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Abstract: PO1222

Sildenafil Inhibits ADPKD-Derived Cyst Growth in 3D Culture and Induces Apoptosis in the Han/SPRD Cy+/Cy+ Rat Model of Renal Cystic Disease

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Bacallao, Robert L., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Xu, Weimin, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Rhodes, George, Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

In prior studies we found that human renal cyst cells express high levels of cGMP phosphodiesterase 5, 6 and 9 as compared to normal human kidney cells. In breast cancer, inhibition of cGMP phosphodiesterases induces apoptosis. We tested the hypothesis that sildenifil blocks cell proliferation and/or induces apoptosis in cyst epithelia.

Methods

PKD Q4004X cells were grown in Matrigel and treated with vehicle or sildenifil for 7 days. Cyst size was assayed by light microscopy. Differences in cyst area were analyzed by two-way ANOVA. In separate experiments, male cy+/cy+ rats were given either vehicle or sildenifil in their water supply for 7 or 28 days. Kidneys were harvested and apoptosis was assayed by staining with anti-M30, a monoclonal antibody that binds to apoptosis mediated cleavage of cytokeratin 18 in epithelial cells.

Results

In 3D cultures of PKD Q4004X cells, cultures treated with sildenafil (1, 2 and 4 ug/ml) are significantly smaller than vehicle-treated cultures (p<0.05 for all doses). Further analysis showed that cysts larger than 150 um2 <span style="font-size:10.8333px">were not observed in any sildenafil treated cultures.
Figure 1 shows M30 staining in sildenafil (20 ug/kg/day x 7 to treated rats (panel B , red channel merged with the green autofluorescence channel) versus vehicle treated animals. M30 positive cells are found predominantly around cyst lumens. Apoptotic changes in cyst epithelia were also observed in rats treated with sildenafil for 40 days.

Conclusion

Sildenafil inhibits human kidney cyst cyst growth in 3D culture. In male cy+/+ rats, sildenafil at a dose of 20 mg/kg/day for 7 or 40 days results in apoptosis of cyst epithelia.

Seven days of sildenafil treatment induces apoptosis in cystic kidneys. Sections stained with anti-cytokeratin 8/18 (red channel). Nuclei are labeled with Hoechst 33342. Green channel is autofluorescence. A: Vehicle treated male rats. Note the absence of cytokeratin 8/18. B: Vehicle + 20 mg/kg sildenafil treated for 7 days.. Arrows point to cytokeratin 8/18 positive (red cytosolic stain) in cyst epithelia.

Funding

  • Private Foundation Support