cDNA representational difference analysis used in the identification of genes related to the aging process in rat kidney

cDNA representational difference analysis used in the identification of genes related to the aging process in rat kidney

Mechanisms of Ageing and Development 126 (2005) 882–891 www.elsevier.com/locate/mechagedev cDNA representational difference analysis used in the iden...

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Mechanisms of Ageing and Development 126 (2005) 882–891 www.elsevier.com/locate/mechagedev

cDNA representational difference analysis used in the identification of genes related to the aging process in rat kidney Bokyung Sung a, Kyung Jin Jung a, Hyun Seok Song b, Myung Jin Son b, Byung Pal Yu c, Hae Young Chung a,* a

College of Pharmacy, Aging Tissue Bank, Pusan National University, Jangjeon-dong, Geumjeong-ku, Busan 609-735, South Korea b Department of Molecular Biology, Pusan National University, Jangjeon-dong, Geumjeong-ku, Busan 609-735, South Korea c Department of Physiology, The University of Texas Health Science Center at San Antonio, TX 78229-3900, USA Accepted 22 March 2005 Available online 11 May 2005

Abstract Aging is a complex physiological process by which the functions of many organ systems deteriorate. Growing evidence shows that agerelated changes and damage are causally related to oxidative stress and inflammatory responses from reactive species. The aim of this study was to identify differentially expressed genes in old and young kidneys of Fisher 344 male rats during the aging process using complementary DNA representational difference analysis (cDNA RDA). cDNA RDA is a subtractive technique for identifying a focused set of differentially expressed genes. The distinctive advantage of this technique is its capability of detecting differences in gene expressions at less than one copy per cell and identifying genes not previously described in the database. Reverse transcription-polymerase chain reaction with specific primers was applied to confirm the differences found by RDA. Twenty-one putative differentially expressed genes were identified. Sixteen genes were up-regulated during aging and were associated with stress-response and inflammatory reactions, while five genes were down-regulated. These data suggested that the inflammatory process is a plausible cause of the aging process. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Aging; cDNA RDA; Inflammation; Oxidative stress; Gene expression

1. Introduction A general characteristic of the aging process is a progressive imbalance in the intrinsic control of regulatory systems. The reduced or impaired regulatory systems of aging organisms have adverse reactions to both internal and environmental stresses, such as an attenuated ability to maintain homeostasis, resulting in decreased immune function responsiveness and in increased vulnerability to disease and morbidity (Sherman and Goldberg, 2001; Masoro and Austad, 2001). Although the precise molecular mechanism of this multifaceted aging process has been extensively studied, it remains unclear. Accumulative evidence documents that oxidative damage caused by reactive species is in a large part responsible for the * Corresponding author. Tel.: + 82 51 510 2814; fax: + 82 51 518 2821. E-mail address: [email protected] (H.Y. Chung).

functional deterioration associated with aging (Yu, 1996; Finkel and Holbrook, 2000; Droge, 2002). The oxidative stress hypothesis of the aging process proposed that redox imbalance due to the net effect of oxidative stress and an insufficient counter-acting, anti-oxidative force is responsible for the disruption of normal cellular functions, characteristic of the aging process (Yu, 1996). Our recent review on the agerelated inflammatory process presented evidence showing that the involvement of the oxidative stress in the inflammatory response may be what links the aging process to many age-related diseases (Chung et al., 2000). Based on these available findings, we proposed a new term, ‘molecular inflammation’ to distinguish and emphasize the importance of molecular alterations and their reaction mechanisms that are distinct from chronic and fully expressed inflammatory phenomena (Bodamyali et al., 2000; Chung et al., 2002). The identification of genes in which expression correlated with age can provide valuable insight into the molecular

0047-6374/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2005.03.009

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mechanisms of the aging process. Recent studies emphasize the importance of such evidence in aging research (Vijg and Suh, 2003), and increasing efforts are expected to focus on categorizing the expression patterns of transcripts, linking altered to the determination of gene expression of the respective cellular responses. The elucidation of factors involved in gene expression and their functional roles in relation to aging is an important undertaking in the identification of underlying molecular mechanisms and provide opportunities for anti-aging studies. In recent years, a number of analytical techniques have been developed for the study of differential gene expression in an organism or in cells as to identify the genes involved in a given process or treatment (Diatchenko et al., 1996; Liang et al., 1994; Mathieu-Daude et al., 1999; Lukyanov et al., 1997; Brown et al., 1999; Wada et al., 1997). These techniques are all very powerful, allowing for the detection of changes in expression of mRNAs by selective enrichment. The selection of a suitable technique should depend on individual experimental needs and requirements. Representational difference analysis (RDA) is a PCRbased subtractive enrichment procedure. Originally developed for the identification of differences between complex genomes (Lisitsyn and Wigler, 1993), this technique is now adapted to enable the isolation of genes with an altered expression for comparison among various tissues or cell samples (cDNA RDA) (Hubank and Schatz, 1994). Briefly, the procedure relies on generation of representations of cDNA fragments from two different mRNA populations digested with a four-cutting restriction endonuclease followed by linker ligation and PCR amplification. The generated representations are then subjected to iterative steps of subtractive cross-hybridization and selective PCR amplification, to enrich the fragments of cDNA that are more abundant in one population. Unlike other currently available gene analyses, the RDA technique offers several advantages over other approaches including the isolation of false positives. Unwanted difference products can be competitively eliminated, and genes producing rare transcripts that may not be represented in currently available databases can be detected. Indeed, RDA can be more sensitive than other subtractive cDNA methods, and has the potential of identifying small differences in transcript levels (Hubank and Schatz, 1994). However, RDA methods have some limitations. For instance, the technique involves slightly more manipulation and is prone to minor impurities in samples. The limited size of the differential products, 250–300 base pairs, makes inconvenient for other applications. Expressed sequence tags (ESTs) and non-functional regions are often included, masking valuable information (Zabarovsky, 2000; Carulli et al., 2000). Although there have been several studies on gene expression profiles of brain, liver, and skeletal muscle using microarray technology (Prolla, 2002; Cao et al., 2001; Blalock et al., 2004), however, no reports show gene

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expression of aged kidney using cDNA RDA. In the present study, we report cDNA RDA analysis on the identification of genes differentially expressed in rat kidney during the aging process.

2. Experimental procedures 2.1. Animals Rat maintenance procedures for specific pathogen-free status and the dietary compositions for chow used in this study have been previously reported (Bertrand et al., 1999). This study complied with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (Publication No. 85-23) and was approved by an Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio. Briefly, specific pathogen-free Fischer 344 male rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% a-methionine, 0.2% choline chloride, 5% salt mix and 2% vitamin mix, and 3% Solka-Floc. Rats at 6, 12, 18, and 24 months of age were sacrificed by decapitation and the kidneys were quickly removed and rinsed in iced-cold buffer (pH 7.4, containing 100 mM Tris, 1 mM EDTA, 0.2 mM phenylmethyl-sulfonylfluoride (PMSF), 1 mM pepstatin A, 2mM leupeptin, 80 mg/L trypsin inhibitor, 20 mM bglycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate). Tissues were then immediately frozen in liquid nitrogen and stored at 80 8C. Close attention was given to histopathological examination of the aged kidney, and no evidence of nephrotic lesions was found in these soy protein-fed Fischer rats even at the advanced age of 24 months as shown by Iwasaki et al. (1988). 2.2. Preparation of RNA and cDNA synthesis Total RNA was extracted from rat kidney at ages 6, 12, 18, and 24 months (n = 5 per each group) using Trizol (Gibco BRL, Grand Island, NY, USA) and electrophoresed on a 1% agarose/MAE buffer (pH 7.0, containing 50% formamide, 2.2 M formaldehyde, 1 mM 4-morpholinopropanesulfonic acid (MOPS), 0.4 M sodium acetate, 0.05 mM EDTA) gel to examine for degradation. Six- and 24-monthold rat kidney RNAs were pooled and used for RDA. Poly(A)+ mRNA was isolated from 250 mg of total RNA using Oligotex mRNA Kit (Qiagen, Hilden, Germany) and cDNA was generated using the Superscript II reverse transcriptase (Gibco BRL, Grand Island, NY, USA) as outlined in the manufacturer’s protocol. 2.3. Representational difference analysis (RDA) RDA was performed as previously described (Hubank and Schatz, 1994). Each cDNA pool was digested with

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DpnII (NEB, Hertsfordshire, UK) and ligated to adaptors to allow PCR amplification of this material. To generate driver cDNA, the adaptors were removed from the amplified cDNA with DpnII digestion. To form tester cDNA, new adaptors were ligated to the amplified cDNA after DpnII digestion. The first round of subtraction was performed using tester and driver at a ratio of 1:100. After denaturation at 95 8C and renaturation at 67 8C for 24 h, PCR was performed using tester-specific primers to produce difference product 1 (DP1). DP1 was then digested with DpnII and new adaptors were added. This was then mixed with driver at a ratio of 1:1000 to generate difference product 2 (DP2). Bands were isolated from a 1.2% gel and ligated into pCR4-TOPO vector (TOPO TA Cloning1 Kit for Sequencing, Invitrogen). Plasmid DNA was sequenced by ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). The resulting sequences were then matched to existing sequences in the GenBank database. 2.4. Reverse transcription-polymerase chain reaction (RT-PCR)

reaction. Aliquots of the reverse transcripts (2 mL) were then amplified in a 25 ml PCR reaction, containing 1.5 U Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA) and 0.2 mM specific oligonucleotide primers. Electrophoresis was performed in 1.2% agarose gel. After staining in ethidium bromide solution achieved, the gel was observed under UV transilluminator.

3. Results 3.1. Amplicon formation To analyze differentially expressed genes with age, cDNA were obtained from young (6-month-old) and old (24month-old) F344 male rat kidneys. Fig. 1 describes the subtraction strategy. Initially, amplicons were prepared from each sample as outlined in Fig. 1. After isolation of mRNA from each population of total RNA, cDNA was prepared. Each cDNA sample was digested with DpnII. This is expected to result in fragments. 3.2. cDNA RDA of young and old rat kidney mRNAs

Semi-quantitative RT-PCR was carried out to compare levels of differentially expressed mRNAs in young and aged rat kidney. Briefly, total RNA was extracted as described above using Trizol and 2 mg of RNA was reverse transcribed using 200 units of Superscript reverse transcriptase II (Gibco BRL, Grand Island, NY, USA) and 500 ng of random primer (Promega, Madison, WI, USA) as primer per 25-mL

cDNA RDA was performed using young and old kidney mRNA samples as described in Section 2. Hybridization and subtraction was performed as outlined in Fig. 1. Young and old samples were derived from pools of tissue specimens obtained from a mixture of five individual young and old rats. To isolate the genes more actively transcribed in both

Fig. 1. Representative difference analysis (RDA) hybridization scheme for identifying differentially expressed genes during aging process. The RDA starts with total RNA of young and old rat kidney, after conversion to cDNA, with each result in a tester and driver cDNA population. For complete RDA, two separate comparisons are made. Tester (young, Y)/diver (old, O): identifies up-regulated messages, which are present in great abundant in cDNA population of young relative to old rats. Tester (O)/driver (Y): identifies down-regulated messages, which are abundant in cDNA population of old relative to young rats.

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young and old samples, we used cDNA from young and olds as the drivers and testers. Two rounds of subtraction were used to generate difference product 2 (DP2) in both instances, because this method has previously been shown to remove unwanted background and for enriching differentially expressed fragments (O’Neill and Sinclair, 1997). Fig. 2 shows the agarose gel electrophoresis of cDNA derived from young and old kidneys, which appeared as a smear ranging from 0.5 to 2 kbp. After restriction digestion with DpnII, DNAs were ligated to adaptor linkers, and the representative amplicons were then subjected to cDNA

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RDA. Two rounds of cDNA RDA were used to generate difference product (DP), and Fig. 2B shows amplification products for DPs. 3.3. Sequence analysis of subtraction products Difference products for first and second subtractions were characterized by DNA sequence analysis. After purification by agarose gel electrophoresis, difference products were cloned into the pCR4-TOPO vector and sequenced using an ABI automated sequencer and the appropriate sequencing primers. The sequence results were then used to interrogate publicly accessible nucleotide database (GenBank) and identify any sequence homologies with previously sequenced genes and expressed sequence tags (ESTs). One hundred clones were isolated in young and old kidneys. Analysis of these sequences indicated that 21 different sequences were represented in the young and old kidneys. Other sequences were identified as similar to known genes, human or mouse ESTs, and some sequences overlapped identified sequences. Tables 1 and 2 show the summary of identified sequences up- and down-regulated genes during aging process. Genes up-regulated with age are categorized by their biological functions, inflammatory and stress responses, energy metabolism, and signal transductions. On the other hand, only five biological functions were noted for the down-regulated genes. 3.4. Expression analysis of individual transcripts

Fig. 2. Gel electrophoresis of double-stranded cDNA and representative amplicons derived from young and old rat kidneys. (A) The ethidium bromide staining of agarose gel electrophoresis of cDNA derived from young and old rat kidneys appear as a smear ranging from 0.1 to 2 kbp. (B) After restriction enzyme digestion with DpnII, DNAs were ligated to adaptors, and then the representative amplicons were prepared. The representative amplicons show the smaller size, that is, 0.3–2 kbp (lanes 1 and 2). Lanes 3–6 shows differential products (DP), DP1 after one round of subtraction/amplification (lanes 3 and 4), and DP2 after two rounds of subtraction/amplification (lanes 5 and 6).

Because subtractive enrichment procedures have been shown to yield false positives, the expression pattern of individual clones in young versus old was further verified by semi-quantitative RT-PCR. A semi-quantitative RT-PCR was performed with a glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as a control for equal loading. To confirm upand down-regulated expressions of the isolated genes, we used four age groups, 6, 12, 18, and 24 months, to observe differential expression of each age group. Fig. 3 shows upregulated genes with age. Among them Ppib (cyclophilin B), urinary plasminogen activator (Plau), endothelin-1 (ET-1), TGF-1b, g-glutamyl transpeptidase (g-GT), ferritin subunit H (Fth), and heat shock protein-70 (HSP 70) markedly increased with age; then again, pro-a2(I) collagen (COL-1), receptor for advanced glycated end products (RAGE), Umodulin, and xanthine dehydrogenase (XDH) changed a little during the aging process. Fig. 4 shows the mRNA levels of five identified down-regulated genes with age; g-glutamylcystein synthetase (GS) and senescence marker protein-30 (SMP-30) were greatly reduced with age.

4. Discussion Advances in molecular biology have led to the development of powerful, high-throughput methods for

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Table 1 Up-regulated genes with aging in rat kidney by cDNARDA Functional category

Gene name

GenBank accession no.

Description

Inflammatory response

Rattus norvegicus cyclophilin B (Ppib)

NM_022536

Rattus norvegicus pro-a2(I) collagen (COL1A2) Rat receptor for advanced glycosylation end products (RAGE) Rattus norvegicus TGF-1b Rattus norvegicus endothelin-1 (ET-1) Rat urinary plasminogen activator, urokinase (uPA) Rattus norvegicus Tamm-Horsfall protein (THP/Urmodulin)

AF004877 L33413 NM_021578 NM_012548 NM_013085 NM_017082

Proinflammatory factor for T lymphocytes Inflammation/extracelluar matrix Amplification of inflammatory responses Inflammation Inflammation Tissue degradation and cell migration Activation of cytokine expression

Rattus norvegicus xanthine dehydrogenase (Xdh), mRNA Rattus norvegicus Ferritin subunit H (Fth1), mRNA

NM_017154 NM_012848

Rattus norvegicus g-glutamyl transpeptidase Rattus norvegicus heat shock 70 kD protein 8 (Hspa8)

M33821 NM_031165

Oxidative stress response Oxidative stress response Iron storing molecule Oxidative stress/redox Chaperone

Energy metabolism

Rat ATPase, Na+K+ transporting, a2 polypeptide (Atp1a2) Rattus norvegicus NADH-ubiquinone oxidoreductase

NM_012505 D86215

Cation transporter Mitochondrial OXPHOS

Signal transduction

Rat 11b-hydroxysteroid dehydrogenase type 1 gene (HSD11b1) Rattus norvegicus Ste-20 related kinase SPAK mRNA Rattus norvegicus Fas-associated factor (Faf)

NM_017080 NM_019362 NM_130406

Interconvert active glucocorticoids Serine/threonine kinase activity Apoptosis

Stress response

the analysis of differential gene expression (Carulli et al., 1998). The differential cloning method, cDNA RDA is reported to have advantages over the differential display method (Liang and Pardee, 1992) as used here, and is a powerful technique for the isolation of differentially expressed genes. Recently, new experimental approaches have been used to search for age-related changes in gene expression. These techniques included differential display, microarrays, and DNA chips. The common advantage of these three techniques is screening of a large number of mRNA species under various conditions with no a priori. These techniques were useful in the detection of expected and unexpected metabolic or signaling pathways in aging brain, pituitary, muscle, and liver (Cao et al., 2001; Grillari et al., 2000; Welle et al., 2001; Horikawa et al., 2001; Weindruch and Prolla, 2002). Published microarray studies reported stress response, and inflammatory related genes were up-regulated with age (Cao et al., 2001; Weindruch and Prolla, 2002; Terao et al., 2002). We compared the gene expressions found in this study with those reported from previous studies using liver, muscle, and brain of aged animals (Cao et al., 2001; Weindruch and Prolla, 2002; Terao et al., 2002) and found that our data agreed with those of other studies. We used cDNA RDA to isolate differentially expressed transcripts in young and old rat kidney. Isolation of

differentially expressed genes may also be attributed to the quality of mRNA, which is unstable in the presence of RNase, which is active in various tissues, including kidney. To prevent the RNA degradation and the loss of rarely expressed genes, whole kidneys were directly subjected to RNA isolation rather than isolating glomeruli or tubular segments. Furthermore, we used semi-quantitative RT-PCR analysis to verify the RDA output. Our cDNA RDA technique identified twenty-one putative differentially expressed genes during aging process. Among them, five genes were down-regulated with age. Thus, most of the isolated genes, which were up-regulated during the aging process, fall into four categories: (a) inflammatory response, (b) stress response, (c) energy metabolism, and (d) signal transduction. This study is, to our knowledge, the first to report on differentially expressed genes with age using the subtractive method. The following are descriptions of gene expression related to physiological and functional roles during aging. 4.1. Up-regulated genes during aging 4.1.1. Genes related to the inflammatory response Our recent review on the age-related inflammatory process presented evidence showing that the oxidative

Table 2 Down-regulated genes in aging process Gene name

GenBank accession no.

Description

Rattus sp. cytochrome oxidase subunit I (COX-1) Rattus norvegicus Aquaporin-2 (Aqp2), mRNA Rat Peptide methionine sulphoxide reductase (PMSR) Rat gamma-glutamylcysteine synthetase (g-GCS) Rat senescence marker protein (SMP-30)

S79304 NM_012909 AY005464 J05181 NM_031546

Miochondrial OXPHOS Renal water channel DNA repair GSH synthesis Calcium signaling

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process involved in the inflammatory response may be the major link bridging normal aging and many age-related degenerative diseases (Chung et al., 2000). In this study, we detected several genes related to inflammatory reactions were up-regulated with age. Cyclophilin B (CyPB) belongs to the immunophilin family and its biological fluid levels closely correlate with inflammation (Allain et al., 1999). For instance, urokinase plasminogen activator (uPA), which plays an important role in fibrinolysis and in the activation and chemotaxis of neutrophils and lymphocytes in kidney (Roelofs et al., 2003), was increased during the aging process as we detected in the present study. Two extracellular proteins, Tamm-Horsfall protein (THP/ uromodulin), known to enhance cytokine expression in monocytes, and pro-a2(I) collagen (COL1A2), increased in both acute and chronic inflammatory status regulated by NFkB (Lawrance et al., 2003) were observed. Correlated to the increase of COL1A2, the expression of transforming growth factor-1b (TGF-1b) was shown to increase. Because progressive fibrosis is a histological hallmark of the aging kidney, expression of fibrosis-related genes, COL1A2, pro a(III), collagen (COL-III), and TGF-1b and -3b, are shown to increase during aging (Gagliano et al., 2000). Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide and regulator of blood flow and plays an important role in cardiovascular disease caused by chronic kidney disease and end-stage renal failure (Amann et al., 2003; Virdis and Schiffrin, 2003). In addition, both mRNA and protein levels of ET-1 are up-regulated with age (Goettsch et al., 2001). Activation of the ET-1 with aging may promote the development of age-dependent diseases such as glomerulosclerosis, hypertension, and atherosclerosis (Goettsch et al., 2001; Barton et al., 2000). Similar to ET-1, the receptor for advanced glycation endproducts (RAGE) was found to increase in aged kidney. The accumulation of AGE occurs in euglycemia and during aging from oxidants and inflammation. Several studies have elucidated that RAGE activation caused inflammatory disorders, for example, atherosclerosis, inflammatory bowel disease, diabetes, and renal failure, which are all closely related to aging (Foell et al., 2003; Basta et al., 2002; Schmidt and Stern, 2000; Aronson, 2002). It was recently reported (Simm et al., 2004) that RAGE increases with age and contributes to the dysfunction in the heart. These data strongly indicate that the up-regulation of RAGE is involved in age-related renal inflammation. 4.1.2. Stress response genes In the current study, stress response genes, xanthine dehydrogenase (XDH), g-glutamyl transpeptidase (g-GT), ferritin subunit H, and heat shock protein 70 (HSP70) were found to increase with age. HSP70 protein is a well known molecular chaperone and is induced in all cells in response to various environmental stresses, heat and oxidative stress, and other stresses (Bartling et al., 2003). Previously, increased HSP70 expression during aging has been demonstrated to protect the cells or organism in response

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to oxidative stress (Wheeler et al., 1999; Maiello et al., 1998). Xanthine dehydrogenase (XDH) is a complex enzyme that catalyzes oxidation of hypoxathine to xanthine, subsequently producing uric acid. The enzyme has interconvertible forms, xanthine oxidase (XO) that is one of the major cellular sources of superoxide production. The conversion of XDH to XO increases during the aging process (Chung et al., 1999). This increased conversion of XDH to XO with age may be an important contributing factor to increased renal oxidative stress during aging (Chung et al., 1999). The role of g-GT (EC 2.3.2.2) in the glutathione (GSH) metabolism is to recover cysteine from extracellular GSH (Eschwe`ge et al., 1997). Previous studies on g-GT provided limited information on the enzyme activity during the aging process. The g-GT activity shows tissue differences as reduces in the muscle, but increases in kidney and liver during aging. In the present study, we observed that the mRNA expression of g-GT increased with age. Recent studies have shown that g-GT plays an active pro-oxidant role under certain conditions that enhance oxidative stress (Pieri et al., 2003; Cutrin et al., 2000; Dominici et al., 2003). Therefore, the up-regulation of g-GT in aged rat kidney might contribute to age-related oxidative stress. Ferritin has been suggested to be an antioxidant protein. Ferritin is composed of two subunit types, termed H and L, and both are involved in the reduction of oxidative stress. Transcription of the ferritin gene is shown to increase under conditions such as aging and oxidative stress (Rikans et al., 1997; Cairo et al., 1995; Orino et al., 2001). Furthermore, the overexpression of ferritin is shown to reduce oxidativerelated cell death (Orino et al., 1999). Therefore, the ferritin increase observed in our results, also could indicate a high renal oxidative status that may be partly responsible for agerelated to glomerular deterioration (Moriguchi et al., 2003). 4.1.3. Energy metabolism related genes The up-regulation of Na+K+-ATPase, a2 (Atp1a2), and NADH-ubiquinone oxidoreductase was observed during the aging process. Na+K+-ATPase (sodium pump) is a heterodimer protein that plays a key role in the maintenance of cell homeostasis by regulating the membrane potential and cation transport across the sarcolemmal membrane (Ostadal et al., 2003). In addition, there is evidence that oxygen-free radicals and the aging process decrease the Na+K+-ATPase activity and gene expression (Dhalla et al., 1999; Asghar and Lokhandwala, 2004), although conflicting evidence exists. For instance, Bu¨ ssemaker et al. (2003) reported that mRNA expression and activity of the Na+K+-ATPase subunit increased in aged rat. Also, Na+K+-ATPase activity increased in alcohol-induced and diabetic rat kidney (Rodrigo et al., 2002; Baines and Ho, 2002). In this study, we observed Na+K+-ATPase expression increased with age. NADH:ubiquinone oxidoreductase (NUO) is a major component of the mitochondrial energy generating system. We observed that NUO expression increased with age in rat

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Fig. 3. Verification of identified genes up-regulated with age by semi-quatitative RT-PCR analysis. The genes were categorized by their functions: (A) inflammatory response, (B) stress response, (C) energy metabolism, and (D) signal transduction. RT-PCR analysis performed on RNA isolated from F344 rat kidneys, ages 6, 12, 18, and 24 months. GAPDH primers were used in separate PCR reactions to control for efficiency of cDNA synthesis in each sample.

kidney. NUO also increased in cardiomyocytes under ischemia-reperfusion (Yeh et al., 2004) and in MPTPtreated mice (Gu et al., 2003). NADH:ubiquinone oxidoreductase (complex I) consists of more than 42 subunits. The precise roles of all subunit of NUO are not well understood. 4.1.4. Genes involved in signal transduction In the present study, we observed the increase of 11bhydroxysteroid dehydrogenase type 1 (11b-HSDl), Ste-20 related kinase SPAK, and Fas-associated factor 1 (FAF 1) in

aged kidney. FAF1 is a Fas-associating molecule that enhances Fas-mediated apoptosis (Chu et al., 1995). During aging, the altered regulation of cell death, such as Fas/Fas L system, leads to apoptosis (Pahlavani and Vargas, 2001). The expression of Fas was recently shown to greatly increase in aged liver (Pinti et al., 2003; Zhang et al., 2002); however, no data are available in the relationship between FAF 1 and aging. We observed the up-regulation of FAF 1 with age in the present. Stress kinase SPAK is a serine/threonine kinase that belongs to the SPS1 subfamily of STE20 kinases and is expressed in various tissues including kidney (Johnston et al., 2000). SPAK plays an important role in the activation of MAP kinase pathway such as with p38 phosphorylation. Our previous report on the activation of stress kinases in aged kidney (Kim et al., 2002) supports SAPK induction in the aging process. 11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1) is known as a lip-hydroxycorticosteroids accessing regulator in cortisone/cortisol shuttle. 11b-HSD1 is proposed as a compensatory response to inflammatory stimuli, such as IL1b and TNF-a, to promote availability of anti-inflammatory glucocorticoids (Yong et al., 2002; Escher et al., 1997). 4.2. Down-regulated genes during aging

Fig. 4. Verification of identified genes down-regulated with age by semiquatitative RT-PCR analysis. RT-PCR analysis performed form RNA isolated from F344 rat kidneys, ages 6, 12, 18, and 24 months. GAPDH primers were used in separate PCR reactions to control for efficiency of cDNA synthesis in each sample.

In the current study, only five down-regulating genes were detected by cDNA RDA in aged rat kidney. Among them, two genes, peptide methionine sulphoxide reductase (PMSR) and senescence maker protein-30 (SMP-30) were

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observed. PMSR expresses in kidney and liver, and its expression and activity decrease with age. It has been suggested that the down-regulation of PMSR can contribute to the accumulation of oxidized protein associated with the aging process (Petropoulos et al., 2001). The other aging marker, SMP-30, is expressed specially in the liver and kidney and progressively decreases during aging (Fujita et al., 1992). A recent study (Fujita et al., 1999) showed that SMP-30 modulates the activity of the Ca2+ pump, suggesting that the decline of SMP-30 leads to the dysregulation of Ca2+ homeostasis, which may result in the age-related deterioration via the disturbance of intercellular signaling system. Water channel, aquaorin-2 (Aqp-2) decreased during aging process as monitored in the current study. A previous study reported that the decrease of Aqp-2 with aging is closely associated with the reduction of papillary osmolality (Combet et al., 2001). This drop in Aqp-2 expression in the distal part of the nephron could be the main cause for the fall in the concentrating ability of the kidney and for the agerelated impaired control of hydration (Teillet et al., 1999). Declining g-glutamylcysteine synthetase (g-GCS) with the age was observed in the current study. GCS is the ratelimiting enzyme in the novo GSH synthesis, and reports show an age-related decrease of its activity and expression (Liu, 2002; Iantomasi et al., 1993). Liu (2002) suggested that a decrease of GCS might contribute to the age-associated declining of GSH content and underlie the increased oxidative damage of macromolecules in the aged animals. Cytochrome oxidase subunit I (COX-1), mitochondrialencoded enzyme, decreased with age. A previous study reported a mitochondrial cytochrome oxidase activity that is closely associated with oxidative stress, decreased in brain, heart, liver, and kidney of aged mice (Navarro et al., 2004). However, the mRNA level of COX shows differences depending on the subunit types and tissues (Barazzoni et al., 2000). Our data shows a down-regulation of COX-1 in aged rat kidneys, which may be contributed by oxidative stress during aging. In summary, we have identified genes that are up- and down-regulated during the aging process. We showed that several genes involved in stress and inflammatory response genes are preferentially expressed in aged rats. Although only a limited number of genes were identified according to their functionality, the information gained from our study could provide additional database on the physiological changes that occur with age, such as increased stress and inflammatory responses. In addition, the characterization of the roles of up- and down-regulated genes during the aging process could provide clues to the new information for future studies on aging.

Acknowledgements This study was financially supported by Pusan National University in the program, Post-doc. 2005. This work is

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supported by a grant from the Plant Diversity Center, 21st Frontier Research Program, Ministry of Science and Technology (PF0320801-00).

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