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.

Acute renal failure (ARF) 3 occurs in up to 5% of hospital admissions, and is a leading cause of morbidity and mortality (1). A common cause of ARF is sepsis, which results from overwhelming infection (2, 3). Although a variety of bacterial products may cause the diffuse inflammatory response seen in sepsis, one of the most important is endotoxin (LPS), a component of the cell wall of Gram-negative bacteria. Injection of LPS into animals reproduces many of the manifestations of sepsis, including ARF. Cytokines such as TNF are thought to be key early mediators of this syndrome. In previous work, we have shown that mice deficient in TNFR1 are resistant to LPS-induced ARF, and that TNFR1 mediates LPS-induced ARF within the kidney (4).

In recent years, much has been learned about the immediate events following LPS exposure. Various bacterial products, including LPS, signal through a family of transmembrane proteins known as the Toll-like receptors (TLRs) (5). The importance of the TLRs in facilitating innate immunity is underscored by their highly conserved presence in organisms ranging from Drosophila to humans. TLR4 has been found to be the primary molecule through which LPS activates cells, leading to the rapid release of cytokines such as TNF and IL-1. In addition, TLR4 binds an endogenous ligand, heat shock protein 60, and this interaction may mediate inflammation seen in response to cellular damage (6). TLR4 is most highly expressed in leukocytes, but is also present in a variety of organs, including the kidney (7, 8). The key role for TLR4 in vivo was illustrated when the C3H/HeJ strain of mice, long known to be resistant to LPS-induced mortality, was found to be homozygous for a naturally occurring single base pair mutation in the TLR4 gene, leading to a complete absence of functional protein (9). When bone marrow-derived cells from these mice were transferred to irradiated wild-type control mice, relative protection against LPS-induced mortality resulted, implying that leukocyte TLR4 signaling is necessary to cause the systemic effects of LPS (10).

Nevertheless, TLR4-independent pathways of LPS action have also been described (11, 12), including possible signaling through TLR2 (13, 14). Additionally, in vitro work has documented effects of LPS on renal tubular and mesangial cells (15, 16, 17, 18). We therefore undertook the following study to examine the role and site of action of TLR4 in LPS-induced ARF.

C3H/HeJ and C3H/HeOuJ (wild-type control) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). In all experiments, 8- to 10-wk-old male mice were injected i.p. with 0.25 mg of Escherichia coli LPS (Sigma-Aldrich, St. Louis, MO). This dose of LPS is higher than the 0.15 mg dose used in previous studies, as preliminary work showed that the C3H/HeOuJ strain is somewhat less sensitive to LPS than the C57BL/6 strain. Blood was obtained via retroorbital bleeding at the time of injection and at various later time points. Blood urea nitrogen (BUN) concentrations were used to determine renal function and were measured with a Beckman CX5CE autoanalyzer (Beckman Coulter, Fullerton, CA). Mice were sacrificed at 6, 24, or 48 h, with collection of blood as above and harvest of kidney tissue. The above procedures were done under anesthesia using a continuous inhalational isoflurane/oxygen mixture. The animals were maintained and experiments were performed in accordance with the guidelines set by the University of Chicago Institutional Animal Care and Use Committee.

For routine histologic analysis, kidneys were sectioned coronally, fixed in methyl Carnoy’s solution at 4°C for 48 h, embedded in paraffin, and stained with periodic acid-Schiff base. Sections were scored for tubular injury in a blinded fashion, as previously described (4).

For immunohistochemistry, 4-μm kidney cryostat sections were fixed with ether/ethanol, incubated with 0.3% H2O2 for 30 min, and blocked with dilute horse serum. Sections were stained for neutrophils by sequential incubation with rat anti-mouse neutrophil (anti-Gr-1; BD PharMingen, San Diego, CA) at 1/60 for 30 min, followed by HRP-conjugated rabbit anti-rat IgG (Sigma-Aldrich) at 1/60 for 30 min, and diaminobenzidine reagent (Vector Laboratories, Burlingame, CA) for 10 min. A blinded observer counted the number of neutrophils per high power field and recorded the average of 10 fields for each sample.

Tissue sections were stained for apoptotic nuclei via the TUNEL technique, using a commercially available kit (Trevigen, Gaithersburg, MD), according to manufacturer instructions. In brief, kidney cryosections were cut into 10-μM sections and mounted on charged slides. Slides were dried overnight, rehydrated through graded alcohols to PBS, and fixed in 10% Formalin for 10 min at room temperature. This was followed by proteinase K treatment (20 μg/ml) for 15 min at 37°C and incubation in 3% H2O2/methanol for 5 min. Specimens were incubated with TdT/Br-dNTP mixture at 37°C for 60 min, followed by anti-5-bromo-2′-deoxyuridine at 1/150 for 60 min at 37°C. This was followed by incubation in streptavidin-HRP at 1/500 for 30 min, followed by detection with TACS blue label for 5 min. Sections were counterstained with nuclear fast red for 10 min, washed, and mounted under coverslips.

At time of sacrifice, kidneys were cut into thirds and snap frozen in liquid nitrogen. From this, genomic DNA was purified by means of the DNeasy DNA purification system (Qiagen, Valencia, CA), according to the manufacturer’s instructions, and was quantitated spectophotometrically. The extent of DNA laddering was amplified and detected via LM-PCR using a commercially available kit (Clontech Laboratories, Palo Alto, CA), according to the manufacturer’s instructions, as follows. DNA isolated from each animal was incubated with supplied primer targets and T4 DNA ligase for 18 h at 16°C. A total of 20 μg of this ligated DNA was then used as the template for PCR, using supplied primers and Advantage DNA polymerase (Clontech Laboratories) for 21 cycles at 94°C (1 min)/72°C (3 min). The reaction product for each animal was electrophoresed through a 1.3% agarose gel, and ethidium bromide-stained bands were detected with UV light illumination. To ensure an equivalent amount of genomic DNA template was used for each animal, standard PCR for the gene En-2 was performed, using primers contained in the above kit.

TNF levels were determined from sera obtained at baseline and 2 and 6 h after LPS administration using a commercially available ELISA kit for mouse TNF (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. The limit of sensitivity of this assay was 10 pg/ml.

A portion of whole kidney obtained at sacrifice from each mouse was frozen in liquid nitrogen. This was later placed in TRIzol reagent (Life Technologies, Grand Island, NY), from which total RNA was purified according to the manufacturer’s instructions. To remove all traces of genomic DNA, samples were then treated with RNase-free RQ1 DNase (Promega, Madison, WI; 1 U/4 μg RNA) in 10 μl of reaction buffer (final concentration 40 mM of Tris-HCl, 10 mM of MgSO4, 1 mM of CaCl2, pH 8.0), at 37°C for 30 min. This was followed by addition of 1 μl of 20 mM EGTA, pH 8.0, to stop the reaction, and incubation at 65°C for 10 min to inactivate the DNase. cDNA was generated from RNA using random hexamers as primers with the SuperScript first-strand synthesis kit (Life Technologies), according to the manufacturer’s instructions, and diluted 5-fold before analysis.

Real-time PCR was performed using the Prism 7700 reactor and the SybrGreen intercalating dye method with HotStar DNA polymerase (Applied Biosystems, Foster City, CA). Each reaction was conducted in a total volume of 50 μl with primers at 200 nM, 1 mM of dNTPs, 3 mM of MgCl2, and 10 μl of sample or standard cDNA. PCR was conducted with a hot start at 95°C (5 min), followed by 45 cycles at 95°C (15 s)/60°C (30 s). For each sample, the number of cycles required to generate a given threshold signal (Ct) was recorded. Using a standard curve generated from serial dilutions of kidney cDNA, the ratio of ICAM-1 expression relative to GAPDH expression was calculated for each experimental animal, and normalized relative to an average of ratios from the control group. Measurements of TNF mRNA expression were performed in an analogous fashion, except that the annealing temperature was 58°C. Products of each reaction yielded a single band when run on agarose gel, confirming specific amplification. Primers were synthesized by Integrated DNA Technologies (Coralville, IA), with sequences as follows: GAPDH forward primer, 5′-GGC AAA TTC AAC GGC ACA GT-3′; GAPDH reverse primer, 5′-AGA TGG TGA TGG GCT TCC C-3′; TNF forward primer, 5′-CCG ATG GGT TGT ACC TTG TC-3′; TNF reverse primer, 5′-GTG GGT GAG GAG CAC GTA GT-3′; ICAM-1 forward primer, 5′-CGC AAG TCC AAT TCA CAC TGA-3′; ICAM-1 reverse primer, 5′-CAG AGC GGC AGA GCA AAA G-3′.

Selected organs were isolated from each animal at sacrifice and snap frozen in liquid nitrogen. These were later homogenized in a buffer consisting of 200 mM of NaCl, 10 mM of Tris-HCl (pH 7.0), 5 mM of EDTA, 10% glycerol, 1 mM of PMSF, 20 μM of pepstatin, 20 μM of leupeptin, and 0.1 μM of aprotinin. The total protein concentration in each homogenate was determined by means of the bicinchoninic acid method (Pierce, Dallas, TX). Samples (20 μg/lane) were electrophoresed through a 10% SDS-PAGE gel under nonreducing conditions. Proteins were transferred to an Immobilon-P nitrocellulose membrane (Millipore, Bedford, MA), and blocked in 5% milk/TBST overnight. This membrane was then incubated with goat anti-mouse TNF (R&D Systems) at 1/1000, followed by HRP rabbit anti-goat IgG (Sigma-Aldrich) at 1/2000, and activity was detected using the Supersignal West Pico chemiluminescent kit (Pierce). Three animals were randomly selected from each group for analysis.

To define the level of action of the TLR4, single kidney transplants were performed between C3H/HeJ and wild-type mice, and their response to LPS injection was studied. To provide adequate controls, four groups of mice were studied: C3H/HeJ to wild type, wild type to C3H/HeJ, C3H/HeJ to C3H/HeJ, and wild type to wild type. Each individual mouse was used only as a recipient or a donor, at an age of 10 wk. Mice were anesthetized with 65 mg/kg i.p. pentobarbital. The left kidney of donor mice was perfused via the renal artery with 0.3 ml of cold saline, and resected with artery, vein, and ureter attached. The kidney was stored at 4°C until time of anastomosis. Next, the recipient underwent midline abdominal incision, followed by suprarenal clamping of aorta and inferior vena cava. The donor kidney was placed in the right flank, and its artery and vein were attached with 10-0 nylon suture via side-to-end anastomoses with the recipient aorta and inferior vena cava. The bladder was punctured with a 21-gauge needle, and the ureter was sewn in place. Both native kidneys were then resected. Animals were allowed to recover 10 days after surgery before LPS injection. Preliminary dosing experiments showed that mouse kidney transplant recipients have a stable, mild elevation of BUN and creatinine, but are somewhat more sensitive to LPS; thus, a dose of 0.125 mg was used in these studies.

Data were analyzed with Minitab software (State College, PA). Unless noted otherwise, data are given as mean ± SEM. Groups were compared by two-tailed t test, or ANOVA using the Dunn-Sidak correction for multiple comparisons in cases in which more than two groups were compared. When BUN levels pre- and post-LPS were compared within a given group of mice, a paired t test was used. When comparing the severity of ARF between two groups, the slope of BUN vs time was determined for each individual animal by least squares regression, and the individual slopes in the two groups were compared with the Mann-Whitney rank sum test. A p value ≤0.05 was considered significant.

To determine whether the protection against LPS-induced mortality previously observed in C3H/HeJ mice extends to LPS-induced ARF, C3H/HeJ mice and wild-type controls were injected with a sublethal dose of LPS, and BUN was measured at various time points up through 48 h. Mice of the C3H/HeOuJ strain, which are homozygous for a fully functional TLR4, were used as the wild-type control group. Although wild-type controls developed severe ARF manifested by an abrupt rise in BUN, C3H/HeJ mice showed absolutely no evidence of ARF (24-h BUN of 131.6 ± 24.8 mg/dl vs 28.8 ± 1.8 mg/dl, p < 0.001, n = 4 per group; Fig. 1,A). In addition, renal histology of wild-type controls at time of sacrifice showed a moderate degree of tubular injury (Fig. 1,D), while renal histology in C3H/HeJ mice was completely normal (Fig. 1 E).

FIGURE 1.

C3H/HeJ mice are resistant to LPS-induced ARF. A, In contrast to the acute rise in BUN seen in wild-type mice, BUN levels were completely unchanged in C3H/HeJ mice after LPS injection. n = 4 per group. Normal kidney histology is shown for wild-type and C3H/HeJ mice at baseline in B and C, respectively. D, By 48 h after LPS injection, pathology in wild-type mice showed modest tubular injury, consisting of tubular dilation and flattening (asterisk) and degenerated tubules with small vacuoles (arrows). E, In C3H/HeJ mice, renal pathology after LPS injection was indistinguishable from baseline. Magnification ×200, periodic acid-Schiff stain.

FIGURE 1.

C3H/HeJ mice are resistant to LPS-induced ARF. A, In contrast to the acute rise in BUN seen in wild-type mice, BUN levels were completely unchanged in C3H/HeJ mice after LPS injection. n = 4 per group. Normal kidney histology is shown for wild-type and C3H/HeJ mice at baseline in B and C, respectively. D, By 48 h after LPS injection, pathology in wild-type mice showed modest tubular injury, consisting of tubular dilation and flattening (asterisk) and degenerated tubules with small vacuoles (arrows). E, In C3H/HeJ mice, renal pathology after LPS injection was indistinguishable from baseline. Magnification ×200, periodic acid-Schiff stain.

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Subsequent experiments focused on events occurring in the first 6 h after LPS injection, during which ARF becomes established. In both wild-type and C3H/HeJ mice, serum levels of TNF were undetectable at baseline. Two hours after LPS injection, there was a profound increase in serum TNF levels in wild-type mice, which was completely absent in C3H/HeJ mice (1.24 ± 0.16 ng/ml vs 0.00 ng/ml, p < 0.001; Fig. 2). By 6 h, these levels had returned to near baseline in wild-type mice, and remained undetectable in C3H/HeJ mice (data not shown). In parallel with this extreme difference in TNF release, all wild-type mice had developed ARF at 6 h after LPS injection, while C3H/HeJ mice had no evidence of ARF (6-h BUN of 61.7 ± 5.6 mg/dl in wild-type mice vs 32.3 ± 1.1 mg/dl in C3H/HeJ mice, n = 8 per group, p < 0.001). This supports our hypothesis that TLR4-mediated TNF release has a primary pathogenic role in LPS-induced ARF.

FIGURE 2.

Serum TNF release after LPS administration. Serum TNF levels as determined by ELISA are shown. At baseline, the amount of TNF in both C3H/HeJ and wild-type mice was undetectable. Two hours after LPS injection, there was a profound increase in serum TNF in wild-type mice, but not in C3H/HeJ mice. n = 8 per group.

FIGURE 2.

Serum TNF release after LPS administration. Serum TNF levels as determined by ELISA are shown. At baseline, the amount of TNF in both C3H/HeJ and wild-type mice was undetectable. Two hours after LPS injection, there was a profound increase in serum TNF in wild-type mice, but not in C3H/HeJ mice. n = 8 per group.

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Our previous work has shown an influx of neutrophils into the kidney 48 h after LPS administration. Therefore, we hypothesized that C3H/HeJ mice would be resistant to LPS-induced neutrophil infiltration. Six hours after LPS administration, a robust neutrophilic infiltration into renal parenchyma was observed in wild-type mice, illustrating that the neutrophilic infiltration occurred early and coincident with development of renal functional impairment. This infiltration was present diffusely throughout the renal cortex, especially prominent in venules, but also observed in peritubular areas and occasionally in glomeruli (Fig. 3, AC). The extent of renal neutrophil infiltration in C3H/HeJ mice was significantly less than in wild-type controls (6.4 ± 1.0 vs 11.4 ± 1.3 neutrophils per high power field, p = 0.05), and the magnitude of the change from baseline was also less (Fig. 3 D). Interestingly, C3H/HeJ mice did exhibit an increase from 3.5 ± 0.7 neutrophils per high power field at baseline to 6.4 ± 1.0 neutrophils per high power field after LPS, although this did not reach statistical significance (p = 0.23).

FIGURE 3.

Renal neutrophil infiltration following LPS administration. A, At baseline, few neutrophils were present in renal tissue. After LPS administration to wild-type mice, abundant neutrophils were found diffusely throughout the renal cortex, especially in renal venules (B) and peritubular capillaries (C, arrows). A similar distribution of neutrophils was observed in C3H/HeJ mice, although this was quantitatively less. D, Quantitation of neutrophil infiltration in the four groups is shown. ∗, p < 0.05 vs wild-type baseline, p = 0.23 vs C3H/HeJ baseline; ∗∗, p ≤ 0.05 vs all other groups. Magnification ×200 in A and B, and ×400 (high power field) in C.

FIGURE 3.

Renal neutrophil infiltration following LPS administration. A, At baseline, few neutrophils were present in renal tissue. After LPS administration to wild-type mice, abundant neutrophils were found diffusely throughout the renal cortex, especially in renal venules (B) and peritubular capillaries (C, arrows). A similar distribution of neutrophils was observed in C3H/HeJ mice, although this was quantitatively less. D, Quantitation of neutrophil infiltration in the four groups is shown. ∗, p < 0.05 vs wild-type baseline, p = 0.23 vs C3H/HeJ baseline; ∗∗, p ≤ 0.05 vs all other groups. Magnification ×200 in A and B, and ×400 (high power field) in C.

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The influx of inflammatory leukocytes into a variety of tissues after endotoxin administration has been attributed to LPS-induced up-regulation of various adhesion molecules and chemotactic chemokines (19, 20). One of the most important molecules in this adhesion process is ICAM-1 (21). To investigate the role of TLR4 in ICAM-1-mediated neutrophil infiltration, whole kidney taken at sacrifice 6 h after LPS administration was analyzed for ICAM-1 mRNA by means of real-time PCR. This showed a profound increase in renal ICAM-1 transcription in wild-type mice after LPS administration (20.5 ± 2.9-fold increase, p < 0.001; Fig. 4). The level of ICAM-1 mRNA in C3HeJ mice after LPS was much less, although was definitely elevated compared with baseline (2.9 ± 0.4-fold increase, p < 0.05).

FIGURE 4.

Renal ICAM-1 expression after LPS administration. Real-time PCR was used to determine ICAM-1 mRNA levels in whole kidney 6 h after LPS administration. Each data point represents an individual animal, and the dotted line represents the average baseline expression for wild-type mice. Although ICAM-1 expression was significantly increased in both wild-type and C3H/HeJ mice after LPS injection compared with baseline, this up-regulation was markedly greater in wild-type mice. ∗, p < 0.01 vs baseline; ∗∗, p < 0.01 vs all other groups.

FIGURE 4.

Renal ICAM-1 expression after LPS administration. Real-time PCR was used to determine ICAM-1 mRNA levels in whole kidney 6 h after LPS administration. Each data point represents an individual animal, and the dotted line represents the average baseline expression for wild-type mice. Although ICAM-1 expression was significantly increased in both wild-type and C3H/HeJ mice after LPS injection compared with baseline, this up-regulation was markedly greater in wild-type mice. ∗, p < 0.01 vs baseline; ∗∗, p < 0.01 vs all other groups.

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In previous studies, we have also shown that LPS administration causes renal cell apoptosis in vivo, and that the extent of renal apoptosis strongly correlates with the degree of renal failure (4). In this study, we again observed minimal apoptosis at baseline on TUNEL-stained kidney cryosections, in contrast to a clear increase in apoptotic bodies seen in wild-type mice 6 h after LPS, early in the course of LPS-induced ARF (Fig. 5, A and B). Apoptotic nuclei were primarily seen in both tubular and peritubular locations; the latter may represent apoptosis of peritubular capillary cells, consistent with what has been observed in other vascular beds after LPS administration (22). We further quantitated renal apoptosis in C3H/HeJ and wild-type mice by means of LM-PCR, which amplifies the characteristic DNA laddering seen in apoptotic cells. In contrast to the scant amount of apoptosis seen at baseline in both C3H/HeJ and wild-type mice (Fig. 5,C), which probably reflects normal cell turnover, apoptosis was markedly increased in wild-type mice after LPS (Fig. 5,D). This stands in contrast to the significantly lesser amount of apoptosis seen in C3H/HeJ kidney following LPS (Fig. 5 D).

FIGURE 5.

Renal apoptosis after LPS administration. As depicted in A, TUNEL staining of wild-type kidney cryosections revealed only rare apoptotic nuclei at baseline. In contrast, LPS injection induced scattered apoptosis (arrows), as depicted in B. Renal apoptosis was further quantitated by LM-PCR, shown in C and D. Each lane represents whole kidney taken from an individual animal 6 h after LPS injection. C, There is little apoptosis at baseline in either wild-type or C3H/HeJ kidney, in contrast to wild-type kidney following LPS injection (far right lane). As shown in D, LPS-induced apoptosis was greater in wild-type mice as compared with C3H/HeJ mice.

FIGURE 5.

Renal apoptosis after LPS administration. As depicted in A, TUNEL staining of wild-type kidney cryosections revealed only rare apoptotic nuclei at baseline. In contrast, LPS injection induced scattered apoptosis (arrows), as depicted in B. Renal apoptosis was further quantitated by LM-PCR, shown in C and D. Each lane represents whole kidney taken from an individual animal 6 h after LPS injection. C, There is little apoptosis at baseline in either wild-type or C3H/HeJ kidney, in contrast to wild-type kidney following LPS injection (far right lane). As shown in D, LPS-induced apoptosis was greater in wild-type mice as compared with C3H/HeJ mice.

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When put in the context of our previous work, the above data support the hypothesis that LPS acts through TLR4 to trigger systemic release of TNF, which reaches the kidney through the circulation and acts through renal TNFR1 to cause ARF (4). However, because TLR4 is expressed in the kidney, it is conceivable that LPS could be working through renal TLR4 as well. It has been demonstrated that TNF is synthesized in the kidney following LPS administration (23). In support of this, real-time PCR showed a profound increase in renal TNF mRNA 6 h after LPS administration in wild-type mice (19.5 ± 2.3-fold increase, p < 0.01), which was significantly less in C3H/HeJ mice (Fig. 6,A). Western blotting substantiated this at the protein level, showing that while the primary sites of TNF protein production 6 h after LPS administration are the liver and small intestine, there is also a significant amount that is synthesized in the kidney (Fig. 6 B).

FIGURE 6.

Renal TNF synthesis. A, Real-time PCR was used to determine TNF mRNA levels in whole kidney 6 h after LPS administration. Each data point represents an individual animal, and the dotted line represents the average baseline expression of wild-type kidney. Although TNF expression was significantly increased in both wild-type and C3H/HeJ mice after LPS injection compared with baseline, this up-regulation was markedly greater in wild-type mice. ∗, p < 0.01 vs baseline; ∗∗, p < 0.01 vs all other groups. B, Western blots of various organs 6 h after LPS injection. After LPS administration, the majority of TNF was induced in liver and small intestine, but considerable renal synthesis was also seen in wild-type mice.

FIGURE 6.

Renal TNF synthesis. A, Real-time PCR was used to determine TNF mRNA levels in whole kidney 6 h after LPS administration. Each data point represents an individual animal, and the dotted line represents the average baseline expression of wild-type kidney. Although TNF expression was significantly increased in both wild-type and C3H/HeJ mice after LPS injection compared with baseline, this up-regulation was markedly greater in wild-type mice. ∗, p < 0.01 vs baseline; ∗∗, p < 0.01 vs all other groups. B, Western blots of various organs 6 h after LPS injection. After LPS administration, the majority of TNF was induced in liver and small intestine, but considerable renal synthesis was also seen in wild-type mice.

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To determine the site at which TLR4 mediates LPS-induced ARF, single kidney transplants were performed between C3H/HeJ and wild-type mice, and their response to LPS injection was studied. C3H/HeJ kidneys in wild-type recipients still developed severe ARF after LPS injection (n = 11), while wild-type kidneys in C3H/HeJ recipients were largely resistant to LPS-induced ARF (p < 0.01, n = 9) (Fig. 7). Additionally, tubular injury scores showed significantly greater pathologic injury in C3H/HeJ kidneys in wild-type recipients vs wild-type kidneys in C3H/HeJ recipients (tubular injury scores 3.5 ± 0.4 vs 1.7 ± 0.2, p < 0.01). This proves that the TLR4 that mediates LPS-induced ARF is located in a primarily extrarenal location. However, it is notable that even in C3H/HeJ recipients, wild-type kidneys had a significant increase in BUN after LPS administration (41.4 ± 2.5 mg/dl at baseline, vs 61.0 ± 8.9 mg/dl 12 h after LPS, p < 0.05 by paired t test) before declining to baseline levels, unlike the C3H/HeJ→C3H/HeJ control group, which had no increase in BUN above baseline at any time point.

FIGURE 7.

TLR4 mediates LPS-induced ARF primarily from outside the kidney. Although C3H/HeJ kidneys implanted into wild-type recipients (C3H/HeJ→WT, n = 11) sustained severe ARF after LPS injection, wild-type kidneys implanted into C3H/HeJ recipients (WT→C3H/HeJ, n = 9) were protected. ∗, p < 0.001 for wild-type recipients vs C3H/HeJ recipients. Nevertheless, even wild-type kidneys in C3H/HeJ recipients had a transient rise in BUN at 12 h after LPS in comparison with baseline (p < 0.05). Syngeneic transplants of WT→WT (n = 7) and C3H/HeJ→C3H/HeJ (n = 6) mice are depicted as controls.

FIGURE 7.

TLR4 mediates LPS-induced ARF primarily from outside the kidney. Although C3H/HeJ kidneys implanted into wild-type recipients (C3H/HeJ→WT, n = 11) sustained severe ARF after LPS injection, wild-type kidneys implanted into C3H/HeJ recipients (WT→C3H/HeJ, n = 9) were protected. ∗, p < 0.001 for wild-type recipients vs C3H/HeJ recipients. Nevertheless, even wild-type kidneys in C3H/HeJ recipients had a transient rise in BUN at 12 h after LPS in comparison with baseline (p < 0.05). Syngeneic transplants of WT→WT (n = 7) and C3H/HeJ→C3H/HeJ (n = 6) mice are depicted as controls.

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The above data build upon our previous work and strengthen our understanding of how LPS causes ARF. In this paradigm, administered LPS acts through extrarenal TLR4, leading to the rapid release of TNF into the circulation within the first several hours. In turn, this circulating TNF acts through renal TNFR1 to cause ARF, through a variety of mechanisms that may involve renal neutrophil infiltration and renal apoptosis (4). In addition, given that LPS injection causes transient ARF in wild-type kidneys transplanted into C3H/HeJ recipients, there appears to be a novel, albeit lesser role for intrarenal TLR4 in this LPS-induced ARF. Thus, LPS also acts in part through renal TLR4, presumably leading to local TNF synthesis, as shown above, followed by paracrine action on renal TNFR1 and subsequent ARF.

The above data are consistent with our previous study in suggesting an important role for the mechanisms of renal apoptosis and neutrophil infiltration. Previously, we focused on each of these two processes 48 h after LPS exposure, while in this study we show that both renal apoptosis and neutrophil infiltration are well underway 6 h after LPS injection, during the period when ARF is initiated. The fact that these processes were found to correlate with LPS-induced ARF in both the C3H/HeJ and C57BL/6 mouse strains also strengthens their likely importance.

Apoptosis of kidney cells is a mechanism of cellular death that is difficult to discern with routine histology, but that may contribute importantly to loss of renal function. Because apoptotic cells are rapidly and efficiently cleared by neighboring cells, a small amount of apoptosis may add up over the course of time to a significant amount of cellular loss. TNF administration to a variety of cell types, including renal epithelial cells, has been shown to initiate apoptosis (24, 25). TNF may trigger apoptosis in a receptor-mediated fashion, via TNFR1 activation of initiator caspase-8 and caspase-10. Additionally, activation of TNFR1 may cause apoptosis indirectly through various inflammatory events, such as NO release, local hypoxia, and reactive oxygen species released by invading neutrophils, all of which may bring about apoptosis through the mitochondrial pathway (26, 27, 28).

Infiltration of various tissues such as liver, lung, and kidney by neutrophils and other inflammatory leukocytes has been well demonstrated to occur after LPS administration (21, 29, 30). LPS, as well as cytokines such as TNF, are known to up-regulate chemotactic chemokines and adhesion molecules, such as ICAM-1, which promote leukocyte adhesion and migration (19, 20, 21). Once in the tissue, these inflammatory cells may cause tissue injury via release of injurious proteases and reactive oxygen species (31). Additionally, there is evidence that neutrophils may mediate changes in local vascular tone by signaling through ICAM-1 or by releasing reactive oxygen species (32). In this study, we demonstrate invasion of the renal parenchyma by neutrophils soon after LPS administration, in parallel with a profound increase in ICAM-1 expression. Additionally, LPS and cytokines have been shown to directly activate neutrophils, priming them for adhesiveness and activity (33).

Interestingly, the LPS-resistant C3H/HeJ mice also had increased neutrophilic infiltration into the kidney after LPS. It was previously reported that a closely related strain of TLR4-deficient mice showed an exaggerated influx of neutrophils into the peritoneal cavity after LPS injection, demonstrating that LPS triggers neutrophil chemotaxis in part via a TLR4-independent pathway (14). The mechanism for this neutrophil chemotaxis in TLR4-deficient mice is not known. However, the fact that we observed renal neutrophil infiltration without any elevation in BUN in C3H/HeJ mice shows that the mere presence of this number of neutrophils is not sufficient to cause LPS-induced ARF. It is likely that additional TLR4-dependent events, such as the stimulation of the neutrophil respiratory burst or the involvement of key adhesion molecules or chemokines, are also necessary for neutrophils to cause tissue damage. Alternatively, renal neutrophil infiltration may simply be an associated finding without a direct pathogenic role.

Given the fact that mice lacking a functional TLR4 are fully resistant to LPS-induced mortality as well as LPS-induced ARF, TLR4 clearly plays a primary role in the response to LPS in vivo. However, it is noteworthy that renal TNF and ICAM-1 expression were significantly increased after LPS administration in C3H/HeJ mice, albeit much less so than in wild-type mice. This implies that there is some response to LPS through TLR4-independent pathways, as has been described elsewhere (11, 12). Although early studies seemed to clearly show that LPS also interacts and signals through TLR2 (13, 34), a subsequent study ascribes the effects of TLR2 to contamination of commercial LPS preparations with other inflammatory bacterial products (35). Although we cannot rule out that a component of the increases in TNF and ICAM-1 expression that we observed was due to LPS impurities, it seems unlikely that all the changes we observed were entirely due to this artifact.

In conclusion, the above experiments show that extrarenal TLR4 is crucial in mediating LPS-induced ARF, via systemic cytokine release and subsequent intrarenal events such as renal cell apoptosis and renal neutrophil infiltration. The distal events occurring in the kidney are likely to be complex and interrelated, and the extent to which apoptosis and neutrophil infiltration individually contribute to LPS-induced ARF, as well as other possible effects of TNF on the kidney, is the subject of ongoing work. It is hoped that a better understanding of this process will lead to therapies that can effectively prevent or reverse the ARF associated with sepsis.

1

This work was supported by National Institutes of Health Grants R01DK41873, R01DK55357, and K08DK61375.

3

Abbreviations used in this paper: ARF, acute renal failure; BUN, blood urea nitrogen; LM, ligase mediated; TLR, Toll-like receptor.

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