Klotho-Dependent Cellular Transport Regulation.

Klotho is a transmembrane protein that in humans is encoded by the hKL gene. This protein is known to have aging suppressor effects and is predominantly expressed in the distal convoluted tubule of the kidney, parathyroid glands, and choroid plexus of the brain. The Klotho protein exists in both full-length membrane form and a soluble secreted form, which exerts numerous distinct functions. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it functions as ß-glucuronidase and a hormone. Soluble Klotho is a multifunction protein present in the biological fluids including blood, urine, and cerebrospinal fluid of mammals. Klotho deficiency leads to multiple organ failure accompanied by early appearance of multiple age-related disorders and early death, whereas overexpression of Klotho results in the opposite effects. Klotho, an enzyme and hormone, has been reported to participate in the regulation of cellular transport processes across the plasma membrane either indirectly through inhibiting calcitriol (1,25(OH)2D3) formation or other mechanism, or by directly affecting transporter proteins, including ion channels, cellular carriers, and Na(+)/K(+)-ATPase. Accordingly, Klotho protein serves as a powerful regulator of cellular transport across the plasma membrane. Importantly, Klotho-dependent cellular transport regulation implies stimulatory or inhibitory effects. Klotho has been shown to play a key role in the regulation of multiple calcium and potassium ion channels, and various cellular carriers including the Na(+)-coupled cotransporters such as NaPi-IIa, NaPi-IIb, EAAT3, and EAAT4, CreaT1 as well as Na(+)/K(+)-ATPase. These regulations are parts of the antiaging function of Klotho, which will be discussing throughout this chapter. Clearly, further experimental efforts are required to investigate the effect of Klotho on other transport proteins and underlying molecular mechanisms by which Klotho exerts its effect.

Intracellular signaling of the aging suppressor protein Klotho.

The Klotho protein deficiency is known to participate in premature aging. As an aging suppressor, Klotho is an important molecule in aging processes and its overexpression results in longevity. Due to many reasons, the insulin/insulin-like growth factor-1 (IGF-1) has been considered as a key pathway in aging research. The Klotho gene is closely related to this pathway. The Klotho gene encodes a transmembrane protein that after cleavage is also found as a secreted protein. Importantly, its overexpression suppresses insulin/IGF-1 signaling and thus extends the lifespan. In addition, Klotho participates in the regulation of several other intracellular signaling pathways, including regulation of FGF23 signaling, cAMP, PKC, transforming growth factor-ß (TGF-ß), p53/p21, and Wnt signaling. The aim of this review is to summarize current literature that shows the involvement of Klotho in the regulation of several intracellular pathways. The results of our review clearly indicate that Klotho participates in several intracellular signaling pathways, and by regulating them, Klotho is involved in aging and longevity.

Akt2/PKBbeta-sensitive regulation of renal phosphate transport.

AIM: The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. METHODS: The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na(+) phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKBbeta knockout mice (akt2(-/-)) and corresponding wild-type mice (akt2(+/+)). Transporter protein abundance was determined using Western blotting and phosphate transport by (32)P uptake into brush border membrane vesicles. RESULTS: The phosphate-induced current in NaPi-IIa-expressing Xenopus oocytes was significantly increased by the coexpression of Akt/PKB. Phosphate excretion [micromol per 24 h per g BW] was higher by 91% in akt2(-/-) than in akt2(+/+) mice. The phosphaturia of akt2(-/-) mice occurred despite normal transport activity and expression of the renal phosphate transporters NaPi-IIa, NaPi-IIc and Pit2 in the brush border membrane, a significantly decreased plasma PTH concentration (by 46%) and a significantly enhanced plasma 1,25-dihydroxyvitamin D(3) concentration (by 46%). Moreover, fractional renal Ca(2+) excretion was significantly enhanced (by 53%) and bone density significantly reduced (by 11%) in akt2(-/-) mice. CONCLUSIONS: Akt2/PKBbeta plays a role in the acute regulation of renal phosphate transport and thus contributes to the maintenance of phosphate balance and adequate mineralization of bone.

Triggering of suicidal erythrocyte death by radiocontrast agents.

BACKGROUND: According to in vitro observations, gadolinium-containing magnetic resonance (MRT) contrast agents stimulate suicidal cell death or apoptosis. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the erythrocyte surface. Eryptosis is triggered by increased cytosolic Ca2+-activity. This study explored whether gadolinium-containing MRT contrast agents stimulate eryptosis. MATERIALS AND METHODS: Annexin V-binding reflecting PS exposure and forward scatter reflecting cell volume were determined in erythrocytes within freshly drawn blood from patients (8female symbol, 3male symbol, 29-72 years) prior to and 10 min after administration of gadoterate meglumine (0.1 mmol kg(-1) b.w. Dotarem; six patients) or gadobenate dimeglumine (0.05 mmol kg(-1) bw Multi Hance; five patients). In a separate series, eryptosis was determined prior to and following in vitro incubation of erythrocytes from 16 blood donors for 4 h with gadoterate meglumine (5 mM Dotarem) or gadobenate dimeglumine (5 mM Multi Hance). Finally, eryptosis and Fluo3 fluorescence reflecting cytosolic Ca2+ were determined in vitro following exposure to Gd3+. Data were analysed using paired t-test or anova with Tukey's test as post-test. RESULTS: The MRT contrast agents such as gadoterate meglumine (Dotarem) and gadobenate dimeglumine (Multi Hance) significantly increased the percentage of eryptotic cells. Moreover, in vitro exposure to gadoterate meglumine (5 mM), gadobenate dimeglumine (5 mM) or Gd3+ (1.9 microM) stimulated eryptosis in vitro. The effect of Gd3+ was paralleled by increase in cytosolic Ca2+-activity. CONCLUSIONS: MRT contrast agents may stimulate suicidal erythrocyte death or eryptosis in vitro and in vivo.