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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：HarmPeters, SebastianMartini, RaikoWoydt, MatthiasRückert, FuijoShimizu, HiroshiKawachi, LutzLiefeldt, StephanieKrämer,  Hans-H.Neumayer作者单位：1 Division of Nephrology, Charité, Campus Mitte,Humboldt-University, D-10098 Berlin, Germany; and Department of Cell Biology and Institute of Nephrology,Niigata University Graduate School of Medical and Dental Sciences,Niigata 951-851 Japan
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      【摘要】
   Moderate alcohol consumption has shownbeneficial effects in experimental and human cardiovascular disease.With the use of rat models of acute and chronic progressive anti-thy1glomerulonephritis (GN), we tested the hypothesis that moderate alcoholintake is protective in renal fibrotic disease. In acute anti-thy1 GN,untreated nephritic rats showed marked mesangial cell lysis and induced nitric oxide production at day 1 and high proteinuria,glomerular matrix accumulation, and transforming growth factor(TGF)- 1, fibronectin, and plasminogen activatorinhibitor (PAI)-1 expression at day 7 after diseaseinduction, respectively. In animals 15 wk after induction of chronicprogressive anti-thy1 GN, disease was characterized by significantlyreduced renal function, persisting albuminuria as well as increasedglomerular and tubulointerstitial matrix expansion,TGF- 1, fibronectin, and PAI-1 protein expression. Inboth anti-thy1 GN models, an ethanol intake of ~2 ml per day andanimal was achieved, however, disease severity was not significantly altered by moderate alcohol consumption in any of the protocols. Inconclusion, moderate alcohol intake does not influence renal matrixprotein production and accumulation in acute and chronic progressiveanti-thy1 glomerulofibrosis. The study suggests that, in contrast tocardiovascular disorders, moderate alcohol consumption might notprovide specific protection in renal fibrotic disease.
      【关键词】 fibrosis transforming growth factor inducible nitric oxideproduction beer
   INTRODUCTION
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NUTRITION PLAYS amajor role in human health and disease. Modifying food and fluid intakewas probably one of the first approaches humankind used to prevent,influence, or treat its various diseases. For renal disorders,beneficial effects of dietary modifications have been documented,mainly in experimental settings, for the intake of calories, proteins,certain amino acids, lipids, minerals, and vitamins ( 2, 3, 22 ). In cardiovascular disease, positive effects of moderatealcohol intake have been documented in numerous experimental and humanstudies ( 12, 20, 21, 36 ), whereas only little is knownabout its action on the course of kidney disorders.+ }7 m' Q* D8 V
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As for cardiovascular disease, pathological expansion of extracellularmatrix proteins is a hallmark of acute and chronic renal disease( 4, 17 ). Glomerular and tubulointerstitial matrixaccumulation result from an increase in the production of matrixproteins such as fibronectin, biglycan, and collagens; a decrease ofmatrix protein degradation by increased production of proteaseinhibitors such as plasminogen activator inhibitor (PAI)-1; and anoverexpression of matrix-binding integrins on the cell surface( 4, 25 ). Overproduction of the cytokine transforminggrowth factor (TGF)- has been identified as a key feature of tissuefibrosis wherever it occurs. While in acute renal disease TGF- overexpression and matrix deposition are transient and reversible,chronic renal disease is characterized by ongoing tissue injuryresulting in persisting TGF- overproduction and progressive renalfibrosis and insufficiency ( 4 ). This concept is reflectedexemplarily in the rat model of anti-thy1 glomerulonephritis. Inanimals with two kidneys, anti-thy1 antibody injection leads to acuteand reversible mesangioproliferative glomerulonephritis ( 1 ), whereas in uninephrectomized rats, injection ofanti-thy1 antibody results in persisting glomerulosclerosis andprogressive tubulointerstitial fibrosis ( 23 ), which may berelated to a continuous hyperfiltration injury of the remaining kidney.

Because cardiovascular and kidney disorders share a number ofsimilarities at the cellular and molecular level, we hypothesized thatmoderate alcohol intake may limit TGF- overexpression and matrixexpansion in renal disease. To test this hypothesis, we administered 40 ml beer/day to rats with acute or chronic progressive anti-thy1glomerulonephritis. In acute anti-thy1 glomerulonephritis, alcoholactions on the initial mesangial cell injury and subsequent matrixexpansion were determined ( day 1 and day 7 afterantibody injection, respectively). In chronic anti-thy1glomerulonephritis, ethanol's effects on renal function, glomerularsclerosis, and tubulointerstitial fibrosis were analyzed 15 wk afterdisease induction.

METHODS
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Materials

Unless otherwise indicated, materials, chemicals, orculture media were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Animals
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Male Wistar rats (180-250 g) obtained from Charles River(Sulzfeld, Germany) were fed a normal protein diet (22.5% protein, Altromin, Lage, Germany) for at least 3 days before the start of theexperiment to allow equilibration. Animal care and treatment were inconformity with the guidelines of the American Physiological Societyand approved by local authorities. Animals were housed in aconstant-temperature room with a 12:12-h light-dark cycle. Body weightwas determined at the beginning and end of each experiment. Food, beer,and water intakes were monitored daily.! q& }' E& `7 z3 n3 g5 k; ^, x
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Induction of Acute and Chronic Progressive Anti-Thy1Glomerulonephritis0 R) e) B* O- O$ y" u& G9 l
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Acute anti-thy1 glomerulonephritis was induced by tail veininjection of the monoclonal antibody OX-7 (1 mg/kg body wt in PBS) aspreviously described ( 29 ). For chronic progressiveglomerulonephritis, one kidney was surgically removed and themonoclonal antibody mAb 1-22-3 (4 mg/kg body wt in PBS) wasintravenously injected 3 days later. In the kidney, OX-7 and mAb1-22-3 antibodies bind to a thy1-like antigen (although todifferent epitopes) on the surface of mesangial cells and causecomplement- and nitric oxide (NO)-dependent cell lysis ( 1, 23, 24 ). Control animals were injected with equal volumes of PBS only.
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Production of OX-7 and mAb 1-22-3* t6 s6 g8 T  J! z9 @9 Q+ a; ]

OX-7 and mAb 1-22-3 were produced from hybridoma celllines as previously described ( 29 ). The antibodies werediluted in PBS (pH 7.4) and stored at 70°C until use.
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Moderate Alcohol Consumption
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A moderate alcohol intake was achieved by supplying 40 ml beer(4.9% ethanol, 23 bitter units, expressing the bitterness of thebeer's taste) per day and rat. The beer was provided every lateafternoon, and in general the animals started drinking immediately. Inaddition, the animals had free access to tap water. This approach results in a daily intake of ~2 ml ethanol/animal, which, for rats,constitutes moderate alcohol intake ( 9 ).
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Experimental Design

In protocol 1, the action of moderate alcohol intakeon early mesangial cell lysis ( protocol 1A, injury phase, day 1 ) and on the subsequent matrix expansion( protocol 1B, matrix expansion phase, day 7 ) wasanalyzed in rats following the induction of acute anti-thy1glomerulonephritis. In protocol 2, the effect of moderatealcohol consumption was investigated in rats with chronic progressiveanti-thy1 glomerulonephritis (progression from acute glomerular tochronic tubulointerstitial fibrosis) 15 wk after disease induction./ K5 t* ?( |( `7 w9 S9 _, y, r

In experiments 1B and 2, the histological gradingof renal matrix accumulation was paralleled by protein measurements ofthe key fibrosis mediator and marker TGF-. In addition, renalexpression of the matrix protein fibronectin was measured as anindicator of matrix protein production. The protease inhibitor PAI-1was used as a sensitive marker of the matrix-degrading system.TGF- 1, fibronectin, and PAI-1 expression were measuredat the protein level in the supernatant of cultured glomeruli or mincedcortical tissue harvested from individual animals. The interaction ofethanol with inducible NO production was tested in vivo in protocol 1A (NO-mediated mesangial cell damage) and in vitroin protocols 1B and 2 (stimulation of culturedglomeruli of cortical tissue with LPS).) r' a& [* B3 _! ^: [
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Protocol 1A
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Effect of moderate alcohol intake on the injury phase of acuteanti-thy1 glomerulonephritis (day 1 after antibody injection). Five days before antibody injection, Wistar rats were assigned to thefollowing groups: 1 ) PBS-injected controls (control; n = 4); 2 ) anti-thy1 antibody-injectedanimals, no treatment (aGN; n = 8); and anti-thy1antibody-injected rats plus moderate alcohol intake (aGN C 2 H 5 OH; n = 8).1 |& K2 G* C! X; W5 G0 `* i5 F

One day after antibody injection, the histological degree ofmesangial cell lysis as well as the release of basal and LPS-stimulated nitrite production of cultured glomeruli were analyzed. At this point,mesangial cell lysis is complete and inducible glomerular NO productionis markedly increased ( 28 ).

Protocol 1B+ R( i& l1 [! _8 w7 ^5 b* b

Effect of moderate alcohol intake on the matrix expansion phaseof anti-thy1 glomerulonephritis (day 7 after antibody injection). One day after antibody injection, when the mesangial cell lysis hadoccurred and the fibrotic response had started ( 28 ), Wistar rats were assigned to the following groups: 1 )control ( n = 4); 2 ) aGN ( n = 8); and 3 ) aGN C 2 H 5 OH( n = 8).4 k  e  n' W' t) u# Y
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Seven days after disease induction, histological glomerularmatrix accumulation and production of nitrite, TGF- 1,fibronectin, and PAI-1 of cultured glomeruli were determined. In acuteanti-thy1 glomerulonephritis, the fibrotic response peaks 7 days afterantibody injection and provides a large "therapeutic window"between normal and disease levels ( 29 ).2 k3 ~5 M. O. A/ c, a6 H7 _) O/ \: H
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Protocol 2
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Effect of moderate alcohol intake on progression from acute tochronic progressive anti-thy1 glomerulonephritis (15 wk after antibodyinjection). Five weeks after uninephrectomy and antibody injection, Wistar ratswere treated as follows: 1 ) uninephrectomized, PBS-injected controls (control; n = 4); 2 )uninephrectomized, anti-thy1 antibody-injected animals, no treatment(cGN; n = 10); and 3 ) uninephrectomized, anti-thy1 antibody-injected rats plus moderate alcohol intake (cGN C 2 H 5 OH; n = 10).
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Fifteen weeks after induction of chronic anti-thy1 glomerulonephritis,parameters of renal function [glomerular filtration rate (GFR), serumcreatinine, and blood urea nitrogen (BUN)] and indexes of glomerularand tubulointerstitial matrix accumulation (histological matrix score,glomerular and cortical protein expression of TGF- 1,fibronectin, and PAI-1) were determined. In addition, basal andLPS-stimulated nitrite production were assessed in cultured glomerularand cortical tissue., G; n# u' M  }3 v  ?3 n

Measurement of Systolic Blood Pressure and Albuminuria9 n6 D1 n3 d2 N+ x! x. k" t6 b

In animals with chronic anti-thy1 glomerulonephritis, systolicblood pressure was measured 2 days before death in conscious animals bythe tail-cuff method as previously described ( 29 ). Becauseacute anti-thy1 glomerulonephritis has been shown to be normotensive( 29 ), blood pressure was not measured in this model. Inboth acute and chronic anti-thy1 glomerulonephritis, a 24-h urine wascollected from each rat the day before death, using metabolic cages.Albuminuria was measured using a microplate technique and a rabbitanti-rat albumin peroxidase-conjugated antibody ( 19 ). Albuminuria is expressed as milligrams of protein per 24 hours.# f; J8 m7 i0 C0 n9 d
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At the end of each experiment, the animals were anesthetizedwith ether. After a midline abdominal incision, 5-10 ml blood weredrawn from the abdominal aorta and the kidneys were subsequently perfused with 30 ml ice-cold PBS. For histological examination, cortical tissue was fixed in 10% neutral buffered formalin.

Measurement of Renal Function and Serum Ethanol Concentrations

Serum and urine creatinine, BUN, and serum ethanolconcentrations were measured spectrophotometrically in enzyme-basedassays. GFR was calculated on the basis of serum and urinary creatinine concentration and the corresponding urinary volume., X- l) A6 T7 \& n3 P9 d
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Production of TGF- 1, Fibronectin, and PAI-1 byGlomeruli or Cortical Tissue in Culture9 q" H3 H& V! D* c3 c. g8 A; n

In acute and chronic progressive anti-thy1 glomerulonephritis,glomeruli from individual rats were isolated by a graded sieving technique (150-, 125-, 106-, and 75-µm mesh metal sieves) asdescribed previously ( 29 ). In chronic anti-thy1 animals, apiece of cortical tissue was weighed and minced extensively with arazor blade. Glomeruli or cortical tissue was suspended in DMEMsupplemented with 0.1 U/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. For stimulation of inducible NO synthase (iNOS),10 µg LPS/ml from Escherichia coli (serotype 0127:B8) permilliliter were added. Glomeruli were cultured in a density of 2,000/mlfor 48 h and minced cortical tissue at a density of 10 mg/ml,respectively. After 48-h incubation at 37°C and 5% CO 2,supernatants were harvested and stored at 70°C until analysis ofTGF- 1, fibronectin, PAI-1, or nitrite content.

Light Microscopy
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All microscopic examinations were performed in a blindedfashion. Three-micrometer sections of paraffin-embedded tissue were stained with periodic acid-Schiff (PAS). For calculation of mesangial cell lysis, the number of the remaining cell nuclei was counted in 30 glomeruli of 80- to 100-µm diameter from each animal. Glomerular matrix expansion was evaluated by rating the mesangial matrix-occupying area of 30 glomeruli from each rat using the following scoring system:1 = 0-25%, 2 = 26-50%, 3 = 51-75%, and4 = 76-100%. For estimation of renal matrix expansion inchronic progressive anti-thy1 glomerulonephritis, a combined glomerularand tubulointerstitial fibrosis score was used. Glomerular matrixaccumulation was rated as described above. Tubulointerstitial matrixdeposition was assayed in 20 randomly selected cortical areas persample observed at ×250 magnification using the following scale:0 = normal, 1 = lesions involving 50%, respectively. The individual renal fibrosisscore was derived by adding the mean glomerular and tubulointerstitialmatrix index of each animal.

Measurement of TGF- 1, Fibronectin, and PAI-1
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TGF- 1 content of culture supernatant was measuredafter acid activation using a commercially available ELISA kit(TGF- 1 Duoset, R&D Systems, Wiesbaden, Germany)according to the manufacturer's instructions. Fibronectin and PAI-1levels were measured with modified inhibitory enzyme-linkedimmunoassays (ELISA) according to published methods ( 31 ).Three samples from each rat were analyzed.

Measurement of Nitrite( H. g! m* X" @- Y( B' t. z' H

Nitrite is a stable end-product of NO and served as an indicatorof endogenous NO synthesis ( 25 ). Nitrite levels in culture supernatant were measured by the Griess reaction ( 13 ).Briefly, 100 µl of sample were mixed with 100 µl Griess reagent[0.05% N -(1-naphthyl) ethylene diamine dihydrochloride,0.5% sulfanilamide in 45% glacial acetic acid] in 96-well plates.After 10-min incubation in the dark, absorbance was read at 546 nm inan automated plate reader (MRX II, Dynex Technologies, Frankfurt/Main,Germany). Standard samples were prepared with sodium nitrite.
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Statistical Analysis: [% I0 [" x* t+ w5 {4 ~7 g5 a

Data are expressed as means ± SE. Statistical analysisbetween the groups was performed by one-way ANOVA and subsequent t -test with Bonferroni correction for multiple comparison. A P value significant.

RESULTS
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Body Weight and Ethanol Intake; I0 M4 w- k! @) |
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In protocols 1A and 1B, there were nosignificant differences in body weight gain between the groupsinvestigated. In protocol 2, the body weight in week15 was significantly lower in both groups of nephritic rats (cGN:515 ± 11 g, cGN C 2 H 5 OH:509 ± 14 g) compared with the normal controls (574 ± 12 g, P This finding is probably areflection of chronic renal disease and insufficiency in these twogroups. In all three experiments, animals drank the 40 ml beer (4.9%ethanol) provided each day. This corresponds to a daily ethanol intakeof 1.96 ml/rat, which, in the acute anti-thy1 animals, resulted in anapproximate ethanol intake of 8-10 ml/kg body wt and, in thechronic anti-thy1 rats, of ~4-6 ml/kg body wt according to theirweight gain over time, respectively. Because the term moderate alcoholintake covers a range of alcohol intakes and the 40 ml provided eachday are close to the usual daily drinking volume of the rats, theamount of beer was not increased in protocol 2. In bothprotocols, ethanol concentrations in the blood taken the morning beforedeath were below a detection limit of 0.02 per thousand. This confirmsthat the alcohol intake actually achieved was moderate.
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Protocol 1A7 T8 g8 u! l3 k; f2 I, `; S9 n

Effect of moderate alcohol intake on the injury phase of acuteanti-thy1 glomerulonephritis. Compared with the normal control animals, injection of anti-thy1antibody resulted in a significantly reduced glomerular cell number(60.5 ± 1.8 vs. 45.8 ± 1.0, P 1 ) as well as basal (1.1 ± 0.2 vs. 9.2 ± 1.5 nmol nitrite/ml, P Fig. 2 A ) and LPS-stimulatedglomerular NO production (3.5 ± 0.9 vs. 43.8 ± 3.9 nmol/ml, P 2 B ), indicating the level of iNOS expression. Compared with the nephritic animals, the 6-day alcohol administration showed no significant action on disease activity[glomerular cell number: 46.3 ± 1.5, basal and LPS-stimulated glomerular NO production: 8.0 ± 1.2 and 40.7 ± 4.3 nmolnitrite/ml, respectively, all P = not significant (NS)vs. aGN; Figs. 1 and 2 ].; s9 t, g8 X" B& t: u- {
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Fig. 1. Effect of moderate alcohol intake( C 2 H 5 OH) on the glomerular cell numberexpressing mesangial cell lysis in rats 1 day after induction of acuteanti-thy1 glomerulonephritis (aGN). Normal control animals (control)were injected with PBS (* P
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Fig. 2. Effect of moderate alcohol intake ( C 2 H 5 OH)on the basal ( A ) and LPS-stimulated nitrite production( B ) of cultured glomeruli (2,000 glomeruli/ml and 48 h). Glomeruli were harvested 1 day after injection of anti-thy1antibody (aGN). Normal control animals (control) received an injectionwith PBS (* P

Protocol 1B% G$ z5 _+ ]* K7 V& A

Effect of moderate alcohol intake on the matrix expansion phase ofacute anti-thy1 glomerulonephritis. Seven days after injection of anti-thy1 antibody, disease wascharacterized by a significant increase in albuminuria (46.8 ± 12.3 mg/24 h; Fig. 3 ), histologicalmatrix accumulation (matrix score 2.9 ± 0.1), and glomerularproduction of TGF- 1 (673 ± 53 pg/ml), fibronectin(5,747 ± 338 ng/ml), and PAI-1 (1,095 ± 103 ng/ml, P 4, A - D ). Providing a 6-day moderate alcohol supply did not significantly limit albuminuria (46.1 ± 9.6 mg/24 h) or reduce the fibrotic response (matrixscore 3.0 ± 0.2, TGF- 1 680 ± 110 pg/ml,fibronectin 5,730 ± 735 ng/ml, PAI-1 1,253 ± 92 ng/ml, all P = NS vs. aGN; Figs. 3 and 4 ). Inducible NO productionof glomeruli in culture was not different between nephritic rats withand without alcohol feeding (aGN: 3.9 ± 1.0 nmol nitrite/ml vs.aGN C 2 H 5 OH 4.4 ± 1.0 nmolnitrite/ml, P = NS).
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Fig. 3. Effect of moderate alcohol intake( C 2 H 5 OH) on albumin excretion 7 days afterinduction of acute anti-thy1 glomerulonephritis (aGN). Moderate alcoholsupply was started 24 h after injection of anti-thy1 antibody.Normal control animals (control) received a PBS injection. Urine wascollected for 24 h using metabolic cages (* P' w' o$ P9 k% ?1 ^+ H' g( p$ L4 c

Fig. 4. Effect of moderate alcohol intake ( C 2 H 5 OH)on glomerular matrix accumulation ( A ) and glomerularproduction of transforming growth factor (TGF)- 1 ( B ), fibronectin ( C ), and plasminogen activatorinhibitor (PAI)-1 ( D ) 7 days after induction of acuteanti-thy1 glomerulonephritis (aGN). Controls received PBS (control).Treatment was started 1 day after disease induction. Glomeruli wereharvested from individual animals and cultured at a density of 2,000/mlfor 48 h (* P; _' _/ B# {/ e! {

Taken together, the results of protocol 1 show that moderatealcohol consumption does not limit mesangial cell injury or subsequent glomerular TGF- overexpression and matrix accumulation in acute anti-thy1 glomerulonephritis.
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Protocol 2
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Effect of moderate alcohol intake on the progression from acute tochronic progressive anti-thy1 glomerulonephritis. As depicted in Fig. 5, chronic anti-thy1glomerulonephritis shows glomerular sclerosis and a progress of thematrix expansion into the tubulointerstitial space. Compared with thenonnephritic controls, fibrotic disease in chronic anti-thy1glomerulonephritis was characterized by moderately elevated bloodpressure (124 ± 2 vs. 138 ± 7 mmHg, P ± 11 vs. 128 ± 31 mg/24 h; Fig. 6 ), increased serumcreatinine (0.5 ± 0.1 vs. 1.0 ± 0.2 mg/dl; Fig. 7 A ) and BUN (57 ± 4 vs.102 ± 25 mg/dl; Fig. 7 B ), and reduced GFR (2.3 ± 0.1 vs. 1.3 ± 0.3 ml/min; Fig. 7 C; all parameters P matrix score wasmarkedly increased (control 1.2 ± 0.3 vs. cGN 3.5 ± 0.6, P 5 ). In addition, nephritic rats showed significantly higher glomerular expression of TGF- 1 (79 ± 17 vs. 170 ± 31 pg/ml; Fig. 8 A ), fibronectin (6,442 ± 413 vs. 12,632 ± 2,905 ng/ml; Fig. 8 B ), and PAI-1(194 ± 14 vs. 430 ± 60 ng/ml; Fig. 8 C; P the tubulointerstitial level, protein expression was increased significantly for TGF- 1 (37 ± 3 vs. 157 ± 35 pg/ml; Fig. 9 A ), fibronectin(5,514 ± 485 vs. 9,909 ± 1,356 ng/ml; Fig. 9 B ),and PAI-1 (443 ± 33 vs. 855 ± 139 ng/ml; Fig. 9 C; P parameters).

Fig. 5. Effect of moderate alcohol intake ( C 2 H 5 OH)on renal matrix accumulation 15 wk after induction of chronic anti-thy1glomerulonephritis (cGN). Moderate alcohol supply was started 5 wkafter disease induction (uninephrectomy and anti-thy1 antibodyinjection) and continued for 10 wk. Normal control animals (control)received an injection with PBS. Renal matrix score ( A ) isthe sum of a combined glomerular and tubulointerstitial matrix score(0-4, each) (# P B ), chronic anti-thy1 animals without( C ), and with moderate alcohol intake ( D ).

Fig. 6. Effect of moderate alcohol intake( C 2 H 5 OH) on albumin excretion 15 wk afterinduction of chronic anti-thy1 glomerulonephritis (cGN). Moderatealcohol supply was started 5 wk after disease induction and continuedfor 10 wk. Normal control animals (control) received a PBS injection.Urine was collected for 24 h using metabolic cages(# P

Fig. 7. Effect of moderate alcohol intake ( C 2 H 5 OH)on renal function 15 wk after induction of chronic anti-thy1glomerulonephritis (cGN). Moderate alcohol supply was started 5 wkafter disease induction and continued for 10 wk. Normal control animals(control) received a PBS injection. Serum creatinine ( A ),blood urea nitrogen (BUN; B ), and glomerular filtration rate(GFR; C ) are shown (# P

Fig. 8. Effect of moderate alcohol intake ( C 2 H 5 OH)on glomerular TGF- 1 ( A ), fibronectin( B ), and PAI-1 ( C ) production 15 wk afterinduction of chronic anti-thy1 glomerulonephritis (cGN). Moderatealcohol supply was started 5 wk after disease induction and continuedfor 10 wk. Normal control animals (control) received a PBS injection.Glomeruli were harvested from individual animals and cultured at adensity of 2,000/ml for 48 h (# P* y$ o* x2 ~2 m! D5 m, X$ r

Fig. 9. Effect of moderate alcohol intake ( C 2 H 5 OH)on cortical TGF- 1 ( A ), fibronectin( B ), and PAI-1 production ( C ) 15 wk afterinduction of chronic anti-thy1 glomerulonephritis (cGN). Moderatealcohol supply was started 5 wk after disease induction and continuedfor 10 wk. Normal control animals (controls) were injected with PBS.Renal cortical tissue was extensively minced and cultured at a densityof 10 mg/ml for 48 h (# P

In chronic anti-thy1 glomerulonephritis, moderate alcohol intake had nosignificant influence on disease severity as shown for albuminuria(125 ± 23 mg/day); renal function (creatinine 0.9 ± 0.3 mg/dl, BUN 104 ± 23 mg/dl, GFR 1.5 ± 0.3 ml/min);histological renal matrix accumulation (renal matrix score 3.6 ± 0.6), glomerular (TGF- 1 175 ± 32 pg/ml,fibronectin 12,098 ± 2,506 ng/ml, PAI-1 453 ± 68 ng/ml);and cortical matrix protein expression (TGF- 1 139 ± 28 pg/ml, fibronectin 10,593 ± 1,274 ng/ml, PAI-1 819 ± 80 ng/ml) (Figs. 5-9; all P = NS vs. cGN).Systolic blood pressure was slightly but not significantly lower inalcohol-fed animals (134 ± 3 mmHg) compared with untreateddisease controls (138 ± 7 mmHg). In vitro stimulation with LPSdid not result in significantly different inducible NO production ofcultured glomeruli (cGN: 0.7 ± 0.2 nmol nitrite/ml vs. cGN C 2 H 5 OH 0.9 ± 0.3 nmol nitrite/ml) orcortical tissue (cGN: 3.8 ± 0.4 nmol nitrite/ml vs. cGN C 2 H 5 OH 3.5 ± 1.2 nmol nitrite/ml)." w. P9 F- K% B3 a4 [" k# z
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Thus the data gathered in chronic anti-thy1 glomerulonephritis areconsistent with the results in acute anti-thy1 animals. Taken together,both protocols show that moderate alcohol consumption neither limitsnor aggravates matrix expansion and iNOS expression in the model ofacute and chronic anti-thy1 glomerulofibrosis.$ v' |- w. t+ ~( @
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DISCUSSION9 }1 C$ b/ w& G$ A6 i. N

Although alcohol consumption is common among the generalpopulation, only little is known about its effect on the course of renal disease. This contrasts with cardiovascular disorders, where several epidemiological human studies have found that moderate alcoholintake, ranging from one to two drinks per day, is associated with alower risk of coronary heart disease, ischemic stroke, dementia, and total mortality, especially in elderly men and women ( 12, 20, 21, 32 ). In experimental studies, furthermore, moderate ethanol intake reduces blood pressure and subsequent renalvascular injury in spontaneously hypertensive rats ( 36 ). In a rabbit model of vascular balloon injury, moderate alcohol intakehas been found to limit neointimal hyperplasia in apressure-independent manner involving less local chemokine expression( 10 ).

To extend these findings to renal disease, the present study employedthe rat model of anti-thy1 glomerulonephritis to test in vivo thehypothesis that moderate alcohol consumption protects from acute andchronic renal matrix accumulation and renal insufficiency. It must beemphasized that experimental circumstances were varied in many ways toallow detection of even small protective effects. These variationsincluded 1 ) the use of acute anti-thy1 glomerulonephritis, amodel in which glomerular matrix expansion is fast and marked and, asrecently shown, even small antifibrotic actions can be detected( 29 ); 2 ) the use of chronic progressiveanti-thy1 glomerulonephritis, in which fibrosis slowly progresses fromthe glomerulus into the tubulointerstitium and little beneficialactions mount up to a detectable level over time ( 23 ); 3 ) determination of the key fibrosis mediatorTGF- 1, which, as shown recently, is a valid andsensitive marker of renal matrix expansion ( 26, 29 ); 4 ) measurement of the matrix protein fibronectin and theprotease inhibitor PAI-1 to allow detection of potential antifibroticactions independent of TGF-; and 5 ) analysis of renalfunction and urinary protein excretion. However, in neither of the twomodels nor on any parameter was moderate alcohol intake associated witha beneficial effect. Because the mechanisms of matrix expansion invarious renal diseases are rather common, the findings of this studymay be relevant for fibrotic renal disease in general. However, the present study does not exclude potential benefits of moderate alcoholintake in other renal models or human kidney disease.
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The precise mechanism of how alcohol intake influences cardiovasculardisease is not fully understood. Several pathways have been proposedand may be involved. Epidemiological studies suggest that moderatealcohol consumption influences cardiovascular risk factors, primarilyblood pressure, but also plasma cholesterol and triglyceride levels,platelet function, and fibrinolytic parameters, thereby preventinginitiation and progression of atherosclerosis ( 12, 20, 21 ). At the molecular and cellular level, ethanol's actionshave been associated with the inhibition of proliferation of many celltypes ( 5, 6, 11, 18 ), suppressed postprandial vascularsmooth muscle cell hypertrophy, a downregulation of PAI-1 expression( 15 ), increased expression of vascular endothelial growthfactor and subsequent angiogenesis ( 16 ), and stimulation of endothelial NO production and action ( 35, 37 ). Takentogether, most of these mechanisms involved in the beneficial actionsof moderate alcohol intake converge in effects that would limit tissue matrix protein production and accumulation. For the present study, wetherefore decided to test the effect of moderate alcohol intake onrenal matrix expansion as a common downstream pathway rather than on anupstream "surrogate" parameter. Because matrix accumulation incardiovascular and in kidney disease shares many similarities ( 4 ), the lack of any benefit of moderate alcohol intake in renal fibrosis is surprising. The reason for this important difference is not clear. One explanation could be that endothelial dysfunction, inwhich many protective effects of alcohol converge as well, may be lessimportant for the course and progression of fibrotic renal disease.2 D5 O$ ]. v% J/ o; y: K1 N9 e
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In addition to matrix expansion, the present study analyzed the effectof moderate alcohol intake on renal inducible NO production for thereason that ethanol has been found to inhibit iNOS in several celltypes in vitro ( 14, 33, 34 ). Induction of iNOS is a keyinjurious stimulus in several models of renal disease, including theinjury phase of acute anti-thy1 glomerulonephritis, acute tubularnecrosis, renal transplant rejection, and lupus nephritis of theMRL/lpr mouse strain ( 27 ). As a consequence of tissueinjury, inducible NO production results in subsequently increasedmatrix expansion, as shown in acute anti-thy1 glomerulonephritis andchronic MRL/lpr lupus nephritis ( 28, 30 ). In the present study, moderate alcohol intake showed no effect on the in vivo induction of iNOS in the injury phase of anti-thy1 glomerulonephritis or on its in vitro activation in cultured glomerular and cortical tissue from fibrotic anti-thy1 animals. This solid finding is highlyconsistent with the results on renal matrix expansion in acute andchronic anti-thy1 glomerulonephritis.( n) F. Z7 H2 n) \  A
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Other than the amount of alcohol, some studies have suggested thatthere might be an association between the type of alcoholic beverageand prevention of cardiovascular disease. A strong case has been madefor what is called the "French paradox." This term refers to thefact that the cardiovascular mortality rate in France is justapproximately half of that of other Western countries, although theprevalence of risk factors is not very different ( 12 ).This phenomon has been related to the high consumption of red wine inFrance. Red wine contains polyphenol compounds, which are strongantioxidants and may mediate protective actions ( 12, 21 ).Furthermore, a recent study showed that red wine directly reducesvascular endothelin expression ( 7 ), which is a key growthfactor in atherosclerosis. In the present study, beer was used tosupply alcohol to rats with fibrotic renal disease. Beer contains highamounts of flavonoids, which are strong antioxidants as well ( 8, 12 ). However, it has to be pointed out that a possiblesuperiority of one type of alcoholic beverage has never beeninvestigated systematically, and most cohort studies do not support anassociation between the preferred kind of drink and the prevention ofcardiovascular disease ( 12 ). Thus, in addition to themoderate alcohol intake achieved in this study, the choice of beer asan alcoholic beverage is probably of less importance for theinterpretation of its results.
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In conclusion, moderate alcohol intake does not influence renal matrixexpansion in anti-thy1 models of acute and chronic progressiveglomerulofibrosis. The effect of alcohol intake in various doses, inother renal models and human kidney disease warrants further investigation.% w, H5 Q2 c0 `
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ACKNOWLEDGEMENTS: R0 O9 s. f6 l* C- B
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The technical assistance of T. Loof and A. Stössel is highly appreciated.
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