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Abstract: TH-PO487

mTORC1 Regulates Renal Proximal Tubular Megalin Function

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Kunke, Madlen, Christian-Albrechts University zu Kiel, Kiel, Germany
  • Zanon rodriguez, Luis, Christian-Albrechts University zu Kiel, Kiel, Germany
  • Schwaerzer, Gerburg, Christian-Albrechts University zu Kiel, Kiel, Germany
  • Theilig, Franziska, Christian-Albrechts University zu Kiel, Kiel, Germany
Background

Renal proximal tubular cells constantly recycle nutrients to ensure minimal loss of essential substrates into the urine. Thus, endocytosis is a hallmark of the proximal tubule. Protein uptake is mediated by the scavenger receptors megalin and cubilin, followed by internalization of the ligand-receptor complex via the clathrin-mediated pathway. The mTORC1 complex is a principle regulator of proximal tubular function, including endocytosis. However, the effect of mTORC1 on megalin function remains unknown.

Methods

Tubular deletion of mTORC1 was created by crossbreeding Raptorfl/fl with Pax8rt-TA and TetOCre mice. Phosphoproteomics were performed to analyze the phosphorylation of megalin in the renal cortex. Endocytosis was detected in proximal tubules using Alexa555-labelled lactoglobulin. In vitro, we induced mTORC1 activity through transient transfection with Rheb, used megalin minireceptor 2 (MMR2) to introduce mutations in the respective phosphosite S4577A, S4577D, S4577E, and followed endocytosis through Alexa555-labelled albumin.

Results

mTORC1-deficient mice showed normal megalin expression and distribution within proximal tubules compared to wildtype mice. Interestingly, receptor-mediated endocytosis of Alexa555-labelled lactoglobulin was blocked in mTORC1-deficient mice. Phosphoproteomics of mTORC1 deficient mice revealed a strongly reduced phosphorylation at S4577 of the C-terminus of megalin, which was confirmed by transient transfection of MDCKII cells with Rheb stimulating mTORC1 activity. Transfection of wildtype or mutated MMR2 in MDCKII cells caused no difference in megalin expression and its cellular distribution. However, endocytosis was increased in the presence of S4577D mutant compared to megalin wildtype

Conclusion

mTORC1 complex is an important regulator of proximal tubular function and phosphorylates megalin to influence megalin function.

Funding

  • Private Foundation Support