β-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2 Open Access

Klotho is a transmembrane protein expressed in several tissues with particular strong expression in kidney and choroid plexus [1, 2]. The extracellular domain of the Klotho protein with β-glucuronidase activity may be cleaved off and released into blood and cerebrospinal fluid [3, 4, 5, 6, 7]. Klotho has a powerful impact on ageing [8, 9]. Klotho deficiency results in severe growth retardation, accelerated appearance of several age related disorders, and dramatic shortening of the life span [9]. Conversely, klotho over-expression extends the life span [9, 10].

Klotho is required for the inhibitory effect of FGF23 on 1α-hydroxylase and thus 1,25(OH)₂D₃ formation [2, 8, 11, 12, 13, 14]. Effects of 1,25(OH)₂D₃ include stimulation of klotho expression [15] as well as intestinal and renal Ca²⁺ and phosphate absorption [16, 17]. Klotho deficiency is followed by excessive 1,25(OH)₂D₃ formation [2, 13, 14] leading to increase of plasma Ca²⁺[18] and phosphate [17] concentrations as well as vascular calcification [19]. Besides its effect on 1,25(OH)₂D₃ formation, klotho modifies the function of several transport proteins including Ca²⁺ channels [20], Na⁺,phosphate cotransport [4, 21], Na⁺/K⁺ ATPase [22], renal outer medullary K⁺ channels [23], Kv1.3 K⁺ channels [24], KCNQ1/KCNE1 [25], the creatine transporter CreaT [26], and the excitatory amino acid transporters EAAT1, EAAT2, EAAT3 and EAAT4 [27, 28].

Carriers impacting on ageing include the peptide transporter PEPT1 [32]. Intestinal peptide transport is a major source of nutritional amino acids and is thus decisive for growth and development as well as signaling regulating metabolism and ageing [32]. The possibility was thus considered that klotho may influence peptide transport which in turn may affect ageing.

The present study thus explored, whether Klotho impacts on the function of the peptide transporters 1 (PEPT1) and/or 2 (PEPT2), which accomplish electrogenic cellular uptake of di- and tripeptides [29, 30, 31] including peptide-like drugs [30, 31].

All experiments conform with the 'European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes' (Council of Europe No 123, Strasbourg 1985) and were conducted according to the German law for the welfare of animals. The surgical procedures on the adult Xenopus laevis frogs were reviewed and approved by the respective government authority of the state Baden-Württemberg (Regierungspräsidium) prior to the start of the study (Anzeige für Organentnahme nach §36).

Constructs encoding rabbit wild-type PEPT1 and PEPT2 [33, 34], were used for generation of cRNA as described previously [35, 36].

Xenopus oocytes were prepared as previously described [37, 38]. Ten ng cRNA encoding PEPT1 or Twenty ng cRNA encoding PEPT2 were injected on the same day after preparation of the oocytes. The oocytes were maintained at 17°C in ND96-A, a solution containing (in mM): 88.5 NaCl, 2 KCl, 1 MgC1₂, 1.8 CaC1₂, 2.5 NaOH, 5 HEPES (pH 7.4), 5 sodium pyruvate (C₃H₃NaO₃), Gentamycin (100 mg/l), Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), and Theophiline (90 mg/l) [39, 40]. Where indicated, 30 ng/ml ß-Klotho protein and/or 10 µM β-glucuronidase inhibitor DSAL were added to the respective solutions. The voltage clamp experiments were performed at room temperature 3 days after the first injection of cRNA encoding PEPT1 and 4 days after the injection of PEPT2 [41, 42]. Glycine-glycine induced currents were taken as a measure of glycine-glycine transport [43, 44]. The holding potential was -70mV. The data were filtered at 10 Hz and recorded with a Digidata A/D-D/A converter (1322A Axon Instruments) and Clampex 9.2 software for data acquisition and analysis (Axon Instruments) [33, 45]. The control superfusate (ND96-B) contained (in mM): 93.5 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl_2, 2.5 NaOH and 5 HEPES (pH 7.4). The flow rate of the superfusion was approx. 20 ml/min, and a complete exchange of the bath solution was reached within about 10 s [46, 47]. For kinetic analysis the glycine-glycine induced-current (I_Glu) was plotted against the respective glycine-glycine concentration (s) and maximal current (I_max) as well as concentration required for half maximal current (k_m) calculated using the equation I_Glu= I_max∙s/(k_m+ s).

All animal experiments were conducted according to the German law for the welfare of animals and according to the guidelines of the American Physiological Society and were approved by local authorities (Regierungspräsidium Tübingen). Experiments were performed using jejunal segments from 6-week old female klotho-deficient mice (kl^-/-) and wild-type mice. The origin of the mice, breeding and genotyping were described previously [9]. Prior to the experiments mice had access to food (1314, Altromin, Heidenau, Germany) and water ad libitum, and were kept under constant humidity (55 ± 10%), temperature (22 ± 2°C) and 12h light-dark cycle conditions. Mice were fasted for 6 hours prior to experiments. Peptide transport rate was estimated from glycine-glycine or from glycine-Sar induced current across the intestinal epithelium. After removing the outer serosal and the muscular layer of jejunum under a microscope, tissues were mounted onto a custom-made mini-Ussing chamber with an opening diameter of 0.99 mm and an opening area of 0.00769 cm². Transepithelial potential difference (V_te) was determined continuously and transepithelial resistance (R_te) estimated from the voltage deflections (ΔV_te) elicited by imposing rectangular test currents of 1 µA and 1.2 s duration at a rate of 8/min. R_te was calculated according to Ohm's law [48]. The serosal and luminal perfusate contained (in mM): 145 NaCl, 1 MgCl₂, 2.6 Ca-gluconate, 0.4 KH₂PO₄, 1.6 K₂HPO₄, 5 glucose. To assess peptide transport, glycine-glycine (10 mM) or glycine-Sar (10 mM) was added to the luminal perfusate.

Data are provided as means ± SEM, n represents the number of replicates. All voltage clamp experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. Data were tested for significance using ANOVA or Student's t-test, as appropriate. Results with p < 0.05 were considered statistically significant.

The present study explored whether soluble recombinant β-Klotho protein modifies the electrogenic glycine-glycine transport by the peptide transporters PEPT1 and PEPT2. To this end, PEPT1 or PEPT2 expressing Xenopus laevis oocytes were left untreated or were treated with β-Klotho protein (30 ng/ml) and glycine-glycine-induced inward current (I_Gly) measured by dual electrode voltage clamp taken as a measure of electrogenic glycine-glycine transport.

As illustrated in Fig. 1, I_Gly was negligible in water-injected oocytes indicating that the oocytes did not express significant endogenous electrogenic glycine-glycine transport. In contrast, glycine-glycine (2 mM) triggered a sizable I_Gly in PEPT1 expressing Xenopus laevis oocytes. The treatment of PEPT1 expressing oocytes with β-Klotho protein (30 ng/ml) was followed by a significant decrease of I_Gly.

In order to test whether β-Klotho protein modifies the maximal I_Gly and/or the affinity of PEPT1, untreated or β-Klotho protein (30 ng/ml) treated PEPT1 expressing Xenopus laevis oocytes were exposed to glycine-glycine concentrations ranging from 10 µM to 5000 µM. As illustrated in Fig. 2, I_Gly was a function of the extracellular glycine-glycine concentration. Maximal I_Gly was significantly (p<0.05) lower in β-Klotho protein treated (44.3 ± 3.8 nA, n = 9) than in untreated (62.4 ± 6.8 nA, n = 11) PEPT1 expressing Xenopus laevis oocytes. The concentration required for half maximal I_Gly (apparent K_m) tended to be lower in β-Klotho protein treated (367.9 ± 40.1 µM, n = 9) than in untreated (271.9 ± 79.9 µM, n = 11) PEPT1 expressing Xenopus laevis oocytes, a difference, however, not reaching statistical significance.

A further series of experiments explored whether the effect of β-Klotho protein is related to its β-glucuronidase activity. To this end PEPT1 expressing Xenopus laevis oocytes were treated with β-Klotho protein in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). As illustrated in Fig. 3, the effect of β-Klotho protein on electrogenic glycine-glycine transport in PEPT1 expressing Xenopus laevis oocytes was virtually abolished by DSAL (10 µM).

An additional series of experiments explored whether klotho was similarly effective in PEPT2 expressing Xenopus laevis oocytes. As illustrated in Fig. 4, glycine-glycine (2 mM) triggered a sizable I_Gly in PEPT2 expressing Xenopus laevis oocytes. Treatment with β-Klotho protein (30 ng/ml) of PEPT2 expressing oocytes was followed by a significant decrease of I_Gly.

In order to test whether klotho sensitivity plays a role in vivo, glycine-glycine (10 mM)-induced current was measured in intestinal epithelia utilizing Ussing chambers. As illustrated in Fig. 5, the intestinal glycine-glycine induced current (I_Gly) was significantly larger in klotho deficient (kl^-/-) mice than in wild-type mice (kl^+/+). Similar observations were made with the peptide Gly-Sar (Fig. 6)

The present study uncovers a novel function of β-Klotho protein, i.e. the down-regulation of the peptide transporters PEPT1 and PEPT2. Treatment of either, PEPT1 and PEPT2 expressing oocytes with human recombinant β-Klotho significantly decreased the glycine-glycine-induced inward current (I_Gy). Kinetic analysis revealed that β-Klotho was in large part effective by decreasing the maximal transport rate of the carriers. The large scatter of the calculated concentrations required for half maximal current precludes safe conclusions about effects of klotho on carrier affinity. The effect of β-Klotho on I_Gly was reversed in the presence of the β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate DSAL. Thus, β-klotho was apparently effective as β-glucuronidase. Klotho belongs to the β-glycosidase family [49]. Klotho has previously been shown to hydrolyze extracellular sugar residues of target membrane proteins [49]. As shown for the epithelial Ca²⁺ channel TRPV5 [49] and the excitatory amino acid transporters [28], klotho may stabilize the transport proteins in the cell membrane. In contrast to its effect on TRPV5 and excitatory amino acid transporters, klotho down-regulates the peptide carriers. The present observations do not rule out that klotho modifies peptide transport in PEPT1 or PEPT2 expressing oocytes by mechanisms other than β -glucuronidase activity and other than destabilization of the carrier proteins in the cell membrane. Whatever mechanism involved, the effect of klotho obviously affects intestinal peptide transport. Presumably due to lacking down-regulation by klotho of peptide transporters, the peptide transport was markedly enhanced in klotho deficient mice. We can, however, not rule out the involvement of more indirect mechanisms modifying peptide transport in klotho deficient mice.

In view of the impact of peptide transporters on ageing [32], it is tempting to speculate that the down-regulation of intestinal peptide transport contributes to the impact of klotho on ageing. Even though up-regulation of peptide transporters is expected to accelerate ageing [32], the dramatic acceleration of ageing in klotho deficient mice is, however, obviously in large part the result of deranged mineral metabolism and tissue calcification [19].

In conclusion, β-Klotho down-regulates the activity of the peptide transporters PEPT1 and PEPT2. The effect apparently modifies intestinal peptide transport and may contribute a small part to the impact of klotho on ageing.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic and technical support by Elfriede Faber. This study was supported by the Deutsche Forschungsgemeinschaft, GRK 1302, SFB 773 B4/A1, La 315/13-3, Open Access Publishing Fund of Tuebingen University and the EMBO Long Term Postdoctoral fellowship (ATLF 20-2013 to M.S.S).

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

A. Abousaab and J. Warsi contributed equally thus shared the first authorship.