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Urolithiasis (2014) 42:387–394DOI 10.1007/s00240-014-0676-x

ORIGINAL PAPER

Increased oxidative DNA damage seen in renal biopsies adjacent stones in patients with nephrolithiasis

Wipawee Kittikowit · Uraiwan Waiwijit · Chanchai Boonla · Preecha Ruangvejvorachai · Chaowat Pimratana · Chagkrapan Predanon · Supoj Ratchanon · Piyaratana Tosukhowong

Received: 13 December 2013 / Accepted: 9 June 2014 / Published online: 15 July 2014 © Springer-Verlag Berlin Heidelberg 2014

calcium phosphate) and uric acid stones was not sig-nificantly different. Increased leukocyte infiltration was observed in renal tissues from patients with nephrolithi-asis. Exposure of HK-2 cells to COM caused increased intracellular reactive oxygen species and nuclear expres-sion of 8-OHdG. To our knowledge, this is the first report of increased 8-OHdG expression in renal tubular cells of patients with nephrolithiasis. In vitro, COM crystals were capable of inducing oxidative damage of DNA in the proximal renal tubular cells.

Keywords Kidney stone · ROS · DNA damage · 8-OHdG · Immunohistochemistry · Calcium oxalate

Introduction

Several lines of evidence including in vitro, animal, and human studies have been suggested that nephrolithiasis is a disease mediated by oxidative stress [1]. Increased mito-chondrial superoxide and decreased glutathione (GSH) production are demonstrated in proximal (LLC-PK1) and distal (MDCK) tubular cells treated with calcium oxalate monohydrate (COM) [2]. Injury of renal tubular cells by oxalate and calcium oxalate (CaOx) crystals is shown to be mediated by reactive oxygen species (ROS) production [3], and such oxidative injury is attenuated by citrate [4] and antioxidants [5]. Additionally, NRK52E cells exposed to apatite crystals cause increased oxidative stress and lipid peroxidation [6]. In animal models, an imbalance of oxidant–antioxidant causing excessive oxidative stress was shown in ethylene glycol (EG)-treated rats [7]. Green tea treatment is capable of increasing super oxide dismutase activity and decreasing CaOx deposit in the kidneys of rats with EG-induced nephrolithiasis [8]. The significant

Abstract Urinary excretion of 8-hydroxydeoxyguano-sine (8-OHdG), a marker of oxidative DNA damage, is significantly higher in nephrolithiasis patients than in healthy individuals, indicating that these patients have higher degree of oxidative stress. In the present study, we investigated 8-OHdG expression in renal biopsies of patients with nephrolithiasis and in renal tubular cells (HK-2 cells) exposed to calcium oxalate monohydrate (COM). We performed immunohistochemical staining for 8-OHdG in renal biopsies adjacent stones obtained from 28 patients with nephrolithiasis. Controls were noncan-cerous renal tissues from nephrectomies of patients with renal cancer. 8-OHdG was overexpressed in the nucleus of renal tubular cells in patients with nephrolithiasis com-pared with controls. Only one nephrolithiasis biopsy was negative for 8-OHdG, whereas in 19 cases 8-OHdG was highly expressed. The level of expression of 8-OHdG among patients with calcium oxalate (mostly mixed with

W. Kittikowit · P. Ruangvejvorachai Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

U. Waiwijit · C. Boonla · P. Tosukhowong (*) Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailande-mail: piyaratana_t@yahoo.com

C. Boonla e-mail: chanchai.b@chula.ac.th

C. Pimratana · C. Predanon Department of Surgery, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

S. Ratchanon Unit of Urology, Surgical Department, Khon Kaen Hospital, Khon Kaen 40000, Thailand

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increase in nitrosative stress in rats with EG-induced neph-rolithiasis can be diminished by dietary supplements of l-arginine [9]. Vitamin E also shows antilithogenic activity in EG-treated rats [10]. The bioflavonoid quercetin is capa-ble of inhibiting oxidative stress and crystal deposits in rats treated with sodium oxalate [11]. In human studies, signifi-cant increases in markers of lipid peroxidation, including urinary thiobarbituric acid-reactive substances and malon-dialdehyde (MDA), are found in patients with renal CaOx stones compared with normal volunteers [12]. Tungsanga et al. [13] found that patients with renal stones had higher plasma MDA, erythrocyte MDA, urinary MDA, urinary protein, and urinary N-acetyl-β-glucosaminidase activity, but lower erythrocyte GSH, erythrocyte glutathione peroxi-dase activity, plasma protein thiol, and plasma vitamin E, than the normal controls suggesting greater oxidative stress and renal tubular cell damage in the patients. We demon-strated that patients with nephrolithiasis excreted signifi-cantly more urinary 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, than healthy controls [14]; this cause of renal tubular injury was confirmed in a subsequent study [15]. In addition, an elevated urinary 8-OHdG was independently associated with an increased renal tubular damage. This evidence suggests that patients with kidney calculi have increased oxidative DNA damage. Increased 8-OHdG expression was demonstrated in patients with acquired cystic disease of the kidney [16]. Renal glo-merular and interstitial fibrosis is associated with increased cytosolic expression of 8-OHdG [17]. To our knowledge, intrarenal expression of 8-OHdG lesions in nephrolithiasis patients has not hitherto been investigated.

Although oxidative stress is well recognized to mediate the pathogenesis of kidney stones, most evidence is deduced from experimental models. Evidence in humans has remained limited. In the present study, we investigated the expression of 8-OHdG in renal biopsies adjacent to stones obtained from patients with kidney calculi. Additionally, we determined ROS generation and 8-OHdG lesions in renal tubular cells challenged with lithogenic milieu in vitro.

Patients and methods

Patients and renal biopsies

Twenty-eight patients with kidney stones, who were admitted to Khon Kaen Hospital, Khon Kaen province, Thailand, and underwent open surgery for stone removal, were recruited for the study. Renal biopsy specimens from the stone-bearing kidneys were collected in accordance with standard proce-dures (wedge resection at surgery) by urologists (C.Pi. and C.Pr.). Renal tissues near the stones (stone adjacent renal tis-sues) were biopsied from the patients for research purposes

only. Written informed consent was obtained from all par-ticipants before specimen collection. The research protocol was approved by the Ethics Committee, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

The biopsied tissue was immersed in buffered 10 % for-malin, and processed according to routine histological pro-tocols. Serial paraffin-embedded sections were cut at 4 µM for hematoxylin and eosin (H&E) and immunohistochemi-cal staining.

H&E staining was performed to evaluate intrarenal inflammation according to the relative amount of leukocyte infiltration. The leukocyte infiltrated area, which reflected the degree of inflammation, was graded into five categories, namely 0 or negative: 0–5 %, 1+ 6–25 %, 2+ 26–50 %, 3+ 51–75 %, and 4+ 76–100 % (evaluated by W.K.). At least 10 microscopic fields were examined. The area infil-trated was estimated and expressed as percentage of the total examined area (100 %).

Immunohistochemistry

Expression of 8-OHdG in renal tissues was investigated by immunoperoxidase staining [16, 17]. After deparaffiniza-tion, rehydration and antigen retrieval, renal sections were incubated in 3 % H2O2 for 5 min to inactivate endogenous peroxidase. The sections were incubated in 3 % normal horse serum for 20 min to block nonspecific binding, and subsequently incubated with 1:200 anti-8-OHdG antibody (Clone N45.1, JaICA) overnight at 4 °C. After washing, the sections were incubated with horseradish peroxidase-conjugated secondary antibody for 30 min. Specific immu-nocomplexes were visualized by diaminobenzidine (DAB), and slides were counterstained with Mayer’s hematoxylin. Expression of 8-OHdG was evaluated and graded according to percentage of positive cells by pathologist (W.K.) as fol-lowed: negative; no expression, 1+ 1–25 %, 2+ 25–50 %; 3+ 50–75 %, and 4+ >75 %.

Intracellular ROS production

Human proximal tubular cell line (HK-2 cell) was pur-chased from the American Tissue Culture Collection. The cells were maintained in high glucose Dulbecco’s modified Eagle’s Medium (DMEM), 10 % fetal bovine serum, and 0.1 % streptomycin/penicillin, at 37 °C, 5 % CO2. Intracel-lular ROS production was assayed using dichloro-dihydro-fluorescein diacetate (DCFH-DA) [18]. In brief, confluent HK-2 cells were incubated with serum-free DMEM con-taining 0.1 mM DCFH-DA for 30 min in the dark. After washing, serum-free DMEM containing various concentra-tions of COM (1, 10, 50, 100, and 200 µg/cm2) were added. Fluorescence was measured using a microplate reader at the beginning (0 min) and at the end (60 min). Excitation

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and emission filters were set at 485 and 535 nm, respec-tively. The arbitrary fluorescent unit (AFU) was expressed as a ratio of fluorescent intensity at 60–0 min.

Immunocytochemistry

Immunoperoxidase staining of 8-OHdG in treated HK-2 cells was conducted according to a previously described protocol [19]. Round coverslips were placed into 24-well plate to grow cells on the coverslips. Confluent cells were treated with 100 µg/cm2 COM for 24 h. Cells were fixed with cold acetone for 10 min, treated with RNAse A (100 g/mL) at 37 °C for 1 h, denatured by incubation with 70 mM NaOH/0.14 M NaCl in 40 % ethanol at 4 °C for 5 min, and permeabilized with 0.1 % Triton X-100 at 4 °C for 5 min. After blocking with 5 % normal horse serum, coverslips were incubated with 1:200 anti-8-OHdG antibody at 4 °C overnight. After washing, the coverslips were incubated with HRP-conjugated secondary antibody for 30 min, washed with PBS, visualized by DAB, and counterstained with Mayer’s hematoxylin.

Statistical analysis

The data were presented as the mean (standard deviation). Two independent groups were compared using a two-sam-ple t test. Difference among the three independent groups was assessed by an ANOVA, followed by Tukey’s multi-ple comparison test. Analyses were conducted using Stata, SPSS, and GraphPad Prism software. P < 0.05 was consid-ered to indicate statistical significance.

Results

Overexpression of 8-OHdG in renal biopsies of kidney stone patients

We recruited 28 patients with kidney calculi aged 53.7 ± 12.8 years old (range 24–73 years old). There were 12 men (42.9 %) and 16 women (57.1 %). Of 28 patients, 20 had stones available for mineral analysis (by Fourier transform infrared spectroscopy). Calcium oxalate (CaOx), calcium phosphate (CaP), and uric acid (UA) stones accounted for 60 % (12/20), 10 % (2/20), and 30 % (6/20), respectively (Table 1).

Varied degrees of 8-OHdG expression in renal tissues were found (Table 2). Figure 1 shows representative his-tological micrographs of 8-OHdG staining. Only one case was found to be negative for 8-OHdG. In positive cases, 1+ expression was found in four cases, 2+ in 4, 3+ in 6, and 4+ in 13. In five control renal sections, there was no or very low expression of 8-OHdG (Fig. 1a). We did not

see the significant difference of 8-OHdG expression among patients with different stone types (CaOx, CaP, and UA) (Fig. 2). Fisher’s exact test showed no statistical difference in the distribution of 8-OHdG expression among patients with CaOx and UA stones. Over 60 % of the patients with either CaOx, CaP, or UA stones had high expression of 8-OHdG (3+ and 4+). Therefore, our current histological data showed that oxidative DNA damage was increased in renal tissues adjacent to the stones regardless of the type of stone.

Most of kidney cells that were labeled with 8-OHdG antibody were renal tubular cells. 8-OHdG was exclu-sively positive in the nucleus of renal tubular cells. This indicated that renal tubular cells of nephrolithiasis patients were under oxidative stress, and the formation of oxidative DNA lesions was increased. We speculated that ROS in these cells is overwhelmingly generated by the lithogenic surroundings.

H&E staining was conducted to assess inflammation of nephrolithiasis intrarenal tissues. The majority (19/28, 67.9 %) of nephrolithiasis renal tissues had significant infil-tration of mononuclear cells (Fig. 3). Nine renal sections (32.1 %) had no sign of inflammation, whereas six sections were inflamed and graded as 1+, three sections as 2+, six sections as 3+, and four sections as 4+.

Increased ROS generation in HK-2 cells treated with COM

To confirm that ROS in renal tubular cells was overpro-duced under lithogenic conditions, intracellular ROS was determined in HK-2 cells exposed to various concentra-tions of COM. We found that exposure of HK-2 cells to 200 µg/cm2 COM for 1 h significantly caused increased ROS production compared with untreated controls (Fig. 4).

8-OHdG lesion was increased in HK-2 cells exposed to COM

We investigated whether COM-induced ROS caused 8-OHdG lesions in renal tubular cells. We found that expo-sure of HK-2 cells to 100 µg/cm2 COM for 24 h caused significantly increased formation of 8-OHdG lesions com-pared with untreated controls (Fig. 5). Our data clearly demonstrated that COM induced ROS generation in the HK-2 cells leading to the formation of oxidative DNA lesions and 8-OHdG.

Discussion

It is well known that lithogenic milieu such as oxalate, urate, CaP crystals, and CaOx crystals stimulate ROS generation in renal tubular cells, and the role of ROS in

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the kidney stone formation has been widely investigated, mainly in the experimentally nephrolithic rats [1]. ROS rapidly reacts with cellular macromolecules resulting in the

impairment of normal cell function, which consequently causes cell injury, and eventually cell death. Such oxida-tive injury in turn promotes the deposit of crystals in the kidneys. Furthermore, ROS induces production of various inflammatory mediators, and inflammation is activated to accelerate the lithogenic process further [20, 21]. ROS can directly attack DNA and causes oxidative DNA lesions, mainly 8-OHdG. Previously, we reported an elevated uri-nary excretion of 8-OHdG in nephrolithiasis patients com-pared with the nonstone forming controls [14, 22]. Herein, we report that expression of 8-OHdG lesions in stone-forming renal tissues was markedly increased relative to the nonstone forming renal tissues. These data indicate an increase in intrarenal oxidative stress in nephrolithiasis

Table 1 Demographic and clinical characteristics of the studied nephrolithiasis patients

M male, F female, BMI body mass index, CaOx calcium oxalate stone, UA uric acid stone, CaP calcium phosphate stone, Uni. unilateral stone formation, Bil. bilateral stone formation, eGFR estimate glomerular filtration rate, Cr creatinine, P plasma, U urine, vol. volume, ND no dataa Staghorn stoneb Hydronephrosis evaluated from intravenous pyelogramc Mixed with calcium phosphated Mixed with magnesium ammonium phosphate

Cases Age (years)

Sex BMI (kg/m2)

Stone formed

Stone type

P-Cr (mg/dL)

eGFR (mL/min/1.73 m2)

U-vol. (mL)

U-Cr (mg/dL)

RKT116 71 M 20.45 ND UA 1.2 58.7 600 114.9

RKT117 73 M 24.97 Uni.a,b 1.2 61.5 1,100 86.2

RKT119 26 F 21.78 Uni.b UA 1.0 75.2 2,100 82.9

RKT121 67 M 23.88 Uni.a,b UA 1.15 73.1 600 96.7

RKT124 65 M 28.96 Uni.a,b 1.5 47.4 1,000 159.2

RKT130 53 M ND Uni.a,b 0.8 102.0 900 113.1

RKT131 37 F 17.93 Uni.a UA 2.4 25.0 1,200 180.0

RKT132 52 M 28.03 Uni.a CaOxc 1.2 72.8 2,200 10.0

RKT133 63 F 20.89 Uni.b CaOxc 1.1 56.7 1,200 31.5

RKT134 72 M 15.57 Uni.a CaOxc 1.1 69.0 1,500 5.5

RKT148 49 F 21.30 Uni.a,b CaOxc 1.2 52.1 1,200 67.1

RKT149 48 F 23.11 Bil.a CaOx 0.9 73.9 2,200 116.9

RKT154 56 M ND ND 0.8 98.9 1,000 61.3

RKT155 52 M 19.14 Uni.a,b 0.9 99.7 1,200 35.1

RKT165 53 F 23.81 Uni.b CaOxc 1.1 55.0 1,100 32.1

RKT166 47 F 24.44 Bil.a,b CaOxd 0.96 80.7 3,300 10.2

RKT168 54 F 26.22 Bil.b CaOxc 1.6 36.3 1,200 18.7

RKT172 24 M 23.61 ND CaP 3.2 26.1 1,200 89.2

RKT173 58 M 24.75 Bil.a,b UA 0.9 94.7 1,300 ND

RKT174 49 F 38.81 Bil.a,b CaOxc 1.2 53.2 1,200 55.7

RKT175 36 F 26.63 Bil.b CaP 1.3 54.9 1,200 79.4

RKT176 63 F 21.76 Uni.a,b CaOxc 1.0 60.1 1,000 73.2

RKT177 60 F 37.78 Bil.a UA 1.0 59.9 1,200 49.5

RKT181 42 F 22.31 Bil.b CaOxc 1.3 50.7 1,200 17.5

RKT182 ND F ND Uni.a,b CaOxc ND ND 1,200 28.4

RKT187 53 F 27.56 ND 2.4 22.4 1,000 61.5

RKT188 68 F 27.77 ND 1.5 35.3 1,000 35.9

RKT190 58 M 23.44 ND 4.6 13.2 1,200 45.6

Table 2 Degree of 8-OHdG expression in renal biopsies of nephro-lithiasis patients

8-OHdG expression Number of patients %

Total 28 100

Negative 1 4

1+ 4 14

2+ 4 14

3+ 6 21

4+ 13 46

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patients. Additionally, we found that patients with CaOx and UA did not have a significantly different degree of 8-OHdG expression (Fig. 2). The majority of cases showed a high degree of 8-OHdG expression. Furthermore, two cases with CaP stones had a very high expression of this DNA lesion. Our previous data also demonstrated that patients with different stone types (CaOx, struvite, and UA) had comparable levels of urinary 8-OHdG [14]. Sev-eral lines of evidence showed that exposure of renal cells to oxalate or COM or CaP or urate caused increased oxi-dative stress [1]. Therefore, based on our current data, we concluded that stone-forming kidneys of nephrolithiasis patients manifested oxidative damage to DNA, irrespective of the types of stones. Blockage of this intrarenal oxidative damage in stone-forming patients may attenuate kidney

dysfunction and might also prevent the development of oxi-dative stress-mediated comorbidities, e.g., cardiovascular disease and chronic kidney disease [23]. We recently dem-onstrated that 3-month treatment with our in-house lime powder regimen (having high antioxidant activity) effec-tively attenuated tubular injury and oxidative damage in patients with nephrolithiasis [24].

We hypothesized that oxidative DNA damage in the renal tubular cells of stone-bearing kidneys of nephrolithi-asis patients was induced by lithogenic surroundings, such as lithogenic ions and crystals. In the present in vitro exper-iment, we showed that ROS was overproduced by COM, and that the generated ROS reacted with guanosine bases to form 8-OHdG lesions. Although it is well known that COM or oxalate is capable of inducing ROS production in renal tubular cells [2, 5, 11, 25], here we provide additional information regarding 8-OHdG formation in the COM-treated cells. It is of interest to explore further the DNA repairing mechanism that is used to remove this oxidative lesion as well as the consequences once cells have exces-sive oxidative DNA damage because of chronic stimula-tion by lithogenic substances. Because oxidative 8-OHdG lesions are also increased in patients with CaP and UA stones, oxidative DNA damage induced by CaP and UA crystals should be further investigated.

ROS generation was significantly increased in HK-2 cells treated with 200 µg/cm2 (213.3 µg/mL) COM for 1 h (Fig. 4). However, this change was rather small (approx. 1.4-fold). Thamilselvan et al. [26] showed that exposure of LLC-PK1 and MDCK cells to oxalate (1 mM) and/or COM (500 µg/mL) for 2 h caused significant increases in super-oxide production (approx. two to threefold). Habibzadegah-Tari et al. [21] also showed that superoxide production was significantly increased in HK-2 cells treated with COM (67, 133, and 267 µg/cm2) for 2 h. Superoxide anion was

Fig. 1 Expression of 8-OHdG in renal tissues of nephrolithi-asis patients. 8-OHdG was negative in the control renal section, noncancerous/nonstone forming renal tissues (a). Various degrees of 8-OHdG expression, i.e., 1+ (b), 2+ (c), 3+ (d), and 4+ (e) were found in the renal sections of nephrolithiasis patients. The 8-OHdG

antibody mainly labeled the nuclei of renal tubular cells (brown color), indicating the site of oxidative DNA damage. The expressions of 8-OHdG were 1+ in 4 of the tissues examined, 2+ in 4, 3+ in 6, and 4+ in 13. One nephrolithiasis renal section was negative for 8-OHdG. Magnification ×400

Fig. 2 Expression of 8-OHdG among patients with different stone types (CaOx, CaP and UA). Significant difference in the 8-OHdG expression level among patients with CaOx and UA stones was not observed. The majority of the patients had high expression of 8-OHdG (3+ and 4+) regardless of the types of stones. Numbers above the bars indicate percents within each group. 0 = negative

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significantly increased in HK-2 cells after 2 h oxalate treat-ment, but the change was not significant at 1 h treatment [27]. Rashed et al. [25] reported that intracellular ROS pro-duction (by DCFH-DA assay) was significantly increased

in the LLC-PK1 cells exposed to 1 mM oxalate for 3 h. Therefore, the small change in ROS generation observed in the present in vitro study might be the result of a shorter exposure time, and we consider that increasing the duration of exposure might generate oxidative damage resembling that seen in vivo (Fig. 1).

In addition to oxidative stress, inflammation is known to play a significant role in the lithogenesis [20]. We have shown various degrees of intrarenal inflammation and fibrosis in the stone-containing kidneys of nephrolithiasis patients [28]. Intrarenal mRNA expression of inflamma-tory mediators (monocyte chemoattractant protein-1 and interleukin-6) is associated with renal impairment [22]. Our recent proteomic data also show an abundance of inflam-matory and fibrotic proteins in urine and stone matrices obtained from patients with nephrolithiasis [29]. In the cur-rent study, we confirmed that stone-containing kidney tis-sue of nephrolithiasis patients appears to have robust signs of inflammations. The actual role of this inflammatory acti-vation, as a cause or consequence of kidney stone forma-tion, remains to be elucidated.

Limitations of the current study include a lack of data regarding urinary 8-OHdG to assess whether urinary 8-OHdG is a predictor of intrarenal oxidative damage in patients with nephrolithiasis. The sample size of control renal tissues was relatively small. However, we were con-fident that there was no or very low expression of 8-OHdG in the nondiseased renal tissues. Study by Huang et al. [16] showed no positive staining of 8-OHdG in 13 normal kidneys.

Fig. 3 H&E staining of renal tissues of nephrolithi-asis patients. Nineteen of 28 nephrolithiasis renal sections showed infiltration of mononu-clear cells to varying degrees. a No leukocyte infiltration was found in control renal sections. b Representative micrograph of inflamed nephrolithiasis renal section (3+). Magnification ×200

Fig. 4 Renal tubular cells (HK-2) exposed to lithogenic COM crys-tals showed increased intracellular ROS generation as determined by assay with DCFH-DA fluorescent probes. Significant increase in ROS production was observed after exposure to COM (200 µg/cm2) for 1 h. Bars and error bars indicate mean and standard error of the mean, respectively. *P < 0.05 vs. control

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In conclusion, to our knowledge, this is the first study demonstrating an increase in 8-OHdG lesions in renal tubu-lar cells of stone-containing kidneys in patients with nephro-lithiasis. Our in vitro experiment showed that COM crystals induce ROS generation and formation of 8-OHdG lesions in the renal tubular cells. Perhaps an increased 8-OHdG expression in the stone-containing kidneys of nephrolithia-sis patients is induced, at least in part, by lithogenic crystals.

Acknowledgments U.W. received a 72nd Birthday Anniversary of His Majesty the King’s Scholarship from Chulalongkorn University (Ratchadaphiseksomphot Endowment Fund). We are grateful for the assistance of the Biochemistry and Molecular Biology of Metabolic Diseases Research Unit.

Conflict of interest The authors declare that there are no conflicts of interest.

References

1. Khan SR (2013) Reactive oxygen species as the molecular modu-lators of calcium oxalate kidney stone formation: evidence from clinical and experimental investigations. J Urol 189:803–811

2. Khand FD, Gordge MP, Robertson WG, Noronha-Dutra AA, Hothersall JS (2002) Mitochondrial superoxide production dur-ing oxalate-mediated oxidative stress in renal epithelial cells. Free Radic Biol Med 32:1339–1350

3. Khan SR (2005) Hyperoxaluria-induced oxidative stress and anti-oxidants for renal protection. Urol Res 33:349–357

4. Byer K, Khan SR (2005) Citrate provides protection against oxa-late and calcium oxalate crystal induced oxidative damage to renal epithelium. J Urol 173:640–646

5. Thamilselvan S, Khan SR, Menon M (2003) Oxalate and calcium oxalate mediated free radical toxicity in renal epithelial cells: effect of antioxidants. Urol Res 31:3–9

6. Escobar C, Byer KJ, Khaskheli H, Khan SR (2008) Apatite induced renal epithelial injury: insight into the pathogenesis of kidney stones. J Urol 180:379–387

7. Huang HS, Ma MC, Chen J, Chen CF (2002) Changes in the oxi-dant-antioxidant balance in the kidney of rats with nephrolithiasis induced by ethylene glycol. J Urol 167:2584–2593

8. Itoh Y, Yasui T, Okada A, Tozawa K, Hayashi Y, Kohri K (2005) Preventive effects of green tea on renal stone formation and the role of oxidative stress in nephrolithiasis. J Urol 173:271–275

9. Pragasam V, Kalaiselvi P, Sumitra K, Srinivasan S, Varalakshmi P (2005) Counteraction of oxalate induced nitrosative stress by sup-plementation of l-arginine, a potent antilithic agent. Clin Chim Acta 354:159–166

10. Huang HS, Chen J, Chen CF, Ma MC (2006) Vitamin E attenu-ates crystal formation in rat kidneys: roles of renal tubular cell death and crystallization inhibitors. Kidney Int 70:699–710

11. Park HK, Jeong BC, Sung MK, Park MY, Choi EY, Kim BS, Kim HH, Kim JI (2008) Reduction of oxidative stress in cultured renal tubular cells and preventive effects on renal stone formation by the bioflavonoid quercetin. J Urol 179:1620–1626

12. Huang HS, Ma MC, Chen CF, Chen J (2003) Lipid peroxidation and its correlations with urinary levels of oxalate, citric acid, and osteopontin in patients with renal calcium oxalate stones. Urol-ogy 62:1123–1128

13. Tungsanga K, Sriboonlue P, Futrakul P, Yachantha C, Tosuk-howong P (2005) Renal tubular cell damage and oxidative stress in renal stone patients and the effect of potassium citrate treat-ment. Urol Res 33:65–69

14. Boonla C, Wunsuwan R, Tungsanga K, Tosukhowong P (2007) Urinary 8-hydroxydeoxyguanosine is elevated in patients with nephrolithiasis. Urol Res 35:185–191

15. Boonla C, Youngjermchan P, Pumpaisanchai S, Tungsanga K, Tosukhowong P (2011) Lithogenic activity and clinical relevance of lipids extracted from urines and stones of nephrolithiasis patients. Urol Res 39:9–19

16. Huang XB, Ito F, Nakazawa H, Toma H (1999) Increased expres-sion of 8-hydroxydeoxyguanosine in acquired cystic disease of the kidney. Nephron 81:458–459

17. Tsai JP, Liou JH, Yeh KT, Tai HC, Cheng YW, Chang HR (2011) Intensity of cytosol expression of 8-OHdG in normal renal tubules is associated with the severity of renal fibrosis. Swiss Med Wkly 141:w13268

18. Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27:612–616

Fig. 5 Immunocytochemistry to detect 8-OHdG expression in COM-treated HK-2 cells. No expression of 8-OHdG in the untreated control cells (a). Nuclear expression of 8-OHdG was increased (arrows) in

HK-2 cells challenged with 100 µg/cm2 COM for 24 h (arrow head) (b). Magnification ×400

394 Urolithiasis (2014) 42:387–394

1 3

19. Yao X, Zhong L (2005) Genotoxic risk and oxidative DNA dam-age in HepG2 cells exposed to perfluorooctanoic acid. Mutat Res 587:38–44

20. Khan SR (2004) Crystal-induced inflammation of the kidneys: results from human studies, animal models, and tissue-culture studies. Clin Exp Nephrol 8:75–88

21. Habibzadegah-Tari P, Byer KG, Khan SR (2006) Reactive oxy-gen species mediated calcium oxalate crystal-induced expression of MCP-1 in HK-2 cells. Urol Res 34:26–36

22. Boonla C, Hunapathed C, Bovornpadungkitti S, Poonpirome K, Tungsanga K, Sampatanukul P, Tosukhowong P (2008) Mes-senger RNA expression of monocyte chemoattractant pro-tein-1 and interleukin-6 in stone-containing kidneys. BJU Int 101:1170–1177

23. Khan SR (2012) Is oxidative stress, a link between nephrolithi-asis and obesity, hypertension, diabetes, chronic kidney disease, metabolic syndrome? Urol Res 40:95–112

24. Tosukhowong P, Yachantha C, Sasivongsbhakdi T, Ratchanon S, Chaisawasdi S, Boonla C, Tungsanga K (2008) Citraturic, alka-linizing and antioxidative effects of limeade-based regimen in nephrolithiasis patients. Urol Res 36:149–155

25. Rashed T, Menon M, Thamilselvan S (2004) Molecular mecha-nism of oxalate-induced free radical production and glutathione redox imbalance in renal epithelial cells: effect of antioxidants. Am J Nephrol 24:557–568

26. Thamilselvan S, Byer KJ, Hackett RL, Khan SR (2000) Free rad-ical scavengers, catalase and superoxide dismutase provide pro-tection from oxalate-associated injury to LLC-PK1 and MDCK cells. J Urol 164:224–229

27. Huang MY, Chaturvedi LS, Koul S, Koul HK (2005) Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells. Kidney Int 68:497–503

28. Boonla C, Krieglstein K, Bovornpadungkitti S, Strutz F, Spittau B, Predanon C, Tosukhowong P (2011) Fibrosis and evidence for epithelial-mesenchymal transition in the kidneys of patients with staghorn calculi. BJU Int 108:1336–1345

29. Boonla C, Tosukhowong P, Spittau B, Schlosser A, Pimratana C, Krieglstein K (2014) Inflammatory and fibrotic proteins prot-eomically identified as key protein constituents in urine and stone matrix of patients with kidney calculi. Clin Chim Acta 429:81–89