Dehydroepiandrosterone Pretreatment Alters the Ischaemia/Reperfusion-Induced VEGF, IL-1 and IL-6 Gene Expression in Acute Renal Failure

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Original Paper

Kidney Blood Press Res 2009;32:175–184 DOI: 10.1159/000224629

Dehydroepiandrosterone Pretreatment Altersthe Ischaemia/Reperfusion-Induced VEGF, IL-1 and IL-6 Gene Expression in Acute Renal Failure

Ádám Vannay a, b Andrea Fekete c Róbert Langer d Tibor Tóth e Erna Sziksz c

Barna Vásárhelyi a, c Attila J. Szabó c György Losonczy f Csaba Ádori h

Anikó Gál g Tivadar Tulassay a–c András Szabó c

a Research Laboratory of Paediatrics and Nephrology, Hungarian Academy of Sciences, b János Szentágothai Knowledge Centre, c 1st Department of Paediatrics, d Department of Transplantation and Surgery, e 2nd Department of Pathology, f Department of Pulmonology and g Clinical and Research Centre forMolecular Neurology, Semmelweis University, and h Laboratory of Neurochemistry and Experimental Medicine, National Institute of Psychiatry and Neurology, Budapest , Hungary

creased at T 2 and T 24 compared to T 0 kidneys in both groups. VEGF protein levels were lower at T 2 and T 24 in G DHEA than in G PG . Conclusion: We found that DHEA pretreatment alters renal IL-1 � , IL-6 and VEGF synthesis. Moreover, contrary changes in VEGF mRNA and protein levels suggest that VEGF synthesis – distinct from other organs – might be primarily posttranscriptionally regulated in postischaemic rat kid-neys. Copyright © 2009 S. Karger AG, Basel

Introduction

Ischaemia/reperfusion (I/R)-induced acute renal fail-ure (ARF) is a frequent clinical syndrome, which is asso-ciated with high morbidity and mortality rates. ARF may occur during different clinical circumstances, including renal transplantation or suprarenal aortic surgery. ARF is, however, potentially reversible if the patient survives

Key Words

Dehydroepiandrosterone � Interleukin 1 � � Interleukin 6 � Ischaemia/reperfusion � Vascular endothelial growth factor

Abstract

Background: Beneficial effects of dehydroepiandrosterone (DHEA) pretreatment were reported in ischaemia/reperfu-sion (I/R)-induced kidney damage. Methods: To investigate the mechanism of DHEA pretreatment during renal I/R inju-ry, the left renal pedicles of DHEA- [G DHEA ; 4.0 mg/kg/day DHEA dissolved in propylene glycol (PG)] and PG-pretreated male Wistar rats (G PG ) were clamped for 55 min followed by 2 (T 2 ) and 24 h (T 24 ) of reperfusion. Sham-operated, non-clamped animals (T 0 ) served as controls in both groups. Re-nal function, kidney morphology and interleukin 1 � (IL-1 � ), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) expression were determined in the kidneys of both groups. Results: Renal functional parameters and kidney structure did not differ in G DHEA versus G PG at any time point. Renal mRNA expression of IL-1 � was lower at T 0 , while IL-6 at T 2 was lower in G DHEA than in G PG . While renal VEGF mRNA expression remained unchanged, protein levels were in-

Received: September 25, 2008 Accepted: March 26, 2009 Published online: June 11, 2009

Ádám Vannay, MD, PhD 1st Department of Paediatrics, Semmelweis University Bókay J. u. 53–54HU–1083 Budapest (Hungary) Tel. + 36 1 334 3186, Fax +36 1 313 8212, E-Mail [email protected]

© 2009 S. Karger AG, Basel1420–4096/09/0323–0175$26.00/0

Accessible online at:www.karger.com/kbr

Ádám Vannay and Andrea Fekete have equally contributed to this work.

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Kidney Blood Press Res 2009;32:175–184176

the initial insult [1] . Previously we have demonstrated that sex hormones strongly influence the outcome of I/R-induced ARF. While oestrogens are believed to be protec-tive [2] , androgens are supposed to be harmful [3, 4] fac-tors in renal I/R injury. Recently another steroid hor-mone, dehydroepiandrosterone (DHEA), has been shown to protect the brain [5] and kidney [6] against I/R injury. Although its physiological role is still unclear, exogenous-ly administered DHEA has been revealed to alter vascu-lar haemodynamics [7, 8] and endothelial cell functions [9] . Reduced vascular flow in the postischaemic kidney leads to the swelling of endothelial cells [10] and results in the ‘no-reflow’ phenomenon [11] , which may initiate and extend renal dysfunction [12, 13] . Recent findings in-dicate that a network of interacting cytokines including interleukin (IL) 1 � , IL-6 and vascular endothelial growth factor (VEGF) [14–16] regulates vascular haemodynam-ics [17–22] by inducing the synthesis of vasodilating agents such as nitric oxide and prostacycline [19, 21–23] . VEGF also alters postischaemic survival of endothelial [24] and renal tubular epithelial [25] cells. Previously we have shown that renal synthesis of VEGF is rapidly in-creasing during renal I/R injury [26, 27] .

Since DHEA is known to be protective in renal I/R in-jury and it also influences vascular haemodynamics, in the present study we elucidated the effects of DHEA pre-treatment on postischaemic kidney function and struc-ture and on the renal synthesis of IL-1 � and IL-6 and VEGF in a rat model of renal I/R injury.

Materials and Methods

Animals and Treatment Protocol Experiments were performed on male (200–220 g) Wistar rats.

Animals were housed in a temperature-controlled (22 8 1 ° C) room with alternating light and dark cycles. Animals had free ac-cess to standard rat chow and water. The institutional committee on animal welfare approved all experiments.

Rats were randomized into 2 treatment groups according to Lohman et al. [7] . In the DHEA-pretreated group (G DHEA ), ani-mals were subcutaneously injected with DHEA (4.0 mg/kg body weight/day) dissolved in 0.1 ml propylene glycol (PG; Sigma Chemical Co., St. Louis, Mo., USA), first 25 and then 1 h before the surgical procedure. After the surgery, animals were main-tained on the same daily dosage during the whole experiment. PG-pretreated animals (G PG ) were identically treated (0.1 ml/day) and served as only vehicle-treated controls of DHEA pretreat-ment.

Ischaemia Protocol and Study Design Animals were anaesthetized by intraperitoneal injection of

pentobarbital sodium (50 mg/kg body weight; Nembutal, Abbott Laboratories, Abbott Park, Ill., USA). Animals were placed on a

thermocontrolled pad to maintain rectal temperature at 37 ° C. After a midline abdominal incision, the left renal pedicles were occluded with an atraumatic microvascular clamp for 55 min. Before the end of the ischaemic period, the right, contralateral kidneys were removed. After removing the microvascular clamp, the abdomen was closed, and the animals remained on thermo-controlled pads until complete recovery. Sham-operated animals (T 0 ), who underwent the same surgical procedure except clamp-ing of the left renal pedicle, served as controls.

All groups of animals were sacrificed by bleeding from the abdominal aorta after isolation, either before clamping the left renal pedicle (n = 6), at 2 (n = 6) or 24 h (n = 6) of reperfusion. Blood samples were collected for serum preparation, and the left kidneys were removed. Kidney segments were immediately fro-zen in liquid nitrogen or fixed in formalin (4%, pH = 7.4; fig. 1 ).

Serum Biochemistry Serum creatinine and blood urea nitrogen concentrations

were photometrically determined with commercially available test kits (F. Hoffmann-La Roche, Basel, Switzerland) on a Hitachi 712 spectrophotometer.

Serum DHEA and dehydroepiandrosterone sulphate (DHEA-S) levels were determined by direct radio-immunoassay as de-scribed previously [28] .

Histological Analysis Paraffin sections of kidneys fixed in formalin (4%, pH = 7.4)

were stained with haematoxylin-eosin. Samples were coded and examined in a blinded fashion. Tubular, glomerular and intersti-tial lesions were scored from 0 to 3 (0 = none, 1 = mild, 2 = mod-erate, 3 = severe).

TUNEL and Caspase-3 Double Labelling For the identification of apoptotic and necrotic cell popula-

tions, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and caspase-3 (C-3) double labelling were performed on 5- � m-thin sections of formalin-fixed (10%) paraf-fin-embedded rat kidney tissue blocks. For TUNEL detection of fragmented DNA, deparaffinated sections were pretreated with 20 � g/ml proteinase K in Tris-HCl for 20 min at 37 ° C. Then sam-ples were incubated in phosphate-buffered saline (PBS) for 3 ! 5 min and in TUNEL reaction mixture (Roche Diagnostics, Basel, Switzerland), containing fluorescein-labelled nucleotides (dUTP) and terminal deoxynucleotidyl transferase, for 60 min at 37 ° C. After extensive washes in PBS, sections were incubated with rab-bit active C-3 antibody (1: 100; Santa Cruz Biotechnology Inc., Cal-if., USA) in PBS for 60 min at 37 ° C. The fluorescence-labelled secondary antibody was Alexa fluor 568 goat anti-rabbit IgG(1: 100; Molecular Probes Inc., Oreg., USA) in PBS for 40 min at 37 ° C. Sections were covered with Vectashield (Vector Laborato-ries Inc., Burlingame, Calif., USA), and immunofluorescence la-belling was evaluated using a laser scanning confocal microscope (Bio-Rad MRC 1024/Nikon Diaphot, Hercules, Calif., USA) with a krypton-argon laser system (for excitation 488 nm; for emission 522 and 585 nm, TUNEL and C-3, respectively).

Images for morphometry analysis were made on 10 (0.15 mm 2 ) randomly selected non-overlapping areas of the kidneys. All im-ages were taken with the same settings of confocal microscopy. The number of TUNEL-positive, active C-3-immunoreactive and double-labelled renal tubular cells was counted.

DHEA Pretreatment Alters I/R-Induced ARF

Kidney Blood Press Res 2009;32:175–184 177

RNA Isolation and Reverse Transcription Polymerase Chain Reaction Total RNA was extracted from kidney samples by an RNeasy

total RNA isolation kit (Qiagen GmbH, Hilden, Germany), ac-cording to the instructions of the manufacturer. One microgram of total RNA was reverse-transcribed with Superscript II RNase H – (Gibco/BRL, Eggenstein, Germany) to generate first-strand cDNA. PCR was performed in a final volume of 50 � l containing 10% 10 ! PCR buffer, 2 m M MgCl 2 , 0.2 m M dNTPs, 1.5 units Ampli Taq DNA polymerase (Gibco/BRL) and sense and anti-sense primer, 0.5 � M of each. Specific primer pairs for IL-1 � , IL-6 [29] , VEGF [26] and glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) [30] are shown in table 1 . The conditions of PCRs were as follows: 15 s at 94 ° C (denaturing); 15 s at 57 ° C (IL-1 � ), at 66 ° C (IL-6), at 56 ° C (VEGF) and at 55 ° C (GAPDH; annealing),

and 30 s at 72 ° C (extension) for 35 cycles. PCR products were separated by electrophoresis on 2.5% agarose gels and visualized by staining with ethidium bromide. The mRNA expression of each gene was determined by densitometric comparison with GAPDH as internal control from the same sample.

Western Blot Analysis Kidney samples were solubilized in a buffer containing 10 � g/

ml leupeptin, 10 � g/ml aprotinin, 1% Triton X-100, 0.1 M Tris-HCl (pH = 8.0), 1 m M ethylene glycol-bis(2-aminoethylether),N,N,N�,N�-tetra-acetic acid, 5 m M sodium fluoride, 1 m M phenylmethyl-sulphonyl fluoride and 10 m M sodium orthovanadate. The lysed samples were centrifuged (10,000 g, 5 min, 4 ° C) to pellet cellular fragments. The protein concentration of the supernatants was de-termined by the Bradford assay (Bio-Rad Laboratories, Calif.,

Kidney removal 24 h after 55 min of ischaemia (T24)(n = 6/group)

Kidney removal 2 h after 55 min of ischaemia (T2)(n = 6/group)

Kidney removal following pretreatment only (T0)(n = 6/group)

GPG

GDHEA

1st treatment 2nd treatment

GPG

GDHEA

1st treatment 2nd treatment

Beginning of the ischaemia

GPG

GDHEA

1st treatment 2nd treatment 3rd treatment

Beginning of the ischaemia

Fig. 1. Treatment protocol and study design. The animals were subcutaneously injected with DHEA (4.0 mg/kg body weight/day dissolved in 0.1 ml PG) or PG, first 25 and then 1 h before the sur-gical procedure. After the surgical procedure, the animals were maintained on the same daily dosage during the experiment.

They were sacrificed by bleeding from the abdominal aorta, either before clamping of the left renal pedicle (T 0 ; n = 6), at 2 (T 2 ; n = 6) or 24 h (T 24 ; n = 6) of reperfusion. Blood and kidney samples were collected for further analysis.

Gene Primer pairs Product length

IL-1� SenseAntisense

5�-GCA CTG CAG GCT TCG AGA TGA-3�5�-GGT GGG TGT GCC GTC TTT CA-3�

220 bp

IL-6 SenseAntisense

5�-CTT CCA GCC AGT TGC CTT CT-3�5�-TCC CGA CCA TTG CTG TTT CC-3�

496 bp

VEGF SenseAntisense

5�-GTG CAC TGG ACC CTG GCT TTA C-3�5�-TTT TCT GGC TTT GTT CTA TCT TTC-3�

398 bp

GAPDH SenseAntisense

5�-GGT GAA GGT CGG AGT CAA CG-3�5�-CAA AGT TGT CAT GGA TGA CC-3�

496 bp

Table 1. Specific primer pairs for IL -1�, IL-6, VEGF and GAPDH

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Kidney Blood Press Res 2009;32:175–184178

USA). 10 � g of protein samples were separated by 12% SDS poly-acrylamide gel electrophoresis at 120 V and 40 mA for 90 min (Penguin TM Dual-Gel Water-Cooled Systems, Owl, N.H., USA). Prestained protein mixture (Benchmark TM , Gibco/BRL) was used as marker of molecular mass. The separated proteins were trans-ferred to nitrocellulose membrane (Hybond TM ECL TM , AP Bio-tech, UK) in transfer buffer containing Tris, glycine and metha-nol at 70 V, 220 mA for 80 min (Minitank TM electroblotter, Owl, N.H., USA). Non-specific binding sites were blocked for 1 h (23 ° C) in a solution containing 5% non-fat dry milk, Tween 20 and 10 ! PBS followed by 60 min (23 ° C) incubation with a monoclonal an-tibody for VEGF [VEGF-A (C-1), Santa Cruz Biotechnology Inc.] diluted to 1: 500. Blots were washed and then incubated with per-oxidase-conjugated secondary anti-mouse IgG antibodies diluted to 1: 1,000 for 30 min (23 ° C) (Sigma Chemical Co.). Immunoreac-tive bands were visualized using the enhanced chemilumines-cence Western blotting detection protocol (AP Biotech). The val-ues were normalized to an internal standard and expressed as the relative optical density.

Statistical Analysis Survival of the animals was analysed by Kaplan-Mayer analy-

sis (log-rank test). Histological scores are expressed as medians and ranges. Histological changes were analysed using the Kruskal-Wallis test followed by multiple pairwise comparisons according to the Mann-Whitney U test. Other data are expressed as means and standard deviations. Statistical analysis was performed by one-way analysis of variance followed by multiple pairwise com-parisons according to the Newman-Keuls test. Data were consid-ered to be significantly different if p was less than 0.05.

Results

Serum Biochemistry Fifty-five minutes of warm ischaemia induced acute

renal insufficiency in both groups as reflected by in-creased serum creatinine (p ! 0.001, T 2 and T 24 vs. T 0 )

and blood urea nitrogen (p ! 0.001, T 24 vs. T 0 ) concentra-tions. There was, however, no significant difference in the serum creatinine or blood urea nitrogen concentrations of G PG versus G DHEA animals at any investigated time points ( table 2 ).

Serum DHEA and DHEA-S levels of G DHEA animals were higher at each investigated time point (T 0, T 2 and T 24 ) compared to G PG rats ( table 2 ; p ! 0.001, G DHEA vs. G PG ).

In G DHEA animals, serum DHEA levels were higher at T 24 compared to T 0 and T 2 values (p ! 0.05, T 24 vs. T 0 ;p ! 0.01, T 24 vs. T 2 ), and serum DHEA-S levels were high-er at T 2 compared to T 0 values (p ! 0.001, T 2 vs. T 0 ) and at T 24 compared to T 2 and T 0 values (p ! 0.001, T 24 vs. T 0 and T 2 ).

In G PG animals there was no difference in serum DHEA or DHEA-S levels between the investigated time points.

Histological Analysis Histological analysis of T 0 kidneys revealed normal

kidney structure without glomerular, tubular or intersti-tial lesions in any of the groups [G DHEA : median 0.0 (range 0–0); G PG : 0.0 (0–0); fig. 2 ]. In T 2 kidneys, mild-moderate tubular lesions were observed in both groups [G DHEA : 1.5 (1–2), G PG : 1.5 (1–2); p ! 0.05, T 2 vs. T 0 ]. In T 24 kidneys of both groups, moderate-severe tubular lesions were pres-ent [G DHEA : 3 (2–3), G PG : 3 (2–3); p ! 0.05, T 24 vs. T 0 and T 2 ]. Flattening of the tubular epithelial cells was evident in T 2 and T 24 kidneys of both groups. There was no evi-dent glomerular or interstitial lesion in T 2 and T 24 kid-neys in any of the groups. However, there was no signifi-cant difference between kidneys of G PG and G DHEA ani-

Table 2. Serum creatinine, urea nitrogen (BUN), DHEA and DHEA-S levels

Serum constituent Treatment group T0 T2 T24

Creatinine, �M GDHEA 55.5083.39 94.2585.95a 380.33827.11a, c

GPG 58.882.84 99.4287.48a 378.60832.46a, c

BUN, mM GDHEA 5.6580.56 7.3680.83 39.6286.55a, c

GPG 5.7880.97 7.8280.71 35.6684.57a, c

DHEA, nM GDHEA 49.3185.72e 44.95811.06e 57.4786.87b, d, e

GPG 0.8980.07 1.4080.24 1.2580.34DHEA-S, nM GDHEA 107.60813.86e 158.03815.23a, e 194.33824.64a, c, e

GPG 7.5080.76 8.3781.08 6.9281.54

Data are expressed as means 8 standard deviation.a p < 0.001, b p < 0.05 versus T0; c p < 0.001, d p < 0.01 versus T2; e p < 0.001 versus GPG.

DHEA Pretreatment Alters I/R-Induced ARF

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ba

c d

e f

Fig. 2. Cross-sections from rat kidneys stained with haematoxy-lin-eosin. a , b G DHEA and G PG , respectively: renal tubular epithe-lial cells in T 0 kidneys of both groups reveal normal kidney struc-ture. c , d G DHEA and G PG : tubular epithelial cells show focal tubu-lar necrosis in T 2 kidneys of both groups. e , f G DHEA and G PG : the tubular epithelial cell death and the desquamated debris formed

tubular casts (asterisks) which are evident in T 24 kidneys of both groups. The flattening of renal tubular epithelial cells is present in all postischaemic kidney samples (arrowheads). a–f Original magnification ! 400. Histological changes were analysed using the Kruskal-Wallis test followed by multiple pairwise compari-sons according to the Mann-Whitney U test.

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Kidney Blood Press Res 2009;32:175–184180

mals in any histological parameters investigated at any time points.

TUNEL and C-3 Double Labelling Fifty-five minutes of warm ischaemia resulted in the

increasing number of TUNEL (p ! 0.05, T 2 vs. T 0 and T 24 vs. T 2 ; p ! 0.01, T 24 vs. T 0 ) or C-3 (p ! 0.05, T 2 vs. T 0 and T 24 vs. T 2 ; p ! 0.01, T 24 vs. T 0 ) in immunopositive tubular epithelial cells both in G DHEA and G PG groups ( fig. 3 a and c or b, respectively). However, there was no significant difference in the number of C-3- and TUNEL-immu-nopositive tubular epithelial cells in the postischaemic kidneys of G DHEA versus G PG animals.

Renal IL-1 � mRNA Expression Renal IL-1 � mRNA expression was unchanged in T 2

and T 24 compared to T 0 kidneys of both G DHEA and G PG groups. IL-1 � mRNA expression was lower in the T 0 kid-neys of G DHEA than G PG animals (p ! 0.05, G DHEA vs. G PG ).

A tendency of lower IL-1 � mRNA expression was also present in T 2 and T 24 kidneys of G DHEA animals ( fig. 4 ).

Renal IL-6 mRNA Expression Renal IL-6 mRNA expression was increased in T 2 kid-

neys of G PG and G DHEA animals compared to T 0 values(p ! 0.001, T 2 vs. T 0 in both groups). In T 24 kidneys IL-6 mRNA expression almost returned to the T 0 values in both groups (p ! 0.001, T 2 vs. T 24 in G PG ). In T 2 kidneys, IL-6 mRNA expression was lower in G DHEA than in G PG animals (p ! 0.05, G DHEA vs. G PG ; fig. 5 ).

Renal VEGF mRNA Expression There was no difference in the renal mRNA expres-

sion of VEGF at T 0 , T 2 and T 24 either in G DHEA or in G PG animals. However, the renal mRNA expression of VEGF tended to be higher in G DHEA than G PG animals at T 2 and T 24 ( fig. 6 ).

T0 T2 T4a

0b

20

40

60

80

a

a, b100

T0 T2 T24

TUN

EL-p

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cells

(n/0

.15

mm

2 )

GPG

GDHEA

0

10

20

30

40

50

60

a

a, b70

T0 T2 T24C

-3-p

osit

ive

cells

(n/0

.15

mm

2 )

GPG

GDHEA

c

Fig. 3. Effect of DHEA pretreatment on the number of necrotic and apoptotic cells in the rat kidneys. a Necrotic and apoptotic cells were stained using the TUNEL kit (green in the online ver-sion) and the apoptotic cells with active C-3 antibody combined with Alexa 568 fluorochrome (red in the online version). Renal ischaemia resulted in an increasing number of TUNEL- and C-3-positive tubular epithelial cells in the postischaemic kidney sam-ples. Original magnification ! 400. b , c The numbers of necrotic

and apoptotic cells were determined before the 55-min warm ischaemia and at 2 and 24 h of reperfusion (T 0 , T 2 and T 24 kidneys, respectively). Data are expressed as means 8 SD of 6 animals at each time point of each group. Analysis of significance was per-formed by one-way analysis of variance followed by multiple pair-wise comparisons according to the Newman-Keuls test. a p ! 0.05, T 2 versus T 0 and T 24 versus T 2 ; b p ! 0.01, T 24 versus T 0 in both groups.

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DHEA Pretreatment Alters I/R-Induced ARF

Kidney Blood Press Res 2009;32:175–184 181

0

0.5

1.0

1.5

2.0

3.0

2.5

IL-1

�/G

APD

H (a

rbit

rary

un

its)

T0

*

T2 T24

GPG

GDHEA

Fig. 4. Effect of DHEA pretreatment on the mRNA expression of IL-1 � in rat kidneys. Renal IL-1 � mRNA expression was deter-mined before the 55-min warm ischaemia and at 2 and 24 h of reperfusion (T 0 , T 2 and T 24 kidneys, respectively). The optical density of the PCR products was corrected for GAPDH. Data are expressed as means 8 SD of 6 animals at each time point of each group. Analysis of significance was performed by one-way analy-sis of variance followed by multiple pairwise comparisons accord-ing to the Newman-Keuls test. * p ! 0.05 versus G PG .

0

5

10

15

20

25

30

40

35

IL-6

/GA

PDH

(arb

itra

ry u

nit

s)

T0 T2

a, b

a, c

T24

GPG

GDHEA

Fig. 5. Effect of DHEA pretreatment on the mRNA expression of IL-6 in rat kidneys. Renal IL-6 mRNA expression was determined before the 55-min warm ischaemia and at 2 and 24 h of reperfu-sion (T 0 , T 2 and T 24 kidneys, respectively). The optical density of the PCR products was corrected for GAPDH. Data are expressed as means 8 SD of 6 animals at each time point of each group. Analysis of significance was performed by one-way analysis of variance followed by multiple pairwise comparisons according to the Newman-Keuls test. a p ! 0.001, T 2 versus T 0 ; b p ! 0.001, T 2 versus T 24 ; c p ! 0.05 versus G PG .

0

1

2

3

4

5

T0

VEG

F/G

APD

H (a

rbit

rary

un

its)

T2 T24

GPG

GDHEA

Fig. 6. Effect of DHEA pretreatment on the mRNA expression of VEGF in rat kidneys. Renal VEGF mRNA expression was deter-mined before the 55-min warm ischaemia and at 2 and 24 h of reperfusion (T 0 , T 2 and T 24 kidneys, respectively). The optical density of the PCR products was corrected for GAPDH. Data are expressed as means 8 SD of 6 animals at each time point of each group. Analysis of significance was performed by one-way analy-sis of variance followed by multiple pairwise comparisons accord-ing to the Newman-Keuls test.

0

50

100

150

250

200

300

T0

VEG

F (a

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GDHEA

GPG GDHEA

T0T0

GPG GDHEA

T2T2

GPG GDHEA

T24T24

T2

a

c, d

T24

a

b, d

Fig. 7. Effect of DHEA pretreatment on VEGF protein levels in rat kidneys. The VEGF protein level was determined under reducing conditions before the 55-min warm ischaemia and at 2 or 24 h of reperfusion (T 0 , T 2 and T 24 kidneys, respectively). Data for VEGF protein levels were obtained by computerized analysis of the Western blots. Data are expressed as means 8 SD of 6 animals at each time point of each group. Analysis of significance was per-formed by one-way analysis of variance followed by multiple pair-wise comparisons according to the Newman-Keuls test. a p ! 0.001, b p ! 0.01, c p ! 0.05 versus T 0 ; d p ! 0.01 versus G PG .

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VEGF Protein Level VEGF protein levels were increased in T 2 and T 24 com-

pared to T 0 kidneys of G DHEA (p ! 0.05, T 2 vs. T 0 ; p ! 0.01, T 24 vs. T 0 ) and G PG (p ! 0.001, T 2 and T 24 vs. T 0 ) animals. Renal VEGF protein levels were lower at T 2 and T 24 in G DHEA than in G PG animals (p ! 0.01, G DHEA vs. G PG ). A tendency of lower VEGF protein level was also present in T 0 kidneys of G DHEA versus G PG animals ( fig. 7 ).

Discussion

In the present study we investigated the effect of short-term DHEA pretreatment on I/R-induced ARF, which is one of the major determinants of acute and chronic renal transplant dysfunction.

Renal function, morphology and the number of apo-ptotic tubular epithelial cells were assessed at 2 and 24 h of reperfusion (T 2 and T 24 , respectively). Postischaemic (T 2 and T 24 ) increase in serum creatinine and blood urea nitrogen concentrations, severity of renal tubular dam-age and the number of TUNEL- or C-3-positive cells were similar in both treatment groups.

During renal I/R injury, exogenously administered DHEA, which is a precursor of testosterone and 17 � -oes-tradiol, may have both deleterious and beneficial effects. On the one hand, DHEA treatment reportedly increased the number of apoptotic cells in vitro and in vivo [31, 32] . On the other hand, DHEA pretreatment protects I/R-in-duced brain [5] , spinal cord [33] , muscle flap [8] or kidney [6] damage. This dual pro- or antioxidant effect of DHEA pretreatment seems to be dose dependent. High doses of DHEA pretreatment exert pro-oxidant effects; however, this effect of DHEA completely disappears when it is ad-ministered at lower doses [34] . In our study we applied a dose that has been previously demonstrated to exert pro-tective effects [6, 7, 35] .

Different mechanisms may participate in the positive effects of DHEA pretreatment on renal I/R injury. DHEA may exert its beneficial effects as a powerful antioxidant [5, 6] . Indeed Aragno et al. [6] observed a reduced hydro-gen peroxide level and increased glutathione peroxidase and superoxide dismutase activity in the postischaemic kidneys of DHEA-pretreated animals compared with controls. In addition to its antioxidant properties, DHEA reportedly alters postischaemic vascular haemodynam-ics [7, 8] and endothelial cell functions [9] .

Recovery of the kidney from I/R-induced ARF relies on different processes, including restoration of vascular haemodynamics or regeneration of endothelial and epi-

thelial cells [36, 37] . Although haemodynamic and mor-phological changes have been described [38] , the molecu-lar bases of the recovery of the postischaemic kidney are not understood.

To investigate the molecular mechanism of short-term DHEA pretreatment, we studied the renal synthesis of IL-1 � , IL-6 and VEGF during I/R injury. IL-1 � , IL-6 and VEGF are important regulators of vascular haemody-namics and several other endothelial and epithelial cell functions [17–19, 21, 22, 24, 25, 39] and have a pivotal role in the development of postischaemic renal damage [25, 26, 40] .

In the present study, we found lower renal IL-1 � and IL-6 mRNA expression in DHEA-pretreated than in G PG rats, which indicates that DHEA pretreatment has a sig-nificant impact on the renal synthesis of these cytokines. Decreased IL-6 mRNA expression in the kidney is in line with previous in vitro data demonstrating that exoge-nously administered DHEA decreases IL-6 synthesis [41] . However, there have been no previous data about the im-pact of DHEA pretreatment on the synthesis of IL-1 � . Previously, it has been shown that exogenously adminis-tered DHEA inhibits nuclear-factor- � B-dependent tran-scription in vitro [42, 43] . Since IL-1 � [44] and IL-6 [45, 46] are considered as members of the nuclear-factor- � B -regulated genes, reduced nuclear factor � B activation may explain the decreased IL-1 � and IL-6 synthesis in the postischaemic kidneys of DHEA-treated animals.

Changes in VEGF mRNA expression and protein lev-els were similar in both DHEA-pretreated and G PG ani-mals. While mRNA expression of VEGF did not change significantly, the VEGF protein level was increased in the postischaemic kidneys of both groups. Moreover, the VEGF protein level was lower in the postischaemic kid-ney of DHEA-pretreated than G PG animals.

Although we cannot fully explain the mechanism of DHEA pretreatment on renal VEGF synthesis, presum-ably altered IL-1 � and IL-6 synthesis may participate in the regulation of VEGF synthesis. Since previous data suggest an interconnection in the synthesis of these cyto-kines [15, 16] , we hypothesize that the simultaneously de-creased IL-1 � and IL-6 mRNA expression and VEGF protein level might be interrelated in the rat kidney. IL-1 � induces VEGF synthesis mediated by hypoxia-inducible factor 1 [47] and IL-6 phosphorylates the eukaryotic ini-tiation factor 4E [48] , which increases the translation of VEGF [49] . However, other mechanisms can also be in-volved in the effect of exogenously administered DHEA on VEGF synthesis.

DHEA Pretreatment Alters I/R-Induced ARF

Kidney Blood Press Res 2009;32:175–184 183

In accordance with previous results, our data [25–27] also suggest that VEGF synthesis might be regulated rather at a posttranscriptional than at a transcriptional level in the postischaemic rat kidney. Different mecha-nisms have been suggested to participate in the posttran-scriptional regulation of VEGF synthesis. It has been shown that hypoxia leads to the stabilization of VEGF transcript, by binding to the RNA-binding protein HuR [50, 51] . Moreover, an internal ribosomal entry site of the 5 � -untranslated region of VEGF ensures the translation of VEGF even under hypoxic conditions [52] .

In conclusion, here we report a potential molecular mechanism of the previously reported beneficial effects

of DHEA pretreatment. Altered synthesis of IL-1 � , IL-6 and VEGF may participate in the protective effects of DHEA pretreatment on I/R-induced ARF. However, fur-ther investigations with different doses and pretreat-ment/treatment periods are needed in both female and male animals to elucidate the impact of exogenously ad-ministered DHEA during renal I/R injury.

Acknowledgements

This work was supported by OTKA F-048842, F042563, 71730, ETT 184/2003, 435/2006 and GVOP 3.3.3 grants. Andrea Fekete is a recipient of a Bolyai scholarship.

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