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Leptospira Outer Membrane Protein Activates NF-B and Downstream Genes Expressed in Medullary Thick Ascending Limb Cells

CHIH-WEI YANG^*, MAI-SZU WU^*, MING-JENG PAN, JENN-JYE HONG^*, CHUN-CHEN YU^*, ALAIN VANDEWALLE and CHIU-CHING HUANG^*

^* Division of Nephrology, Chang Gung Memorial Hospital, Taipei, Taiwan, Republic of China
Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
National Institute of Health and Medical Research (INSERM), Unit 478, Faculty of Medicine, Xavier Bichat, Paris, France.

Correspondence to Dr. Chih-Wei Yang, Division of Nephrology, Chang Gung Memorial Hospital, 199 Tun-Hwa North Road, Taipei, 105, Taiwan. Phone: 886 3 3285386; Fax: 886 3 3282173; E-mail: cwyang{at}ms1.hinet.net

	Abstract

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Abstract. Tubulointerstitial nephritis is the main manifestation of acute renal damage caused by leptospirosis, but the mechanism remains unexplored. Patients infected with Leptospira shermani in Taiwan disclosed tubular dysfunction particularly in the medullary thick ascending limb of loop of Henle (mTAL), and the related renal damage seems to be underestimated. To elucidate the mechanism of tubular damage, outer membrane protein extract from Leptospira was administered to a model of cultured mTAL cells derived from normal mice. The addition of outer membrane protein extract from L. shermani to cultured mTAL cells induced a significant nuclear DNA binding of the NF- B transcription factor by electrophoresis mobility shift assay. Forty-eight h after adding the outer membrane protein extract (0.2 µg/ml) to the cultured cells, the expression of inducible nitric oxide mRNA increased by 4.2-fold, monocyte chemoattractant protein-1 by 3-fold, and tumor necrosis factor- by 2.4-fold when compared with untreated cells examined by reverse transcription competitive-PCR. Supernatant nitrite, monocyte chemoattractant protein-1, and tumor necrosis factor- protein levels also increased by 1.8-, 7.1-, and 5-fold, respectively. An antiserum raised against L. shermani largely prevented these effects. Outer membrane protein extract from L. bratislava induced fewer effects than L. shermani, and the avirulent nonpathogenic L. biflexa serovar patoc did not induce significant effects in the mTAL cells. In conclusion, L. shermani infection may cause mTAL cell damage and inflammation through the NF- B—associated pathway. Findings of this study may be important in understanding the pathogenesis of tubulointerstitial nephritis caused by these organisms.

	Introduction

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Leptospirosis is a widespread zoonosis caused by members of the genus Leptospira. These highly invasive spirochetal pathogens infect a broad range of mammalian hosts through either direct contact with infected animals or indirect contact with soil or water contaminated with leptospira (1). Most leptospirosis is mild with self-limited flu-like illness. Five to 10% of cases of leptospirosis induce multiple organ damage with characteristic kidney, liver, and lung lesions (1). The infection may lead to fatal illness after the appearance of hemorrhage, jaundice, and acute renal failure (Weil syndrome). Endemic and epidemic spread of leptospirosis has caused morbidity and mortality worldwide. Leptospirosis seems to be underestimated as a cause of acute renal failure in Taiwan (2). An increasing number of leptospirosis cases have been reported since 1996 (3). Among 24 serogroups of pathogenic leptospires, L. santarosai serovar shermani (L. shermani) is the most frequently encountered serovar in Taiwan both in humans and in animals (4) and may be associated with medullary thick ascending limb cell (mTAL) dysfunction (5).

Several viscera may be affected in leptospirosis. The most commonly involved organ is the kidney. Direct invasion of the organism into the kidney induces tubulointerstitial nephritis and acute renal failure (1,6). The characteristic renal lesions include interstitial edema, infiltrates of lymphocytes, monocytes, plasma cells, and neutrophils (7). These changes may be reversible but may lead to a chronic carrier state of the disease, in which leptospira localize and remain viable in the renal tubules despite the presence of humoral or cellular immunity of the host (8).

Pathogenic Leptospira outer membrane contains lipopolysaccharide, glycolipid, and lipoproteins that determine virulence and are the main targets for immunity. Leptospira endotoxin derived from the outer membrane from virulent strains of leptospira may be a possible mechanism for the pathogenesis of leptospirosis and has been the focus of current leptospiral research. The leptospira endotoxin differs from Gram-negative bacteria in that it lacks 2-Keto-3-deoxyoctonic acid (KDO), an authentic chemical component of endotoxin, inducing less pyrogenicity and less lethality when administered to animals, whereas it may induce necrosis of mammalian cells (9). In contrast, the outer membrane of avirulent leptospira does not contain endotoxin-like components. Peptidoglycan, a protein extracted from the outer membrane endotoxin of L. interrogans, activates macrophages (10) and induces the release of tumor necrosis factor- (TNF-) from human monocytes (11).

Further elucidation of the kidney as a primary target of leptospirosis has shown that virulent L. interrogans may adhere to renal epithelial cells when studied in vitro, whereas the avirulent strain of saprophytic L. biflexa serovar patoc adheres nonspecifically to a glass surface (12). Pathogenic leptospira antigen has been detected in the kidney of infected mice by immunoperoxidase staining near blood vessels, within inflammatory infiltrates, and intraluminal in proximal and distal portions of the nephron (13). More recent, several outer membrane proteins were cloned and localized to the tubule and interstitium during acute tubulointerstitial nephritis of hamsters (14).

Although leptospirosis is an important cause of acute renal failure worldwide, mechanism of renal injury caused by this organism is not yet fully studied and understood. To elucidate the mechanism of tubulointerstitial injury caused by L. shermani infection, we analyzed the effect of the leptospira outer membrane protein on cultured mouse mTAL cells in relation to the expression of a variety of genes related to tubular injury and inflammation. This study suggests a possible mechanism of how an infecting organism may induce a renal injury.

	Materials and Methods

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Preparation of Outer Membrane Protein Extract of Leptospira
Two commonly encountered pathogenic leptospira serovars, L. shermani and L. bratislava, and a nonpathogenic L. biflexa serovar patoc were obtained from American Type Culture Collection (Manassas, VA) and cultured in 10% EMJH leptospiral enrichment medium (Difco, Detroit, MI). Leptospires were enumerated by dark-field microscopy as described by Miller (15). Protein extract from the outer membrane of leptospires was obtained as described previously (16). Briefly, leptospira cells were cultured for 5 to 7 d at 28°C until a cell density of 10⁸ per milliliter was obtained. A total of 200 ml of cultured cells were centrifuged at 15,000 x g for 30 min and resuspended in 8 ml of 1 M NaCl. The bacteria were observed under dark-field microscopy until conversion to spherical form, centrifuged again at 15,000 x g for 30 min, and resuspended in 8 ml of distilled water. Treatment of this cell suspension with 0.04% sodium dodecyl sulfate solubilized the outer membrane. The cells were removed by centrifugation, and the supernatant was filtered through a 0.45- µm membrane filter and lyophilized. Protein concentration was measured by the Bradford method (Protein Assay, Bio-Rad Laboratories, Hercules, CA). Each extraction procedure yielded approximately 0.5 mg of protein per serovar.

Mouse mTAL Cell in Culture
Experiments were carried out on subcultured cells derived from isolated mTAL microdissected from the kidney of a 1-mo-old normal mouse as described previously (17). Cultured mTAL cells were grown in a modified culture medium (Dulbecco's modified Eagle's medium: F-12 nutrient mixture (Ham; GIBCO BRL, Rockville, MD) 1:1 vol/vol; 60 nM sodium selenate; 5 µg/ml transferrin; 2 mM glutamine; 5 µg/ml insulin; 50 nM dexamethasone; 1 nM triiodothyronine; 10 ng/ml epidermal growth factor; 2% fetal calf serum; 20 mM Hepes, pH 7.4) at 37°C in 5% CO₂-95% air atmosphere. All experiments were performed on sets of confluent cells (6th and 15th passages) grown on Petri dishes. The cultured cells were shifted to a serum-free medium 24 h before the experiment. Outer membrane protein extract from three leptospira serovars was added to the cell culture medium for 48 h. Total RNA was extracted for reverse transcriptase-PCR, and supernatant was collected for nitrite and protein measurement. Nuclear protein was extracted for electrophoresis mobility shift assay. All measurements were done at least in triplicate assays.

Cytotoxicity
Cell viability was estimated by a tetrazolium-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) (18) to determine the nonspecific cytotoxicity of the outer membrane protein extract of L. shermani and L. bratislava to mTAL cells. Cells were plated in 96-well plates (Corning Co., Corning, NY). After culturing for 3 d, cells were exposed to various concentrations (0.1 to 1 µg/ml) of outer membrane protein extract. After a 48-h incubation, 40 µ1 of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well. After 2 h at 37°C, the cells were lysed by adding 100 µ1 of 20% (wt/vol) sodium dodecyl sulfate and 50% (vol/vol) N,N-dimethylformamide (pH 4.7) and incubated overnight at 37°C. The absorbance at 570 nm was measured for each well using a Dynex microplate reader. The reported cell viability was the percentage of viable cells in comparison with the control wells. Duplicate measurements were made for each on at least two separate occasions.

Limulus Assay
For detecting endotoxin, activity to gelate the amoebocyte lysate of the horseshoe crab Limulus polyphemus was tested by using the E-Toxate Limulus amebocyte lysate (Sigma, St. Louis, MO) according to the instructions of the manufacturer.

RNA Extraction and RT-PCR
RNA was extracted from confluent mTAL cells using the guanidinium thiocyanate-phenol-chloroform method using RNA-zol (Cinna/Biotecx Laboratories International Inc., Friendwood, TX). The total RNA concentration was treated with RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) at 37°C for 30 min, and RNA concentration was evaluated by spectrophotometry. RNA (1 µg) was reverse-transcribed with avian myeloblastosis virus reverse transcriptase (RT AMV, Boehringer Mannheim) at 42°C for 60 min. Complementary DNA was amplified for 30 to 42 cycles in 100 µ1 total volume containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 10 mM dNTP, 1.5 to 3.0 mM MgCl₂, 1 unit Taq polymerase, and 10 pmol of specific PCR primers. The thermal cycling protocol was as follows: 94°C for 1 min, 60°C for 1 min, and 72°C for 3 min. Amplification products were separated on a 4% agarose gel with ethidium bromide and then photographed.

Primer Construction
The primers constructed for RT-PCR were as described previously (19,20). The nitric oxide synthase (iNOS) primer pair was 5' gtg ttc cac cag gag atg ttg 3' (sense) and 5' tct ggt cga tgt cat gag caa agg 3 ' (antisense) and yielded a 508-bp PCR product. The monocyte chemoattractant protein-1 (MCP-1) primer pair was 5' agg tcc ctg tca tgc tcc tgg 3' (sense) and 5' gtc act cct aca gaa gtg ctt g 3 ' (antisense) and yielded a 424-bp PCR product. The TNF- primer pair was 5' atg agc aca gaa agc atg atc cgc 3' (sense) and 5' cca aag tag acc tgc ccg gac tc 3' (antisense) and yielded a 692-bp PCR product. The transforming growth factor-ß1 (TGF-ß1) primer pair consisted of sense 5' ata cag ggc ttt cga ttc agc 3' and antisense 5' gtc cag gct cca aat ata gg 3' and yielded a 360-bp PCR product. The laminin B1 primer pair was 5 ' caa gct tga gag gaa cgt gg 3' (sense) and 5' tta cct tgg tca ccg agc 3' (antisense) and yielded a 443-bp PCR product. The intercellular adhesion molecular-1 (ICAM-1) primer pair was 5 ' aac ata aga ggc tgc cat cac g 3' (sense) and 5' tcg gag gat cac aaa cga agc 3' (antisense) and yielded a 432-bp PCR product. The osteopontin primer pair was 5' aca ctt cac tcc aat cgt cc 3' (sense) and 5' tgc cct ttc cgt tgt tgt cc 3' (antisense) and yielded a 245-bp PCR product. The cyclo-oxygenase 2 (COX-2) primer pair was 5' gga gag aag gaa atg gct gc 3' (sense) and 5' tta cag ctc agt tga acg cc 3' (antisense) and yielded a 411-bp PCR product. The ß-actin primer pair was 5' tct agg cac caa ggt gtg 3' (sense) and 5' tca tga ggt agt ccg tca gg 3' (antisense) and yielded a 460-bp PCR product.

Quantitation of PCR Products: Competitive PCR Assay
The PCR products were analyzed initially by amplification at the exponential phase. For those mRNA levels that showed changes in the ratio to ß-actin mRNA by optical density obtained by scanning densitometer, competitive PCR assays were performed for more accurate quantitation. Competitive PCR was performed for the measurement of iNOS, MCP-1, TNF-, TGF-ß1, and ß-actin. The test template for all PCR reactions was an aliquot of cDNA collected from cell culture. To quantitate test cDNA, various amounts of mutant cDNA templates were added to compete with test cDNA on an equimolar basis, as described (20,21). For iNOS, MCP-1, TNF-, TGF-ß1, and ß-actin, deletion cDNA mutant templates were developed to create 39-, 87-, 124-, 78-, and 103-bp deletions in the middle of the molecules, resulting in mutant cDNA of 469, 339, 568, 282, and 357 bp, respectively. After agarose gel electrophoresis, amplification bands stained by ethidium bromide were quantitated from the film negative by scanning densitometry. As previously reported (21), the ratio of mutant to wild-type band density was calculated for each lane and plotted as a function of the amount of initial mutant template added to the reaction. The amount of cDNA was derived from linear regression analysis with duplicate or triplicate assays. The mean values for assays were expressed as a percentage change to the control.

Nitrite Production Assay
Accumulation of nitrite in the culture supernatant was determined by a Griess reagent and was taken as an index of nitric oxide production (22). All samples were kept in -20°C until analysis when 1 ml of culture supernatant was added to the Griess reagent. The Griess reagent (1 ml) was composed of equal amounts of 0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% concentrated H₃PO₄, which were added right before reaction. The reaction was performed when the Griess reagent reacted with nitrite in the culture supernatant (1 ml) and formed a pink to dark color after incubation for 15 min and then were read by spectrophotometer at 546 nm. NaNO₂ was included as standard.

Enzyme-Linked Immunosorbent Assay for MCP-1 and TNF-
The supernatant from cultured mTAL cells was subjected to measurement of peptide levels after exposure to 0.2 µg/ml outer membrane protein of L. shermani and L. bratislava for 48 h. Mouse MCP-1 and TNF- peptide levels were measured by the enzyme-linked immunosorbent assay (ELISA) method using commercially available kits (Quantikine, R&D Systems, Minneapolis, MN).

Antiserum for L. Shermani
Antiserum against L. shermani was prepared in New Zealand white rabbits by immunizing outer membrane protein as described previously (23). A total of 400 µg of detergent-soluble protein was mixed with Freund's complete adjuvant and injected intradermally into various sites along the back of the rabbit. A booster of the same dose was given 2 wk later. The animals were bled 2 wk after the last inoculation. The titer of the antiserum was determined by a microscopic agglutination test and revealed a titer of 1:10,000.

Nuclear Protein Extraction
Nuclear proteins were prepared according to Satriano and Schlondorff (24) with modifications. After the medium was removed, mTAL cells in 75-cm² flasks were harvested with trypsin and pelleted after centrifugation. Pellets from cells were washed with ice-cold phosphate-buffered saline and resuspended with buffer A containing 10 mM Hepes (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 3.5 mM dithiothreitol (DTT), H₂O, and protease inhibitor (Complete^TM, Boehringer Mannheim). Cells were incubated on ice for 10 min and centrifuged for 5 min at 650 x g. The pellets were resuspended with the same buffer A containing 0.5% Nonidet P-40, lysed by vortexing, and allowed to swell on ice for 20 min. The nuclear fractions were pelleted for 5 min at 6,000 x g and resuspended with buffer B containing 5 mM Hepes (pH 7.9), 26% glyceral, 1.5 mM MgCl₂, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM DTT, 0.4 M NaCl, H ₂O, and complete. After incubation on ice for 30 min, the nuclear fractions were centrifuged for 10 min at 12,000 x g. The supernatants containing nuclear protein were divided into aliquots and stored at -80°C for subsequent use. Protein concentrations were determined by a Bradford assay using the Bio-Rad protein assay.

Electrophoretic Mobility Shift Assay
Whole-cell nuclear extracts were subjected to assays for NF- B binding activity using NF- B consensus oligonucleotide (5'-AGT TGA GGG GAC TTT AGG C-3'; Promega, Madison, WI) radiolabeled with [-³²P] dATP by T₄ polynucleotide kinase 3 (Amersham Pharmacia Biotech, Uppsala, Sweden). A total of 10 µg of nuclear protein was incubated with 70 to 80 kcpm of ³²P-labeled NF-B oligonucleotide in a binding mixture (50 mM Hepes [pH 7.9], 20% glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 0.5 µg poly dI-dC [Pharmacia Biotech], and H ₂O), to a final volume of 15 µl. After incubation at room temperature for 20 min, the protein-DNA complexes were resolved on native 4% polyacrylamide gel in 0.5x Tris-borate-EDTA buffer system and run at 200 V for 1.5 h in a 4°C cold room. Gels were transferred to Whatmann paper, dried, and exposed to Kodak XR5 film (Rochester, NY) in film holders for 4 to 16 h at -80°C.

Specific competition control of unlabeled oligonucleotide at 100-fold excess was added to the binding reaction mixture for 10 min before the addition of the labeled B probe. To confirm the specificity of binding reaction, a 100-fold unrelated unlabeled oligonucleotide (SP-1) was added to the binding reaction mixture 10 min before the addition of the labeled B probe.

Supershift assays were performed using 2 µl of rabbit polyclonal antibodies (100 µg/ml) to the NF-B subunits p50, p65, and c-Rel (Santa Cruz Biotechnology, Santa Cruz, CA) for 10 min at room temperature followed by adding the labeled B probe. Antibodies to individual NF-B protein subunits may cause either supershift or deplete homodimeric or heterodimeric complexes that bind the oligonucleotides as indicated in several studies (25).

Statistical Analyses
Differences between groups were analyzed by unpaired t test. All measurements were done in at least triplicate experiments, and values were expressed as means ± SEM. Statistical significance was set at P < 0.05.

	Results

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Cytotoxicity
To ensure that the observed changes induced by outer membrane protein extract of L. shermani were not related to cell damage, cell viability, using as an index of cell injury (18), was analyzed for L. shermani-treated (0.1 to 1 µg/ml L. shermani outer membrane protein extract for 48 h at 37°C) and untreated mTAL cells. The percentage of viable cells was not significantly different between treated and untreated cells (0.1 µg/ml, 96 ± 4%; 0.2 µg/ml, 99 ± 5%; 0.3 µg/ml, 95 ± 8%; 0.5 µg/ml, 102 ± 12%; 1 µg/ml, 97 ± 6%; n = 4 versus control). Most mTAL cells remained viable after 48 h of L. shermani outer membrane extract incubation. Untreated mTAL cells were incubated in either serum-free or serum-rich condition for 24 h to evaluate cell viability at serum-free condition. The experiments suggested that 24 h of serum-free incubation did not affect the cell viability and growth of mTAL cells (serumfree, 124 ± 3%; serum-rich, 123 ± 3%; n = 6 versus control at hour 0).

Limulus Assay
Gelation activity in the Limulus test was observed at an outer membrane protein extract concentration over 100 ng/ml. The outer membrane protein extract was active in the limulus lysate gelation test for L. shermani and L. bratislava but not for the avirulent nonpathogenic serovar L. patoc.

iNOS, MCP-1, and TNF- mRNA Levels after Exposure to L. Shermani Outer Membrane Protein
mRNA for iNOS, MCP-1, TNF-, and ß-actin were measured by RT-competitive PCR, and the results were expressed as percentage change of controls. Forty-eight h after adding outer membrane protein to mTAL cells, the iNOS mRNA showed a 3.4-, 3.8-, 4.2-, 4.3-, and 4.4-fold increase at doses of 0.1, 0.2, 0.3, 0.5, and 1 µg/ml, respectively, when compared with the control (P < 0.01). The MCP-1 mRNA showed a 1.6-, 2.1-, 3.0-, 3.1-, and 3.1-fold increase at doses of 0.1, 0.2, 0.3, 0.5, and 1 µg/ml, respectively, when compared with the control (P < 0.05 at doses of 0.1 and 0.2 µg/ml; P < 0.01 at doses of 0.3, 0.5, and 1 µg/ml). The TNF- mRNA showed a 1.5-, 2.4-, 2.5-, 3.0-, and 3.2-fold increase at doses of 0.1, 0.2, 0.3, 0.5, and 1 µg/ml, respectively, when compared with the control (P < 0.05 at doses of 0.1, 0.2, and 0.3 µg/ml; P < 0.01 at doses of 0.5 and 1 µg/ml) (Figure 1).

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Reverse transcriptase-PCR (RT-PCR) results of nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor- (TNF-), and ß-actin mRNA 48 h after adding outer membrane protein of Leptospira shermani to medullary thick ascending limb (mTAL) cells (A). The iNOS mRNA showed a 3.4-, 3.8-, 4.2-, 4.3-, and 4.4-fold increase at doses of 0.1, 0.2, 0.3, 0.5, and 1 µg/ml when compared with the control. The MCP-1 mRNA showed a 1.6-, 2.1-, 2.4-, 2.5-, and 2.3-fold increase at doses of 0.1, 0.2, 0.3, 0.5, and 1 µg/ml when compared with the control. The TNF- mRNA showed a 1.5-, 2.4-, 2.5-, 3.0-, and 3.2-fold increase at doses of 0.1, 0.2, 0.3, 0.5, and 1 µg/ml when compared with the control. No significant changes in mRNA levels were found in ß-actin (B). M, marker; C, control; *P < 0.05; **P < 0.01.

Pathogenic and Nonpathogenic Leptospira: L. Shermani, L. Bratislava, and L. Patoc
In addition to L. shermani, the outer membrane protein extract from another common pathogenic serovar, L. bratislava, and from a nonpathogenic serovar, L. patoc, was added to the mTAL cells for a comparison study. Outer membrane protein extract (0.2 µg/ml) was administered to mTAL cells for 48 h. Significant changes in mRNA levels were found in iNOS, MCP-1, and TNF- but not in the control ß-actin mRNA. By competitive RT-PCR, the outer membrane protein induced a 4.2-fold increase in iNOS mRNA by L. shermani (P < 0.01) and a 1.5-fold increase (P < 0.05) by L. bratislava. MCP-1 mRNA showed a 3-fold increase by L. shermani (P < 0.01) and a 1.8-fold increase by L. bratislava (P < 0.05). TNF- mRNA showed a 2.4-fold increase (P < 0.05) by L. shermani and a 1.5-fold increase (P < 0.05) by L. bratislava (Figure 2). Administration of L. patoc outer membrane protein extract did not induce changes in any mRNA levels. For COX-2, osteopontin, laminin B1, TGF-ß1, and ICAM-1 mRNA, the changes in the levels were compared by optical density obtained by scanning densitometer of the PCR product at the exponential phase. None of these genes were upregulated by any of the leptospira outer membrane proteins (data not shown).

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Representative RT-competitive PCR results of iNOS, MCP-1, TNF-, and ß-actin by leptospira outer membrane proteins (A). The outer membrane protein (0.2 µg/ml) induced a 4.2-fold increase in iNOS mRNA by L. shermani and a 1.5-fold increase by L. bratislava. MCP-1 mRNA showed a 3-fold increase by L. shermani and a 1.8-fold increase by L. bratislava. TNF- mRNA showed a 2.4-fold increase by L. shermani and a 1.5-fold increase by L. bratislava (B). Outer membrane protein from avirulent L. patoc did not give significant changes in the mRNA. LS, L. shermani; LB, L. bratislava; LP, L. patoc; *P < 0.05; **P < 0.01.

Supernatant Nitrite, MCP-1, and TNF- Peptide Levels
Outer membrane protein from L. shermani stimulated nitrite production 1.7-fold in the supernatant when compared with controls (70 ± 9 versus 39 ± 6 ng/ml, P < 0.05). Outer membrane protein from L. bratislava did not stimulate significant different levels of nitrite (46 ± 8 versus 39 ± 6 ng/ml). The supernatant MCP-1 levels were significantly increased 7.1-fold by L. shermani (754.4 ± 96.7 versus 105.7 ± 36.2 pg/ml, P < 0.001). L. bratislava also increased MCP-1 production by 3.2-fold (346.3 ± 75.7 versus 105.7 ± 26.2 pg/ml, P < 0.05). The supernatant TNF- levels were significantly increased 5-fold by L. shermani (75.3 ± 10.4 versus 15.0 ± 4.4 pg/ml, P < 0.001). L. bratislava also increased TNF- production by 3.2-fold when compared with controls (36.1 ± 8 versus 15.0 ± 4.4 pg/ml, P < 0.05) (Figure 3).

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Supernatant nitrite and peptide levels of MCP-1 and TNF- . Outer membrane protein from L. shermani stimulated nitrite production 1.7-fold in the supernatant when compared with controls. Outer membrane protein from L. bratislava did not stimulate significantly different levels of nitrite. The supernatant MCP-1 levels were significantly increased 7.1-fold by L. shermani and 3.2-fold by L. bratislava. The supernatant TNF- levels were significantly increased 5-fold by L. shermani and 3.2-fold by L. bratislava. *P < 0.05; ***P < 0.001.

Heat Inactivation and Protein Digestion
Heat treatment of the L. shermani outer membrane protein extract by boiling (100°C for 30 min) reduced the mRNA levels by 35% in iNOS (P < 0.05), 34% in MCP-1 (P < 0.05), and 23% in TNF- (P < 0.05). Outer membrane protein extract digested with proteinase K (10 mg/ml) for 30 min at 37°C reduced the mRNA levels by 51% in iNOS (P < 0.05), 58% in MCP-1 (P < 0.05), and 66% in TNF- (P < 0.05) (Figure 4). These results indicate that the active component of the outer membrane protein extract is heat labile and can be digested by proteinase K.

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Representative RT-competitive PCR results of heat treatment, protein digestion, and antiserum effect by L. shermani outer membrane protein extract in mTAL cells (A). Heat treatment of the L. shermani outer membrane protein extract reduced the mRNA levels by 35% in iNOS, 34% in MCP-1, and 23% in TNF- . Outer membrane protein extract digested with proteinase K reduced the mRNA levels by 51% in iNOS, 58% in MCP-1, and 66% in TNF- . These results indicate that the active component of the outer membrane protein extract is heat labile and can be digested by proteinase K. Outer membrane protein preincubated with antiserum to L.shermani reduced the mRNA levels by 85% in iNOS, 74% in MCP-1, and 89% in TNF-, indicating the specificity of the effect (B). No significant reduction was seen by preincubation of outer membrane protein with preimmune serum. HT, heat treatment; PK, proteinase K digestion; Ab, antiserum preincubation; S, preimmune serum preincubation. *P < 0.05; **P < 0.01.

Inhibition by Antiserum
To clarify the specificity of the increased mRNA levels, a rabbit antiserum raised to the outer membrane protein of L. shermani as well as preimmune rabbit serum were incubated with the outer membrane protein from L. shermani, respectively, before adding to the cell culture. In 48-h incubation, the increment of iNOS, MCP-1, and TNF- mRNA was inhibited significantly by the antiserum but not by preimmune rabbit serum. Outer membrane protein of L. shermani preincubated with antiserum to L. shermani reduced the mRNA levels by 85% in iNOS (P < 0.01), 74% in MCP-1 (P < 0.01), and 89% in TNF- (P < 0.01). Outer membrane protein of L. shermani preincubated with preimmune rabbit serum somewhat reduced mRNA levels of iNOS, MCP-1, and TNF- by 25%, 29%, and 20%, respectively, but did not reach statistical significance (Figure 4). Adding antiserum alone did not influence the levels of expression in mRNA (data not shown).

NF-B Nuclear DNA Binding
Significant nuclear DNA binding of NF-B transcriptional factor was observed at 90 min under the influence of outer membrane protein extract by L. shermani and, to a lesser degree, by L. bratislava but not by L. patoc (Figure 5). At 48 h, significant nuclear DNA binding of NF-B transcriptional factor was observed by L. shermani and to a lesser degree by L. bratislava (Figure 6A). The NF-B nuclear DNA binding is specific as shown by the inhibition by 100-fold excess of cold NF-B probe but not by an irrelevant SP-1 probe. In a supershift assay, the NF-B complex is composed of at least p50, p65 peptides subunits but not c-Rel (Figure 6B).

View larger version (61K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Significant nuclear DNA binding of NF- B transcriptional factor is observed in the mTAL cell cultured under the influence of virulent L. shermani and L. bratislava but not avirulent L. patoc outer membrane protein as shown by the electrophoretic mobility shift assay study at 90 min.

View larger version (65K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Significant NF- B nuclear DNA binding persisted at 48 h by L. shermani and L. bratislava (A). The specificity was shown by the inhibition by 100-fold excess of cold NF- B probe but not by an irrelevant SP-1 probe. In the supershift assay, the NF- B complex is composed of at least p50, p65 subunits but not c-Rel (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Clinical and pathologic observations suggest that a toxin may play a role in the pathogenesis of leptospirosis. Previous reports have shown that the main factors in the pathogenesis of the human leptospirosis are related to the presence of organisms elicited by their migration and to elaboration of their virulent toxins, including products released by lysis of the microorganism (26).

Identification of outer membrane proteins, which are involved in the pathogenesis of leptospirosis, is an important step for current leptospiral research (14). Leptospiral outer membrane proteins are likely to be relevant to host—pathogen interactions. Leptospiral endotoxins, located on the outer membrane, seem to be the major antigens that result in immunity to L. interrogans and might be responsible for renal dysfunction (27). By immunoelectron microscopy, gold-labeled leptospiral antigen was found adjacent to cell membranes of the hepatocyte, kidney tubular cells, and endothelial cells of the interstitial capillary in animal studies (28). Leptospiral glycolipoprotein expression was also detected in renal tubules and vascular lumen of interstitium and paralleled with tubulointerstitial nephritic changes (29). Moreover, glycolipoprotein extracted from L. interrogans is a potent inhibitor of Na⁺-K⁺-ATPase in renal epithelial cells (30). Therefore, a disturbed tubular function may be elicited by the leptospiral outer membrane protein components.

In this study, outer membrane protein from pathogenic leptospira, particularly L. shermani, induced increased expression of iNOS, MCP-1, and TNF- of mTAL cells both in mRNA and in the end products including nitrite and peptide levels. Conversely, outer membrane protein extract from avirulent nonpathogenic serovar L. patoc did not induce changes in the gene expression. These findings are compatible with the notion that saprophytic avirulent Leptospira does not induce clinical manifestations as demonstrated by the lack of characteristic leptospira endotoxin, a different pattern of outer membrane antigen expression (31), and its readily being phagocytosed by human monocyte and polymorphonuclear cells (32,33). That only pathogenic leptospira adhere to renal epithelial cells in culture but not avirulent nonpathogenic serovar L. biflexa (12) indicates that the wide variety of gene expressions may be induced by outer membrane protein from virulent leptospira. These results, for the first time, demonstrate the relationship between leptospira infection and the possible mechanism of tubulointerstitial nephritis.

The induction of mRNA seems to be specific to the outer membrane protein because antiserum raised against the outer membrane protein reduced the effects, whereas preimmune serum has no effect. Consistent with previous reports that the outer membrane protein may be heat modifiable (34), the induction of mRNA can be inhibited by heat treatment and may be reduced by protein digestion of the leptospiral outer membrane components.

In this study, endotoxin activity was found in the outer membrane components from pathogenic leptospiras but not from nonpathogenic leptospira. That heating and protein digestion reduced the ability in mRNA upregulation of mTAL cells by pathogenic leptospira outer membrane highly suggests that protein component may play an important role. Outer membrane proteins were known to be expressed along with lipopolysaccharide in the outer membrane of leptospira (14), and both leptospira lipopolysaccharide-like substance and protein contents in the pathogenic leptospira outer membrane have been shown to be toxic to mammalian cells. The outer membrane extraction method used in this study may harvest protein components and leptospira lipopolysaccharide-like substance from pathogenic leptospira. This is consistent with a previous description showing that extract of glycolipoprotein from the outer membrane of pathogenic leptospira induced the gelation of Limulus lysate because it contains a small amount of lipopolysaccharide-like substance. However, glycolipoprotein was responsible for major toxicity by leptospira (27). The extraction method used in this study does not obtain endotoxin activity from nonpathogenic leptospira outer membrane. It is possible that nonpathogenic leptospira contains neither sufficient leptospira endotoxin in the outer membrane nor pathogenic outer membrane protein. Thus, it seems that the outer membrane protein could be part of the leptospira endotoxin.

The mTAL segment plays a vital role in the regulation of solute transport, intrarenal hemodynamics, and renal immune reaction. Recent evidence indicates that nitric oxide has potent effects on renal function, including modulation of renal and glomerular hemodynamics, renin secretion, tubuloglomerular feedback, and sodium excretion (35). The presence of iNOS mRNA expression was found on the mTAL and is one of the major sources of nitric oxide production under both basal and stimulated conditions (36). L. shermani, a serovar belonging to pathogenic L. interrogans, stimulated nitric oxide production by its outer membrane protein in mTAL cells. The production of nitric oxide in mTAL cells may participate in the regulation of renal medullary oxygenation and play an important role in the prevention of medullary hypoxic injury. Conversely, in stimulated states, nitric oxide has potent proinflammatory effects. It is possible that through the interaction with superoxide and the generation of peroxynitrite, nitric oxide may further contribute to tissue injury (37). Thus, increased production of nitric oxide may act as a double-edged sword during leptospirosis renal injury.

The characteristic feature of tubulointerstitial nephritis caused by leptospira infection is the infiltration of inflammatory cells including mononuclear cells (8). One of the major findings in this study is the upregulated expression of MCP-1 by leptospira outer membrane protein. Chemokine expression is upregulated in the kidney during various forms of glomerular and interstitial injury (38,39). Among the chemotactic factors, MCP-1 plays an active role in renal injury and accounts for approximately 70 to 80% of the monocyte chemotactic activity produced by cultured human mesangial cells (40), renal cortical epithelial cells (41), proximal tubular cells (42), and bovine glomerular endothelial cells (43). It is clear that intrinsic renal cells can produced a large amount of chemotactic peptides. Thus, it can be speculated from this in vitro study that induced chemokine synthesis by cultured cells may be involved in renal chemokine production in vivo.

TNF- is a proinflammatory cytokine produced by monocytes/macrophages (44) and by resident renal cells (45). Cinco et al. (11) found that a preparation of outer membrane peptidoglycan of L. interrogans serovar copenhageni induced the release of TNF- from peripheral blood mononuclear cells. The TNF- gene contains NF-B—binding sequence in its promoter (46). TNF- itself stimulates NF-B activation (47) and may further lead to increased TNF- synthesis through further NF-B activation in an autocrine amplification manner. In this study, we demonstrated that the outer membrane protein stimulates TNF- expression in mTAL cells with an associated increased nuclear DNA binding of NK-B.

NF-B activation has been shown to be important in various nephritis models and plays a central role of inflammation both in glomerulonephritis and in tubulointerstitial nephritis (47). In this study, outer membrane protein from Leptospira induced marked nuclear DNA binding of NF-B and an associated increase in downstream genes, including iNOS, MCP-1, and TNF-. These effects were more prominently induced by L. shermani than by L. bratislava but not by the avirulent non-pathogenic L. patoc, and the effects were consistently shown in the downstream genes. This result is consistent with previous studies in mTAL cells showing that iNOS production may be stimulated by lipopolysaccharide through NF-B activation (48).

The NF-B in this study is a complex composed of p50 and p65 but not c-Rel subunits by the supershift assay. Similar NF-B isotypes were observed in mTAL cells stimulated by lipopolysaccharide (48). Whether mTAL cells respond to different stimuli with similar pathways of activation deserves further observation. It is interesting to note that the NF-B downstream COX-2, ICAM-1 genes were not induced by leptospira outer membrane protein in mTAL cells. Therefore, a differential expression may exist in the activation of NF-B downstream genes in this model. No evidence was found in this study to suggest that leptospira outer membrane protein induces the synthesis of genes related to tubulointerstitial cell activation such as osteopontin. From animal and human biopsy studies, there is evidence that chronic infection of leptospirosis may induce chronic interstitial nephritis and fibrosis (49). However, the growth factor and extracellular matrix genes examined in this study, namely TGF-ß1 and laminin B1, did not show significant changes. Therefore, direct evidence linking the outer membrane protein to the induction of fibrosis in this cell type is lacking. Further study is needed to evaluate the role of leptospira infection in fibrosis in other cell types, in particular, interstitial fibroblast could be a reasonable candidate to study these effects.

In conclusion, outer membrane protein in pathogenic leptospira may be responsible for renal tubular injury and inflammation through NF-B—associated gene expression as indicated in this study. As proximal tubular dysfunction is the major form of injury in most serovars, this study indicated that L. shermani infection might also induce mTAL injury. Finally, these results may be important in understanding the pathogenesis of tubulointerstitial nephritis caused by this organism.

	Acknowledgments

 
This study was supported by grants from the National Health Research Institute and the National Science Council, Taiwan, Republic of China. The authors thank Yi-Ching Ko, Chiung-Tseng Huang, Ya-Hui Huang, and Hsiao-Mei Yu for their excellent and dedicated technical support.

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