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Abstract: TH-PO647

Using the T30H Mouse to Investigate the Role of Myocardin in Obstructive Uropathy

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 501 Development, Stem Cells, and Regenerative Medicine: Basic

Authors

  • Milmoe, Natalie J., UCL GOS Institute of Child Health , London, United Kingdom
  • Stuckey, Daniel J., UCL, London, United Kingdom
  • Rahim, Ahad A., University College London, London, United Kingdom
  • Harvey, Robert J., University of the Sunshine Coast, Sippy Downs, New South Wales, Australia
  • Winyard, Paul, UCL GOS Institute of Child Health , London, United Kingdom
Background

Obstructive uropathies account for 20% of paediatric end stage renal failure. Irreparable damage often occurs to the kidneys before surgical correction of obstruction is possible. There are few in vivo models of this process. T30H mice were generated many years ago and have a balanced, heritable chromosomal translocation between chromosomes 2 and 11. The exact translocation point was previously unknown. They die soon after birth with large non-emptying bladders, hydronephrosis and reduced nephron numbers, despite there being no physical obstruction. Nevertheless, they are still a useful model for many features of obstructive uropathy.

Methods

We have utilised next generation sequencing, histology, RT-PCR and cell culture to examine gene expression and the bladder phenotype.

Results

We have investigated the T30H genome and discovered the exact translocation point, it does not span recognised genes, but is upstream of myocardin, a master regulator of smooth muscle.

In the bladder, markers such as α SMA and Calponin are absent or expression is severely reduced. Urothelial markers such as Uroplakin 3a are unchanged. The translocation has no effect on all other systems involving smooth muscle, organs such as the heart and gut appear normal.

The splice variants of myocardin present in wild type and T30H mice at birth are similar, suggesting expression levels of these splice variants are more important than whether or not they are expressed. Quantitative PCR will be required to confirm this hypothesis.

We have isolated smooth muscle cells from wild type bladders at E14, then transfected them with lentivirus containing myocardin shRNA. This gene knockdown causes both reduced growth and expression of smooth muscle specific genes, including αSMA, consistent with the bladder phenotype in vivo.

We are investigating the bladder phenotype in utero; the defect is identifiable using ultrasound from E16. In future we will uncover the developmental point when the defect is instigated, and whether the defect arises as a failure of muscle development, or whether the muscle grows and then is not maintained.

Conclusion

Understanding bladder smooth muscle regulation will better our knowledge of urinary tract development, and enable us to develop improved therapies for treating smooth muscle complications arising from urinary tract malformations.

Funding

  • Private Foundation Support