Bacterial endotoxin (LPS) is responsible for much of the widespread inflammatory response seen in sepsis, a condition often accompanied by acute renal failure (ARF). In this work we report that mice deficient in TNFR1 (TNFR1−/−) were resistant to LPS-induced renal failure. Compared with TNFR1+/+ controls, TNFR1−/− mice had less apoptosis in renal cells and fewer neutrophils infiltrating the kidney following LPS administration, supporting these as mediators of ARF. TNFR1+/+ kidneys transplanted into TNFR1−/− mice sustained severe ARF after LPS injection, which was not the case with TNFR1−/− kidneys transplanted into TNFR1+/+ mice. Therefore, TNF is a key mediator of LPS-induced ARF, acting through its receptor TNFR1 in the kidney.

Toll-like receptor 4 (TLR4) is present on monocytes and other cell types, and mediates inflammatory events such as the release of TNF after exposure to LPS. C3H/HeJ mice are resistant to LPS-induced mortality, due to a naturally occurring mutation in TLR4. We therefore hypothesized that LPS-induced acute renal failure (ARF) requires systemic TNF release triggered by LPS acting on extrarenal TLR4. We injected C3H/HeJ mice and C3H/HeOuJ controls with 0.25 mg of LPS, and sacrificed them 6 h later for analysis of blood urea nitrogen (BUN) and kidney tissue (n = 8 per group). In contrast to C3H/HeOuJ controls, C3H/HeJ mice were completely resistant to LPS-induced ARF (6-h BUN of 32.3 ± 1.1 vs 61.7 ± 5.6 mg/dl). C3H/HeJ mice released no TNF into the circulation at 2 h (0.00 vs 1.24 ± 0.16 ng/ml), had less renal neutrophil infiltration (6.4 ± 1.0 vs 11.4 ± 1.3 neutrophils per high power field), and less renal apoptosis, as assessed by DNA laddering. Transplant studies showed that C3H/HeJ recipients of wild-type kidneys (n = 9) were protected from LPS-induced ARF, while wild-type recipients of C3H/HeJ kidneys (n = 11) developed severe LPS-induced ARF (24-h BUN 44.0 ± 4.1 vs 112.1 ± 20.0 mg/dl). These experiments support our hypothesis that LPS acts on extrarenal TLR4, thereby leading to systemic TNF release and subsequent ARF. Renal neutrophil infiltration and renal cell apoptosis are potential mechanisms by which endotoxemia leads to functional ARF.

In this study, we demonstrate that mice deficient in TNFR1 (TNFR1 -/-) were resistant to LPSinduced encephalopathy. Systemic administration of lipopolysaccharide (LPS) induces a widespread inflammatory response similar to that observed in sepsis. Following LPS administration TNFR1 -/-mice had less caspase-dependent apoptosis in brain cells and fewer neutrophils infiltrating the brain (p<0.039), compared to control C57Bl6 (TNFR1 +/+ ) mice. TNFR1-dependent increase in aquaporin (AQP)-4 mRNA and protein expression was observed with a concomitant increase in water content, in brain (18% increase in C57Bl6 mice treated with LPS vs those treated with saline), similar to cerebral edema observed in sepsis. Furthermore, absence of TNFR1 partially but significantly reduced the activation of astrocytes, as shown by immunofluorescence and markedly inhibited iNOS mRNA expression (p<0.01).Septic Encephalopathy is a devastating complication of sepsis. Although, considerable work has been done to identify the mechanism causing the pathological alterations in this setting, the culprit still remains an enigma. Our results demonstrate for the first time that endotoxemia leads to inflammation in brain, with alteration in blood-brain barrier, up-regulation of AQP4 and associated edema, neutrophil infiltration, astrocytosis, as well as apoptotic cellular death, all of which appear to be mediated by TNF-α signaling through TNFR1. KeywordsRodent; Septic Encephalopathy; Lipopolysaccharide; Apoptosis; Aquaporin 4 Neuroimmunology Sepsis is a condition characterized by uncontrolled infection and affects many organs including brain (Green et al., 2004), with an attendant high mortality rate. Administration of bacterial endotoxin (LPS), a cell wall component of gram-negative bacteria, to mice cause pathogenesis, mimicking what occurs in clinical sepsis (Gardenfors et al., 2002;Radzivil et al., 1990). Septic encephalopathy could be due to multiple factors including inflammatory cells and their mediators, reduced cerebral blood flow, disruption of the blood-brain barrier (BBB), cerebral edema and inflammation (Papadopoulos et al., 2000). One of the mediators of inflammation that play a key role in sepsis, tumor necrosis factor (TNF-α), is increased in circulation following LPS administration (Tsao et al., 2001;Merrill and Benveniste, 1996). Serum TNF-α is an acute phase response and does not correlate with the alterations in brain, Address correspondence and reprint requests to Dr. Jessy J. Alexander, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC5100, Chicago, IL 60637. jalexand@medicine.bsd.uchicago.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered w...

Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration perm-selectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse.

Abstract. In previous work, it was demonstrated that apoptosis occurs in the kidney during LPS-induced acute renal failure (ARF). However, the relative importance of apoptosis in LPSinduced ARF remained unproven. Because the caspase enzyme cascade is responsible for carrying out apoptosis, it was hypothesized that treatment with a caspase inhibitor would protect mice from LPS-induced ARF. C57BL/6 mice received an injection of LPS and were treated with either the broad-spectrum caspase inhibitor z-VAD-fmk or vehicle and compared with unmanipulated mice. LPS induced a significant increase in caspase-3 activity in vehicle-treated mice, which was significantly inhibited by z-VAD. Mice that were treated with z-VAD were protected from ARF and demonstrated significantly less apoptosis as measured by both terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and DNA laddering.