Recombinant tumor necrosis factor stimulates interleukin-1 production in glomerular cultures from rats with nephrotoxic serum nephritis

We have investigated the effect of recombinant tumor necrosis factor (r-TNF) on the release of interleukin-1 (IL-1) in glomerular cultures from rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN). Freshly isolated glomeruli from the NTSN rats were incubated for 24 h in the presence of r-TNF. When the glomeruli were activated by r-TNF substantial amounts of IL-1 could be detected in the supernatants as measured by the mouse thymocyte assay. The r-TNF-induced IL-1 activity was significantly higher in rats with NTSN than those in normal controls and the other control group, consisting of preimmunized rats (rabbit IgG), then given normal rabbit globulin instead of NTS. To avoid the effect of prostaglandins on the IL-1 assay, we cultured the glomeruli with addition of indomethacin and assayed IL-1 activity in the culture supernatants. This cyclooxygenase inhibitor augmented r-TNF-induced IL-1 production. Our data suggest that r-TNF can serve as a potent stimulator of IL-1 synthesis in glomerular cultures from rats with NTSN.     

LTB4 in nephrotoxic serum nephritis in rats

We studied leukotriene B4 (LTB4) synthesis in isolated glomeruli of rats with nephrotoxic serum nephritis. This nephritis was induced in male Sprague Dawley rats by injecting one proteinuric dose of nephrotoxic serum (rabbit anti-rat-GBM serum) after prior immunization of the rats with rabbit IgG. Histological and analytical examinations were performed in kidneys perfused until free of blood 6, 12, 24, 48 and 72 hours after induction of the disease. To investigate LTB4 production, glomeruli were isolated and incubated for one hour in the presence of Ca++-ionophore A23187. The supernatants were analyzed for LTB4. The peak comigrating on reverse-phase high performance liquid chromatography (RP-HPLC) with reference LTB4 was isolated. The ethyl ester trimethylsilyl ether derivative of this peak was analyzed by gaschromatography-mass spectrometry (GC/MS). Identical spectra of the glomerular samples and of reference LTB4 in the positive and in the negative ion chemical ionization mode provided unequivocal evidence that the substance released from the nephritic glomeruli was indeed LTB4. Six hours after injection of nephrotoxic serum, glomerular LTB4 release was highest with 5.52 +/- 0.50, then declining to 2.20 +/- 0.10 ng/mg glomerular protein at 12 hours. At 24, 48 and 72 hours no statistically significant difference from control animals was found. No metabolism of LTB4 to 20-hydroxy- or 20-carboxy-LTB4 was detected during the incubation period. Albuminuria developed during the first 24 hours after nephrotoxic serum challenge and rose steadily throughout the observation period up to 277 +/- 25 mg/24 hr after 72 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renal dendritic cells stimulate IL-10 production and attenuate nephrotoxic nephritis

The role of renal dendritic cells (DCs) in glomerulonephritis is unknown. This question was addressed in nephrotoxic nephritis, a murine model of human necrotizing glomerulonephritis, which is dependent on CD4(+) Th1 cells and macrophages. DCs in nephritic kidneys showed signs of activation, accumulated in the tubulo-interstitium, and infiltrated the periglomerular space surrounding inflamed glomeruli. In ex vivo coculture experiments with antigen-specific CD4(+) T cells, DCs stimulated the secretion of IL-10, which is known to attenuate nephrotoxic nephritis, and the Th1 cytokine IFNgamma. Endogenous renal CD4(+) T cells produced both of these cytokines as well, but those from nephritic mice secreted increased amounts of IL-10. Renal DCs were found to express ICOS-L, an inducer of IL-10. To evaluate the in vivo role of renal DCs in disease, CD11c(+) DCs were depleted on days 4 and 10 after the induction of nephritis by injecting CD11c-DTR/GFP mice with diphtheria toxin. Sparing DCs until day 4 did not affect the autologous phase of nephritis. The number of renal DCs was reduced by 70% to 80%, the number of renal macrophages was unchanged, and periglomerular infiltrates were eliminated. On days 11 to 14, we observed aggravated tubulointerstitial and glomerular damage, reduced creatinine clearance, and increased proteinuria. These findings demonstrate that renal DCs exert a renoprotective effect in nephrotoxic nephritis, possibly by expressing ICOS-L and/or by inducing IL-10 in infiltrating CD4(+) Th1 cells.     

Urinary eicosanoids and the assessment of glomerular inflammation.

Nephrotoxic nephritis, a model system for glomerulonephritis, is characterized by glomerular inflammation, proteinuria, and a marked increase in ex vivo glomerular eicosanoid production. This study addressed whether urinary eicosanoids might serve as noninvasive markers for glomerular inflammation and damage with nephrotoxic nephritis and its accelerated variant. Accelerated nephritis, relative to simple nephritis, was characterized by more substantial glomerular inflammation, particularly that due to neutrophils. Correspondingly, accelerated nephritis was accompanied by greater proteinuria and more marked elevations in glomerular eicosanoids generated ex vivo. With respect to urinary eicosanoids, thromboxane, but not leukotriene B4, was detected in the urine of normal animals. After the induction of nephrotoxic nephritis, urinary thromboxane was moderately elevated (twofold) and urinary leukotriene B4 was variably present (three of seven animals). In accelerated nephritis, urinary thromboxane was more markedly elevated (sixfold) and leukotriene B4 was consistently present. The presence of urinary leukotriene B4 was confirmed by gas chromatography/mass spectrometry. Urinary eicosanoids together correlated with glomerular leukocyte numbers and proteinuria by linear regression. Urinary leukotriene B4 individually correlated with glomerular neutrophil numbers. Renal metabolism of leukotriene B4 to omega oxidation products by the rat kidney was not apparent. These data validate that the enhanced glomerular eicosanoid metabolism seen in nephrotoxic nephritis takes place in vivo and additionally suggest that both urinary thromboxane and leukotriene B4 may serve as noninvasive markers for glomerular inflammation and damage. In light of these and prior studies, urinary thromboxane may be a general marker of glomerular inflammation and leukotriene B4 may be a more specific index of acute inflammation.     

Interferon-beta reduces proteinuria in experimental glomerulonephritis

Interferon-beta (IFN-beta) is a multifunctional cytokine with immunomodulatory properties. We examined the effect of IFN-beta in three separate rat models of glomerular injury and in cultured human glomerular endothelial cells and podocytes. In nephrotoxic nephritis in WKY rats, recombinant rat IFN-beta started either at induction or after establishment of disease significantly reduced 24-h proteinuria by up to 73% and 51%, respectively, but did not affect serum creatinine. There was a slight reduction in numbers of glomerular macrophages, but no difference in glomerular or tubulointerstitial scarring. In Thy-1 nephritis in Lewis rats, IFN-beta started at induction of disease reduced proteinuria by up to 66% with no effect on numbers of glomerular macrophages, but a reduced number of proliferating cells. In puromycin nephropathy in Wistar rats, IFN-beta started at induction of disease reduced proteinuria by up to 93%, but had no effect on glomerular histology. In cultured cells, human IFN-beta-1a had a dramatic effect on barrier properties, increasing electrical resistance across monolayers of either glomerular endothelial cells or podocytes and decreasing trans-monolayer passage of albumin. In conclusion, these results show that IFN-beta reduces proteinuria in three different rat models of glomerular injury and that its anti-proteinuric action may result from direct effects on cells that comprise the glomerular filtration barrier. These data indicate that IFN-beta may have potential as a therapeutic agent in proteinuric renal disease.     

Prevention and treatment of experimental crescentic glomerulonephritis by blocking tumour necrosis factor-alpha.

BACKGROUND: The mechanisms controlling progression of glomerulonephritis are poorly understood, but there is increasing evidence that tumour necrosis factor-alpha (TNF-alpha) plays a central role in many aspects of glomerular inflammation and scarring. We investigated the role of TNF-alpha in an experimental model of crescentic glomerulonephritis in Wistar Kyoto (WKY) rats by continuously blocking endogenous TNF-alpha, using its soluble receptor sTNFr p55, both before and after establishment of nephritis. METHODS: Glomerulonephritis was induced by a single intravenous injection of 0.1 ml nephrotoxic serum. In the first experiment, rats were pre-treated with sTNFr p55 2 mg/kg intraperitoneally 1 hour before induction of nephritis and on a daily basis thereafter until day 4. In the second experiment, a similar protocol was followed, but treatment with sTNFr p55 was continued until day 10. In the third experiment, treatment with sTNFr p55 was delayed until 4 days after induction of nephritis and continued until day 10. The effects of treatment on renal function, renal histology, cellular infiltration, activation and proliferation, and IL-1beta expression were assessed by standard methods. RESULTS: In the first experiment, short-term treatment with sTNFr p55 caused a marked reduction in albuminuria and fibrinoid necrosis. It also reduced glomerular cell infiltration, activation and proliferation. In the second experiment, prolonged treatment with sTNFr p55 caused a sustained reduction in albuminuria and all histological and cellular parameters of glomerular inflammation; in particular it completely prevented the development of crescents. In the third experiment, delayed therapy of established nephritis with sTNFr p55 significantly reduced albuminuria and glomerular inflammation, including the prevalence of crescent formation. In both long-term experiments, there was less glomerular expression of IL-1beta and lower serum concentrations of IL-beta in sTNFr p55-treated rats. CONCLUSIONS: This study shows that neutralization of endogenous TNF-alpha is effective in preventing acute glomerular inflammation and crescent formation, and in treating established disease, in a rat model of crescentic nephritis. These results may have therapeutic implications for human glomerulonephritis.     

Fifteen-S-hydroxyeicosatetraenoic acid (15-S-HETE) specifically antagonizes the chemotactic action and glomerular synthesis of leukotriene B4 in the rat.

In models of experimental glomerulonephritis, there is temporal concordance between the shift in the glomerular cellular infiltrate from neutrophils (PMN) to macrophages/monocytes and the suppression of glomerular leukotriene B4 (LTB4) generation. Since macrophages are a rich source of 15-lipoxygenase (15-LO) products, we investigated whether the principal product of arachidonate 15-lipoxygenation, 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), was capable of antagonizing the proinflammatory actions of LTB4 in the rat. PMN exhibited chemotaxis to LTB4 in a dose dependent manner with an LC50 of 10(-8) M. When rat neutrophils were pre-treated with 15-S-HETE, chemotaxis to LTB4 was inhibited in a dose dependent manner (maximal at 30 microM 15-S-HETE) but, the same concentration did not inhibit chemotaxis to n-formyl-1-methionyl-1-phenylalanine (FMP). 12-S-HETE (30 microM) did not inhibit chemotaxis to LTB4. Glomeruli from rats injected with nephrotoxic serum three hours earlier generated increased levels of LTB4; prior exposure of such glomeruli to 15-S-HETE totally normalized LTB4 production. The glomerular production of 15-S-HETE and LTB4 was also determined 3 hours, 72 hours and 2 weeks after administration of nephrotoxic serum. Whereas there was an early, short-lived, burst of LTB4 followed by a return to baseline levels, the production of 15-S-HETE increased steadily over the two week period and was present in amounts fivefold greater than LTB4. Thus, these studies assign a role for locally generated 15-LO derivatives in arresting LTB4-promoted PMN infiltration and suppressing LTB4 synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of plasminogen activator inhibitor on nephrotoxic nephritis and its modulation by tumor necrosis factor.

To evaluate the suggested imbalance between coagulation and fibrinolysis in the development of glomerulonephritis, plasminogen activator inhibitor (PAI) activity was studied in the plasma of rats with nephrotoxic nephritis. PAI activity rose within 1 h of the injection of nephrotoxic globulin (NTG), peaked at 2 h and returned to the normal range within 24 h. PAI activity was dependent on the dose of NTG and increased significantly during passage through the kidney. PAI activity was also detected in the culture supernatant from isolated glomeruli with nephrotoxic nephritis. Intracapillary fibrin deposits were formed within 2 h; their numbers increased gradually over 24 h. Tumor necrosis factor (TNF) also induced a progressive increase in PAI activity in normal rats. The injection of TNF to rats with NTG synergistically accelerated both the increase in PAI activity and the prevalence of fibrin deposits. These results suggest that PAI may be released from the glomeruli affected by nephrotoxic nephritis and imply that PAI may play a role in the local coagulation in the capillaries of the nephritic kidneys, although this is probably not the only mechanism which explains the continued formation of the glomerular fibrin deposits.     

Role of interleukin-10 in rat mesangioproliferative glomerulonephritis

SUMMARY: Interleukin-10 (IL-10) has been recognized as a growth factor for rat mesangial cells in vitro ; however, its role in mesangioproliferative glomerulonephritis is unknown. We studied the expression of IL-10 mRNA in the rat anti-Thy-1 model of mesangioproliferative glomerulonephritis (experiment 1) and, subsequently, the effects of blocking IL-10 during anti-Thy-1 nephritis using the IL-10 inhibitor, AS101 (experiment 2). In experiment 1, PCR analysis failed to detect IL-10 mRNA in normal rat kidney, however, a clear signal for IL-10 mRNA was evident on day 6 of anti-Thy-1 nephritis. In situ hybridization showed IL-10 mRNA expression in focal glomerular areas in anti-Thy-1 nephritis. Combined in situ hybridization and immunohistochemistry showed that glomerular IL-10 mRNA was expressed by both macrophages and mesangial cells. In experiment 2, treatment with AS101 significantly downregulated renal IL-10 gene expression, as demonstrated by semiquantitative PCR. However, the induction of glomerular hypercellularity, mesangial proliferation (PCNA+ cells), mesangial cell activation (α-SMA expression) and macrophage accumulation (ED1+ cells) seen in saline-treated anti-Thy-1 nephritis was unaffected by AS101 treatment. In conclusion, renal IL-10 gene expression is upregulated during pathological mesangial cell proliferation in rats with anti-Thy-1 nephritis. However, the inability of IL-10 suppression with AS101 to prevent anti-Thy-1 disease suggests that IL-10 is not essential for pathological mesangial cell proliferation.     

Indomethacin reduces proteinuria in passive Heymann nephritis in rats

Indomethacin has been used to lower proteinuria in human glomerular diseases with controversial results. The mechanism of indomethacin beneficial effects has not been established. A possible explanation is that indomethacin reduces proteinuria by inhibiting the synthesis of renal prostaglandins (PGs); however, appropriate studies to address this issue have never been done. The objectives of the present study were: to investigate whether indomethacin influences protein excretion in an experimental model of immunologically-mediated glomerular disease; to establish if the possible favorable effect of indomethacin on proteinuria is related to a reduction in glomerular filtration rate (GFR); to establish the possible association between the antiproteinuric effect of indomethacin and its inhibitory effect on arachidonic acid (AA) metabolites of renal or extrarenal origin; and to further investigate the relationship between proteinuria and renal thromboxane (Tx) synthesis previously demonstrated in experimental models of nephrotoxic nephritis and adriamycin (ADR) nephrosis. To this purpose we used an experimental immune-complex disease, passive Heymann nephritis (PHN) which was induced in the rat by a single intravenous (i.v.) injection of heterologous serum directed against a brush border component (gp 330 antigen). Indomethacin at a dose of 6 mg/kg intraperitoneally (i.p.) administered for four consecutive days to PHN animals during the period of heavy proteinuria, effectively reduced urinary protein excretion. The reduction in proteinuria does not appear to be a consequence of a reduction in GFR as documented by inulin clearance. Glomerular synthesis and urinary excretion of vasodilatory prostacyclin (PGI2) and PGE2 were decreased or unchanged in PHN animals in respect to control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional significance of leukotriene B4 in normal and glomerulonephritic kidneys.

Leukotriene B4 (LTB4) is the major 5-lipoxgenase product released during early experimental glomerulonephritis. To test its functional relevance, its actions in the normal rat kidney and its influence on renal function in the heterologous phase of mild nephrotoxic serum-induced glomerular injury were examined. Intrarenal administration of leukotriene B4 resulted in mild vasorelaxant and natriuretic responses which were shared by 12(R)-hydroxyeicosatetraenoic acid but not 12(S)-leukotriene B4 or 12(S)-hydroxyeicosatetraenoic acid, suggesting activation of a common recognition site with a requirement for 12(R) stereochemistry. The polymorphonuclear cell-specific activator, N-formyl-Met-Leu-Phe, stimulated leukotriene B4 production from isolated perfused kidneys harvested from nephrotoxic serum-treated rats to a significantly greater degree than from control animals treated with nonimmune rabbit serum. The renal production of leukotriene B4 correlated directly and strongly (r = 0.79, P less than 0.01) with renal myeloperoxidase activity, suggesting interdependence of leukotriene B4 generation and polymorphonuclear cell infiltration. In vivo, intrarenal administration of leukotriene B4 to rats with mild nephrotoxic serum-induced injury was associated with an increase in polymorphonuclear cell infiltration, reduction in renal plasma flow rate, and marked exacerbation of the fall in glomerular filtration rate, the latter correlating strongly with the number of infiltrating polymorphonuclear cells/glomerulus, whereas inhibition of 5-lipoxygenase led to preservation of glomerular filtration rate and abrogation of proteinuria. Thus, although devoid of vasoconstrictor actions in the normal kidney, increased intrarenal generation of leukotriene B4 during early nephrotoxic serum-induced glomerular injury amplifies leukocyte-dependent reductions in glomerular perfusion and filtration rates, likely due to enhancement of polymorphonuclear cell recruitment/activation.     

Pentoxifylline attenuates experimental mesangial proliferative glomerulonephritis.

BACKGROUND: Accumulation of glomerular macrophages, proliferation of mesangial cells (MCs), and deposition of extracellular matrix proteins are pathobiological hallmarks of glomerulonephritis. We previously reported that a clinically available nonselective inhibitor of cyclic 3',5'-nucleotide phosphodiesterase, pentoxifylline (PTX), inhibits proliferation of cultured rat MCs, as well as collagen production by these cells. In this study, we investigated the in vivo effects of PTX on rat anti-Thy1 disease, a model of mesangial proliferative nephritis. METHODS: Anti-Thy1 nephritis was induced in Sprague-Dawley rats by injecting mouse anti-rat Thy1 antibodies intravenously. Nephritic rats were randomly assigned to receive PTX (0.1 g/kg/day) or vehicle (phosphate-buffered saline) and were sacrificed at various time points. Paraffin kidney sections were stained with hematoxylin and periodic acid-Schiff reagents for glomerular histology. Frozen kidney sections were stained by monoclonal antibodies against proliferating cell nuclear antigen, ED-1, and alpha-smooth muscle actin and were visualized by color development from a horseradish peroxidase reaction. Monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and various extracellular matrix mRNAs were analyzed by Northern blotting. Urine protein concentrations were determined by Lowry's method. RESULTS: Nephritic rats treated with PTX excreted less urinary protein on day 5 of nephritis than vehicle-treated nephritic rats. In periodic acid-Schiff-stained kidneys from PTX-treated nephritic rats, there was attenuation of both glomerular cellularity and glomerular sclerosis compared with vehicle-treated nephritic rats. PTX decreased the augmented glomerular mRNA levels of MCP-1 and ICAM-1 at two hours and on day 1 of nephritis. Immunoreactive staining showed that PTX reduced the number of proliferating glomerular macrophages on days 1, 2, and 3, but not at two hours of nephritis, compared with vehicle-treated nephritic rats. On day 5, PTX decreased the number of activated proliferating MCs and attenuated the glomerular mRNA levels of type I (alpha1), type III (alpha1), and type IV (alpha1) collagen and fibronectin compared with vehicle-treated nephritic rats. CONCLUSION: The administration of PTX to rats with anti-Thy1 disease reduces accumulation and proliferation of glomerular macrophages, attenuates proteinuria, suppresses activation and proliferation of MCs, and ameliorates glomerular sclerosis. These results suggest that PTX may have a suppressive effect in acute phases or relapses of mesangial proliferative glomerulonephritis.     

Induction of urokinase receptor expression in nephrotoxic nephritis.

The urokinase receptor (uPAR) is a multifunctional molecule involved in pericellular, fibrinolytic, and proteolytic activities, as well as in cell adhesion and chemotaxis and may play a role in the pathogenesis of tissue remodeling occurring during glomerulonephritis. We analyzed sequentially the expression of uPAR by immunohistochemistry and in situ hybridization in an accelerated model of nephrotoxic nephritis in rats. A strong induction of uPAR mRNA expression was observed in glomeruli as soon as 1 h after nephrotoxic serum injection. The intensity of glomerular uPAR mRNA and antigen expression increased and peaked at 24 h. At that time, numerous glomerular fibrin deposits, monocyte/marcrophage infiltration, and heavy proteinuria were observed. Fibrin deposition was detected at 6 h, peaked at 24 h, and progressively declined over the next 3 weeks, while uPAR antigen expression remained elevated until the end of the study (3 weeks). By double labeling, we showed that the expression of uPAR was mediated by both intrinsic glomerular cells and infiltrating macrophages. Severe podocytic lesions developed within 3 days after antiserum injection, and glomerulosclerosis rapidly progressed within 2-3 weeks. These results show that glomerular uPAR expression is induced in nephrotoxic nephritis and suggest that uPAR may promote local proteolysis and also tissue remodeling, leading to the late development of glomerulosclerosis.     

Tubulointerstitial nephritis during the heterologous phase of nephrotoxic serum nephritis

This study was designed to characterize the immunopathology of acute tubulointerstitial disease in nephrotoxic serum nephritis in nonsensitized rats. Groups of Lewis rats were studied at 12 time periods ranging from 10 min to 28 days after nephrotoxic serum injection. Nephritic rats developed interstitial nephritis during the acute heterologous phase of renal injury. Coincident with the focal deposition of nephrotoxic antibodies along tubular basement membranes at 24 h, an influx of polymorphonuclear cells and macrophages was evident. The most prominent infiltrate, present between days 3 and 7, was dominated by macrophages with smaller numbers of lymphocytes that were mainly cytotoxic T cells. Dual-labeling studies demonstrated the colocalization of linear tubular basement membrane deposits of the nephrotoxic antibody with focal clusters of interstitial lymphohemopoietic cells. Increased complement deposition was not evident along the tubular basement membranes; moreover, C3 depletion with cobra venom factor failed to attenuate the interstitial inflammation. During the late autologous phase of glomerulonephritis, tubular basement membrane deposits of rat IgG did not appear and the interstitial disease resolved. The results of this study demonstrate that the heterologous phase of nephrotoxic serum nephritis is an antibody-mediated disease directed against the basement membranes not only of the glomeruli but also of some tubules. Antibody deposition is followed by an acute influx of phagocytic cells to both regions of the kidney. These cells may play an important role in the genesis of acute interstitial injury and chronic interstitial fibrosis associated with antiglomerular basement membrane nephritis.     

Glomeruli synthesize nitrite in experimental nephrotoxic nephritis

Activated macrophages synthesize nitric oxide (NO) from L-arginine. In culture, the major stable end product is nitrite (NO2). Activated macrophages accumulate in glomeruli and are responsible for injury in experimental immune complex glomerulonephritis. We examined NO2- production by isolated glomeruli and urinary NO2- in accelerated nephrotoxic nephritis in the rat. Normal glomeruli did not produce NO2- spontaneously or when stimulated with lipopolysaccharide (LPS) (1 microgram/ml) or A23187 (2 microgram/ml). Cultured mesangial cells at first or seventh passage did not produce NO2- spontaneously or when stimulated. Nephritic glomeruli spontaneously produced NO2 at all times studied; this production was maximal at 24 hours after induction of glomerulonephritis (158.4 +/- 8.4 nmol/48 hr/ml, N = 3). The production of NO2- was inhibited 75 to 100% by NG-monomethyl-L-arginine (L-NMMA), and this inhibition was reversed by L-arginine, indicating NO2- production from L-arginine via NO. The production of NO2- was increased by LPS (1 microgram/ml) at 2, 7 and 21 days. NO2- was undetectable in normal rat urine; however, it was present in urine of rats with glomerulonephritis (Day 0 to 1:8161 +/- 2605 nmol/24 hr. N = 12). The production of NO in nephritic glomeruli may have implications for both the mechanism of glomerular injury and glomerular hemodynamics.     

Adjuvant-induced macrophage-dominant nephrotoxic serum nephritis in rats

Background To investigate cellular mechanisms responsible for the development of crescentic glomerular lesions, we have studied the effects of complete adjuvant (CA) on subnephritogenic nephrotoxic serum (NTS) nephritis (NTS-N) in Brown Norway rats.Methods A subnephritogenic dose of NTS, 0.02ml per rat, was intravenously injected into rats that were previously (14 days earlier) treated intradermally with CA, incomplete adjuvant, or PBS.Results Proteinuria developed by day 2 only in CA-treated rats. Also, severe glomerular lesions associated with cellular crescent formation developed by day 2 only in the CA-treated rats. The glomerular extent of infiltration of IL-1-positive cells, MCP-1 expression level, and the number of ED1 and PCNA double-positive activated macrophages peaked on day 4 and decreased thereafter. The numbers of T lymphocytes and polymorphonuclear neutrophils did not significantly increase throughout the experiments.Conclusions These results indicate that CA can potently induce crescentic NTS-N characterized by the infiltration of activated macrophages, which presumably bind to rabbit IgG on the glomerular basement membrane and may play a critical role in the development of severe NTS-N associated with crescent formation.     

Production of reactive oxygen species and scavenger treatment in nephrotoxic nephritis]

Role of reactive oxygen species (ROS) was studied in accelerated nephrotoxic nephritis (NTN) in rats. In this experimental model, histological examination, and luminol amplified chemiluminescence (CL) assay of peripheral polymorphonuclear neutrophils (PMN), peritoneal macrophages (M phi), and isolated glomeruli were performed time-sequentially. Effect of ROS scavengers were also examined in this experiment. Daily dosages of bovine liver catalase and SOD were 550,000 and 1,000 units respectively. After nephrotoxic IgG injection, CL of glomeruli increased strikingly attaining peak at day 1, and remained high until the end of the experiment. This increase of CL may have reflected the release of ROS by glomerular cells and/or infiltrating cells stimulated in situ. In fact, peripheral PMN and peritoneal M phi showed no increase of CL after nephrotoxic IgG injection. Glomerular cells increased as early as 3 hours after induction of nephritis. Accumulation of PMN was noted for the first three days, whereas that of M phi became prominent after 4 days. Favourable effect was obtained in terms of proteinuria by administration of catalase, only when catalase was given at initial 3 days of nephritis. The data suggest that generation of ROS reflected by increase of CL in glomeruli of NTN rats is attributable to the PMN and M phi infiltrated in glomeruli as well as glomerular resident cells per se. It is also suggested that glomerular PMN increasing in the early phase of NTN plays a considerable role in glomerular injury.     

A simple method to identify NBT-positive cells in isolated glomeruli

Background. Reactive oxygen radicals are probably involved in the pathogenesis of human and experimental models of renal disease, yet current methods are inadequate to quantify and identify the cells producing reactive oxygen radicals. Methods and Results. We used the nitroblue tetrazolium reaction to determine superoxide anion production in glomerular cells in phorbol myristate-stimulated glomerular suspensions and in isolated glomeruli from rats with nephrotoxic nephritis, ureteral obstruction, and puromycin aminonucleoside nephrosis. We were also able to identify these nitroblue tetrazolium+cells using specific appropriate antibodies. When the technique was tested in conditions known to increase reactive oxygen radicals, as phorbol myristate-stimulated glomeruli and glomeruli from animals with nephrotoxic nephritis and ureteral obstruction, increased number of nitroblue tetrazolium+cells were found. These cells were identified as glomerular intrinsic cells (Thy-1 +) or infiltrating leukocytes (leukocyte common antigen+ or antineutrophil+). Conclusions. This method may be useful to determine cells participating in glomerular damage induced by reactive oxygen radicals.