Bradykinin reduces growth factor-induced glomerular ERK1/2phosphorylation - 《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 13:32 |只看该作者 |倒序浏览 |打印

作者：EricCellier, MarilyneMage, JohanDuchêne, ChristianePécher, RéjeanCouture, Jean-LoupBascands,  Jean-PierreGirolami作者单位：1 Institut National de la Santé et de laRecherche Médicale U38 IFR Institut Louis Bugnard,31403 Toulouse Cedex  France; and Department of Physiology, Faculté deMédecine, Université de Montréal, Montreal, Quebec,Canada H3C 3J7
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      【摘要】
   Several experimental data report bothmitogenic and antimitogenic effects of bradykinin (BK). To conciliatethese apparent opposite effects, we hypothesized that, depending oncell context activation, BK could reduce the mitogenic effect of growthfactors. Therefore, in the present study we assessed the existence ofpossible negative cross talk between BK and potential pathogenic growth factors in freshly isolated rat glomeruli (IG). Next, we determined whether this cross talk could be pharmacologically recruited during angiotensin-converting enzyme (ACE) inhibition in the diabetic rat. InIG from normal rats, BK, via activation of the B 2 kinin receptor (B 2 R), causes a transient stimulation of ERK1/2phosphorylation, whereas it inhibits ERK1/2 phosphorylation induced byIGF-1, PDGF-BB, VEGF, or basic FGF. The reduction of growthfactor-induced ERK1/2 phosphorylation is abolished by an inhibitor oftyrosine phosphatase. In glomeruli from diabetic rats, hyperglycemiaincreased the phosphorylation level of ERK-1/2 as well as oxidativestress. The reversal of these events by ACE inhibition is mediated viaB 2 R activation. These observations are consistent with apotential therapeutic role of BK and B 2 R during glomerulosclerosis.
      【关键词】 kinin B receptor growth factors mitogenactivatedprotein kinases angiotensinconverting enzyme inhibition
   INTRODUCTION3 B( a9 S, n1 N9 y8 V& ]4 A  s: [* ~

DURING DIABETIC NEPHROPATHY (DN), hyperglycemia triggers numerous deleteriousbiological responses, such as extracellular matrix protein secretion,cell proliferation, and growth factor activation, including IGF-1,PDGF-BB, VEGF, and their receptors ( 11, 23, 41, 53, 56 ).These growth factors are suggested to be involved in the hyperplasiaand extracellular matrix accumulation associated with acute or chronicglomerulosclerosis ( 1, 8, 23, 36, 46 ). The effects ofthese growth factors are likely occurring via the phosphorylation ofthe MAPK ERK1/2 ( 3, 13, 14 ). Such phosphorylation of thisMAPK occurs in the glomerulus and mesangial cells at an early phase ofvarious pathologies such as DN, mesangioproliferativeglomerulosclerosis, or high-salt diet-induced nephropathy ( 5, 9, 30, 31 ). The ERK1/2 phosphorylation is suggested to play animportant role in the establishment of the hyperproliferative state( 33, 35 ). Finally, hyperglycemia-induced MAPK activationcan be considered as an early biochemical signaling event, which is thestarting point of a deleterious signaling cascade. The upregulation ofgrowth factor activity will emphasize MAPK activation and accelerate the progression of DN. On this basis, control of MAPK activity can bean innovative therapeutic strategy to decelerate the worsening of DN.1 ?! f% y# L7 Z: b& w) n
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The regulation of mitogenic activity by G protein-coupledreceptors, particularly regarding the MAPK pathway, has been largely investigated. With respect to the kinin receptors, bradykinin (BK), theagonist of the kinin B 2 receptor (B 2 R), hasbeen shown to induce proliferation in glomerular mesangial cells( 6, 20 ) and fibrosis in vascular smooth muscle cells( 18 ). The profibrogenic effects of BK are associated withthe phosphorylation of ERK1/2, which is a prerequisite for theactivation of this MAPK ( 18 ). Finally, the activation ofERK1/2 by BK has been demonstrated in various cells lines: A431,mesangial, and vascular smooth muscle cells ( 20, 25, 50 ).
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Whereas the initial studies conducted only with quiescent cellsdemonstrated mainly mitogenic effects ( 6, 20 ), more recent studies by several groups report inhibitions of both cell proliferation and ERK1/2 phosphorylation by BK. Dixon and Dennis ( 15 )evidenced an inhibition of mitogenesis by BK in arterial smooth musclecells stimulated with PDGF-AB. Our laboratory recently demonstrated that B 2 R activation reduces serum-stimulated mitogenesis inrat mesangial cells ( 2 ). Moreover, Graness et al.( 26 ) have shown that EGF-induced ERK1/2 activation wasinhibited by BK via tyrosine phosphatase activation, and Tsuchida etal. ( 49 ) suggested an antihypertrophic role forB 2 R on the renal vasculature. However, it is not knownwhether this cross talk prevails in vivo and might extend to othergrowth factors involved in glomerulosclerosis.
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A substantial amount of experimental and clinical studies have reportedthat angiotensin-converting enzyme (ACE) inhibitors are renoprotective,notably in DN ( 29, 38 ). ACE inhibitors prevent thegeneration of angiotensin II, which exerts well-known profibrogenic andproliferative effects ( 54 ). Nevertheless, ACE inhibitorsalso favor the accumulation of kinins by preventing their degradation( 10 ). In addition, compelling evidence suggests theinvolvement of kinins in the renal effects of ACE inhibitors ( 22, 39 ), and Tschöpe et al. ( 48 ) have shown thatkidney B 2 R is upregulated in streptozotocin (STZ)-diabeticrats. Nevertheless, the roles of BK and B 2 R in theprotective effects of ACE inhibitors during the development of DNremain to be established.

The aim of the present study was threefold: 1 ) toinvestigate in freshly isolated rat glomeruli (IG) the effect ofB 2 R activation on the phosphorylation of ERK1/2 induced byIGF-1, PDGF-BB, VEGF and basic (b)FGF; 2 ) to explore themechanism involved in the cross talk between BK and growth factors; and 3 ) to assess whether this cross talk could bepharmacologically recruited in a physiopathological state by exploringthe effect of ACE inhibition on the phosphorylation level of glomerularERK1/2 and on 4-hydroxynonenal (4-HNE) protein derivatization, an indexof the oxidative stress in STZ-diabetic rats with regard to theputative involvement of B 2 R.  V0 X  u2 C( X. N

MATERIALS AND METHODS9 J) q$ Q9 ?0 E& V3 S
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Animal Use and Care
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Male Sprague-Dawley rats (12 wk old; Harlan; n = 108) were housed under controlled conditions in a room with a 12:12-hlight-dark cycle and standard rat chow and tap water available adlibitum. Rats were food-starved 18 h before kidneys werecollected. Experimental procedures and protocols were ethicallyapproved by the Midi-Pyrenees regional administration in strictcompliance with the guiding principles for animal research (US).
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Drugs and Compounds
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The commercial sources of products were as follows. BK,des-Arg 9 -BK (DBK), IGF-1, PDGF-BB, VEGF, bFGF,orthovanadate, N G -nitro- L -argininemethyl ester ( L -NAME), ouabain, EGTA, SDS,glycerol, PMSF, soybean trypsin inhibitor (SBTI), aprotinin, leupeptin, -mercaptoethanol, poly(Glu-Tyr), bacitracine, BSA, genistein, DTT,TCA, ammonium molybdate, isobutanol, and toluene were from Sigma-Aldrich (St. Quentin Fallavier, France). NaCl, RPMI 1640, andbromophenol blue were from Merck Eurolab (Strasbourg, France). Tris andglycine were from GIBCO BRL (Cergy-Pontoise, France). [ 33 P]ATP was from Amersham Biosciences (Saclay, France),PBS from Biochrom (Berlin, Germany), EDTA from ICN, and STZ fromPharmacia and Upjohn (St. Quentin Yvelines, France). Ramipril andHOE-140 were generously provided by Aventis Pharma (Frankfurt,Germany), whereas losartan was a kind gift from Merck (Rahway, NJ).0 l" t' B7 @1 |+ \: `

Ex Vivo Experiments

Isolated glomerular preparation. Glomeruli were isolated as routinely performed in the laboratory bygraded sieving ( 6 ). Briefly, rats were anesthetized intraperitoneally with 65 mg/kg pentobarbital sodium (Sanofi, Montpellier, France) and killed by exsanguination, and the kidneys werequickly removed. The cortex was forced through three consecutive steelsieves with decreasing pore sizes (180, 125, and 75 µm) 90% of theglomeruli appeared to be decapsulated and free of surrounding tubulesand arterioles. Glomeruli were resuspended in RPMI 1640 culture mediumand redistributed in experimental tubes containing ~5,000glomeruli/tube. After the appropriate incubation time at 37°C, and inthe presence of BK, DBK, IGF-1, PDGF-BB, VEGF, bFGF, L -NAME, ouabain, and the tyrosine phosphatase inhibitororthovanadate (OV), the incubation was stopped by adding 1 ml ofice-cold PBS containing 1 mM OV. The dose of BK (100 nM) and of thedifferent growth factors was the maximal response dose according to adose-response curve performed in pilot experiments. Then tubes werecentrifuged (15,000 rpm, 4°C, 2 min), and the supernatant wasdiscarded. The pellet containing the glomeruli was resuspended in 100 µl of lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM OV, 0.36 mg/ml PMSF, 10 µg/ml SBTI, 1 µg/ml aprotinin, 1 µg/mlleupeptin, 0.1% SDS, pH 7.5), sonicated for 10 s, and centrifuged(15,000 rpm, 4°C, 15 min). Insoluble material was discarded, and theproteins of the soluble extract were boiled in Laemmli buffer (32 mMTris, 1% SDS, 5% glycerol, 0.0005% bromophenol blue, 2.5% -mercaptoethanol, pH 6.8) for 6 min and stored frozen untilSDS-PAGE. Protein concentration was determined by the Bradford protein assay.2 `: C. A2 G; e3 b, o

In Vivo Experiments

Diabetes was induced by an intraperitoneal injection of 65 mg/kgSTZ freshly dissolved in 0.05 M citrate buffer, pH 4.5. Age-matched control rats received the vehicle only. Once diabetes was established (4-5 days afterward), diabetic rats were randomly divided into five groups. Rats belonging to the first group received no other treatment. Insulin was given to the rats in the second group as asubcutaneous implant delivering 2 U/24 h (Linshin, Scarborough, ON).Rats in group 3 received 1 mg · kg 1 · day 1 of the ACEinhibitor ramipril in their drinking water. Rats from group4 received ramipril and in addition were subcutaneously injecteddaily with 0.25 mg/kg of the B 2 R-selective antagonist HOE-140. Rats from group 5 received 10 mg · kg 1 · day 1 of theAT 1 -receptor antagonist losartan in their drinking water. The selected doses of ramipril (1 mg · kg 1 · day 1 ) andlosartan (10 mg · kg 1 · day 1 ) havebeen previously demonstrated to reverse many functional andmorphological events of DN ( 24, 55 ). For its part, the dose of HOE-40 (0.25 mg/kg) is twofold that of a dose demonstrated toinhibit the hypotensive effects of BK in vivo ( 40 ). Seven days after the initiation of these different treatments, glycemia wasmeasured with a EuroFlash LifeScan glycometer (Issy-les Moulineaux, France), the rats were killed and the kidneys removed for glomerular protein extraction.- y  v( g% P  U" X- A
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SDS-PAGE and Western blotting. Equal amounts of proteins (25 µg) were separated by SDS-PAGE inTris-glycine buffer under a 150-V, 30-mA current in a Bio-Rad miniaturetransfer gel apparatus (Mini-Protean, Bio-Rad Laboratories, Richmond,CA) on a 10% SDS-polyacrylamide gel. Proteins were then transferred toa nitrocellulose membrane (Amersham, Orsay, France) inTris-glycine-methanol buffer under a 100-V, 300-mA current in a Bio-Radminiature transfer gel apparatus (Mini-Protean, Bio-Rad Laboratories).The membrane was blotted with the appropriate antibody. Proteins werevisualized using a horseradish peroxidase-conjugated goat anti-rabbitimmunoglobulin (Amersham) and an enhanced chemiluminescence (ECL) kit (Amersham).( C2 P4 Q8 V' k$ A, J9 ~- |- ^
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MAPK phosphorylation, MAPK phosphatase-1 expression, and 4-HNEprotein derivatization. ERK1/2 phosphorylation was assessed by Western blotting withantiphospho-ERK antibodies (dilution 1:3,000; Promega, Madison, WI)that recognize the active forms of ERK1 [molecular wt (MW) = 44]and ERK2 (MW = 42). In a preliminary study, we established thatunder these experimental conditions the detection of ERK1/2 phosphorylation by this method was highly correlated with the incorporation of radioactive phosphorus in the myelin basic protein. Similarly, MAPK phosphatase-1 (MPK-1) expression was studied with ananti-MKP-1 polyclonal antibody (1:250; Santa Cruz Biotechnology, LePerray, France). 4-HNE protein derivatization was estimated with apolyclonal antibody directed against 4-HNE Michael adducts (1:2,000;Calbiochem, Nottingham, UK). The amount of total ERK was alsovisualized as a control using an antibody that recognizes total ERK1protein (1:1,500; Santa Cruz Biotechnology) independently of its levelof phosphorylation.6 a8 h- s* ^3 D; o
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Tyrosine phosphatase activity. The poly(Glu-Tyr) substrate was phosphorylated with[ 33 P]ATP as described earlier ( 42 ).Glomeruli were isolated and incubated with 100 nM BK for various timesas described earlier. After IG lysis, 10 µg of the protein extractwere incubated in 500 µl PTP buffer (50 mM Tris · HCl, 0.5 mg/ml bacitracin, 0.1% BSA, 50 mM DTT, pH 7.5) with 30,000 counts/minof 33 P-labeled poly(Glu-Tyr) for 10 min at 30°C. Thereaction was stopped by addition of 1 volume of ice-cold 30% TCA andincubated 30 min on ice. After centrifugation at 13,000 rpm for 10 minat 4°C, 1 volume of ammonium molybdate was added to 1 volume ofsupernatant, and the mixture was incubated for 10 min at 30°C. Then,2 volumes of isobutanol/toluene (50:50) were added, and the solutionwas thoroughly mixed. The amount of inorganic - 33 Pextracted using this method was counted with a liquid scintillation counter (Packard Instruments, Groningen, The Netherlands). Results areexpressed as the percentage of tyrosine phosphatase activity in theabsence of BK ( time 0 ).1 j1 f, X5 l- n/ L

Statistical Analysis
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Data are expressed as means ± SE of n independent experiments. Body weight, glycemia, and tyrosinephosphatase activity results were analyzed by one-way ANOVA, followedby either Student's t -test for paired data or Dunnett'stest for multiple comparisons. A Kruskal-Wallis test and post hocWilcoxon-Mann-Whitney test were used for Western blot densitometricanalysis. Only P considered significant.All analyses were performed with SigmaStat 1.0 software (Jandel).

RESULTS

Ex Vivo Experiments

Effects of BK, IGF-1, PDGF-BB, VEGF, and bFGF on glomerular ERK1and -2 phosphorylation. Before studying the possible interaction between B 2 R andgrowth factor receptor signaling, we studied the effect on ERK1/2 phosphorylation of separate activation of these receptors in IG. TheB 2 R agonist BK at 100 nM induced a time-dependent andtransient phosphorylation of ERK1/2 (p44 and p42) that peaked at 2 min, reaching 320% of control value ( P levels at 10 min (Fig. 1 A, lanes2-6 ). IGF-1 (65 nM) alsoelicited a transient phosphorylation of ERK1/2 that peaked between 2 and 5 min (250% of control P at 10 min (Fig. 1 B, lanes8-12 ). PDGF-BB (8 nM) induced a transient ERK1/2phosphorylation that peaked at 2 min of incubation (330% of control; P 1 C, lanes 14-18 ). VEGF (25 nM) and bFGF (30 nM) induced an increase in ERK1/2 phosphorylation that peaked at 2 min( P returned to basal levels at 20 min (Fig. 1, D and E, lanes 20-24 and 26-30, respectively). Total ERK1 expression remainedunchanged during all these stimulations.
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Fig. 1. Time-dependent effect of 100 nM bradykinin (BK; A ), 65 nM IGF-1 ( B ), 8 nM PDGF-BB ( C ), 25 nM VEGF( D ), and 30 nM basic (b)FGF ( E ) on ERK1/2phosphorylation in isolated glomeruli (IG). IG were incubated in thepresence of either peptide during different times (from 30 s to 20 min). ERK1/2 phosphorylation was measured by Western blotting with anantibody against the phosphorylated forms. Analysis were performed onan equal amount of protein (25 µg) as measured with an antiserumagainst total ERK1 (phosphorylated and nonphosphorylated forms). TheERK1/2 phosphorylation (P-ERK) was expressed as the fold-increase ofthe P-ERK vs. total ERK1 ratio compared with control ( time0 ), and results are shown as means ± SE of the scanningdensitometry of 5 experiments. ** P

B 2 R activation reduced growth factor-induced glomerularERK1 and -2 phosphorylation. Next, we studied the effect of a pretreatment with BK andB 1 -kinin receptor agonist DBK on IGF-1-, PDGF-BB-, VEGF-,and bFGF-induced ERK1/2 phosphorylation. As shown in Fig. 2 A, the phosphorylation ofERK1/2 in IG in the presence of IGF-1 was inhibited by BK ( lane 5 vs. 4; P DBK ( lane 6 vs. 4 ),which was devoid of any effect by its own ( lane 3 ).Moreover, as shown in Fig. 2, B-D, the phosphorylation of ERK1/2 induced by PDGF-BB, VEGF, and bFGF was also inhibited in thepresence of BK ( lanes 13 vs. 12, 19 vs. 18, and 25 vs. 24; P ( lanes 14 vs. 12, 20 vs. 18, and 26 vs. 24 ), which was without effect when used alone ( lanes 11, 17, and 23 ). Moreover, incubation with an equimolarconcentration of angiotensin II did not reduce IGF-1-inducedphosphorylation of ERK1/2 (Fig. 2 A, lane 8 vs. 4 ), whereas angiotensin II did stimulate ERK1/2phosphorylation when used alone (Fig. 2 A, lane7 ). In all these experiments, the level of the total form ofERK1 remained unchanged.
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Fig. 2. Effect of BK, des-Arg 9 -BK (DBK), and angiotensin II onIGF-1 ( A )-, PDGF-BB ( B )-, VEGF ( C )-,and bFGF-induced ( D ) ERK1/2 phosphorylation in IG. IG wereincubated with IGF-1, PDGF-BB, VEGF, or bFGF for 2 min, whereas BK,DBK, and angiotensin II were added 18 min beforehand. Western blotanalysis was performed as described in the legend of Fig. 1.** P n = 5).
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Negative cross talk between B 2 R and growth factorreceptors involves tyrosine phosphatase activation. The inhibitory effect of BK on IGF-1- or PDGF-BB-induced ERK1/2activation (Fig. 3, A and B, lanes 4 vs. 3 and 11 vs. 10; P inhibitor OV (Fig. 3, A and B, lanes 8 vs. 7 and 14 vs. 13 ). Moreover, similar inhibitory effects of BK were alsodemonstrated on VEGF- and bFGF-induced ERK1/2 phosphorylation (data notshown). Nevertheless, one could argue that this blockade by OV is infact related to a nonspecific inhibition of theNa -K -ATPase transporter by OV. However, theinhibitory effect of BK on IGF-1-induced ERK1/2 phosphorylationwas not blocked by the Na -K -ATPase-specificinhibitor oubain, thus excluding such a hypothesis (Fig. 3 C, lane 20 vs. 19 ). In all these experiments, thelevel of the total form of ERK1 remained unchanged.

Fig. 3. Blockade of the inhibitory effect of BK on IGF-1 ( A )-and PDGF-BB ( B )- induced ERK1/2 phosphorylation in IG withthe tyrosine phosphatase inhibitor orthovanadate (OV) or with either N G -nitro- L -arginine methyl ester( L -NAME) or ouabain ( C ). IG were incubated withIGF-1 or PDGF-BB for 2 min, whereas BK (18 min), OV (50 min), L -NAME (50 min), and ouabain (20 min) were addedbeforehand. Western blot analysis was performed as described in thelegend of Fig. 1. ns, Not significant. ** P n = 5).
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To provide more direct evidence for the involvement of a tyrosinephosphatase, we measured the tyrosine phosphatase activity inglomerular extract after stimulation with BK. The data shown in Fig. 4 demonstrated that BK induced atime-dependent increase in tyrosine phosphatase activity, reaching asignificant 250% maximum increase after 10-min stimulation with 100 nMBK ( P 0.01).2 i* L4 `- [3 g' _* v9 _2 Q: v+ [
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Fig. 4. BK-induced increase in total tyrosine phosphataseactivity. IG were incubated with 100 nM BK for the time indicated.Total tyrosine phosphatase activity was determined using 33 P-labeled poly(Glu-Tyr). Data are expressed as thepercentage of free 33 P observed in nontreated IG( time 0 ). Basal total tyrosine phosphatase activity innontreated IG was 52 cpm · min 1 · 10 µgprotein 1, where cpm is counts/min. Results are expressedas means ± SE of 3 independent experiments. ** P time 0 ).
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MKP-1 is not involved in the negative cross talk betweenB 2 R and IGF-1R. Because the induction of the immediate early gene MKP-1 is known to beinvolved in the reduction of growth factor-induced MAP kinaseactivation ( 7 ), we investigated this possibility bystudying the effect of BK on the expression of MKP-1 by Western blotanalysis. As shown in Fig. 5, theconstitutive expression of MKP-1 was unchanged by incubation in thepresence of either IGF-1 (2 min) or BK (20 min) ( lanes 2 and 3 ).
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Fig. 5. Effect of IGF-1 and BK on MAPK phosphatase-1 (MKP-1)protein expression and ERK1/2 phosphorylation in IG. IG were incubatedwith IGF-1 for 2 min, whereas BK was added 18 min beforehand. Westernblot analysis was performed as described in the legend of Fig. 1, withantibodies directed against MKP-1 and P-ERK1/2 ( n = 6).** P lanes2 and 3 ).3 e+ K2 s8 b0 n! z! D1 G: l& y

Nitric oxide synthesis is not involved in the negative cross talkbetween B 2 R and IGF-1R. B 2 R activation is known to trigger nitric oxide (NO)release from endothelial cells, and NO is known to be antiproliferative ( 12 ). However, the prior incubation of IG with 100 µM L -NAME did not alter the inhibition of IGF-1-induced ERK1/2activation by BK (Fig. 3 C, lane 17 vs. 16; P involvement of NO in the negative cross talk between B 2 Rand IGF-1R in IG.' P7 ^1 w+ t. K6 F5 @) ~, o2 K4 z

In Vivo Experiments2 A  ?: M8 t  q* K

Physiological parameters of STZ-diabetic and control rats. As shown in Table 1, STZ-treated ratsexhibited a significant loss of body weight (about 15%; P hyperglycemia (336 mg/dl blood glucose; P controlrats and thus could be considered as diabetic. Insulin administrationnormalized blood glucose and body weight (108 ± 16 mg/dl bloodglucose and 379 ± 18 g body wt) in rats under ramipril andlosartan treatments, but both hyperglycemia and body weight losspersisted.

Table 1. Data for rats included in in vivo experiments
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B 2 R activation reduces diabetes-induced glomerularERK1/2 phosphorylation. As it can be observed in Fig. 6,STZ-treated rats exhibited glomerular ERK1/2 phosphorylation 280% ofcontrol value compared with the untreated control group ( lane2 vs. 1; P witheither insulin or the ACE inhibitor ramipril abolished this activation( lanes 3 and 4 vs. 2; P of ramipril wasblunted by the blockade of B 2 R with HOE-140 ( lane 5 vs. 4; P blocker losartan did not affectthe diabetes-induced increase in glomerular ERK1/2 phosphorylation( lane 6 ). In all the groups of rats, the level of the totalform of glomerular ERK1 remained unchanged.1 k2 U. ~; C/ u3 L( {) l8 x5 R
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Fig. 6. Effect of B 2 receptor (B 2 R)blockade on ERK1/2 phosphorylation in IG of streptozotocin(STZ)-diabetic rats. STZ-diabetic rats were either untreated orreceived insulin (2 U/day), an angiotensin-converting enzyme (ACE)inhibitor (ramipril; 1 mg · kg 1 · day 1 ) and/or theB 2 R antagonist HOE-140 (0.25 mg · kg 1 · day 1 ) and anangiotensin AT 1 receptor blocker (losartan; 10 mg · kg 1 · day 1 ). Westernblot analysis was performed as described in the legend of Fig. 1 ( n = 4). ** P lane 2 ). §§ P

B 2 R activation reduces diabetes-induced oxidativestress in glomeruli. As shown in Fig. 7, STZ-treated ratsdemonstrated an increased level of 4-HNE protein derivatization, themajor increase being observed in the range of 70 kDa compared withuntreated control ( P n = 4).Such enhancement in 4-HNE protein derivatization is an indication ofincreased oxidative stress. Treatment with insulin, the angiotensinII-receptor blocker losartan, or the ACE inhibitor ramipril abolishedthis modification ( P n = 4).This inhibitory effect of ramipril was blunted by the blockade ofB 2 R with HOE-140 ( P n = 4). In all groups of rats, the level of the totalform of glomerular ERK1 remained unchanged.
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Fig. 7. Effect of B 2 R blockade on 4-hydroxynonenal(4-HNE) protein derivatives labeling in IG of STZ-diabetic rats.STZ-diabetic rats were either untreated or received insulin (2 U/day),losartan (10 mg · kg 1 · day 1 ), ramipril(1 mg · kg 1 · day 1 ), and/orHOE-140 (0.25 mg · kg 1 · day 1 ). Westernblot analysis was performed as described in the legend of Fig. 1 withan antibody directed against 4-HNE Michael protein adducts( n = 4). ** P lane 2 ). §§ P
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DISCUSSION

This paper reports for the first time the inhibition by BK ofERK1/2 phosphorylation triggered in rat IG by different growth factors,namely, IGF-1, PDGF-BB, VEGF, and bFGF. This negative cross talk occursselectively via B 2 R activation and involves tyrosinephosphatase activation. The second original finding is that chronic ACEinhibition reduces the early diabetes-induced glomerular ERK1/2phosphorylation as well as oxidative stress in vivo through endogenousB 2 R activation. Hence, the data suggest the involvement ofBK in the therapeutic effects of ACE inhibitors during the developmentof DN and also underscore, during in vivo ACE inhibition, thepharmacological recruitment of the negative cross talk betweenB 2 R and growth factor receptors as seen ex vivo.$ T! k% ?! E; G; |$ t

Our ex vivo observation of the negative modulation by BK of variousgrowth factor signaling is consistent with the inhibition by BK ofmesangial cell proliferation ( 2 ). This negative cross talkappears to be specific for BK and B 2 R because angiotensin II, another ligand for a G protein-coupled receptor, is unable toreduce IGF-1-induced ERK1/2 phosphorylation. Graness et al. ( 26 ) also observed an inhibition of EGF signaling aftertyrosine phosphatase activation by BK in A431 cells. Therefore, thefact that BK can inhibit ERK1/2 phosphorylation triggered by receptors of different growth factors, IGF-1, PDGF-BB, VEGF, bFGF, and EGF, strongly suggests that the inhibitory action of BK occurs at a commondownstream level of growth factor signaling, most likely tyrosinephosphatase activation directed on ERK1/2. Such a hypothesis isconsistent with our finding of BK-induced tyrosine phosphatase activityin IG. The involved tyrosine phosphatase is unlikely to be MKP-1.Indeed, it is acknowledged that the activity of this early gene productis essentially regulated at its expression level ( 5, 7 ).Therefore, our observation that BK does not modify glomerular MKP-1protein expression argues against such involvement of MKP-1 in theinhibitory effect of BK on growth factor signaling. On the other hand,our group recently demonstrated in vitro that B 2 R fixationby BK triggers the activation of the protein tyrosine phosphatase SHP-2via a direct protein-protein interaction resulting in the inhibition ofcell proliferation ( 19 ). Therefore, SHP-2 is an obviouscandidate, although the in vivo demonstration of its involvement in thenegative cross talk between BK and growth factors in IG is stillhampered by the lack of a specific inhibitor.
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The inhibition of growth factor-induced ERK1/2 phosphorylation in IG byBK could be correlated with an inhibition of a downstream effect suchas cell proliferation, which is believed to involve ERK1/2 activation.The control of cell proliferation can occur at a distinct level eitherby reducing mitogenesis or by increasing cell death. The present datafavor an inhibitory action on the proliferative pathway. In thisrespect, another study has shown that BK reduced smooth muscle cellproliferation induced by PDGF via an unknown mechanism( 15 ). Several other studies have demonstrated anantiproliferative effect of BK in different cell lines without anyproposed mechanism ( 2, 43 ).+ K. p$ m. G* U, s7 b5 ?( L5 N
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On the other hand, contrasting evidence has shown that BK inducesproliferation in glomerular mesangial cells ( 20 ).Moreover, the activation of ERK1/2 by BK has been demonstrated invarious cells lines: A431, mesangial, and vascular smooth muscle cells ( 20, 25, 50 ). It is noteworthy that the proliferativeeffect of BK has been essentially demonstrated in quiescent mesangial cells with high concentrations of BK. Therefore, it can be suggested that the mitogenic action of BK might depend on the level of cell activation by a growth factor. In a starving condition, BK might promote cell proliferation and ECM protein secretion, whereas theopposite effect becomes preferential during proliferation states, suchas glomerulosclerosis, after activation by several growth factors." ~& W2 e  R) i' @, g6 E

The inhibition by B 2 R activation of IGF-1, PDGF-BB, VEGF,and bFGF signaling might be of physiopathological relevance as the involvement of all these growth factors has been evoked in the progression of glomerulosclerosis, notably during DN but also in renalfibrogenesis. IGF-1 increases glucose uptake in mesangial cells byaugmenting the expression of GLUT1 ( 4, 34 ) and stimulates the secretion of collagen I and IV and proliferation by mesangial cells( 14, 21 ). However, other evidence contests the existence of a role for IGF-1 in the establishment of DN. Indeed, Doi et al.( 17 ) have shown that transgenic mice overexpressing IGF-1 do not exhibit glomerulosclerosis and tubular atrophy, which are hallmarks of DN. Nevertheless, since that initial work, a larger numberof reports suggest a role for growth factors in the progression of DNand thereby may be of interest for the future development of new drugsuseful in the treatment of diabetic kidney disease ( 23 ).Interestingly, the proliferative activity of IGF-1 is amplified byprior stimulation with PDGF ( 16 ). PDGF-BB, secreted byglomerular cells as well as activated platelets andmacrophages, is the most potent mitogen for mesangial cells invitro and in vivo ( 36 ). Moreover, PDGF has been shown tobe induced by TGF- and to mediate TGF- -induced accumulation ofcollagen IV and fibronectin ( 32 ). Also, VEGF has beenreported to enhance collagen synthesis via the activation of ERK( 3 ). bFGF was acknowledged to elicit the proliferation ofboth fibroblasts and mesangial cells ( 44, 46 ) and therebyis involved in renal fibrogenesis. Because these four growth factorsare involved in the development of DN, it is conceivable that thenegative modulation of their signaling by BK may be of therapeuticalrelevance in DN.- ]& {5 r: {; |. q3 X

Next, we demonstrated in vivo that the increased phosphorylation ofERK1/2 and oxidative stress assessed by the detection of 4-HNE proteinderivatization, a well-established index of oxidative stress indiabetic glomerular lesions ( 47 ), in glomeruli of STZ-diabetic rats is reversed by ACE inhibition via B 2 Ractivation. This confirms the physiopathological relevance of our exvivo observations of negative cross talk between BK and growth factor receptors in IG from normal rats. The increased phosphorylation ofglomerular ERK1/2 at an early phase of STZ-induced diabetes was alsoobserved by Awazu et al. ( 5 ) and Haneda et al.( 31 ). Awazu et al. ( 5 ) ascribed thediabetes-induced activation of ERK1/2 to decreased phosphatase activityand MKP-1 protein expression. This hypothesis is further supported bythe fact that tyrosine phosphatase inhibition with OV mimics thediabetic phenotype in mesangial cells, i.e., increased cellproliferation, activation of protein kinase C, tyrosine phosphorylationof intracellular proteins, and induction of PDGF-B chain geneexpression ( 52 ). In addition, we now demonstrate thathyperglycemia plays a primary role in glomerular ERK1/2 phosphorylationand oxidative stress during diabetes because strict glycemic controlwith insulin abolished MAPK phosphorylation as well as 4-HNE proteinderivatization. Early activation of glomerular ERK1/2 in diabetes issuggested to play an important role in the progression of DN ( 33, 35 ) and is consistent with a combined effect of high glucose andvarious growth factors, including IGF-1, PDGF-BB, and VEGF ( 23, 54 ). Moreover, oxidative stress may play an important role inthe progression of DN and emphasize the phosphorylation of ERK1/2( 27, 28 ).
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One may note that strict glycemic control with insulin, which isobviously the more appropriate initial therapy for type I diabetes, isas efficient as ACE inhibitors to reduce diabetes-induced phosphorylation of ERK1/2 and 4-HNE protein derivatization. However, the diabetes-induced phosphorylation of ERK1/2 and oxidative stress arereversed by ACE inhibition, without any effect on glycemia, suggestingthe involvement of a glycemia-independent mechanism. Therefore, strictglycemic control with insulin and ACE inhibitors may exert independentand additive effects and thereby may be successfully associated todelay or stabilize the rate of progression of renal disorderassociated with diabetes, as recently recommended ( 37 ).Such an effect of ACE inhibition is consistent with the renoprotectiveeffects of this treatment during diabetes mellitus ( 23, 29, 38 ). Furthermore, it was shown that ACE inhibition favors theaccumulation of BK ( 10 ). Hence, according to present evidence, ACE inhibition potentiates BK concentration and reduces phosphorylation of ERK1/2 and oxidative stress through B 2 Ractivation, because blockade of the B 2 R abolished theeffect of ACE inhibitors. Moreover, in vitro stimulation of IG fromuntreated diabetic rats with BK reduced the enhanced ERK1/2 level,confirming the involvement of B 2 R (data not shown). Incontrast to ramipril, losartan did not reduce diabetes-induced ERK1/2phosphorylation although it reduced 4-HNE protein derivatization. Thisresult is consistent with the effect of angiotensin II shown in Fig. 2 A, in which angiotensin II did not inhibit IGF-I-inducedERK1/2 phosphorylation. Although both ACE inhibitors andAT 1 receptor blockade are renoprotective, notablyconcerning oxidative stress, it seems that the inhibition of growthfactor-induced ERK1/2 phosphorylation is specific to BK. The presentobservation supports the existence of a protective action of BK and theB 2 R against the deleterious effects of high glucose andgrowth factors present in the glomeruli during diabetes mellitus. Thehypothesis of a tonic-protective role of BK, at least via theB 2 R, against the severity of renal complication associatedwith diabetes mellitus is in agreement with a recent report usingtransgenic mice for ACE (34a). This report demonstrates that modest genetically determined increases in plasma ACE levels, which decrease BK concentration without significantly affecting angiotensin II ( 45 ) result in severe renal complicationsin the diabetic mouse.
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In conclusion, the activation of the B 2 R inhibits IGF-1-,PDGF-BB-, VEGF-, and bFGF-induced ERK1 and -2 phosphorylation in IGfrom normal rats, via the activation of a tyrosine phosphatase. Thisnegative cross talk could be pharmacologically recruited during ACEinhibition, demonstrating that chronic activation of B 2 Runder such treatment inhibits the phosphorylation of ERK1/2 as well asoxidative stress in glomeruli of STZ-diabetic rats. These inhibitoryactions in the glomeruli are consistent with a renoprotective action ofBK and B 2 R during diabetes mellitus and support theexistence of a role for this autacoid in the beneficial effects of ACEinhibition during the development of DN. Such findings open newperspectives concerning the treatment of glomerulosclerosis, notablyduring diabetes mellitus.4 m6 d5 M8 G2 c9 R! x& y2 G" t
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ACKNOWLEDGEMENTS
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The authors acknowledge Aventis Pharma (Germany) for providingramipril and HOE-140 and Merck (US) for losartan. The authors alsoacknowledge the technical assistance of Denis Calise in managing theinsulin implants as well as Dr. Joost P. Schanstra for helpful suggestions during the writing of the manuscript.
      【参考文献】% Z; d# O8 q- h! Q
1. Abboud, HE. Growth factors in glomerulonephritis. Kidney Int 43:252-267,1993  .& G6 G/ `- ~! z& D& E: D
4 ^3 x; U" I7 x. V; Q

% Z+ P( ?3 P" G8 f& o
2. Alric, C,Pecher C,Bascands JL,andGirolami JP. Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells. Immunopharmacology 45:57-62,1999  .

, U( q: `8 Z: h2 [3 P* U

3. Amemiya, T,Sasamura H,Mifune M,Kitamura Y,Hirahashi J,Hayashi M,andSaruta T. Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells. Kidney Int 56:2055-2063,1999  .* `6 L" l! i( ^2 v& A
6 g  ?! A; s0 e5 h/ B. E* z) k

8 n& I2 q/ O. w" G: c. ?
4. Asada, T,Ogawa T,Iwai M,Shimomura K,andKobayashi M. Recombinant insulin-like growth factor I normalizes expression of renal glucose transporters in diabetic rats. Am J Physiol Renal Physiol 273:F27-F37,1997 .

1 Z0 h2 O+ E0 `$ P7 r( G
5. Awazu, M,Ishikura K,Hida M,andHoshiya M. Mechanisms of mitogen-activated protein kinase activation in experimental diabetes. J Am Soc Nephrol 10:738-745,1999 .
0 y3 H/ ~; p, j
  S& k) O* k, ?' Y+ }
% ?+ h- s9 y" V
6. Bascands, JL,Pecher C,Rouaud S,Emond C,Tack Y,Bastie MJ,Burch R,Regoli D,andGirolami JP. Evidence for existence of two distinct bradykinin receptors on rat mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 264:F548-F556,1993 .
7 L* W) o& l5 R  l# ^- e/ g0 [2 N  O
2 F8 P' r5 Z4 R& I; E

7. Beltman, J,McCormick F,andCook SJ. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem 271:27018-27024,1996 .
" _( H2 G9 F7 Q
* m% j3 i/ m; w! w- H- e
  L5 b" r2 ^0 E4 k" [% s* |
8. Blazer-Yost, BL,Goldfarb S,andZiyadeh FN. Insulin, insulin-like growth factors, and the kidney.In: Hormones, Autacoids, and the Kidney, edited by Goldfarb S,Ziyadeh FN,and Stein JH.. New York: Churchill Livingstone, 1991, p. 339-363.

7 K. N) G& n4 q2 o0 h
9. Bokemeyer, D,Ostendorf T,Kunter U,Lindemann M,Kramer HJ,andFloege J. Differential activation of mitogen-activated protein kinases in experimental mesangioproliferative glomerulonephritis. J Am Soc Nephrol 11:232-240,2000 .
; `" D; R; w3 L. |7 \
8 a, j9 I3 ^0 z3 w" U: x

10. Campbell, DJ,Kladis A,andDuncan AM. Bradykinin peptides in kidney, blood, and other tissues of the rat. Hypertension 21:155-165,1993 .  d4 m8 s# [$ j8 D

11. Cha, DR,Kim NH,Yoon JW,Jo SK,Cho WY,Kim HK,andWon NH. Role of vascular endothelial growth factor in diabetic nephropathy. Kidney Int 58:S104-S112,2000.7 @$ H: v7 p. \( g' X, B

! m7 q+ T! s" P3 ~
) ?' t' u% B1 O7 q3 u& A2 C
12. Chin, TY,Lin YS,andChueh SH. Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase. Eur J Biochem 268:6358-6368,2001  .
" }! V2 L$ [* f4 l4 V" O! H9 V

13. 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 .
/ i. ^: e" ?4 H- H  B

14. Coolican, SA,Samuel DS,Ewton DZ,McWade FJ,andFlorini JR. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem 272:6653-6662,1997 .5 H" Q( d5 d4 x
& ]  \( K+ l! n# l
+ Z8 W" @  S& b  V3 s
, w/ ~& [; V, {; T
15. Dixon, BS,andDennis MJ. Regulation of mitogenesis by kinins in arterial smooth muscle cells. Am J Physiol Cell Physiol 273:C7-C20,1997 .

16. Doi, T,Striker LJ,Elliot SJ,Conti FG,andStriker GE. Insulin-like growth factor-1 is a progression factor for human mesangial cells. Am J Pathol 134:395-404,1989 .' u: X, k5 {: m1 ^8 Q8 @8 E

# t: i4 t+ R2 _. `! g
7 u% C! z2 {) {( v+ |) Q& x$ E& ]
17. Doi, T,Stricker LJ,Gibson CC,Agodoa LYC,Brinster RL,andStricker GE. Glomerular lesions in mice transgenic for growth hormone and insulin like growth factor-I. Am J Pathol 137:541-552,1990 .$ y4 P# F8 G5 S1 t; s" q. \5 n

18. Douillet, CD,Velarde V,Christopher JT,Mayfield RK,Trojanowska ME,andJaffa AA. Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF- and MAPK. Am J Physiol Heart Circ Physiol 279:H2829-H2837,2000 .

/ t. P. g; ]& R- j, d2 v& U6 r& {
; z. g2 l( h, B& G% R
19. Duchêne, J,Schanstra JP,Pécher C,Pizard A,Susini C,Esteve JP,Bascands JL,andGirolami JP. A novel protein-protein interaction between a G-protein coupled receptor and phosphatase SHP-2 is involved in bradykinin-induced inhibition of cell proliferation. J Biol Chem 277:40375-40383,2002 .# l8 g1 e/ D2 Q( O+ z
+ [/ Q+ G4 `! o

20. El-Dahr, SS,Dipp S,andBaricos WH. Bradykinin stimulates the Erk-Elk-1-Fos/AP-1 pathway in mesangial cells. Am J Physiol Renal Physiol 275:F343-F352,1998 .
5 R7 `2 a+ X" P' {! ^9 V. P

1 L  x) `3 {: B
21. Feld, SM,Hirschberg R,Artishevsky A,Nast C,andAdler SG. Insulin-like growth I induces mesangial proliferation and increases mRNA and secretion of collagen. Kidney Int 48:45-51,1995  .
& P- E1 I2 K) i/ f) d; y
* D5 b8 `8 r7 L$ Y6 M+ [3 N7 V# d
  B3 Z- G1 L5 k+ v% f
22. Fenoy, FJ,Scicli G,Carretero O,andRoman RJ. Effect of an angiotensin II and a kinin receptor antagonist on the renal hemodynamic response to captopril. Hypertension 17:1038-1044,1991 .
5 F! x  R4 o3 j4 x( `9 g& e

6 D; q8 _8 k3 {( m3 E
23. Flyvberg, A. Putative pathophysiological role of growth factors and cytokines in experimental diabetic kidney disease. Diabetologia 43:1205-1233,2000  .3 A/ U  k8 V4 V9 X- }0 T" d  g
7 T; D: e: @: T4 h, R- n  J
7 Y! U6 |0 M# |) z
0 L' w8 u  l2 o4 `9 l' ]: ?+ h
24. Gilbert, RE,Cox A,Wu LL,Allen TJ,Hulthen UL,Jerums G,andCooper ME. Expression of transforming growth factor- 1 and type IV collagen in the renal tubulointerstitium in experimental diabetes. Effects of ACE inhibition. Diabetes 47:414-422,1998 .

2 U! F7 @0 N( @+ B
8 \' }& D' I# ?- Y+ A3 Q2 [
25. Graness, A,Adomeit A,Heinze R,Wetzker R,andLiebmann C. A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a G q/11 protein, phosphatidylinositol 3-kinase, and protein kinase C. J Biol Chem 273:32016-32022,1998 .; }3 u% g5 K* ]
5 G+ e& V! j: v9 e8 {

26. Graness, A,Hanke S,Boehmer FD,Presek P,andLiebmann C. Protein-tyrosine-phosphatase-mediated transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells. Biochem J 347:441-447,2000  .
9 `7 P$ W# M2 s" |* e* r

27. Ha, H,andKim KH. Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C. Diabetes Res Clin Pract 45:147-151,1999  .
. j" x! H' i1 c' g2 g, `
6 U: z+ }& K* z+ N4 X0 v+ A# \

28. Ha, H,andLee HB. Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Kidney Int 58:S19-S25,2000.
  s1 U/ N( l8 z. [% _+ p" [

29. Hajinazarian, M,Cosio FG,Nahman NS,andMahan JD. Angiotensin-converting enzyme inhibition partially prevents diabetic organomegaly. Am J Kidney Dis 23:105-117,1994  .4 O1 @7 k" s$ ^( c0 ~
: w7 d1 g* \* ]4 T" B
- W8 F- s+ `' m9 r+ ]* w, W0 J
" x, E; h$ T7 [+ E
30. Hamaguchi, A,Kim S,Izumi Y,andIwao H. Chronic activation of glomerular mitogen-activated protein kinases in Dahl salt-sensitive rats. J Am Soc Nephrol 11:39-46,2000 .

31. Haneda, M,Araki S,Togawa M,Sugimoto T,Isono M,andKikkawa R. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes 46:847-853,1997 ./ r2 M& q1 d$ U6 q

+ |& ^3 c. h8 I7 L. C2 l# n* \

32. Hänsch, GM,Wagner C,Bürger A,Dong W,Staehler G,andStoeck M. Matrix protein synthesis by glomerular mesangial cells in culture: effects of transforming growth factor (TGF ) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA. J Cell Physiol 163:451-457,1995  .
5 w* z, W+ h/ X! X5 O% Z( m
' y; i+ W2 \6 v

33. Hayashida, T,Poncelet AC,Hubchak SC,andSchnaper HW. TGF- 1 activates MAP kinase in human mesangial cells: a possible role in collagen expression. Kidney Int 56:1710-1720,1999  .4 G  j2 q, h* i/ ?
5 N* z9 C# S# g' w

( ~% s- y3 y% L+ w/ n8 F8 L
34. Heilig, CW,Liu Y,England RL,Freytag SO,Gilbert JD,Heilig KO,Zhu M,Concepcion LA,andBrosius FC, III. D -Glucose stimulates mesangial cell GLUT1 expression and basal and IGF-1-sensitive glucose uptake in rat mesangial cells: implications for diabetic nephropathy. Diabetes 45:1030-1039,1997., e" A  v, _. M0 @/ J) o

, ]3 Z- X0 ~7 ]0 F$ s
9 r4 u9 }& y) S6 w" {4 G  `2 \
34a. Huang, W,Gallois Y,Bouby N,Bruneval P,Heudes D,Belair MF,Krege JH,Meneton P,Marre M,Smithies O,andAlhenc-Gelas F. Genetically increased angiotensin I-converting enzyme level and renal complications in the diabetic mouse. Proc Nat Acad Sci USA 98:13330-13334,2001 .6 p4 o) e( ^4 `7 j$ ?$ S4 r
+ _$ i) w6 b$ [) [+ u+ |

- b$ i4 {$ Q& ]. d# V6 x
35. Isono, M,Iglesias-De La Cruz MC,Chen S,Hong SW,andZiyadeh SN. Extracellular signal-regulated kinase mediates stimulation of TGF- 1 and matrix by high glucose in mesangial cells. J Am Soc Nephrol 11:2222-2230,2000 .
( g) _1 e1 r' Z" _0 g$ x

36. Johnson, R,Ida H,Yoshimura A,Floege J,andBowen-Pope DF. Platelet-derived growth factor: a potentially important cytokine in glomerular disease. Kidney Int 41:590-594,1992  .
  I3 N# ?' {4 M3 v1 M+ H8 p% A( M

37. Keane, F. Metabolic pathogenesis of cardiovascular disease. Am J Kidney Dis 38:1372-1375,2001  .; M, a/ o) m# q4 {
, r* j) E/ m3 {& u7 m: e
, ~: ^& t0 [5 J9 T0 I: L% ^( D
) {0 u# U+ k- z
38. Lewis, EJ,Hunsicker LG,Bain RP,andRohde RD. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N Engl J Med 329:1456-1462,1993 .6 S6 K7 i7 R* m  x; p
6 \$ W; Y, s" [- ^( P

39. Mattson, DL,andRoman RJ. Role of kinins and angiotensin in the renal hemodynamic response to captopril. Am J Physiol Renal Fluid Electrolyte Physiol 260:F670-F679,1991 .

3 B/ T- h5 a  Y/ r' g
% o- k- X! n# v' Y3 s/ w6 z# r
40. Mukai, H,Fitzgibbon WR,Ploth DW,andMargolius HS. Effect of chronic bradykinin B 2 receptor blockade on blood pressure of conscious Dahl salt-resistant rats. Br J Pharmacol 124:197-205,1998  .

. Q: D' Q) P0 H1 A; p, s: K0 f

41. Nakamura, T,Fukui M,Ebihara I,Osada S,andNagaoka I. mRNA expression of growth factors in glomeruli from diabetic rats. Diabetes 42:450-456,1993 .$ `7 f6 a& x1 |1 e8 E3 F
$ Y7 j# P9 N4 s  S) U: F

8 ?  q# b9 u! d% ~0 p. B
42. Olcese, L,Lang P,Vely F,Cambiaggi A,Marguet D,Blery M,Hippen KL,Biassoni R,Moretta A,Moretta L,Cambier JC,andVivier E. Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases. J Immunol 156:4531-4534,1996 .

; m. i5 ?8 m$ E4 v. S

43. Patel, KI,andSchrey MP. Inhibition of DNA synthesis and growth in human breast stromal cells by bradykinin: evidence for independent roles of B 1 and B 2 receptors in the respective control of cell growth and phospholipid hydrolysis. Cancer Res 52:334-340,1992 .* Z$ z3 R- ]) ~! \8 g

' P& r/ x  l" p9 M

44. Shankland, SJ,Pippin J,Flanagan M,Coats SR,Nangaku M,Gordon KL,Roberts JM,Couser WG,andJohnson RJ. Mesangial cell proliferation mediated by PDGF and bFGF is determined by levels of the cyclin kinase inhibitor p27 Kip1. Kidney Int 51:1088-1099,1997  .
% V" ~; d8 t; v

45. Smithies, O,Kim HS,Takahashi N,andEdgell MH. Importance of quantitative genetic variations in the etiology of hypertension. Kidney Int 58:2265-2280,2000  .
% L, e" }4 }& r* e; }( H
7 T7 I: e  s3 I' p9 L

46. Strutz, F,Zeisberg M,Hemmerlein B,Sattler B,Hummel K,Becker V,andMüller GA. Basic fibroblast growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast proliferation. Kidney Int 57:1521-1538,2000  .! ?) L' V8 L, C. K) ^! t
3 c. `4 \/ @# l; {. P; r. r, c0 l

5 }* F" f& U: q) ~& l6 d% I2 V
47. Suzuki, D,Miyata T,Saotome N,Horie K,Inagi R,Yasuda Y,Uchida K,Izuhara Y,Yagame M,Sakai H,andKurokawa K. Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions. J Am Soc Nephrol 10:822-832,1999 .

* _0 v2 ^: v8 L
5 e- E) W. N% Y* q6 r
48. Tschöpe, C,Reinecke A,Seidl U,Yu M,Gavriluk V,Riester U,Gohlke P,Graf K,Bader M,Hilgenfeldt U,Pesquero JB,Ritz E,andUnger T. Functional, biochemical, and molecular investigation of renal kallikrein-kinin system in diabetic rats. Am J Physiol Heart Circ Physiol 277:H2333-H2340,1999 ./ U, o* {1 V5 ]1 t5 |; U
& ~  @6 e" _6 B8 o3 K
) r8 e7 s6 ^( I* _2 h9 N

49. Tsuchida, S,Miyazaki Y,Matsusaka T,Hunley TE,Inagami T,Fogo A,andIchakawa I. Potent antihypertrophic effect of the bradykinin B 2 receptor system on the renal vasculature. Kidney Int 56:509-516,1999  .
/ u. M( D( [: C- K% L

: m, J, `5 s, _5 u) P8 w
50. Velarde, V,Ullian ME,Morinelli TA,Mayfield RK,andJaffa AA. Mechanism of MAPK activation by bradykinin in vascular smooth muscle cells. Am J Physiol Cell Physiol 277:C253-C261,1999 .8 H0 W! h0 |; q' o9 g; _1 N
+ i" `; T+ s: Q4 U

$ [+ c( q$ Y1 h/ w, ~1 l
52. Wenzel, UO,Fouqueray B,Biswas P,Grandaliano G,Choudhury GG,andAbboud HE. Activation of mesangial cells by the phosphatase inhibitor vanadate. J Clin Invest 95:1244-1252,1995  .- o2 |" E2 t* \) h" z

53. Werner, H,Shen-Orr Z,Stannard B,Burguera B,Roberts CT,andLeroith D. Experimental diabetes increases insulin like growth factor I and II receptor concentration and gene expression in kidney. Diabetes 39:1490-1497,1990 .# k0 W- R+ R7 N1 ^# Z: H* @; [9 b( Z
, Z2 C4 {9 L5 \9 X0 d
# J$ d4 m; E" r

54. Wolf, G,andZiyadeh FN. Molecular mechanisms of diabetic renal hypertrophy. Kidney Int 56:393-405,1999  .
9 g5 K$ z$ [4 F2 m, D7 p
2 V$ h3 p  g! C; _9 k2 r0 p
- d# M: _" ~; D
55. Yavuz, DG,Ersoz O,Kucukkaya B,Budak Y,Ahiskali R,Ekicioglu G,Emerk K,andAkalin S. The effect of losartan and captopril on glomerular basement membrane anionic charge in a diabetic rat model. J Hypertens 17:1217-1223,1999  .
8 M7 U7 {/ g. ~# S" \) B" t

56. Young, BA,Johnson RJ,Alpers CA,Eng E,Gordon K,Floege J,andCouser WG. Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int 47:935-944,1995  .

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