Upregulation of the Creatine Transporter Slc6A8 by Klotho

Klotho is expressed mainly in kidney and choroid plexus [1,2]. The extracellular domain of the Klotho protein may function as enzyme with β-glucuronidase activity and, after being cleaved off, as hormone [3,4,5,6,7]. Klotho expression is a decisive determinant of ageing and life span [8,9]. As shown in mice, Klotho deficiency leads to severe growth retardation and accelerates the appearance of several age related disorders resulting in shortening the life span to less than 5 months [8]. Conversely, Klotho overexpression extends substantially the life span [8,9].

Klotho is required for the inhibitory effect of FGF23 on 1α-hydroxylase and thus 1,25(OH)₂D₃ formation [2,9,10,11]. 1,25(OH)₂D₃ stimulates Klotho expression [12] as well as intestinal and renal Ca²⁺ and phosphate transport [13,14]. Lack of Klotho in Klotho-deficient mice [2,10,11] leads to excessive 1,25(OH)₂D₃ formation with increase of plasma Ca²⁺[15] and phosphate [14] concentration, vascular calcification [16] and growth deficit [2]. In addition to its influence on 1,25(OH)₂D₃ formation [2,9,10,11,17,18] and thus 1,25(OH)₂D₃ dependent regulation of Ca²⁺ and phosphate transport Klotho influences more directly Ca²⁺ channels [19] and Na⁺, phosphate cotransport [4,20]. Moreover, Klotho up-regulates the Na⁺/K⁺ ATPase [21], renal outer medullary K⁺ channels ROMK [22] voltage gated channels, KCNQ1/KCNE1 [23] and excitatory amino acid transporters EAAT 3 and EAAT4 [24]. Thus, Klotho exerts some of its effects by regulating transport mechanisms in the cell membrane.

Carriers expressed in both, brain and kidney include the widely expressed creatine transporter CreaT (Slc6A8) [25,26,27,28], a member of the Na⁺,Cl^- coupled transporter superfamily for neurotransmitters (e.g. dopamine, GABA, serotonin and norepinephrine), amino acids (e.g. glycine) [29,30,31] as well as the organic osmolytes betaine [32] and taurine [33]. Slc6A8 activity is regulated by AMP activated kinase [34], cyclosporine A [35], mTOR [36], serum and glucocorticoid inducible kinase isoforms [37], PIKfyve [38], as well as Src [39], and its expression is increased by growth hormone [40]. Slc6A8 is further regulated by extracellular and cytosolic creatine levels [41,42].

The present study explored whether Klotho participates in the regulation of creatine transporter CreaT (Slc6A8). To this end, cRNA encoding Slc6A8 was injected into Xenopus oocytes either without or with additional injection of cRNA encoding Klotho. Moreover, Slc6A8 and Klotho expressing Xenopus oocytes were treated with β-glucuronidase inhibitor DSAL (10 μM). To estimate creatine transporter CreaT (Slc6A8) activity, the creatine induced current was determined utilizing dual electrode voltage clamp.

Murine full-length Klotho was subcloned from pCR-XL-TOPO vector (Imagenes, Berlin, Germany) into pSGEM, a Xenopus oocyte expression vector using XhoI - SpeI restriction sites. For generation of cRNA [43,44] constructs were used encoding Klotho [20] and CreaT (Slc6A8) [30,45]. The constructs were used for the generation of cRNA as described previously [46,47,48].

Xenopus oocytes were prepared as previously described [49,50]. Xenopus oocytes were injected with DEPC-water and 15 ng cRNA encoding Slc6A8 with or without additional co-injection of 10 ng cRNA encoding Klotho protein on the same day of preparation of the Xenopus oocytes. The oocytes were maintained at 17 °C in ND96 solution containing: 88.5.mM NaCl, 2mM KCl, 1mM MgC1₂, 1.8mM CaC1₂, 5mM HEPES. Tretracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Refobacin (100 mg/l) and Theophylin (90 mg/l) as well as sodium pyruvate (5mM) were added to the ND96, and the pH was adjusted to 7.5 by addition of NaOH. The control superfusate (ND96) contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂ and 5 mM HEPES. The pH was adjusted to 7.4 by addition of NaOH [51,52]. Where indicated, Brefeldin A (5µM, Sigma), recombinant human alpha Klotho protein (30ng/ml, 5334-KL-R&D Systems) and D-saccharic acid 1,4-lactone monohydrate (DSAL, 10µM, Sigma) were added. All experiments were performed at room temperature (about 22°C) 3-5 days after injection. Pipettes were filled with 3 M KCl and had resistances of 0.3-3.0 MΩ. The substrate (creatine) was added to the solutions at a concentration of 2 mM unless otherwise stated. The flow rate of the superfusion was approximately 20 ml/min, and a complete exchange of the solution in the recording bath was reached within about 10 sec. Where indicated, brefeldin A at the concentration of 5µM was added to the solutions in order to test for alterations of CreaT protein stability in the plasma membrane. Brefeldin A is involved in plasma membrane trafficking and the treatment of Xenopus oocytes with brefeldin A prevents further insertion of new carrier protein into the plasma membrane through the vesicular pathway. Therefore, the consequent decay of carrier activity could be taken as a measure of carrier protein clearance from the plasma membrane. Two-electrode voltage-clamp recordings were performed as described previously [53,54] at a holding potential of -60 mV. The data were filtered at 10 Hz and recorded with a GeneClamp 500 amplifier, a DigiData 1300 A/D-D/A converter and the pClamp 9.2 software packages for data acquisition and analysis (Axon Instruments, Foster City, CA, USA) [53,55]. The offset potentials between both electrodes were zeroed before sealing. The applied voltages refer to the cytoplasmic face of the membrane with respect to the extracellular space. The outward currents, defined as flow of positive charge from the cytoplasmic to the extracellular membrane face, are positive currents and depicted as upward deflections of the original current traces.

Data are provided as means ± SEM, n represents the number of experiments. All oocyte experiments were repeated with at least 2 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using ANOVA, as appropriate, and results with p < 0.05 were considered statistically significant.

The present study explored whether Klotho protein participates in the regulation of the Na⁺-Cl^-coupled creatine transporter CreaT (Slc6A8). To this end, cRNA encoding Slc6A8 was injected into Xenopus oocytes with or without additional injection of cRNA encoding Klotho protein. The Slc6A8 activity was estimated from the creatine induced current generated by electrogenic creatine transport. As shown in Fig. 1, creatine did not generate an appreciable current in water-injected Xenopus oocytes or in oocytes expressing Klotho protein alone, indicating that Xenopus oocytes do not express appreciable endogenous electrogenic creatine transporters. In contrast, creatine induced a large current in Xenopus oocytes expressing Slc6A8. The additional coexpression of Klotho was followed by a significant increase of the creatine induced current (Fig. 1).

In order to elucidate whether Klotho protein coexpression modifies the maximal creatine induced current or the affinity of the carrier, Xenopus oocytes expressing Slc6A8 without or with additional expression of Klotho protein were exposed to different concentrations of creatine for kinetic analysis. As shown in Fig. 2, the creatine induced current was a function of the extracellular creatine concentration. The maximal creatine induced current was higher in Xenopus oocytes expressing Slc6A8 together with Klotho (29.5 ± 3.5 nA, n = 8) than in Xenopus oocytes expressing Slc6A8 alone (20.2 ± 3.7 nA, n = 10). The concentration required for halfmaximal creatine induced current was not significantly different between Xenopus oocytes expressing Slc6A8 together with Klotho (37.4 ± 9.0 µM, n = 10) and Xenopus oocytes expressing Slc6A8 alone (34.4 ± 8.3 µM, n = 8). The increased maximal transport rate pointed to an increase of creatine transporter CreaT abundance in the plasma membrane of Scl6A8 expressing Xenopus oocytes following coexpression of Klotho.

In theory, Klotho could modify carrier protein abundance in the cell membrane by either, accelerating insertion of carrier protein into the cell membrane or delaying retrieval of carrier protein from the cell membrane. In order to discriminate between those two possibilities, Xenopus oocytes expressing either both, Slc6A8 and Klotho, or Slc6A8 alone, were treated with brefeldin A (5 µM), an inhibitor of protein insertion into the cell membrane. As shown in Fig. 3, brefeldin A treatment was followed by a decay of the current in both groups, an effect, which was faster in Xenopus oocytes expressing Slc6A8 alone than in Xenopus oocytes expressing both Slc6A8 and Klotho. The ratio of the decay of creatine induced current in Slc6A8 and Klotho expressing Xenopus oocytes over the current in Xenopus oocytes expressing Slc6A8 alone, increased from 0.96 ± 0.40 (n = 11) in the absence of brefeldin A to 2.01 ± 0.57 (n = 11) following a 12 hours treatment with brefeldin A. The observations disclose an effect of klotho in the absence of carrier insertion and thus strongly suggest that the effect of klotho is secondary to stabilization of carrier protein within the cell membrane. A further series of experiments elucidated whether the β-glucuronidase activity of Klotho is required for its effect on Slc6A8 activity. To this end, Xenopus oocytes expressing both, Slc6A8 and Klotho were treated with β-glucuronidase inhibitor DSAL (10 μM) for 48 h in order to suppress β-glucuronidase activity of expressed klotho. As shown in Fig. 4A, the treatment of Slc6A8 and Klotho-expressing Xenopus oocytes with DSAL reversed the effect of Klotho coexpression on the creatine induced inward current.

Additional experiments were performed to test, whether the effect of Klotho coexpression on creatine induced current was mimicked by treatment of Slc6A8-expressing Xenopus oocytes with recombinant human alpha Klotho protein (30 ng/ml). To possibly disclose a stabilizing effect of klotho on the carrier protein in the cell membrane, the oocytes were exposed for 24 h or 48 h to klotho protein. As shown in Fig. 4B, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein resulted in a significant increase of creatine induced inward current.

The present observations disclose a novel function of Klotho, i.e. the up-regulation of the creatine transporter CreaT (Slc6A8). Klotho increases the maximal transport rate of the carrier presumably by stabilizing the carrier protein in the cell membrane. The Klotho concentration required was within the range of concetrantions encountered in vivo [56]. The effect of Klotho was reversed in the presence of the β-glucuronidase inhibitor DSAL added from the beginning of Klotho expression in order to continuously suppress β-glucuronidase activity of expressed Klotho. The reversal of the effect of Klotho indicates that the effect of Klotho on Slc6A8 indeed requires β-glucuronidase activity of Klotho. Previous studies suggested that Klotho may regulate ion channels by removing terminal sialic acids [5,6].

Klotho further up-regulates the Na⁺/K⁺ATPase [21], which maintains the electrochemical gradient for Na⁺ coupled transport [57]. Thus, Klotho could modify Na⁺ coupled creatine transport in part by up-regulation of Na⁺,K⁺ ATPase activity. The Na⁺/K⁺ ATPase further maintains the intracellular K⁺ concentration, which is in turn decisive for cell survival [58,59,60,61,62].

Slc6A8 is critically important for proper function of the brain, as genetic defects of Slc6A8 lead to mental retardation with seizures [63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80]. Slc6A8 deficient mice suffer from learning and memory deficits resembling human Slc6A8 deficiency [63]. Consequences of Klotho deficiency include degeneration of mesencephalic dopaminergic neurons [81], an effect, however, reversed by vitamin D restriction [81] and thus presumably not due to direct influence of Klotho on Slc6A8. Klotho deficiency further leads to cognitive deficits [82]. Klotho is required for proper maturation of rat primary oligodendrocytic progenitor cells [82] and Klotho deficiency is paralleled by lack of oligodendrocytes with decreased abundance of major myelin protein [82]. Downregulation of Klotho in the aged brain may contribute to a decrease of white matter and myelin abnormalities during cerebral ageing [83]. To which extent compromised up-regulation of Slc6A8 by Klotho contributes to the deterioration of cerebral function in Klotho deficiency or in the aging brain, remains to be shown. Clearly, additional studies will be required to define the in vivo relevance of Klotho sensitive creatine transport.

Klotho confers cardioprotection [84], an effect in large part due to down-regulation of TRPC6 channels. It is noteworthy, however, that Slc6A8 deficiency may impair cardiac function [85] and that decreased Slc6A8 abundance is observed in the failing heart [86]. Thus, at least in theory, up-regulation of Slc6A8 could contribute to cardioprotection.

The present study demonstrates that Klotho is a powerful stimulator of the creatine transporter CreaT (Slc6A8). Klotho dependent regulation of Slc6A8 may contribute to neurodegeneration and cardiac failure in Klotho deficiency.

The authors of this manuscript state that they do not have any conflict of interests and nothing to disclose.

The authors acknowledge the technical assistance of E. Faber. The manuscript was meticulously prepared by Tanja Loch and Ali Soleimanpour. This study was supported by the Deutsche Forschungsgemeinschaft (GK 1302) and the Open Access Publishing Fund of Tuebingen University.

A. Almilaji and M. Sopjani contributed equally to this manuscript and thus share first authorship