STIMULATION OF RAT TYPE IIA SODIUM DEPENDENT PHOSPHATE COTRANSPORTER (NAPI-2) BY SERUM AND GLUCOCORTICOID-INDUCED KINASE SGK1 AND NA+/H+ EXCHANGE REGULATING FACTOR NHERF2

STIMULATION OF RAT TYPE IIA SODIUM DEPENDENT PHOSPHATE COTRANSPORTER (NAPI-2) BY SERUM AND GLUCOCORTICOID-INDUCED KINASE SGK1 AND NA⁺/H⁺ EXCHANGE REGULATING FACTOR NHERF2

HAMDY M. EMBARK

Animal Physiology Department, Faculty of Vet. Medicine, South Valley University, Qena 83523, Egypt, Phone: +2 011/00657030,   Fax: +2 096/5211223.

Email: h.embark@vet.svu.edu.eg; atutohm@yahoo.com                                   Assiut University web-site: www.aun.edu.eg

INTRODUCTION

The maintenance of Pi homeostasis is a critical event to the appropriate growth and well-being of both young and adult animals (Adedokun and Adeola, 2013), because it is necessary for the development, maintenance, and repair of bone and tissues (Mulroney et al., 2004). The major regulation of Pi homeostasis occurs at the kidney (Tenenhouse and Murer, 2003). The homeostasis of Pi is maintained via renal glomerular filtration followed by tightly controlled tubular reabsorption (Prasad and Bhadauria, 2013). The renal tubular reabsorption of phosphate requires cellular phosphate uptake across the brush border and exit at the basolateral cell membrane (Biber and Murer, 1994). One of the key components involved is sodium dependent phosphate cotransporter system localized in the brush border membrane of the tubular epithelium which mediates the uptake of Pi against the electrochemical gradient from primary urine into the cell (Forster et al., 1997).

The sodium dependent phosphate cotransporter systems mediating the transport have been classified in three different types according to both molecular and functional characteristics (Albano et al., 2015). Type I cotransporter (NaPi-1) induces an anion channel (Yanagawa et al., 1999), type IIa is the renal brush border sodium dependent phosphate cotransporter and type IIb is found apically in many tissues including the small intestine and the lung, but not the kidney (Murer et al., 2000). Type III cotransporters are being found every where including renal tubules (Werner and Kinne, 2001).

The isoforms of the type II sodium dependent phosphate cotransporters from several mammalian species have been cloned such as the rat (NaPi-2) and human (NaPi-3) (Magagnin et al., 1993), opossum kidney (NaPi-4) (Sorribas et al., 1994), flounder (NaPi-5) (Forster et al., 1997), rabbit (NaPi-6) (Verri et al., 1995) and murine (NaPi-7) (Hartmann et al., 1995). The type II cotransporters in the renal brush border are rate limiting for renal tubular phosphate reabsorption in the respective species (Murer, 1992).

The type II sodium dependent phosphate cotransporters are tightly regulated by parathyroid hormone (PTH) (Traebert et al., 2000) which inhibits proximal tubular phosphate reabsorption by stimulation of NaPi-internalization and degradation (Pfister et al., 1998). Further hormones regulating renal tubular phosphate transport include insulin (Li et al., 1996) and growth hormone (Costanzo et al., 1974), which mediates its effect through IGF-1 (Hammerman et al., 1980). Insulin (Bandyopadhyay et al., 1999) and insulin like growth factor IGF-1 (Vanhaesbroeckand Alessi, 2000) are both known to stimulate PI3 kinase, which has previously been demonstrated to enhance insertion of NaPi into the cell membrane (Pfister et al., 1999). Downstream targets of PI3 kinase include phosphoinositol dependent kinase 1 (PDK1) (Park et al., 1999), which activates the protein kinase B (PKB) (Vanhaesbroeck and Alessi, 2000) and Serum and glucocorticoid-inducible kinase SGK1 (Kobayashi and Cohen, 1999).

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins (Yun et al., 2002). The C-terminal tail of NaPi-2 contains a PDZ binding motif, which may bind to the PDZ domains of NHE regulating factors NHERF1 or NHERF2. Both, NHERF1 and NHERF2 are expressed in the proximal renal tubule (Shenolikar and Weinman, 2001). Moreover, it has been shown that NHERF1 bind to the NaPi-2 and play a role in the apical expression of these cotransporters (Hernando et al., 2002). Thus, the possibility was considered that NHERF2 also participates in the regulation of NaPi-2 activity.

Recent experiments disclosed a role of serum and glucocorticoid dependent kinase SGK1 in the interaction of NHE3 with NHERF2 (Yun, 2003; Yang et al., 2014). SGK1 has originally been cloned as a glucocorticoid sensitive gene from rat mammary tumor cells (Firestone et al., 2003). The human isoform has been identified as a cell volume regulated gene (Waldegger et al., 1997). Subsequent studies revealed the genomic regulation of SGK1 by aldosterone (Cowling and Birnboim, 2000), 1,25 (OH)₂D₃ (Akutsu et al., 2001), TGFß (Waldegger et al., 1999) and a variety of further cytokines (Lang and Cohen, 2001).

SGK1 is expressed in a wide variety of human epithelial tissues including intestine, kidney and placenta (Waldegger et al., 1997; Wallace et al., 2011). Thus, they may participate in the regulation of transport in those tissues. Similarly NaPi-2 and NHERF2, SGK1 is expressed in the rat proximal renal tubule and OK cells (Fuster et al., 2007) and may well participate in the regulation of NaPi-2.

The present experiments have been performed to explore whether rat type IIa sodium dependent phosphate cotransporter (NaPi-2) is regulated by SGK1 and NHERF2. To this end, cRNA encoding wild-type rat NaPi-2 has been injected with or without cRNA encoding NHERF2 and/or wild-type SGK1 into Xenopusleavis oocytes.

MATERIALS and METHODS

cRNA synthesis

cRNA encoding the wild-type rat sodium dependent phosphate cotransporter NaPi-2 (Werner et al., 1991), the Serum and glucocorticoid-inducible kinase SGK1 (Kobayashi et al., 1999), and the Na⁺/H⁺ exchange regulating factor NHERF2 (Yun et al., 2002) were synthesized in vitro as described previously (Busch et al., 1995; Forster et al., 1997; Wagner et al., 2000).

Expression in Xenopus laevis oocytes

Dissections of Xenopuslaevis ovaries, collection, and handling of the oocytes were done as described by Wagner et al., 2000. Oocytes were injected with 7.5 ng SGK1 and/or 5ng NHERF2 cRNA on the first day after preparation of the oocytes. On the second day after preparation 1ng wild-type rat NaPi-2 was injected. Control oocytes were injected with H₂O. All steps were performed at room temperature 3-8 days after injection of the respective cRNAs.

Voltage-clamp analysis

As shown in Fig. 1, two electrode voltage clamp recordings were performed at a holding potential of -50 mV. The data were filtered at 10 Hz and recorded with MacLab digital to analog converter and software for data acquisition and analysis (AD Instruments, Castle Hill, Australia). The control bath solution (ND96) contained 96 mMNaCl, 2 mMKCl, 1.8 mM CaCl₂, 1 mM MgCl₂ and 5 mM HEPES, pH 7.4. Where indicated, phosphate was added at 1 mmol/l. The final solutions were titrated to the pH 7.4 using HCl or KOH. The flow rate of the superfusion was 20 ml/min and a complete exchange of the bath solution was reached within about 10 s. In two electrode voltage clamp experiments substrate induced currents were recorded following the application of phosphate. The magnitude of the induced currents varied two- to fivefold, depending on the time period after cRNA injection and on the batch of oocytes (from different animals). Thus, comparisons were made only within the same batch of oocytes.

Fig. 1: The two-electrode voltage-clamp. The voltage recording electrode e_V monitors the membrane potential; this is compared with a command voltage V_c, and the magnified difference is applied to a current injection electrode, e_I. A bath electrode e_B serves as the return path for the injected current.

Statistical analysis

Data are provided as means ± SEM, n represents the number of oocytes investigated. All experiments were repeated with at least 3 batches of oocytes; in all repetitions qualitatively similar data were obtained. All data were tested for significance using the Student t-test, and only results with P< 0.05 were considered as statistically significant.

RESULTS

NaPi-2 induced inward currents

Addition of phosphate (1 mM) to Xenopusleavis oocytes expressing NaPi-2 led to an inward current (I_P) approaching -4.7 ± 0.5 nA (n = 14) at a holding potential of -50 mV (Fig. 2). Water injected oocytes, not injected with NaPi-2, did not show any electrogenic phosphate transport and the phosphate induced inward currents were negligible (-0.3 ± 0.1 nA, n = 8).

Fig. 2: Phosphate induced inward current (IP) in Xenopus oocytes expressing NaPi-2. Xenopuslaevis oocytes were injected with either water (H₂O), or cRNA encoding wild-type rat type IIa sodium dependent phosphate cotransporter (NaPi-2) alone. Addition of 1 mM phosphate into ND96 solution induced inward current in Xenopus oocytes expressing NaPi-2. In contrast, very small phosphate-induced inward current was observed in H₂O-injected oocytes may be due to expression of endogenous proteins. * indicates statistically significant difference to current in H₂O-injected oocytes.

Stimulation of NaPi-mediated currents by coexpression of SGK1

As shown in Fig. 3, coexpression of SGK1 led to a marked increase of phosphate induced inward currents in NaPi-2 expressing oocytes. Significantly (p < 0.05) higher phosphate induced inward currents were observed in oocytes injected with SGK1 and NaPi-2 (-7.4 ± 0.9 nA, n = 14) than the respective value in oocytes expressing NaPi-2 alone.

Fig. 3: Phosphate induced inward current (I_P) in Xenopus oocytes expressing NaPi-2 with SGK1. Xenopuslaevis oocytes were injected with either cRNA of encoding wild-type rat type IIa sodium dependent phosphate cotransporter (NaPi-2) alone or together with SGK1. Coexpression of SGK1 with NaPi-2 significantly stimulated the phosphate induced inward current (I_P) in contrast to coexpression of NaPi-2 + H₂O (*p < 0.05). * indicates statistically significant difference to current in Xenopus oocytes expressing NaPi-2 alone.

Stimulation of NaPi-mediated currents by coexpression of NHERF2

Coexpression of NHERF2 together with NaPi-2 increased significantly phosphate-induced inward currents in NaPi-2 expressing oocytes (Fig. 4). Significantly (p < 0.05) higher phosphate induced inward currents were observed in oocytes injected with NHERF2 and NaPi-2 (-8.4 ± 0.9 nA, n = 14) than phosphate induced inward currents in oocytes expressing the cotransporter alone.

Fig. 4: Stimulation of NaPi-2 transport activity by NHERF2. Xenopuslaevis oocytes were injected with cRNA of wild-type rat type IIa sodium dependent phosphate cotransporter (NaPi-2) alone or together with NHERF2. Coexpression of NHERF2 with NaPi-2 significantly increased phosphate induced inward currents in contrast to coexpression of NaPi-2 + H₂O (*p < 0.05). * indicates statistically significant difference to current in Xenopus oocytes expressing NaPi-2 alone.

NHERF2 further increase the effect of SGK1on the NaPi-mediated currents

As shown in Fig. 5, upon coexpression of NaPi-2 with both, SGK1 and NHERF2, phosphate induced inward currents were significantly larger than phosphate induced inward currents in oocytes expressing NaPi-2 with SGK1 or NaPi-2 with NHERF2 alone. In Xenopus oocytes coexpressing NaPi-2 together with both, NHERF2 and SGK1, phosphate induced inward currents approached -12.9 ± 1.5 nA, n = 14).

Fig. 5: Phosphate induced inward current (I_P) in Xenopus oocytes expressing NaPi-2 with or without coexpression of SGK1. and/or NHERF2. Xenopuslaevis oocytes were injected with cRNA encoding wild-type rat IIa sodium dependent phosphate cotransporter (NaPi-2) alone or NaPi-2 together with cRNA encoding SGK1 and/or NHERF2. Coexpression of SGK1 enhances Phosphate induced inward current (I_P), an effect potentiated by additional expression of NHERF2 (*p < 0.05). * indicates significant difference between expression of NaPi-2 alone, or with SGK1, or NHERF2 and coexpression of NaPi-2 together with both, SGK1 and NHERF2.

DISCUSSION

The present experiments disclose two completely novel mechanisms involved in the regulation of renal tubular phosphate transport, i.e., the regulation by NHERF2 and by the protein kinase SGK1. The kinase increases the NaPi-2 activity and stimulates NaPi-2 mediated phosphate transport. As shown for SGK1, the effect is potentiated by additional coexpression of NHERF2. The effect is at least partially due to stimulation of the insertion of the carrier into the cell membrane and delaying the endocytotic retrieval of NaPi-2 cotransporter (Fig. 6).

Fig. 6: Proposed model of NaPi-2 cotransporter regulated by SGK1 and NHERF2. The SGK1/NHERF2 stimulates the insertion of new NaPi-2 cotransporters into the plasma membrane and delay the endocytoticretrival of NaPi-2 cotransporters.

In this respect the action of the kinase mimicks that of PI3 kinase (Pfister et al., 1999). In analogy, the serum and glucocorticoid dependent kinase (SGK1) (Webster et al., 1993; Waldegger et al., 1997), another downstream target of PI3 kinase (Kobayashi and Cohen, 1999) stimulates Na⁺ channel activity similarly by fostering of channel insertion into the cell membrane (De la Rosa et al., 1999). The SGK1 thus presumably mediates the stimulating effect of IGF-1 on epithelial Na⁺ channel activity (Blazer-Yost and Cox, 1988; Blazer-Yost et al., 1992; Blazer-Yost et al., 1996).

SGK1 is not constitutively active but requires activation by phosphorylation. The upstream kinase is the phosphoinositol dependent kinase PDK1 which is in turn activated by IGF-1 through PI3 kinase (Kobayashi and Cohen, 1999; Park et al., 1999). IGF-1 has indeed been shown to stimulate phosphate transport (Caverzasio et al., 1985; Caverzasio and Bonjour, 1988) and inhibition of PI3 kinase leads to internalization and subsequent lysosomal degradation of NaPi (Pfister et al., 1999).

CONCLUSIONS

Rat type IIa sodium dependent phosphate cotransporter (NaPi-2), Serum and glucocorticoid-inducible kinase 1 (SGK1), and Na⁺/H⁺ exchange regulating factor 2 (NHERF2) are expressed in rat proximal renal tubules. Coexpression of SGK1 enhances NaPi-2 activity and stimulates phosphate transport through NaPi-2, an effect potentiated by coexpression of NHERF2. SGK1 may mediate the effect of IGF-1 and insulin on renal tubular phosphate transport which stimulates SGK1 through PI3 kinase and PDK1. Thus, the present observations unravel a novel signaling pathway in renal tubular transport regulation.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the assistance and input provided by all members and professors working for the institute of physiology, faculty of medicine, EberhardKarls University, Tuebingen, Germany, where the present work was performed.

REFERENCES

Adedokun, S.A. and Adeola, O. (2013): Calcium and phosphorus digestibility: Metabolic limits. J. Appl. Poult. Res. 22:600-608.

Akutsu, N.; Lin, R.; Bastien, Y.; Bestawros, A.; Enepekides, D.J.; Black, M.J. and White, J.H. (2001): Regulation of gene Expression by 1alpha,25-dihydroxyvitamin D3 and Its analog EB1089 under growth-inhibitory conditions in squamous carcinoma Cells. Mol. Endocrinol. 15(7): 1127-1139.

Albano, G.; Moor, M.; Dolder, S.; Siegrist, M.; Wagner, C.A.; Biber, J.; Hernando, N.; Hofstetter, W.; Bonny, O. and Fuster, D.G. (2015): Sodium-dependent phosphate transporters in osteoclast differentiation and function. PLoS One. 10(4): e0125104.

Bandyopadhyay, G.; Standaert, M.L.; Sajan, M.P.; Karnitz, L.M.; Cong, L.; Quon, M.J. and Farese, R.V. (1999): Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta. Mol. Endocrinology 13(10): 1766-1772.

Biber, J. and Murer, H. (1994): Renal epithelial apical Na/Pi cotransporter. Cell. Physiol. Biochem. 4(5-6): 185-197.

Blazer-Yost, B.L. and Cox, M. (1988): Insulin-like growth factor 1 stimulates renal epithelial Na⁺ transport. Am. J. Physiol. 255(3 Pt 1): C413-7.

Blazer-Yost, B.L.; Shah, N.; Jarett, L.; Cox, M. and Smith, R.M. (1992): Insulin and IGF-1 receptors in a model renal epithelium: receptor localization and characterization. Biochem. Int. 28(1): 143-53.

Blazer-Yost, B.L.; Record, R.D. and Oberleithner, H. (1996): Characteriz of hormone stimulated Na⁺ transport in a high-resistance clone of the MDCK cell line. Pflugers Arch. 432(4): 685-691.

Busch, A.E.; Wagner, C.A.; Schuster, A.; Waldegger, S.; Biber, J.; Murer, H. and Lang, F. (1995): Properties of electrogenic Pi transport by a human renal brush border Na⁺/Pi transporter. J. Am. Soc. Nephrol. 6(6): 1547-1551.

Caverzasio, J.; Brown, C.D.A.; Biber, J.; Bonjour, J.P. and Murer, H. (1985): Adaptation of phosphate transport in phosphate deprived LLC-PK1 cells. Am. J. Physiol. 248(1 Pt 2): F122-F127.

Caverzasio, J. and Bonjour, J.P. (1988): Influence of recombinant IGF-I (somatomedin C) on sodium-dependent phosphate transport in cultured renal epithelium. Prog. Clin. Biol. Res. 252: 385-386.

Costanzo, L.S.; Sheehe, P.R. and Weiner, I.M. (1974): Renal action of vitamin D in D-deficient rats. Am. J. Physiol. 226(6): 1490-1495.

Cowling, R.T. and Birnboim, H.C. (2000): Expression of serum- and glucocorticoid-regulated kinase (sgk) mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes. J. Leukoc. Biol. 67(2): 240-248.

De la Rosa, D.A.; Zhang, P.; Naray-Fejes-Toth, A.; Fejes-Toth, G. and Canessa, C.M. (1999): The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes. J. Biol. Chem. 274(53): 37834-37839.

Firestone, G.L.; Giampaolo, J.R. and O'Keeffe, B.A. (2003): Stimulus-dependent regulation of the serum and glucocorticoid inducible protein kinase (Sgk) transcription, subcellular localization and enzymatic activity. Cell. Physiol. Biochem. 13(1): 1-12.

Forster, I.C.; Wagner, C.A.; Busch, A.E.; Lang, F.;Biber, J.; Hernando, N.; Murer, H. and Werner, A. (1997): Electrophysiological characterization of the flounder type II Na⁺/Pi cotransporter (NaPi-5) expressed in Xenopuslaevis oocytes. J. Membr. Biol. 160(1): 9-25.

Fuster, D.G.; Bobulescu, I.A.; Zhang, J.; Wade, J. and Moe, O.W. (2007): Characterization of the regulation of renal Na⁺/H⁺ exchanger NHE3 by insulin. Am. J. Physiol. Renal. Physiol. 292(2): F577-585.

Hammerman, M.R.; Karl, I.E. and Hruska, K.A. (1980): Regulation of canine renal vesicles Pi transport by growth hormone and parathyriod hormone. Biochim. Biophys. Acta. 603(2): 322-335.

Hartmann, C.M.; Wagner, C.A.; Busch, A.E.; Markovich, D.; Biber, J.; Lang, F. and Murer, H. (1995): Transport characteristics of a murine renal Na/Pi-cotransporter. Pflügers Arch. 430(5): 830-836.

Hernando, N.; Déliot N.; Gisler, S.M.; Lederer, E.; Weinman, E.J.; Biber, J. And Murer, H. (2002): PDZ-domain interactions and apical expression of type IIa Na/Pi cotransporters. Proc. Natl. Acad. Sci. USA 99(18): 11957-11962.

Kobayashi, T. and Cohen, P. (1999): Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem. J. 339(Pt 2): 319-328.

Kobayashi, T.; Deak, M.; Morrice, N. and Cohen, P. (1999): Characterization of the structure and regulation of two novel isoforms of serum and glucocorticoid-induced protein kinase. Biochem. J. 344(Pt 1): 189-197.

Lang, F. and Cohen, P. (2001): Regulation and physiological roles of serum- and glucocorticoidinduced protein kinase isoforms. Sci STKE 2001(108): RE17.

Li, H.; Ren, P.; Onwochei, M.; Ruch, R.J. and Xie, Z. (1996): Regulation of rat Na⁺/Pi cotransporter-1 gene expression: the roles of glucose and insulin. Am. J. Physiol 271(6 Pt 1): E1021-E1028.

Magagnin, S.; Werner, A.; Markovich, D.; Sorribas, V.; Stange, G.; Biber, J. and Murer, H. (1993): Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc. Natl. Acad. Sci. USA 90(13): 5979-5983.

Mulroney, S.E.; Woda, C.B.; Halaihel, N.; Louie, B.; McDonnell, K.; Schulkin, J.; Haramati, A. and Levi, M. (2004): Central control of renal sodium-phosphate (NaPi-2) transporters. Am. J. Physiol. Renal Physiol. 286(4): F647-652.

Murer, H. (1992): Cellular mechanisms in proximal tubular Pi reabsorption: some answers and more questions. J. Am. Soc. Nephrol. 2(12): 1649-1665.

Murer, H.; Hernandez, N.; Forster, I. and Biber, J. (2000): Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol. Rev. 80(4): 1373-1408.

Park, J.; Leong, M.L.; Buse, P.; Maiyar, A.C.; Firestone, G.L. and Hemmings, B.A. (1999): Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J. 18(11): 3024-3033.

Pfister, M.F.; Ruf, I.; Stange, G.; Ziegler, U.; Lederer, E.; Biber, J. and Murer, H. (1998): Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi contrasporter. Proc. Natl. Acad. Sci. USA 95(4): 1909-1914.

Pfister, M.F.; Brunskill, N.J.; Forgo, J.; Stange, G.; Biber, J. and Murer, H. (1999): Inhibition of phosphatidylinositide 3-kinase in OK-cells reduces Na/Pi- cotransport but does not interfere with its regulation by parathyroid hormone. Pflügers Arch. 438(3): 392-396.

Prasad, N. and Bhadauria, D. (2013): Renal phosphate handling: Physiology. Indian J. Endocrinol. Metab. 17(4):620-627.

Shenolikar, S. and Weinman, E.J. (2001): NHERF: targeting and trafficking membrane proteins. Am. J. Physiol. Renal Physiol. 280(3): F389-F395.

Sorribas, V.; Markovich, D.; Hayes, G.; Stange, G.; Forgo, J.; Biber, J. and Murer, H. (1994): Cloning of a Na/Pi cotransporter from opossum kidney cells. J. Biol. Chem. 269(9): 6615-21.

Tenenhouse, H.S. and Murer, H. (2003): Disorders of renal tubular phosphate transport. J. Am. Soc. Nephrol. 14(1): 240-248.

Traebert, M.; Roth, J.; Biber, J.; Murer, H. and Kaissling, B. (2000): Internalization of proximal tubular type II Na-P(i) cotransporter by PTH: immunogold electron microscopy. Am. J. Physiol. Renal Physiol. 278(1): F148-F154.

Vanhaesbroeck, B. and Alessi, D.R. (2000): The P13K-PDK1 connection: more than just a road to PKB. Biochem. J. 346(Pt 3): 561-576.

Verri, T.; Markovich, D.; Perego, C.; Norbis, F.; Stange, G.; Sorribas, V.; Biber, J. and Murer, H. (1995): Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate. Am. J. Physiol. 268(4 Pt 2): F626-633.

Wagner, C.A.; Friedrich, B.; Setiawan, I.; Lang, F. and Broeer, S. (2000): The use of Xenopuslaevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell. Physiol. Biochem. 10(1-2): 1-12.

Waldegger, S.; Barth, P.; Raber, G. and Lang, F. (1997): Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc. Natl. Acad. Sci. USA 94(9): 4440-4445.

Waldegger, S.; Klingel, K.; Barth, P.; Sauter, M.; Rfer, M.L.; Kandolf, R. and Lang, F. (1999): h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine. Gastroenterology 116(5): 1081-1088.

Wallace, K.; Long, Q.; Fairhall, E.A.; Charlton, K.A. and Wright, M.C. (2011): Serine/threonine protein kinase SGK1 in glucocorticoid-dependent transdifferentiation of pancreatic acinar cells to hepatocytes. J. Cell. Sci. 124(Pt 3): 405-413.

Webster, M.K.; Goya, L.; Ge, Y.; Maiyar, A.C. and Firestone, G.L. (1993): Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol. Cell. Biol. 13(4): 2031-2040.

Werner, A.; Moore, M.L.; Mantei, N.; Biber, J.; Semenza, G. and Murer, H. (1991): Cloning and expression of cDNA for a Na/Pi cotransport system of kidney cortex. Proc. Nati. Acad. Sci. USA 88(21): 9608-9612.

Werner, A. and Kinne, R.K. (2001): Evolution of the Na-P(i) cotransport systems. Am. J. PhysiolRegul. Integr. Comp Physiol 280(2): R301-R312.

Yanagawa, N.; Pham, C.; Shih, R.N.; Miao, S. and Jo, O.D. (1999): Chloride dependency of renal brush-border membrane phosphate transport. Am. J. Physiol 277(4 Pt 2): F506-F512.

Yang, J.; Singh, V.; Chen, T.E.; Sarker, R.; Xiong, L.; Cha, B.; Jin, S.; Li, X.; Tse, C.M.; Zachos, N.C. and Donowitz, M. (2014): NHERF2/NHERF3 protein heterodimerization and macrocomplex formation are required for the inhibition of NHE3 activity by carbachol. J. Biol. Chem. 289(29): 20039-20053.

Yun, C.C.; Chen, Y. and Lang, F. (2002): Glucocorticoid activation of Na⁺/H⁺ exchanger isoform 3 revisited. The roles of SGK1 and NHERF2. J. Biol. Chem. 277(10): 7676-7683.

Yun, C.C. (2003): Concerted roles of SGK1 and the Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2) in regulation of NHE3. Cell. Physiol. Biochem. 13(1):29-40.

تحفيز الناقل المشترک للصوديوم والفوسفات-٢ في الفئران بواسطة انزيم الکيناز المحدث

بالسيرم والجلوکوکورتيکويد-١ والعامل المنظم-٢

حمدي أمبارک

Email: h.embark@vet.svu.edu.eg; atutohm@yahoo.com           Assiut University web-site: www.aun.edu.eg

يتم إعادة امتصاص الفوسفات الغير عضوي أساساً في الأنابيب الکلوية الملتوية القريبة عبر الناقل المشترک للصوديوم والفوسفات-٢أيه في الفئران. لقد تم استنساخ أشکال مماثلة من الناقل المشترک للصوديوم والفوسفات-٢أيه من کائنات حية مختلفة مثل الناقل المشترک للصوديوم والفوسفات-٢ في الفئران. الانزيم الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١ متزامن بشکل کبير في التعبير في نفس المکان مع الناقل المشترک للصوديوم والفوسفات-٢ في الفئران في خلايا الأنابيب الکلوية الملتوية القريبة. أهمية انزيم الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١ في تنظيم الناقل المشترک للصوديوم والفوسفات-٢ ما زالت حتى الآن بعيد المنال والدراسة. ومن ناحية أخري يحتوي الطرف الکربوکسيلي للناقل المشترک للصوديوم والفوسفات-٢ على معلومات عن التعبير الطرفي ويتفاعل عن طريق الأحماض الأمنية الثلاثة الطرفية للناقل مع عدة بروتينات التي تحتوي على نطاق الـ بي دي زد مثل العامل المنظم-١ والعامل المنظم-٢ .کل من العامل المنظم-١ والعامل المنظم-٢ تعدل استهداف ونقل العديد من البروتينات إلى غشاء الخلية. يتم التحکم في نقل مبادل الصوديوم والهيدروجين-٣ بواسطة کل من العامل المنظم-٢ وانزيم الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١. لاختبار احتمال تورطهم في تحفيز وتنشيط هذا الناقل تم حقن الحمض النووي الربي الخاص بالناقل المشترک للصوديوم والفوسفات-٢ في البويضات مع أو بدون حقن الحمض النووي الربي الخاص بإنزيم الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١ أو العامل المنظم-٢ أو کليهما معاً. باستخدام طريقة ثنائي الإلکترود ذات الجهد الکهربائي المشبک، يمکن حساب کمية الکهرباء کنشاط لهذا الناقل باعتبارها الرکيزة الحالية التي يسببها.التعرض للفوسفات ١ ملم يحدث تيارا کهربائي في البويضات المعبرة للناقل المشترک للصوديوم والفوسفات-٢ ولکن ليس في البويضات المحقونة بالماء بدون الناقل. إضافية انزيم الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١ في البويضات المعبرة للناقل المشترک للصوديوم والفوسفات-٢ أدى إلى تحفيز بشکل کبير کمية الکهرباء التي يسببها نقل الفوسفات بوساطة الناقل المشترک للصوديوم والفوسفات-٢ في البويضات. وعلاوة على ذلک، أيضا إضافية العامل المنظم-٢ قد حفز بشکل کبير کمية الکهرباء التي يسببها نقل الفوسفات بوساطة الناقل المشترک للصوديوم والفوسفات-٢ في البويضات. تأثير انزيم الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١ على الناقل المشترک للصوديوم والفوسفات-٢ في الفئران يزداد بواسطة إضافية العامل المنظم-٢. هذه الملاحظات تشير إلى أن الکيناز المحدث بالسيرم والجلوکوکورتيکويد-١ والعامل المنظم-٢ ينظمان نشاط الناقل المشترک للصوديوم والفوسفات-٢ وهکذا فإن من المرجح أنهم يشارکان معاً في تنشيط تأثير بعض الهرمونات مثل هرمون النمو والأنسولين على نقل الفوسفات الکلوي. هذه النتائج الحالية تکشف عن آليات مبتکرة وجديدة لتنظيم نشاط الناقل المشترک للصوديوم والفوسفات-٢ ونقل الفوسفات الکلوي التي قد تکون ذات أهمية بالنسبة لتنظيم توازن الفوسفات فى جسم الکائن الحي.