Abstract: FR-OR059

Angiomotin Knockout in Rats Causes Proteinuria

Session Information

Category: Glomerular

  • 1003 Glomerular: Cell Biology

Authors

  • Zhang, Yaochun, National University of Singapore, Singapore, Singapore
  • Ward, Jerrold m., Global VetPathology, Montgomery Village, Maryland, United States
  • Hossain, Zakir, National University of Singapore, Singapore, Singapore
  • Foo, Sik Yin, National University of Singapore, Singapore, Singapore
  • Liu, Isaac, National University of Singapore, Singapore, Singapore
  • Chan, Chang-Yien, National University of Singapore, Singapore, Singapore
  • Sun, Zi Jin, National University of Singapore, Singapore, Singapore
  • Yap, Hui Kim, National University Hospital, Singapore, Singapore
  • Ng, Kar Hui, National University of Singapore, Singapore, Singapore
Background

Angiomotins were originally identified as angiostatin binding proteins and implicated in the regulation of endothelial cell migration and tube formation. Recent studies have shown that Angiomotion plays a central role in tight junction maintenance via the complex formed with ARHGAP17, which acts by regulating the uptake of polarity proteins at tight junctions. This study aimed to investigate the renal functions of Angiomotin with an Angiomotin knockout (Amot KO) rat model.

Methods

Using CRISPR/Cas9 system, we created an Amot KO founder rat with a 5 bp insertion in the coding region, which produced a premature stop codon, resulting in a truncated protein of 63 amino acids. The founder was crossed with wild type rats to establish Amot KO rat line. The phenotypes of Amot KO (n = 11) male rats were studied and compared with wild type control (n = 11). Body weight, serum and urine albumin:creatinine ratios were monitored monthly. Total RNA were extracted from peripheral blood mononuclear cells and sent for RNA-Seq. The rats were euthanized at different time points. Kidney cortex samples were obtained for pathological examinations.

Results

Increased urine albumin:creatinine ratio was found as early as 4 month age in the Amot KO rats. The ratio increased progressively with age and reached 547.7 ± 254.6 µg/mg in the 6 month old Amot KO rats, while the ratio is 150.4 ± 117.4 µg/mg for the wild type control (p < 0.01). Pathological changes in the rat kidneys were systemically examined with H&E and PAS staining. While wild type control rats showed normal renal glomeruli and proximal tubules, prominent thickened GBMs, thickened Bowman’s capsule, casts or crystal in distal tubule, thicker tubular basement membrane and hyperplastic epithelium as well as podocyte atypia can be seen in the 6 and 10 month old Amot KO rats. RNA-Seq results showed more than 300 differentially expressed genes including Smad7, Rac1 and pdlim1. Analysis using IPA revealed altered effector pathways in glomerular injury, renal necrosis, and nephritis.

Conclusion

AMOT appears to play important roles in regulating renal functions. AMOT may exert its function through Rich1/Gap complex affecting activation status of Rho GTPases or via interactions with slit diaphragm and actin cytoskeleton proteins.

Funding

  • Government Support - Non-U.S.