Role of MAPK pathways in light chain-induced cytokineproduction in human proxima - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：SuleSengul, CraigZwizinski,  VecihiBatuman,作者单位：1 Section of Nephrology, Department of Medicine,Tulane Medical Center, Tulane Cancer Center, and Veterans Administration Medical Center, New Orleans,Louisiana 70112


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      【摘要】9 u5 Z/ X; @3 h8 {4 J
   We previously demonstrated that lightchain (LC) endocytosis by human proximal tubule cells (PTCs) leads toproduction of cytokines through activation of NF- B. Here, weexamined the role of MAPK pathways in these responses using fourspecies of myeloma LCs ( 1, 2, 3, and 1 ) previously shown to inducecytokine production by PTCs. Among these, 1 -LC, whichyielded the strongest cytokine responses, was selected for detailedstudies. Activation of MAPKs was probed by Western blot analysis forthe active kinases, ERK 1/2, JNK 1/2, and p38 in 1 -LC-exposed human PTCs. To evaluate the functional roleof MAPKs in LC-induced cytokine responses, we tested the effects ofU-0126, an ERK inhibitor; SP-600125, an inhibitor of JNK; SB-203580, ap38 inhibitor; and curcumin, a JNK-AP-1 inhibitor, all added to mediabefore 4-h exposure to 1.5 mg/ml 1 -LC. IL-6 and monocytechemotactic protein-1 (MCP-1) were determined by ELISA. Both LCand human serum albumin (HSA) activated ERK, although the HSA effectwas weaker. 1 -LC stimulated all three MAPKs, althoughphosphorylation of ERK was more pronounced and sustained than others.Inhibitors of ERK, JNK, and p38 reduced LC-induced IL-6 and MCP-1production. These findings suggest that activation of MAPKs plays arole in LC-induced cytokine responses in PTCs. Activation of MAPKs maybe involved in cytokine responses induced by other proteins as well asLCs and may be pivotal in the pathophysiology of tubulointerstitialinjury in proteinuric diseases. : v7 X2 x/ E" Q! `& w
      【关键词】 intracellular signaling mechanisms interleukin MCP progression of renal diseases myeloma proteinuria5 h  }/ x4 \8 o2 Q% O/ S
   INTRODUCTION4 C5 z5 [& b& ?$ s
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INCREASED ENDOCYTOSIS of filtered proteins in the proximal tubule has been shown to inducecytokine production by proximal tubule cells (PTCs), possibly mediatingtubulointerstitial injury ( 1, 33, 47, 48 ). This has beenproposed as a major mechanism in the progression of kidney disease inproteinuric states. In multiple myeloma, excessive production andluminal delivery of light chains (LCs) to PTCs cause increasedendocytosis of LCs via the endocytic receptors cubilin and megalin andresult in eventual degradation within the lysosomes ( 3, 4 ). We previously demonstrated that increased endocytosis ofmyeloma LCs by human PTCs induces inflammatory cytokine productionthrough activation of NF- B ( 38 ). However, possiblesignaling mechanisms mediating these inflammatory responses and,specifically, the potential role of intracellular signaling pathwayssuch as MAPKs have not been elucidated in response to LC endocytosis.2 ^0 b7 [+ R' i

The present study was designed to investigate the cellular signalingmechanisms mediating these responses in cultured human PTCs. For thispurpose, we first determined the phosphorylation of the three majorMAPKs, ERK 1/2, JNK 1/2, and p38, in 1 -LC-exposed humanPTC by immunoblotting using phospho-specific antibodies. To evaluatethe functional role of phosphorylation of these MAPKs, we tested theeffects of pharmacological inhibitors of MAPKs, U-0126, an inhibitor ofERK ( 11 ); SP-600125, an inhibitor of JNK ( 5, 16 ); SB-203580, an inhibitor of p38 ( 7, 46 ); andcurcumin, an inhibitor of the JNK-AP1 pathway ( 23, 27 ), on 1 -LC-induced IL-6 and MCP-1 production. Our resultssuggest that MAPK pathways are involved in LC-induced cytokine responses.

MATERIALS AND METHODS" V. t- B; V- d( ?5 w
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Isolation and purification of myeloma LC. Myeloma LCs isolated from the urine of myeloma patients and previouslyshown to induce cytokine production through activation of NF- B inhuman PTCs were used in these experiments. LCs were isolated andpurified by a slight modification of the method previously reportedfrom our laboratory ( 30, 38 ). Briefly, LCs wereprecipitated from urine with ammonium sulfate (55 to 90% saturationdetermined empirically), extensively dialyzed against distilled waterand lyophilized. The precipitated LCs were purified by dissolving thelyophilized desalted crude protein in buffer at pH 6.0, followed bychromatography on carboxymethyl-Sephadex (C-50; Pharmacia, Piscataway,NJ). Under these conditions, the LCs were bound to the column,whereas the contaminants were not. Bound LC was eluted with 0.6 mol/lof NaCl, redialyzed against distilled water, and lyophilized. Thepurity of LCs was confirmed by SDS-PAGE ( 21 ) and theimmunological identity reported from the clinical laboratory wasconfirmed by Western blotting using goat anti-human and -LC antibodies.) S* I) E9 ?+ }4 l; M, S5 f1 V
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We tested our LC preparation for endotoxin using the chromogenic Limulus amebocyte lysate (LAL) test (Charles River Labs, Charleston, SC). We found that our LCs used in these experiments wereessentially endotoxin free ( used inthese experiments did not contain measurable quantities of IL-6 andTNF- (sensitivity of the ELISA kits 0.7 and 4.4 pg/ml, respectively), indicating that cytokine contamination does not occurduring purification ( 38 ).0 ^" |, O1 V* F$ m: ^, L1 Q

LCs were collected from myeloma patients with modest renalinsufficiency without albuminuria and no evidence of glomerular involvement. Thus the LCs studied here are considered tubulopathic. Kidney biopsies were not performed in the patients.

Cell cultures. In all experiments, SV40 immortalized human PTCs were used. Grown as amonolayer, these cells show marker brush-border enzymes and havebiochemical and morphological characteristics similar to other widelyused PTC lines, including LLC-PK 1, OK, HK-2, and human PTCsin stable culture ( 31 ). Brush-border enzymes and phloridzin-inhibitable glucose transport were similar to other established PTC models. This transformed cell line's response toinflammatory stimuli was extensively compared with parental PTCs andwas found similar ( 14 ). Cells were routinely grown inDRM-23E medium supplemented with 0.5% (vol:vol) fetal bovine serum inT-75 flasks (Falcon, Becton Dickinson Labware) at 37°C in ahumidified atmosphere of 95% air-5% CO 2 and refed atintervals of 2 or 3 days. At confluence, the culture medium wasaspirated, the cultures were rinsed with HBSS, and the cells wereremoved by trypsin/ethylene-diamine tetraacetic acid digestion,reseeded into T-75 flasks containing the complete medium, and cultured to confluence.! ?7 Y) \% g+ X# z4 L; i8 ]
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Preparation of whole cell lysates. Cells planted onto 35-mm sterile tissue culture dishes (Corning GlassWorks, Corning, NY) or six-well tissue culture plates were grown at37°C in serum-free medium in an incubator for 24 h. After eachexperiment, medium was removed and cells were washed with PBS. Thefollowing steps were done on ice: 200 µl of RIPA buffer consisting of50 mM Tris · HCl (pH 7.4), 1% NP-40(IGEPAL-CA630), 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA (EGTA)(pH 8.0), protease inhibitors (aprotinin, leupeptin, and pepstatin, 1 µg/ml each, and freshly added), 1 mM Na 3 VO 4,and 1 mM PMSF (added immediately before use) were added to the dishes.After 10 min of incubation, dishes were scraped with a cell scraper andthen lysates were transferred to 1.5-ml microcentrifuge tubes usingsyringes fitted with 21-gauge needles. Lysates were passed through a21-gauge needle to shear DNA and centrifuged at 13,000 g for10 min at 4°C. Finally, supernatants were harvested as whole celllysates and used in immunoblotting studies.

Protein concentrations were determined in whole cell lysates preparedfrom PTCs by using Pierce BCA Protein Assay (Rockford, IL).

Western blot analysis for MAPKs. Equal amounts of proteins were separated by NuPAGE Bis-Tris gel byusing precast gels (10% acrylamide) and a minigel apparatus (Novex).Electrophoresis was performed according to manufacturer's recommendations. Separated proteins were electrophoreticallytransferred to nitrocellulose membranes for 1 h at 30 V by using asemi-dry blotting apparatus (Novex). Proteins were probed withpolyclonal phospho-specific antibodies against ERK 1/2, JNK 1/2, andp38 (1:5,000, 1:2,000, and 1:5,000 dilutions, respectively) by using aWestern Breeze Chemiluminescent Kit according to the manufacturer's protocol (Novex). Integrity of phospho-specific antibodies against MAPKs was tested using sorbitol and nerve growth factor-treated PC12cell extracts provided by the antibody supplier (Promega, Madison, WI).We determined total MAPKs using primary antibodies (1:1,000 dilution,Cell Signaling, Beverly, MA) to confirm equal protein loading in thegels for each experiment. Representative blots from one of at leastthree separate experiments were selected for illustration.7 c5 R" v4 f& w' T" K, ]
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We examined the effects of four different myeloma LCs( 1, 2, 3, and 1 ) and human serum albumin (HSA) on ERK activation. Forthis experiment, myeloma LCs (1.5 mg/ml) and HSA (1.5 mg/ml, Sigma)were dissolved in serum-free regular media. Confluent monolayers ofserum-deprived PTCs were treated with protein solutions for 10 min insix-well plates. At the end of exposure, cell lysates were prepared andblotted for both phosphorylated and total forms of ERK (Fig. 1 ). A 1 -LC (first LC onFig. 1 ) that we previously demonstrated to yield the strongest cytokineresponses ( 38 ) was selected for detailed studies.

Fig. 1. Effects of different light chains (LCs) and human serumalbumin (HSA) on ERK activation. The effects of 4 myeloma LCs( 1, 1, 2, and 3, LC-1 to -4, respectively) and HSA on ERK activationwere determined. Cells were treated with 4 different LCs and HSA for 10 min, cell lysates were then probed for phospho-specific( top ) and total ERK ( bottom ). The effect of LCson ERK activation was more marked than HSA. These LCs were the sameproteins used in our previous reports ( 30, 38 ) and shownto induce strong cytokine responses in proximal tubule cells (PTCs),whereas HSA had no effect ( 38 ). (Representative figurefrom 3 different experiments is shown.)
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For time course studies, 1 -LC (1.5 mg/ml, ~50µM) dissolved in serum-free medium was added to confluent monolayersof cells for 10, 20, 40, 60, 120, and 240 min, and then cell lysateswere prepared and used in immunoblotting studies for phosphorylated andtotal forms of MAPKs (Fig. 2, A - C ).

Fig. 2. Effects of 1 -LC on MAPK activation. HumanPTCs were exposed to 1.5 mg/ml (~50 µM) of 1 -LC forthe indicated times. A : there was marked activation of ERK1/2 in LC-exposed cells, detected as early as 10 min, decreasing butpersisting over 4 h. Both JNK 1/2 and p38 were also activated. B : phospho-JNK 1/2 bands seemed to fade completely after 40 min of exposure to myeloma LC. C : phospho-p38 band startedto fade after 40 min but was sustained up to 240 min. A - C, bottom : total MAPKs correspondingto each experiment. (Representative blots from 3 different experimentsare shown.)

Because the most marked response appeared with the ERK pathway, we alsoperformed dose-response experiments with 1 -LC(0.4-0.025 mg/ml) on ERK activation. In this experiment, cellswere treated with decreasing concentrations of 1 -LC for10 min and cell lysates were probed for ERK. For comparison, total ERKwas also shown for each experiment (Fig. 3 ).2 N5 j# q( D  W; r: l3 d, c+ K' A

Fig. 3. Effects of varying doses of 1 -LC on ERKactivation. Confluent monolayers of PTCs were exposed to decreasingconcentrations of 1 -LC (0.4- 0.025 mg/ml) for 10 min. Top : effect of 1 -LC on ERK activationappeared to be dose dependent and was observed at concentrations as lowas 0.025 mg/ml (~1 µM) at 10 min. Bottom : total ERK.(Representative blots from 3 different experiments are shown.)

Effects of pharmacological inhibitors on activation of MAPKs. To test the effects of MAPK inhibitors, after 24-h serum deprivation,confluent monolayers of cells were pretreated with the MAPK inhibitors(U-0126, SP-600125, and SB-203580) for 1 h at 1, 5, and 15 µMconcentrations and then treated with 1.5 mg/ml (50 µM) of 1 -LC in the continued presence of inhibitors. These inhibitors are considered selective for their respective pathways bymany investigators ( 5, 7, 11, 16, 23, 27, 46 ). After 10 min of exposure, cell lysates were prepared using the method describedabove. Blotting studies were performed using primary antibodiesagainst phosphorylated and total forms of MAPK pathways (Fig. 4, A - C ).
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Fig. 4. Effects of pharmacological inhibitors on LC-induced activation ofMAPKs. A : effects of U-0126 on 1 -LC-inducedactivation of ERK. Cells were pretreated with different concentrations(1, 5, and 15 µM) of U-0126, a pharmacological inhibitor of ERK, for1 h and then treated with 1 -LC (1.5 mg/ml, ~50µM) in the continued presence of U-0126 for 10 min. Top :U-0126 inhibited 1 -LC-induced activation of ERK. Bottom : total ERK was not affected. (Representative blotsfrom 3 different experiments are shown.) B : effects ofSP-600125 (SP) on 1 -LC-induced activation of JNK. Cellswere pretreated with different concentrations (1, 5, and 15 µM) ofSP, a pharmacological inhibitor of JNK, for 1 h and then exposedto 1 -LC (1.5 mg/ml, ~50 µM) for 10 min in thepresence of SP. Top : activation of JNK was inhibited by SP. Bottom : total JNK was not affected. (Representative blotsfrom 3 different experiments are shown.) C : effects ofSB-203580 (SB) on 1 -LC-induced activation of p38. After1-h pretreatment with SB (1, 5, and 15 µM), a pharmacologicalinhibitor of p38, cells were exposed to 1 -LC (1.5 mg/ml,50 µM) for 10 min. Top : 1 -LC-inducedactivation of p38 was suppressed by SB. Bottom : total p38was not affected. (Representative blots from 3 different experimentsare shown.)2 z4 o$ ?! o9 ?+ |6 B" ?

Effect of MAPK inhibitors on IL-6 and MCP-1 production. For MAPK inhibitor experiments, serum-deprived (24 h) cells werepretreated for 1 h with MAPK inhibitors (Figs. 5-8 ). Concentrations of the inhibitors (1-15 µM forU-0126, SP-600125, curcumin, and SB-203580) used in these experiments weredetermined from published literature ( 5, 7, 11, 16, 23, 27, 46 ) and pilot studies in our laboratory. These concentrations ofinhibitors had negligible effects on basal cytokine production (see RESULTS ). Cell viability was determined by trypan blueexclusion assays; in all experiments, at least 85% of cells remainedviable ( 7 ). After pretreatment with inhibitors, cells wereincubated with 1 -LC (1.5 mg/ml, ~50 µM) for 4 hin the presence and absence of the MAPK inhibitors (Figs. 5-8 ).After exposure, culture supernatants were harvested and stored at 70°C for ELISA assays.5 M3 s& }# t0 h
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Fig. 5. Effect of ERK inhibition on basal and 1 -LC-induced IL-6 and monocyte chemotactic protein(MCP-1) production. Cells were pretreated with ERK inhibitor U-0126(1-15 µM) for 1 h before exposure to 1 -LC(1.5 mg/ml, ~50 µM) for 4 h. Basal secretion of both IL-6( A ) and MCP-1 ( C ) is normalized to 1. 1 -LC-stimulated IL-6 ( B ) and MCP-1( D ) production is normalized to 1 and results are expressedas fold-increase over basal or LC-stimulated cytokine production. A : 1 -LC-stimulated IL-6 production by nearly4-fold compared with controls [from 121.01 ± 1.35 (normalized to1) to 436.02 ± 45. 9 pg/10 6 cells]. U-0126 alone hadno effect on IL-6 production. B : LC-induced IL-6 productionwas significantly inhibited by U-0126 (up to 75% with 15 µM U-0126). C : 1 -LC-stimulated MCP-1 production by 6-foldcompared with controls [from 65.44 ± 1.08 (normalized to 1) to406.69 ± 15.3 pg/10 6 cells]. D : U-0126significantly inhibited MCP-1 production. Results are presented asmeans ± SE ( n = 3, * P 0.05 vs.control, *** P 1 -LC).- e' ^/ `8 H% p6 E/ _

Fig. 6. Effect of JNK inhibition on basal and 1 -LC-induced IL-6 and MCP-1 production. Cells werepretreated with JNK inhibitor SP (1-15 µM) for 1 h beforeexposure to myeloma LC (1.5 mg/ml, ~50 µM) for 4 h. Basalsecretion of both IL-6 ( A ) and MCP-1 ( C ) isnormalized to 1. 1 -LC-stimulated IL-6 ( B ) andMCP-1 ( D ) production is normalized to 1 and results areexpressed as fold-increase over basal or LC-stimulated cytokineproduction. A : in this experiment, LC induced ~3-foldincrease in IL-6 production compared with control (from 133.05 ± 2.54 to 375.1 ± 1.00 pg/10 6 cells). Unlike previousexperiments, SP alone also inhibited basal IL-6 production. B : LC-induced IL-6 production was significantly inhibited bySP. C : SP alone had no effect on MCP-1 production, but 1 -LC-induced MCP-1 production was significantlyinhibited by SP ( D ) at all 3 concentrations. Results arepresented as means ± SE ( n = 3, * P 0.05 vs. control, *** P 1 -LC).
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Fig. 7. Effect of curcumin (Curc) on basal and 1 -LC-induced IL-6 production. Cells were pretreated withCurc, an inhibitor of JNK-AP1 (1-15 µM), for 1 h beforestimulation with myeloma LC (1.5 mg/ml, ~50 µM) for 4 h. Basalsecretion of both IL-6 ( A ) and MCP-1 ( C ) isnormalized to 1. 1 -LC-stimulated IL-6 ( B ) andMCP-1 ( D ) production is normalized to 1 and results areexpressed as fold-increase over basal or LC-stimulated cytokineproduction. A - C : Curc alone had no effect oncytokine production. B - D : Curc significantlyinhibited 1 -LC-induced IL-6 and MCP-1. Results arepresented as means ± SE ( n = 3, * P 0.05 vs. control, ** P 1 -LC, *** P 1 -LC).
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Fig. 8. Effect of p38 inhibition on basal and 1 -LC-induced IL-6 and MCP-1 production. Cells werepretreated with p38 inhibitor SB (1-15 µM) for 1 h beforestimulation with myeloma LC (1.5 mg/ml, ~50 µM) for 4 h. Basalsecretion of both IL-6 ( A ) and MCP-1 ( C ) isnormalized to 1. 1 -LC-stimulated IL-6 ( B ) andMCP-1 ( D ) production is normalized to 1 and results areexpressed as fold-increase over basal or LC-stimulated cytokineproduction. SB alone had no effect on basal secretion of either IL-6( A ) or MCP-1 ( C ). LC-induced IL-6 production wassignificantly inhibited by SB at only 15-µM concentration( B ). Similarly, LC-induced MCP-1 production wassignificantly inhibited by SB ( D ) at all 3 concentrations.Results are presented as means ± SE ( n = 3, * P 0.05 vs. control, *** P 1 -LC).
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Measurement of IL-6 and MCP-1 levels by ELISA. IL-6 and MCP-1 were measured in the supernatants using commercial humanELISA kits (Quantikine; R&D Systems) according to the manufacturer'sprotocol. The sensitivity of the ELISA is 0.7 pg/ml for IL-6 and 5 pg/ml for MCP-1 assays. Cytokine concentrations in the unknown sampleswere determined by comparison with a standard curve developed withknown amounts of recombinant human cytokines provided with the kits.Experiments were conducted in triplicate using 96-well microplates, andresults were read in a microplate reader. Cells were trypsinized andcounted and expressed as picograms of cytokine per 10 6 cells. Results were presented as fold-increase over either basal orLC-stimulated responses normalized to 1.! n2 W1 |6 I! C9 e- _0 `

Reagents and antibodies. Cell culture products and all other reagents were obtained from Sigma(St. Louis, MO) unless otherwise specified. Antibodies against active(phosphorylated) ERK 1/2, JNK 1/2, and p38 were purchased from Promega.MAPK inhibitors (U-0126 and SB-203580) were purchased from Calbiochem(San Diego, CA). SP-600125 was purchased from Biomol (Plymouth Meeting,PA). Antibodies against total MAPKs were purchased from Cell Signaling.
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Statistical analysis. Results were expressed as means ± SE. Multiple comparisons weremade by ANOVA and Bonferroni's multiple-comparison tests. Statisticalanalyses, curve fitting, and calculations were done using GraphPadPrism, version 3 for Windows NT (1999, GraphPad Software, SanDiego, CA). Statistical significance was defined as P

RESULTS

LCs induce activation of ERK in PTCs. Four different LCs ( 1, 2, 3, and 1 ) at 1.5-mg/ml concentrationinduced activation of ERK 1/2 compared with control (Fig. 1 ). There wasa weaker response to HSA (1.5 mg/ml) also, although this protein didnot elicit cytokine responses in human PTCs ( 38 ).3 p- r; O  h; |) G- l' d

1 -LC induces activation of ERK, JNK, and p38 inPTCs. Human PTCs exposed to 1 -LC (1.5 mg/ml, ~50 µM) for10-240 min resulted in marked activation of ERK 1/2, JNK 1/2, andp38. ERK activation was detected as early as 10 min, decreasing but persisting over 4 h (Fig. 2 A ). The 1 -LC-induced phosphorylation of both JNK 1/2 and p38 wasagain discernible at 10 min and sustained for up to 4 h, althoughphospho-JNK 1/2 bands seemed to fade after 40 min (Fig. 2, B and C ). Blots for total MAPKs remained constant throughoutthe duration of the experiments.

Because the most marked response appeared with ERK 1/2, we determinedthe effect of varying doses of 1 -LC on ERKphosphorylation. We found the effect of this LC to be dose dependentand effective at concentrations as low as 0.025 mg/ml (~1 µM) (Fig. 3 ).
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Effects of pharmacological inhibitors on activation of MAPKs. We tested the effects of MAPK inhibitors at the concentrations (1, 5, and 15 µM) used in these experiments on respective MAPK pathways.These agents inhibited phospho-MAPKs at the same concentrations demonstrated to inhibit IL-6 and MCP-1 production, although the degreeof apparent MAPK inhibition did not always seem proportionate to thedegree of inhibition in the cytokine response. These inhibitors had noeffect on total MAPKs (Fig. 4, A - C ).2 P8 H: z- |, _

MAPK inhibitors suppress 1 -LC-induced IL-6 and MCP-1production. We tested the effect of the pharmacological inhibitors of ERK, JNK,JNK-AP-1, and p38 pathways on production of IL-6 and MCP-1 in controlcells and in cells exposed to 1 -LC, 1.5 mg/ml for 4 h.
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Myeloma 1 -LC increased IL-6 production by about fourfoldfrom baseline compared with control cells (from 121.01 ± 1.35 to 436.02 ± 45.9 pg/10 6 cells, P 5 A ). This same LC induced an approximately sixfold increase in MCP-1 production compared with control cells (from65.44 ± 1.08 to 406.69 ± 15.3 pg/10 6 cells, P 5 C ). The pharmacologicalinhibitor of ERK, U-0126, showed a marked and dose-dependent inhibitionof IL-6 in LC-exposed cells but had no effect on basal IL-6 and MCP-1 secretion in control cells not exposed to LC (Fig. 5, A - C ). U-0126 also inhibited MCP-1 production(Fig. 5 D ). However, the inhibitory effect of this inhibitorof ERK on MCP-1 production in LC-exposed cells was not as marked as itseffect on IL-6 (Fig. 5 ).

SP-600125, an inhibitor of JNK, and curcumin, an inhibitor ofJNK-AP1, also inhibited both IL-6 and MCP-1 in 1 -LC-exposed cells (Figs. 6 and 7 ). 1 -LCagain induced marked stimulation of IL-6 (~3- to 4-fold; Figs. 6 A and 7 A ). These inhibitors did not havesignificant effects on basal secretion of cytokines in control cells,except for the experiment with SP-600125, which showed a significantinhibition of IL-6 (Fig. 6 A ). 1 -LC-induced IL-6 and MCP-1 production was also significantly inhibited by curcumin(Fig. 7, B and D ).& d4 {7 q. U: r. g0 V4 o  g6 j

Finally, we examined the effect of SB-203580, an inhibitor of p38.These experiments showed that p38 blockade caused a significant inhibition of both IL-6 and MCP-1 production in LC-exposed cells (Fig. 8, B and D ). However, the effect of thisinhibitor on IL-6 production was evident only at 15 µM and was notseen at lower concentrations (Fig. 8 B ). SB-203580 did nothave a significant effect on basal secretion of either IL-6 or MCP-1(Fig. 8, A and C ).

DISCUSSION
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Cytokine production in response to filtered serumproteins has recently attracted increasing attention as a majormechanism of progression of kidney diseases in proteinuric states( 1, 32, 33, 43, 47, 48 ). We recently showed that excessive LC endocytosis causes IL-6, MCP-1, and IL-8 production through activation of NF- B in cultured human PTCs and proposed that this maybe an important mechanism of tubulointerstitial disease commonly seenin multiple myeloma ( 38 ). However, the potential role of signaling pathways such as MAPKs in these responses is unknown.0 l; f( L% Z* t! @8 i, A& Q

MAPKs are important mediators of cellular stress responsesphosphorylating selected intracellular proteins, includingtranscription factors when challenged with a wide variety of stimuli( 12, 37 ). Three major MAPK families have been identifiedin mammalian cells: ERK 1 and 2, also known as p42/44 MAP kinase; JNK,also known as SAPK 1; and p38 MAP kinase, also known as SAPK 2 ( 28, 34 ). They have been shown to be activated as earlyresponses to a variety of stimuli, including peptide mitogens, growthfactors, oxidants, proinflammatory-inflammatory cytokines, lipidmediators, complement, albumin, and physical stressors in many culturedcell systems ( 8-10, 13, 15, 18, 19, 22, 26, 35, 36, 41, 45 ). Increased production of inflammatory cytokines through activation of different MAPKs has been described in response to IL-1, TNF-, and physical pressure in both cultured mesangial andPTCs ( 22, 35, 40 ). Many transcription factors, such as c-Jun, ELK-1, ATF1, ATF2, NF- B, and cAMP response element binding protein, have been shown to be regulated by MAPK pathways ( 20, 44 ). MAPKs could act via the transcription factorsand they could directly or indirectly regulate cytokine genetranscription ( 17, 41 ).
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We recently observed that inflammatory cytokine production in responseto excessive LC endocytosis in cultured human PTCs is mediated viaactivation of NF- B ( 38 ). In the present study, wedemonstrated that LC-induced cytokine responses involve activation ofMAPKs. Among these MAPKs, ERK 1/2 appeared to play the dominant role inLC-exposed cells. However, all three MAPKs appeared to participate,suggesting that LC-induced cytokine responses involve complex andmultiple mechanisms., {- V0 d% e- P/ E. Z
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Our studies provide two lines of evidence supporting these conclusions.First, we found that phosphorylated MAPKs are upregulated in LC-exposedcells. Immunoblotting experiments showed activation of all three MAPKsstudied here. The most robust and sustained responses were seen withERK at exposures as early as 10 min and decreasing over time butpersisting as long as 4 h (Fig. 2 A ). Second, theinhibitors of the three major MAPKs as well as the JNK-AP-1 inhibitorcurcumin all resulted in significant inhibition of LC-induced IL-6 andMCP-1 production (Figs. 5-8 ). Taken together, these two lines ofevidence show that MAPKs are involved in LC-induced cytokine productionin human PTCs.
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Although the ERK pathway appeared to be more prominent than otherkinases, all three MAPKs tested here were clearly involved. That allthree signaling cascades might participate in cytokine responses is notsurprising, because it is well documented that there may beconsiderable overlap and perhaps "cross talk" among these MAPKs( 41 ). Furthermore, cell stress responses are well known touse multiple effector pathways.
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The effects of pharmacological inhibitors of MAPKs should beinterpreted with caution, however. Although many investigators considerthese inhibitors specific for their respective pathways ( 5, 7, 11, 16, 23, 27, 46 ), it has been shown that some of these agentsmay have either variable or additional effects that may be relevant tothe responses observed here. For example, SB-203580 can directlyinhibit thromboxane synthase and cyclooxygenase-1 and -2 ( 6, 25 ). Curcumin, considered a selective AP-1 inhibitor, at higherconcentrations (40-60 µM) has been shown to inhibit TNF-,phorbol ester-, and hydrogen peroxide-mediated NF- B activation at astep before I- B phosphorylation in a myeloid cell line( 39 ). In another study, curcumin was shown to inhibit AP-1, NF- B, as well as Egr-1 in cultured endothelial cells( 29 ). At lower concentrations, i.e., 10 µM, curcumin wasdemonstrated to inhibit endogenous VCAM-1 expression in humanmicrovascular endothelial cells independent of and without an effect onNF- B. In this study, antibodies against c-Jun and c-Fos inhibitedNF- B activation, suggesting an interaction between AP-1 and NF- B( 2 ). Although these studies were conducted in differentcell lines and used inhibitors at higher concentrations than used inour studies, they raise the possibility that some of thecytokine-blocking effects of these pharmacological inhibitors of MAPKsmay be mediated through other pathways and may involve overlappingmechanisms. In our experiments, the apparent effects of the MAPKinhibitors on their respective MAPKs were not always proportionate tothe degree of cytokine inhibition, and this is also consistent with thepossibility that the effects of MAPK inhibitors on cytokine responsesmay involve additional mechanisms.
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We recently demonstrated that LC-induced cytokine production wasthrough the activation of NF- B, similar to the observations reportedby investigators attributing such responses to increased proteinendocytosis ( 1, 32, 33, 48 ). It is of interest that LCeffects were seen at concentrations as low as 25 µg/ml, concentrations that can easily occur in typical patients with myelomaand even in proteinuric patients without myeloma ( 38 ).
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LCs are well documented to undergo vigorous endocytosis in PTCs via theendocytic receptors cubilin-megalin ( 3, 4 ). Otherinvestigators who demonstrated albumin endocytosis as a proinflammatoryevent also proposed that albumin endocytosis is accomplished throughthe tandem cubilin-megalin system ( 42 ). The interactionbetween LC ligand with the endocytic receptors cubilin-megalin suggestsan attractive speculation into how endocytosis can result in activationof MAPKs and the subsequent or simultaneous activation of nucleartranscription factors causing increased production of inflammatorycytokines in PTCs. Whether LC preferentially binds to cubilin and isendocytosed by its "chaperone" megalin or binds to megalindirectly, the cytoplasmic domain of megalin, which contains NPXY motifs( 42 ), could easily initiate the required phosphorylationcascade through tyrosine phosphorylation, progressing via ERK 1/2 andeventually resulting in phosphorylation of I- B, the penultimate stepin the activation of NF- B. Our experiments suggested a significantrole for MAPKs in LC-mediated cytokine responses. It is relevant thatMAPKs have been demonstrated to have a key role in endocytosis( 24 ), lending credence to our thesis that LC endocytosisactivates MAPKs.

Previous studies on the endocytosis-induced cytokines in PTC havefocused almost exclusively on albumin ( 1, 33, 47, 48 ).These studies demonstrated that albumin exposure in various PTC linesresulted in production of cytokines through activation of NF- B.Albumin has also been demonstrated by Dixon and Brunskill ( 10 ) to stimulate p44/42 in opossum kidney PTCs; however,whether this results in cytokine production has not been examined. Our observations with myeloma LCs have similarities to these findings butalso differ in fundamental ways. With the use of human PTCs and HSAeven at concentrations exceeding those reported in the literature, wefailed to elicit cytokine responses ( 38 ). In the presentstudy, we did observe activation of ERK by HSA, although this effectwas much weaker compared with LCs. Taken together, our observations onLCs and HSA and the previously published studies on albumin suggestthat endocytosis of all filtered proteins may have inflammatoryconsequences. Our results suggest that LCs may be more potent thanalbumin in inducing cytokines in PTCs. Furthermore, LC effects wereobserved at very low concentrations (~1 µM) that can occur even inpatients without myeloma, suggesting a potential role of LCs in theprogression of kidney disease in other proteinuric diseases.. h- C( i! b; ~$ c) X  P$ v
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This study reveals clues that may improve our understanding of howproteinuria and excessive endocytosis of serum proteins can causeinflammatory responses in PTCs. A comprehensive description of the keysteps that start with endocytosis resulting in production of cytokineswill require additional experiments. Elucidation of the precisemechanisms of cytokine responses involved in protein endocytosis mayopen up novel areas probing therapeutic interventions that may behelpful in intervening with the progression of kidney disease in proteinuria.
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ACKNOWLEDGEMENTS
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These studies were supported by a Merit Review Grant from theDepartment of Veterans Affairs. Partial support was also provided fromdevelopmental funds of the Tulane Cancer Center (research fellowshipaward to S. Sengul). Parts of this study are presented as anabstract at the 2002 annual meeting of the American Society ofNephrology in Philadelphia, PA.
      【参考文献】
1. Abbate, M,Zoja C,Corna D,Capitanio M,Bertani T,andRemuzzi G. In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation. J Am Soc Nephrol 9:1213-1224,1998 .% w; l4 Z; d$ c# q/ v, Y4 \

1 f% P: P# t4 a0 S
2 B: h, R4 j+ x3 [  \, U( l
2. Ahmad, M,Theofanidis P,andMedford RM. Role of activating protein-1 in the regulation of the vascular cell adhesion molecule-1 gene expression by tumor necrosis factor-. J Biol Chem 273:4616-4621,1998 .4 c6 j: y# b% V* @* _

% c9 @  _/ E# V: P7 S

3. Batuman, V,andGuan S. Receptor-mediated endocytosis of immunoglobulin light chains by renal proximal tubule cells. Am J Physiol Renal Physiol 272:F521-F530,1997 .

1 q$ p; M4 `8 ~, o  N8 c
3 P8 @! R6 q( \; n, E4 L
4. Batuman, V,Verroust PJ,Navar GL,Kaysen JH,Goda FO,Campbell WC,Simon E,Pontillon F,Lyles M,Bruno J,andHammond TG. Myeloma light chains are ligands for cubilin (gp280). Am J Physiol Renal Physiol 275:F246-F254,1998 .
) J' a2 }" Q* z) A
9 T& {( b2 l+ \, m( I- k8 B& g
4 I- c' S1 p9 @1 ?7 i$ x
5. Bennett, BL,Sasaki DT,Murray BW,O'Leary EC,Sakata ST,Xu W,Leisten JC,Motiwala A,Pierce S,Satoh Y,Bhagwat SS,Manning AM,andAnderson DW. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98:13681-13686,2001 .# n3 U7 U& E6 D0 q: i6 |9 s& R

; s* e9 j; ~2 _
6. Borsch-Haubold, AG,Pasquet S,andWatson SP. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase. J Biol Chem 273:28766-28772,1998 .
; h9 d2 o( ]- @, ~3 I8 y+ b- v

7. Carter, AB,Monick MM,andHunninghake GW. Both Erk and p38 kinases are necessary for cytokine gene transcription. Am J Respir Cell Mol Biol 20:751-758,1999 .

4 _1 M  Q: z5 P' E+ w
% U- A( x" r' v7 J3 d6 f. s
8. Choudhury, GG,Karamitsos C,Hernandez J,Gentilini A,Bardgette J,andAbboud HE. PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF. Am J Physiol Renal Physiol 273:F931-F938,1997 .
6 I. z9 L8 Z' t& _) {5 Z

, o' ]7 f+ A6 u9 W# n% n7 Y5 g
9. Cybulsky, AV,Papillon J,andMcTavish AJ. Complement activates phospholipases and protein kinases in glomerular epithelial cells. Kidney Int 54:360-372,1998  .
6 W) q5 f) U% r- F

- J7 e7 i: q1 h$ A
10. Dixon, R,andBrunskill NJ. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen- activated protein kinase in opossum kidney proximal tubular cells. Clin Sci (Lond) 98:295-301,2000 .: j* _  |; ~; ]3 v* \  s6 {
4 V2 h* n" N) D9 V

11. English, JM,andCobb MH. Pharmacological inhibitors of MAPK pathways. Trends Pharmacol Sci 23:40-45,2002 .

- K& T; R3 H/ s3 c1 |$ {
. d3 V6 i5 P. i( v: _7 I% B
12. Fanger, GR,Gerwins P,Widmann C,Jarpe MB,andJohnson GL. MEKKs, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-Jun amino-terminal kinases? Curr Opin Genet Dev 7:67-74,1997  .1 j/ v) F$ n" W9 Z
* N. \6 I! \% G- S% @: J
# |+ B$ w2 z  O' |) y' j- Y( _
" V' F! ?7 c+ v! ~
13. Gaits, F,Salles JP,andChap H. Dual effect of lysophosphatidic acid on proliferation of glomerular mesangial cells. Kidney Int 51:1022-1027,1997  ./ h7 x0 e( M( y

% ]. Q- t' R3 W& N- T# j: u) w

14. Gerritsma, JS,van Kooten C,Gerritsen AF,Mommaas AM,van Es LA,andDaha MR. Production of inflammatory mediators and cytokine responsiveness of an SV40-transformed human proximal tubular epithelial cell line. Exp Nephrol 6:208-216,1998  .- C3 }2 d8 b8 H# ~# y

! X% q5 }/ O8 H5 K

15. Guo, YL,Baysal K,Kang B,Yang LJ,andWilliamson JR. Correlation between sustained c-Jun N-terminal protein kinase activation and apoptosis induced by tumor necrosis factor- in rat mesangial cells. J Biol Chem 273:4027-4034,1998 .

6 u- Z8 z6 W, \( n: p% f3 _
16. Han, Z,Boyle DL,Chang L,Bennett B,Karin M,Yang L,Manning AM,andFirestein GS. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 108:73-81,2001  .9 ^3 m' s8 L. A( E( ]

' h+ M/ t$ U# y3 T5 ?. C( h4 W8 N

17. Hulleman, E,Bijvelt JJ,Verkleij AJ,Verrips CT,andBoonstra J. Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle. J Cell Physiol 180:325-333,1999  .' p& b4 v/ l( {# _& f2 |- N5 d) N

18. Huwiler, A,Stabel S,Fabbro D,andPfeilschifter J. Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: comparison of hypertrophic and hyperplastic agonists. Biochem J 305:777-784,1995 .
' A9 w8 {. x5 h' n$ c. z
$ ~5 M/ v  \% H3 n
% J3 Z7 H6 p  L& v( X/ k: L; O
19. Ingram, AJ,Ly H,Thai K,Kang M,andScholey JW. Activation of mesangial cell signaling cascades in response to mechanical strain. Kidney Int 55:476-485,1999  .$ U7 e7 {3 Y( \1 `0 D

20. Karin, M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem 270:16483-16486,1995 .

. Q) L2 E5 t, o( T, q$ c! z3 M$ j
+ S2 O9 n& F8 k; M, E9 E
21. Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685,1970 .
) @# o) z2 {' u% X2 R$ `

22. Leonard, M,Ryan MP,Watson AJ,Schramek H,andHealy E. Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells. Kidney Int 56:1366-1377,1999  .
. f; C  e+ [3 W3 `, T
' P0 f, c! i+ T) Y

23. Li, WQ,Dehnade F,andZafarullah M. Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway. J Immunol 166:3491-3498,2001 .

24. McPherson, PS,Kay BK,andHussain NK. Signaling on the endocytic pathway. Traffic 2:375-384,2001  .

- c5 T9 ?: H2 B0 O' E' }

25. Minuz, P,Gaino S,Zuliani V,Tommasoli RM,Benati D,Ortolani R,Zancanaro C,Berton G,andSantonastaso CL. Functional role of p38 mitogen activated protein kinase in platelet activation induced by a thromboxane A2 analogue and by 8-iso-prostaglandin F2. Thromb Haemost 87:888-898,2002  .
9 ^" G8 A) {+ c2 r, \# C

; b2 X$ k- z7 Q
26. Mondorf, UF,Piiper A,Herrero M,Bender M,Scheuermann EH,andGeiger H. Lipoprotein (a) stimulates mitogen activated protein kinase in human mesangial cells. FEBS Lett 441:205-208,1998  .* f  j8 X  y& _+ I

27. Nakayama, K,Furusu A,Xu Q,Konta T,andKitamura M. Unexpected transcriptional induction of monocyte chemoattractant protein 1 by proteasome inhibition: involvement of the c-Jun N-terminal kinase-activator protein 1 pathway. J Immunol 167:1145-1150,2001 .8 _8 {# o# J% K# O, q. H6 I# g

  h. Q6 i3 Q. z$ V
28. Pearson, G,Robinson F,Beers Gibson T,Xu BE,Karandikar M,Berman K,andCobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22:153-183,2001 .0 \7 \* r7 `3 e: o  d

% t3 H3 `6 S: Z1 X

29. Pendurthi, UR,Williams JT,andRao LV. Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin. Suppression of activation of transcription factors Egr-1, AP-1, and NF- B. Arterioscler Thromb Vasc Biol 17:3406-3413,1997 .
/ M: h. u4 K+ c9 _

3 R* m9 M! _; y  o5 @. |+ U
30. Pote, A,Zwizinski C,Simon EE,Meleg-Smith S,andBatuman V. Cytotoxicity of myeloma light chains in cultured human kidney proximal tubule cells. Am J Kidney Dis 36:735-744,2000  .# L; c8 p$ h# ^; i! e
8 H( U3 e4 Q6 e) E; i
' m& D+ y6 |7 z. u
2 K$ j/ V: a9 M& L. @
31. Racusen, LC,Monteil C,Sgrignoli A,Lucskay M,Marouillat S,Rhim JG,andMorin JP. Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines. J Lab Clin Med 129:318-329,1997  .

2 N- a( w' ]* _2 b6 s
/ Z  P  I5 r+ M% E& K4 \
32. Rangan, GK,Wang Y,Tay YC,andHarris DC. Inhibition of nuclear factor- B activation reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int 56:118-134,1999  .  u6 P3 _! ?- m- |% m
3 _1 W8 V7 E0 \
" t+ j6 {0 M$ u# B) N

33. Remuzzi, G,andBertani T. Pathophysiology of progressive nephropathies. N Engl J Med 339:1448-1456,1998 .
6 ^' X' Q2 `4 J6 b; m# [

+ J9 y& e8 q. p
34. Robinson, MJ,andCobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9:180-186,1997  .

; u7 A6 H: [4 K

35. Rovin, BH,Wilmer WA,Danne M,Dickerson JA,Dixon CL,andLu L. The mitogen-activated protein kinase p38 is necesssary for interleukin 1 -induced monocyte chemoattractant protein 1 expression by human mesangial cells. Cytokine 11:118-126,1999  .

36. Schramek, H,Schumacher M,andPfaller W. Sustained ERK-2 activation in rat glomerular mesangial cells: differential regulation by protein phosphatases. Am J Physiol Renal Fluid Electrolyte Physiol 271:F423-F432,1996 .

7 q8 }* A, {+ @' f' N, o* B/ o3 |
37. Seger, R,andKrebs EG. The MAPK signaling cascade. FASEB J 9:726-735,1995 .
8 C8 P* ?& ]5 ^, x

9 _) U- N7 I; g
38. Sengul, S,Zwizinski C,Simon EE,Kapasi A,Singhal PC,andBatuman V. Endocytosis of light chains induces cytokines through activation of NF- B in human proximal tubule cells. Kidney Int 62:1977-1988,2002  .
5 @2 j1 r' q3 e% s/ Z  `& H

39. Singh, S,andAggarwal BB. Activation of transcription factor NF- B is suppressed by curcumin (diferuloylmethane). J Biol Chem 270:24995-25000,1995 .$ ~3 k! u, _; J) `7 y% G# C

9 e# G% a* @! \9 ?4 p/ L
40. Suda, T,Osajima A,Tamura M,Kato H,Iwamoto M,Ota T,Kanegae K,Tanaka H,Anai H,Kabashima N,Okazaki M,andNakashima Y. Pressure-induced expression of monocyte chemoattractant protein-1 through activation of MAP kinase. Kidney Int 60:1705-1715,2001  .( r/ `8 m8 F; t: g: R" o
, _- [* g8 S* m! S/ I
5 X, N6 g  m: n9 u$ b/ |. R: o

41. Tian, W,Zhang Z,andCohen DM. MAPK signaling and the kidney. Am J Physiol Renal Physiol 279:F593-F604,2000 .

42. Verroust, PJ,Birn H,Nielsen R,Kozyraki R,andChristensen EI. The tandem endocytic receptors megalin and cubilin are important proteins in renal pathology. Kidney Int 62:745-756,2002  .
4 e8 i3 z  B2 B: N' M, Y
! Q9 D$ R2 L  t
/ Z0 i9 N# _3 r, j: |
43. Wang, Y,Rangan GK,Tay YC,andHarris DC. Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor B in proximal tubule cells. J Am Soc Nephrol 10:1204-1213,1999 ." {  W) C9 a: ~; |9 x0 G, ~* R
, E7 m) z5 b* E! _2 T5 p

6 U; R# x2 \' F7 H
44. Wesselborg, S,Bauer MK,Vogt M,Schmitz ML,andSchulze-Osthoff K. Activation of transcription factor NF- B and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J Biol Chem 272:12422-12429,1997 .
9 ]  U' |' P( V' e" `
, b7 p1 J0 a# {3 h6 U
# W" @( Q  X- g6 h
45. Wilmer, WA,Tan LC,Dickerson JA,Danne M,andRovin BH. Interleukin-1 induction of mitogen-activated protein kinases in human mesangial cells. Role of oxidation. J Biol Chem 272:10877-10881,1997 .& o7 U% H4 a) v% K- |& B
* O3 b5 w4 C+ J4 q  [0 @
, c( g! |' e1 j& T# k

46. Zhang, SL,Tang SS,Chen X,Filep JG,Ingelfinger JR,andChan JS. High levels of glucose stimulate angiotensinogen gene expression via the P38 mitogen-activated protein kinase pathway in rat kidney proximal tubular cells. Endocrinology 141:4637-4646,2000 .3 p- n! {# M$ e! \

47. Zoja, C,Benigni A,andRemuzzi G. Protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators. Exp Nephrol 7:420-428,1999  .8 ^1 p3 @5 @( F
2 L1 ~2 i5 \* m" M; [

3 \# ?6 v% m/ ~  W5 L; r
48. Zoja, C,Donadelli R,Colleoni S,Figliuzzi M,Bonazzola S,Morigi M,andRemuzzi G. Protein overload stimulates RANTES production by proximal tubular cells depending on NF- B activation. Kidney Int 53:1608-1615,1998  .

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