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E17 Opening of mitochondrial permeability transition pores induces kidney stone formation
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wednesday 7 september 2011 / european urology supplements 10 (2011) 461–474
E17 Opening of mitochondrial permeability transition pores induces kidney stone formation K. Niimi, T. Yasui, K. Taguchi, Y. Fujii, Y. Hirose, T. Kobayashi, S. Hamamoto, M. Hirose, A. Okada, Y. Itoh, K. Tozawa, K. Kohri. Nagoya City University Graduate School of Medical Sciences, Dept. of Nephro-Urology, Nagoya, Japan Introduction and Objectives: Renal tubular epithelial cell injury induced by oxidative stress has been considered very important in the initial process of kidney stone formation. We previously reported the efficacy of green tea or catechin with anti-oxidative function for the prevention of kidney stone formation. Moreover, we recently discovered mitochondrial collapse, which is regarded as a cause of oxidative stress and renal tubular epithelial cell injury. It has been considered that mitochondrial collapse is produced by opening of mitochondrial permeability transition pores (MPTP) which is inhibited by cyclosporine A (CsA). In this study, we evaluated whether MPTP opening induces kidney stone formation through renal tubular cell injury, and the possibility of preventing kidney stone formation by CsA in vitro and in vivo studies. Material and Methods: In vitro study: Cultured renal epithelial (NRK-52E) cells were incubated with 2 mM CsA for 10 min and then a suspension of calcium oxalate monohydrate (COM) crystals (100 mg/cm2 ) was homogeneously layered over the cells for different durations. Mitochondrial function and oxidative stress were visualized by mitochondrial fluorescent assays using tetramethylrhodamine ethyl ester perchlorate (TMRE) and Western blotting, respectively, and cytosolic cytochrome c and caspase 3 activities as markers of cell apoptosis were evaluated by Western blotting. In vivo study: Rat models of kidney stones caused by ethylene glycol (EG) received CsA (2.5–10.0 mg/kg/day) for 14 days. Kidney stone formation, oxidative markers, and the structure of mitochondria within renal epithelial cells were evaluated by Pizzolato staining, immunohistochemical staining and transmission electron microscopy (TEM) respectively. Results: In vitro study: COM crystals diminished the mitochondrial TMRE intensity and decreased superoxide dismutase (SOD) as an oxidative marker, whereas CsA maintained the level of TMRE and SOD. CsA decreased the COM crystals-induced expression of cytosolic cytochrome c and caspase 3 activities. In vivo study: CsA decreased crystal formation and oxidative stress in rats administered EG. Furthermore, TEM showed that CsA also maintained the mitochondrial microstructure in renal tubular epithelial cells. Conclusions: This study showed that mitochondrial collapse by the opening of MPTP is an inducing factor in the initial process of kidney stone formation. We also showed that CsA inhibited the opening of MPTP and prevented kidney stone formation. E18 The interaction of the calcifying nanoparticles (CNP) with human renal tubular epithelial cells (HK-2) in vitro Y.L. Deng, D.D. Meng, C.Y. Li. The First Hospital of Guangxi Medical University, Dept. of Urology, Nanning, China Introduction and Objectives: To observe the interaction of the calcifying nanoparticles (CNP) with human renal tubular epithelial cells (HK-2) in vitro. To observe and investigate the mechanisms of HK-2 injury induced by CNP, and to explore the potential role of CNP in the formation of Calcium oxalate kidney stones. Material and Methods: Human renal tubular epithelial cells were cultured in vitro and the culture medium was added with CNP, the cell-crystal reaction was detected by light microscopy and transmission electron microscopy (TEM). To investigate the
oxidative stress, NADPH oxidase inhibitor apocynin was chosed as the intervener. We assessed the levels of LDH, MDA, HA in the mediums after 24 hours. Results: CNP can induce changes of the HK-2, have observed the adhesion and phagocytosis of CNP by the HK-2 under TEM. After 24 hours, the level of LDH, MDA, HA were significantly different among the 4 groups (P < 0.05). Conclusions: HK-2 has abilities of adhering and phagocyting with CNP; and CNP can cause the damage induced by oxidative stress of HK-2. E20 The buffer capacity of urine. A new aspect for differential diagnostics of urolithiasis E.V. Khomutova1 , E.V. Khomutov2 , V.M. Bilobrov1 , N.M. Bogdan1 . 1 Institute of Physical-Organic & Coal Chemistry, Dept. of Nucleophilic Reactivity Investigation, Donetsk, 2 Donetsk National Medical University, Dept. of Biochemistry, Donetsk, Ukraine Introduction and Objectives: Urolithiasis is one of the most common urological diseases that is the most often diagnosed in people between the ages of 30 and 60 years. Despite the fact that extensive researches are directed to diagnostics and treatment of urolithiasis, the growth tendency of incidence of this disease is observed. Therefore to improve the quality of life of patients it is necessary to reveal calculus before complications with further choice of optimal methods of treatment and programs for patients rehabilitation. However, in our opinion, the solution of this problem is not only in accumulation and analysis of the clinical researches data, but also in understanding physicochemical processes proceeding in urine of the healthy people and urolithiasis patients. The present work was aimed at finding the basic physicochemical processes proceeding in urine that leads to damage of the urine’s phase stability, and in particular at determination of the role of ions interaction on a course of a calculus formation. Material and Methods: The effect of ionic strength, concentration and the ratio of albumin, magnesium, calcium, citrate, phosphate and oxalate ions concentrations on the buffer properties and phase state was investigated by method of potentiometric titration of the solutions modeling urine. The buffer capacity of morning urine of 10 healthy volunteers and 30 urolithiasis patients was determined. The results of the model studies were analyzed together with the theoretical calculations and the data received for urine. Results: It was found that at the moment of the microheterogeneous phase formation the system’s buffer capacity increases in the quite certain intervals of pH that depend on composition of the heterogeneous phase. It was shown, that albumin serves both as the inhibitor and the promotor of biomineralization. On the one hand, it prevents formation of calcium phosphate by means of binding calcium ions, on the other hand – if albumin concentration exceeds 1 g/l it will promote formation of the heterogeneous phase due to interacting with polycarboxilic acids that are natural chelates for calcium. The dependences of urinary buffer capacity of normal volunteers and patients with urolithiasis on pH were received and analyzed. Conclusions: It was established, that the distinctiveness of urinary buffer capacity of patients with urolithiasis is determined by competition between complexing and heterogeneous phase formation. Findings could be used for developing of the noninvasive pre-surgical diagnostic tests of urolithiasis types.
wednesday 7 september 2011 / european urology supplements 10 (2011) 461–474
E17 Opening of mitochondrial permeability transition pores induces kidney stone formation K. Niimi, T. Yasui, K. Taguchi, Y. Fujii, Y. Hirose, T. Kobayashi, S. Hamamoto, M. Hirose, A. Okada, Y. Itoh, K. Tozawa, K. Kohri. Nagoya City University Graduate School of Medical Sciences, Dept. of Nephro-Urology, Nagoya, Japan Introduction and Objectives: Renal tubular epithelial cell injury induced by oxidative stress has been considered very important in the initial process of kidney stone formation. We previously reported the efficacy of green tea or catechin with anti-oxidative function for the prevention of kidney stone formation. Moreover, we recently discovered mitochondrial collapse, which is regarded as a cause of oxidative stress and renal tubular epithelial cell injury. It has been considered that mitochondrial collapse is produced by opening of mitochondrial permeability transition pores (MPTP) which is inhibited by cyclosporine A (CsA). In this study, we evaluated whether MPTP opening induces kidney stone formation through renal tubular cell injury, and the possibility of preventing kidney stone formation by CsA in vitro and in vivo studies. Material and Methods: In vitro study: Cultured renal epithelial (NRK-52E) cells were incubated with 2 mM CsA for 10 min and then a suspension of calcium oxalate monohydrate (COM) crystals (100 mg/cm2 ) was homogeneously layered over the cells for different durations. Mitochondrial function and oxidative stress were visualized by mitochondrial fluorescent assays using tetramethylrhodamine ethyl ester perchlorate (TMRE) and Western blotting, respectively, and cytosolic cytochrome c and caspase 3 activities as markers of cell apoptosis were evaluated by Western blotting. In vivo study: Rat models of kidney stones caused by ethylene glycol (EG) received CsA (2.5–10.0 mg/kg/day) for 14 days. Kidney stone formation, oxidative markers, and the structure of mitochondria within renal epithelial cells were evaluated by Pizzolato staining, immunohistochemical staining and transmission electron microscopy (TEM) respectively. Results: In vitro study: COM crystals diminished the mitochondrial TMRE intensity and decreased superoxide dismutase (SOD) as an oxidative marker, whereas CsA maintained the level of TMRE and SOD. CsA decreased the COM crystals-induced expression of cytosolic cytochrome c and caspase 3 activities. In vivo study: CsA decreased crystal formation and oxidative stress in rats administered EG. Furthermore, TEM showed that CsA also maintained the mitochondrial microstructure in renal tubular epithelial cells. Conclusions: This study showed that mitochondrial collapse by the opening of MPTP is an inducing factor in the initial process of kidney stone formation. We also showed that CsA inhibited the opening of MPTP and prevented kidney stone formation. E18 The interaction of the calcifying nanoparticles (CNP) with human renal tubular epithelial cells (HK-2) in vitro Y.L. Deng, D.D. Meng, C.Y. Li. The First Hospital of Guangxi Medical University, Dept. of Urology, Nanning, China Introduction and Objectives: To observe the interaction of the calcifying nanoparticles (CNP) with human renal tubular epithelial cells (HK-2) in vitro. To observe and investigate the mechanisms of HK-2 injury induced by CNP, and to explore the potential role of CNP in the formation of Calcium oxalate kidney stones. Material and Methods: Human renal tubular epithelial cells were cultured in vitro and the culture medium was added with CNP, the cell-crystal reaction was detected by light microscopy and transmission electron microscopy (TEM). To investigate the
oxidative stress, NADPH oxidase inhibitor apocynin was chosed as the intervener. We assessed the levels of LDH, MDA, HA in the mediums after 24 hours. Results: CNP can induce changes of the HK-2, have observed the adhesion and phagocytosis of CNP by the HK-2 under TEM. After 24 hours, the level of LDH, MDA, HA were significantly different among the 4 groups (P < 0.05). Conclusions: HK-2 has abilities of adhering and phagocyting with CNP; and CNP can cause the damage induced by oxidative stress of HK-2. E20 The buffer capacity of urine. A new aspect for differential diagnostics of urolithiasis E.V. Khomutova1 , E.V. Khomutov2 , V.M. Bilobrov1 , N.M. Bogdan1 . 1 Institute of Physical-Organic & Coal Chemistry, Dept. of Nucleophilic Reactivity Investigation, Donetsk, 2 Donetsk National Medical University, Dept. of Biochemistry, Donetsk, Ukraine Introduction and Objectives: Urolithiasis is one of the most common urological diseases that is the most often diagnosed in people between the ages of 30 and 60 years. Despite the fact that extensive researches are directed to diagnostics and treatment of urolithiasis, the growth tendency of incidence of this disease is observed. Therefore to improve the quality of life of patients it is necessary to reveal calculus before complications with further choice of optimal methods of treatment and programs for patients rehabilitation. However, in our opinion, the solution of this problem is not only in accumulation and analysis of the clinical researches data, but also in understanding physicochemical processes proceeding in urine of the healthy people and urolithiasis patients. The present work was aimed at finding the basic physicochemical processes proceeding in urine that leads to damage of the urine’s phase stability, and in particular at determination of the role of ions interaction on a course of a calculus formation. Material and Methods: The effect of ionic strength, concentration and the ratio of albumin, magnesium, calcium, citrate, phosphate and oxalate ions concentrations on the buffer properties and phase state was investigated by method of potentiometric titration of the solutions modeling urine. The buffer capacity of morning urine of 10 healthy volunteers and 30 urolithiasis patients was determined. The results of the model studies were analyzed together with the theoretical calculations and the data received for urine. Results: It was found that at the moment of the microheterogeneous phase formation the system’s buffer capacity increases in the quite certain intervals of pH that depend on composition of the heterogeneous phase. It was shown, that albumin serves both as the inhibitor and the promotor of biomineralization. On the one hand, it prevents formation of calcium phosphate by means of binding calcium ions, on the other hand – if albumin concentration exceeds 1 g/l it will promote formation of the heterogeneous phase due to interacting with polycarboxilic acids that are natural chelates for calcium. The dependences of urinary buffer capacity of normal volunteers and patients with urolithiasis on pH were received and analyzed. Conclusions: It was established, that the distinctiveness of urinary buffer capacity of patients with urolithiasis is determined by competition between complexing and heterogeneous phase formation. Findings could be used for developing of the noninvasive pre-surgical diagnostic tests of urolithiasis types.