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The effect of indomethacin on hyperoxaluria-induced renal tubular epithelial injury/Hiperoksalurinin neden oldugu renal tubuler epiteliyal hasar uzerine indometazinin etkisi.

Renal tubular damage is the most important step for the formation of renal stones. [1,2] It has been shown in numerous experimental studies that oxalate ions form oxidative stress either on their own or in the form of calcium oxalate (CaOx) crystals in the renal tubular cells leading to the formation of free oxygen radicals and reactive oxygen species (ROS) which causes either cell proliferation at lower concentrations or cell death. [1,3] On the other hand, recent studies have shown that this injury may be limited by some antioxidant agents. [4,5] Although the underlying mechanism leading to renal epithelial cell injury in hyperoxaluria is not well established, some authors have focused on the hyperoxaluria-dependent inflammatory changes as a triggering factor for epithelial hazards. ROS are produced during the interactions between the crystals and renal cells, and are responsible for the various cellular responses. CaOx crystals generally form in the renal tubules. Exposure of renal epithelial cells to CaOx crystals results in the increased synthesis of osteopontin, bikunin, heparan sulphate, monocyte chemoattractant protein (MCP)-l, and prostaglandin (PG) E2, which are known to participate in inflammatory processes and in extracellular matrix production. [3]

It has also been claimed that CaOx crystals activate the lysosomal enzymes, followed by the development of anti-inflammatory effect together with the increase in the synthesis and expression of the macromolecules. [3] In a recent study, oxaluria-dependent inflammatory responses were defined as the key regulators of development for nephrolithiasis. [6] Last but not least, experimental studies have pointed out that the initial lesion appears to be crystal formation along the brush border of the proximal tubule, with eventual crystal deposition in collecting ducts and papillary interstitium, and eventual tubule obstruction, interstitial inflammation, and fibrosis. [7]

Indomethacin as a potent anti-inflammatory agent is a nonselective inhibitor of cyclooxygenase (COX) l and 2 enzymes that participate in prostaglandin synthesis from arachidonic acid. Prostaglandins are hormone-like molecules normally found in the body, where they have a wide variety of effects, some of which lead to pain, fever, and inflammation. [8]

Taking the possible role of inflammatory changes being formed in renal interstitium after hyperoxaluria induction, in this present animal study, we aimed to evaluate the possible effect of indomethacin on the hyperoxaluria-induced crystal deposition and apoptosis in renal tubular epithelial cells.

Material and methods

Fifty New Zealand white male rabbits weighing 2 to 4 kg were studied. The animals were fed a standard ration at room temperature. The animals were divided into 3 groups; the animals in the first 2 groups were fed with hyperoxaluric diet [0.75% ethylene glycol (EG) in distilled drinking water] to induce hyperoxaluria, animals in the last group received no additional diet or procedure. In Group 1, animals were fed with EG diet and sacrificed on the 7th (n=l0) and 28th (n=l0) days. In addition to EG diet, the rabbits in Group 2 were ingested indomethacin 2 mg/kg/day for 7 days, and sacrificed on the 7th (n=l0) and 28th (n=l0) days. No particular procedure or treatment was applied for Group 3 and animals in this group served as the control (n=l0).

Animals were anesthetized with ketamine hydrochloride (25 mg/kg) at the end of the periods designed for the study groups; kidneys were removed with bilateral flank incision, and sent for histopathological evaluation. Histology of the renal tissue and crystal deposition were assessed on the light microscope (10x10, 10x40) subsequent to the performance of haematoxylin-eosin staining. Grading with respect to crystallization and/or calcification was determined in 1 [cm.sup.2] area.

According to the grading; [5] (+) means minimal (crystallization and/or calcification in l-3 tubules), (++) means moderate (crystallization and/or calcification in 4-7 tubules), and (+++) means severe (crystallization and/or calcification in >7 tubules).

Detection and scoring of apoptosis

The in situ detection of renal tubular cells with DNA strand breaks in paraffin-embedded parenchymal sections was achieved by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick and labeling (TUNEL) method using an ApopTag kit (Oncor, Gaithersburg, MD, USA). Briefly, after deparaffinization and rehydration, tissue sections were incubated with 200 [micro]g/mL proteinase K (Oncor, Gaithersburg, MD, USA) for 20 min at room temperature, washed in distilled water and then treated with 3% hydrogen peroxide in phosphate buffered salina (PBS) for 10 min at room temperature to quench endogenous peroxidase activity. Sections were incubated with TdT and dioxigenin-1, l-dUTP in a humidified chamber at 37[degrees]C for 1 h and then treated with antidioxigenin-peroxidase for 30 min at room temperature. Subsequently, sections were exposed to 0.05% substrate for 7 min, washed with distilled water and PBS, and then counterstained for 10 min. Sections were then dehydrated in 100% butanol, cleared in xylene, and mounted with Entellan (Merck Scientific, Fairlacon, NJ, USA). Negative controls were obtained by omitting the TdT enzyme, and the same volume of distilled water was used. The Apoptag kit used during the study contained the positive controls. For the quantification of apoptosis in tubular cells to quantitate the incidence of apoptosis at each time point, the percentage of the number of TUNEL-positive cell nuclei within a renal tubule cross section was calculated after counting 1,000 tubular cells in each sample. Scoring the degree of apoptosis was defined as follows; [5] (+) means limited (apoptosis less than 5%), (++) means moderate (apoptosis ranging from 5% to 10%), and (+++) means evident (apoptosis >10%).

Paired-t and Wilcoxon rank tests were used for the statistical evaluation of the data obtained at the end of the study and p<0.05 values were considered statistically significant at 95% confidence level.


Crystal formation

Crystal formation in the renal tubules during the early and late periods were found to be significantly more remarkable in the first 2 groups fed with EG compared with the control group (p<0.05). Crystal formation of indomethacin receiving rabbits was also compared with their non-receiving counterparts (only EG group). Indomethacin was found to decrease the presence and degree of crystallization when compared with only EG receiving group in both early and late period (p=0.034 and p=0.015, respectively) (Table 1). Figures 1 and 2 show the degree of the crystallization in Groups 1 and 2, respectively.

Apoptotic changes

The evaluation of the apoptotic changes during both early and late follow-up phase showed that evident tubular apoptosis could be seen in tissue specimens obtained from animals undergoing hyperoxaluria induction (Groups 1 and 2) (Table 2). Limited or no apoptotic changes were present in control group animals. As compared with Group 1, limited apoptotic changes were seen in indomethacin-treated Group 2 with respect to the mean apoptotic index evaluation both during early as well as late follow-up (Table 2). The severity of the apoptosis was shown in Figures 3 and 4 for Groups 1 and 2, respectively.


Hyperoxaluria is being accepted as the main risk factor for the formation of idiopathic CaOx stones as demonstrated in several clinical studies. [1,9] Experimental studies have clearly shown the oxalate ion (and/or CaOx crystals)-induced injury in the renal tubular cells which seems to play a critical role in the formation of urinary calculi. [10,11] The initiation of the oxalate-dependent tubular injury process triggered by the membraneous lipid peroxidation, as a result of formation of free oxygen radicals, cause injury and necrosis in the renal tubular cells. [12] On the other hand, formation of CaOx crystals activate the lisosomal enzymes, followed by the development of anti-inflammatory effect together with the increase in the synthesis and expression of the macromolecules. Thus, the initial lesion appears to be crystal formation along the brush border of the proximal tubule, with eventual crystal deposition in collecting ducts and papillary interstitium, and eventual tubule obstruction, interstitial inflammation and fibrosis. [3,13] Finally, it has been well demonstrated that the response of the renal epithelial cells to oxalate ions and/or CaOx crystals was biphasic where mitogenic effect occurred at low concentrations, and toxic effect occurred at high concentrations. [1]





CaOx crystals are agents, free radicals, and ROS that play a critical role at the molecular level in the dependent cell injury. ROS, formed dependent to the oxidative stress, are generally short-lived. Their most important products are malondialdehyde, isoprostanes, and oxidized lipids reacting with lipids, amino acids, proteins, carbohydrates and nucleic acids at the cellular level. [4] In recent clinical studies the urinary level of the mentioned ROS was demonstrated to rise as an indication of the oxidative stress and renal epithelial injury. [14] Thamilselvan et al. [15] determined increase at the levels of urinary lipid peroxidase and LDH in the ethylene glycol-induced hyperoxaluric rats with nephrolithiasis. This effect was proved to decrease with antioxidant vitamin E. [15] Huang et al. [16] demonstrated that the oxidative stress could be decreased, level of glutathione concentration in the renal tissue could be increased significantly, and level of thiobarbituric acid could be decreased with losartan, an angiotensin converting enzyme (ACE) inhibitor.

In numerous animal experiments, tubular ischemia at hyperoxaluric basis was demonstrated to increase the production of ROS. Koul et al. [17] displayed that c-myc gene expression, an apoptotic marker due to oxalate-dependent oxidative stress, increased. In analogous studies, occurrence of cell deaths concurrent with cellular proliferation and concentration as a result of interaction between the oxalate ions and renal tubular epithelium, were stated. [17-19] Sarica et al. [5] demonstrated that the apoptotic changes in renal tubular cells induced by hyperoxaluria might be limited by allopurinol, an antioxidant agent, and vitamin E.

Although the renal tubular epithelial hazards are caused by direct injury or ROS formation, the eventual inflammation formation is highly expected. In an animal study with experimental CaOX nephrolithiasis, production of chemoattractants leading to migration of monocytes and macrophages which can cause to localized interstitial inflammation and fibrosis to sites of interstitial crystal deposition were shown. [20] Also, several in vitro experiments showed the eventual inflammation and fibrosis formation in crystal deposition areas as a fate of CaOX dependent injury. [21-23]

We investigated the effect of indomethacin, an anti-inflammatory agent, on the injury induced in the renal tubular cells by hyperoxaluria-dependent ischemia and inflammatory changes in the interstitium. In the hyperoxaluric diet treated subjects, crystallization was observed to develop during early (7th day) and late (28th day) periods and to become more severe on the basis of tubular ischemia. While the mean apoptotic index was significantly high in hyperoxaluric diet treated group, indomethacin application was found to decrease tubular crystal deposition and apoptotic alterations significantly.

In the light of these findings, it seems that oxalate ions cause tubular cell injury either by themselves or in the form of CaOX crystals. Taking the ischemia formation and subsequent inflammatory changes into account, anti-inflammatory medication (indomethacin in this study) may limit these changes to some extend. Thus, in experimental conditions, this kind of medication can limit these apoptotic changes which ultimately cause stone formation.

Submitted (Gelis tarihi): 13.10.2009

Accepted after revision (Duzeltme sonras? kabul tarihi): 16.11.2009


(1.) Khan SR. Calcium oxalate crystal interaction with renal tubular epithelium, mechanism of crystal adhesion and its impact on stone development. Urol Res 1995;23:71-9.

(2.) Sarica K, Yagci F, Bakir K, Erbagci A, Erturhan S, Ucak R. Renal tubular injury induced by hyperoxaluria: evaluation of apoptotic changes. Urol Res 2001;29:34-7.

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

(4.) Khan SR. Hyperoxaluria-induced oxidative stress and antioxidants for renal protection. Urol Res 2005;33:349-57.

(5.) Sarica K, Erbagci A, Yagci F, Bakir K, Erturhan S, Ucak R. Limitation of apoptotic changes in renal tubular cell injury induced by hyperoxaluria. Urol Res 2004;32:271-7.

(6.) Umekawa T, Chegini N, Khan SR. Oxalate ions and calcium oxalate crystals stimulate MCP-1 expression by renal epithelial cells. Kidney Int 2002;61:105-12.

(7.) Worcester EM, Chuang M, Laven B, Orvieto M, Coe FL, Evan AP, et al. A new animal model of hyperoxaluria and nephrolithiasis in rats with small bowel resection. Urol Res 2005;33:380-2.

(8.) Mitchell JA, Akarasereenont P, Thiemermann C, Flower RJ, Vane JR. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase. Proc Natl Acad Sci USA 1993;15:11693-7.

(9.) Hackett RL, Shevock PN, Khan SR. Cell injury associated with calcium oxalate crystalluria. J Urol 1990;144:1535-8.

(10.) Hackett RL, Shevock PN, Khan SR. Madin-Darby canine kidney cells are injured by exposure to oxalate and to calcium oxalate crystals. Urol Res 1994;22:197-203.

(11.) Lieske JC, Norris R, Swift H, Toback FG. Adhesion, internalization and metabolism of calcium oxalate monohydrate crystals by renal epithelial cells. Kidney Int 1997;52:1291-301.

(12.) Khan SR, Shevock PN, Hackett RL. Acute hyperoxaluria, renal injury and calcium oxalate urolithiasis. J Urol 1992;147:226-30.

(13.) Khan SR. Role of renal epithelial cells in the initiation of calcium oxalate stones. Nephron Exp Nephrol 2004;98:55-60.

(14.) Tungsanga K, Sriboonlue P, Futrakul P, Yachantha C, Tosukhowong P. Renal tubular cell damage and oxidative stress in renal stone patients and the effect of potassium citrate treatment. Urol Res 2005;33:65-9.

(15.) Thamilselvan S, Hackett RL, Khan SR. Lipid peroxidation in ethylene glycol induced hyperoxaluria and CaOx nephrolithiasis. J Urol 1997;157:1059-63.

(16.) Huang H-S, Ma M-C, Chen J, Chen CF. Changes in the oxidant-antioxidant balance in the kidneys of rats with nephrolithiasis induced by ethylene glycol. J Urol 2002;167:2584-93.

(17.) Koul H, Kennington L, Nair G, Honeyman T, Menon M, Scheid C. Oxalate-induced initiation of DNA synthesis in LLC-PK1 cells, a line of renal epithelial cells. Biochem Biophys Res Commun 1994;205:1632-7.

(18.) Koul S, Fu S, Menon M, Koul H. Oxalate exposure induces apoptosis in renal proximal tubular epithelial cells (LLCPK1 and HK-2 cells) in culture. Urolithiasis 2000, 9th International Symposium on Urolithiasis; Proceedings. p. 247.

(19.) Scheid C, Koul H, Hill WA, Luber-Narod J, Kennington L, Honeyman T, et al. Oxalate toxicity in LLC-PK1 cells: role of free radicals. Kidney Int 1996;49:413-9.

(20.) Umekawa T, Iguchi M, Uemura H, Khan SR. Oxalate ions and calcium oxalate crystal-induced up-regulation of osteopontin and monocyte chemoattractant protein-1 in renal fibroblasts. BJU Int 2006;98:656-60.

(21.) Knoll T, Steidler A, Trojan L, Sagi S, Schaaf A, Yard B, et al. The influence of oxalate on renal epithelial and interstitial cells. Urol Res 2004;32:304-9.

(22.) Khan SR, Kok DJ. Modulators of urinary stone formation. Front Biosci 2004;9:1450-82.

(23.) Worcester EM, Chuang M, Laven B, Orvieto M, Coe FL, Evan AP, et al. A new animal model of hyperoxaluria and nephrolithiasis in rats with small bowel resection. Urol Res 2005;33:380-2.

Faruk Yencilek [1], Sakip Erturhan [2], Onder Canguven [3], Bulent Erol [4], Hakan Koyuncu [1], Cemal Goktas [3], Kemal Sarica [1]

[1] Yeditepe University Medical Faculty, Department of Urology, Istanbul, Turkey

[2] Gaziantep University Medical Faculty, Department of Urology, Gaziantep, Turkey

[3] Kartal Training and Research Hospital, Department of Urology, Istanbul, Turkey

[4] Karaelmas University Medical Faculty, Department of Urology, Zonguldak, Turkey

Correspondence (Yazisma): Yard. Doc. Dr. Faruk Yencilek. Barbaros Mah. Halk Cad. Acelya Sok. Moontown Sitesi 2/2F, D2/2 Blok, No: 50, Atasehir 34746 ?stanbul, Turkey.

Phone: +90 216 578 40 00 e-mail:
Table 1. Degree of crystallization between three groups in the
early (7th day) and late (28th day) period (number of specimens
with crystallization/total number of specimens)

 Early period
 (first week)

 + ++ +++

Group 1 (EG only) 2/10 4/10 3/10
Group 2 (EG + indomethacin) 1/10 3/10 1/10
Group 3 (control) -- -- --
p value 0.034

 Late period
 (first month)

 + ++ +++

Group 1 (EG only) 2/10 4/10 4/10
Group 2 (EG + indomethacin) -- 2/10 1/10
Group 3 (control) -- -- --
p value 0.015

EG: Ethylene glycol (hyperoxaluric diet).

Table 2. Mean apoptotic index (min-max) of three groups in the
early (7th day) and late (28th day) period

 Early period Late period
 (first week) (first month)

Group 1 (EG only) 17.3 (15.2-19.2) 20.9 (20.4-21.8)
Group 2 (EG + indomethacin) 11.2 (9.1-12.0) 14.3 (13.1-17.0)
Group 3 (control) 2.9 (2.4-3.2) 2.4 (1.7-2.6)
p value <0.001 <0.001

EG: Ethylene glycol (hyperoxaluric diet)
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Title Annotation:Endourology/Endouroloji
Author:Yencilek, Faruk; Erturhan, Sakip; Canguven, Onder; Erol, Bulent; Koyuncu, Hakan; Goktas, Cemal; Sari
Publication:Turkish Journal of Urology
Article Type:Report
Geographic Code:7TURK
Date:Dec 1, 2009
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