Renal Na/P-cotransporters

Transcription

1 Kidney International, Vol. 49 (1996), pp EPITHELIAL SODIUM-DEPENDENT Pi TRANSPORTERS Renal Na/P-cotransporters JURG BIBER, MARIA CUSTER, SIMONA MAGAGNIN, GILI HAYES, ANDREAS WERNER, MARIUS LOTSCHER, BRIGITFE KAISSLING, and HEINI MURER Institute of Physiology, University Zurich, Zurich, Switzerland Renal proximal tubular reabsorption of phosphate (P1) contributes to the maintainance of phosphate homeostasis. Uptake of P at the apical cell surface is obligatory dependent on the presence of sodium (Na/P-cotransport). Transport across the basolateral membrane involves multiple pathways such as an anion exchange mechanism and sodium-dependent P1-cotransport but is different from that in the apical membrane. The rate of proximal Pr reabsorption is adjusted acutely and chronically to the homeostatic needs of the body. In vivo and in vitro studies have provided evidence that physiological regulation of proximal tubular P1- reabsorption is most likely related to alterations in the transport capacity of apical Na/P-cotransport [1 3]. In this article the basic characterization of recently identified renal Na/P-cotransport proteins will be described. For a more detailed discussion of some functional and physiological and pathophysiological aspects, the reader is referred to additional articles in this issue. Identification of proximal tubular apical Na/P1-cotransporters Originally, a Na/P1-cotransport system of rabbit kidney cortex (NaPi-1) was identified by expression cloning using oocytes of Xenopus laevis and tracer flux studies using [32P]-inorganic phosphate [4]. Homologous proteins have subsequently been identified in mouse and human kidney cortex [5 7]. By a new round of expression cloning starting from rat and human kidney cortex edna libraries, additional Na/P-cotransporters have been identified: NaPi-2 and Napi-3 [8]. Amino acid comparison revealed that the Na/P1-cotransport systems NaPi-1 and NaPi-213 share weak overall homology (20% identity). Therefore, renal Na/P,- cotransporters identified so far have been classified in type I (NaPi-1 related) and a type II (NaPi-2 related) Na/P-cotransporters [9]. Additional type II Na/P-cotransporters were identified by homology screening using a NaPi-2 cdna probe: NaPi-4 from OK-cells [10], NaPi-5 from flounder kidney [11], NaPi-6 from rabbit kidney cortex [12] and NaPi-7 from mouse kidney cortex [13, 14]. Furthermore, a type II Na/P-cotransporter has also been identified in bovine renal NBL-1 cells [15]. Based on sequence comparison, similarity of type I cotransporters to a hypothetical protein (C38C10) of Caenorhahditis elegans was noticed. Since the hydropathy profile of the latter is almost identical to the one of type I cotransporters, it seems likely that this protein exhibits transport functions similar to the NaPi-1 Na/P-cotransportcr. From cerebellar granule cells cloning of a 1996 by the International Society of Nephrology Na/P1-cotransport system has recently been described [16]. The identified protein is of 30% identity to the type I Na/P1-cotransporter and hydropathy analysis suggests that this brain-specific Na/P1-cotransporter may belong to the class of type I cotransporters. Thus far, sequence comparison of type II Na/P1-cotransporters with current protein sequence databases revealed no significant homologies to other proteins. Type I Na/P-cotransporters are of approximately 465 and type II cotransporters of approximately 635 amino acids in length with predicted unglycosylated masses of 55 and 68 kda, respectively [4, 8, 9]. The putative secondary structures of the type I and type II Na/P1-cotransporters as predicted by hydropathy analysis are depicted in Figure 1. Both types of cotransporters contain several putative N-glycosylation sites and consensus phosphorylation sites for protein kinase C, which will be discussed below. Tissue and cellular localization of type I and type II Na/P-cotransporters Northern blot analysis demonstrated expression of type I cotransporter mrna in kidney cortex and in liver tissue [4, 17]. Expression of type II mrna was exclusively found in kidney cortex with the exception of a related transcript found in human lung tissue [8]. Notably, by Northern blot analysis no cross reaction with mrna isolated from small intestine was observed, suggesting that the small intestinal apical Na/P1-cotransporter may represent an additional type of Na/P-cotransporters. Detailed analysis using microdissected nephron segments and amplification of mrna after reverse transcription (RT-PCR) and in situ hybridization demonstrated uniform proximal tubular expression of type I as well as type II mrna [5, 18, 19]. Immunohistochemical studies revealed that both types of renal Na/P-cotransport systems are expressed at the brush border membrane of proximal tubular cells; no immunoreactivity was detected at the basolateral membrane surface [19, 20; Lötscher et a!, this issuel. Under normal physiological conditions, the type I cotransporters are expressed uniformly in apical membranes of proximal tubules of superficial and deep nephrons [20]. Expression of type II cotransporters (NaPi-2) was found to be more heterogeneous. Strongest immunoreactivity of anti-(napi-2) antibodies was observed in Si-segments of deep nephrons and gradually decreased towards the S3-segments [19]. Immunoelectron microscopy documented uniform distribution of the type II cotransporter along the microvilli (M. Lötscher, personal communication). 981

2 982 Biber et al: Renal Na/Ps transporters COOH 465 H2N COOH 637 Fig. 1. Predicted secondaiy structures of cloned renal type I and type II NaIP-cotransport systems (A) NaPi/rabbit and (B) NaPi-2/rat. Role of type I and type II Na/P1-cotransporters in the control of P-homeostasis Three lines of evidence suggest that the type II Na/P1-cotransporter is largely involved in proximal tubular P1-reabsorption and represents a target for physiological regulation of proximal tubular P1-reabsorption, such as by dietary P-intake and parathyroid hormone. As will be discussed the role of type I Na/P-cotransporters in proximal P1-reabsorption is currently less clear [Busch et al, this issue]. Functional characterization Kinetic characterization of sodium-dependent P. Transport expressed in oocytes of X laevis by 32P tracer flux demonstrated that the type II Na/P1-cotransport system exhibits kinetic parameters similar to the ones obtained in earlier studies perfomed with isolated proximal tubular brush border-membranes vesicles: Km(Pi) 0.15 mm, Km(Na) 50 ms, Hill coeficient around 2 [8, 10, 12, 14]. Similar values were also obtained after expression of the type II cotransporter (NaPi-2) in insect Sf9 cells [21] and in MDCK cells transfected with type II Na/P1-cotransporter [22]. Furthermore, expressed type II NaIP1-cotransport activity exhibits the characteristic ph-dependence (higher transport rate at alkaline ph) as described for proximal P-reabsorption [1, 2, 23, 24]. Detailed analysis of the ph-dependence and evidence that the type II cotransporter likely interacts with both di- and monovalent P has recently been described [14, 25]. In contrast, no ph dependence of Na/P1-cotransport related to the type I cotransporter could be demonstrated thus far (unpublished observations and A. Busch, personal communication). Superfusion of oocytes injected with either type I or type II crna with a phosphate containing solution provokes an inwardly sodiumdependent current, suggesting that sodium-dependent transport of P, by both NaIP-cotransporters is electrogenic [14, 25; Busch et al, this issue]. Electrophysiological studies further demonstrated that the type II Na/P-cotransporter is highly specific for phosphate ions but also transports arsenate. However the oxyanion 5042 is not a substrate for the type II Na/P1-cotransporter [14, 26; Busch et al, this issue]. Recent studies revealed evidence that the type I Na/P1- cotransporter (NaPi-1) exhibits anion channel properties and is able to transport organic anions [Busch et al, this issue].

3 Biber et al: Renal Na/P, transporters 983 Regulation of proximal apical NaIP,-cotransport (type II) by dietary P,-intake Restriction of dietary phosphate is associated with an increase of the overall proximal tubular capacity to reabsorb P In vivo and in vitro studies demonstrated that the increase of apical proximal Na/P1-cotransport is manifested by an increase of the maximal transport activity, Vmax [1, 2]. Under in vivo conditions apical Na/P1-cotransport is increased already within two hours of dietary P-restriction (acute adaptation) and lasts for several days (chronic adaptation) [2, 27 29; Levi et al, this issue]. Using antibodies and cdna probes of type I and II Na/P1-cotransporters, it has been demonstrated that the abundance of type II Na/P1-cotransporter mrna and protein is altered by changes of P1-intake, whereas the type I cotransport system (both at the mrna and protein level) was not affected by P1-restriction [12, 29, 30]. Figure 2 summarizes the results obtained with brush border membranes isolated from kidney cortex of rabbits adpated chronically to a low P1 diet. As illustrated, increased Na/P1-cotransport in membrane vesicles of adapted animals was paralleled by an increase of the abundance of the type II (NaPi-6) cotransporter but not of the type I cotransporter (NaPi-1). A Type II cotransporter is regulated by parathyhroid hormone Parathyroid hormone (PTH) produces a decrease in urinary P1 excretion due to inhibition of brush-border Na/P1-cotransport. Evidence was provided that the phosphaturic effect of PTH involves endocytic retrieval of Na/P1-cotransport systems from the apical membrane [1 3]. With antibodies directed against the type II cotransporter (NaPi-2), by immunohistochemistry it has been demonstrated that PTH provoked a decrease of type II cotransporters within the apical membrane, which was paralleled by a change of brush-border membrane Na/P1-cotransport [31]. In proximal tubular cells of PTH treated rats increased immunoreactivity was observed in a subapical region, suggesting that PTH activates an endocytic mechanism which results in an internalization of type II Na/P1-cotransporters (NaPi-2) [LOtscher et al and Kempson, this issue]. If PTH eventually also regulates the abundance of type I cotransporters within the proximal apical membrane has not yet been investigated. Structural/functional aspects of type II cotransporters As illustrated in Figure 1, type II Na/P1 cotransporters may span the membrane eight times. Needless to say, currently no clear structural information is available. In the following we will discuss briefly some structure/function relationships as obtained so far. N-glycosylation Amino acid sequences of type II cotransporters contain four or five potential N-glycosyaltion sites [4, 8, 9]. By site directed mutagensis of the asparagine residues 298 and 328 of the rat NaPi-2 cotransporter (Fig. 1) in in vitro translation and in oocyte expression experiments demonstrated that the mutated NaPi-2 crna results in an unglycosylated form of the NaPi-2 protein NaPI-6 8OkD NaPI-1 6OkD Actin 45kD Fig. 2. Sodium-dependent P-transport (A) and Western blot analysis (B) of renal brush-border membrane vesicles isolated from rabbits fed either a high [32]. This indicates that the mature type II cotransporter protein (1.2% P.) or low (0.1% P1) P,-diet for sic days. is N-glycosylated in the extracellular loop between the transmembrane regions M3 and M4. Tracer flux and elctrophysiological with oocytes injected with wild-type type II crna, injection of studies with oocytes injected with mutated crna demonstrated mutated crna resulted in a reduced rate of Na/P1-cotransport, that the transport characteristic (Kmp,, K,Na and ph dependence) suggesting an impaired delivery of unglycosylated transporters to was independent of the glycosylation state. However, compared the oolemma [32]. B LPD LPD HPD HPD

4 984 Biber et al: Renal NaIP, transporters IDf I anti-n I Phosphotylation In vitro studies revealed evidence that inhibition of type II related Na/P-cotransport is mediated by activation of a regulatory cascade involving protein kinase A and C activities [2]. Although several consensus sites for protein kinase C are found within the amino acid sequence of the type II Na/P1-cotransporter, a direct relationship of type II transporter phosphorylation and parathyroidhormone action has yet to be determined. Interestingly, treatment of oocytes injected with type II crna with phorbol ester resulted in inhibition of Na/P1-cotransport [33]. Site directed mutagenesis of predicted consensus sites for protein kinase C (serine residues at positions 5, 91, 462 and 625 and threonine residue number 508) did not, however, prevent phorbole esterinduced inactivation. This suggests that additional but unpredictable phosphorylation sites of the type II Na/P1-cotransporter may be involved, or that phosphorylation of another protein mediates the inhibitory action of phorbol esters. Western blots detection of type II cotransporters anti-c Fig. 3. Detection of the type II Na/Pi-cotransporter (NaPi-2lrat) in isolated renal brush-border membranes by polyclonal antibodies raised against the N- or C-terminus. Electrophoretic separation of brush-border membrane proteins was performed under non-reducing or reducing (100 nm DTT) conditions. On Western blots of electrophoretically separated brush-border membrane proteins, and using antibodies raised against a synthetic N-terminal or C-terminal peptide of the type II cotransporter (NaPi-2), a protein of approximately 85 kda is detected under non-reducing conditions [19]. Yet under reducing conditions additional bands of molecular weights 50 kda (detected with the anti-n-terminal antibody) 40 kda (detected with the anti-cterminal antibody) were observed (Fig. 3). This observation indicates that in isolated brush border membranes the type II cotransporter exists in a cleaved form but is held together via a disulfide bridge. Although the same observation was made with freshly microdissected proximal tubules (unpublished data), it remains to be determined if such a cleavage of type II Na/P1- cotransporters represents a preparative artifact, or occurs in vivo and therefore may play a role in the regulation of Na/P,-cotransport. Summary Two non-homologous proximal tubular apical Na/P1-cotransport systems (type I and type II) have been identified thus far by expression cloning. Subsequent studies provided evidence that the type II Na/P1-cotransporter represents a target for the physiological and pathophysiological regulation of proximal reabsorption of phosphate. The exact role of the type I Na/P1-cotransporter in proximal P-reabsorption and eventually also in the renal handling of other substrates, such as organic anions, is currently less clear and needs further investigation. Evidence was obtained that acute changes of brush border membrane Na/P-cotransport involves endo- and exocytic movement of type II NaIP1-cotransporters. In particular, we elucidated if and how phosphorylation reactions are involved and defined the intracellular structures of the endo/exocytic apparatus involved. At the level of the gene it will be necessary to elucidate its organization in order to understand the mechanisms involved in chronic regulations of Na/P1-cotransport related to the type II Na/P1-cotransporter. Furthermore, for structural investigations of these integral membrane proteins, they have to be isolated in sufficient quantities. Thus far the type II cotransporter (NaPi-2) has been expressed in Sf9 insect cells [20], which may eventually allow a purification of this protein. Acknowledgments The authors acknowledge the continous financial support by the Swiss National Fonds (Grant No to H.M. and Grant No to J.B.). Reprint requests to Dr. J. Biber, Institute of Physiology, University Zurich- Irchel, Winterthurerstrasse 190, CI-I-8057 Zurich, Switzerland. Biber@physioLunizh.ch References 1. BERNDT TJ, Kr'tox FG: Renal regulation of phosphate excretion, in The Kidney, Physiology and Pathophysiology, edited by SELDIN GW, GIEBIscH G, New York, Raven Press, 1992, pp MURER H, WERNER A, RESHKIN S, WUARIN F, BIBER 1: Cellular mechanisms in proximal tubular reabsorption of inorganic phosphate. 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