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Abstract: PO1509

Elevated Expression of CaMK4 Contributes to mTOR-dependent Cell Proliferation and Cyst Growth of ADPKD Cells

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Zhang, Yan, University of Kansas Medical Center, Kansas City, Kansas, United States
  • Daniel, Emily A., University of Kansas Medical Center, Kansas City, Kansas, United States
  • Reif, Gail, University of Kansas Medical Center, Kansas City, Kansas, United States
  • Wallace, Darren P., University of Kansas Medical Center, Kansas City, Kansas, United States
Background

Mammalian target of rapamycin (mTOR), a central integration site for pathways involved in cell growth, is abnormally activated in renal cyst-lining cells in human ADPKD and PKD mice. mTOR inhibition reduces cell proliferation and cyst growth in PKD mice. A better understanding of the pathways responsible for elevated mTOR activation is important for the development of new therapies. Calcium/calmodulin-dependent kinase type IV (CaMK4) was recently identified as an important upstream regulator for mTOR activation in multiple cells. CaMK4 is fully activated by binding of Ca2+/Calmodulin(CaM) and phosphorylation by Ca2+/CaM-dependent kinase kinases β (CaMKKβ). However, the role of CaMK4 on mTOR signaling and cyst growth in ADPKD remains unclear.

Methods

We stained tissue sections of human ADPKD kidneys and normal human kidneys (NHKs) with an antibody to CaMK4. We also analyzed levels of CaMK4 in primary human ADPKD and NHK cells, 30-weeks old Pkd1RC/RC (BALB/c background) and Pkd1RC/+ (normal) mouse kidneys. We determined the effects of W7, a calmodulin inhibitor, STO-609, a CaMKKβ inhibitor, and KN-93, a CaMK4 inhibitor, on mTOR signaling, cell proliferation, and in vitro cyst growth of ADPKD cells. To confirm a role of CaMK4 on mTOR regulation, we knocked down CaMK4 using a lentiviral shRNA approach in human ADPKD cells.

Results

We found moderate levels of CaMK4 in nuclei of tubule cells in NHK; by contrast, there were elevated levels of CaMK4 in the cytosol and nuclei of cystic epithelial cells of human ADPKD kidneys. CaMK4 level was 2.5-fold higher in human ADPKD cells compared to NHK cells. We also found a 2.8-fold increase of CaMK4 expression in Pkd1RC/RC kidneys compared to normal kidneys. Inhibition of CaMK4 using KN-93 caused a remarkably decreased in levels of P-S6K and P-S6 in a dose-dependent and time-dependent manner. Inhibition of CaMK4 activation using W7 and STO-609 reduced P-S6 in ADPKD cells. CaMK4 knock down decreased levels of mTOR, P-S6K and P-S6 confirming regulation of CaMK4 on mTOR signaling in ADPKD cells. W7, STO-609, and KN-93 significantly inhibited cell proliferation and in vitro cyst growth of ADPKD cells within a collagen matrix.

Conclusion

The aberrant expression of CaMK4 appears to contribute to elevated mTOR-dependent proliferation of cystic cells and may be a potential target to slow cyst growth in PKD.

Funding

  • NIDDK Support