Amino acids induce indicators of response to injury in glomerular mesangial cell - 《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 13:38 |只看该作者 |倒序浏览 |打印

作者：Rick L. Meek, Sheryl K. Cooney, Stephanie D. Flynn, Robert F. Chouinard, Maria H. Poczatek, Joanne E. Murphy-Ullrich,  Katherine R. Tuttle作者单位：1 Research Department, The Heart Institute of Spokane, Spokane, Washington 99204; and Division of Molecular and Cellular Pathology, Department of Pathology, and Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 , X# w9 y1 U6 S% S* t2 `: a
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      【摘要】
   High-protein diets exacerbate glomerular hyperfiltration and the progression of diabetic nephropathy. The purpose of this study was to determine whether amino acids also produce nonhemodynamic injury in the glomerulus. When rat mesangial cells were cultured with an amino acid mixture designed to replicate the composition in plasma after protein feeding, production of mRNA (Northern blot analysis) and/or protein (ELISA or Western blot analysis) for transforming growth factor- 1 (TGF- 1 ), fibronectin, thrombospondin-1 (TSP-1), and collagen IV were enhanced in a manner comparable to a culture with high glucose (30.5 mM). The bioactive portion of total TGF- (NRK assay) increased in response to amino acids. The TSP-1 antagonist LSKL peptide reduced bioactive TGF- and fibronectin, indicating the dependence of TGF- 1 activation on TSP-1. DNA synthesis ([ 3 H]thymidine incorporation), an index of cellular proliferation, increased in response to amino acids and was further enhanced by culture with increased levels of both amino acids and glucose. TGF- 1 and matrix proteins increased when mesangial cells were cultured with excess L -arginine (2.08 mM) alone. Although L -arginine is the precursor of nitric oxide (NO), such responses to amino acids do not appear to be mediated through increased NO production. NO metabolites decreased in the media, and these responses to mixed amino acids or L -arginine were not prevented by NO synthase inhibition. In conclusion, amino acids induce indicators of response to injury in mesangial cells, even when hemodynamic stress is absent. In conditions associated with increased circulating amino acids, such as diabetes and/or a high-protein diet, direct cellular effects could contribute to glomerular injury. ( c4 ?9 I1 a4 W8 z* A4 ^- N
      【关键词】 transforming growth factor thrombospondin extracellular matrix proteins mesangial cell proliferation hyperglycemia- A0 F2 D# `6 `4 M+ j" g
   HIGH - PROTEIN DIETS, RESULTING in increased circulating amino acids, produce glomerular hyperfiltration and hypertension, major mechanisms of progressive renal injury ( 5, 47 ). In the clinical setting of diabetes, this is a particular concern because high-protein diets have been promoted to modulate hyperglycemia or to promote weight loss. Furthermore, poor glycemic control also increases plasma amino acid concentrations ( 10 ). As such, hyperaminoacidemia is common in diabetes and could exacerbate renal injury. The glomerular hyperfiltration associated with increased circulating amino acids is similar to that induced by hyperglycemia ( 5, 47 ). In clinical studies, we have demonstrated that people with poorly controlled type 1 or type 2 diabetes have an augmented glomerular hyperfiltration response to amino acids, which can be corrected by strict glycemic control ( 41 - 43 ). The mechanism(s) of the interaction between hyperglycemia and amino acids is unknown. Hormonal mediators have been proposed, but none has been identified ( 43 ). Whether amino acids have direct cellular effects that enhance or cause injury is unexplored." y1 f6 e" y3 j9 g
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The mesangial cell is a key cell involved in the control of glomerular hemodynamics, as well as in the response to injury. Recognized causes of injury, such as hyperglycemia, have both hemodynamic and direct cellular effects. In vitro models utilizing mesangial cell culture have shown that cellular stretch (a model of glomerular hypertension) or high-glucose media (a model of hyperglycemia) induce production of transforming growth factor- (TGF- ) ( 19, 31, 48 ). TGF- is a profibrotic cytokine that is a seminal cellular mediator of response to injury ( 36 ). TGF- is secreted in an inactive form, which can be activated by diverse mechanisms, including interactions with the matrix protein thrombospondin-1 (TSP-1) ( 20 ). In mesangial cells cultured with high-glucose media, TSP-1 expression is also elevated and enhances TGF- bioactivity ( 28, 39 ). The potential influence of amino acids on these processes has not been evaluated.
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Either a mixture or certain individual amino acids could have renal effects. L -Arginine is a plausible candidate because it is the precursor of nitric oxide (NO). NO has many important functions in the kidney, including promoting vasodilation, which may enhance glomerular hyperfiltration, as well as direct cellular effects, which may be protective or injurious. Depending on the experimental system, increased NO has been variably reported to either decrease or increase TGF- production and fibrosis ( 9, 21, 37 ). In mesangial cells cultured with a high-glucose concentration, stimulation of NO production through endogenous (interleukin-1 ) or exogenous (NO donor) methods prevented TGF- induction ( 9 ). Furthermore, suppression of NO by hyperglycemia has been reported to increase TGF- activation and mesangial matrix production ( 40, 44 ). In mesangial cells, it is unclear to what extent L -arginine functions as an NO precursor without cytokine stimulation of inducible NO synthase, and possible relationships among amino acids, NO, and TGF- have not been studied.
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The overall goal of this study was to determine whether amino acids, in the absence of hemodynamic stress, produce mesangial cell responses associated with the type of glomerular injury observed in diabetes. A mixture of amino acids, designed to replicate the composition in plasma after protein feeding, was evaluated for effects on expression of TGF- 1 and matrix proteins as well as on mesangial cell proliferation and viability (other indicators of response to injury). These effects were explored with and without a concomitant high-glucose concentration. Whether TSP-1 influences amino acid-induced TGF- bioactivity was assessed by use of an inhibitory peptide. The effects of L -arginine on NO and expression of TGF- 1 and matrix proteins were also determined.6 b% x# _( n; U0 T# v7 F6 {% n
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METHODS) ]* ^1 l* @& U4 v2 X

Mesangial Cell Isolation and Culture

Rat mesangial cells were recovered from glomeruli isolated by sieving the cortex of kidneys excised from 6-mo-old female Sprague-Dawley rats. Mesangial cells were grown in DMEM (Life Technologies, Gaithersburg, MD), supplemented with penicillin-streptomycin (100 U/ml) and 10% heat-inactivated fetal bovine serum (Summit Technologies, Ft. Collins, CO) in a humidified atmosphere of 5% CO 2 at 37°C. Cells were passaged at confluence using 0.025% trypsin (Life Technologies) in PBS and used for experiments between passages 4-6 and 14-19.; p* X# v4 o- P' @# I
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Experimental Conditions
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Increased levels of amino acids and glucose. Mesangial cells were seeded into 100-mm dishes or 24-well plates (Nunclon, Cambridge, MA) at 10,000 cells/cm 2. When cells reached confluence, they were made quiescent in serum-free DMEM for 48 h. Cells were then exposed to experimental conditions for 48 h ( Table 1 ): 1 ) control, serum-free DMEM; 2 ) increased amino acids, serum-free DMEM supplemented with 10% Travasol mixed amino acid solution (Baxter, Deerfield, IL) and L -arginine; addition of Travasol and L -arginine, which increased individual amino acid concentrations 1.5- to 6-fold and raised osmolarity 13.3 mosM, relative changes comparable to those observed in plasma after a protein meal ( 3 ); 3 ) high-glucose, serum-free DMEM containing 30.5 mM glucose; 4 ) a combination of increased amino acids and high glucose; and 5 ) addition of mannitol to raise osmolarity 13.3 mosM.
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Table 1. Amino acid and glucose concentrations in cell culture media for experimental groups5 c8 K5 N5 z) C
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Increased levels of amino acids with TSP-1 inhibitory peptide. In separate experiments, LSKL peptide (AnaSpec, San Jose, CA), a selective antagonist of TSP-1, and SLLK peptide (AnaSpec), an inert control, were used to evaluate effects of TSP-1 on TGF- bioactivity ( 30 ). Confluent mesangial cells were cultured with serum-free DMEM for 48 h. Cells were then exposed to the following conditions for 48 h: 1 ) control, serum-free DMEM; 2 ) control plus LSKL peptide (5 µM); 3 ) control plus SLLK peptide (5 µM); 4 ) increased amino acids ( Table 1 ); 5 ) increased amino acids plus LSKL peptide (5 µM); and 6 ) increased amino acids plus SLLK peptide (5 µM). The conditioned media were removed and 2 µg/ml each of the protease inhibitors pepstatin, leupeptin, antipain, and aprotinin (Sigma, St. Louis, MO) were added. The media were then stored at -70°C until assayed for TGF- bioactivity and fibronectin protein.
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Increased level of L -arginine and NO synthase inhibition. Confluent mesangial cells were made quiescent in serum-free DMEM for 48 h. Cells were then placed in experimental conditions for 48 h ( Table 1 ): 1 ) control, serum-free DMEM; 2 ) increased amino acids; and 3 ) L -arginine (2.08 mM). Each of these conditions was also prepared with the addition of the NO synthase inhibitor N G -monomethyl- L -arginine ( L -NMMA; 1 mM) to examine whether NO synthesis contributed to the experimental outcomes.
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Outcome Measurements

mRNA. Total RNA was isolated from mesangial cell monolayers utilizing Tri Reagent (Molecular Research Center, Cincinnati, OH) and quantified by UV spectrophotometry (Beckman DU 660, Beckman Coulter, Fullerton, CA).

Probes specific for rat sequences published in the GenBank database were obtained by RT-PCR. TGF- 1 cDNA is from nucleotides 818-1315 of sequence X52498 . Fibronectin cDNA is from nucleotides 5372-5636 of sequence X15906  (EIIIa splice variant associated with TGF- bioactivity). 1 (IV) Collagen is from nucleotides 11-421 of sequence AA924749 . TSP-1 cDNA has been previously described ( 15 ). GAPDH is from nucleotides 469-987 of sequences X02231  and X00972. The cDNAs were radiolabeled with [ 32 P]dCTP (Oligolabeling or Ready-to-Go kit, Amersham Pharmacia, Piscataway, NJ).

RNA (5 µg) was denatured, separated by formaldehyde-agarose gel electrophoresis, transferred to charged nylon membranes (Schleicher and Schuell, Keene, NH), and baked at 80°C. Blots were hybridized in Quickhyb (Stratagene, La Jolla, CA) using 32 P-labeled cDNA at 2 x 10 6 cpm/ml (with cpm being counts/min) for 2 h at 68°C and washed at 60°C with 0.1 x SSC (15 mM NaCl, 1.5 mM sodium citrate) containing 0.1% SDS. Most of the Northern blots were quantified from digital images obtained with the Cyclone Storage Phosphor System and Optiquant Image Analysis Software (Packard Instruments, Meriden, CT). In several early experiments, Northern blots were exposed to Kodak Biomax MS-1 film (Eastman Kodak, Rochester, NY) for multiple timed exposures. Digital images were obtained with a Duoscan 1200 scanner (AGFA, Wilmington, MA) and analyzed with Kodak 1D Image Analysis Software (Eastman Kodak). Both techniques for quantifying Northern blots yielded comparable results. Quantities of TGF- 1, fibronectin, 1 (IV) collagen, and TSP-1 mRNA were expressed as the ratio to GAPDH mRNA.

Proteins. Cell culture media were collected at the end of the experimental periods. Protease inhibitors were added, and media were frozen at -70°C. TGF- 1 protein was quantified by ELISA using a Duoset kit (R&D Systems, Minneapolis, MN), and total TGF- protein was measured by bioassay (see TGF- bioactivity ). Fibronectin was measured by competition ELISA using rat plasma fibronectin and anti-rat fibronectin antibodies (Chemicon International, Temecula, CA). Standards or unknown samples (100 µl) were incubated with an equal volume of 1:2,000 dilution of rabbit anti-fibronectin antibody in PBS blocking buffer with 3% gelatin (Sigma) for 2 h. The antibody/antigen mixture (100 µl) was transferred to 96-well plates (Rainin Instruments, Oakland, CA), which had been coated overnight with fibronectin (100 ng in 100 µl of 0.5 M NaHCO 3, pH 9.5). The plates were incubated for 2 h, rinsed, and incubated with a 1:20,000 dilution of biotinylated anti-rabbit antibody (Sigma) for 2 h. After the plates were rinsed, they were incubated with a 1:30,000 dilution of streptavidin-horseradish peroxidase (HRP; Rockland Immunochemicals, Gilbertsville, PA) in PBS. HRP was detected with a substrate reagent (R&D Systems). Total media protein was measured with a Protein Assay Kit (Bio-Rad, Hercules, CA). Absorbance for the ELISA and total protein assays was measured with a Bio-Tek FL-600 plate reader (Bio-Tek Instruments, Winooski, VT). TGF- 1 (pg/µg) and fibronectin (ng/µg) were quantified as ratios to total media protein.
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TSP-1 was measured by Western blot analysis using mouse monoclonal anti-human TSP-1 (antibody 133, cross-reactive to rat TSP-1) ( 33 ). Conditioned media were electrophoresed through denaturing SDS-PAGE (4-20% gradient) gels. Separated proteins were electrophoretically transferred to nitrocellulose membrane, blocked (5% nonfat dry milk, 0.9% NaCl, 10 mM Tris · HCl, pH 7.4), and incubated with anti-human TSP-1 antibody (1:20,000) for 1 h. After the blots were washed, they were incubated with HRP-conjugated secondary antibody (1:20,000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h and detected by enhanced chemiluminescence (Pierce, Rockford, IL). Digitized images of exposed X-ray films were analyzed using Optiquant software (Packard Instruments). TSP-1 was quantified as the ratio to total media protein (density units/µg).
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Collagen IV protein was not measured because an antibody capable of recognizing the rat protein was not available. Multiple antibodies to human or mouse collagen IV from commercial sources were tested, but none had cross-reactivity with rat collagen IV in our ELISA and Western blot systems.  i* t0 R. ~1 x# R
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TGF- bioactivity. Both total and bioactive TGF- in conditioned media were measured by colony formation of normal rat kidney cells (NRK-49F, American Type Culture Collection, Manassas, VA) in soft agar assay, as previously described ( 33 ). Baseline colony formation was measured in wells incubated with 2.5 ng of epidermal growth factor in fresh media.4 N& v* d+ @( d- `* p! U

Mesangial cell proliferation. Incorporation of [ 3 H]thymidine was used to measure DNA synthesis as an index of mesangial cell proliferation. Cells were grown in 24-well plates and exposed to 4 µCi [ 3 H]thymidine/ml of media (NEN Life Sciences, Boston, MA) for the last 4-8 h of treatment. Cells were washed three times and incubated with ice-cold 10% trichloroacetic acid for 15 min. Fixed cell layers were solubilized with 0.4 M NaOH at 60°C for 10 min and neutralized with 0.2 M glacial acetic acid. Each 100-µl sample was diluted in 0.8 M NaOH and vacuum blotted onto a nylon membrane (Immobilon NY  , Millipore, Bedford, MA), air-dried, and exposed to a 3 H-sensitive phosphor screen. Data were collected from the screen with the Cyclone Phosphor Storage system and analyzed using Optiquant software (Packard Instruments). Total cellular protein was measured with a DC Protein Assay Kit (Bio-Rad). Absorbance was measured on a Bio-Tek FL-600 plate reader (Bio-Tek Instruments). Data were reported in digital light units per square millimeter per microgram of cell protein.

NO metabolites. As an index of NO in conditioned media, nitrite and nitrate were measured using a fluorometric NO assay kit (Calbiochem, San Diego, CA). Fluorescence was determined with a Bio-Tek Fl-600 fluorescence plate reader (Bio-Tek Instruments) using an excitation wavelength of 365 nm and an emission wavelength of 460 nm. Nitrate/nitrite were expressed as the ratio to total media protein (nmol/µg).% l- Z, y# ~: F% u: c  U, r
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Cell viability. Cell viability was assessed using a In Vitro Toxicology Assay Kit (Sigma) for lactate dehydrogenase (LDH). Cells were grown in 24-well plates and treated as previously described. Conditioned media were collected at the end of experimental periods, and the remaining cell layer was lysed with kit reagent. LDH values obtained from the media and the lysed cell layer were combined for total LDH. Media LDH was expressed as a percentage of total LDH.
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Statistics
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Data are expressed as means ± SE. One-way ANOVAs were used to evaluate the various outcome measurements in response to experimental conditions. Two-way ANOVAs were used to evaluate differences between the group with increased amino acids and control and TSP-1 peptide groups for total and bioactive TGF-. Specific tests between conditions were conducted using a priori contrasts. Statistics were computed using SPSS, version 10 (SPSS, Chicago, IL). Probabilities P statistically significant.

RESULTS
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TGF- 1 and Matrix Proteins in Response to Increased Amino Acids, High-Glucose, or the Combination Condition

Increased amino acids enhanced expression of all genes in a manner similar to high glucose ( Fig. 1 ). The combination condition of increased amino acids and high glucose increased mRNA for TGF- 1, 1 (IV) collagen, and TSP-1. A similar trend was noted for fibronectin, but the change from control did not reach statistical significance ( P = 0.06). The effects of the combination condition were not significantly different from either elevated levels of glucose or amino acids alone. Mannitol treatment, designed to produce levels of osmolarity similar to experimental conditions, did not increase mRNA for the various proteins (data not shown). TGF- 1 (ELISA), fibronectin, and TSP-1 proteins increased in parallel with the mRNA results ( Table 2 ).
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Fig. 1. Increased amino acid, high-glucose, and combination conditions elevate mesangial cell mRNA for transforming growth factor- 1 and matrix proteins. A : representative Northern blot analysis. Experimental groups were the following: lane 1, control; lane 2, increased amino acids; lane 3, high glucose; and lane 4, combination. B : quantitative measurements of Northern blot analysis ( n = 8-11). Values are means ± SE. * P P  Y2 ], {3 x* e$ M
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Table 2. Transforming growth factor- 1 and matrix proteins in response to experimental conditions
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TSP-1-Induced Activation of TGF- by Increased Amino Acids) [2 J3 g9 K. i( K! L

Both total and bioactive TGF- (NRK assay) were elevated in the conditioned media of the increased amino acids group ( Fig. 2, A and B ). Total TGF- increased 1.88 ± 0.34-fold ( P = 0.006), whereas bioactive TGF- increased 2.68 ± 0.21-fold ( P was proportionally greater in the group that received increased amino acids compared with control [48.9 ± 6.3 vs. 30.5 ± 1.7% ( P = 0.021)]. A parallel increase in media fibronectin protein was observed [4.77 ± 0.30 and 2.67 ± 0.20 ng/µg ( P and control groups, respectively. The TSP-1 antagonist LSKL peptide decreased the increment in bioactive TGF- without changing total TGF- in the increased amino acids group ( Fig. 2, A and B ). Therefore, the proportion of bioactive TGF- in the LSKL group was reduced compared with the group that received only increased amino acids [38.7 ± 5.5% ( P = 0.014)]. Fibronectin protein was similarly reduced in response to LSKL peptide [2.80 ± 0.20 ng/µg ( P = 0.001)]. The inert SLLK peptide did not decrease total or bioactive TGF- ( Fig. 2, A and B ) or fibronectin protein [3.92 ± 0.29 ng/µg ( P = 0.115 compared with increased amino acids group)] in conditioned media.7 _5 C/ s' G' y. d' k3 S; ]

Fig. 2. Effects of increased amino acids and thrombospondin-1 peptides (antagonist LSKL and inert SLLK) on total and bioactive transforming growth factor- in mesangial cells. Conditioned media were assayed (NRK bioassay) for total transforming growth factor- ( A ) and bioactive transforming growth factor- ( n = 10; B ). Values are means ± SE. * P P  x3 L/ U: n& K' v

TGF- 1 and Matrix Proteins in Response to Increased L -Arginine and NO Synthase Inhibition0 A% z# p6 Y. ?3 y! A: N6 K, k/ h0 y

Increased L -arginine raised mRNA and protein levels for TGF- 1 (ELISA), fibronectin, and TSP-1, and mRNA for 1 (IV) collagen in a manner comparable to the amino acid mixture group ( Table 3 ). Both conditions also resulted in decreased NO metabolites in the conditioned media: 160 ± 18 vs. 125 ± 10 and 101 ± 10 nmol/µg ( P = 0.015 and P = 0.025) in control, amino acid mixture, and L -arginine groups, respectively. Addition of the NO synthase inhibitor L -NMMA did not consistently influence TGF- 1 or matrix proteins in either the amino acid mixture or L -arginine condition ( Table 3 ). When the amino acid mixture group also received L -NMMA, mRNA and protein for TGF- 1 and fibronectin and 1 (IV) collagen mRNA remained above control levels. For TSP-1, the protein level was greater than control, but the mRNA level was not. None of these values was significantly different from those in the amino acid mixture group that did not receive NO synthase inhibition. When L -NMMA was added to the L -arginine group, TGF- 1 and fibronectin proteins were greater than control, but mRNA levels were not. To the contrary, TSP-1 and 1 (IV) collagen mRNA remained greater than control. Although TSP-1 protein was numerically larger than control, it was not statistically significant ( P = 0.074). None of these was significantly different from the respective values in the group treated with only L -arginine. In summary, no overall pattern of response to support a role for increased NO production was observed.

Table 3. Effects of the amino acid mixture or L-arginine alone, with or without nitric oxide synthase inhibition, on TGF- 1 and matrix proteins) G+ W+ s8 r( m
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Effects of Amino Acids on Mesangial Cell Proliferation and Viability
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The culture of mesangial cells with increased amino acids raised [ 3 H]thymidine incorporation in a manner comparable to that induced by high glucose ( Fig. 3 ). The combination condition increased [ 3 H]thymidine incorporation more than either condition alone. The mixture of amino acids did not adversely affect mesangial cell viability, as determined by LDH release into the media [11 ± 4 vs. 6 ± 1% in control ( P = 0.142)]. However, the high-glucose and combination conditions did increase media LDH compared with control [23 ± 2 and 24 ± 5%, respectively ( P vs. control)]., |* `" H+ k, F2 B( n

Fig. 3. Increased amino acids, high-glucose, and combination conditions enhance mesangial cell [ 3 H]thymidine incorporation ( n = 7). Values are means ± SE. * P P P

DISCUSSION

The present data demonstrate that amino acids have adverse effects beyond the renal hemodynamic changes long associated with excess protein in the diet. In glomerular mesangial cells, production and activation of TGF- and expression of matrix proteins (fibronectin, collagen IV, and TSP-1) occurred in response to increased amounts of amino acids designed to replicate those in plasma after protein feeding. The effects of increased amino acids were similar in magnitude to those induced by the high-glucose condition. L -Arginine alone enhanced expression of TGF- 1 and matrix proteins to the same extent as the amino acid mixture and high-glucose conditions. Even though L -arginine is an NO precursor, NO metabolites decreased in the conditioned media of both the L -arginine alone and amino acid mixture groups. In addition, increased levels of amino acids augmented DNA synthesis as much as observed in response to a high-glucose level and, with the combination condition, this index of cellular proliferation was even more pronounced.1 F3 h" J9 u) X/ L, T4 f" \0 w

High-protein diets exacerbate progressive renal disease, particularly in diabetes ( 5, 47 ). Conversely, diets low in protein reduce the progression and, most importantly, decrease the risk of end-stage renal disease and death in diabetic nephropathy ( 14, 24, 47 ). Furthermore, when combined with renin-angiotensin system blockade, a low-protein diet has the additive effect of reducing proteinuria ( 11, 26, 32 ). These reports and others indicate that dietary protein has effects other than those related to glomerular hypertension or renin-angiotensin system activation, but the mechanisms are still unclear ( 11, 23, 26, 32 ). Our data provide new evidence that amino acids have direct cellular effects associated with glomerular injury. However, we cannot exclude involvement of the renin-angiotensin system in the responses of mesangial cells to amino acids. Mesangial cells can produce angiotensin II, especially when stretched or cultured with an increased amount of glucose ( 4, 38 ). In addition, in cultured mesangial cells, we recently found that increased amino acids approximately doubled the amount of mRNA for the angiotensin II type 1 receptor and reduced expression of aminopeptidase A, a metalloprotease that degrades angiotensin II ( 8 ). These responses could potentiate effects of angiotensin II, including stimulation of TGF- production and mesangial cell proliferation ( 2, 18, 29 ).$ E! l" W& e0 Z: r+ s% }

Increased mesangial cell DNA synthesis in response to amino acids is another new finding in this study. However, unlike the high-glucose condition, the amino acid mixture did not adversely affect mesangial cell viability, nor did it worsen the reduced cell viability associated with high glucose. In addition to mesangial matrix expansion, cell growth is a common feature of diabetic nephropathy ( 45 ). The augmented DNA synthesis in response to the combination condition suggests that, when high circulating levels of glucose and amino acids coexist, a common occurrence in diabetes, mesangial cell proliferation could be exacerbated in an additive fashion. In a recent preliminary report, similar findings were observed for the single amino acid glutamine, alone or in combination with a high-glucose level ( 35 ). In our experimental system, glutamine was not further increased by the addition of mixed amino acids because it is not present in the standard solution for hyperalimentation (Travasol). This solution was chosen to produce an in vitro condition similar to clinical hyperaminoacidemia. Nevertheless, even without further increasing glutamine, increases in other amino acids augmented mesangial cell DNA synthesis. These data suggest that common features among amino acids could lead to this consequence.

TGF- is secreted in an inactive form due to a noncovalent association with the latency-associated peptide. Although the mechanism of activation is not fully understood, TSP-1 can interact with the latency-associated peptide to liberate active TGF- ( 20, 30, 33 ). Either short- or long-term exposure of mesangial cells to a high-glucose level increases TGF- bioactivity by such a TSP-1-dependent mechanism ( 28, 46 ). In the present study, TSP-1 induced by amino acids also activated TGF- and enhanced production of the matrix protein fibronectin. Of particular interest, amino acid-induced TGF- production and activation and matrix protein (fibronectin, collagen IV, and TSP-1) expression were nearly identical to responses produced by the high-glucose condition. However, the combination of elevated levels of amino acids and glucose did not further enhance expression of TGF- 1 or these matrix proteins. Taken together, the available data suggest that the mechanism(s) leading to amino acid- or glucose-induced responses may be redundant or shared.
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High-glucose concentrations have previously been reported to decrease NO and promote TSP-1-dependent TGF- bioactivity in mesangial cells ( 40, 44 ). Similarly, we found that NO metabolites in the conditioned media decreased when mesangial cells were cultured with either the amino acid mixture or L -arginine. This may seem paradoxical because L -arginine is the precursor for NO. In addition, production of TGF- 1 and matrix proteins increased as much in response to L -arginine as with the amino acid mixture. No clear directional effect of L -NMMA on these outcomes was observed, further supporting the contention that the mechanism(s) in this system is not likely to be dependent on increased NO production. The increase in TGF- itself could conceivably reduce production of NO by inhibiting transcription of inducible NO synthase, as has been observed in vascular smooth muscle cells stimulated by interleukin-1 ( 25 ). However, in mesangial cells, the contribution of inducible NO synthase to the production of NO is uncertain in the absence of cytokine stimulation. The quenching of NO by reactive oxygen species could also explain the decrease in NO metabolites in the conditioned media ( 6, 16 ). Indeed, glutamine has recently been reported to enhance superoxide production by neutrophils ( 27 ). Similarly, in recent experiments performed in our laboratory, we have found an increase in hydrogen peroxide, an indicator of oxidative stress, in response to the culture of mesangial cells with the same amino acid mixture used in the present study (Meek R and Tuttle K, unpublished observations). Treatment with the antioxidant vitamin E prevented the amino acid-induced increases in TGF- 1 and fibronectin proteins and 1 (IV) collagen mRNA (Meek R and Tuttle K, unpublished observations).; ?& o" F2 N/ m
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Because many effects of amino acids are remarkably similar to those of glucose, a potentially unifying hypothesis is that an excess of either constituent can lead to formation of advanced glycation end products (AGE). AGE are produced by nonenzymatic glycation of free amino groups, leading to generation of a Schiff base and Amadori products ( 22 ). The process of AGE formation is closely linked to oxidative stress, as both a cause and a consequence ( 1, 12, 22 ). Reactive oxygen species signal expression of TGF- by activating the protein kinase C pathway ( 7, 12, 13, 17, 34 ). Until recently, AGE formation was only explored in the setting of hyperglycemia. However, emerging data indicate that alternate pathways can also produce AGE. In a neutrophil cell culture system, a mixture of free amino acids with normal-glucose concentration increased carboxymethyllysine, a prominent AGE, by a hydrogen peroxide-dependent mechanism ( 1 ). Mesangial cells can produce hydrogen peroxide and may form AGE when exposed to increased levels of amino acids.* b$ t( r4 w; O: d, U

Our study has several limitations. The relative increases in amino acid levels in the cell culture media were comparable to those in plasma after a protein meal or infusion of mixed amino acids, but in vivo studies will be required to delineate more clearly the physiological relevance of our findings ( 3, 42 ). It is also uncertain whether a general increase in amino acids (e.g., availability of free amino groups for AGE formation) or specific amino acids were responsible for the mesangial cell responses. Nevertheless, our in vitro model, which was free of influences from other hormonal/metabolic disturbances or hemodynamic stress, has established that an amino acid profile typical of protein feeding produces direct effects associated with injury in mesangial cells. Future studies will be directed toward elucidating the specific mechanisms of these processes, including dose and time dependency of responses.; @2 w. o3 t- @7 T
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In conclusion, increased levels of amino acids enhanced expression of TGF- 1 and matrix proteins by glomerular mesangial cells. TGF- bioactivity was further enhanced via a mechanism dependent on TSP-1. The changes in TGF- 1 and matrix proteins were comparable to those induced by culture of mesangial cells with a high-glucose concentration. Whether mesangial cells were cultured with the amino acid mixture or L -arginine alone, TGF- 1 was induced even though NO metabolites decreased in the conditioned media. Increased amino acid levels also promoted mesangial cell DNA synthesis, and, when combined with high glucose, this index of cellular proliferation was further augmented. Emerging data suggest that either excess amino acids or glucose may lead to production of AGE and oxidative stress, which could be a mechanism for quenching NO and promoting injury via the TGF- pathway. We have demonstrated that amino acids may directly cause or enhance mesangial cell responses associated with fibrosis and progressive renal disease. Such effects could add to glomerular injury, especially in diabetes, a condition where high levels of circulating amino acids, as well as hyperglycemia, are common metabolic disturbances.2 q; B5 b9 H" {2 m: @9 k
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ACKNOWLEDGMENTS. i! r2 M: o3 ^  N8 R+ [9 U9 p- z
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The authors are grateful to Robert J. Anderberg for technical assistance, Marcia J. Hauger for graphics and word processing, and Drs. E. Carolyn Johnson and G. Dennis Clifton for constructive critiques of the manuscript.5 k: [; c* H1 h9 Z# g# c

Research support was provided by The Heart Institute of Spokane and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-54624 (J. E. Murphy-Ullrich).' @3 L  ?$ z$ d
      【参考文献】' R( N" v; G  k4 I0 C
Anderson MM, Requena JR, Crowley JR, Thorpe SR, and Heinecke JW. The myeloperoxidase system of human phagocytes generates N - -(carboxymethyl)lysine on proteins. A mechanism for producing advanced glycation end products at sites of inflammation. J Clin Invest 104: 103-113, 1999.

4 R+ o; T" D7 ^  y5 {+ c

Anderson PW, Zhang XY, Tian J, Correale JD, Xi XP, Yang D, Graf K, Law RE, and Hsueh WA. Insulin and angiotensin II are additive in stimulating TGF- 1 and matrix mRNAs in mesangial cells. Kidney Int 50: 745-753, 1996.
' H: m7 G# k+ Q1 m: ?: {& U4 _
% j6 U. y& H4 B8 E0 }
; h: @) ?2 H: `2 p' g
Aoki TT, Brennan MF, Muller WA, Soeldner SJ, Alpert JS, Saltz SB, Kaufmann RL, Tan HM, and Cahill GF. Amino acid levels across normal forearm muscle and splanchnic bed after a protein meal. Am J Clin Nutr 29: 340-350, 1976.

Becker BN, Yasuda T, Kondo S, Vaikunth S, Homma T, and Harris RC. Mechanical stretch/relaxation stimulates a cellular renin-angiotensin system in cultured rat mesangial cells. Exp Nephrol 6: 57-66, 1998.6 M3 X. ^; K$ h+ A) V

, ]5 w& [7 R2 t
  z& R: v8 E, l( Y+ V6 ~
Brenner BM, Meyer TW, and Hostetter TH. Dietary protein intake and the progressive nature of kidney disease: the role of hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation, and intrinsic renal disease. N Engl J Med 307: 652-659, 1982.
5 p3 o* C- w0 w& g( R- y- ~- J

Bucala R, Tracey KJ, and Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest 87: 432-438, 1991.7 d+ w- r- Z9 r( \7 b% \1 O8 S

5 t5 o3 V" Q4 B* Y3 x

Chen S, Cohen MP, Lautenslager GT, Shearman CW, and Ziyadeh FN. Glycated albumin stimulates TGF- 1 production and protein kinase C activity in glomerular endothelial cells. Kidney Int 59: 673-681, 2001.

& l/ D2 q4 N1 B: F3 F0 ^

Chouinard RF, Meek RL, Cooney SK, and Tuttle KR. Effects of amino acids and glucose on mesangial cell aminopeptidase A and angiotensin receptors. Kidney Int 61: S106-S109, 2002.* V) w' R& L* ^/ P; ]

) n8 g8 S3 X, h+ N- A& A
Craven PA, Studer RK, Felder J, Phillips S, and DeRubertis FR. Nitric oxide inhibition of transforming growth factor- and collagen synthesis in mesangial cells. Diabetes 46: 671-681, 1997.: M3 d. J) i$ d$ j

% ~  i% D' D0 J

Felig P, Wahren J, Sherwin R, and Palaiologos G. Amino acid and protein metabolism in diabetes mellitus. Arch Intern Med 137: 507-513, 1977.6 V) C8 m7 j  l. i2 A% P2 p
4 T4 L0 k" `+ h! o, X

$ n2 P) ^0 L% ^
Gansevoort RT, de Zeeuw D, and de Jong PE. Additive antiproteinuric effect of ACE inhibition and a low-protein diet in human renal disease. Nephrol Dial Transplant 10: 497-504, 1995.
0 x3 @+ q$ y: x
7 ^  i4 z" O  ^- y% B+ N
$ F* M% @: B. b( h7 T  s. e
Ha H and Lee HB. Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Kidney Int 77: S19-S25, 2000.
4 ~4 l" v0 p2 i  p; N( t# a

8 M  A  k" T2 N" m" d. g4 ]2 B
Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, and Kikkawa 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.+ z) R8 ]- `; `7 E

6 O8 r8 q! C$ A+ I- w9 l" O
Hansen HP, Tauber-Lassen E, Jensen BR, and Parving HH. Effect of dietary protein restriction on prognosis in patients with diabetic nephropathy. Kidney Int 62: 220-228, 2002.$ X* z$ I# Z% I. V" d+ e, m$ }* v; [

& Q% r1 U. `$ ~7 q3 [) y. v

Hugo C, Pichler R, Meek R, Gordon K, Kyriakides T, Floege J, Bornstein P, Couser WG, and Johnson RJ. Thrombospondin 1 is expressed by proliferating mesangial cells and is up-regulated by PDGF and bFGF in vivo. Kidney Int 48: 1846-1856, 1995.
" K. g8 }& z7 ~/ I) _6 o4 a  `

- H0 m' P; m- V3 r" w. m+ |& E
Ishii N, Patel KP, Lane PH, Taylor T, Bian K, Murad F, Pollock JS, and Carmines PK. Nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus. J Am Soc Nephrol 12: 1630-1639, 2001.
7 [7 h$ ~! F0 J7 H4 k+ F1 L

! ~! I7 V  U$ h) s; V& Q+ M
Isono M, Cruz MC, Chen S, Hong SW, and Ziyadeh FN. Extracellular signal-regulated kinase mediates stimulation of TGF- 1 and matrix by high glucose in mesangial cells. J Am Soc Nephrol 11: 2222-2230, 2000.1 z. ?/ v$ A, T6 a3 Y& y

Kagami S, Border WA, Miller DE, and Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 93: 2431-2437, 1994.0 j$ K' x: h% h0 H
- S+ x6 a% H+ }8 w0 a
1 H4 I4 ~; O. T" T) b; }3 @9 v

Murphy M, Godson C, Cannon S, Kato S, Mackenzie HS, Martin F, and Brady HR. Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J Biol Chem 274: 5830-5834, 1999.
$ a* f" r( ^, W% K9 \2 |
1 ?% \8 C6 B* X$ G. o

Murphy-Ullrich JE and Poczatek M. Activation of latent TGF- by thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev 11: 59-69, 2000.- w  A! x& ~9 o4 e
, ?! s7 W, R6 r: y1 c  [+ c

! q1 x. \) w$ D7 ~1 ^' e9 y2 g
Narita I, Border WA, Ketteler M, Ruoslahti E, and Noble NA. L -Arginine may mediate the therapeutic effects of low protein diets. Proc Natl Acad Sci USA 92: 4552-4556, 1995.. l7 u5 ]2 S5 b" L3 P
: b$ A# F) k) V# ]

- ~9 Y0 o# X4 k4 y6 A4 A! G6 L7 V
Nishikawa T, Edelstein D, and Brownlee M. The missing link: a single unifying mechanism for diabetic complications. Kidney Int 77: S26-S30, 2000.

O'Donnell MP, Kasiske BL, Schmitz PG, and Keane WF. High protein intake accelerates glomerulosclerosis independent of effects on glomerular hemodynamics. Kidney Int 37: 1263-1269, 1990.
  }7 R/ Z. H! @

+ C8 j* n* g; \& z
Pedrini MT, Levey AS, Lau J, Chalmers TC, and Wang PH. The effect of dietary protein restriction on the progression of diabetic and nondiabetic renal diseases. A meta-analysis. Ann Intern Med 124: 627-632, 1996.1 Q& D/ `4 k6 U6 R

( k6 T( W5 S5 |3 L+ r  r
Perrella MA, Yoshizumi M, Fen Z, Tsai JC, Hsieh CM, Kourembanas S, and Lee ME. Transforming growth factor- 1, but not dexamethasone, down-regulates nitric oxide synthase mRNA after its induction by interleukin-1 in rat smooth muscle cells. J Biol Chem 269: 14595-14600, 1994.
$ b4 r, f! o/ p& |1 r+ f0 k0 h$ g0 l

Peters H, Border WA, and Noble NA. Angiotensin II blockade and low-protein diet produce additive therapeutic effects in experimental glomerulonephritis. Kidney Int 57: 1493-1501, 2000.( X3 s# E# k7 k# e2 E  K
- k/ e$ i$ g; y) F4 H8 t, I

Pithon-Curi TC, Levada AC, Lopes LR, Doi SQ, and Curi R. Glutamine plays a role in superoxide production and the expression of p47 phox, p22 phox and gp91 phox in rat neutrophils. Clin Sci (Colch) 103: 403-408, 2002.

# |6 P# _  q( Q2 O) z' a4 n
Poczatek MH, Hugo C, Darley-Usmar V, and Murphy-Ullrich JE. Glucose stimulation of transforming growth factor- bioactivity in mesangial cells is mediated by thrombospondin-1. Am J Pathol 157: 1353-1363, 2000.# _# U5 k6 `& B8 k) h9 F
4 u- J6 |' y' q- v& V

# e( Z9 \- K. N+ g0 r* n
Ray PE, Bruggeman LA, Horikoshi S, Aguilera G, and Klotman PE. Angiotensin II stimulates human fetal mesangial cell proliferation and fibronectin biosynthesis by binding to AT 1 receptors. Kidney Int 43: 177-184, 1994.8 Z7 Z$ G9 I0 L# {; O4 b

: ~" V0 O0 T: I: Y
Ribeiro SM, Poczatek M, Schultz-Cherry S, Villain M, and Murphy-Ullrich JE. The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-. J Biol Chem 274: 13586-13593, 1999.- P+ e3 X9 U2 R# A, G
9 Q3 ?/ F) D) w6 k6 D
8 h. R: {  s. U

Riser BL, Cortes P, Heilig C, Grondin J, Ladson-Wofford S, Patterson D, and Narins RG. Cyclic stretching force selectively up-regulates transforming growth factor- isoforms in cultured rat mesangial cells. Am J Pathol 148: 1915-1923, 1996., q. ~. V: V" u, L1 [% h
* `2 n" z4 q  }" M* r. A
+ V- s8 h3 C% j  t4 {! K

Ruilope LM, Casal MC, Praga M, Alcazar JM, Decap G, Lahera V, and Rodicio JL. Additive antiproteinuric effect of converting enzyme inhibition and a low protein intake. JAmSoc Nephrol 3: 1307-1311, 1992.3 E3 B6 c1 [& P" |+ I8 z7 X+ f! {
4 G# m; y& t9 c5 X6 u  B5 L

  \/ Q* r" F: i/ {8 V
Schultz-Cherry S and Murphy-Ullrich JE. Thrombospondin causes activation of latent transforming growth factor- secreted by endothelial cells by a novel mechanism. J Cell Biol 122: 923-932, 1993./ e4 W0 W* m. v  [" }: u

6 v% ]% k7 z7 u! `+ I( ^
Scivitaro V, Ganz MB, and Weiss M. AGEs induce oxidative stress and activate protein kinase C- II in neonatal mesangial cells. Am J Physiol Renal Physiol 278: F676-F683, 2000.
! J. u0 U" r+ p; G4 h, g" Q

Sellitti DF, Pithon-Curi TC, Hirszel P, and Doi SQ. Glutamine synergistically increases mesangial cell proliferation induced by high glucose levels (Abstract). J Am Soc Nephrol 13: 297A, 2002.

Sharma K and Ziyadeh FN. Hyperglycemia and diabetic kidney disease. The case for transforming growth factor- as a key mediator. Diabetes 44: 1139-1146, 1995.

Shihab FS, Yi H, Bennett WM, and Andoh TF. Effect of nitric oxide modulation on TGF- 1 and matrix proteins in chronic cyclosporine nephrotoxicity. Kidney Int 58: 1174-1185, 2000.

! a% j* }* _- q' ~$ P
Singh R, Alavi N, Singh AK, and Leehey DJ. Role of angiotensin II in glucose-induced inhibition of mesangial matrix degradation. Diabetes 48: 2066-2073, 1999.$ f; H1 h4 C0 e' q
' I6 V' w3 D) G0 }& N

. U+ k" \* L! u; @& W
Tada H and Isogai S. The fibronectin production is increased by thrombospondin via activation of TGF- in cultured human mesangial cells. Nephron 79: 38-43, 1998.

) T) F6 k" m" K1 s  U+ G/ p( Z+ ?3 [
  c7 ^" L8 @2 {6 K7 ]' s5 U
Trachtman H, Futterweit S, and Crimmins DL. High glucose inhibits nitric oxide production in cultured rat mesangial cells. J Am Soc Nephrol 8: 1276-1282, 1997.
1 D. r5 ?% X' q) f# f" K! z
# Z7 A% b: G& F1 b5 U8 s3 y

Tuttle KR and Bruton JL. Effect of insulin therapy on renal hemodynamic response amino acids and renal hypertrophy in non-insulin-dependent diabetes. Kidney Int 43: 167-173, 1992.
: I1 |; q9 s( V4 y2 y& [, U& V
# E  F0 r- k  x$ \4 j0 x# f

Tuttle KR, Bruton JL, Perusek MC, Lancaster JL, Kopp DT, and DeFronzo RA. Effect of strict glycemic control on renal hemodynamic response to amino acids and renal enlargement in insulin-dependent diabetes mellitus. N Engl J Med 324: 1626-1632, 1991.
' n/ S6 a$ M& v2 S4 n4 R9 f! D. o$ D2 T
5 x& u+ x. {2 C8 t- F  A5 _7 q
" Z3 K$ s9 J0 D, m
Tuttle KR, Puhlman ME, Cooney SK, and Short RA. Effects of amino acids and glucagon on renal hemodynamics in type 1 diabetes. Am J Physiol Renal Physiol 282: F103-F112, 2002.! o9 ?! Y" Q4 u3 a! P

6 T% c; _4 C. U# Y6 ?6 A( I

Wang S, Shiva S, Poczatek MH, Darley-Usmar V, and Murphy-Ullrich JE. Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor- activation in mesangial cells. J Biol Chem 277: 9880-9888, 2002.
* y5 W0 T/ ^  x( t. {
" O* Q5 t4 G) x

Wolf G. Cell cycle regulation in diabetic nephropathy. Kidney Int 77: S59-S66, 2000.3 U& G& R8 m6 c7 d$ L' e9 O  T* W

8 v, V$ D- y0 L9 _& o/ t6 \' L
# N6 I  M) S# o8 f
Yevdokimova N, Wahab NA, and Mason RM. Thrombospondin-1 is the key activator of TGF- 1 in human mesangial cells exposed to high glucose. J Am Soc Nephrol 12: 703-712, 2001.# B" t% j) P7 |9 c6 q. G
6 l/ c# ?' }9 _2 z

Zatz R, Meyer TW, Rennke HG, and Brenner BM. Predominance of hemodynamic rather than metabolic factors in the pathogenesis of diabetic glomerulopathy. Proc Natl Acad Sci USA 82: 5963-5967, 1985.7 v7 {$ h4 h+ T: {; N" {4 Z
3 l* U) U' s; F  Y7 t4 B' U2 ~
7 v7 N, f7 C/ v! a: s0 {4 s

Ziyadeh FN, Sharma K, Ericksen M, and Wolf G. Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-. J Clin Invest 93: 536-542, 1994.

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