Angiotensin II mediates LDL-induced superoxide generation in mesangial cells - 《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 13:47 |只看该作者 |倒序浏览 |打印

作者：So Yeon Park, Chi Young Song, Bong Cho Kim, Hye Kyoung Hong,  Hyun Soon Lee作者单位：Department of Pathology, Seoul National University College of Medicine, Seoul 110-79 Korea

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      【摘要】9 u0 Y$ ?; f. B3 C3 \1 ~
   Lipid abnormalities and activation of the local renin-angiotensin system (RAS) may be involved in the pathogenesis of chronic glomerular disease. This study investigated whether low-density lipoprotein (LDL) activates local RAS in cultured human mesangial cells (HMC) and, at the same time, whether ANG II mediates LDL-induced mesangial cell proliferation, hypertrophy, and superoxide ( ) generation. Quiescent HMC were exposed to 50 to 200 µg/ml of LDL or 10 -7 to 10 -10 M ANG II for 0.5 to 24 h in the presence or absence of 10 -6 M losartan, an ANG II type I (AT 1 ) receptor antagonist, or 10 -5 M diphehylendieodonium (DPI) or 10 -4 M apocynin, inhibitors of nicotinamide adenine dinucleotide phosphate oxidase. LDL induced an up to threefold increase in the ANG II levels in the culture medium of HMC. LDL upregulated AT 1 receptor and angiotensinogen mRNA expression in HMC. LDL incubated with HMC increased production by up to 3.3 times compared with the level of control cells. The LDL-induced, increased generation was suppressed by losartan, DPI, or apocynin. LDL significantly increased mesangial [ 3 H]thymidine or [ 3 H]leucine incorporation, whereas these processes were abrogated by losartan. In conclusion, LDL increases ANG II production by mesangial cells, which in turn results in increased production, and cell proliferation and hypertrophy, these effects of ANG II being mediated by the AT 1 receptor.
      【关键词】 lipids AT receptor nicotinamide adenine dinucleotide phosphate oxidase hyperplasia hypertrophy7 L; v3 B+ @2 y3 k/ }% F
   LOW - DENSITY LIPOPROTEIN (LDL) is a major cholesterol-carrying lipoprotein and is extremely susceptible to oxidative damage. Elevated levels of LDL are a major risk factor in atherosclerosis, but oxidative modification of LDL seems to enhance the atherogenesis ( 18 ). Hypercholesterolemia itself does not initiate a glomerular injury, but it could aggravate the original glomerular injury, leading to glomerulosclerosis. As in the case of atherosclerosis, both LDL and oxidative stress appear to be involved in the pathogenesis of progressive glomerulosclerosis ( 16, 25 ). In support of this view, oxidized LDL has been demonstrated in the lesions of glomerulosclerosis in both humans ( 26 ) and rats ( 23 ). Furthermore, dietary cholesterol supplementation accelerated and antioxidant reduced the severity of renal injury in experimental glomerulosclerosis ( 23 ).

There is growing evidence that LDL communicates with the renin-angiotensin system (RAS) in the development of atherosclerosis ( 17, 18, 31, 34 ). ANG II is a major active product of the RAS. Angiotensinogen mRNA or ANG II has been found in cultured mesangial cells ( 3, 21 ), suggesting the presence of a local tissue RAS in mesangial cells. Type 1 ANG II (AT 1 ) receptor mRNA or protein is expressed on cultured mesangial cells ( 5, 9 ), and most known effects of mesangial ANG II are mediated through the AT 1 receptor ( 1 ). In cultured vascular smooth muscle cells (VSMC), LDL stimulates AT 1 receptor gene expression ( 31 ). VSMC are closely related to mesangial cells in origin, histochemistry, and contractility. Currently, the effects of LDL on angiotensinogen, ANG II, and AT 1 receptor expression in mesangial cells are still unknown.

Activation of local tissue RAS induces vascular oxidative stress ( 12 ). ANG II enhanced superoxide ( ) generation through the activation of membrane-bound nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidases in VSMC ( 10 ) and mesangial cells ( 14 ), which may function as second messengers for cell hyperplasia and hypertrophy. In cultured mesangial cells, LDL stimulates release ( 7 ) and cell proliferation ( 11, 16, 25 ) and hypertrophy ( 11 ). However, the mechanism by which these effects occur is still unknown.% [7 G- E& i% N, }
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In the present study, we examined the effects of LDL on ANG II, angiotensinogen mRNA, and AT 1 receptor mRNA expression in human mesangial cells (HMC). We also examined whether LDL-induced mesangial generation and cell proliferation/hypertrophy are mediated by ANG II.9 e# F3 z4 U- @  F: P) Q# m. k+ E' z
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MATERIALS AND METHODS
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Reagents. ANG II, lucigenin, and diphehylendieodonium (DPI) were purchased from Sigma (St. Louis, MO). Losartan was kindly provided by DuPont Merck (Rahway, NJ). ANG II ELISA kits were purchased from Peninsula Laboratories (San Carlos, CA). A dual-quantitative RT-PCR kit was purchased from Maxim Biotech (San Francisco, CA). Other reagents were obtained from sources mentioned in our previous reports ( 24, 25 ).

Culture of HMC. HMC were obtained from adult nephrectomy specimens, as previously described ( 27, 35 ). The culture medium was made of DMEM supplemented with 20% fetal calf serum, 200 mM L -glutamine, and antibiotics. For the present experiments, cells from between passages 5 and 7 were used.

Preparation of LDL. Human LDL (density: 1.019-1.063) was isolated from the plasma of normal volunteers by the method of sequential ultracentrifugation, as we previously described ( 27 ). Isolated LDL was dialyzed for 24 h at 4°C against buffer A, which contained 0.15 M NaCl and 0.24 mM EDTA at pH 7.4. After dialysis, LDL was stored at 4°C under nitrogen and was used within 14 days.

Experimental conditions. HMC were grown to confluency. The cells were synchronized to quiescence in serum-free DMEM containing 5 µg insulin-transferrin-selenite/ml for 48 h. After synchronization, cells were treated with 50 to 200 µg/ml LDL or 10 -7 to 10 -10 M ANG II for a time period varying from 0.5 to 24 h. In certain experiments, 10 -6 M losartan, an AT 1 receptor antagonist, 10 -5 M DPI or 10 -4 M apocynin, inhibitors of NAD(P)H oxidase, or 10 -4 M allopurinol, an inhibitor of xanthine oxidase, was added to the cells 1 h before LDL or ANG II administration. In a given experiment, simultaneous control monolayers were treated with serum-free DMEM alone.
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Assessment of DNA synthesis. HMC grown in 96-well plates were synchronized and exposed to 200 µg/ml LDL or 10 -7 to 10 -10 M ANG II for 24 h in the presence or absence of losartan, DPI, apocynin, or allopurinol. During the last 18 h of the incubation period, [ 3 H]thymidine (1 mCi/well) was added to all of the wells. At the end of the pulsing period, the cells were washed, dissolved in 1% SDS/0.3 N NaOH solution, and placed in 4 ml of a scintillation cocktail to measure the total radioactivity, using a previously described method ( 25 ).! \2 C9 i1 T% h6 K" K3 {2 |

Assessment of cell hypertrophy. HMC grown in six-well plates were synchronized and exposed to 200 µg/ml LDL for 48 h in the presence or absence of losartan, DPI, apocynin, or allopurinol. During the last 30 h of the incubation period, [3 H]leucine (1 µCi/ml) was added to all of the plates. At the end of the pulsing period, the cells were washed and solubilized overnight with 1.5 ml 0.1% SDS. The content was transferred to a tube containing 60 µl 10% BSA. Proteins were precipitated with 300 µl 20% tricholoroacetic acid and left at room temperature for 2 h. Samples were then spun, the supernatant was discarded, and pellet was resuspended in 0.5 N NaOH. Duplicate aliquots were removed and counted in a scintillation counter, as described by Jaimes et al. ( 14 ).

Determination of production. The level of production was determined by the chemiluminescence of lucigenin (bis- N -methylacridium nitrate), which emits light upon reduction and interaction with, as described by Ohara et al. ( 32 ). Briefly, HMC grown in six-well plates were synchronized and incubated with 200 µg/ml LDL or 10 -7 to 10 -10 M ANG II at 37°C for a period ranging from 30 min to 2 h in the presence or absence of losartan, DPI, apocynin, or allopurinol. The cells were then trypsinized, pelleted by centrifugation, and resuspended in 1 ml Krebs-HEPES buffer. The cell suspensions were transferred to scintillation vials containing dark-adapted 0.25 mmol/l lucigenin. The chemiluminescence of lucigenin was detected by means of a chemiluminometer (Berthold Multi-Biolumit LB 9505 C, Pforzheim, Germany). Photon emission was measured every minute for 15 min at room temperature in the out-of-coincidence mode. A buffer blank was subtracted from each reading, and generation was calculated by comparison with a standard curve generated using xanthine/xanthine oxidase. Protein content was measured using bicinchoninic acid with BSA as a standard.
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Quantification of ANG II by ELISA. At the end of the incubation period, the conditioned medium was centrifuged and stored at -70°C until being assayed. The quantitative measurement of ANG II antigen was carried out according to the manufacturer's instructions, using ELISA kits. Briefly, the samples or standards were incubated with anti-ANG II antibody and biotinylated ANG II in 96-well plates. After incubation, the unbound biotinylated ANG II was removed by washing, and the immobilized anti-ANG II antibody/biotinylated ANG II complex was determined by means of its reaction with streptavidin-horse radish peroxidase in the wells using 3,3',5,5'-tetramethyl benzidine dihydrochloride and H 2 O 2 as a substrate. The reaction was terminated with 2 N HCl, and the color intensity in each well was measured at 450 nm using an ELISA microtiter plate reader (Vmax, Molecular Devices). The amount of ANG II in each well was calculated from the standard curve.8 x# |  I: m) U8 x8 y" O- o

Generation of AT 1 receptor cDNA. Total cellular RNA was isolated from cultured HMC and used to synthesize cDNA by reverse transcription (RT) using reverse transcriptase (Gibco BRL). Oligonucleotide primers were chosen in homologous parts of the coding region of the rat AT 1 A and AT 1 B receptor genes. With the use of the cDNA mixture together with the reverse primer (5'-GCA CAA TCG CCA TAA TTA TTC-3', position 739-719 bp) and the sense primer (5'-CAC CTA TGT AAG ATC GCT TC-3', position 295-314 bp), the PCR was performed using the method described by Chansel et al. ( 6 ).3 U7 P: Y6 X9 h6 v
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Northern blot analysis. Total cellular RNA was isolated from confluent experimental and control cultures by using the acid guanidium thiocyanate-phenol-chloroform extraction method. With the use of a Rediprime labeling kit as the random primer DNA labeling system, the cDNA templates were radiolabeled with [ 32 P]deoxycytidine triphosphate. The blotted membranes were incubated with the specific [ 32 P]-labeled cDNA probes. The filters were dried and exposed at -70°C using Agfa film (Agfa-Gevaert N.V., Hortsel, Belgium). The mRNA levels of the AT 1 receptor were expressed as the ratio of the optical density units of AT 1 receptor to those of -actin.' H4 `& ~$ r3 ^2 `, Q* e, ]. m8 X+ s

Dual-quantitative RT-PCR. With the use of the cDNA mixture, angiotensinogen primer (5'-GCT-TTC-AAC-ACCTAC-GTC-CA-3' and 5'-AGC-TGT-TGG-GTA-GAC-TCT-GT-3') ( 21 ) and a dual-quantitative RT-PCR kit supplying GAPDH primer, GAPDH and angiotensinogen mRNAs were amplified at the same time. The PCR products were analyzed using 1% agarose gel electrophoresis.: D! q# n0 F9 C$ q' c: S& i4 m' J, s

Statistical analyses. Results were expressed as means ± SD. Results were analyzed by two-way analysis of variance for three groups or by Wilcoxon's rank sum test between two groups. A P value of
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RESULTS5 t0 Q( E3 I. d) z4 w3 c

LDL increases ANG II secretion in mesangial cells. The concentration of ANG II in the medium of the control HMC was 0.14 ± 0.01 ng/ml (means ± SD), when it was measured using ELISA kits. Thirty minutes after the addition of 50 to 200 µg/ml of LDL, the level of ANG II antigen present in the medium increased by two to three times compared with that of the control cells but not necessarily in a dose-dependent manner ( Fig. 1 ).
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Fig. 1. Expression of ANG II in culture media of human mesangial cells (HMC) exposed to 50 to 200 µg/ml low-density lipoprotein (LDL) for 30 min. Values are means ± SD of 3 comparable experiments. * P
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LDL and ANG II increase AT 1 receptor mRNA expression in mesangial cells. Thirty minutes to 4 h after the addition of 200 µg/ml of LDL to HMC, AT 1 receptor mRNA expression increased to 1.2 and 1.4 times the levels in the control cells ( Fig. 2 )./ z1 T% Y0 V; r. h( L6 K, L, S
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Fig. 2. Northern blot analysis of ANG II type I (AT 1 ) receptor mRNA in HMC. Cells were incubated with serum-free DMEM alone ( lane 1 ) or with the addition of 200 µg/ml LDL for 0.5 h ( lane 2 ), 2 h ( lane 3 ), or 4 h ( lane 4 ). The blots were hybridized with [ 32 P]-labeled cDNA for AT 1 receptor (AT 1 R; A ) and -actin ( B ). C : quantitative expression of AT 1 R mRNA abundance after correcting for the -actin signal. The AT 1 R mRNA levels of treated HMC are expressed as percent increases compared with the mRNA levels of untreated controls. Values are means ± SD of 3 separate experiments. * P; k6 S  r* H: g1 W$ E
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When HMC were exposed to 10 -10 M ANG II for 0.5 to 4 h, AT 1 receptor mRNA expression was significantly increased compared with controls ( Fig. 3 ).1 A) a/ q% N% V

Fig. 3. Northern blot analysis of AT 1 R mRNA in HMC. Cells were incubated with serum-free DMEM alone ( lane 1 ) or with the addition of 10 -10 M ANG II for 0.5 h ( lane 2 ), 2 h ( lane 3 ), or 4 h ( lane 4 ). The blots were hybridized with [ 32 P]-labeled cDNA for AT 1 R ( A ) and -actin ( B ). C : quantitative expression of AT 1 R mRNA abundance after correcting for the -actin signal. Data are expressed as described in Fig. 2 legend. Values are means ± SD of 3 separate experiments. * P

Angiotensinogen mRNA expression in LDL-treated cells. Thirty minutes or 5 h after the addition of 200 µg/ml of LDL to HMC, angiotensinogen mRNA expression was significantly increased, when corrected for GAPDH expression, compared with the control cells ( Fig. 4 ).9 J. G% m/ t& O0 n1 I$ F
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Fig. 4. Electrophoresis of dual-quantitative RT-PCR products to detect angiotensinogen (ANGN) mRNA in HMC. Cells were incubated with serum-free DMEM alone ( lane 1 ) or with the addition of 200 µg/ml LDL for 0.5 h ( lane 2 ) or 5 h ( lane 3 ).
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generation in LDL- and ANG II-treated cells. The chemiluminescence produced by lucigenin increased with time and reached a plateau within 15 min. production, estimated by measuring the chemiluminescence 10 min after exposure to lucigenin, was 3.3-fold higher in HMC exposed to 200 µg/ml of LDL for 1 h than in the control cells ( Fig. 5 ).

Fig. 5. Time course of O 2 - production estimated by lucigenin chemiluminescence in controls and LDL-treated cells. HMC were exposed to 200 µg/ml LDL for 1 h, with or without pretreament with losartan, diphehylendieodonium (DPI), or apocynin. Data are expressed as means ± SD of 6 different experiments.
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When HMC were exposed to 10 -7 to 10 -10 M ANG II for 1 h, the production was significantly increased compared with controls but not necessarily in a dose-dependent manner ( Fig. 6 ).
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Fig. 6. Time course of O 2 - production in ANG II (AII)-treated cells. HMC were exposed to 10 -7 to 10 -10 M ANG II for 1 h. Data are expressed as means ± SD of 6 comparable experiments.
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AT 1 receptor antagonist abrogates LDL- and ANG II-induced, increased generation. To determine whether ANG II was responsible for the LDL-induced production in HMC, the cells were exposed to 10 -6 M losartan for 1 h before LDL treatment. The treatment with losartan was not accompanied by increased cell lethality as assessed by trypan blue exclusion. The increased production induced by LDL was blocked with losartan ( Fig. 5 ), suggesting that ANG II plays a role in the process of generation mediated by the AT 1 receptor.) }; D+ h  K# h" r% t4 s8 D# a

Pretreatment of losartan (10 -5 M) also inhibited the 10 -7 M ANG II-induced production ( Fig. 7 ).
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Fig. 7. Effects of losartan (Los) and DPI on ANG II-induced production by HMC. Cells were exposed to 10 -7 M ANG II for 1 h, with or without pretreatment with 10 -5 M losartan or 10 -5 M DPI. Data are expressed as means ± SD of 3 comparable experiments. * P
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NAD(P)H oxidase inhibition attenuates LDL- and ANG II-induced production. To examine whether mesangial NAD(P)H oxidase activation was involved in the LDL-induced production, the cells were exposed to an NAD(P)H oxidase inhibitor, DPI (10 -5 M) or apocynin (10 -4 M), for 1 h before the LDL treatment. Treatment with DPI or apocynin was not accompanied by increased cell lethality, as assessed by trypan blue exclusion. Both DPI and apocynin significantly inhibited the effects of LDL on production ( Fig. 5 ), suggesting that these effects of LDL are mediated by the activation of NAD(P)H oxidase. The addition of allopurinol also reduced the LDL-induced production but to a smaller extent than DPI or apocynin (data not shown).
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Pretreatment of DPI (10 -5 M) also blocked the ANG II-induced production ( Fig. 7 ).
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Cell proliferation in LDL- and ANG II-treated cells. When the HMC were incubated with LDL for 24 h, there was a significant increase in [ 3 H]thymidine incorporation into mesangial DNA ( Fig. 8 ).
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Fig. 8. Effects of LDL on [ 3 H]thymidine incorporation in HMC. HMC were exposed for 24 h to 200 µg/ml LDL in the presence or absence of losartan or DPI. Data represent the means ± SD of 6 different experiments. * P ** P  C3 B0 J4 H8 f" `6 l1 e" P2 d4 N
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HMC exposed to 10 -7 to 10 -10 M of ANG II for 24 h also significantly increased cell proliferation as measured by [ 3 H]thymidine incorporation. The cell proliferation tended to be increased in a dose-dependent manner (10 -8 to 10 -10 M). However, the cell proliferation was reduced when 10 -7 M ANG II was used ( Fig. 9 ).. k# V. E7 f1 j+ r( m% D
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Fig. 9. Effects of ANG II on [ 3 H]thymidine incorporation in HMC. HMC were exposed to 10 -7 to 10 -10 M ANG II for 24 h. Data represent the means ± SD of 6 separate experiments. * P

AT 1 receptor antagonist inhibits LDL-induced mesangial cell proliferation. To determine whether ANG II was responsible for the LDL-induced cell proliferation in HMC, the cells were exposed to 10 -6 M losartan for 1 h before LDL treatment. The addition of losartan to LDL-treated cells significantly reduced DNA synthesis ( Fig. 8 ), suggesting that ANG II is involved in the process of mesangial cell proliferation mediated by the AT 1 receptor.* i- N2 E( l* x( `- x
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However, the addition of DPI ( Fig. 8 ), apocynin, or allopurinol to LDL-treated cells did not significantly reduce DNA synthesis.5 ]' q/ w: c$ u0 H' g! g; X
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Cell hypertrophy in LDL-treated cells. When the HMC were incubated with LDL for 48 h, there was a significant increase in protein synthesis as measured by [ 3 H]leucine incorporation ( Fig. 10 ).  O5 H' U* Z$ |- u

Fig. 10. Effects of LDL on [ 3 H]leucine incorporation in HMC. HMC were exposed for 48 h to 200 µg/ml LDL in the presence or absence of losartan, DPI, apocynin (apo), or allopurinol (allo). Data represent the means ± SD of 4 experiments. * P
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The addition of losartan, DPI, apocynin, or allopurinol to LDL-treated cells significantly reduced [ 3 H]leucine incorporation ( Fig. 10 ).

DISCUSSION

This study demonstrates, to the best of our knowledge for the first time, that LDL increases angiotensinogen mRNA and ANG II expression in HMC with upregulation of AT 1 receptor mRNA. Furthermore, this study shows that LDL stimulates mesangial cell proliferation/hypertrophy and NAD(P)H oxidase-dependent production mediated via ANG II.3 b5 l' Y! F% D, m
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We observed that LDL significantly increased angiotensinogen mRNA and ANG II expression in HMC, suggesting that LDL induces a local RAS activation in mesangial cells. Local glomerular ANG II formation might exert both hemodynamic (leading to increased glomerular capillary pressure) and nonhemodynamic effects (stimulation of cellular hyperplasia/hypertrophy and extracellular matrix accumulation) on the mesangial cells ( 1 ). Increased signals for renin, angiotensinogen, and angiotensin-converting enzyme mRNAs were detected in glomerular mesangial cells of patients with chronic nephritides, suggesting that local RAS may be involved in the progression of renal disease ( 21 ).1 d3 L4 j. W. S- X) F7 _

We also demonstrated that the incubation of HMC with LDL or ANG II for between 0.5 and 4 h upregulates AT 1 receptor mRNA expression. In support of our observation, LDL induced an upregulation of AT 1 receptor mRNA expression in VSMC ( 31 ). The AT 1 receptor upregulation could lead to an elevated functional response of HMC on ANG II stimulation. Yet some authors reported that ANG II did not induce any change in AT 1 receptor mRNA levels ( 5 ) or even downregulated its expression in cultured mesangial cells ( 28 ) or VSMC ( 22, 30 ). The reason for the downregulation of AT 1 receptor induced by ANG II is unclear. Nickenig et al. ( 31 ) argued that a principal mechanism underlying this downregulation might be the inducible destabilization of AT 1 receptor mRNA. Furthermore, they suggested that LDL causes AT 1 receptor upregulation in VSMC by means of AT 1 receptor mRNA stabiliziation.8 b4 l: I  d1 Q2 h7 d4 S
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We found that LDL induced overproduction in HMC, most likely via the NAD(P)H oxidase mechanism. This excess generation was blocked by losartan, suggesting that the effects of LDL on production are mediated by the AT 1 receptor in association with local RAS activation. Furthermore, our dose-effect curve of production against ANG II concentration shows that endogenous ANG II induced by LDL is responsible for the production of observed in Fig. 5. Previous studies showed that activation of the local RAS enhances the vascular production of the reactive oxygen species ( 12 ) through the activation of membrane-bound NAD(P)H oxidases ( 10, 12, 29 ). In cultured mesangial cells, ANG II-induced generation was also mediated by the NAD(P)H oxidase system ( 14 ). Stimulation of the AT 1 receptor activates G protein-coupled phospholipase C and hydrolyzes membrane phosphoinositides, thus leading to the activation of protein kinase C (PKC) and the elevation of intracellular calcium level ( 33 ). PKC seems to activate NAD(P)H oxidase in mesangial cells exposed to ANG II ( 14 ). As yet, it is not clear whether PKC, activated by LDL ( 24, 35 ) or oxidized LDL ( 2 ), can also lead to NAD(P)H oxidase activation in HMC.
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generated by LDL may react with nitric oxide (NO) to yield peroxynitrite and other oxidants. This, in turn, may lead to the destruction of NO in mesangial cells, by causing oxidation of the cofactor tetrahydrobiopterin ( 20 ). NO has an antiproliferative ( 8 ) and antifibrotic effect on mesangial cells ( 19 ). Thus it is tempting to speculate that the destruction of NO in mesangial cells by LDL-induced may lead to mesangial cell proliferation and extracellular matrix deposition.

In confirmation of previous studies ( 11, 16, 25 ), LDL was found to stimulate mesangial cell proliferation in this study. This LDL-induced mesangial hyperplasia was attenuated by losartan, demonstrating that the AT 1 receptor plays a role in this process. Furthermore, our dose-effect curve of cell proliferation against ANG II concentration demonstrates that endogenous ANG II produced by LDL is partly responsible for the cell proliferation observed in Fig. 8. ANG II induces growth-stimulatory effects principally mediated through AT 1 receptors ( 10, 14 ), although binding of ANG II to type 2 receptors has opposite effects including inhibition of proliferation ( 36 ). The stimulatory effects of ANG II on cell growth appear to occur via synthesis of ( 10, 14 ) and growth factors such as platelet-derived growth factors ( 37 ) and transforming growth factor- ( 15 ). In the present study, DPI, apocynin, or allopurinol had no effect on LDL-induced cell growth, suggesting that growth factors other than are involved in the process of cell proliferation mediated by ANG II.8 T4 G% M4 x- l" Y

We also found that LDL stimulates [ 3 H]leucine incorporation in HMC. This LDL-induced mesangial hypertrophy was attenuated by losartan, suggesting that the AT 1 receptor plays a role in this process. Furthermore, DPI, apocynin, or allopurinol inhibited the LDL-induced cell hypertrophy, suggesting that is involved in the process of cell hypertrophy mediated by ANG II.( y0 `6 }3 b1 R( Q1 j/ \% {7 e) w

AT 1 receptor blocker losartan has an important renoprotective effect in patients with diabetic ( 4 ) or nondiabetic nephropathy ( 13 ). Most patients with chronic renal disease show lipid abnormalities. Thus the antioxidative or anti-proliferative properties of losartan on LDL-stimulated HMC demonstrated in this study provide support for its therapeutic role in patients with chronic renal disease. Further studies are required to confirm whether these effects of losartan, which were observed in an in vitro system on cultured HMC, can also occur in vivo.

In summary, LDL stimulates ANG II expression in HMC, which may in turn mediate mesangial production and cell proliferation/hypertrophy via the AT 1 receptor. These results suggest that interactions between LDL and local RAS in mesangial cells play an important role in the progression of glomerular disease.' x: D: l7 m% C2 _. B5 H9 o, M" ~
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DISCLOSURES' U1 l) m5 i7 C+ e" L; Q) J1 C

This study was supported by grants from the Korea Research Foundation.% j6 E* p1 z4 [* G
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