Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C102
Acetylsalicylic acid impairs early renal development in cultured embryonic metanephros through a COX-independent mechanism
A. S. Carvalho1, S. J. Welham1, M. J. Elmes1
1. Nutritional Sciences, University of Nottingham, Loughborough, United Kingdom.
The maternal ingestion of Non-Steroidal Anti-Inflammatory drugs (NSAIDs) during pregnancy has increased over the past few decades and has been widely associated with renal failure and abnormal renal development in the offspring at birth. NSAIDs inhibit the COX enzymes, which catalyse the formation of prostaglandins from arachidonic acid, hence implicating a role for prostaglandins in renal development. This study aimed to evaluate the effects of the non-selective NSAID acetylsalicylic acid (ASA), also known as Aspirin, on ex vivo embryonic kidney growth and development, and to establish whether ASA treated metanephroi can be rescued by exogenous prostaglandin E2 (PGE2). Mice, at 8 weeks of age or older, were timed mated and culled at day 12 of gestation. Embryos were quickly removed and decapitated. Whole metanephroi (n=8-12) were microdissected from the embryos and cultured for 7 days in DMEM: F-12 serum free medium supplemented with insulin, transferrin and selenium. Contra-laterals were cultured in ASA concentrations ranging from 0.04mg/ml - 0.4mg/ml. Images were taken at 5x magnification on the day of dissection (day 0) and every 24hrs thereafter. Metanephroi cross-sectional areas were measured using Image Pro Plus v5.1. Analysis through paired t-test, showed that ASA concentrations as low as 0.1mg/ml produced significantly smaller metanephroi by day 4 of culture compared to controls (P<0.025), while the “physiological” dose of 0.2mg/ml produced smaller metanephroi by day 3 of culture (P<0.05). An ASA concentration of 0.4mg/ml had a detrimental effect on size as well as structure from day 1 of culture (P<0.005). In addition, 0.2 mg/ml ASA significantly reduced the size of the zone of nephrogenesis (P<0.005), and 0.2 mg/ml - 0.4mg/ml ASA halved the number of ureteric bud branches after 48-hours of culture (P<0.005). These results suggest that ASA has dose dependent adverse effects on early stage ex vivo renal growth and development. Prostaglandin rescue experiments using PGE2 were then performed to determine whether the phenotype observed was as a result of COX inhibition and the absence of prostaglandin synthesis. Metanephroi were cultured with either 0.2mg/ml or 0.4mg/ml of ASA for the first 2 days of culture, and then media replaced for the remainder of the experiment with either 10µM PGE2 alone, or in combination with each ASA dose. PGE2 alone rescued metanephros back to control size by 7 days of culture when treated for 2 days with either 0.2mg/ml or 0.4mg/ml ASA. However, metanephroi cultured with PGE2 in combination with either ASA dose for the full 7 days in culture did not rescue metanephros size. In conclusion the adverse affects of ASA on early renal development may be occurring through a COX-independent pathway, and future work will focus on elucidating the possible mechanisms.
Where applicable, experiments conform with Society ethical requirements