Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

Am J Physiol Renal Physiol. 2010 Jul;299(1):F167-77. doi: 10.1152/ajprenal.00162.2010. Epub 2010 May 12.

Abstract

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na(+)-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten V(max) parameter. [(14)C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism*
  • Aminoimidazole Carboxamide / analogs & derivatives
  • Aminoimidazole Carboxamide / pharmacology
  • Animals
  • Biotinylation
  • Blotting, Western
  • Cell Line, Transformed
  • Cell Polarity
  • Creatine / metabolism*
  • Energy Metabolism
  • Enzyme Activation
  • Enzyme Activators / pharmacology
  • Epithelial Cells / drug effects
  • Epithelial Cells / enzymology*
  • Humans
  • Immunohistochemistry
  • Intracellular Signaling Peptides and Proteins / antagonists & inhibitors
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / drug effects
  • Kidney Tubules, Proximal / enzymology*
  • Kinetics
  • Male
  • Membrane Potentials
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Mice
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Oocytes
  • Patch-Clamp Techniques
  • Plasma Membrane Neurotransmitter Transport Proteins / genetics
  • Plasma Membrane Neurotransmitter Transport Proteins / metabolism*
  • Protein Kinase Inhibitors / pharmacology
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism
  • Rats
  • Ribonucleotides / pharmacology
  • Sirolimus / pharmacology
  • Sodium / metabolism
  • TOR Serine-Threonine Kinases
  • Xenopus laevis

Substances

  • Enzyme Activators
  • Intracellular Signaling Peptides and Proteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Plasma Membrane Neurotransmitter Transport Proteins
  • Protein Kinase Inhibitors
  • Ribonucleotides
  • creatine transporter
  • Slc6a8 protein, rat
  • Aminoimidazole Carboxamide
  • Sodium
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • mTOR protein, mouse
  • mTOR protein, rat
  • Protein-Serine-Threonine Kinases
  • AMP-Activated Protein Kinases
  • AICA ribonucleotide
  • Creatine
  • Sirolimus