Abstract: FR-PO776
Loss of Hypoxia-Regulated MicroRNA-210 Results in Decreased Nephron Number
Session Information
- Development and Organoid Models
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Hemker, Shelby L., University of Pittsburgh - Rangos Research Center, Pittsburgh, Pennsylvania, United States
- Bodnar, Andrew J., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Malta C.S Santos, Debora, UPMC Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Clugston, Andrew Scott, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Ho, Jacqueline, UPMC Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background
Low nephron number increases an individual’s risk for developing hypertension and chronic kidney disease, which affect approximately 30% and 15% of American adults, respectively. Intrauterine growth restriction and fetal hypoxia are significant causes of low nephron number; however, the underlying molecular mechanisms that drive this are unknown. Nephron number, in turn, is determined prior to birth in humans. microRNA-210 (miR-210) is the most consistently induced microRNA in hypoxia and regulates various processes including metabolism, cell cycle progression, and apoptosis. We hypothesize that loss of miR-210 results in abnormal kidney development.
Methods
To test this hypothesis, we obtained a global miR-210 knockout (KO) mouse. We used the “gold standard” physical dissector/fractionator combination method to estimate nephron number at postnatal day 30 (P30). We collected wildtype (WT) and KO littermates right before the burst in nephrogenesis at P0 and right before the end of nephrogenesis at P2. To measure changes in gene expression in nephron progenitors and their derivatives, we performed qPCR, immunofluorescent staining, and Western blot assays.
Results
We found an approximately 35% reduction in nephron number in miR-210 KO male kidneys and a 28% reduction in both WT and KO female kidneys, compared to WT males. Analysis of nephron progenitor proliferation, apoptosis and differentiation markers showed no discernable difference in kidney development at P0. However, we observed decreased expression of the differentiation marker Jag1 by Western blot at P2, as well as fewer total Lef1-, Jag1, and Nicd1-positive differentiating nephron structures. We observed no difference in nephron progenitor proliferation nor apoptosis at P2. However, there was an approximately 35% increase in the total number of cleaved-Casp3-positive cells and its immunofluorescent staining suggests that there is an increase in apoptosis in differentiating nephron structures. Furthermore, we found increased mRNA expression of several pro-apoptotic miR-210 target genes (Casp8AP2/FLASH, Ptpn2, and Bnip3).
Conclusion
miR-210 KO kidneys have a nephron deficit, which is associated with decreased differentiating nephron structures and increased apoptosis within those structures.
Funding
- NIDDK Support