Angiotensin II Upregulates Toll-Like Receptor 4 on Mesangial Cells
Gunter Wolf*,
Jürgen Bohlender*,
Tzvetanka Bondeva*,
Thierry Roger,
Friedrich Thaiss and
Ulrich O. Wenzel
* Klinik für Innere Medizin III, University of Jena, Jena, Germany; Infectious Diseases Services, Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; and Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany
Address correspondence to: Dr. Gunter Wolf, Klinik für Innere Medizin III, University Hospital Jena, Erlanger Allee 101, D-07747 Jena, Germany. Phone: +49-3641-9324301; Fax: +49-3641-9324302; E-mail: gunter.wolf{at}med.uni-jena.de
Received for publication July 10, 2005.
Accepted for publication March 20, 2006.
Angiotensin II (AngII) mediates proinflammatory properties byactivating NF-B transcription factor nuclear translocation andinducing the expression of chemokines. For examination of whetherAngII modulates the expression of Toll-like receptor 4 (TLR4),a key element of the innate immune system that senses LPS, mousemesangial cells (MMC) were treated with AngII. AngII upregulatedTLR4 mRNA and protein in MMC, and this effect was mediated throughAngII type 1 receptors. Reporter gene experiments indicate thatan activating protein-1 (AP-1) as well as an E-26 specific sequence(Ets) binding site in the TLR4 promoter are responsible forthe AngII-stimulated transcriptional activity of the TLR4 gene.Preincubation of MMC with AngII enhanced LPS-induced NF-B activationand chemokine expression. Immunohistochemical analyses revealedthat double-transgenic rats that overexpressed human renin andangiotensinogen expressed higher levels of glomerular TLR4 comparedwith normal Sprague-Dawley rats. In vivo, infusion with AngIIbut not with norepinephrine into rats for 7 d also enhancedglomerular NF-B activation after systemic application of LPS,suggesting that the effects are independent of concomitantlyinduced hypertension. Together, these observations suggest thatAngII leads to an activation of the innate immune system bya novel mechanism involving the upregulation of TLR4. Our datacontribute to a better understanding of how exogenous infectionsmay trigger renal autoimmune processes, particularly in pathophysiologicsituations with high renal AngII concentrations. Because TLR4binds endogenous ligands (e.g., extracellular matrix components)in addition to microbial products, AngII-mediated upregulationof TLR4 also could be relevant for the development of inflammationin many noninfectious renal diseases.
Acute or chronic inflammation is a hallmark of many kidney diseases.Although this pathophysiologic process is apparent in glomerulonephritis,tubulointerstitial nephritis, and vasculitis, there is clearevidence that renal infiltration with leukocytes also occursin primarily non–immune-mediated diseases such as diabeticnephropathy and hypertension and after reduction of renal mass(1). It is assumed that primary renal injury, either immunologicor nonimmunologic, leads to a local induction of chemokinesand cytokines that, in turn, mediates the migration of immunecells into the kidney. Clinically, it is well appreciated thatinfections with bacteria or viruses can induce flare-ups ofautoimmune syndromes (2). For example, infections are thoughtto trigger the onset and exacerbation of IgA nephropathy, immunecomplex nephritis, and vasculitis (e.g., systemic lupus erythematosus,anti-neutrophil cytoplasmic antibody–positive vasculitis[3]). It is obvious that dysregulation of the immune system,which normally detects and eliminates invading microorganismsby discriminating between self and nonself, is involved in suchpathophysiologic processes. Principally, the immune system canbe divided into adaptive and innate immunity. Much is knownof the complex processes of the adaptive immune system in whichB cells and T cells detect nonself structures by means of antigen-specificreceptors.
Whereas the adaptive immune system exists only in vertebrates,the innate immune system is present in all multicellular organisms,indicating that it is an ancestral system (4). This system representsthe first line of host defense against microbial pathogens.Detection of invading pathogens by innate immune cells relieson the sensing of molecular structures that are common to manypathogens but not expressed by the host. These structures aredetected through germ-line encoded receptors or molecules, collectivelynamed "pattern recognition receptors." One important class ofpattern recognition receptors that was identified recently inmammals consists of members of the Toll-like receptor (TLR)family. TLR were discovered during studies of the search forgenes with sequence homology with the intracellular domain ofthe IL-1 receptor and Drosophila Toll molecules. DrosophilaToll originally were characterized as a protein involved inthe establishment of the dorsoventral polarity in developingfly embryos, then in the resistance of adult flies to fungalinfections. Currently, 13 TLR genes have been identified inthe mouse and human genome. The first report involving a TLRin host response to a microbial product was published in 1998,when missense and null mutations of the mouse TLR4 gene werelinked with the unresponsiveness phenotype to LPS, the majorproinflammatory cell wall component of Gram-negative bacteria(5). Since then, TLR have been shown to recognize a broad rangeof microbial structures. Upon activation, most TLR induce acommon intracellular signaling pathway that culminates in activationof the NF-B, a key transcription factor involved in the inductionof cytokines, chemokines, and cell-surface molecules such asadhesins, selectins, integrins, and co-stimulatory molecules(6–8). It is interesting that kidney mesangial as wellas tubular cells express TLR4 (9,10).
There is accumulating evidence that angiotensin II (AngII) exertsproinflammatory effects in the kidney by locally stimulatingchemokine expression by a NF-B–dependent mechanism (11,12).Our study tested the whether AngII influences TLR4 expressionin mesangial cells. Our in vitro and in vivo data indicate thatAngII leads to an upregulation of TLR4 on mesangial cells thathas functional consequences, such as an increase in the magnitudeof LPS-induced NF-B activation and chemokine expression.
Cell Culture
Mouse mesangial cells (MMC) are a cell line that originallywas derived from SJL mice (13). These cells have been used bymany investigators and have been characterized extensively (14).They express many features of mesangial cells, including expressionof AngII type 1 (AT1) receptors (13). Cells were grown in DMEM(Life Technologies-BRL, Eggenstein, Germany) with 10% FCS in5% CO2 at 37°C. MMC were passaged every 4 to 5 d. Beforestimulation with AngII or LPS, MMC were rested for 24 h in DMEMwithout FCS.
Northern Blots
MMC were stimulated with AngII (10–10 to 10–6 M)for 24 h. Some cells also received the AT1 receptor antagonistlosartan (10–6 M; gift from MSD, Munich, Germany) or theAT2 receptor blocker PD123177 (10–6 M; Sigma, Deisenhofen,Germany). For the induction of chemokines, cells either werepreincubated for 24 h with 10–7 M AngII or directly received10 ng/ml LPS (Escherichia coli type O111:B4; Sigma) for 30 min.After washing in RNAse-free PBS, cells were lysed directly withacid guanidinium thiocyanate, and total RNA was isolated. Equalamounts of total RNA (15 µg per lane) were denatured informamide-formaldehyde at 65°C and electrophoresed througha 1.2% agarose gel that contained 2.2 M formaldehyde. Blotting,hybridization, and washing conditions were as described previously(12). A 0.5-kb cDNA fragment encoding murine TLR4 was synthesizedby PCR, subcloned into pCR-TOPO (Invitrogen, Karlsruhe, Germany),sequenced, and used as a probe (5). For chemokine detection,the cDNA probes used were the following: monocyte chemoattractantprotein-1 (MCP-1)/CCL2 (a 577-bp EcoRI fragment), RANTES/CCL5(an EcoRI-XhoI fragment of the murine RANTES clone pMuR3 [14,15]).For control hybridizations, a 2.0-kb cDNA insert of the plasmidpMCI encoding the murine ribosomal 18S band was applied. Autoradiographieswere scanned with Fluor-S multi-imager (Bio-Rad Laboratories,Hercules, CA), and data were analyzed with the computer programMulti-Analyst (Bio-Rad) (15). TRL4 signals were normalized to18S band signals and expressed relative to the ratio, set at1.0, that was obtained from control cells (no AngII). Northernblots were repeated four to five times.
Western Blots
MMC were incubated with various concentrations of AngII (10–10to 10–6 M; Sigma) for 24 h. After washing in ice-coldPBS, cells were lysed on ice in 150 µl of a buffer thatcontained 2% SDS and 60 mmol/L Tris-HCl (pH 6.8) supplementedwith a cocktail of protease inhibitors (Complete; BoehringerMannheim, Mannheim, Germany; contains antipain-HCl, chymostatin,leupeptin, bestatin, pepstatin, phosphoramidon, aprotinin, andEDTA). In addition, for selected experiments, glomeruli thatwere isolated from rats that received an infusion of PBS, AngII,or LPS (see below) were lysed directly in this buffer or intothe buffer. The protein content was measured by a modificationof the Lowry method. Proteins (70 µg in 5% glycerol/0.03%bromophenol blue/10 mmol/L dithiothreitol [DTT]) were boiledfor 5 min. They then were separated under denaturing conditionson a 7% NuPAGE precast gel (Invitrogen) and subsequently electroblottedonto a nitrocellulose membrane (Hybond-N; Amersham, Braunschweig,Germany) in transfer buffer (50 mmol/L Tris-HCl [pH 7.0], 380mmol/L glycine, 0.1% SDS, and 20% methanol). The blots wereblocked in 5% nonfat dry milk in PBS with 0.1% Tween 20 for1 h at 22°C. For the detection of TLR4, a 1:100 dilutionof a goat polyclonal IgG antibody that was generated againstan epitope that mapped within an extracellular domain of mouseTLR4 was used (Santa Cruz Biotechnology, Santa Cruz, CA). Washes,incubation with horseradish peroxidase–conjugated anti-goatsecondary antibodies (Santa Cruz Biotechnology), and detectionusing the ECL reagent (Amersham) were performed according tothe manufacturers’ recommendations (16). For controllingfor variations in protein loading and transfer, membranes werewashed for 30 min in PBS with 0.1% Tween 20 and then incubatedwith a mouse mAb against -actin (1:2000 dilution; Sigma). TRL4signals were normalized to -actin signals and expressed relativeto the ratio, set at 1.0, that was obtained from the controlcells (no AngII). Western blots were repeated independentlyfour times for cell culture experiments and three times foranimal experiments. Exposed films were analyzed by densitometryas described above.
Gel Shift Assay
MMC either were preincubated for 24 h with 10–7 M AngIIand subsequently received 10 ng/ml LPS for 30 min or were stimulateddirectly with LPS. After incubation, cells were washed twicewith ice-cold PBS, collected, centrifuged, and resuspended inbuffer A, which was composed of 20 mmol/L HEPES, 0.1 mmol/LEDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 1 µg/mlleupeptin, and 1 µg/ml aprotinin. Cells were lysed bythe addition of Nonidet-P40 for 10 s, lysates were centrifugedfor 30 s at 13,000 rpm, and pellets were resuspended in bufferB (30 mmol/L HEPES, 0.5 mol/L NaCl, 0.1 mmol/L EDTA, 0.1 mmol/LEGTA, 1 mmol/L PMSF, 1 µg/ml leupeptin, and 1 µg/mlaprotinin). Supernatants that contained nuclear proteins werealiquotted, frozen, and stored in liquid nitrogen. A total of3.50 pmol of a consensus NF-B DNA binding site double-strandoligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was end labeledwith [-32P]ATP (3000 Ci/mmol; Amersham) using T4 polynucleotidekinase (12). Binding reactions were performed in gel shift bindingbuffer (10 mmol/L Tris-HCl [pH 7.5], 4% glycerol, 1 mmol/L MgCl2,0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 50 µg/mlpoly [dI-dC] poly [dI-dC]) with and without cold competitoroligonucleotides at room temperature for 30 min. OCT1 double-strandconsensus oligonucleotides (5'-TGTCGAATGCAAATCACTAGAA-3') wereused as nonspecific competitors. Reactions were stopped by additionof loading buffer, and samples were run on a nondenaturing 6%polyacrylamide gel. Gels were exposed to x-ray films. Specificbands were quantified by densitometry as described above, andbinding of nuclear proteins from cells without AngII was assigneda value of 1.0. Gel shift analysis was performed independentlyfour times with qualitatively similar results.
Transient Transfection Experiments
The pNF-B-Luc reporter plasmid contains four tandem copies ofthe -enhancer that is fused to the herpes simplex virus thymidinekinase promoter that is linked to the reporter gene luciferase(12). The mouse TLR4 promoter was cloned in the pGL3-basic vector(Promega, Madison, WI). Wild-type, deleted, and mutant constructshave been described in detail previously (17,18). The activatingprotein-1 (AP-1) consensus DNA binding sequence (GTCAGATGAC)in –518 TLR4 promoter construct was disrupted by site-directedmutagenesis (to GTCAGACCAC, construct –518AP-1mut). BecauseEts (E-26 specific sequence) transcription factor was describedrecently to play a role in AngII-mediated inflammation and inductionof MCP-1 (19), we also used a construct in which an Ets sitewas mutated (construct –518Etsdmut). Cells were transfectedwith 10 µg of pNF-B-Luc or mouse TLR4 promoter luciferasereporter vectors together with the same amount of the pSV--galactosidasevector using Lipofectin (Invitrogen). After transfection, themedium was changed (serum-free DMEM) and incubation was prolongedfor 24 h with or without 10–7 M AngII. Transfected cellsthen were challenged with 10 ng/ml LPS for 60 min. At the endof the experiments, cells were washed three times in PBS, andcell layers were lysed. Luciferase and -galactosidase activitieswere measured using standard techniques (12). The ratio betweenluciferase and -galactosidase activities was calculated, anddata from unstimulated control cells were assigned an arbitraryvalue of 1.0. Results are representative of at least three independentexperiments performed in triplicate.
Animal Experiments
For analysis of the role of AngII on TLR4 expression in vivo,male Wistar rats (body weight 200 g) received intraperitonealinfusion for 7 d with either AngII or norepinephrine (Sigma)using osmotic minipumps (Alzet 2002, Sulzfeld, Germany; infusionrate 0.5 µl/h, 250 ng AngII/min, norepinephrine 600 ng/min[20,21]). Control animals received infusion of PBS. SystolicBP was measured on awake, slightly restrained animals on day6 using tail plethysmography. Rats then received 2 mg LPS/kgbody wt intraperitoneally for 4 h. At the end of the experiment,animals were slightly anesthetized with ether, and the kidneyswere perfused in situ via the aorta with 20 ml of ice-cold PBS.Glomeruli were isolated by differential sieving as describedpreviously (22). The final preparation had a purity >90%as judged by light microscopy. Isolated glomeruli were usedfor gel shift experiments. The whole experiment was repeatedthree times with three to six rats in each individual group.
For studying whether TLR4 expression is stimulated in vivo underprolonged conditions of high AngII, double-transgenic rats (dTGR;obtained from the Max-Delbrück Center, Berlin, Germany)that harbored human renin and angiotensinogen genes were used(23). dTGR have high circulating and local renal AngII levelsand develop a renal inflammatory state with infiltration ofmonocytes/macrophages and T cells (24). Immunohistochemicalstaining was performed on paraffin sections with microwave treatmentusing a polyclonal anti-TLR4 antibody generated against C-terminalsequence of the mouse receptor, which cross-reacts with ratTLR4 (Santa Cruz Biotechnology). An anti-goat peroxidase-labeledantibody was used as secondary reagent, and development of thecolor reaction was performed as described (25). The primaryantibody was replaced by nonimmune goat serum as a negativecontrol. An investigator who was uninformed on the various groupingsperformed a semiquantitative score. The intensity of stainingwas scored in a total of 30 glomeruli or x40 power field fortubular staining from three rats in each group. The extent ofexpression was estimated on a semiquantitative scale as describedby Kaneko et al. (26): 0, absent; 1, weak; 2, moderate or focalstaining; or 3, strong or diffuse staining.
Statistical Analyses
All data are presented as means ± SEM. Statistical significancevarious different groups first was tested with the nonparametricKruskal-Wallis test. Individual groups subsequently were testedusing the Wilcoxon-Mann-Whitney test. P < 0.05 was consideredsignificant.
AngII (10–8 to 10–6 M) for 24 h stimulated a significantincrease of TLR4 mRNA (Figure 1) and TLR4 protein (Figure 2)dose-dependently in MMC. Densitometric analysis of Northernand Western blots demonstrated that these changes were statisticallysignificant (Figures 1 and 2). The AT1 receptor antagonist losartanbut not the AT2 receptor antagonist PD123177 blocked AngII-mediatedincrease of TLR4 mRNA expression, indicating that the AT1 receptortransduced the AngII effect (Figure 3).
Figure 1. Northern blot analysis of Toll-like receptor 4 (TLR4) expression by mouse mesangial cells (MMC). Stimulation of cells with 10–8 to 10–6 M angiotensin II (AngII) for 24 h induced a significant increase in TLR4 mRNA expression. *P < 0.05 versus unstimulated controls; n = 5.
Figure 2. Western blot analysis of TLR4 by MMC. For controlling for small variations in protein loading and transfer, the membrane was reincubated with an anti–-actin antibody. AngII (10–9 to 10–6 M) for 24 h significantly increased TLR4 protein in MMC cell lysates. *P < 0.05 versus control; n = 4.
Figure 3. Northern blot analysis of TLR4 expression by MMC. The AngII type 1 (AT1) receptor antagonist losartan but not an AT2 blocker PD123177 antagonized AngII-stimulated TLR 4 mRNA expression in MMC. *P < 0.05 versus controls; n = 3.
For further exploration of potential mechanisms by which AngIIincreased TLR4 mRNA expression, MMC were transiently transfectedwith mouse TLR4 promoter luciferase reporter vectors (17,18).As shown in Table 1, AngII significantly increased –518promoter activity. Because the –518 gene region of themouse TLR4 gene contains a putative AP-1 binding site (18) thatcould mediate the AngII effect, this site was mutated in –518construct (–518AP-1mut) and the activity of the mutantpromoter was compared with that of the wild-type promoter inMMC. AngII failed to stimulate the activity of –518AP-1mut,indicating a pivotal role of the AP-1 site in AngII-mediatedincreased transcriptional activity of TLR4 (Table 1). It Etsalso was described recently as an important transcription factorfor AngII-mediated inflammatory effects, at least in vasculature(19). Therefore, we also studied a different reporter gene constructin which an Ets consensus binding site in the –518 generegion is mutated. Compared with the wild-type –518, constructbasal transcription was already significantly reduced in the–518 Etsdmut construct, indicating that ETS transcriptionfactors are important for TLR4 expression in MMC (Table 1).AngII (10–7 M) completely failed to stimulate reportergene activity in the –518 Etsdmut construct (Table 1).To test whether AngII-mediated stimulation of TLR4 expressionleads to enhanced NF-B activation, we performed gel shift assays.As shown in Figure 4, incubation of MMC with 10 ng/ml LPS for30 min or 10–7 M AngII for 24 h increased NF-B nucleartranslocation. However, the LPS-mediated increase was higherin cells that were pretreated with AngII for 24 h (Figure 4).Densitometric analysis revealed that these changes were statisticallysignificant (Figure 4). For confirmation that NF-B nuclear translocationincreased gene transcription, MMC were transiently transfectedwith an NF-B reporter plasmid. Although LPS as well as AngIIincreased NF-B–mediated transcriptional activity in MMC,MMC preincubation of cells with AngII before addition of LPSenhanced this response (Table 2).
Figure 4. NF-B nuclear translocation in MMC. Nuclear extracts from MMC that were incubated with AngII (10–7 M for 24 h) and then with or without LPS (10 ng/ml for 30 min) were analyzed by electrophoretic mobility shift assay (EMSA) using a consensus NF-B site oligonucleotide. Intensity of the NF-B bands was quantified by densitometry. Although LPS induced NF-B activation, preincubation with AngII led to a significant increase in NF-B binding to consensus nucleotides. *P < 0.05 versus controls; #P < 0.05 versus LPS alone; n = 4.
Because NF-B is an important regulator of the expression ofchemokines (14), we used MCP-1 and RANTES mRNA expression asa readout to investigate whether AngII enhanced LPS-stimulatedexpression of these chemokines. As shown in Figure 5, preincubationof MMC for 24 h with 10–7 M AngII significantly increasedLPS-induced MCP-1 and RANTES mRNA as compared with cells thatwere not pretreated with AngII.
Figure 5. Northern blot analysis of monocyte chemoattractant protein-1 (MCP-1) and RANTES mRNA expression in MMC. Preincubation with 10–7 M AngII enhanced LPS-mediated increase in chemokine transcripts compared with LPS challenge of cells that were grown without AngII. Representative of three independent experiments with qualitatively similar results.
For investigation of whether similar mechanisms are operativein vivo, AngII or norepinephrine was infused by osmotic minipumpsin rats for 7 d. Systolic BP increased significantly comparedwith controls, but systolic BP was not significantly differentbetween the AngII and norepinephrine infusion groups (AngIIinfusion 198 ± 11*; norepinephrine 164 ± 7*; controls112 ± 3 mmHg; n = 6 to 10, *P < 0.01 versus controls,NS between AngII and norepinephrine). Kidneys were removed 4h after intraperitoneal injection of LPS, and nuclear proteinswere prepared from isolated glomeruli. Compared with PBS infusion,AngII infusion increased LPS-induced NF-B activation in isolatedglomeruli (controls 1.0; AngII 1.1 ± 0.1, LPS 8.4 ±1.2*; LPS+AngII 15.9 ± 1.8 relative increase in NF-Bactivation; *P < 0.01 versus controls, P < 0.05 versusLPS only; n = 3; see Figure 6A). However, infusion with norepinephrineto increase the systolic BP failed to enhance LPS-induced NF-Bactivation (Figure 6B). In fact, norepinephrine infusion reducedLPS-induced NF-B activation by 30% compared with AngII (twoindependent experiments; Figure 6B). In addition, isolated glomeruliwere lysed from control and AngII-infused rats, and Westernblots were performed for detection of TLR4 protein expression.As shown in Figure 7, AngII infusion for 7 d but not LPS injectionfor 4 h significantly increased glomerular TLR4 protein expression.
Figure 6. EMSA for NF-B binding using nuclear proteins from isolated glomeruli. (A) LPS injection into rats that received infusion for 7 d with AngII exhibited in isolated glomerular significantly more nuclear proteins binding to NF-B sequences compared with control animals the received infusion with PBS only (quantification; see Results). (B) For testing whether these effects were mediated by AngII-induced hypertension, rats received infusion with norepinephrine for 7 d. However, norepinephrine infusion failed to increase LPS-mediated NF-B activation. This experimental series was performed three times (A) and twice (B) with pooled kidneys (three to six rats).
Figure 7. Western blot analysis of TLR4 protein expression in isolated glomeruli from rats that received PBS (controls), AngII (250 ng AngII/min) infusions for 7 d by osmotic minipumps, or 2 mg LPS/kg body wt intraperitoneally for 4 h (LPS). Densitometric analysis was normalized to -actin. AngII infusion for 7 d but not LPS injection for 4 h caused a significant upregulation of TLR4 protein expression in isolated glomeruli. *P < 0.05; n = 4.
Four-week-old dTGR that harbored human renin and angiotensinogengenes with high intrarenal AngII concentrations revealed a glomerularupregulation of TLR4 protein compared with age-matched Sprague-Dawleyrats (controls 0.88 ± 0.11; dTGR 1.38 ± 0.20 stainingscore; P < 0.01; Figure 8, A through C). In addition, tubularTLR4 expression was increased in dTGR (controls 1.25 ±0.09; dTGR 1.93 ± 0.11 staining score; P < 0.001;Figure 8, D and E).
Figure 8. Immunohistochemical staining for renal TLR4 protein in 4-wk-old Sprague-Dawley (A and D) or aged-matched double-transgenic rats (dTGR; B and C) with high local AngII concentrations. There is an increase in mesangial (B) but also tubular (E) TLR4 protein expression. Substitution of the primary antibody to nonimmune goat serum served as a negative control (C). This experiment was performed independently three times. Magnifications: x800 in A through C; x200 in D and E.
Many clinical observations have suggested that infections maytrigger the onset and/or worsening of glomerulonephritis andrenal vasculitis. For example, in a Japanese study, 68% of renalbiopsy specimens from patients with IgA nephropathy, an envelopeantigen of Staphylococcus aureus, was co-localized with an IgAantibody in glomeruli (27). In Wegener’s granulomatosis,respiratory tract infections frequently precede or accompanyinitial symptoms of upper airway involvement (3). Treatmentwith co-trimoxazole reduces the incidence of relapses in patientswith Wegener’s granulomatosis in remission (28). However,whether Gram-negative bacteria as a major source of LPS areinvolved in triggering human glomerulonephritis and vasculitisis unknown.
We previously reported that experimentally induced glomerulonephritishas a much more severe course in rats with Goldblatt (two kidneys,one clip) hypertension, a condition with high local AngII concentrations(29). There also is experimental evidence that AngII itselfexerts proinflammatory effects, mainly by inducing chemokinessuch as MCP-1 and RANTES and by upregulating the expressionof endothelial adhesion molecules that initiate the transferof circulating leukocytes into the renal tissue (22,30).
Our study shows that AngII upregulates TLR4 mRNA and proteinexpression by mesangial cells both in vitro and in vivo. Thisupregulation had functional consequences as revealed by thefact that LPS-mediated NF-B activation and LPS-induced chemokinegene transcription were enhanced in MMC that were preincubatedwith AngII. Ruiz-Ortega et al. (30) described that AngII alonefor 3 to 6 h but not for 24 h induced MCP-1 mRNA. Our resultsthat AngII alone for 24 h in the preincubation group failedto stimulate MCP-1 is in excellent agreement with these findings.Reporter gene experiments indicated that AngII increased TLR4promoter activity, an effect that was dependent on the presenceof a functional AP-1 as well as on Ets sites. The protein productsof c-fos and c-jun form a heterodimer that binds to AP-1 sites.Because we and others previously demonstrated that AngII stimulatesthe expression of c-fos and c-jun (30,31), it is likely thatAngII promotes TLR4 expression by increasing AP-1–dependentTLR4 gene transcription in MMC. Zhan et al. (19) recently reportedthat Ets is a critical regulator of AngII-mediated vascularinflammation and remodeling. In this study, MCP-1 expressionwas reduced in Ets1 –/– mice compared with controlmice in response to AngII (19). We observed a marked reductionalready in basal transcriptional activity with the Ets mutantconstruct. AngII was ineffective in stimulating transcriptionalactivity of this construct, suggesting a major role of the Etssite in TLR4 gene transcription. These data are in agreementwith observations in macrophages demonstrating a pivotal roleof the Etsd site in mouse TLR4 transcription under basal conditions(18). In addition, our reporter gene experiments may shed lighton a new angle of the results of Zhan et al. Because the TLR4promoter contains several Ets sites (18) and the Ets familyof transcription factors comprises >35 members, further experimentswill have to address the exact role of the individual siteson overall TLR4 mRNA expression.
Other TLR, such as TLR9, which detects microbe-specific unmethylatedcytosine-guanosine rich DNA, have been implicated in systemicimmune disorders (32,33). It therefore remains to be exploredwhether AngII modulates the expression of other TLR besidesTLR4, especially TLR9. A link between the Tamm-Horsfall glycoprotein(THP), which is expressed abundantly at the luminal surfaceof tubular cells and excreted into the urine and is known forits critical role in antibacterial host defenses, and activationof TLR4 was described recently (34). THP activates myeloid dendriticcells via TLR4 to acquire a fully mature phenotype. Therefore,THP links innate immune cell activation with adaptive immunity.We have not tested whether AngII may induce the upregulationof TLR4 on dendritic cells, but it is tempting to speculatethat this mechanism might function by enhancing adaptive immunity.Although we focused on mesangial cells in this study, we observedtubular upregulation of TLR4 in transgenic rats that expresshigh local AngII concentrations. Semiquantitative analysis ofimmunohistochemical stainings demonstrated that tubular TLR4expression was even stronger than glomerular staining in dTGR.This interesting issue and a potential relationship to THP willbe investigated further in future studies.
Although TLR4 recognizes primarily LPS, it seems to recognizeendogenous ligands as well. For example, the immune-stimulatoryactivity of heat-shock protein 60 is mediated by TLR4 (4). Itis interesting that extracellular matrix components such asfibronectin, hyaluronic acid, and fragments of heparan sulfatehave been reported to activate TLR4 (4,35). Recently, Schaeferet al. (36) found that the matrix component biglycan is proinflammatoryand signals through TLR2 and TLR4. Possibly in conditions suchas rapid progressive glomerulonephritis, a destruction of theglomerular ultrafiltration barrier with the potential releaseof extracellular matrix components and mesangial depositionof fibrin may lead to activation of locally upregulated TLR4,thereby enhancing glomerular inflammation. Indeed, a strikingconcurrence of biglycan overexpression and enhanced numbersof infiltrating cells has been described in the kidney, albeitin a model of tubulointerstitial inflammation (37).
It is interesting that a recent study suggested that Asp299GlyTLR4 polymorphism in renal transplant recipients is associatedwith a lower risk for acute rejection (38). This mutation affectsthe extracellular domain of TLR4 and impairs its function (38).Because activation of AT1 receptors has been linked to refractoryrejection, part of this effect may be mediated through the upregulationof TLR4 (39). In addition, exogenous or endogenous ligands (e.g.,fibronectin) for TLR4 may trigger further acute and chronicrejections, particularly subsequent to ischemia-perfusion injury(40).
Other pathophysiologic situations in which our results may beof relevance are acute renal failure and sepsis (41). AngIIconcentrations are very high in sepsis and septic shock (42).Consequently, induction of TLR4 by AngII would trigger furtherinflammation in the kidney, thereby contributing to the developmentof acute renal failure in sepsis.
AngII stimulates transcription of TLR4 mRNA through activationof AP-1 and Ets-dependent TLR4 promoter activity in mesangialcells. This results in an upregulation of TLR4 protein withenhanced NF-B signaling and induction of chemokines. Our findingsprovide new insight into the process by which AngII contributesto inflammation: By modifying innate immunity.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft(Wo 460/2-7), the Interdisziplinäres Zentrum für KlinischeForschung Jena, and a grant from the Leenaards Foundation toT.R.
Part of this work was presented at the Annual Meeting of theAmerican Society of Nephrology; November 2 to 17, 2003; SanDiego, CA, and was published in abstract form (J Am Soc Nephrol14: 635A, 2003).
We thank Katharina Jablonski, Regine Schröder, Isolde Lohr,and Stefan Gatzemeier for excellent technical help.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
B transcription factor nuclear translocation and inducing the expression of chemokines. For examination of whether AngII modulates the expression of Toll-like receptor 4 (TLR4), a key element of the innate immune system that senses LPS, mouse mesangial cells (MMC) were treated with AngII. AngII upregulated TLR4 mRNA and protein in MMC, and this effect was mediated through AngII type 1 receptors. Reporter gene experiments indicate that an activating protein-1 (AP-1) as well as an E-26 specific sequence (Ets) binding site in the TLR4 promoter are responsible for the AngII-stimulated transcriptional activity of the TLR4 gene. Preincubation of MMC with AngII enhanced LPS-induced NF-
-actin (1:2000 dilution; Sigma). TRL4 signals were normalized to 
-32P]ATP (3000 Ci/mmol; Amersham) using T4 polynucleotide kinase (12). Binding reactions were performed in gel shift binding buffer (10 mmol/L Tris-HCl [pH 7.5], 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 50 µg/ml poly [dI-dC] poly [dI-dC]) with and without cold competitor oligonucleotides at room temperature for 30 min. OCT1 double-strand consensus oligonucleotides (5'-TGTCGAATGCAAATCACTAGAA-3') were used as nonspecific competitors. Reactions were stopped by addition of loading buffer, and samples were run on a nondenaturing 6% polyacrylamide gel. Gels were exposed to x-ray films. Specific bands were quantified by densitometry as described above, and binding of nuclear proteins from cells without AngII was assigned a value of 1.0. Gel shift analysis was performed independently four times with qualitatively similar results. 
