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

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 ϩ -dependent creatine uptake into CRTexpressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten Vmax 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-␤-Dribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-␤-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. proximal tubule; metabolism; Xenopus oocytes; target of rapamycin; SLC6A8 AMP-ACTIVATED PROTEIN KINASE (AMPK) is a ubiquitous metabolic-sensing kinase that exists as an ␣,␤,␥ heterotrimer and is activated by cellular energy depletion (elevated ratios of intracellular AMP to ATP concentration) and other cellular stresses (e.g., Ca 2ϩ stress). AMPK activation involves phosphorylation of the catalytic ␣-subunit at Thr 172 (pThr 172 ) by upstream AMPK kinases that include the LKB1 complex and the Ca 2ϩ

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria

“…In contrast, CRT1 was reported to be a brush border protein. 12 Our findings of basolateral MCT12 localization in the kidney are consistent with the basolateral localization reported for CD147, which is an accessory protein for several monocarboxylate transporters of the SLC16 family transporters, including MCT12. 3 Individuals with MCT12 mutation displayed unaltered fractional excretion of creatine, but increased fractional excretion of guanidinoacetate.…”

Creatine: a miserable life without it

“…In proximal kidney tubules, however, activation of AMPK inhibits creatine transporter activity, by down-regulation of the transport and/or insertion of the CrT protein into the apical membrane (Li et al 2010). This down-regulation of creatine transport could be important for reducing cellular energy expenditure for creatine reabsorption by the proximal tubules.…”

“…This down-regulation of creatine transport could be important for reducing cellular energy expenditure for creatine reabsorption by the proximal tubules. Together with absorbed sodium, which would increase the energetic load on the Na + /K + -ATPase, this would be unfavorable under conditions of local and whole-body metabolic stress (Li et al 2010). Curiously, it is still not known whether AMPK, in fact, interacts directly with and phosphorylates CrT, or whether these effects are indirect via another protein kinase in the signaling cascade.…”

Hypoxia decreases creatine uptake in cardiomyocytes, while creatine supplementation enhances HIF activation

“…2011) and others (Li et al. 2010) have demonstrated that activation of AMP‐activated kinase (AMPK), the cell's energy master regulator (Hardie et al. 2012), modulates Cr transport in a tissue‐specific manner.…”