INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Vol. 23, no. 2,523-533 (2010)

SUPPLEMENTATION WITH ESSENTIALAMINO ACIDS IN MIDDLE AGE MAINTAINSTHE HEALTH OF RAT KIDNEY

G. CORSETTI, A. STACCHIOTTI, G. D'ANTONA" E. NISOLI2, F.S. DIOGUARDI3ANd R. REZZANI

Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, (Jniversity ofBrescia, Brescia; Division of Human Physiology, Department of Experimental Medicine, Universityof Pavia, Pavia; 2Department of Pharmacology, Chemotherapy and Medical Toxicology, University

of Milano, Milano; 3Department of Internal Medicine, University of Milano, Milano, Italy

Received November 26, 2009 - Accepted March 16, 2010

Chronic kidney diseases are a social and economic problem, and diet has long been recognized as afundamental modulator of kidney health in human and experimental models. Age-dependent alterationsin mitochondrial function play a crucial role in the development of diseases of aging, and mitochondrialdisorders have been observed in experimental models of kidney failure. Recently, the beneficial dietaryeffect of a specific mixture of essential amino acids (EAA) has been studied in elderly subjects, but nodata were collected from the kidney. The aim of this study was to assess whether daily supplementationof the diet with EAA at the beginning of senescence could preserve renal health. We used middle-aged(18-month-old) male Wistar rats fed a standard diet and water ad libitum (M-aged group) or a diet withadded EAA (1.5 g/kg per day) dissolved in drinking water for 3 months (M-aged+EAA group). Young (2-month-old) rats fed a standard diet for 3 months were used as controls. Mitochondrial morphology andmarkers for collagen, cyt-c-oxidase, HSP60, GRP75, eNOS, iNOS, Baxo Bct2 and VEGF were analyzed inglomeruli and tubules. EAA supplementation limited fibrosis and increased the capillary tuft area in theglomeruli of M-aged rats. VEGF and eNOS were enhanced in glomeruli and the peritubular space withthe EAA-supplemented diet. Mitochondrial cyt-c oxidase, Bcl2, and chaperones increased in the distattubules of the EAA group to levels similar to those observed in the young group. Mitochondrial areaand density after EAA intake did not differ from young groups. The results suggest that prolonged EAAintake could represent a strategy for maintaining the healthy status of the kidney in M-aged animals.

Chronic kidney diseases (CKD) and theirprogression to end-stage renal disease (ESRD) areconsiderable social and economic problems in allindustrialized countries. Each year in Italy, morethan 18,000 patients are diagnosed as having CKD(1). The incidence of CKD is strictly linked to age:in patients over 75 years it was almost 7 times that

Mailing address: Dr Giovanni Corsetti,Division of Human Anatomy,Department of Biomedical Sciences and Biotechnology,University of Brescia,viale Europa, ll 25124 Brescia, ItalyTel: ++39 0303717484, Fax: ++39 0303717486e-mail address: corsetti@med.unibs.it

in patients aged 20-39 years and more the twicethat in patients aged 40-59 years (2). Therefore,interventions that may prevent the development ofCKD in middle age are very important for publichealth.

Reactive oxygen species (ROS) and nitrogenspecies are responsible for human aging

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Key words: kidney, mitochondria, essential amino acids supplementation, aging, rat

523

s24 G. CORSETTI ETAL.

independently of specific pathologies (3). Oxidativedamage and mitochondrial dysfunction are alsodirectly involved in several chronic disorders such asnephrotic syndrome, glomerulosclerosis, and ESRD.Altered bioavailability of antioxidant superoxidesand nitric oxide (NO) are major factors in vascularand glomerular changes in aging (4).

Diet has long been recognized as a fundamentalmodulator of kidney health in both human andexperimental models (5-6). In particular, caloricrestriction (CR) retards the progression ofmany age-related molecular, physiological, andpathological processes when initiated at early agesin mammalian models (7). Recently, in rat kidney, itwas demonstrated that initiation of CR at l8 months,before the development of significant age-relatedchanges, reduces renal damages (8).

Age-dependent alterations in mitochondrialfunction play a crucial role in the development ofdisease in aging (9-10). Thus, the maintenance ofhigh mitochondrial activity in middle-aged andelderly subjects would be useful for preventing orat least retarding the development of age-relatedkidney diseases.

The beneficial dietary effect of a specific mixtureof essential amino acids (EAA) (I2-L-Amin@,Professional Dietetics, Milan) has recently beenstudied in elderly subjects, in both experimentalanimals and in clinical trials. EAA supplementationsignificantly increases the number and volume ofmitochondria and sarcomeres in skeletal musclesand hearts of old mice, counteracts age-inducedsarcopenia, improves mitochondrial ATp production,and enhances physical performance in aged ratsFurthermore, EAA supplementation has beneficialeffects in hypercatabolic syndrome and attenuatesmetabolic and functional damage in the diabetic ratheart, improving mitochondrial activity (l l).

These results suggest that long-term EAAsupplementation could represent a novel nutritionalapproach to maintaining the health of organs in agedsubjects. EAA supplementation retains intrinsicmitochondrial activity, and its effects are very similarto the advantages obtained with CR. However, mostprevious studies have been carried out on skeletaland cardiac muscle. Here, we examined the effectof EAA intake on the kidneys of middle-agedrats by examining changes in markers related to

mitochondrial, vascular, and tubular wellness.

MATERIALS AND METHODS

AnimalsThe experimental protocol was approved and

conducted in accordance with the ltalian Ministry ofHealth and complied with the National Animal protectionGuidelines. Seventeen male Wistar rats were used:5 young (2-month-old) animals, and 12 middle-aged(M-aged; l8-month-old) animals. We chose these agesbecause mitochondrial enzyme abnormalities are rare atl8 months, whereas by 24 months, mitochondrial enzymealterations are seen in animals on a standard diet (8).

The animals were caged separately. M-aged animalswere divided into 2 groups: control group (M-aged, n :6) and EAA treated group (M-aged+EAA, n : 6). Theanimals were placed in a quiet room with the temperatureand humidity controlled. A l2ll2-h light/dark cycle wasmaintained (lights on from 7 a.m. to 7 p.m.). The rats werefed a standard diet ad libitum (18.8% protein content;Dottori Piccioni, Gessate, Milan, Italy) with water adlibitum (control M-aged and young groups) or a dietsupplemented with EAA solution (M-aged+EAA group)that provided 1.5 g/kg per day of EAA (I2-L-Amin@,Professional Dietetics, Milan, Italy) in the drinkingwater for 90 days. The concentration of the EAA in thedrinking water was adjusted to the average daily waterconsumption of the rats (about 25 mL) and mimickedthe recommended daily dose for humans (12). Thecomposition of the EAA supplement is shown in Table I.The body weight and water or EAA consumption of eachanimal were monitored daily.

At the end of the treatments, the animals weresacrificed under deep ether anesthesia. The kidneys werequickly removed and placed in an ice-cold saline solution.The samples used for histochemical analysis werecarefully mounted in Tissue-tek@ ocr (Sakura FinetekEurope, the Netherlands) embedding medium before theywere frozen in liquid nitrogen and stored at -80oC.

Trans m is s ion e I e c tron micros copyOne kidney from each animal was removed, fixed with

3o/o gltúaraldehyde in cacodylate bufîer (pH 7.4, 0.2M),and postfixed for t hour with I %o OsO4in the same buffer.The samples were processed with standard procedures forembedding in Araldite (Sigma-Aldrich Chemical Co,Milan, Italy) and polymerized at 60.C for 72 h. Thicksections (about I pm) were stained with Epoxy TissueStain (#14950, Electron Microscopy Sciences, pA, USA).ultrathin sections (70 nm) were stained with a saturatedaqueous solution of uranyl acetate and lead citrate and

Int. J, Immunopathol. Pharmacol. 525

examined with a Philips CM10 electron microscope(Royal Philips Electronics, Amsterdam, the Netherlands)

at 80 KV.Data on mitochondria were collected from 30 randomly

selected areas from each sample at a final enlargement ofx8900 using electron microscope film (Kodak electron

film 4489, 6.5 x 9 cm, New York, USA); 5 different

section levels of each sample were examined. The total

area examined was about 90,000 pm2 in each group. The

number of cortical glomeruli examined for morphometrywas about 180 in each group.

MorphometryAll measurements were obtained using standard

morphometric techniques, as previously described (13).

We calculated the area of the cytoplasm (Acyl), the area

of mitochondria (Amit), and the number of mitochondria(Nzir). The number of glomeruli (Ng/o), the mean area ofglomerular capillaries (Aglo), the mean area of the renal

corpuscle (Acor), and total area of the renal parenchyma

(Atot)were evaluated on thick plastic sections stained withtoluidine blue. From the mitochondrial data we calculated

the ratio between Amit and Acyt (AmitlAcyt) and the

number of mitochondria in 100 pm2 of cytoplasm, also

called the mitochondrial density (Nmit/l00 pmr). Fromglomerular data we calculated the ratio between Aglo and

Atot (AglolLtot) and the number of glomeruli per unitarea, also called the glomerular density (Ngio/mmr).

HistochemistrySirius Red. Collagen deposition and fibrosis were

evaluated by a Sirius Red staining method using a modifiedpicrosirius procedure as previously described (14). Briefly,the sections were deparaffinized, rehydrated in distilledwater, and immersed in l% phosphomolybdic acid(Sigma-Aldrich Chemical Co, Milan, Italy) for 5 min and

then covered with 0.1% (w/v) Sirius Red F3B (C.1.35780

Science Lab, Huston, TX, USA) in saturated picric acid

solution for I h at room temperature. The sections were

then washed in water and rapidly dehydrated, cleared inxylene, and mounted. All sections stained with Sirius Red

were analyzed using a light microscope (Olympus BX50,Tokyo, Japan) under normal light and polarized lightobtained with a polarizer filter (Olympus U-ANI Tokyo,Japan) in order to analyze the initial collagen organizationand the fibrosis, respectively. Under these conditions,collagen fibres of different thickness are coloured

differently. Whereas the birefringent (anisotropic) colouris more a measure of collagen fibre size than of collagen

type, usually the thicker and denser type I collagen fibres

are detected as orange to red, whereas the thinner type IIIcollagen fibres appear yellow to green (15).

Cytochrome-c oxidase (Cyt-c-ox). Cyt-c-ox activiry

which reflects mitochondrial density (16), was determined

in situ on cryostat sections by histochemical methods.

Briefly, frozen renal samples were cut on a cryostat

to a thickness of 5 pm. The sections were incubated

for t h at 37"C with 20 mg 3,3'diaminobenzidine-tetrahydrochloride (DAB), 0.1 M acetate buffer pH 5.5,

0)% MnCl' and 0.1 ml HrO, and then dehydrated and

mounted with DPX. The positive staining was brown.

ImmunohistochemistrySections were incubated overnight with primary

anti-NOS O{OS2-N20-sc651), anti-eNOS (NOS3-C20-

sc654), anti-VEGF (C-1-sc7269), anti-Bcl2 (sc-492), otanti-Bax (sc-526) polyclonal antibodies from Santa Cruz

Biotechnology Inc. (Santa Cruz, CA, USA) diluted 1:

100 and monoclonal anti-HSP-60 (spa-806 Stressgen,

Assay Designs Inc., MI, USA) and anti-CRP-75 (sps-825,

Stressgen) diluted l:300 with PBS. The sections were

processed in accordance with the manufacturers' protocols,

visualized with a rabbit ABC-peroxidase staining system

kit (Santa Cruz Biotechnology), and mounted with DPX.

The reaction product was visualized using 0.3% H.,O, and

DAB at room temperature. The immunohistochemistry

control was performed by omitting the primary antibody in

the presence of isotype-matched IgGs. To exclude incorrect

interpretation of the immunostaining due to the presence ofendogenous biotin (17), we also carried out experiments

using the peroxidase-anti-peroxidase detection system,

but obtained similar results. Each set of experiments was

performed in triplicate, with each replicate always carried

out under the same experimental conditions.

The staining intensity on both histochemical and

immunohistochemical slides was evaluated using an optical

Olympus BX50 microscope equipped with an image

analysis program (lmage Pro Plus rM 4.5.1, Immagini& Computer, Milano, Italy) and analyzed quantitatively.

The integrated optical density (IOD) was calculated forarbitrary areas, by measuring 10 fields for each sample

using a 40x objective. Data were pooled to obtain a mean

value, and a statistical analysis was applied to compare the

results obtained from different experimental groups.

StatisticsMorphometric data are expressed as mean + SD

unless otherwise stated. The statistical significance of the

differences between means was assessed with one-way

ANOVA followed by the Student-Newman-Keuls test

or with a Student /-test. A probability of less than 50ó

(P < 0.05) was considered significant.

RESULTS

Body weight, kidney weight, and daily

526 G. CORSETTI ETAL.

Table I. Composition and relative percentages of theamîno acids in the EAA dietary supplement.

Essential AminoAcid

o//o

Leucine 31.25Lysine 16.25

Isoleucine 15.62

Valine t5.62Threonine 8.75Cysteine 3.7 5

Histidine 3.7 5

Phenvlalanine 2.6Methionine t.25

Tyrosine 0.7 sTryptophan 0.5

consumption of water and EAA solution aresummarized in Table II. No significant differencesin body or kidney weight between M-aged and M-aged+EAA animals were observed. The water andEAA solution intake did not differ between groups.

MorphometryGlomeruli. The data are summarized in Table

III. We did not observe any significant differencesof glomerular number (Ng/o/mm2) among groups.However, M-aged rats had significantly smallerglomerular capillary networks (Aglo), reflected ina reduction in AglolAtor of about 40% comparedto young rats. Animals in the M-aged+EAA groupwere not statistically different from young animalson these measures.

Mitochondria. The data are summarized inTable IV. In M-aged animals, the Nmitll00 pm2inside the distal tubules decreased significantly(by about 55%) compared to young animals,whereas in M-aged+EAA animals the Nlzitll00pm2 decreased about 30% compared to the valuesin young rats. The AmitlAcyt ratio decreased about30% in M-aged animals, whereas in the EAA-fedanimals the ratio was not different from that inyoung animals.

In distal tubular cells of young animals, themitochondria were mainly seen in the basal side

of tubular cells and were elongated and orientedparallel to one another. In contrast, the mitochondriain the distal tubules of M-aged animals wererounded, surrounded by large cytoplasmic areas,and distributed in both the basal and apical sidesof cells. Furthermore, the cristae were very tighto sothe mitochondrial matrix appears uniformly electrondense. The mitochondria of M-aged+EAA animalswere clustered only inside the basal side of cellsand were oriented parallel to each other and theywere elongated, with easily identifiable cristae (datanot shown). The morphology of mitochondria fromEAA-supplemented M-aged animals thus appearedto be very similar to that of young animals.

Histology and histochemistry .

Sirius Red staining. The M-aged animalsoccasionally showed signs of interstitial fibrosis ofmoderate intensity (data not shown). In contrast,the M-aged animals supplemented with EAA didnot show these morphological signs of incipientsenescence.

Cyt-c-ox staining. The most intense brownreaction was detected in the proximal and distaltubules in the renal cortex and outer medulla of theyoung and M-aged+EAA groups. The staining wasmainly concentrated inside the basal side of cells.M-aged standard-diet animals showed a strongdecrease of cyt-c-ox expression and therefore veryfaint histochemical staining (Fig. I ). The IODvalues of staining in the three experimental groupsare shown in Table V.

ImmunohistochemistryWe performed immunohistochemical staining for

various markers in the three experimental groups.The values are summarized in Table V.

Vascular marker (VEGF)M-aged animals showed significantly lower

VEGF staining compared to young animals. Inparticular, the glomerular capillary endothelia andBowman's capsule cells of M-aged animals didnot show VEGF staining. In contrast, the animalssupplemented with EAA showed intense VEGFstaining mainly in glomerular capillaries and to a

lesser extent inside the Bowman's capsule cells,such as podocytes (data not shown).

Int. J. Immunopathol. Pharmacol.

Table lI. Body and kidney weights and mean daily consumption of liquid ín Young and M-aged animals.

Body weights

(e)

Kidney weights

(e)

liquid consumption u

(ml/day)

Young (n : 5) 416+120 3.4 + 0.2 21.48 + 3.6

M-aged (n: A 553+27* 4.1 + 0.1 * 25.3t + 4.1

M-aged+EAA (r: A 577+15* 4.2 + 0.3 * 23 .02 + 6.8

F value F : 104.86 F:8.66 F :2.57

' Young and M-aged rats consumed water M-aged+EAA consumed EAA dissolved in waterb Values are given as mean + SD.* P < 0.05 versus young animals, Student-Newman-Keuls test.

Table lll. Glomeruli. Densíty of glomeruli (Nglo/mm2)and ratío between glomerular orea and total area (Aglo/Atot) in the three experimental groups.

Nglolmm2 AglolAtot

Young 20.1 + 3.1 0.07 + 0.01

M-aged 17.4 + 6.2 0.04 + 0.02 *

M-aged + EAA 18.7 + 8.6 0.06 + 0.02 "

F value n.s. F:12.54

Values are given as mean +.îD.{< P < 0.05 versus young; " P < 0.05 versus M-aged,S tuden t - l{ ewman - K eul s t es t.

Nítrosactive markersEndothelial nitric oxide synthase GNOS). Inside

the tubular cells of young animals, eNOS staining wasvery strong. In the M-aged animals, eNOS stainingdecreased, whereas EAA supplementation restored theintense immunostaining in the tubular compartment tolevels similar to those in the young group. No or veryfaint immunostaining was seen inside the glomeruli inall experimental groups (Fig. 2).

527

Inducible nitric oxide synthase (LNOS). Younganimals showed intense iNOS staining only in somecells of the distal tubules, whereas animals in the M-aged group showed increased staining in glomerularcapillaries and in the distal and proximal tubules.M-aged animals supplemented with EAA showedintense iNOS staining restricted to cells of the distaltubules and, as in young rats, no immunostainingwas seen inside the glomeruli or in other tubularcells (data not shown).

Ant i/pro - ap op t o t ic morkersBcl2. Young and M-aged+EAA animals showed

intense Bcl2 staining in all tubular cells in thecytoplasm. The immunostaining was particularlyintense inside the distal tubular cells, but moremoderate and difftrse inside the proximal tubularcells. Very faint immunostaining was seen inside theglomeruli. In contrast, the M-aged animals showedreduced Bcl2 staining in all tubular cells, and insome cases moderate staining was observed onlyinside the distal tubular cells (Fig. 3).

Bax. Young and M-aged+EAA animals showeda faint-to-moderate signal in the cytoplasm ofproximal tubules, whereas the M-aged animalsshowed stronger staining in proximal tubules. Noreaction was seen in the glomeruli (data not shown).

528 G. CORSETTI ETAL.

Table IY. Mitochondria. Density of mitochondria (lVmit/100 trtm2) and ratio between mitochondrial area andcytoplasmic area (Amit/Acyt) in distal tubules in the threeexperímental groups.

Nmitll00pm' AmitlAcyt

Young 3.7 + 0.68 0.217 + 0.06

M-aged 1.7 +0.37 * 0.15 + 0.06 *

M-aged + EAA 2.2 + 1.43 "I 0.221+ 0.04 "

F value F:24.35 F:10.99

Values are gíven as mean + SD* and Í P < 0.05 versus young; " P < 0.05 versus M-aged, Student-Newman-Keuls tes t.

Mit o c ho ndrial chap eronesHsp-60. Young animals had moderate to strong

immunostaining mainly concentrated inside the cellsof Bowman's capsule, in the mesangial cells, and inthe basal zone of epithelial tubular cells. In the M-aged group Hsp-60 staining was significantly reduced;however, it still persisted at moderate levels insidethe apical zone (near the tubular lumen) of proximaltubular cells. The M-aged+EAA animals showed strongHsp-60 staining in the glomerulus, Bowman's capsulecells, and tubular cells like the young animals (Fig. a).Moreover, the immunostaining of this mitochondrialchaperone in tubular cells was particularly concentratedin the basal zone of the cells.

Grp-75 (Glucose Regulated Protein 75). Younganimals showed intense Grp-75 staining inside thecells of Bowman's capsule and on the basal side oftubular cells. Moderate staining was seen also insidethe glomeruli. In M-aged animals, Grp-75 stainingwas much lower. Indeed, the immunostaining wasfaint inside tubular cells and absent inside Bowman'scapsule and glomeruli. In contrast, the M-aged+EAAanimals showed evident Grp-75 staining, mainly inglomeruli. The staining of tubular cells was faint andoccasionally detected inside the Bowman's capsulecells (data not shown).

DISCUSSION

In the aged kidney progressive glomerulo-

sclerosis, reduced glomerular filtration rate, andvascular hyalinization all occur concomitantlywith the loss of functioning nephrons (18), so thatelderly kidneys aÍe more susceptible to failurewhen other insults are superimposed.

Diet is a crucial modulator of health in humansand experimental models (6, 19). In healthysubjects, high protein intake in the Western diethas no detrimental effects, even though restrictedprotein intake is widely used in subjects affectedby CKD (20). Increased intake of EAA has hadhealthy effects in aging human muscle (21) andin hemodialysis patients (22).Hormones and dietwith balanced EAA implementation greatly restoremitochondrial content and function in aerobictissues, such as skeletal and cardiac muscles, andcounteract sarcopenia in aged experimental animalsand humans (23).

We demonstrate here that in M-aged rats chronicEAA supplementation could prevent the onset ofmitochondrial, vascular, and morphological changesduring the early stages of senescence. Although ourstudy lasted only 3 months, we observed trends in theglomeruli and renal tubules of M-aged rats fed EAAsimilar to those obtained with CR (24). In particular,we detected reduced glomerular tufts in the M-agedgroup that were restored after EAA supplementationto the form in younger rats.

Both reduced glomerular filtration rate andblood flow are strictly linked to aging kidney (25).Although we did not analyse functional glomerularefficiency, the observed VEGF enhancement mightcorrelate with major sprouting of new vessels inglomeruli obtained after the EAA supplementation.Regular blood flow in the glomerular tuft is essentialto assure normal energy metabolism for renalfunction, whereas reduced blood availability affectsmitochondria and results in senescence-relateddiseases.

NO is a gas generated from L-arginine by twoisoforms of NOS, eNOS and inducible (iNOS)forms, that significantly influence glomerularhemodynamics and renin release (26).The balanceof NOS isoforms regulates the vasoconstrictiveactions of angiotensin II and diminishes angiotensinIl-driven hypertrophy and matrix production (27).iNOS is calcium-independent and exacerbatesrenal oxidative damage, whereas eNOS is calcium-

Table Y. Integated optical density valuesexperimental groups.

lnt. J. Immunopathol. Pharmacol.

(+ sD) of histochemical and immunohistochemical staining in the

529

three

Young

n:5

M-aged

n:6

M-aged+EAA

n:6

F value

Cyt-c-ox 26 + 2.4 9+ 1.3 * 22 + 1.5 "* 62.s

VEGF 14+2 4+0.6* ll+1.4'* 124.2

eNOS 53 + 4.1 20 + 2.3 *. 45 + 3.7 "* 55.99

iNOS 5+1.1 13+1.8* 6+0.80 38.52

Bcl-Z 32 + 2.2 18+2* 25 + 3.2 "* 13s.86

Bax 9+0:6 22+l.g* l1+ 1.2 0* 44.35

Hsp-60 16 + 3.5 3 +0.4 {< 15+2.3" 122.r6

GRP-75 3l + 1.8 7 + 1.1 * 19 + 1.6 '* 333.8I

* P < 0.05 versus young; o P < 0.05 versus M-aged, Student-Newman-Keuls

dependent and localizes in vessels to antagonizehypertension (28). In our model we observedenhanced eNOS but almost undetectable iNOS inglomeruli and tubules after EAA intake relative tothe levels in the M-aged group; together, these maycontribute to a cumulative increase in NO. Thisaugmentation was independent of specific EAAintake, because our formula was devoid of free L-arginine and L-glutamine, well known sources of NO(29).Large scale NO production may be beneficialto counteract the impaired NO consumptioncommonly reported in aged kidney (30). Notably,eNOS-dependent NO has been shown to increasemitochondrial biogenesis and functions in severaltissue and cell types (31). The present results suggesta link between eNOS induction and mitochondriarestoration in M-aged rats to health status seen inyoung animals.

Apoptosis is also involved in aging, and theintrinsic pathway of apoptosis, which is linked tomitochondria, is strictly modulated by members ofthe Bcl2 family (32).The expression of the anri-apoptotic Bcl2 family members Bcl-Z, and pro-apoptotic molecule Bax in M-aged rats fed EAA are

in agreement with those reported by Lee ef al. (24)after CR. It has been suggested that the benefit ofCR may partly derive from decreased mitochondrialROS production and oxidative damage (33).Similarly, CR promotes biogenesis of mitochondriaand energetic efficiency (34), thereby preventing theage-related decline in oxidative capacity (35).

To protect themselves from increased ROSproduction, mitochondria maintain an arsenal ofhighly conserved proteins that act as chaperonesto prevent membrane dysfunction (36). The mainmitochondrial chaperones are Hsp-60 and Grp-75 ormortalin, which can be upregulated during stressfulconditions. In particular, a direct role for Hsp-60 insuppressing apoptosis and cyt-c release has beenreported (37).Grp-75 is essential for transportingmitochondrial proteins from the cytoplasm intomitochondria (38) and is strongly implicated in cellaging (39). Chaperone activity is generally reducedin aging, leading to decreased membrane potentialand mitochondrial fragility (40). The faint Hsp-60expression detected here in the tubular cells of M-aged rats fed a standard diet correlated well with a

low Bcl2 signal and cyt-c-ox activify in mitochondria

Young

530 G. CORSETTI ETAL.

Fig. 1. Hístochemistryfor cit-c-ox (counterstainedwith hematoxytin). The M-aged animals showed a marked reductionof cyt-c-ox expression. The EAA supplementation restored the enzyme expression to that of young animals. Scale bari100 pm.

Young M-aged Maged + EAA

Fig. 2. eNOS immunohistochemistry (counterstained with hematoxylin). Young animals strongly expressed eNOS insidethe tubular cells, whereas the expression decreased strongly in the M-aged animals. EAA supplementation restored theimmunostaining ín the tubular compartment to a level similar to that in the young group. Immunostaining was absent orveryfaint inside the glomeruli. Scale baa 100 pm.

Meged M-aged + EAA

Fig. 3. Bcl-2 immunohistochemistry ftounterstaíned with hematoxylin). Young and EAA-fed M-aged animals showedBcl2 expression inside all the tubular cells and strong expressíon inside the dístal tubules cells (arcows). Very faintexpression is visible inside the glomeruli. In contrast, the M-aged animals show reduced Bcl2 expression in the tubularcompartment, although moderate-to-intense expressìon was observed ínside the basal zone of diital tubular cells. Scalebaa 200 pm.

M-aged M-aged + EAA

Fig. 4. Hsp-60 ímmunohistochemistry (counterstained with hematoxylin). Young animals showed moderate-to-strongstaíning mainly concentrated inside the cells of Bowmanb capsule (arrows), in the mesangial cells, and in the basalzone of the tubular cells. The M-aged animals fed a standard diet did not show Hsp-60 expressíon. The EAA-fed M-agedanimals showed strong expression inside the glomeruli and Bowmanb capsule and less inside the tubular cells. Scale bar100 pm.

M-aged M-aged + EAA

Young

Young

Int. J. Immunopathol. Pharmacol. 531

(41). In contrast, after EAA supplementation,increased chaperone expression in the M-aged groupmay indicate the recovery of regular mitochondrialfunction, which is also supported by the restoredcyt-c-ox activity. Together, the upregulation ofcyt-c-ox, Hsp-60, and Grp-75 observed after EAAsupplementation strongly suggests the restoration ofmitochondrial function to a level similar to that seenin juvenile rats.

Nutritional approaches other than CR have beenreported to limit renal aging and to maintain healthymitochondrial activity (42).As noted above, theefficacy of low-protein diets against hypertensionand proteinuria in cDK is controversial, occasionallyit has been considered dangerous (43-44), even ifEAA supplementation has been recently shown toimprove anabolism and suggested to be adoptedas an integral part of the therapeutical approach inchronic hemodialysis patie nts (22).

In conclusion, our data show that prolongedadministration of a balanced EAA mixture may havebeneficial effects on kidney metabolism of M-agedrats, mainly improving mitochondrial function andretarding the typical alterations seen in senescence.Although to date there are no data regarding theeffects of EAA mixture supplementation on renalhealth in aged human, we suggest that balancedEAA mixture supplementation in the diet could bea promising nutritional approach for maintaininga healthy renal status to prevent chronic kidneydiseases and improve the quality of life. However,even if these data are promising, further biochemicaland functional studies are required to corroborate theutility of EAA mixture to retard the onset of aging_associated changes in the kidney.

ACKNOWLEDGEMENTS

The authors would like to thank prof. FrancoConti for providing the antibodies and reagents, andfor his helpful advice. This work was supported bythe Università degli Studi di Brescia (to G.C.) andMinistero dell'lstruzione, dell'università e dellaRicerca (E.N.) grants.

Disclosure: The authors who contributed to this publicationdeclare to have no fìnancial arrangement or affiliation with acorporate organization or a manufacturer of any commercialproduct discussed in this article. Francesco S. Dioguardi has

received reimbursements from professional Dietetics for tripexpenses for speeches on the clinical uses of amino acids.

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