Toxic effects and mechanisms of action of renal cancerogenic substances in vitro

The natural compounds Aristolochic Acid (AA), Ochratoxin A (OTA) and Cycasin are nephrotoxic and carcinogenic in rodents. AA and OTA exposure in humans are known and human exposure to Cycasin is suspected. Despite exposure, toxic effects in humans are only proven for AA. However, the observed effects of the genotoxic substance AA in humans were different to those seen in rodents, suggesting a species-specific mechanism of action. In vitro-models could serve as a powerful tool to evaluate species-specific toxicity. Especially the use of human primary cells could enable characterisation and evaluation of possible human-specific effects.It was postulated, that in vitro-systems, in addition to in vivo-Studies, are a valuable tool to investigate species-specific toxicity of AA, OTA and Cycasin. The aim of the study was to examine the susceptibility of different kidney cortex cell models (continuous cell lines, primary cells) to the toxin in question and therefore their value for studying species-specific effects.In this study, the cytotoxicity of AA, OTA and MAMAc was evaluated and used for a pre-selection of cells (cell line, primary cells) sufficiently susceptible to the toxin in question. Furthermore, effects of the genotoxic compounds AA and MAMAc on the cell cycle and the induction of DNA-strand breaks and DNA-repair were examined. Finally, the effects of AA and OTA on gene-expression were studied.Cytotoxic response was generally higher in primary cells as in the corresponding cells lines, possibly reflecting the higher status of differentiation in primary cells. The observed reduction of cell proliferation and the observed induction of cdkn1a-gene-expression indicate a genotoxic effect of AA in vitro, which seems to reflect sufficient susceptibility of the used cell model. In contrast to this, AA and OTA had only a small effect on gene-expression. However, the low degree of deregulation as well as the small number of deregulated genes could be a result of the low toxin concentrations used. Nevertheless, deregulation of toxin-relevant genes was observed. In this study, species-specific differences in cytotoxicity of AA and MAMAc were shown, with toxicity being highest in kidney cortex cells of porcine origin. These results are in accordance with prior results, showing metabolic activation of AA to be species-dependent, with minipigs having the most effective activation system followed by human and rat. Although cytotoxicity assays can not allow discrimination of distinctive mechanistic responses, it is suspected that the description of constitutive differences in the susceptibility of cells originating of different species can be achieved.From this study, primary kidney cortex cells appear to be a valuable tool to investigate the toxicity of AA, OTA and Cycasin. Furthermore, human primary cells seem to be useful for the evaluation of possible human-specific effects. However, the availability and quality of biopsy material from primary care facilities for the establishment of primary human cell culture is highly variable and commercially available cells are expensive. Given the genetic proximity of human and pigs and the anatomical and physiological similarity of the kidneys, future direction should therefore consider the use of primary porcine cell models as an alternative system

Species-specific Toxicity of Aristolochic Acid (AA) in vitro

Differences in toxicity and carcinogenicity of the nephrotoxic compound aristolochic acid between rodents and humans suggest a species-dependent mechanism of action. The goal of this study was to investigate constitutive differences in the susceptibility of renal cortex cells originating from human, rat and porcine origin in vitro. Effects of 24 and 48 h AA exposure on cell number and MTT reduction were studied. Furthermore, using the effective concentrations causing 20 and 50 % reduction (cell number), cell cycle, 3H-thymidine incorporation and DNA damage analyses were conducted. AA cytotoxicity was observed in all cell types in a time- and concentration dependent manner with species-specific differences, with porcine cells being the most sensitive. AA had a comparable effect on the cell cycle in primary human and porcine cells and the rat NRK-52E cell line following 48 h exposure, also corroborated by the reduced 3H-thymidine incorporation in NRK-52E cells. In addition, DNA unwinding, suggestive of enhanced DNA damage, was observed in primary porcine cells. These results provide an initial insight into the sensitivity and suitability of different in vitro-systems and suggest that primary porcine renal cortex cells could be a valuable in vitro-system to study AA toxicit

A non-interventional comparative study of the 20:1 combination of cafedrine/theodrenaline versus ephedrine for the treatment of intra-operative arterial hypotension: the ‘HYPOTENS’ study design and rationale

<p><b>Objective:</b> To compare the effectiveness of 20:1 cafedrine/theodrenaline approved for use in Germany to ephedrine in the restoration of arterial blood pressure and on post-operative outcomes in patients with intra-operative arterial hypotension of any origin under standard clinical practice conditions.</p> <p><b>Methods and results:</b> ‘HYPOTENS’ is a national, multi-center, prospective, open-label, two-armed, non-interventional study. Effectiveness and post-operative outcome following cafedrine/theodrenaline or ephedrine therapy will be evaluated in two cohorts of hypotensive patients. Cohort A includes patients aged ≥50 years with ASA-classification 2–4 undergoing non-emergency surgical procedures under general anesthesia. Cohort B comprises patients undergoing Cesarean section under spinal anesthesia. Participating surgical departments will be assigned to a treatment arm by routinely used anti-hypotensive agent. To minimize bias, matched department pairs will be compared in a stratified selection process. The composite primary end-point is the lower absolute deviation from individually determined target blood pressure (IDTBP) and the incidence of heart rate ≥100 beats/min in the first 15 min. Secondary end-points include incidence and degree of early post-operative delirium (cohort A), severity of fetal acidosis in the newborn (cohort B), upper absolute deviation from IDTBP, percentage increase in systolic blood pressure, and time to IDTBP.</p> <p><b>Conclusion:</b> This open-label, non-interventional study design mirrors daily practice in the treatment of patients with intra-operative hypotension and ensures full treatment decision autonomy with respect to each patient’s individual condition. Selection of participating sites by a randomization process addresses bias without interfering with the non-interventional nature of the study. First results are expected in 2018. ClinicalTrials.gov identifier: NCT02893241; DRKS identifier: DRKS00010740.</p