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Abstract: PO0623

Dual Tubular Par1a/b cKO Is Protective Against Renal Fibrosis

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Zhou, Vellia, Albert Einstein College of Medicine, Bronx, New York, United States
  • Du, Zhongfang, Albert Einstein College of Medicine, Bronx, New York, United States
  • Brathwaite, Kaye E., Albert Einstein College of Medicine, Bronx, New York, United States
  • Reidy, Kimberly J., Albert Einstein College of Medicine, Bronx, New York, United States
Background

Partitioning defective Par1a/b proteins are highly homologous serine threonine kinases and contribute to kidney development. Tubular Par1a/b expression increases following folic acid and unilateral ureteral obstruction (UUO). Loss of Par1a/b expression during development impairs Notch signaling pathway expression. Notch signaling activation contributes to renal fibrosis. We hypothesized that Par1a/b expression is maladaptive following injury and promotes renal fibrosis.

Methods

Using publically available single cell RNA sequencing data, we examined the cell types where Par1a (Mark3) and Par1b (Mark2) were expressed following UUO. Localization was confirmed using immuno-fluorescence with antibodies specific for Par1a and 1b. Conditional Par1a and 1b flox mice were generated using CRISPR/Cas9 gene editing. Dual tubular conditional Par1a/b knockout (cKO) mice (Pax8-rtTA:tet-O-Cre:Mark2flox:flox:Mark3flox/flox) mice were generated. Deletion of Par1a (Mark3) and Par1b (Mark2) was confirmed following doxycycline induction. UUO was performed in adult (10 week old) male tubular Par1a/b cKO mice and controls; phenotype was examined at 7 days. Tubular Par1a/b deletion was induced by feeding mice doxycycline in chow starting 7 days prior to UUO. Controls were uninduced (-dox) transgenic littermates and doxycycline treated Mark2flox/flox:Mark3flox/flox mice. 6-8 mice/group were studied. To detect renal fibrosis, Picro-Sirius Red staining of collagen was performed. Polarized light and Image J was utilized to quantify fibrosis on 200 x images.

Results

Single cell analysis demonstrated increased expression of Par1a/b in proliferating and injured proximal tubules following UUO. This was confirmed by co-expression of Par1a in ki67 and Sox9 positive tubules following UUO. Dual tubular Par1a/b deletion was protective against fibrosis, with % fibrosis decreasing from 1.6 to 0.65 percent 7 days following UUO (p=0.0048).

Conclusion

Dual Par1a/b knockout in tubules protected against fibrosis. Ongoing studies are examining optimal timing of deletion to promote repair vs. fibrosis. Par1 kinase inhibitors may be potential promising therapeutics for preventing fibrosis in chronic kidney disease.

Funding

  • NIDDK Support