the role of apurinic/apyrimidinic endonuclease on the progression of streptozotocin-induced diabetic...

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Acta Histochemica 114 (2012) 647–652 Contents lists available at SciVerse ScienceDirect Acta Histochemica jou rnal h o mepage: www.elsevier.de/acthis The role of apurinic/apyrimidinic endonuclease on the progression of streptozotocin-induced diabetic nephropathy in rats Jin Nam Kim a,1 , In Youb Chang b,1 , Jin Hwa Kim c , Jung Woo Kim d , Kyeong-Soo Park e , Hyun Il Kim f , Sang Pil Yoon g,a Department of Internal Medicine, Seoulpaik Hospital, Inje University College of Medicine, Seoul, Republic of Korea b Department of Anatomy, College of Medicine, Chosun University, Gwangju, Republic of Korea c Department of Internal Medicine, College of Medicine, Chosun University, Gwangju, Republic of Korea d Department of Anatomy, College of Medicine, Seonam University, Namwon, Jeollabuk-Do, Republic of Korea e Department of Preventive Medicine, College of Medicine, Seonam University, Namwon, Jeollabuk-Do, Republic of Korea f Department of Optometry, College of Health Sciences, Eulji University, Sungnam, Gyeonggi-Do, Republic of Korea g Department of Anatomy, School of Medicine, Jeju National University, Jeju-Do, Republic of Korea a r t i c l e i n f o Article history: Received 28 July 2011 Received in revised form 20 November 2011 Accepted 21 November 2011 Keywords: APE Apoptosis Chitosan oligosaccharide Diabetes Kidney p53 Rat a b s t r a c t Apurinic/apyrimidinic endonuclease (APE) acts as a regulator of p53 or vice versa in the cellular response to oxidative stress. Since oxidative stress-induced apoptosis is suggested in the pathophysiology of diabetic nephropathy, we proposed that APE may have a feasible role in the progression of diabetic complications. We investigated the interrelationship between APE and p53 in streptozotocin-induced diabetic rat kid- neys. Variable parameters on kidneys were checked 12 weeks after streptozotocin administration with or without chitosan oligosaccharide (COS) treatment. Streptozotocin administration caused changes as seen in early diabetic nephropathy with increased kidney size, increased p53, decreased APE, and increased cleaved caspase-3. COS was not suspected as being detrimental to renal measurements, and caused the augmentation of APE after streptozotocin administration. The augmented APE, in association with increased p53, suppressed cleaved caspase-3. 8-OHdG was mainly immunolocalized in the distal tubules, but also in the proximal tubules after streptozotocin administration without COS treatment, while APE was observed in proximal tubules in all groups. These results suggested that p53-dependent apoptosis resulting in suppressed APE might be an underlying mechanism of streptozotocin-induced nephropathy. © 2011 Elsevier GmbH. All rights reserved. Introduction It is well known that early in diabetes the kidney increases in size in association with glomerular hypertrophy by increased glomerular filtration rate, tubular dilatation and accumulation of advanced glycation end products. Though there are many hypothe- ses to explain diabetic nephrotoxicity, hyperglycemia itself also leads to excess free-radical generation and induces oxidative stress (Allen et al., 2005). Excessive oxidative stress may result in oxida- tive damage to proteins, lipids and DNA, and subsequently leads to Abbreviations: APE, apurinic/apyrimidinic endonuclease; BER, base excision repair; COS, chitosan oligosaccharide; RAS, renin–angiotensin system; STZ, strep- tozotocin. Corresponding author at: Department of Anatomy, School of Medicine, Jeju National University, 66 Jejudaehakno, Jeju-Si, Jeju-Do 690-756, Republic of Korea. E-mail address: [email protected] (S.P. Yoon). 1 These authors equally contributed to this study. apoptosis (Allen et al., 2005; Brownlee, 2007). Oxidative stress- induced apoptosis is described in the pathophysiology of streptozotocin (STZ)-induced diabetic nephropathy models (Tesch and Nikolic-Paterson, 2006; Tesch and Allen, 2007). Some reports have provided important concepts regarding the role of p53 in cisplatin-induced nephrotoxicity (Jiang et al., 2007; Jiang and Dong, 2008). This is also true in the case of STZ (Imaeda et al., 2002), because STZ and cisplatin are well recognized as DNA damaging agents. Toxic chemicals, including STZ-induced DNA damage, are considered to be an important trigger of p53 activa- tion. Increased p53 is observed before effector caspase activation and directly activated Bax to induce apoptosis (Chipuk et al., 2005). p53 also participates in base excision repair (BER) against single- base DNA damage due to oxidative stress via its direct interaction with apurinic/apyrimidinic endonuclease (APE) (Zhou et al., 2001). APE is a dual-functional protein that serves as the endonu- clease for an apurinic/apyrimidinic (AP) site in BER and that associates with p53 as a key regulator of apoptosis (Bernstein et al., 2002; Tell et al., 2009). As a consequence, APE acts as a 0065-1281/$ see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.11.011

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Page 1: The role of apurinic/apyrimidinic endonuclease on the progression of streptozotocin-induced diabetic nephropathy in rats

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Acta Histochemica 114 (2012) 647– 652

Contents lists available at SciVerse ScienceDirect

Acta Histochemica

jou rna l h o mepage: www.elsev ier .de /ac th is

he role of apurinic/apyrimidinic endonuclease on the progression oftreptozotocin-induced diabetic nephropathy in rats

in Nam Kima,1, In Youb Changb,1, Jin Hwa Kimc, Jung Woo Kimd, Kyeong-Soo Parke, Hyun Il Kimf,ang Pil Yoong,∗

Department of Internal Medicine, Seoulpaik Hospital, Inje University College of Medicine, Seoul, Republic of KoreaDepartment of Anatomy, College of Medicine, Chosun University, Gwangju, Republic of KoreaDepartment of Internal Medicine, College of Medicine, Chosun University, Gwangju, Republic of KoreaDepartment of Anatomy, College of Medicine, Seonam University, Namwon, Jeollabuk-Do, Republic of KoreaDepartment of Preventive Medicine, College of Medicine, Seonam University, Namwon, Jeollabuk-Do, Republic of KoreaDepartment of Optometry, College of Health Sciences, Eulji University, Sungnam, Gyeonggi-Do, Republic of KoreaDepartment of Anatomy, School of Medicine, Jeju National University, Jeju-Do, Republic of Korea

r t i c l e i n f o

rticle history:eceived 28 July 2011eceived in revised form0 November 2011ccepted 21 November 2011

eywords:PE

a b s t r a c t

Apurinic/apyrimidinic endonuclease (APE) acts as a regulator of p53 or vice versa in the cellular response tooxidative stress. Since oxidative stress-induced apoptosis is suggested in the pathophysiology of diabeticnephropathy, we proposed that APE may have a feasible role in the progression of diabetic complications.We investigated the interrelationship between APE and p53 in streptozotocin-induced diabetic rat kid-neys. Variable parameters on kidneys were checked 12 weeks after streptozotocin administration with orwithout chitosan oligosaccharide (COS) treatment. Streptozotocin administration caused changes as seenin early diabetic nephropathy with increased kidney size, increased p53, decreased APE, and increasedcleaved caspase-3. COS was not suspected as being detrimental to renal measurements, and caused

poptosishitosan oligosaccharideiabetesidney53at

the augmentation of APE after streptozotocin administration. The augmented APE, in association withincreased p53, suppressed cleaved caspase-3. 8-OHdG was mainly immunolocalized in the distal tubules,but also in the proximal tubules after streptozotocin administration without COS treatment, while APEwas observed in proximal tubules in all groups. These results suggested that p53-dependent apoptosisresulting in suppressed APE might be an underlying mechanism of streptozotocin-induced nephropathy.

ntroduction

It is well known that early in diabetes the kidney increasesn size in association with glomerular hypertrophy by increasedlomerular filtration rate, tubular dilatation and accumulation ofdvanced glycation end products. Though there are many hypothe-es to explain diabetic nephrotoxicity, hyperglycemia itself also

eads to excess free-radical generation and induces oxidative stressAllen et al., 2005). Excessive oxidative stress may result in oxida-ive damage to proteins, lipids and DNA, and subsequently leads to

Abbreviations: APE, apurinic/apyrimidinic endonuclease; BER, base excisionepair; COS, chitosan oligosaccharide; RAS, renin–angiotensin system; STZ, strep-ozotocin.∗ Corresponding author at: Department of Anatomy, School of Medicine, Jejuational University, 66 Jejudaehakno, Jeju-Si, Jeju-Do 690-756, Republic of Korea.

E-mail address: [email protected] (S.P. Yoon).1 These authors equally contributed to this study.

065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.acthis.2011.11.011

© 2011 Elsevier GmbH. All rights reserved.

apoptosis (Allen et al., 2005; Brownlee, 2007). Oxidative stress-induced apoptosis is described in the pathophysiology ofstreptozotocin (STZ)-induced diabetic nephropathy models (Teschand Nikolic-Paterson, 2006; Tesch and Allen, 2007).

Some reports have provided important concepts regarding therole of p53 in cisplatin-induced nephrotoxicity (Jiang et al., 2007;Jiang and Dong, 2008). This is also true in the case of STZ (Imaedaet al., 2002), because STZ and cisplatin are well recognized as DNAdamaging agents. Toxic chemicals, including STZ-induced DNAdamage, are considered to be an important trigger of p53 activa-tion. Increased p53 is observed before effector caspase activationand directly activated Bax to induce apoptosis (Chipuk et al., 2005).p53 also participates in base excision repair (BER) against single-base DNA damage due to oxidative stress via its direct interactionwith apurinic/apyrimidinic endonuclease (APE) (Zhou et al., 2001).

APE is a dual-functional protein that serves as the endonu-clease for an apurinic/apyrimidinic (AP) site in BER and thatassociates with p53 as a key regulator of apoptosis (Bernsteinet al., 2002; Tell et al., 2009). As a consequence, APE acts as a

Page 2: The role of apurinic/apyrimidinic endonuclease on the progression of streptozotocin-induced diabetic nephropathy in rats

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egulator of p53 or vice versa. p53 down-regulates APE expres-ion in response to severe DNA damage, thereby promotingpoptosis in human colorectal cancer cells (Zaky et al., 2008)nd in human embryonic kidney 293 cells (Xiong et al., 2008).ecently, we have shown that oxidative DNA damage in ratodels of hydronephrosis may trigger p53-dependent apoptosis

hrough repression of APE (Chang et al., 2011). Therefore, we pro-osed that APE has a possible role in the progression of diabeticephropathy.

Antioxidants such as vitamin C, vitamin E, beta-carotene, taurinend catechin may represent an important defense for the treatmentr prevention of diabetic nephropathy models (Hase et al., 2006;orbes et al., 2008; Wagener et al., 2009). Chitosan oligosaccha-ide (COS) is an antioxidant of natural origin and has anti-diabeticffects via various mechanisms (Lee et al., 2003; Liu et al., 2007;oon et al., 2008; Kim et al., 2009; Yuan et al., 2009). In our previ-us report (Kim et al., 2009), highly deacetylated COS was showno improve the altered glucose metabolism in STZ-induced diabeticats through neogenesis of insulin-producing cells and increasedhe secretory capacity of insulin. As a result, the reductive effect ofOS on HbA1c in our previous report (Kim et al., 2009) is the sames that of an earlier report with metformin (Liu et al., 2008). More-ver, COS has protective effects on glycerol-induced acute renalailure (Yoon et al., 2008) and paraquat-induced nephrotoxicityYoon et al., 2011).

Since nephrotoxicity in association with STZ-induced diabetesay be related to elevated levels of DNA damage, we believe

hat APE may have a feasible role in the progression of dia-etic nephropathy. p53 serves as a key regulator of oxidativetress-induced apoptosis, and we attempted to investigate thenterrelationship between APE and p53 on STZ-induced diabeticat kidneys. In addition, renoprotective effects of COS on diabeticephropathy have not been reported despite several reports on thenti-diabetic effects of COS. Therefore, we also investigated the pro-ective effects of COS in STZ-induced diabetic rat kidneys based onhe activation of APE.

aterials and methods

nimals and treatment

In our previous report (Kim et al., 2009), we fully described thenduction of diabetes as well as preparation and feeding of COS.n brief, low molecular weight chitosan (>98% deacetylated, <10ps viscosity) was purchased from YB bio (Gyungbuk, Republicf Korea) and COS was obtained by the enzymatic method. Maleprague-Dawley rats (8–10 weeks old; Da-mool Science, Daejeon,epublic of Korea) were randomly divided and received 500 mg/kgf COS or 1 ml of distilled water (p.o., once a day). StreptozotocinSTZ; 60 mg/kg, Sigma–Aldrich, St. Louis, MO, USA) was injectedntraperitoneally to the fasted rats. Then rats were grouped as fol-ows; Control, COS, STZ, COS-STZ (n = 5 per group), and continued2 weeks after STZ administration.

All experimental procedures and care of animals were con-ucted in accordance with the guidelines of Chosun University’snimal Care and Use Committee.

easurements

At the end of the experiment, total body weight, kidney weight,

olume ratio of kidney weight per total body weight were checked.lood samples were collected and blood urea nitrogen (BUN) andreatinine were determined by Green Cross Reference LaboratoryGyunggi-Do, Republic of Korea).

ica 114 (2012) 647– 652

Antibodies

The primary antibodies used in this study were: poly-clonal anti-p53 (1:500; Santa Cruz Biotechnology, Santa Cruz,CA, USA), monoclonal anti-APE (1:1000; Santa Cruz Biotechnol-ogy), polyclonal anti-cleaved caspase-3 (1:500; Cell SignalingTechnology, Danvers, MA, USA), monoclonal anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, 1:200; JaICA, Shizuoka, Japan), andpolyclonal anti-�-actin (1:1000; Santa Cruz Biotechnology).

Histology and immunohistochemistry

Kidneys were fixed with 4% paraformaldehyde, embedded inparaffin wax (Tissue-Tek, Sakura, Japan), and then 5 �m-thick tis-sue sections were cut using a Leica RM 2155 rotary microtome(Leica Microsystems, Nussloch, Germany). Randomly selected sam-ples were stained with hematoxylin and eosin (H/E) and periodicacid–Schiff (PAS) using a routine protocol.

Immunohistochemical staining was carried out by a routinemethod. In brief, incubation with primary antibodies was per-formed for 48 h at 4 ◦C. The binding was visualized using anImmPRESSTM avidin–biotin-peroxidase kit (Vector Laboratories,Burlingame, CA, USA) according to the manufacturer’s instructions.Omission of incubation with the primary or secondary antibodyserved as a control for false-positives. Immunolabelled images werecaptured directly using an Olympus C-4040Z digital camera andOlympus BX-50 microscope (Olympus Corp., Tokyo, Japan). Thecaptured images were saved and subsequently processed usingAdobe Photoshop (Adobe System, San Jose, CA, USA). The bright-ness and contrast of the images were adjusted only for the purposeof background consistency.

Western blot analysis

Renal tissues were suspended in cold homogenizing buffer con-taining protease inhibitor cocktail (Roche Diagnostics, Mannheim,Germany), and homogenized using a ultrasonic cell disruptor(Branson Ultrasonics, Danbury, CT, USA) for 30 s three times, witha 30 s interval, and centrifuged at 10,000 × g for 10 min at 4 ◦C.Protein concentration in the supernatants was determined usinga Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). An aliquotof the supernatant (30 �g protein) was suspended in 20 �l of aloading buffer and then boiled for 5 min at 100 ◦C, electrophoresedon 10% SDS-PAGE gels, and transferred to polyvinyldifluo-ridine membranes (GE Healthcare Bio-Sciences, Piscataway,NJ, USA).

Immunoblotting was carried out with each primary anti-body. The horseradish peroxidase-linked secondary antibodies (GEHealthcare Bio-Sciences) were diluted 1:4000. The blotted proteinswere then detected using an Enhanced Chemiluminescence DetectSystem (iNtRON, Biotech, Seoul, Korea). The bands were quanti-fied using ImageQuant 350 (GE Healthcare Korea, Seoul, Republicof Korea), and the data expressed as densitometric units of eachprimary antibody relative to �-actin and in reference to the valueof the control sample for each gel.

Statistical analysis

Data are expressed as mean ± SD. Statistical significance wasassessed by one-way analysis of variance (ANOVA) with Bonferroni

test in four groups with or without COS and/or STZ treatments. Allstatistical analyses were conducted using SPSS, version 12.0 (SPSS,IBM, Chicago, IL, USA). A p value of less than 0.05 was taken asstatistically significant.
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J.N. Kim et al. / Acta Histochemica 114 (2012) 647– 652 649

Table 1Effects of chitosan oligosaccharide (COS) in relation to the measurements with or without streptozotocin (STZ) administration.

Variables Kidney (g)a Kidney/weight (%)b BUN (mg/dl) Creatinine (mg/dl)

Control 1.186 ± 0.113 0.346 ± 0.029 21.88 ± 2.40 0.580 ± 0.084COS 1.020 ± 0.104 0.362 ± 0.055 20.63 ± 1.73 0.567 ± 0.052STZ 1.223 ± 0.230 0.598 ± 0.085 24.25 ± 5.20 0.600 ± 0.063COS-STZ 1.448 ± 0.139 0.639 ± 0.129 25.60 ± 5.69 0.586 ± 0.056

(p < 0. betw

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a Weight of kidney was significantly increased between COS and COS-STZ group

b Volume ratio of kidney weight per total body weight was significantly changedSTZ and COS-STZ) groups (p < 0.05), respectively.

esults

Variable measurements of this study were compared betweenour groups (Table 1). Kidney weight was increased after STZdministration irrespective of COS treatment. Kidney weight wasignificantly increased between COS group and COS-STZ group1.020 ± 0.104 g vs. 1.448 ± 0.139 g, p < 0.05). The volume ratiof kidney weight per total body weight was relatively con-tant in the control group and the COS group (0.346 ± 0.029% vs..362 ± 0.055%). But it was significantly elevated in STZ groupnd COS-STZ group (0.598 ± 0.085% vs. 0.639 ± 0.129%). The vol-me ratio of kidney weight per total body weight was significantly

ncreased between control and diabetic (STZ and COS-STZ) groupsnd between COS and diabetic (STZ and COS-STZ) groups, respec-ively (p < 0.05 each). BUN and creatinine were slightly elevated butignificance was not found.

As indicated in Fig. 1, it made no difference on the cytoar-hitecture of kidney whether there was COS treatment or not.nfiltration of inflammatory cells were transitorily seen 4 weeksfter STZ administration (asterisks), while it was not clearly visiblen case of COS treatment. According to previous reports (Tesch andikolic-Paterson, 2006; Tesch and Allen, 2007) 4 weeks after STZ

dministration was not necessary to develop diabetic nephropathy.hey suggested that it took at least 7–8 weeks, and we continuedo raise animals until 12 weeks after STZ administration. At thend of the experiment, the kidney showed little abnormality but

ig. 1. Photographs of kidneys under diabetic progression after streptozotocin (STZ) admidneys irrespective of STZ treatment. Grossly, the kidney shows little abnormality undnjection (asterisks), while this was not apparent in case of COS treatment. Scale bar = 100

05).een control and diabetic (STZ and COS-STZ) groups (p < 0.05), and COS and diabetic

relatively large lumens of the tubules (tubular dilatations) wereonly seen in COS-STZ group. PAS stain outlines the basement mem-branes of glomeruli and tubules (Fig. 4, upper column). In thecortical labyrinth, proximal tubules generally have a larger diame-ter with brush border than distal tubules have; cross sections of thelumen often appear stellate in paraffin sections. Some changes arefocal glomerulosclerosis and degenerative changes in distal convo-luted tubules on the vascular pole of glomeruli in STZ group.

As demonstrated in Fig. 2, p53 (0.345 ± 0.043 vs. 1), APE(0.549 ± 0.138 vs. 1) and cleaved caspase-3 (0.695 ± 0.292 vs. 1)had slightly weak density in COS group compared to controlgroup. 12 weeks after STZ administration, p53 (1.925 ± 0.515and 2.344 ± 0.208, p < 0.05/each) significantly augmented irrel-evant to COS treatment. APE was decreased in STZ group(0.660 ± 0.177), but significant elevation was observed in COS-STZ group (1.778 ± 0.376, p < 0.05/each) compared to every othergroup. Cleaved caspase-3 significantly increased in STZ group(1.238 ± 0.175, p < 0.05) compared to the COS group, and consid-erably decreased in the COS-STZ group (0.575 ± 0.107, p < 0.05)compared to the STZ group.

8-OHdG (Fig. 3, upper column), a marker for oxidative damage,was immunolocalized mainly in the distal tubules and collect-

ing ducts of the renal cortex in control and COS groups. Theimmunoreactivities were noted across the entire nephron afterSTZ administration (Fig. 4, middle column), while the COS-STZgroup showed a similar distribution of immunolocalized 8-OHdG

inistration. Chitosan oligosaccharide (COS) did not affect the cytoarchitecture ofer H/E stain. Inflammatory cells were transitorily infiltrated at 4 weeks after STZ

�m.

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650 J.N. Kim et al. / Acta Histochemica 114 (2012) 647– 652

Fig. 2. Western blot analysis and densitometric results of p53, APE and cleaved caspase-3 in normal and streptozotocin (STZ)-induced diabetic kidneys. Chitosan oligosac-c 3 in ng or COi mpar

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haride (COS) treatment did not adversely affects the p53, APE and cleaved caspase-roups, on APE in the COS-STZ group, and on cleaved caspase-3 in the STZ groupncreased after STZ administration. *p < 0.05 compared to control group, #p < 0.05 co

o that of control and COS groups. APE (Fig. 3, lower column) wasmmunolocalized in the proximal tubules of the renal cortex of allroups. The immunoreactivities were evident in proximal tubulesf the deep renal cortex (S3 segment, especially), and did nothanged the distribution of immunolocalized APE to STZ adminis-ration. COS-STZ group showed increased APE immunoreactivitiescross the entire nephron compared to the other groups, whichere especially distinct in deep renal cortex (Fig. 4, lower column).

iscussion

This is the first report to show that repressed APE may play a rolen the progression of STZ-induced diabetic nephropathy, and thatOS may have renoprotective effects through augmented APE. Our

esults showed that early stage of diabetic nephropathy was estab-ished after STZ administration with increased p53-dependentpoptosis, and that COS-treated rat kidneys showed the augmentedPE and suppressed cleaved caspase-3. It is noteworthy that the

ig. 3. Distribution of 8-OHdG and APE in normal and streptozotocin (STZ)-induced diabeubules, but across the entire tubules of nephron in STZ group. APE was immunolocalizedfter STZ administration. Chitosan oligosaccharide (COS) did not affect the immunolocalcale bar = 200 �m.

ormal kidneys. Statistical significance was observed on p53 in the STZ and COS-STZS-STZ group. Cleaved caspase-3 negatively related to APE, while p53 constantly

ed to COS group, §p < 0.05 compared to STZ group.

change in APE, irrespective of augmented p53, negatively regulatedthe apoptotic activity in STZ-induced diabetic kidneys.

Oxidative stress has been identified in a variety of dis-eases, and oxidative stress-induced apoptosis is suggested inthe pathophysiology of STZ-induced diabetic nephropathy (Teschand Nikolic-Paterson, 2006; Tesch and Allen, 2007). Major cellu-lar strategies coping with oxidative DNA damage are repair andremoval (Bernstein et al., 2002). Various DNA repair pathways areactivated upon oxidative DNA damage. BER is a major DNA repairpathway protecting cells against single-base DNA damage and canbe initiated through removal of a damaged base by a DNA glycosy-lase. As a result, an AP site can be generated and APE (also calledredox factor) acts as the major AP endonuclease (Bernstein et al.,2002; Tell et al., 2009). Although APE is a pro-survival protein, APEacts as a regulator of p53 and p53 participates in BER via its direct

interaction with APE (Zhou et al., 2001; Bernstein et al., 2002; Xionget al., 2008; Zaky et al., 2008; Tell et al., 2009; Chang et al., 2011).

In this study, we clearly demonstrated that STZ-induced dia-betic nephropathy might be caused by a p53-dependent apoptotic

tic kidneys. 8-OHdG, a marker for oxidative damage, was mainly seen in the distal in the proximal tubules in all groups, but the immunoreactivities were increased

izations in the kidney, but increased APE after STZ injection. Asterisks, glomeruli.

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J.N. Kim et al. / Acta Histochemica 114 (2012) 647– 652 651

Fig. 4. The nephrons in normal and streptozotocin (STZ)-induced diabetic kidneys. Proximal (P), distal (D) and collecting (C) tubules display features that aid in theiridentification under PAS stain. Degenerative changes were seen on the vascular pole (arrow) of glomerulus and distal tubules in STZ group, while chitosan oligosaccharide( of 8-Or

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COS) did not affect the structure of nephrons. The increased immunolocalization

espectively. Scale bar = 50 �m.

athway. The negative correlation between increased p53 andecreased APE was accompanied by increased cleaved caspase-3,n effector for apoptosis. Augmented p53 may directly activate Baxr indirectly via suppressed APE, which can induce the caspaseascades and apoptosis. It was reinforced by the immunohisto-hemistry with 8-OHdG, the marker for oxidative damage. 8-OHdGas mainly immunolocalized in the distal tubules and collectingucts of control, COS and COS-STZ renal cortices, and also observed

n proximal tubules of STZ group only. While STZ administra-ion in the COS treated group caused simultaneous increases of53 and APE, it results in decreased cleaved caspase-3. Since p53ignificantly augmented after STZ administration, p53-dependentpoptosis can be considered to depend on the change of APE inTZ-induced diabetic nephropathy.

In addition to being a regulator of p53, APE acts as a trans-cting factor for repression of the human renin gene (Fuchs et al.,003). The intrarenal renin–angiotensin system (RAS) is upregu-

ated in diabetic nephropathy (Carey and Siragy, 2003; Singh et al.,005; Thomas et al., 2005). Renal tubular apoptosis is attenuatedy angiotensin I converting enzyme inhibitor (Sun et al., 2009)nd increased angiotensin II is related to the p53 (Singh et al.,008). Upregulated RAS causes vasoconstriction through increasedngiotensin II and hypoxic injury to diabetic kidney. This study alsoupported the notion in STZ group that decreased APE might notuppress renin gene expression and then RAS might be activated.PE augmentation, however, was observed in COS-STZ group. In

his group, relatively high levels of APE may block p53-dependentpoptotic pathway. Increased APE could also suppress the reninene expression, inactivate the RAS, and then reduce hypoxic

njury. In addition, previous reports suggested that COS itself hasnhibitory activities for renin (Park et al., 2008) and angiotensin Ionverting enzyme (Park et al., 2003; Huang et al., 2005). Takenogether, it is suggested that COS treatment might cause APE

HdG was seen in STZ group and APE in COS-STZ group compared to other groups,

activation, suppress renin gene expression, and result in positiveeffects against progression of diabetic nephropathy.

In contrast to the STZ group, upregulated APE under COStreatment might result in reduced nephrotoxicity in diabeticrats. STZ-induced DNA damage was extensive in first few hoursafter administration, and it was repaired at a fairly constantrate (Kraynak et al., 1995; Brownlee, 2007; Ku et al., 2009).Simultaneously hyperglycemia under STZ-induced diabetes causesBax-mediated apoptosis (Allen et al., 2005). Since COS treatmentmight have hypoglycemic effects (Lee et al., 2003; Liu et al., 2007;Kim et al., 2009; Yuan et al., 2009) and anti-RAS activities (Parket al., 2003; Huang et al., 2005; Park et al., 2008), nephrotoxicitymight be attenuated with COS treatment in STZ-induced diabeticrats. With reduced oxidative damage the kidneys switch to repairthe damage rather than apoptosis. In our previous report (Yoonet al., 2011), short-term treatments of COS caused basal highlevel of APE in normal rat kidneys, and it acts as a protectivefactor for paraquat-induced nephrotoxicity. In this study, long-term treatments of COS over 12 weeks revealed slightly lowerlevel of APE and significantly increased level of APE after STZadministration. But, COS did not affect the p53 after STZ admin-istration irrespective of COS treatment. It is suggested that COSmight have p53-independent pathway to activate APE under longterm exposure. The exact mechanism for COS treatment should beinvestigated according to acute and chronic responses in furtherstudies.

In conclusion, this study demonstrated that STZ-induced dia-betic nephropathy might be caused by a p53-dependent apoptoticpathway with suppressed APE, and that COS has renoprotective

effects in STZ-induced diabetic rats through APE activation. Sincethe negative correlation between APE and cleaved caspase-3 wasobserved, p53-dependent apoptosis might be depend on the statusof APE in STZ-induced diabetic nephropathy.
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cknowledgement

This study was supported by a research grant from Chosunniversity, 2010.

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