2047 PIOGLITAZONE, A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA@(PPAR-fÁ) ACTIVATOR, POSSIBLY INHIBITS RENAL STONE FORMATION IN RATS

Vol. 185, No. 4S, Supplement, Wednesday, May 18, 2011

THE JOURNAL OF UROLOGY姞

RESULTS: The results showed that the dissolution rates of COM increased linearly by increasing solution undersaturation. In addition dissolution rate was found to be depended on stirring rate for the same undersaturation. This implies that the dissolution mechanism is determined by the diffusion of crystal units to the solution. Furthermore in the present study the effect of pH on dissolution rate was examined and it was found that by decreasing the pH to 4,5 dissolution rate increased by 28 %. SEM showed that synthetic COM crystals developed changes in their morphology during the dissolution process. CONCLUSIONS: In vitro methods based on measurements at constant solution undersaturation provide high accuracy and reproducibility to COM system. Monitoring of the dissolution process at carefully controlled conditions provide information on the initiation, the mechanism and the morphology of the crystals. It was found that under the applied experimental conditions dissolution mechanism is dominated by diffusion from the crystal surface to the undersaturated solution. It is concluded also that at slightly acidic pH dissolution rate of COM crystals increased. The above observations can assist to a better understanding of in vivo dissolution of COM urinary stones. Source of Funding: None

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Source of Funding: None

2047 PIOGLITAZONE, A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA@(PPAR-fÁ) ACTIVATOR, POSSIBLY INHIBITS RENAL STONE FORMATION IN RATS

2046 EFFECT OF RENAL LIPID ACCUMULATION ON THE EARLY PROCESSES OF KIDNEY STONE FORMATION Yasuo Kohjimoto*, Yumiko Sasaki, Nagahide Matsumura, Takeshi Inagaki, Isao Hara, Wakayama, Japan INTRODUCTION AND OBJECTIVES: Although a number of epidemiological studies revealed that metabolic syndrome (MetS) was associated with kidney stone disease, the underlying mechanisms remain unclear. Recent evidence suggests that intracellular lipid accumulation can account for many manifestations of the MetS through cellular dysfunction and injury (lipotoxicity). The present study examined the effect of intracellular accumulation of triglyceride on the early processes of kidney stone formation using cultured renal epithelial cells. METHODS: Madin-Darby canine kidney (MDCK) cells were incubated with albumin-bearing oleic acid (OA) or vehicle (albumin). After varying periods of time, intracellular lipid accumulation was assessed by lipid staining with oil red O and triglyceride measurement. This model of cellular lipotoxicity was provided for the following experiments. (1) Oxalate-induced cellular injury: Cells were exposed to 1 mM sodium oxalate in the culture media for 2 hours. The oxidative stress and cytotoxicity were assessed by measuring hydrogen peroxide (H2O2) and lactate dehydrogenase (LDH), respectively. (2) Calcium oxalate monohydrate (COM) crystal adhesion: After pretreatment with 1mM oxalate for 2 hours, cells were exposed to COM crystal at a concentration of 5␮g/cm2 for 5 minutes. Quantitative analysis of the adherent crystals was performed by measuring the calcium concentration of the cell lysate. RESULTS: Incubation of MDCK cells with OA resulted in doseand time-dependent intracellular accumulation of triglyceride. Oxalateinduced LDH release was significantly increased in the cells incubated with 0.5 mM and 1 mM OA for 12 hours compared with the cells incubated with vehicle (Figure). Also, oxalate-induced H2O2 generation in these cells was increased significantly, but to a minor extent (3.4 – 3.9%, p⬍0.01). COM crystal adhesion to MDCK cells incubated with 1 mM OA was also increased by 24.4 ⫾ 25.1% (p⫽0.05). CONCLUSIONS: The results indicate that lipid accumulation in renal epithelial cells enhances two critical steps in the early processes of kidney stone formation, namely oxalate-induced cellular injury and COM crystal adhesion. Thus, lipotoxicity in renal epithelial cells might account for kidney stone formation in MetS.

Kazumi Taguchi*, Atsushi Okada, Yasuhiro Fujii, Kazuhiro Niimi, Takahiro Kobayashi, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Takahiro Yasui, Keiichi Tozawa, Shoichi Sasaki, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Renal stone disease demonstrates some clinical and pathological characteristics similar to atherosclerosis. Urolithiasis is also considered a metabolic syndrome (MetS) involving inflammation and oxidative stress. MetS is treated with pioglitazone (PGZ), which is a peroxisome proliferatoractivated receptor (PPAR)-gamma activator. PPAR is a member of the nuclear receptor superfamily that regulates lipid, glucose, and amino acid metabolism, and also has anti-inflammatory and antioxidative effects. We hypothesized that PGZ will become a new treatment for renal stone disease. We investigated the effects of PGZ on stone formation by using rat models. METHODS: Eight-week-old male Sprague-Dawley rats were divided into 3 groups: control group, EG group; administered with 1% ethylene glycol (EG), EG⫹PGZ group; and administered with 1% EG and 10 mg/kg PGZ. One percent EG was administered by free drinking. PGZ was administered daily through a gastric tube. The kidneys were extracted at designated time points (days 7, 14, and 28). Twenty-four-hour urine samples were collected in metabolic cages at the same time points. Renal stone formation was detected via polarized light optical microscopy and quantified by an image analyzer (Image Pro Plus). Immunohistochemistry, western blotting (WB), and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were performed for detection of OPN, SOD, and adiponectin expression. RESULTS: The amount of renal stone formation in the EG⫹PGZ group was significantly lower than in that the EG group (Fig). There were no statistically significant differences between the EG and EG⫹PGZ groups in terms of urinary biochemistry values related to stone formation, such as calcium, phosphate, and oxalate. In the EG⫹PGZ group, SOD and adiponectin levels were significantly higher and OPN levels were significantly lower than in the EG group. CONCLUSIONS: Renal stone formation was inhibited by PGZ without differences in oxalate excretion. Our result suggests that the PPAR-gamma activator might suppress stone formation by reducing oxidant stress and renal tubular cell injury. PGZ has the possibility to become a new treatment for renal stones.

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THE JOURNAL OF UROLOGY姞

Vol. 185, No. 4S, Supplement, Wednesday, May 18, 2011

2049 RENAL MACROPHAGE MIGRATION AND CRYSTAL PHAGOCYTOSIS VIA INFLAMMATORY-RELATED GENE EXPRESSION DURING KIDNEY STONE FORMATION AND ELIMINATION IN MICE Atsushi Okada*, Takahiro Yasui, Jun Ichikawa, Kazunori Nakaoka, Yasuhiko Hirose, Kazumi Taguchi, Kazuhiro Niimi, Yasuhiro Fujii, Masayuki Usami, Takahiro Kobayashi, Ryosuke Ando, Shuzo Hamamoto, Masahito Hirose, Yasunori Itoh, Keiichi Tozawa, Kenjiro Kohri, Nagoya, Japan Source of Funding: None

2048 EFFECT OF APOCYNIN ON EXPRESSION OF KIDNEY INJURY MOLECULE-1 & CAOX CRYSTAL DEPOSITION IN HYPEROXALURIC RATS Saeed Khan*, Aslam Khan, Patricia Glenton, Jian Zuo, Gainesville, FL INTRODUCTION AND OBJECTIVES: High oxalate (Ox) and calcium oxalate (CaOx) crystals are injurious to cells and CaOx crystal deposition in kidneys is associated with renal injury and movement of inflammatory cells into the interstitium. We have proposed that Ox and CaOx crystal induced injury is most likely caused by reactive oxygen species (ROS) produced by activation of membrane associated NADPH oxidase. We determined the effect of NADPH oxidase inhibitor apocynin on renal CaOx crystal deposition and the expression of kidney injury molecule-1 (KIM-1), osteopontin (OPN), monocyte chemoattracatant protein-1 (MCP-1), and ED-1. METHODS: Male Sprague-Dawley rats were fed a diet containing 5% HLP and 4mml apocynin to drink for 28 days. Urinary crystals, Ox , Ca, hydrogen peroxide (H2O2), NAG, KIM-1, OPN, and MCP-1 were determined. Paraffin embedded kidneys were stained using H&E, PAS. Calcium oxalate crystals were identified by Pizzzolato staining as well as use of polarizing optics. Kidney sections were processed for immunohistochemistry using specific antibodies against KIM-1, OPN and ED1. Semi-quantitative evaluation of protein in kidneys was performed with computer-assisted densitometric scanning of western blots. RESULTS: Hyperoxaluria produced heavy deposits of CaOx crystals. Renal tubules with the crystals appeared dilated. There was a significant increase in urinary excretion of KIM-1, OPN, H2O2 and MCP-1 and renal expression of KIM-1 and OPN. Staining was seen associated with the crystal deposits as well as in epithelial cells not in apparent contact with the cells. ED-1 positive cells, which were totally absent in the control kidneys, were present in the renal interstitium of hyperoxaluric rats. Apocynin treatment resulted in reduction of crystal deposits, injured and dilated tubules, renal expression of KIM-1, OPN, and ED-1 and urinary excretion of KIM-1, OPN, MCP-1 and H2O2. Apocynin had no effect on the urinary excretion of Ox or NAG. CONCLUSIONS: Data presented here demonstrate that inhibition of NADPH oxidase by apocynin resulted in the reduction of crystal deposition without significant effect upon urinary Ox. Crystal deposition was associated with increased renal expression of KIM-1, OPN, MCP-1, ED-1, markers of injury and inflammation which were also reduced by apocynin treatment. Results provide support to our proposal that renal epithelial injury is critical for crystal retention within the renal tubules and that injury is in part caused by the production of ROS with the involvement of NADPH oxidase. Source of Funding: NIH Grant #RO1-DK078602 and University of Florida Center for the Study of Lithiasis

INTRODUCTION AND OBJECTIVES: We previously established a mouse kidney stone formation model and the generated calcium oxalate crystal deposits could be eliminated after several days, and indicated a susceptibility in which monocyte/macrophage interaction could participate in the phenomenon. To clarify the macrophagerelated factors playing roles in the prevention of crystal formation in mouse kidneys, morphological and expression studies based on microarray pathway analysis were performed. METHODS: Eight-week-old male C57BL/6 mice were intraabdominally administered 80 mg/kg glyoxylate. The kidneys and urine were obtained every 3 days until day 15. Stone formation was confirmed by Pizzolato staining and the amount of crystallization was calculated. Biotin-labeled cRNAs were synthesized from total RNA extracted from kidney specimens, and then hybridized on Affmetrix Mouse Genome 430 2.0 arrays. The fluorescent intensity of each spot was squantified and analyzed with GeneSpring® software. Based on the raw data of microarray analysis, pathway analyses and quantitative PCR-based association analyses were performed. Immunohistochemical staining of macrophage-related proteins and transmission electron microscopy (TEM) were performed. RESULTS: Kidney calcium oxalate crystal depositions increased by day 6, thereafter decreased gradually and had almost disappeared at day 15. In microarray expression data, 18,064 genes demonstrated a significant expression value. Pathway analyses of inflammatory response demonstrated macrophage activation through the increased expression of chemokines. Association analysis of related gene expression values indicated the high association of chemokine (C-C) ligand 2, Cd44, colony stimulating factor 1, fibronectin 1, matrix gla protein, secreted phosphoprotein 1 and TGF-beta 1 both with the amount of renal crystals and F4/80, a macrophage marker. Immunohistochemically, interstitial macrophages increased and CD44 and MHC-class II were upregulated around crystal formation sites. TEM indicated interstitial macrophage migration with the phagocytosis of crystals. CONCLUSIONS: Increased expression of inflammation-related genes of renal tubular cells induced by crystal formation and deposition could induce monocyte/macrophage migration and phagocytosis via the interaction of CD44 to osteopontin and fibronectin. Such crystalremoving ability of macrophages through phagocytosis and digestion might become a new target for the prevention of stone formation. Source of Funding: Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (No. 21791517, No. 217915, No. 21791520, No. 21890220, No. 21592076, No. 20659252, No. 20591887, No. 20591885), the Japan Urological Association (Young Research Grant, 2008), Asahi Kasei Pharma (9th AKUA urology research grant, 2009) and a Suzuken Memorial Foundation Grant, 2009.

2050 HYDROXYPROLINE AND GELATIN DIETS INDUCE LONG-TERM HYPEROXALURIA IN THE PORCINE MODEL Sutchin R. Patel*, Kristina L. Penniston, Lauren Iwicki, Ibrahim Saeed, Thomas D. Crenshaw, Stephen Y. Nakada, Madison, WI INTRODUCTION AND OBJECTIVES: Swine models have been useful in the study of many different disease processes, but the anatomic and physiologic renal similarities between sows and humans make