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作者：MassimoSabbatini, AntonioPisani, FrancescoUccello, GiorgioFuiano, RaffaeleAlfieri, AntonioCesaro, BrunoCianciaruso, Vittorio E.Andreucci作者单位：Departments of Nephrology and Experimental Medicine, University Federico II, 80131 Naples; and Department of Nephrology, Universityof Catanzaro, 88100 Catanzaro, Italy
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4 B* }. f- X+ C( ]' n$ _1 _ 【摘要】
" D* v/ {# Z ^$ m Exogenous arginine slows theprogression of chronic renal failure (CRF) in remnant rats through anitric oxide (NO)-dependent mechanism. We tested whether the inhibitionof arginase could induce similar results through the increasedavailability of endogenous arginine. Three groups of remnant rats werestudied for 8 wk: 1 ) untreated rats (REM); 2 )remnant rats treated with 1% L -arginine (ARG); and 3 ) remnant rats administered a Mn 2 -free diet toinhibit arginase (MNF). Normal rats (NOR) were used as controls. Liverarginase activity was depressed in MNF rats ( 35% vs. REM, P detected among the groups throughout the study; blood pressure wassignificantly lower in MNF vs. ARG and REM rats after 6 wk ( P depressed in REM rats ( 47% vs. NOR, P in ARG and MNF rats ( 40 and 43% vs. REM,respectively, P with comparable changes inrenal hemodynamics. Despite the better GFR, proteinuria was decreasedin both ARG and MNF rats ( 42%, P 57%, P plasma levels, significantly reduced in REM rats ( 41% vs.NOR, P and urinary nitrite excretion, greatly depressed inREM rats ( 76% vs. NOR, P vs. REM, P only slightly depressed in MNFrats ( 18% vs. REM), but intrarenal concentrations of arginine werelower in this latter group ( P MNF rats showed a lowerglomerular sclerosis index ( P REM and ARG).Inhibition of arginase slows the progression of CRF in remnant ratssimilarly to arginine-treated rats; the better histological protectionin MNF rats, however, suggests that additional factors are involved inthese modifications. 7 u1 q5 h( U6 y- | h
【关键词】 remnant rat arginine nitric oxide chronic renal failure7 r0 s; f# ?1 P* t9 h
INTRODUCTION4 s- _& a' [' t S8 y
2 |3 T" ^2 a/ _. H3 \THE METABOLISM OF ARGININE is particularly complex: this semiessential amino acid,in fact, is endogenously synthesized in renal proximal tubules from L -citrulline (Citr) and is then degraded, through distinctenzymatic routes, to several metabolites that may affect both renalfunction and morphology ( 25, 38 ). The first importantpathway is represented by arginase, mostly present in the liver and toa much lesser extent in the kidney, which leads to formation of ureaand ornithine (Orn), the precursor of polyamines and proline involvedin cell-replication turnover and collagen synthesis ( 25 ).The second metabolic route is constituted by the arginine decarboxilase(ADC), active in the brain and in the kidney, which forms agmatine, asubstance able to induce several positive effects on renal function( 18 ). The last important pathway is nitric oxide (NO)synthase (NOS), with at least two different isoforms: endothelial(eNOS) and inducible (iNOS). The former synthetizes small amounts ofNO, which modulate systemic hemodynamics through a paracrine action;conversely, the stimulation of iNOS, mostly represented in inflammatorymigrating cells, produces huge amounts of NO, which enhanceinflammation and generation of free radicals, potentially harmfulto the cells ( 16 ).) R% p8 `: X2 \4 R+ q0 g
: Q/ f# G6 r. j0 ~In the last decade, particular attention has been given bynephrologists to arginine-derived NO because of its role in both regulating renal hemodynamics and modulating inflammatory and proliferating response to various stimuli. Several experimental studies, in fact, have unequivocally demonstrated that it is possible to increase the availability of NO by the administration of exogenous supplements of arginine, as witnessed by the increased plasma andurinary levels of nitrites ( 2, 11, 23 ), or to reduce itsproduction by the use of specific inhibitors of NO synthesis ( 6, 10, 28 ). Promising results have been obtained inarginine-treated rats in nonimmunological models of chronic renalfailure (CRF) ( 2, 11, 15, 27 ) and in other experimentalconditions ( 6, 24, 26, 28 ), which seemed to open new,encouraging perspectives even in curing human renal failure.
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1 `. h6 s. b8 F3 a% BUnfortunately, these experimental results have not been confirmed inimmunological models of renal diseases like glomerulonephritis ( 22 ), in which supplements of arginine were even harmful,nor in patients with chronic renal insufficiency. Indeed, from a unique prospective, randomized trials in which adequate doses of arginine wereadministered for a prolonged period of time to patients either with CRFor with renal transplantation failed to induce any beneficial effect onGFR, hypertension, and proteinuria ( 9, 39 ).
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4 A& h4 o( l; a# r, jThe confounding results of all these studies may be partially explainedby the complex metabolism of arginine, conditioned by the presence ofthe different enzymatic pathways in organs and cells, and by thechanges in such activities in response to diet, hormones, cytokines,and even the type of renal disease ( 38 ). This suggests thepossibility of exogenously modulating the metabolism of arginine in anattempt to modify its impact on renal disease. Because activation ofany of the pathways of arginine catabolism is strictly dependent on theconcentration of both substrate and enzymes into the cells( 21 ), it is reasonable to argue that the partialinhibition of arginase, the main system in degrading arginine, couldoffer higher amounts of arginine to the remaining routes.
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1 u# Q \ L2 V8 b4 ~; E7 UThus this study was undertaken to figure out whether the progression ofCRF in rats was influenced by the inhibition of liver arginase. Wetested this hypothesis in the "remnant" rat, a nonimmunological model of CRF, not only because it shares three critical aspects withhuman CRF (anemia, proteinuria, hypertension) but mostly because thismodel is also characterized by the spontaneous downregulation of iNOS( 1 ). This should allow available arginine to bemetabolized mostly by pathways "beneficial" to the kidney like eNOSor ADC. Arginase inhibition was obtained by the administration of aMn 2 -free diet, because Mn 2 is a key cofactorfor arginase activity; with respect to other inhibitors of arginasecommonly used in in vitro studies (like borate, valine), a dietarydeficiency of Mn 2 can decrease arginase activity to areasonable extent, with no side effects in the young rat( 5 ). Today, new inhibitors are also available, like N G -hydroxy- L -arginine, which ismostly used in cells because of its short half-life and yet has anagonist action on NO synthesis, and S -(2-boronoethil)- L -cysteine, which can be usedin vivo ( 36 ) but was not available to us when we startedthe study.6 E$ e! x& d& i& a+ Y$ q
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MATERIALS AND METHODS
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The study was carried out in 84 male Sprague-Dawley rats(Charles River), with an initial body weight of ~300 g. Chronic renal failure was induced by 5 6 ablation of renal parenchyma, whichinvolved a right nephrectomy and, 1 wk later, ligation of two or threebranches of the left renal artery, both under light pentobarbitalsodium anesthesia (Nembutal, 40 mg/kg). Immediately after completion ofthe surgery, the rats were randomly assigned to one of the followingexperimental groups: REM [untreated remnant rats used as controls( n = 10)]; ARG [remnant rats treated with asupplement of L -arginine (1% wt/vol) in tap waterthroughout the study ( n = 13)]; and MNF [remnant ratstreated with a Mn 2 -free diet throughout the study toinhibit arginase activity ( n = 10)] ( 5 ).Nine unmanipulated littermates were used to assess the normal values ofthis strain of rats (NOR).
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) G# K: E* D: u3 S% t ^The Mn 2 -free diet was similar to the control diet inprotein content (18% casein), the number of calories, vitamins, andminerals, and L -arginine concentration (0.96 g/100 g)(Altromin, Rieper, Italy). Recent studies have shown that a 1-moadministration of a similar diet results in a 26% decrease in arginaseactivity ( 5 ).
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One week after randomization, the rats were placed in individualmetabolic cages and, after a 3-day acclimatization period, a complete,timed 24-h collection of urine was performed for determination ofurinary protein excretion; body weight and food and water intake werealso recorded ( time 0 ). The same metabolic study wasrepeated 4 and 8 wk later. The duration of our experimental study was 8 wk to avoid possible complications resulting from more prolonged Mn 2 deficiency and from the accumulation of ammonia afterinhibition of arginase, which, in mammals, represents the main pathwayof removal of this toxin from the body; preliminary 12-wk studies, however, were executed to evaluate the effects of aMn 2 -free diet in normal rats ( n = 8) andto test the survival of remnant rats on such a diet ( n = 10). On the other hand, a peculiar resistance to retainMn 2 and to detoxify ammonia via glutamine synthesis hasbeen suggested in rats ( 5 ). r/ W; v0 ~5 h2 m( }3 }' i( y# I
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Systolic blood pressure (SBP) was measured by the tail-cuff method 2 wkafter completion of surgery and during week 6 of the observation period: the average of three measurements was used as asingle value for each rat. At the end of the last clearance study( week 8 ), the animals were anesthetized with Inactin (80 mg/kg body wt ip) and prepared for a hemodynamic study, withdetermination of inulin clearance and renal inulin extraction (bloodcollection from the renal vein through a sharpened micropipette), aspreviously reported ( 28 ). At the end of the experiment,bioptic samples for renal histology were collected.
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Six additional remnant rats for each group were anesthetized 8 wk aftersurgery to obtain, after adequate stabilization (60 min), a sterilespecimen of urine under ice (4°C) for determination ofNO 2 /NO 3 urinary excretion. An arterial bloodsample was then collected for determination of plasma concentration ofarginine, Citr, and Orn. Finally, specimens of hepatic and renal tissue were also obtained under sterile conditions for determination ofenzymatic activities. Six normal littermates were also killed to obtainurine, plasma, and hepatic and renal tissue for determination ofnormal, control values of our rat strain (NOR).
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Histology
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1 r& M: {( O; F: X6 JFor the evaluation of the effects of the different regimens onrenal pathology, kidney biopsies were evaluated in a blind fashion.Plastic-embedded 3-µm sections were cut, and hematoxylin-eosin, periodic acid-Schiff (PAS), and Jones staining were performed. Asrecently suggested ( 12 ), histological analysis was madeusing the Banff criteria, grading (0-3) the followingvariables: glomerulosclerosis, arteriosclerosis, and interstitialfibrosis/tubular atrophy. In particular, glomerulosclerosis was definedas mild (score = 1), in the case of an increase in the mesangialmatrix with broadening of mesangial areas up to the diameter of twomesangial cells; moderate (score = 2), in the case of broadeningof mesangial areas by more than the diameter of two mesangial cells;and severe (score = 3), in the case of mesangial sclerosiscorresponding to at least 25% of the glomerular area. The score forarteriolosclerosis was assigned on the basis of severity of thefibrointimal thickening of arteries. A combined score was given tointerstitial fibrosis/tubular atrophy, defined and graded according tothe criteria defined by the Banff group (22a). To increasethe sensitivity of the method, the score of the lesions found in eachglomerulus and artery within each biopsy was recorded and the sum ofthe scores was added, giving a ratio as score/number of glomeruli orarteries in each biopsy ( 12 ). A similar approach was usedfor the evaluation of tubular atrophy/interstitial fibrosis: in thiscase 10 different high-power (×400) microscopic fields within eachbiopsy were examined.5 a, y* w( q( [+ d4 D& i+ I
4 f5 c i0 y) ]' iAnalytic Determinations X% X" U4 X+ E) w$ J" W2 {) x
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Urinary volume was measured gravitometrically in preweighedvials. The concentrations of inulin were measured by the diphenylamine method ( 35 ); urinary protein concentration was assayed bya commercial kit (pyrogallol, Sigma). The urine for determination ofurinary nitrate concentrations was collected in preweighed sterilevials under ice (4°C), filtered through a 0.2-µm filter (Acrodisc,Gelman), and frozen until the time of assay ( 80°C). The dosage wascarried out by a total NO assay utilizing nitrate reductase and theGriess reagent (RD Systems).
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8 `6 J( n8 l' G+ u# k% z( L0 U: X8 \Arginine, Orn, and Citr plasma levels were measured by an amino acidanalyzer (Gold System, Beckman). Arginase activity, both in liver andin kidney specimens, was calculated as the amount of Orn produced inthe presence of excess arginine. Briefly, ~1 g of tissue wasimmediately homogenized in 4 ml of 0.15 mol/l KCl in 0.05 mol/l sodiumphosphate buffer (pH 7.5) at 4°C. Homogenates were centrifuged at 400 g for 10 min at 4°C. The supernatant was then diluted 10 times with 0.025 mol/l Tris · HCl. One hundred microliters of diluted tissue homogenates were incubated for 5 min at37°C with 300 µl of 0.025 mol/l Tris · HCl(pH 7.2) and 100 µl of 0.05 mol/l L -arginine. Afterincubation, the samples were deproteinized by the addition of 1 ml of1.5 mol/l HClO 4 and vortexed. The acidified solution wasneutralized with 0.5 ml of 2 mol/l K 2 CO 3 andcentrifuged at 12.000 g for 1 min. The supernatant wasremoved and diluted 10 times with HPLC-grade water and analyzed for Ornby HPLC ( 5 ).
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# c) c, M; H; a) OStatistics
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) ^ e$ F% h: qOne-way ANOVA was used to compare the different mean values inthe three experimental groups. Bonferroni's test was used to findsignificant differences among the groups under study. Student's t -test for unpaired data was used where indicated.Histological data were analyzed by the Kruskal-Wallis one-way ANOVAfollowed by Dunn's multiple comparison test. A P value statistically significant. Data are expressed asmeans ± SD.2 P6 ~% L c8 V, l
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RESULTS
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. ]& b$ g% o& [: IPreliminary Studies
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The administration of a Mn 2 -free diet did not alterbody weight gain in NOR rats ( n = 8) up to 12 wkcompared with rats on a normal diet (unpublished observations).In these rats, the reduction in liver arginase activity averaged 31%,demonstrating the efficacy of our experimental approach to the problem.A preliminary study in REM rats administered such a diet for up to 12 wk ( n = 10) showed the survival of all but one rat(death during week 3 ).
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Characteristics of the Experimental Model
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Ablation of 5 6 of renal parenchyma resulted in a 37辌rease in GFR values in REM rats compared with NOR rats; thismodification was associated with a rise in mean blood pressure, adecrease in hemaocrit values, and an obvious reduction in total kidneyweight (Table 1 ). Proteinuria, measuredin 24-h samples, was significantly higher in remnant rats at the end of the experimental period (Table 1 ). Arginase activity was similar between REM and NOR rats, but arginine plasma levels were significantly depressed in remnant rats ( 41%, P of Orn and Citr were significantly higher, ascommonly described in CRF. Finally, renal arginase activity was reducedto a significant extent in REM rats ( 53%, P were observed in arginine and Orntissue levels (Table 1 ). Urinary excretion of nitrites was dramaticallyreduced in the REM group (2.28 ± 0.59 vs. 0.67 ± 0.37 nmol · min 1 · gkidney wt 1, P
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. c" K$ L/ `# R# nTable 1. Main hemodynamic and biochemical data of normal rats and remnantuntreated rats1 G. U: R* a; |
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Metabolic Studies# J* b0 v; O5 S9 O6 X8 ~) O: l/ v
( {' D4 b, T3 w) t7 }$ w# q8 U7 gThere was no difference in body weight and food intake (andconsequently of protein, phosphorus, and other nutrients) among thethree groups of remnant rats and NOR rats at the end of the 8-wkexperimental period (Table 2 ).Twenty-four-hour proteinuria, comparable among the groups after 1 wk,was significantly lower in both ARG and MNF rats compared withuntreated REM rats ( 42%, ARG vs. REM, P 57%, MNF vs. REM, P end of theexperimental period (Fig. 1 ).# d& @9 E8 S# K( O ]1 _1 `+ C2 `
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Table 2. Body weight and systolic blood pressure in the different groupsunder study: k! b3 W7 Z2 g: J$ d M) P
2 D# }: Y* t; y6 w7 M% RFig. 1. Twenty-four-hour urinary protein excretion(U prot ) in the 3 groups of remnant rats under study[untreated rats (REM; n = 10; open bars); remnant ratstreated with L -arginine (ARG; n = 13; graybars); remnant rats administered a Mn 2 -free diet (MNF; n = 10; filled bars)]. * P # P: r/ l+ \' w0 ?. ]
2 b* J# z7 |' ]% b1 G- YSBP, which was similar in the three groups of rats after the first 2 wkof observation, was significantly lower in MNF rats during week6 ( P ARG).
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Hemodynamic Studies
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Mean blood pressure was similar in the three groups of remnantrats during the experiments, despite a tendency to be slightly higherin MNF rats [ 13% vs. REM, not significant (NS)] (Table 3 ). GFR, measured as inulin clearance,averaged 0.832 ± 0.17 ml/min in the REM group. Arginine treatmentdetermined a slower progression of renal failure, with a GFRsignificantly higher than in the REM group ( 40%, P observed in the MNF group ( 43% vs.REM, P was explained bycomparable hemodynamic changes: in fact, in both groups we observed areduction in renal vascular resistance (RVR) compared with REM (ARG 36%, P 25%, NS), leading to amarked increase in renal plasma flow (ARG 58% and MNF 53% vs. REM,respectively, both P numerically decreased in ARG and MNF rats ( 10 and 9%,respectively, vs. REM, NS).
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Table 3. Hemodynamic data in the groups under study8 o) m# B0 W1 ^# ?. N, C
6 {8 S& S/ F8 EBiochemical Studies3 ]# P+ v; a. F7 U* p
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Liver homogenates. The Mn 2 -free diet decreased arginase activity in MNF ratswith respect to the other groups ( 35% vs. REM and ARG, both P 0.01); this influenced the tissue concentrationsof Orn, which was lower in MNF rats ( 32% vs. REM, NS, and 45% vs.ARG, P 0.05) (Table 4 ). It is noteworthy that measurablearginine levels could not be detected in liver parenchyma in any of thegroups under study.
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0 K, A3 ]" ^* p% x6 zTable 4. Biochemical parameters in the groups of rats under study
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}2 o! M0 z' A O2 u4 aPlasma and urine. The administration of exogenous arginine (ARG rats) significantlyincreased its plasma level ( 71% vs. REM, P to a higher production of Orn ( 34% vs. REM, NS). In MNF rats, the inhibition of arginase determined a rise in plasma arginine by 38%compared with REM rats (NS). Orn concentrations in MNF rats wassignificantly lower than in the ARG group ( 32%, P 0.05), reflecting both the different baseline plasma levels of arginineand the partial inhibition of arginase.
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Arginase inhibition also determined a marked increase in urinarynitrites in MNF rats (2.05 ± 0.9 nmol · min 1 · gkidney wt 1, P rats after arginineadministration (2.99 ± 0.9 nmol · min 1 · gkidney wt 1, P with the respective plasma levelsof arginine are shown in Fig. 2.% R+ o( o* R' v) _, V) q& D2 I
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Fig. 2. Plasma arginine concentration (µmol/l, open bars) andurinary nitrate excretions(nmol · min 1 · gkidney wt 1, filled bars) in the groups under study( n = 6 rats/group). * P § P
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Kidney homogenates. Renal arginase activity was greatly decreased in all the groups ofremnant rats. Arginine administration slightly increased, and aMn 2 -free diet further decreased this enzymatic activity,with an average difference of 25% between the ARG and MNF groups( P 0.05) (Table 4 ). Intrarenal arginineconcentration was dramatically lower in MNF rats ( 55% vs. REM, NS;and 71% vs. ARG, P nevertheless, Ornconcentration in the MNF group was similar to that in the REM group.) K1 V3 Z9 J0 J0 E7 W
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Histological Results) o; P7 m: t3 g. b4 o
- l3 m' K/ w' G1 B7 TTable 5 depicts the biopsy findingsin the four groups of rats. NOR rats had no sign of histologicaldamage. The morphological changes in the three experimental groups ofrats were of low grade. The score for glomerulosclerosis, however, wassignificantly lower in the MNF group compared with the other twoexperimental groups (both P significantly moresevere in all three groups of rats with remnant kidneys, withoutdifferences among them. The degree of arteriolar sclerosis wasnumerically lower in MNF rats with respect to the other groups.
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" N$ i( e4 U' yTable 5. Histological data in the 4 groups of rats under study: h3 O& S1 ]5 E+ V& T4 a F( l
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Our data demonstrate that the partial inhibition of arginaseactivity in remnant rats is able to slow the progression of CRF, preserving GFR to the same extent as exogenous arginine administration. Inhibition of arginase activity, however, confers an additional histological protection to the kidney, witnessed by the lower degree ofglomerular sclerosis compared with REM and ARG rats.# E" @3 ]2 d! ?% a& z! T$ c/ c
" W& H; P7 c' fAs observed in previous works using the same experimental model( 2, 11, 25, 24 ), our rats treated with arginine show beneficial effects on renal function (GFR 40% vs. REM). In all ofthese studies, the effects of arginine on GFR, proteinuria, and renalhistology were quite variable, mostly depending on the different ratstrain, length of the observation period, and entity of residual renalfunction; one circumstance, however, seems constantly confirmed: ratsadministered low doses of arginine (0.1-0.125% wt/vol of tapwater) have significantly lower values of blood pressure andproteinuria than their untreated remnant controls ( 2, 11, 31 ), whereas higher doses (1%) do not affect blood pressure.This distinction seems very important because inhibition of arginase inour study mimics the condition of "low" arginine supplementation:improved GFR, lower blood pressure, and reduced proteinuria. Moreover,the great increase in urinary nitrite excretion in MNF rats ( 206% vs.REM), despite incompletely restored plasma levels of arginine, stronglysuggests that our experimental model enhances the production and theaction of NO. A further confirmation of the role of NO as mediator ofthese modifications is that the changes in renal hemodynamics afterarginase inhibition overlap those observed after arginineadministration, i.e., a similar improvement in GFR and renal plasmaflow and a slight increase in filtration fraction, factors denotingglomerular vasodilatation.( a+ X, N% k' B; ~
3 S/ }" n8 j o4 a+ pIn contrast to other studies reporting near-normal plasmaconcentrations of arginine in rats with CRF ( 25, 27 ), ourREM rats show significantly lower plasma levels of this amino acid compared with normal rats (Table 1 ). This finding, however, is notsurprising; in the presence of high concentrations of Citr, thesynthesis of arginine by renal tubular cells is conditioned by thenumber of functioning nephrons ( 4 ); its reduction cannot allow a normal cumulative renal release of the amino acid, and, indeed,a 60% reduction in Citr uptake has been observed in the kidneys ofremnant rats ( 25 ). Therefore, normal plasma arginine concentration in CRF must depend on its extrarenal synthesis and release, as demonstrated in patients with CRF with a high protein catabolic rate ( 34 ). This probably occurred in the studyby Reyes et al. ( 27 ), in which plasma arginine levels werenormal or were increased by a high dose (1%) of the amino acid. These rats, in fact, had very extensive renal ablation (7/8) and a very lowbody weight gain throughout the study (~0.7 g/day). Our rats, on thecontrary, have greater residual kidneys, better renal function, and anear-normal weight gain (2.7 g/day), all factors that seem to exclude ahypercatabolic state. Furthermore, arginine administration to rats inthe present study normalizes its plasma values, unmasking a realdeficit in its renal synthesis not compensated for by extrarenal sources.
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The variability of plasma arginine concentration in the differentgroups in our study is particularly intriguing. In fact, despite thefact that intracellular levels of arginine are 10-fold higher than the K m of eNOS ( 21 ), the intracellularpool of the amino acid is poorly accessible to the enzyme, which isthus presently activated by extracellular (plasmatic) arginine( 19 ). As a consequence, when the plasmaconcentration of arginine is somehow raised, this may play a relevantrole in enhancing the synthesis of NO ("arginine-eNOS paradox"), aswell as of other metabolites whose enzymatic pathways are not inhibited(agmatine?).- M3 V& x$ }9 J7 f9 t( T
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A striking finding of this study is the significant preservation of thekidneys from glomerular damage in MNF compared with REM, and even withARG, rats despite the fact that this latter group has a hemodynamicpattern comparable to that of MNF rats. These results can be ascribed,to a great extent, to the lower SBP observed in MNF rats up to 6 wk ofobservation (Table 1 ). The importance of reducing blood pressure toslow the progression of CRF, in fact, is widely recognized in manyexperimental and clinical studies, with the best results obtained withangiotensin-converting enzyme inhibitors, which increase theavailability of NO ( 13, 14, 17, 20 ). All of these studieshave clearly demonstrated the direct correlation between the levels ofSBP and both the functional and histological damage in the kidney; thisstudy further strengthens how lower levels of SBP may positivelyinfluence the course of CRF, mostly when associated to a greateravailability of NO, as in the case of MNF rats.' {1 f0 I) p: V1 V. F, y3 B
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The rise in mean blood pressure observed in MNF rats during thehemodynamic studies is surprising; unexpectedly, these rats afteranesthesia have blood pressure values higher than those observed in theawake state, and even higher than in REM and ARG rats. The lower valuesof proteinuria and the best preservation of renal histology andfunction, however, indicate that this rise in blood pressure is anacute phenomenon, limited to the time of the experiment and possiblyrelated to Mn 2 deficiency. An interaction betweenMn 2 and barbiturate anesthesia has been described in therat ( 8 ); whether this interaction could modify bloodpressure (through altered enzymatic activities), however, remainsspeculative. This increase in blood pressure can explain thenumerically higher values of RVR in MNF rats.! e3 c! U- N1 y/ g& E. w: ]
( I3 z, _' ?1 A: y& k9 \Finally, it is interesting to observe the different behavior of hepaticand renal arginase: the condition of CRF determines a sharp decrease inrenal arginase activity in all the groups of remnant rats, barelyaffected by the high arginine tissue levels in the ARG group; the useof the Mn 2 -free diet, then, does not depress renalarginase activity to the same extent as hepatic arginase. Hepatic andrenal arginase, indeed, represent two different isoforms of the enzyme,encoded by separate genes, with different intracellular locations(cytosolic in the liver and mithocondrial in the kidney), and awell-described isoform selectivity for the binding of both substratesor inhibitors ( 7 ). Despite both arginases requiringMn 2 for their activity, it remains unclear why MNF ratsfailed to show a greater decrease in the renal isoform. Severalpossibilities may be taken into account: 1 ) a lower affinityfor this isoform for Mn 2 ; 2 ) a greater abilityto retain the ion because of the segregation of the renal enzyme insidethe mitochondria; and 3 ) an increased compensatory synthesisof this enzyme secondary to the sharp decrease in liver activity, asdemonstrated in inherited defects of liver arginase ( 33 ).It is certain, however, that the deficiency of Mn 2 inducessome peculiar changes in the kidneys of MNF rats, as shown by the verylow concentrations of arginine in their renal tissue. The disappearanceof arginine from renal tissue, however, is only "virtual" becauseboth the high renal levels of Orn (similar to NOR rats despite 70%lower levels of arginine) and the consistent excretion of urinarynitrites suggest that, conversely, a consistent amount of the aminoacid is present in the kidney. It is possible that arginine ismetabolized very quickly, as occurs in the liver where the highmetabolic rate of the urea cycle makes arginine undetectable( 38 ), which was also observed in this study. It may onlybe hypothesized that the partial inhibition of renal arginase (orMn 2 deficiency) has somehow switched on a tight couplingof enzymatic activities responsible for a more rapid degradation ofarginine, possibly through the ADC pathway, and also because thisenzyme is located in mitochondria and the action of agmatine on renal dynamics is partially mediated by NO. Agmatine, in fact, leads tovasodilatation through stimulation of eNOS ( 30 ) andinhibits the activation of iNOS after cytokine stimulation( 3 ); moreover, agmatine can reduce cellular proliferationby inhibition of Orn decarboxylase (and, indeed, renal Ornconcentration was higher in our MNF rats despite reduced levels ofarginase and of arginine) and can suppress polyamine transport insidethe cells ( 29 ). This is in agreement with the lower indexof glomerular sclerosis in our MNF rats. Although Mn 2 is akey cofactor in many other enzymatic activities (like superoxide dismutase, catalases, or xanthine oxidase), it seems unlikely that ourmodel of Mn 2 deficiency may have greatly influenced thesepathways, which play a relevant role in protecting the cells fromoxidative injury. In fact, as a final result their inhibition shouldresult in worsening of both renal function and morphology, rather thanin the observed beneficial effects.7 t2 C+ R! L* C1 ~8 z
7 W, y' W g# \# ]$ gFurther studies, however, are necessary to determine ADC and eNOSactivities in renal tissue, as well as agmatine concentration, toevaluate whether different inhibitors of arginase are able to elicitsimilar responses in the kidney and to exclude undesired effects due toMn 2 depletion. These efforts could lead to adefinition of a pharmacological approach also potentially useful in humans.
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O0 w( @9 {3 Q" j2 M: OIn conclusion, our in vivo data demonstrate that inhibition of arginaseis capable of producing several beneficial effects in remnant rats withCRF, like preservation of GFR, reduction in proteinuria, decrease inblood pressure, and lowering of histological damage at the glomerularlevel. These changes seem to be linked to a greater availability of NOfrom endothelial cells. With respect to previous models in whicharginine was administered exogenously, our experimental approachsuggests that inhibition of arginase could be a more effective targetto enhance NO production and slow the progression of CRF. This couldopen new perspectives into the treatment of patients with CRF, who havea reduced basal production of NO ( 37 ) but showed nobenefit from exogenous arginine administration. New studies are inprogress to clarify the molecular mechanisms of these interesting,although theoretical, data in experimental animals.
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2 G+ C" r8 g5 q# C3 W8 xACKNOWLEDGEMENTS) u9 d: R9 ^, U6 u/ A4 U: U
4 p7 t6 ~! S$ R3 g8 }) OThis paper was financed in part by Ministero dell'Università e della Ricerca Scientifica e Tecnologica (COFIN 1998)." ^& m+ H% O) q6 y. ?
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