An integrated proteomic approach to studying glomerular nephrotoxicity.

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.

Pulmonary response of fifth component of complement-sufficient and -deficient mice to hyperoxia.

The fifth component of complement, C5, can form fragments that cause neutrophil chemotaxis, oxygen radical production, and lysosomal enzyme release. The purpose of this study was to determine if C5 and these fragments contribute to the inflammation seen in pulmonary oxygen toxicity as defined by histology and analysis of bronchoalveolar lavage fluid (BALF). In addition, the role of C5 in producing mortality in the animals was addressed. Pairs of C5 deficient (C5-) and C5 sufficient (C5+) mice, 6 mo or older, were placed in a chamber and challenged with 95% oxygen at ambient pressure. A significant difference in mortality was observed after 200 h of exposure, i.e., 90% mortality in C5+ mice vs. 25% mortality in C5- mice (P less than 0.001). This difference in mortality was not seen when C5- mice were transfused with plasma that contained C5 derived from C5+ mice. Morphometric analysis of histologic sections with light microscopy revealed earlier pathologic changes in C5+ mice that was characterized by increased cellularity due in part to neutrophil influx into the alveolar-capillary wall. Transmission electron microscopy also confirmed an earlier inflammatory response in the C5+ mice with evidence of injury to alveolar epithelial cells, interstitial edema, and an increase in the cellular component of the interstitium. Analysis of BALF also demonstrated earlier abnormalities in C5+ mice, which included a significantly greater percentage of neutrophils in the C5+ mice at 117 h. Similar studies in younger mice of these strains again showed earlier neutrophil accumulation in C5+ mice, but the time course of the injury was more protracted. This study shows that the presence of C5 is associated with a greater mortality and an earlier influx of neutrophils into murine lungs. However, in the absence of C5, neutrophils will still immigrate into the lung and hyperoxic damage will occur at a later time point, which demonstrates the inherent redundancy of the inflammatory process.

The pulmonary response of C5 sufficient and deficient mice to Pseudomonas aeruginosa.

Neutrophils have been shown to be important in the clearance of Pseudomonas aeruginosa from murine lungs. The mechanisms responsible for the neutrophil influx into the lungs, however, remain poorly defined. This study was undertaken to define the contribution to this inflammatory process by the C5 molecule or its fragments. Congenic C5 sufficient (B10.D2/nSn) and CS deficient (B10.D2/oSn) mice were challenged by intrapulmonary administration of Pseudomonas aeruginosa. Differences in survival of the 2 strains of mice were noted over a 6-day period. In addition, host response was assessed at 6-, 24-, and 48-h time points by histologic examination, analysis of cells from pulmonary lavage, analysis of cells in the peripheral blood and culture of the lungs and blood of challenged mice. Mortality was consistently higher in C5 deficient mice. Neutrophil accumulation within the lung was greater at the 6-h time point in the C5 sufficient mice, and greater at the 48-h time point in the C5 deficient mice as demonstrated by both lavage and histologic examination. Differences in neutrophil accumulation could not be explained by differing blood neutrophils concentrations in the mice before challenge, or a lack of mobilization of neutrophils into the peripheral circulation after challenge in the C5 deficient mice. An early lack of clearance of the bacteria from the lungs of the C5 deficient strain was documented. We conclude that the C5 molecule and its phlogistic fragments are important neutrophil chemotaxins in murine lungs exposed to Pseudomonas aeruginosa, and, quantitatively, may be the most important early stimulus for neutrophil accumulation in this model.

Absence of inflammatory lung injury in rabbits challenged intravascularly with complement-derived chemotactic factors.

Systemic activation of the complement system results in the generation of chemotactic factors that have been suggested to play a role in the pathogenesis of inflammatory pulmonary diseases such as the adult respiratory distress syndrome. This led us to ask whether systemic complement activation by cobra venom factor (CVF) or intravascularly administered zymosan-activated rabbit plasma (ZAP) or rabbit C5a would result in lung injury. As had been described previously for CVF and ZAP, intravenously administered rabbit C5a also caused an acute neutropenia along with sequestration of neutrophils within the pulmonary vasculature. However, no significant lung inflammation as measured by neutrophil emigration or increased vascular permeability occurred with any of the three stimuli. Only when these agents were combined with anesthesia, surgical manipulation, and intubation did significant neutrophil emigration into alveoli occur, but again without any change in vascular permeability. After administration of ZAP, a decrease in dynamic compliance and an increase in pulmonary resistance as well as a transient period of hypoxemia occurred that was not observed after CVF or rabbit C5a treatment. Thus, our studies suggest that changes in lung function after ZAP instillation may not represent changes from complement activation alone in that they are not reproduced with CVF or rabbit C5a. We conclude that complement activation, as an isolated event, may be an insufficient insult in the lung to produce significant lung injury.

Pulmonary microvascular alterations and injury induced by complement fragments: synergistic effect of complement activation, neutrophil sequestration, and prostaglandins.

Fragments of C5 that are generated at, or administered to, extravascular sites in the pulmonary parenchyma induced neutrophil infiltration, edema, tissue damage, and a complete inflammatory response. Generation of C5 fragments within the vascular system induced leukocyte sequestration in the pulmonary vasculature, but without detectable increased vascular permeability or neutrophil migration. By contrast, the combination of short episode of hypoxemia with the intravascular C5 activation led significant increases in pulmonary vascular permeability, mild endothelial alterations, and emigration of neutrophils. Infusion of 10 micrograms PGE2 into animals in which intravascular complement had been activated produced changes in the lungs that were similar to, though less severe than, the combination of hypoxia and complement activation.

The pulmonary response of C5 sufficient and deficient mice to immune complexes.

Evidence based on inhibitor studies has been presented showing that immune complex-induced alveolitis is complement dependent. This study was undertaken to define the contribution to this inflammatory process by the C5 molecule or its fragments. Congenic C5 sufficient (B10.D2/nSn) and C5 deficient (B10.D2/oSn) mice were challenged by intrapulmonary administration of antigen-antibody complexes of bovine serum albumin/rabbit antibovine serum albumin prepared at equivalence. Injury was assessed at 6 and 48 h time points by histologic examination, analysis of cells from pulmonary lavage, and determination of wet/dry weight ratios of both treated and untreated lungs. Histologic findings at both time points revealed a more generalized and severe inflammatory infiltrate in C5 sufficient animals with neutrophil accumulation, edema, and hemorrhage. The differences in neutrophil accumulation were confirmed by pulmonary lavage where C5 sufficient animals had a significantly greater number and percentage of neutrophils at both time points. Wet/dry weight ratios of lung also reflected more severe damage in the C5 sufficient animals. We concluded that the C5 molecule and its phlogistic fragments are important mediators of acute inflammation in immune complex lung injury, and, quantitatively, may be the most important stimuli for neutrophil accumulation in this model.