RESUMO
UNLABELLED: Increased severity of glomerulonephritis in C-C chemokine receptor 2 knockout mice. BACKGROUND: The C-C chemokine receptor 2 (CCR2) is expressed on monocytes and facilitates monocyte migration. CCR2 is a prominent receptor for monocyte chemoattractant protein-1 (MCP-1). This chemokine recruits monocytes to sites of inflammation. It has been suggested that CCR2 and its ligand, MCP-1, play a role in the pathogenesis of glomerulonephritis. The goal of this study was to determine the contribution of CCR2 in a murine model of accelerated nephrotoxic nephritis. We measured the extent of development of renal disease in CCR2 wild-type and knockout mice after the administration of antiglomerular basement membrane antibody. METHODS: Eight groups of animals were treated (N = 10 per group). Four days after IgG immunization, CCR2 wild-type and knockout mice received control serum or nephrotoxic serum. The urinary protein/creatinine ratio was measured on days 1 and 3; plasma and kidneys were collected on days 4 and 7. Kidneys were evaluated by light microscopy, immunohistochemistry, and immunofluorescence. The genotype of mice was confirmed by tissue analysis. RESULTS: Protective effects of CCR2 knockout on the urinary protein/creatinine ratio were observed on day 1, as values for this parameter were significantly lower (35 +/- 3.6) than in nephritic wild-type mice (50 +/- 6.8). There was a marked increase in proteinuria in nephritic wild-type mice on day 1 compared with vehicle-treated, wild-type animals (5 +/- 1.0). On day 3, the ameliorative effects of CCR2 knockout were not observed; the increase in the urinary protein/creatinine ratio was similar in nephritic CCR2 wild-type (92 +/- 11.2) and knockout mice (102 +/- 9. 2). Plasma markers of disease were evaluated on days 4 and 7. At these time points, there were no beneficial effects of CCR2 receptor knockout on plasma levels of urea nitrogen, creatinine, albumin, or cholesterol. On day 7, blood urea nitrogen (248 +/- 19.9 mg/dL) and plasma cholesterol were higher in nephritic CCR2 knockout mice than in wild-type mice (142 +/- 41.7 mg/dL) that received nephrotoxic serum. Histopathologic injury was more severe in nephritic CCR2 knockout mice than nephritic wild-type mice on day 4 (3.1 +/- 0.3 vs. 2.0 +/- 0.3) and day 7 (3.6 +/- 0.2 vs. 2.9 +/- 0.3). By immunohistochemical analysis at day 4, there were significantly fewer mac-2-positive cells, representative of macrophages in the glomeruli of nephritic CCR2 knockout (2.1 +/- 0.6) mice than nephritic wild-type (3.9 +/- 0.5) animals. By indirect immunofluorescence, there was a moderate, diffuse linear IgG deposition of equivalent severity present in glomeruli of both wild-type and CCR2 knockout nephritic mice. CONCLUSION: These results suggest that our strategy was successful in reducing macrophage infiltration, but this model of glomerulonephritis is not solely dependent on the presence of CCR2 for progression of disease. After a transient ameliorative effect on proteinuria, CCR2 knockout led to more severe injury in nephritic mice. This raises the intriguing possibility that a CCR2 gene product ameliorates glomerulonephritis in this murine model. Although effects that occur in chemokine knockout mice are not equivalent to those expected with prolonged use of a chemokine antagonist, this study may nevertheless have implications for consideration of long-term use of chemokine antagonists in renal disease.
Assuntos
Glomerulonefrite/patologia , Receptores de Quimiocinas/genética , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Glomerulonefrite/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Receptores CCR2RESUMO
Transgenic mice (T26) bearing the envelope, regulatory, and accessory genes of HIV- I develop renal disease resembling human HIV-associated nephropathy (HIVAN). Effects of vehicle (VEH) and the angiotensin-converting enzyme inhibitor captopril (CAP) were examined in wild-type (WT) or T26 mice treated from 7 to 100 d of age. Mortality was lower in CAP T26 mice (30 mg/kg: 8%; 100 mg/kg: 12%) than VEH T26 mice (52%). The urinary protein/creatinine ratio was increased in VEH T26 mice (19.5+/-7.60) versus WT mice (6.1+/-0.83), but not in low-dose (7.3+/-0.94) or high-dose (8.2+/-1.02) CAP T26 mice. Blood urea nitrogen was higher in VEH T26 mice (52+/-16.2 mg/dl) than VEH WT mice (24+/-0.8). Blood urea nitrogen was also elevated in CAP WT (high dose: 43+/-2.1 mg/dl) and T26 mice (high dose: 42+/-2.4 mg/dl). Glomerular injury was higher in VEH T26 mice (6.8+/-0.58) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 1.1+/-0.17; high dose: 0.7+/-0.13). Tubulointerstitial injury was also greater in VEH T26 mice (1.1+/-0.10) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 0.4+/-0.10; high dose: 0.3+/-0.10). These data validate recent nonrandomized studies of captopril in HIV-infected patients, and suggest that an angiotensin-converting enzyme substrate is an important mediator in HIVAN. A randomized placebo-controlled trial of captopril in HIVAN may be warranted.
Assuntos
Nefropatia Associada a AIDS/prevenção & controle , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , HIV-1 , Nefropatia Associada a AIDS/patologia , Nefropatia Associada a AIDS/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Expressão Gênica , Genes Virais , HIV-1/genética , HIV-1/patogenicidade , Humanos , Rim/patologia , Masculino , Camundongos , Camundongos Transgênicos , Proteinúria/prevenção & controle , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/genéticaRESUMO
Gadolinium is used as a contrast media for magnetic resonance imaging and, experimentally, to block Kupffer cell phagocytosis. In this study, we utilize electron probe microanalysis to determine the subcellular localization of gadolinium chloride (GdCl3) administered to mice in a short-term toxicology study. Male CD-1 mice were administered 0.0, 2.5, or 8.0 mg/kg GdCl3 iv for 14 consecutive weekdays. Liver-associated enzymes were significantly elevated in high-dose animals only and correlated histologically with multifocal, hepatocellular degeneration associated with a neutrophilic infiltrate. Morphological investigations were performed on high-dose animals. Hepatocytes and Kupffer cells had morphologic features of cellular injury consisting of swollen mitochondria and vesiculated profiles of endoplasmic reticulum. Hepatocytes, Kupffer cells, bile canaliculi, and neutrophils in the liver contained discrete aggregates of electron-dense granular material, as did pulmonary interstitial macrophages, splenic macrophages, and mesangial cells of the renal glomerulus. The intracellular granular material in the liver, lung, spleen, and kidney was confirmed as gadolinium by qualitative electron probe microanalysis. These results document both hepatic and extra-hepatic accumulation of gadolinium in cells of the mononuclear phagocytic system and highlight the importance of electron probe microanalysis in toxicologic assessment.
Assuntos
Gadolínio/farmacologia , Animais , Microanálise por Sonda Eletrônica/métodos , Rim/efeitos dos fármacos , Rim/ultraestrutura , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Masculino , Camundongos , Baço/efeitos dos fármacos , Baço/ultraestruturaRESUMO
Recent reports imply or claim that amylase activity is inhibited in hyperlipemic sera, because diluted samples showed greater activities when assayed by a starch-iodine method. We find that dilutions of both clear and lipemic samples give higher-than-expected activities when assayed by a starch-iodine method, an effect attributable to the variable effects of protein, turbidity, and triglycerides in the hyperlipemic samples. Thus the starch-iodine method is unsuitable for assessing the effects of hyperlipemic samples on amylase activity. To do so, we used an alternative method, in which soluble dyed amylopectin is used as the substrate. This method exhibits apparent zero-order kinetics, and we detected no interfering factors. Plots of sample volume (x) vs. activity (y) for clear and hyperlipemic (triglycerides up to 80 g/liter) sera gave straight lines with y-intercepts of zero. Evidently hypertriglyceridemia does not inhibit amylase activity.
Assuntos
Amilases/sangue , Hiperlipidemias/sangue , Amilopectina/metabolismo , Colorimetria , Iodo , Cinética , Métodos , Amido/metabolismoAssuntos
Etilenodiaminas/farmacologia , Níquel , Animais , Bile/metabolismo , Glicemia/metabolismo , Fezes/análise , Glucagon/sangue , Insulina/sangue , Masculino , Níquel/metabolismo , Níquel/toxicidade , Proteinúria/induzido quimicamente , Radioisótopos , Ratos , Ratos Endogâmicos F344 , Aminoacidúrias Renais/induzido quimicamente , Fatores de TempoAssuntos
Níquel/farmacologia , Proteinúria/induzido quimicamente , Aminoacidúrias Renais/induzido quimicamente , Aminoácidos/análise , Animais , Nitrogênio da Ureia Sanguínea , Feminino , Glomérulos Renais/patologia , Proteínas/análise , Proteinúria/patologia , Ratos , Aminoacidúrias Renais/patologia , Fatores de TempoRESUMO
The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.