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Abstract: FR-PO391

mTORC1 Activity Promotes an Intercalated Cell State Which Is Suppressed by Hes1 to Maintain Principal Cells in a Mature State

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

  • DeRiso, Jennifer, Sanford Research, Sioux Falls, South Dakota, United States
  • Mukherjee, Malini, Sanford Research, Sioux Falls, South Dakota, United States
  • Janga, Madhusudhana R., Sanford Research, Sioux Falls, South Dakota, United States
  • Rothschadl, Morgan Jean, University of South Dakota, Vermillion, South Dakota, United States
  • Bravo, Sabdi, Sanford Research, Sioux Falls, South Dakota, United States
  • Simmons, Alicia L., University of South Dakota, Vermillion, South Dakota, United States
  • Busselman, Brook W., University of South Dakota, Vermillion, South Dakota, United States
  • Chandrasekar, Indra, Sanford Research, Sioux Falls, South Dakota, United States
  • Surendran, Kameswaran, Sanford Research, Sioux Falls, South Dakota, United States
Background

Notch signaling via Hes1 is required to maintain Aquaporin-2 expressing principal cell types in the distal convoluted tubule, connecting tubule and collecting duct segments of the adult mouse kidney in a non-intercalated cell state. Lineage tracing of Hes1-deficient principal cells revealed that Notch signaling suppresses intercalated programs from turning on in mature principal cells. Here we attempt to identify intercalated cell state promoting signals by using unbiased genomic approaches to determine signaling pathways suppressed by Hes1 in principal cells.

Methods

We identified differentially expressed genes in adult mouse kidneys from control littermates (n=9) versus mice with one week of Hes1 inactivation within the kidney epithelial cells (n=7). RNA-sequencing of total kidney RNA was performed to identify differentially expressed genes. Also, we performed chromatin immunoprecipitation (ChIP) using anti-FLAG antibody to identify loci visited by Hes1 in a mature principal cell line expressing Hes1 fused with 3xFLAG. We validated direct targets of Hes1 by chIP-qPCR. We detected mTORC1 acitvity by staining for phosphorylated S6 along with principal and intercalated cell markers. We also traced the fate of wild type versus Tsc2-deficient mature principal cells by genetically labeling them in adult mouse kidneys.

Results

Our results reveal that multiple signaling pathways components, including Wnt, BMP, Hh and mTOR are upregulated soon after inactivation of Hes1. Hes1 suppresses insulin receptor substrate 1 (irs1), a component that mediates mTORC1 activation. Consistent with mTORC1 activity promoting intercalated cell state we observed an increase in phosphorylated S6 ribosomal protein soon after inactivation of Hes1 in principal cells of adult mouse kidneys. Additionally, inactivation of Tsc2, an inhibitor of mTORC1, is sufficient to initiate conversion of principal cells towards a type A intercalated cell state in adult kidneys.

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Conclusion

These studies reveal a role for mTORC1 activity in promoting an intercalated cell state which is suppressed by Notch signaling to maintain principal cells in a mature state.

Funding

  • NIDDK Support