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Suppression of FK506 induced nephrotoxicity in mice by Bacopa monnierii
Abstracts / Toxicology Letters 280S (2017) S137–S140
P-04-04-06 Development of SLC transporters kidney cell models using hTERT-immortalized renal proximal tubule epithelial cells (RPTEC/TERT1) Sara Gualdoni 1 , Chaozhong Zou 2 , Russell McDaniel 2 , Christopher Annesi 2 , Elisabeth Turner 2 1 2
LGC Standards, LGC Standards, Teddington, United Kingdom ATCC Cell Systems, ATCC, Gaithersburg, United States
Backgrounds: Kidney membrane transporters play a key part in drug disposition and renal clearance. Among the SLC families, OAT1, OCT2 and OAT3 are the most important transporters in kidney tissue recommended for drug interaction studies by the FDA and EMA. Unfortunately, primary RPTEC cells lose OAT1, OCT2 and OAT3 transporters expression in culture. Transiently expressing these transporters in primary RPTEC cells show large variations between batches which makes data hard to interpret. Cell line-based models either comes from animal species or are cancerogenic, which means the clinical predictability is greatly compromised. Aims: The aim of this study is to generate a new in vitro kidney toxicity model that have human kidney origin, accurate clinical predictability and consistent data output for initial drug interaction studies. Methods: We have generated transporter cell models using a well characterized hTERT-immortalized Renal Proximal Tubule Epithelial Cells (RPTEC/TERT1) that stably overexpress either the OAT1, OCT2 and OAT3 gene. Results: Immunostaining shows that OAT1, OCT2 and OAT3 are correctly trafficked to the membrane. Clones show epithelial morphology and correct markers expression. The overexpressed transporters have normal transport activities using 5-CF and ASP + (4-(dimethylamino)styryl)-N-methylpyridinium iodide) uptake assays and the uptake can be inhibited by specific inhibitors. Summary: The OCT2/3 and OAT1 modified RPTEC/TERT1 cell lines are a new model for in vitro renal toxicology, which provide human kidney tissue related results, improved consistency over time, and have more predictability for clinical trials versus current in vitro models. http://dx.doi.org/10.1016/j.toxlet.2017.07.388 P-04-04-07 Metabolomics in vitro in kidney cells – A tool for investigation of the nephrotoxicity Barbara Birk 1 , Saskia Sperber 1 , Hans-Albrecht Huener 1 , Andreas Verlohner 1 , Tilmann Walk 2 , Volker Haake 2 , Hennicke Kamp 1 , Ben van Ravenzwaay 1 1
Experimental Toxicology and Ecology, BASF SE, Ludwigshafen am Rhein, Germany 2 Metanomics GmbH, Berlin, Germany In the last few years the development and application of alternatives to animal testing has considerably increased but relevant toxicological endpoints (systemic and reproductive toxicity) can currently not fully be replaced by in vitro methods. Moreover, the interest in the mode of action of toxic compounds is becoming more and more important. BASF has already established successfully metabolomics in vitro in liver cells (HepG2). Now, metabolomics in vitro in kidney cells is a novel approach that might enable the identification of nephrotoxicity including its mode of action. NRK-52e-cells were cultivated on Lumox dishes (Sarstedt) and treated for 48 h with well characterized reference
S139
substance (Bezafibrate). Details about the cultivation, treatment of the cells and the procedure of reproducible metabolite extraction, the metabolome analysis (>200 metabolites) by MS/MS-technology and finally the robustness of the method will be presented. The results of bezafibrate and control samples (DMSO 0.5%), partly obtained within the InnoSysTox Project “Risk-IT” founded by BMBF, Germany, show that this technology is now ready for validation by testing substances with different modes of action. Therewith, metabolomics in vitro in kidney cells might be a new animalfree method to investigate nephrotoxicity including the respective mode of action and might be an important part of the puzzle for the evaluation of systemic toxicity in the future. http://dx.doi.org/10.1016/j.toxlet.2017.07.389 P-04-04-08 Suppression of FK506 induced nephrotoxicity in mice by Bacopa monnierii Atif Abdulwahab A. Oyuoni, Shalini Saggu, Hasibur Rehman, Ehab Toussan, Aziz Al Thabiani Biology, University of Tabuk, Tabuk, Saudi Arabia Calcineurin inhibitors are the backbone of immunosuppressant therapy in organ transplantation. The immunosuppressive property of FK506 is mediated via inhibition of the cellular phosphatase calcineurin. It can cause severe nephrotoxicity and increase free radical formation. This study investigated whether an extract from Bacopa monnierii (BM) could prevent nephrotoxicity caused by FK506. Mice were divided into four groups and treated for one and two weeks as follows: Gr. 1: normal control-treated (saline); Gr. 2: BM extract-treated; Gr 3: FK506 (2 mg/kg, i.p) treated; Gr.4: FK506 plus BM extract. Results of this study revealed that the mice treated with FK506 showed a significant increase in bilirubin, aspartate aminotransferase, alanine transaminase, alkaline phosphatase activities. Pretreatment with the extract reversed almost all the abnormalities in the blood parameters showing protection against FK506 induced toxicity in mice. Inhibitory potential of BM against FK506 induced nephrotoxicity was evaluated in rapports of increased activity of antioxidant enzymes. Renal tissue malondialdehyde level was significantly increased in the FK506 group compared with the control group and was significantly decreased in the FK506-Bacopa treated group. Antioxidant enzyme activities of superoxide dismutase and catalase were significantly suppressed in the FK506 group compared with the control group and were restored when treated with BM. These results demonstrate that FK506 stimulate free radical production in the kidney and the pretreatment with extract minimize nephrotoxicity by scavenging free radicals. Acknowledgment: Financial support from University of Tabuk, S1438-0128. http://dx.doi.org/10.1016/j.toxlet.2017.07.390
P-04-04-06 Development of SLC transporters kidney cell models using hTERT-immortalized renal proximal tubule epithelial cells (RPTEC/TERT1) Sara Gualdoni 1 , Chaozhong Zou 2 , Russell McDaniel 2 , Christopher Annesi 2 , Elisabeth Turner 2 1 2
LGC Standards, LGC Standards, Teddington, United Kingdom ATCC Cell Systems, ATCC, Gaithersburg, United States
Backgrounds: Kidney membrane transporters play a key part in drug disposition and renal clearance. Among the SLC families, OAT1, OCT2 and OAT3 are the most important transporters in kidney tissue recommended for drug interaction studies by the FDA and EMA. Unfortunately, primary RPTEC cells lose OAT1, OCT2 and OAT3 transporters expression in culture. Transiently expressing these transporters in primary RPTEC cells show large variations between batches which makes data hard to interpret. Cell line-based models either comes from animal species or are cancerogenic, which means the clinical predictability is greatly compromised. Aims: The aim of this study is to generate a new in vitro kidney toxicity model that have human kidney origin, accurate clinical predictability and consistent data output for initial drug interaction studies. Methods: We have generated transporter cell models using a well characterized hTERT-immortalized Renal Proximal Tubule Epithelial Cells (RPTEC/TERT1) that stably overexpress either the OAT1, OCT2 and OAT3 gene. Results: Immunostaining shows that OAT1, OCT2 and OAT3 are correctly trafficked to the membrane. Clones show epithelial morphology and correct markers expression. The overexpressed transporters have normal transport activities using 5-CF and ASP + (4-(dimethylamino)styryl)-N-methylpyridinium iodide) uptake assays and the uptake can be inhibited by specific inhibitors. Summary: The OCT2/3 and OAT1 modified RPTEC/TERT1 cell lines are a new model for in vitro renal toxicology, which provide human kidney tissue related results, improved consistency over time, and have more predictability for clinical trials versus current in vitro models. http://dx.doi.org/10.1016/j.toxlet.2017.07.388 P-04-04-07 Metabolomics in vitro in kidney cells – A tool for investigation of the nephrotoxicity Barbara Birk 1 , Saskia Sperber 1 , Hans-Albrecht Huener 1 , Andreas Verlohner 1 , Tilmann Walk 2 , Volker Haake 2 , Hennicke Kamp 1 , Ben van Ravenzwaay 1 1
Experimental Toxicology and Ecology, BASF SE, Ludwigshafen am Rhein, Germany 2 Metanomics GmbH, Berlin, Germany In the last few years the development and application of alternatives to animal testing has considerably increased but relevant toxicological endpoints (systemic and reproductive toxicity) can currently not fully be replaced by in vitro methods. Moreover, the interest in the mode of action of toxic compounds is becoming more and more important. BASF has already established successfully metabolomics in vitro in liver cells (HepG2). Now, metabolomics in vitro in kidney cells is a novel approach that might enable the identification of nephrotoxicity including its mode of action. NRK-52e-cells were cultivated on Lumox dishes (Sarstedt) and treated for 48 h with well characterized reference
S139
substance (Bezafibrate). Details about the cultivation, treatment of the cells and the procedure of reproducible metabolite extraction, the metabolome analysis (>200 metabolites) by MS/MS-technology and finally the robustness of the method will be presented. The results of bezafibrate and control samples (DMSO 0.5%), partly obtained within the InnoSysTox Project “Risk-IT” founded by BMBF, Germany, show that this technology is now ready for validation by testing substances with different modes of action. Therewith, metabolomics in vitro in kidney cells might be a new animalfree method to investigate nephrotoxicity including the respective mode of action and might be an important part of the puzzle for the evaluation of systemic toxicity in the future. http://dx.doi.org/10.1016/j.toxlet.2017.07.389 P-04-04-08 Suppression of FK506 induced nephrotoxicity in mice by Bacopa monnierii Atif Abdulwahab A. Oyuoni, Shalini Saggu, Hasibur Rehman, Ehab Toussan, Aziz Al Thabiani Biology, University of Tabuk, Tabuk, Saudi Arabia Calcineurin inhibitors are the backbone of immunosuppressant therapy in organ transplantation. The immunosuppressive property of FK506 is mediated via inhibition of the cellular phosphatase calcineurin. It can cause severe nephrotoxicity and increase free radical formation. This study investigated whether an extract from Bacopa monnierii (BM) could prevent nephrotoxicity caused by FK506. Mice were divided into four groups and treated for one and two weeks as follows: Gr. 1: normal control-treated (saline); Gr. 2: BM extract-treated; Gr 3: FK506 (2 mg/kg, i.p) treated; Gr.4: FK506 plus BM extract. Results of this study revealed that the mice treated with FK506 showed a significant increase in bilirubin, aspartate aminotransferase, alanine transaminase, alkaline phosphatase activities. Pretreatment with the extract reversed almost all the abnormalities in the blood parameters showing protection against FK506 induced toxicity in mice. Inhibitory potential of BM against FK506 induced nephrotoxicity was evaluated in rapports of increased activity of antioxidant enzymes. Renal tissue malondialdehyde level was significantly increased in the FK506 group compared with the control group and was significantly decreased in the FK506-Bacopa treated group. Antioxidant enzyme activities of superoxide dismutase and catalase were significantly suppressed in the FK506 group compared with the control group and were restored when treated with BM. These results demonstrate that FK506 stimulate free radical production in the kidney and the pretreatment with extract minimize nephrotoxicity by scavenging free radicals. Acknowledgment: Financial support from University of Tabuk, S1438-0128. http://dx.doi.org/10.1016/j.toxlet.2017.07.390