apyrimidinic endonuclease on the progression of streptozotocin-induced diabetic nephropathy in rats

Acta Histochemica 114 (2012) 647–652

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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 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 b

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 kidneys. 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 hypotheses 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 oxidative 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, streptozotocin. ∗ 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. 0065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2011.11.011

apoptosis (Allen et al., 2005; Brownlee, 2007). Oxidative stressinduced 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 activation. 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 singlebase 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 endonuclease 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

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regulator of p53 or vice versa. p53 down-regulates APE expression in response to severe DNA damage, thereby promoting apoptosis in human colorectal cancer cells (Zaky et al., 2008) and in human embryonic kidney 293 cells (Xiong et al., 2008). Recently, we have shown that oxidative DNA damage in rat models of hydronephrosis may trigger p53-dependent apoptosis through repression of APE (Chang et al., 2011). Therefore, we proposed that APE has a possible role in the progression of diabetic nephropathy. Antioxidants such as vitamin C, vitamin E, beta-carotene, taurine and catechin may represent an important defense for the treatment or prevention of diabetic nephropathy models (Hase et al., 2006; Forbes et al., 2008; Wagener et al., 2009). Chitosan oligosaccharide (COS) is an antioxidant of natural origin and has anti-diabetic effects via various mechanisms (Lee et al., 2003; Liu et al., 2007; Yoon et al., 2008; Kim et al., 2009; Yuan et al., 2009). In our previous report (Kim et al., 2009), highly deacetylated COS was shown to improve the altered glucose metabolism in STZ-induced diabetic rats through neogenesis of insulin-producing cells and increased the secretory capacity of insulin. As a result, the reductive effect of COS on HbA1c in our previous report (Kim et al., 2009) is the same as that of an earlier report with metformin (Liu et al., 2008). Moreover, COS has protective effects on glycerol-induced acute renal failure (Yoon et al., 2008) and paraquat-induced nephrotoxicity (Yoon et al., 2011). Since nephrotoxicity in association with STZ-induced diabetes may be related to elevated levels of DNA damage, we believe that APE may have a feasible role in the progression of diabetic nephropathy. p53 serves as a key regulator of oxidative stress-induced apoptosis, and we attempted to investigate the interrelationship between APE and p53 on STZ-induced diabetic rat kidneys. In addition, renoprotective effects of COS on diabetic nephropathy have not been reported despite several reports on the anti-diabetic effects of COS. Therefore, we also investigated the protective effects of COS in STZ-induced diabetic rat kidneys based on the activation of APE.

Materials and methods Animals and treatment In our previous report (Kim et al., 2009), we fully described the induction of diabetes as well as preparation and feeding of COS. In brief, low molecular weight chitosan (>98% deacetylated, <10 cps viscosity) was purchased from YB bio (Gyungbuk, Republic of Korea) and COS was obtained by the enzymatic method. Male Sprague-Dawley rats (8–10 weeks old; Da-mool Science, Daejeon, Republic of Korea) were randomly divided and received 500 mg/kg of COS or 1 ml of distilled water (p.o., once a day). Streptozotocin (STZ; 60 mg/kg, Sigma–Aldrich, St. Louis, MO, USA) was injected intraperitoneally to the fasted rats. Then rats were grouped as follows; Control, COS, STZ, COS-STZ (n = 5 per group), and continued 12 weeks after STZ administration. All experimental procedures and care of animals were conducted in accordance with the guidelines of Chosun University’s Animal Care and Use Committee.

Antibodies The primary antibodies used in this study were: polyclonal anti-p53 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-APE (1:1000; Santa Cruz Biotechnology), polyclonal anti-cleaved caspase-3 (1:500; Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-8-hydroxy-2 deoxyguanosine (8-OHdG, 1:200; JaICA, Shizuoka, Japan), and polyclonal anti-␤-actin (1:1000; Santa Cruz Biotechnology). Histology and immunohistochemistry Kidneys were fixed with 4% paraformaldehyde, embedded in paraffin wax (Tissue-Tek, Sakura, Japan), and then 5 ␮m-thick tissue sections were cut using a Leica RM 2155 rotary microtome (Leica Microsystems, Nussloch, Germany). Randomly selected samples were stained with hematoxylin and eosin (H/E) and periodic acid–Schiff (PAS) using a routine protocol. Immunohistochemical staining was carried out by a routine method. In brief, incubation with primary antibodies was performed for 48 h at 4 ◦ C. The binding was visualized using an ImmPRESSTM avidin–biotin-peroxidase kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Omission of incubation with the primary or secondary antibody served as a control for false-positives. Immunolabelled images were captured directly using an Olympus C-4040Z digital camera and Olympus BX-50 microscope (Olympus Corp., Tokyo, Japan). The captured images were saved and subsequently processed using Adobe Photoshop (Adobe System, San Jose, CA, USA). The brightness and contrast of the images were adjusted only for the purpose of background consistency. Western blot analysis Renal tissues were suspended in cold homogenizing buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and homogenized using a ultrasonic cell disruptor (Branson Ultrasonics, Danbury, CT, USA) for 30 s three times, with a 30 s interval, and centrifuged at 10,000 × g for 10 min at 4 ◦ C. Protein concentration in the supernatants was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). An aliquot of the supernatant (30 ␮g protein) was suspended in 20 ␮l of a loading buffer and then boiled for 5 min at 100 ◦ C, electrophoresed on 10% SDS-PAGE gels, and transferred to polyvinyldifluoridine membranes (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Immunoblotting was carried out with each primary antibody. The horseradish peroxidase-linked secondary antibodies (GE Healthcare Bio-Sciences) were diluted 1:4000. The blotted proteins were then detected using an Enhanced Chemiluminescence Detect System (iNtRON, Biotech, Seoul, Korea). The bands were quantified using ImageQuant 350 (GE Healthcare Korea, Seoul, Republic of Korea), and the data expressed as densitometric units of each primary antibody relative to ␤-actin and in reference to the value of the control sample for each gel. Statistical analysis

Measurements At the end of the experiment, total body weight, kidney weight, volume ratio of kidney weight per total body weight were checked. Blood samples were collected and blood urea nitrogen (BUN) and creatinine were determined by Green Cross Reference Laboratory (Gyunggi-Do, Republic of Korea).

Data are expressed as mean ± SD. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Bonferroni test in four groups with or without COS and/or STZ treatments. All statistical analyses were conducted using SPSS, version 12.0 (SPSS, IBM, Chicago, IL, USA). A p value of less than 0.05 was taken as statistically significant.

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

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

Kidney (g)a

Control COS STZ COS-STZ

1.186 1.020 1.223 1.448

± ± ± ±

0.113 0.104 0.230 0.139

Kidney/weight (%)b 0.346 0.362 0.598 0.639

± ± ± ±

0.029 0.055 0.085 0.129

BUN (mg/dl) 21.88 20.63 24.25 25.60

± ± ± ±

2.40 1.73 5.20 5.69

Creatinine (mg/dl) 0.580 0.567 0.600 0.586

± ± ± ±

0.084 0.052 0.063 0.056

a

Weight of kidney was significantly increased between COS and COS-STZ group (p < 0.05). Volume ratio of kidney weight per total body weight was significantly changed between control and diabetic (STZ and COS-STZ) groups (p < 0.05), and COS and diabetic (STZ and COS-STZ) groups (p < 0.05), respectively. b

Results Variable measurements of this study were compared between four groups (Table 1). Kidney weight was increased after STZ administration irrespective of COS treatment. Kidney weight was significantly increased between COS group and COS-STZ group (1.020 ± 0.104 g vs. 1.448 ± 0.139 g, p < 0.05). The volume ratio of kidney weight per total body weight was relatively constant in the control group and the COS group (0.346 ± 0.029% vs. 0.362 ± 0.055%). But it was significantly elevated in STZ group and COS-STZ group (0.598 ± 0.085% vs. 0.639 ± 0.129%). The volume ratio of kidney weight per total body weight was significantly increased between control and diabetic (STZ and COS-STZ) groups and between COS and diabetic (STZ and COS-STZ) groups, respectively (p < 0.05 each). BUN and creatinine were slightly elevated but significance was not found. As indicated in Fig. 1, it made no difference on the cytoarchitecture of kidney whether there was COS treatment or not. Infiltration of inflammatory cells were transitorily seen 4 weeks after STZ administration (asterisks), while it was not clearly visible in case of COS treatment. According to previous reports (Tesch and Nikolic-Paterson, 2006; Tesch and Allen, 2007) 4 weeks after STZ administration was not necessary to develop diabetic nephropathy. They suggested that it took at least 7–8 weeks, and we continued to raise animals until 12 weeks after STZ administration. At the end of the experiment, the kidney showed little abnormality but

relatively large lumens of the tubules (tubular dilatations) were only seen in COS-STZ group. PAS stain outlines the basement membranes of glomeruli and tubules (Fig. 4, upper column). In the cortical labyrinth, proximal tubules generally have a larger diameter with brush border than distal tubules have; cross sections of the lumen often appear stellate in paraffin sections. Some changes are focal glomerulosclerosis and degenerative changes in distal convoluted 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 control group. 12 weeks after STZ administration, p53 (1.925 ± 0.515 and 2.344 ± 0.208, p < 0.05/each) significantly augmented irrelevant to COS treatment. APE was decreased in STZ group (0.660 ± 0.177), but significant elevation was observed in COSSTZ group (1.778 ± 0.376, p < 0.05/each) compared to every other group. Cleaved caspase-3 significantly increased in STZ group (1.238 ± 0.175, p < 0.05) compared to the COS group, and considerably 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 collecting ducts of the renal cortex in control and COS groups. The immunoreactivities were noted across the entire nephron after STZ administration (Fig. 4, middle column), while the COS-STZ group showed a similar distribution of immunolocalized 8-OHdG

Fig. 1. Photographs of kidneys under diabetic progression after streptozotocin (STZ) administration. Chitosan oligosaccharide (COS) did not affect the cytoarchitecture of kidneys irrespective of STZ treatment. Grossly, the kidney shows little abnormality under H/E stain. Inflammatory cells were transitorily infiltrated at 4 weeks after STZ injection (asterisks), while this was not apparent in case of COS treatment. Scale bar = 100 ␮m.

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Fig. 2. Western blot analysis and densitometric results of p53, APE and cleaved caspase-3 in normal and streptozotocin (STZ)-induced diabetic kidneys. Chitosan oligosaccharide (COS) treatment did not adversely affects the p53, APE and cleaved caspase-3 in normal kidneys. Statistical significance was observed on p53 in the STZ and COS-STZ groups, on APE in the COS-STZ group, and on cleaved caspase-3 in the STZ group or COS-STZ group. Cleaved caspase-3 negatively related to APE, while p53 constantly increased after STZ administration. *p < 0.05 compared to control group, # p < 0.05 compared to COS group, § p < 0.05 compared to STZ group.

to that of control and COS groups. APE (Fig. 3, lower column) was immunolocalized in the proximal tubules of the renal cortex of all groups. The immunoreactivities were evident in proximal tubules of the deep renal cortex (S3 segment, especially), and did not changed the distribution of immunolocalized APE to STZ administration. COS-STZ group showed increased APE immunoreactivities across the entire nephron compared to the other groups, which were especially distinct in deep renal cortex (Fig. 4, lower column).

Discussion This is the first report to show that repressed APE may play a role in the progression of STZ-induced diabetic nephropathy, and that COS may have renoprotective effects through augmented APE. Our results showed that early stage of diabetic nephropathy was established after STZ administration with increased p53-dependent apoptosis, and that COS-treated rat kidneys showed the augmented APE and suppressed cleaved caspase-3. It is noteworthy that the

change in APE, irrespective of augmented p53, negatively regulated the apoptotic activity in STZ-induced diabetic kidneys. Oxidative stress has been identified in a variety of diseases, and oxidative stress-induced apoptosis is suggested in the pathophysiology of STZ-induced diabetic nephropathy (Tesch and Nikolic-Paterson, 2006; Tesch and Allen, 2007). Major cellular strategies coping with oxidative DNA damage are repair and removal (Bernstein et al., 2002). Various DNA repair pathways are activated upon oxidative DNA damage. BER is a major DNA repair pathway protecting cells against single-base DNA damage and can be initiated through removal of a damaged base by a DNA glycosylase. As a result, an AP site can be generated and APE (also called redox factor) acts as the major AP endonuclease (Bernstein et al., 2002; Tell et al., 2009). Although APE is a pro-survival protein, APE acts as a regulator of p53 and p53 participates in BER via its direct interaction with APE (Zhou et al., 2001; Bernstein et al., 2002; Xiong et al., 2008; Zaky et al., 2008; Tell et al., 2009; Chang et al., 2011). In this study, we clearly demonstrated that STZ-induced diabetic nephropathy might be caused by a p53-dependent apoptotic

Fig. 3. Distribution of 8-OHdG and APE in normal and streptozotocin (STZ)-induced diabetic kidneys. 8-OHdG, a marker for oxidative damage, was mainly seen in the distal tubules, but across the entire tubules of nephron in STZ group. APE was immunolocalized in the proximal tubules in all groups, but the immunoreactivities were increased after STZ administration. Chitosan oligosaccharide (COS) did not affect the immunolocalizations in the kidney, but increased APE after STZ injection. Asterisks, glomeruli. Scale bar = 200 ␮m.

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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 their identification under PAS stain. Degenerative changes were seen on the vascular pole (arrow) of glomerulus and distal tubules in STZ group, while chitosan oligosaccharide (COS) did not affect the structure of nephrons. The increased immunolocalization of 8-OHdG was seen in STZ group and APE in COS-STZ group compared to other groups, respectively. Scale bar = 50 ␮m.

pathway. The negative correlation between increased p53 and decreased APE was accompanied by increased cleaved caspase-3, an effector for apoptosis. Augmented p53 may directly activate Bax or indirectly via suppressed APE, which can induce the caspase cascades and apoptosis. It was reinforced by the immunohistochemistry with 8-OHdG, the marker for oxidative damage. 8-OHdG was mainly immunolocalized in the distal tubules and collecting ducts of control, COS and COS-STZ renal cortices, and also observed in proximal tubules of STZ group only. While STZ administration in the COS treated group caused simultaneous increases of p53 and APE, it results in decreased cleaved caspase-3. Since p53 significantly augmented after STZ administration, p53-dependent apoptosis can be considered to depend on the change of APE in STZ-induced diabetic nephropathy. In addition to being a regulator of p53, APE acts as a transacting factor for repression of the human renin gene (Fuchs et al., 2003). The intrarenal renin–angiotensin system (RAS) is upregulated in diabetic nephropathy (Carey and Siragy, 2003; Singh et al., 2005; Thomas et al., 2005). Renal tubular apoptosis is attenuated by angiotensin I converting enzyme inhibitor (Sun et al., 2009) and increased angiotensin II is related to the p53 (Singh et al., 2008). Upregulated RAS causes vasoconstriction through increased angiotensin II and hypoxic injury to diabetic kidney. This study also supported the notion in STZ group that decreased APE might not suppress renin gene expression and then RAS might be activated. APE augmentation, however, was observed in COS-STZ group. In this group, relatively high levels of APE may block p53-dependent apoptotic pathway. Increased APE could also suppress the renin gene expression, inactivate the RAS, and then reduce hypoxic injury. In addition, previous reports suggested that COS itself has inhibitory activities for renin (Park et al., 2008) and angiotensin I converting enzyme (Park et al., 2003; Huang et al., 2005). Taken together, it is suggested that COS treatment might cause APE

activation, suppress renin gene expression, and result in positive effects against progression of diabetic nephropathy. In contrast to the STZ group, upregulated APE under COS treatment might result in reduced nephrotoxicity in diabetic rats. STZ-induced DNA damage was extensive in first few hours after administration, and it was repaired at a fairly constant rate (Kraynak et al., 1995; Brownlee, 2007; Ku et al., 2009). Simultaneously hyperglycemia under STZ-induced diabetes causes Bax-mediated apoptosis (Allen et al., 2005). Since COS treatment might have hypoglycemic effects (Lee et al., 2003; Liu et al., 2007; Kim et al., 2009; Yuan et al., 2009) and anti-RAS activities (Park et al., 2003; Huang et al., 2005; Park et al., 2008), nephrotoxicity might be attenuated with COS treatment in STZ-induced diabetic rats. With reduced oxidative damage the kidneys switch to repair the damage rather than apoptosis. In our previous report (Yoon et al., 2011), short-term treatments of COS caused basal high level of APE in normal rat kidneys, and it acts as a protective factor for paraquat-induced nephrotoxicity. In this study, longterm treatments of COS over 12 weeks revealed slightly lower level of APE and significantly increased level of APE after STZ administration. But, COS did not affect the p53 after STZ administration irrespective of COS treatment. It is suggested that COS might have p53-independent pathway to activate APE under long term exposure. The exact mechanism for COS treatment should be investigated according to acute and chronic responses in further studies. In conclusion, this study demonstrated that STZ-induced diabetic nephropathy might be caused by a p53-dependent apoptotic pathway with suppressed APE, and that COS has renoprotective effects in STZ-induced diabetic rats through APE activation. Since the negative correlation between APE and cleaved caspase-3 was observed, p53-dependent apoptosis might be depend on the status of APE in STZ-induced diabetic nephropathy.

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