and injury. They suggest that complement-dependent ROS generation is the critical initiating event that follows fixation of anti-Thy 1 antibody.

Prostaglandin E1 suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage‐dependent glomerulonephritis

Prostaglandin E1 (PGE1) suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage dependent glomerulonephritis. Macrophages are mediators of glomerular injury in models of proliferative glomerulonephritis. We have recently shown that macrophages in glomerulonephritis have low prostaglandin E2 (PGE) generation, and other evidence implicates eicosanoids as regulators of macrophage activation. Here we have studied in rats the effect of 15(S)‐15‐methyl PGE1 (M‐PGE1) on accelerated nephrotoxic nephritis, a model of acute macrophage‐dependent glomerular injury. M‐PGE1 ameliorated proteinuria (day 4; 61 ± 13 mg/24 h, n=9; vehicle treated, 164.17 mg/24 h, n=11; P<0.002) and glomerular hypercellularity; quantification of infiltrating macrophages by isolating glomerular cells showed reduction in the numbers of macrophages (44.9/glomerulus; vehicle treated, 119.15/glomerulus; P <0.02) with inhibition of Ia antigen expression on infiltrating macrophages (8.5%; vehicle treated 25.4%P < 0.05). Glomerular binding of nephrotoxic globulin and levels of autologous antibodies were not affected by M‐PGE1. Thus the mechanism of suppression involves inhibition of macrophage accumulation and activation. M‐PGE1 administered to normal rats did not affect numbers of resident leucocytes (12.6±1.5/glomerulus; vehicle treated, 13.2±1.3/glomerulus) or alter Ia antigen expression (4.1±0.2 Ia + cells/glomerulus; vehicle treated, 3.9±0.6/glomerulus). This study suggests a therapeutic role for PGE1 in this type of glomerulonephritis, and has implications for the pathophysiology of macrophage‐mediated inflammation.

Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis.

In immune‐induced glomerulonephritis (gn), glomeruli (gl) synthesize nitric oxide (NO), and urinary nitrite (NO2−) excretion is increased. In mammals on a low nitrate (NO3−) diet, urinary NO3− is a measure of endogenous NO3− synthesis. Excretion is increased after administration of macrophage activators, reflecting induction of NO production. To determine whether increased urinary NO2− gn is due to glomerular synthesis we studied urinary NO2−/NO3− in accelerated nephrotoxic nephritis induced by preimmunization with rabbit immunoglobulin G (IgG), followed by rabbit anti‐rat nephrotoxic globulin, and in control rats similarly preimmunized with rabbit IgG, but followed by normal rabbit serum. Both urinary NO2− and NO3− were increased by i.p. preimmunization with rabbit IgG (peak 463 ± 171 nmol NO2−/60.3 ± 9.4 nmol NO3/24 h, P<0.001 for both NO2− and NO3− compared with preimmunization levels). Repeat immunization with i.v. rabbit anti‐rat nephrotoxic globulin (nephritic rats) or normal rabbit globulin (control rats) again increased urinary NO2− and NO3−. There was no statistically significant difference in urinary NO2 and NO3− levels between nephritic rats where globulin had nephrotoxic activity and the control rats injected with normal rabbit globulin, despite increased NO2−synthesis in ex vivo nephritic glomeruli after nephrotoxic globulin (7.9 ± 1.9 nmol/2000 gl/48 h; controls 3.2 ± 1.0 nmol/2000 gl/48 h). Thus neither urinary NO2− nor NO3− levels reflect local activation of the NO pathway in glomeruli. As reported for other stimulants, we show here that systemic stimulation with foreign antigen increased NO synthesis.

Mesangial cell necrosis in Thy 1 glomerulonephritis – an ultrastructural study

Abstract Cell death is central to many physiological and pathological processes. As tissue reactions to the two forms of cell death, necrosis and apoptosis, differ, it is critical to distinguish between them. Although ultrastructure is still the definitive means of assessing this, there are very few in vivo studies. Administration of anti-Thy 1 antibody in rats is a model of acute glomerular mesangial cell death due to their expression of the Thy 1.1 epitope. The nature of this process is unclear; apoptosis was suggested from early morphological studies and recent in vitro effects of anti-Thy 1.1 antibody. We have re-examined the changes by electron microscopy, and identified a process of cell necrosis starting within 30 min of anti-Thy1.1 antibody administration. Although there was chromatin condensation, the necrotic features distinctive from apoptosis were: loss of nuclear membranes, cell swelling and degeneration of cytoplasmic organelles, with liberation of chromatin and organelles into the interstitium causing acute inflammation without phagocytic uptake of apoptotic bodies. These findings accord with the known complement dependence of this model. Ultrastructure is a valuable means of differentiating between in vivo necrosis and apoptosis and this is important for understanding the pathogenesis of injury and subsequent tissue responses.

Glomerular Expression and Cell Origin of Transforming Growth Factor-β1 in Anti-Glomerular Basement Membrane Disease

The glomerular expression (mRNA levels) of transforming growth factor‐β1 (TGF‐β1) was assessed in two forms of rat anti‐glomerular basement membrane (GBM) disease, a macrophage‐independent and a macrophage‐de‐pendent variant. After a single intravenous injection of rabbit anti‐rat GBM immune serum, significant proteinuria and histopathologic changes developed in both variants. Increased TGF‐β1 mRNA levels were found in isolated glomeruli of the macrophage‐dependent variant only in which glomerular infiltration by macrophages also occurred. Macrophages isolated from glomeruli of animals with this variant demonstrated TGF‐β1 mRNA levels comparable to those found in glomeruli isolated at the same time point after injection of the anti‐GBM serum. The observations indicate that in anti‐GBM disease, enhanced glomerular TGF‐β1 expression occurs in the macrophage‐dependent variant and suggest that infiltrating macrophages account for this event.

Induced nitric oxide (NO) synthesis in heterologous nephrotoxic nephritis; effects of selective inhibition in neutrophil-dependent glomerulonephritis.

Increased NO synthesis, due to inducible NO synthase (iNOS) activity, is found in macrophage‐associated glomerulonephritis. Little is known about NO in neutrophil‐dependent immune complex inflammation, and its role remains controversial. We therefore studied early phase heterologous nephrotoxic nephritis (HNTN) induced in rats by nephrotoxic globulin and the effects of selective iNOS inhibition of this model. At 2 h of the model iNOS mRNA was induced and nitrite (NO−2) was generated in glomeruli incubated ex vivo (5.2 ± 1.0 nmol/2000 glomeruli per 24 h). There were 14.7 ± 2.2 polymorphonuclear neutrophils (PMN)/glomerulus (normal controls 0.1 ± 0.1). At 8 h urinary protein was 69 ± 15.3 (normal controls 0.6 ± 0.2 mg/24 h). Peritoneal PMN expressed iNOS and produced significant NO−2 (basal 11.2 ± 0.3 nmol/106 cells per 24 h). Selective iNOS inhibition with L‐N6‐(1‐iminoethyl)‐lysine (L‐NIL) in vitro inhibited nephritic glomerular and PMN NO−2 synthesis. In HNTN L‐NIL in vivo significantly suppressed elevated plasma NO−2/NO−3 levels (representative experiment: 17 ± 2 μm, untreated 40 ± 4 μm,P = < 0.01, normal control 18 ± 2 μm). This inhibition did not affect leucocyte infiltration into glomeruli or induce thrombosis. There was no consistent effect on proteinuria. This is the first demonstration of glomerular iNOS induction and high output NO production in the acute phase of PMN‐dependent acute immune complex glomerulonephritis. Selective iNOS inhibition does not affect the primary mechanism of injury (leucocyte infiltration) in this model.

NO and Glomerulonephritis

The various forms of human glomerulonephritis are a group of diseases of largely unknown etiology, which account for a significant proportion of human renal disease. Most have an immune pathogenesis and injury results from either immune complex deposition or delayed hypersensitivity-type reactions within the glomerulus [1]. In the majority of cases, evidence from animal models suggests that immune complexes form in situ (deposition of circulating preformed immune complexes is now considered a rare event, with the possible exception of acute serum sickness). In these in situ reactions, the antigens may be fixed intrinsic epitopes on glomerular cells or matrix molecules (e.g., the Heymann antigen on glomerular visceral epithelial cells, or the Goodpasture antigen in the glomerular basement membrane), or they can be exogenous antigens which localize due to some particular affinity for glomerular structures (e.g., components of bacteria). In the former case, the corresponding autoantibodies form through the breakdown of self-tolerance. Recently, it has been appreciated through the development of new experimental models that some types of glomerulonephritis may arise without soluble antibody formation and have characteristics of type IV immune reactions, involving sensitized T lymphocytes.

Quinacrine mustard: A fluorescent platelet label. In vitro studies and in vivo localisation in habu venom glomerulonephritis in rats

Abstract Platelets labelled with Quinacrine mustard fluoresce green with ultra-violet light. Quinacrine labelled platelets (QLP) were prepared in vitro and 0.5 ml injected i.v. into rats (concn 7–10 × 105/mm3). No adverse effects were detected. QLP comprised 10–19% of total blood platelets. Frozen sections of lung, liver, kidney and spleen up to 4 hours after i.v. QLP showed no abnormal sequestration. Platelet distribution in blood and tissues was studied in glomerulonephritis (gn) induced by Habu snake venom from 10 minutes up to 4 hours after venom. In the initial phase of thrombocytopenia (10 minutes after venom), QLP localised in lung, liver and glomeruli, glomerular distribution being identical with previous electron microscopic findings. In vitro studies of QLP showed aggregation with sodium arachidonate and collagen, but not with ADP. 5HT levels were within the normal range. Rat platelets labelled in vitro with Quinacrine appear to function well in vitro, and in vivo and can be identified in tissues. This may be a valuable method for studying the fate of platelets activated in vivo.