Proximal Tubular Phosphate Reabsorption: Molecular Mechanisms

Proximal Tubular Phosphate Reabsorption:Molecular Mechanisms

HEINI MURER, NATI HERNANDO, IAN FORSTER, AND JURG BIBER

Institute of Physiology, University of Zurich, Zurich, Switzerland

I. Introduction: Overall Mechanism 1373A. Site of reabsorption 1374B. Cellular mechanism 1374

II. Physiological Regulation 1375A. Major factors 1375B. Other factors 1375

III. Pathophysiological Alterations 1377A. Genetic aspects 1378B. “Acquired” alterations 1378

IV. Phosphate Transport Molecules in Proximal Tubular Cells 1378A. Type I Na-Pi cotransporter 1379B. Type II Na-Pi cotransporter 1380C. Type III Na-Pi cotransporter 1385

V. Type IIa Sodium-Phosphate Cotransporter: The Key Player in Brush-Border MembranePhosphate Flux 1385

A. Transport characteristics 1386B. Altered expression as the basis for altered Pi reabsorption 1389C. Cellular mechanisms in the control of type II Na-Pi cotransporter expression 1393

VI. Summary and Outlook 1397

Murer, Heini, Nati Hernando, Ian Forster, and Jurg Biber. Proximal Tubular Phosphate Reabsorption:Molecular Mechanisms. Physiol Rev 80: 1373–1409, 2000.—Renal proximal tubular reabsorption of Pi is a keyelement in overall Pi homeostasis, and it involves a secondary active Pi transport mechanism. Among the molecularlyidentified sodium-phosphate (Na/Pi) cotransport systems a brush-border membrane type IIa Na-Pi cotransporter isthe key player in proximal tubular Pi reabsorption. Physiological and pathophysiological alterations in renal Pi

reabsorption are related to altered brush-border membrane expression/content of the type IIa Na-Pi cotransporter.Complex membrane retrieval/insertion mechanisms are involved in modulating transporter content in the brush-border membrane. In a tissue culture model (OK cells) expressing intrinsically the type IIa Na-Pi cotransporter, thecellular cascades involved in “physiological/pathophysiological” control of Pi reabsorption have been explored. Asthis cell model offers a “proximal tubular” environment, it is useful for characterization (in heterologous expressionstudies) of the cellular/molecular requirements for transport regulation. Finally, the oocyte expression system haspermitted a thorough characterization of the transport characteristics and of structure/function relationships. Thusthe cloning of the type IIa Na-Pi cotransporter (in 1993) provided the tools to study renal brush-border membraneNa-Pi cotransport function/regulation at the cellular/molecular level as well as at the organ level and led to anunderstanding of cellular mechanisms involved in control of proximal tubular Pi handling and, thus, of overall Pi

homeostasis.

I. INTRODUCTION: OVERALL MECHANISM

Renal handling of Pi determines its concentration inthe extracellular space, the “traffic” place between thetwo major body compartments: skeleton and intracellularspace (37, 46, 101, 102, 216–218, 374). In cells phosphate

participates in energy metabolism and is a constituent ofsignaling molecules, lipids, and nucleic acids. Under “nor-mal” (“steady-state”) physiological conditions, urinary Pi

excretion corresponds roughly to phosphate intake in thealimentary tract, mainly via upper small intestine (37, 94,101, 218). To fulfill the “homeostatic” function, i.e., keep-

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ing extracellular Pi concentration within a narrow range,urinary Pi excretion must be (and is) under strong phys-iological control (37, 101, 102). In contrast to intestinal Pi

absorption, which adjusts rather “slowly” (for review, seeRefs. 94, 290), renal Pi excretion can “adjust” very fast toaltered physiological conditions.

A. Site of Reabsorption

Renal Pi excretion is the balance between free glo-merular filtration and regulated tubular reabsorption. Un-der normal physiological conditions, ;80–90% of filteredload is reabsorbed; renal tubular reabsorption occurs pri-marily in proximal tubules, with higher rates at earlysegments (S1/S2 vs. S3) and in deep nephrons (e.g., Refs.24, 142, 146, 159, 203, 232, 318; for review, see Refs. 37,218, 374). A small fraction of filtered Pi seems to bereabsorbed in the distal tubule (13), but the apparent lossof Pi observed after proximal tubular micropuncture sitescould be most likely explained by the higher reabsorptionin proximal tubules of deep nephrons (for review, see Ref.37). Therefore, a study/analysis of mechanisms participat-ing at the level of the kidney in control of Pi excretion canbe reduced to phenomena occurring in the proximal tu-bule.

B. Cellular Mechanism

The cellular mechanisms involved in proximal tubu-lar Pi reabsorption have been studied by a variety of

techniques including in vivo and in vitro microperfusions(e.g., Refs. 24, 49, 99, 100, 142, 144, 402), tissue-culturetechniques (e.g., Refs. 41, 43, 64, 66, 67, 116, 261, 264), andstudies with isolated brush-border and basolateral mem-brane vesicles (e.g., Refs. 18, 22, 23, 33–35, 44, 52, 53, 55,76, 78, 80, 88, 95, 98, 118, 127, 143, 148, 149, 152, 153, 155,179–182, 204, 239–241, 245, 246, 255, 256, 278, 291, 321,324, 328, 352, 355, 356, 360, 370–372, 375, 392, 400, 401,410, 429–434). We and others have written previouslyseveral comprehensive reviews on cellular mechanismsparticipating in renal tubular handling of Pi and summa-rized the experiments with above-mentioned techniques(e.g., Refs. 37, 46, 100, 101, 138, 149, 278, 282, 283, 291).From these studies a secondary active transport schemeemerged (see Fig. 1, left). Pi is taken up from the tubularfluid by (a) brush-border membrane sodium/phosphate(Na-Pi) cotransporter(s) and leaves the cell via basolat-eral transport pathways. The brush-border entry step isthe rate-limiting step and the target for almost all physi-ological (and pathophysiological) mechanisms altering Pi

reabsorption (see below). Basolateral exit is ill defined,and several Pi transport pathways have been postulatedincluding Na-Pi cotransport, anion exchange, and even an“unspecific” Pi leak (channel?). Basolateral Pi transporthas to serve at least two functions: 1) complete transcel-lular Pi reabsorption in a case where luminal Pi entryexceeds the cellular Pi requirements and 2) guaranteebasolateral Pi influx if apical Pi entry is insufficient tosatisfy cellular requirements. The second can be consid-ered as a “house-keeping” function and might not bespecific for (re)absorptive cells. In this review we sum-

FIG. 1. Scheme for proximal tubular Pi reabsorption. Left: concept of secondary active transport as evidenced bymicroperfusion studies and studies on isolated membrane vesicles. Right: Na-Pi cotransporter molecules in the proximaltubular epithelial cell. For further details and references, see text. [Adapted from Murer et al. (288).]

1374 MURER, HERNANDO, FORSTER, AND BIBER Volume 80

marize the present knowledge on the key transportermolecules involved in proximal tubular transmembrane Pi

movement (apical and basolateral; see Fig. 1, right).

II. PHYSIOLOGICAL REGULATION

As already indicated, regulation of proximal tubularPi reabsorption and thus of brush-border membrane Na-Pi

cotransport codetermines overall Pi homeostasis. Again,many reviews summarizing the regulation of proximaltubular Pi reabsorption at the organ, tubule, cell, andmembrane levels have been written (37, 46, 138, 282–289,292). This information is briefly presented here. In thisreview we focus on the molecular mechanisms underlyingthese regulations.

For a brief overview on regulatory events, we focuson major factors and other factors, and the latter is sub-divided into hormonal and nonhormonal factors control-ling proximal tubular Pi reabsorption (see Ref. 37). Foreach of these regulatory phenomena, a “memory” effectexists, i.e., the changes are induced by adequate pretreat-ment, and after characterization (e.g., by clearance tech-niques) in vivo can then be further analyzed in vitro, e.g.,in microperfusion studies or in studies with isolated mem-brane vesicles (for review, see Refs. 37, 46, 138, 282, 283,287). This memory effect can at present easily be under-stood, as physiological regulation of Pi reabsorption in-volves, as far as they have been studied at the molecularlevel, an altered expression of a brush-border Na-Pi co-transporter protein (type IIa Na-Pi cotransporter; for re-view, see Refs. 39, 242, 284–289, 292; see below). There-fore, they are in all cases, with the possible exception of“fasting” (204), related to changes in maximum velocity(Vmax) of brush-border membrane Na-Pi cotransport ac-tivity in isolated brush-border membrane vesicles (forreview, see Refs. 37, 46, 107, 206, 283).

A. Major Factors

1. Dietary Pi intake

A low dietary Pi intake can lead to an almost 100%reabsorption of filtered Pi, whereas a high dietary Pi in-take leads to a decreased proximal tubular Pi reabsorp-tion (for review, see Refs. 37, 218). These changes canoccur independent of changes in the plasma concentra-tion of different phosphaturic hormones (for review, seeRefs. 37, 218; see also Refs. 7, 9, 316). Thus an “unknown”humoral factor may be involved in the mediation of theseeffects. However, as evidenced by studies on culturedrenal proximal tubular epithelial cells (e.g., OK cells), adirect effect (“intrinsic”) of altered Pi concentration in theextracellular fluid (plasma, glomerular filtrate, culturemedia) also elicits changes in apical (brush-border) mem-

brane Na-Pi cotransport activity (e.g., Refs. 41, 43, 64, 309,339).

2. Parathyroid hormone

Parathyroid hormone (PTH) induces phosphaturia byinhibiting brush-border membrane Na-Pi cotransport ac-tivity; removal of PTH (parathyroidectomy) leads to anincrease in Na-Pi cotransport activity (e.g., Refs. 108, 120,153; for review, see Refs. 37, 46, 101, 138, 206, 218, 283,292). These effects can also be analyzed in a tissue-culturemodel to study cellular/molecular mechanisms involvedin proximal tubular Pi handling, in opossum kidney cells(OK cells; Refs. 67, 85, 86, 261–264, 267, 268, 307, 308, 310,311, 341–345). This in vitro model also provided evidencefor cAMP-dependent and cAMP-independent signalingmechanisms in PTH action (see below; see also Refs. 85,86, 235, 264, 308, 329–333; for review, see Refs. 280, 283,288, 289).

3. Vitamin D

Vitamin D is suggested to increase/stimulate proxi-mal tubular Pi reabsorption. 1,25-Dihydroxycholecalcif-erol treatment of rats was found to stimulate brush-bor-der membrane Na-Pi cotransport (226, 227). It is,however, difficult to discriminate between direct versusindirect effects, as in vivo the vitamin D status is closelyassociated with alterations in plasma calcium and PTHconcentrations (for review, see Refs. 37, 46, 101, 107).Thus, at present, it is not clear whether 1,25-dihydroxyvi-tamin D3 [1,25(OH)2D3] directly regulates mammalianbrush-border membrane Na-Pi cotransport. This is in con-trast to the upper small intestine where 1,25(OH)2D3 stim-ulates brush-border membrane Na-Pi cotransport (for re-view, see Refs. 94, 290). In chicken tubular preparations,administration of 1,25(OH)2D3 increased Pi uptake, aneffect prevented by inhibition of protein synthesis (249,250). However, in these studies in suspended cells, it isnot clear whether the stimulation is related to an in-creased uptake across the brush-border membrane. It hasbeen suggested that the effects of 1,25(OH)2D3 are relatedto changes in the lipid characteritsics of the membrane(114; for review, see Refs. 21, 37). A stimulatory effect of1,25(OH)2D3 was also observed in a subclone of OK cellsand in studies on promoter activation (see sect. VC; Refs.8, 380).

B. Other Factors

1. Hormonal factors

There are additional hormonal factors (e.g., insulin,growth hormone/insulin-like growth factor I/other growthfactors, thyroid and other lipophilic hormones, calcitonin,glucocorticoids, atrial natriuretic peptide, nerve transmit-

October 2000 PROXIMAL TUBULAR PHOSPHATE REABSORPTION 1375

ters, prostaglandins, parathyroid hormone-related pep-tide, phosphatonin, and stanniocalcin) with reported ef-fects on proximal tubular Pi reabsorption, i.e., brush-border membrane Na-Pi cotransport (for review, see Refs.37, 101, 107, 206, 283).

A) INSULIN. Insulin enhances proximal tubular Pi reab-sorption by stimulation of brush-border membrane Na-Pi

cotransport and prevents the phosphaturic action of PTH(e.g., Ref. 155; for review, see Refs. 37, 150, 206). Specificbinding sites for insulin have been identified in basolat-eral membranes of proximal tubular epithelial cells (154,155; for review, see Ref. 150).

B) GROWTH HORMONE/INSULIN-LIKE GROWTH FACTOR I/OTHER

GROWTH FACTORS. Growth hormone, at least in part medi-ated by insulin-like growth factor I (IGF-I; locally pro-duced in the kidney), stimulates proximal tubular Na-Pi

cotransport (e.g., Refs. 65, 153, 281, 335; for review, seeRefs. 37, 150, 206), an effect also observed in OK cells (63,193). Receptors for growth hormone have been identifiedon the basolateral membrane of proximal tubular cellsand appear to activate the phospholipase C pathway(350). Receptors for IGF-I have also been identified inproximal tubular cell membranes, and associated effectsmay involve tyrosine kinase activity (151, 154; for review,see Ref. 150).

Epidermal growth factor (EGF) stimulates Pi reab-sorption in perfused proximal tubules (336, 337) but in-hibits Pi transport in LLC-PK1 and OK cells (15, 140, 314).These effects are independent of cAMP and may involvetyrosine kinase activity and/or phospholipase C activation(see below; for review, see Ref. 206).

Transforming growth factors [i.e., transforminggrowth factor-a (TGF-a)] decrease Na-Pi cotransport ac-tivity in OK cells (233, 314). These effects are independentof cAMP, and the mechanisms might be similar to those inEGF action, sharing the same receptor (TGF-a and EGF;for review, see Refs. 150, 206).

C) THYROID HORMONE/LIPOPHILIC HORMONES. Thyroid hor-mone stimulates proximal tubular Pi reabsorption via aspecific increase in brush-border membrane Na-Pi co-transport (31, 118, 213, 433, 434; for review, see Refs. 37,107). The effect of thyroid hormone can also be observedin primary cultured chick renal cells and in OK cells andis dependent on protein synthesis (298, 367).

There are additional lipophilic hormones with re-ported effects on “renal tubular” Pi transport. All-trans-

retinoic acid (CatRA) specifically increases Na-Pi cotrans-port in OK cells (30; for review, see Ref. 107). On the otherhand, b-estradiol specifically decreases Na-Pi cotransportin brush-border membranes from adequately pretreatedrats (32; for review, see Ref. 107).

D) CALCITONIN. Calcitonin reduces proximal tubularbrush-border membrane Na-Pi cotransport in a PTH- andcAMP-independent manner (36, 430, 436; for review, seeRefs. 37, 206). This effect might be mediated by a rise in

intracellular calcium concentration (for review, see Ref.37).

E) GLUCOCORTICOIDS. Glucocorticoids increase phos-phate excretion by an inhibition of proximal tubularbrush-border membrane Na-Pi cotransport (47, 127; seealso Ref. 411); this effect can occur independent of anincrease in PTH (for review, see Ref. 37). The effects ofglucocorticoids are also apparent in vitro, in primarychick proximal tubular cells (299), and in OK cells (192;see also Refs. 156a, 319, 320). An increase in plasmaglucocorticoid levels may mediate the phosphaturic re-sponse in chronic metabolic acidosis (11, 47, 127; forreview, see Ref. 37).

F) ATRIAL NATRIURETIC PEPTIDE. Atrial natriuretic peptide(ANP) also inhibits proximal tubular brush-border mem-brane Na-Pi cotransport (156, 429). Although a small ef-fect of ANP, mediated by a rise in cGMP, was observed onOK cell Na-Pi cotransport (294), a direct effect on proxi-mal tubular cells is questionable, since receptors for ANPwere not identified in proximal tubular epithelial cells (forreview, see Ref. 37). An increase in renal dopamine pro-duction (see below) could mediate, in the intact organ,the effect of ANP on brush-border membrane Na-Pi co-transport (for review, see Ref. 37; see also Ref. 419).

G) PTH-RELATED PEPTIDE. PTH-related peptide producedby tumors causes phosphaturia. This “PTH analog” causesphosphaturia by mechanisms identical to that involved inPTH action (for review, see Refs. 37, 206; see also Refs.315, 349).

H) PHOSPHATONIN. Studies in patients with tumor-in-duced osteomalacia, with associated hypophosphatemiaand renal Pi wasting, led to the hypothesis that there is anadditional humoral factor controlling serum Pi concentra-tion and renal Pi handling (for review, see Refs. 37, 111,224, 225). This as yet unidentified factor was named phos-phatonin and is suggested to inhibit proximal tubular Pi

reabsorption (60). It was observed that conditioned cul-ture media from tumor cells derived from patients inhib-ited OK cell Na-Pi cotransport. This factor (phosphato-nin?) was suggested to have a proteinous nature and amolecular weight between 8,000 and 25,000. The inhibi-tion of Na-Pi cotransport occurred independently ofchanges in cellular cAMP content. Also, a PTH-receptorantagonist was found (but not identified; PTH related) inthese culture media; it interfered with PTH inhibition ofOK cell Na-Pi cotransport but not with the inhibitoryeffect of phosphatonin (for review, see Refs. 37, 111, 224,225).

I) GLUCAGON. Glucagon administration increases Pi ex-cretion. It was suggested that the effect of pharmacolog-ical doses of glucagon is indirect and related to an in-crease in plasma concentration of liver-derived cAMP (3).

J) STANNIOCALCIN. Two different isoforms of stanniocal-cin (STC) were identified and suggested to be involved incalcium and phosphate homeostasis in fish and in mam-


mals. STC-1 was originally identified in fish and later in ratkidney, in more distally located nephron segments (420).STC-2, ;34% amino acid similarity to STC-1 (189), wasidentified from an osteosarcoma library, and related tran-scripts were found in different tissues including kidney(72, 105, 189). STC-1 stimulates proximal tubular brush-border membrane Na-Pi cotransport (409); STC-2 has atleast in vitro (OK cells), the opposite effect, by a suppres-sion of the type IIa Na-Pi cotransporter (189). Thus STC-1/2 may serve paracrine modulators of Pi reabsorption.

K) PROSTAGLANDIN. Prostaglandins, produced intrare-nally, also modulate renal Pi handling. PGE2 antagonizesthe phosphaturia observed under different physiologicalconditions, e.g., increased PTH levels. This effect is inpart, but not fully, explained by effects on the cAMPsignaling cascade. The latter is illustrated by the observa-tion that inhibition of renal prostaglandin synthesis (byindomethacin) potentiates the cAMP-independent phos-phaturic action of calcitonin (36; for review, see Ref. 37).

L) NERVE TRANSMITTERS. Nerve transmitters also appearto control renal proximal tubular Na-Pi cotransport. Acuterenal denervation increases renal Pi excretion, indepen-dent of the PTH status (for review, see Ref. 37). Theseeffects can be related to the production of dopamineand/or reduced a- or b-adenoreceptor activity. Dopamineand its precursor L-dopa increase Pi excretion (104, 187,188) and inhibit Na-dependent Pi transport in OK cells aswell as in isolated rabbit proximal tubules (19, 79, 104,129, 137, 196). Dopamine can be generated from L-dopaafter brush-border membrane uptake of g-glutamyl-L-dopaand leads in an autocrine/paracrine manner via a stimu-lation of adenylate cyclase to the inhibition of brush-border membrane Na-Pi cotransport (104). Stimulation ofa-adenoreceptors might interfere with hormone-depen-dent stimulation of adenylate cyclase activity (e.g., byPTH) and might therefore lead to an apparent increase inNa-Pi cotransport activity, and explain a hypophosphatu-ric action of a-agonists (70, 403, 422, 423; see also Ref.234). In addition, stimulation of a-adenoreceptors in OKcells blunted the actions of PTH on cAMP production andinhibition of Na-Pi cotransport (77; see also Ref. 103).Serotonin is also synthesized in the proximal tubules andis antiphosphaturic; it stimulates proximal tubular Pi re-absorption (103, 128, 129, 147).

Adenosine infusion in rats stimulates renal Pi reab-sorption (312).

2. Nonhormonal factors

In addition to above hormonal factors, there are sev-eral nonhormonal factors known to affect proximal tubu-lar Na-Pi cotransport.

A) FASTING. Fasting may result in phosphaturia andreverse the effects of a low-Pi diet (for review, see Ref. 37;see also Ref. 28). This effect relates also to a change in

brush-border membrane Na-Pi cotransport (204). In con-trast to dietary Pi-induced changes and other regulatoryconditions, the lowered Pi uptake under fasting condi-tions might be explained by an increase in the apparentMichaelis constant (Km) value for Pi (204). The effect offasting may involve, but cannot be explained by, an in-crease in glucagon levels (for review, see Ref. 37).

B) PLASMA CALCIUM. Changes in plasma calcium lead tochanges in renal proximal tubular Pi reabsorption that areprimarily associated with the corresponding changes inPTH concentration (12, 421; for review, see Ref. 37).However, in vitro data also suggest a direct cellular effectof extracellular calcium on proximal tubular brush-bordermembrane Na-Pi cotransport (e.g., Ref. 301). In isolatedperfused convoluted rabbit proximal tubules, an increasein bath and perfusate calcium concentration provoked anincrease in Pi reabsorption (351). In studies on OK cells,opposite data were obtained: a decrease in medium cal-cium concentration stimulated Na-Pi cotransport (62).These differences are not understood but might be relatedto the time scale used in the experiments. The effects inOK cells required prolonged exposure, were dependenton protein synthesis, and may be related to changes inintracellular Ca21 concentration (see sect. VC4; see alsoRef. 353).

C) ACID BASE. The influence of changes in systemicacid-base status on renal proximal tubular Na-Pi cotrans-port are rather complex and are summarized only briefly.The effects on the kinetic properties of the carrier arediscussed in section VA4; in brief, an alkaline intratubularpH leads to a stimulation of Na-Pi cotransport (14, 328,334, 352; for review, see Refs. 37, 138, 216–218, 283).Acute metabolic acidosis does not significantly interferewith Pi reabsorption. In contrast, chronic metabolic aci-dosis leads to a decrease in Na-Pi cotransport, most likelyrelated to the evaluated glucocorticoid levels (11, 47, 127).These effects are also apparent in OK cells followingappropriate changes in media pH conditions (192, 194).Respiratory acidosis leads to phosphaturia involving cor-responding changes in Na-Pi cotransport. In contrast, re-spiratory alkalosis stimulates proximal tubular Pi reab-sorption (for review, see Ref. 37).

D) VOLUME EXPANSION. Volume expansion of animal in-creases Pi excretion and decreases Na-Pi cotransportrates in isolated brush-border membrane vesicles and inisolated perfused proximal tubules (74, 313, 317, 323, 324;for review, see Ref. 37). It is assumed that the effect ofvolume expansion on proximal tubules is indirect (i.e., viasome humoral factors, in part ANP and/or dopamine).

III. PATHOPHYSIOLOGICAL ALTERATIONS

In addition to the above briefly discussed physiolog-ical regulatory mechanisms, that adjust brush-border


Na-Pi cotransport to the needs of body Pi homeostasis,there are genetically determined alterations in renal Pi

handling and “acquired” alterations in renal Pi reabsorp-tion.

A. Genetic Aspects

The genetic aspects of proximal tubular Na-Pi co-transport have been covered in many reviews (e.g., Refs.338, 384, 390), and we only mention those disorders thathave been characterized at the molecular level. Severalgenetic defects resulting in isolated renal phosphate wast-ing have been described, such as X-linked hypophos-phatemic rickets (XLH; e.g., Refs. 295, 384, 385), autoso-mal dominant hypophosphatemic rickets not associatedwith hypercolcinuria (ADHR, 110, 113), and hereditaryhypophosphatemic rickets with hypercalciuria (HHRH;136, 394). The first is caused by mutations in the PHEX

gene, which has homology to neutral endopeptidasegenes and is hypothesized to process or degrade a circu-lating factor that regulates by an unknown mechanismrenal brush-border membrane Na-Pi cotransport (see be-low; for review, see Refs. 110, 112, 384, 390). A candidategene for ADHR and/or HHRH could be the brush-bordermembrane Na-Pi cotransporter (see below). However, thegene involved in ADHR was recently mapped to chromo-some 12p13 (113), a gene locus different from the brush-border Na-Pi cotransporter (5q35; 222, 223; see below).Although HHRH has the biochemical features of micewith a gene deletion for the brush-border membrane Na-Pi

cotransporter (25; see below), recent studies on a bedouinkindred with HHRH do not support the hypothesis of adirect involvement of the transporter gene in HHRH (A. O.Jones, I. Tzenova, T. M. Fujiwara, D. Frapier, M. Tieder, K.Morgan, and H. S. Tenenhouse, unpublished data). Aninteresting form of a genetically determined reduction inrenal Pi handling is in Dent’s disease, where mutations ina chloride channel (CLC5) lead to an apparent Pi trans-port defect (252; for review, see Ref. 391). How the loss offunction of an endosomal chloride channel leads to adecreased brush-border Na-Pi cotransport needs to bedetermined. Other genetic defects in renal Pi handling aresecondary to changes in vitamin D, PTH, or acid/basemetabolism or are a consequence of more general meta-bolic disorders (for review, see Refs. 109, 216, 217, 338,390).

B. Acquired Alterations

Disturbances in proximal tubular Pi transport seemto be an early indicator of “nonspecific” proximal tubularalterations, occurring as a consequence of “unphysiolog-ical” extrarenal factors (for review, see Refs. 216, 217).This may be explained by the specific kinetic properties of

the brush-border membrane Na-Pi cotransporter (seesect. VH). An example of this may be the observed phos-phaturia when the filtered load of glucose is augmented(in diabetes mellitus), where a “competition” for drivingforce will reduce Na-Pi cotransport rate (22, 392). Moregenerally speaking, when driving forces across the brush-border membrane (Na1 gradient and/or membrane poten-tial) are altered, the transport of phosphate will be re-duced and thus phosphaturia will occur. Furthermore, aspart of its physiological regulation, the transporter pro-tein mediating the rate-limiting Na-Pi cotransport has ahigh turnover. Therefore, “damage” to the brush-bordermembrane or the transporter protein itself will result in amassive reduction in the brush-border membrane contentof Na-Pi cotransporters and thus reduce Pi transport lead-ing to phosphaturia. This may explain, for example, thesensitivity of renal Pi reabsorption to heavy metal intox-ication (see Refs. 4, 141, 169).

Diuretics may inhibit proximal tubular Pi reabsorp-tion when administered to animals or intact tubular prep-arations (for review, see Ref. 37). Because the greatesteffect is produced by acetazolamide, it is assumed thatinhibition is related to an inhibition of carbonic anhy-drase; therefore, the effect is also dependent on the pres-ence of bicarbonate. The effect of other diuretics onproximal tubular Pi reabsorption correlates to some ex-tent with their potency to inhibit carbonic anhydrase.Inhibition of carbonic anhydrase leads to acute and/orchronic changes in systemic and/or tubular pH, which inturn causes the changes in Pi reabsorption.

IV. PHOSPHATE TRANSPORT MOLECULES

IN PROXIMAL TUBULAR CELLS

The cellular scheme for proximal tubular Pi reabsorp-tion given above includes three Na-Pi cotransporters (Fig.1). They have been molecularly identified and have beennamed type I, type II, and type III Na-Pi cotransporters(175; for review, see Refs. 284–289, 377). However, theremay be additional pathways in the brush-border and ba-solateral membranes that have not yet been defined at themolecular level. In heterologous expression systems (e.g.,Xenopus laevis oocytes), the corresponding cRNA/pro-teins augment highly Na1-dependent Pi uptake. The threefamilies of Na-Pi cotransporters share no significant ho-mology at the level of their primary amino acid sequence(Fig. 2 and Table 1). We discuss the structural properties,tissue expression, and functional characteristics of thesethree families of Na-Pi cotransporters. Because the typeIIa Na-Pi cotransporter is the key player (see sect. V), thekinetic properties and the regulatory behavior of the typeIIa transporter are then covered separately and in moredetail.


A. Type I Na-Pi Cotransporter

A cDNA related to the type I Na-Pi cotransporter wasinitially identified by screening a rabbit kidney cortexlibrary for expression of Pi transport activity in X. laevis

oocytes (416). Homologous cDNA (and in part proteins)were then found in human, mouse, and rat kidney cortex,in cerebellar granular cells, and in Caenorhabditis el-

egans (81, 82, 247, 248, 276, 277, 296, 297; for review, seeRef. 414).

The gene encoding the type I cotransporter (NPT 1)maps in humans to chromosome 6 p21.3-p23 (82, 223), inmouse to chromosome 13 close to the Tcrg locus (81,

437), and in rabbits to chromosome 12p11 (223). Thepromoter organization of NPT 1 has been characterized;104 bp upstream of exon 1, a single transcription start sitewas found and a TATA-like sequence at 241 (378).

Type I transporter mRNA has been detected by in situhybridization in mouse kidney proximal tubules and to alesser extent also in distal tubules (81). In rabbits, RT-PCR of microdissected tubular segments localized type ImRNA to the proximal tubules (92). Immunohistochemi-cal experiments and studies with isolated membraneslocalized the type I transporter protein to the proximaltubular brush-border membrane in rabbits and in mice(40; M. Lotscher, J. Biber, and H. Murer, unpublished

FIG. 2. Hydrophobicity predic-tions for the type I, type II, and typeIII Na-Pi cotransporters. Shaded areasindicate potential transmembranesegments. Sequence similarities areindicated by the vertical bars. For fur-ther discussion and references, seetext.

TABLE 1. The three families of Na-Pi cotransporters

Family Name Type I

Type II

Type IIIType IIa Type IIb

Molecule name NaPi-I, rabbit, rat, mouse, orhuman (NaPi-1, NPT1,

Npt1)

NaPi-IIa, mouse, rat,human, rabbit, oropossum (NaPi-2/3/4/6/7)

NaPi-IIb, mouse,human, flounder, orXenopus (NaPi-5)

Glvr-1 (PiT-1)Ram-1 (PiT-2)human, mouse, rat

Chromosomal location(human)

6 5 4 2 (PiT-1)8 (PiT-2)

Amino acids ;465 ;640 ;690 679, 656Predicted

transmembranesegments

6–8 8 8 10

Function (in Xenopus

oocytes)Na-Pi cotransport, Cl channel

activity, interaction withorganic anions

Na-Pi cotransport,electrogenic, pHdependent

Na-Pi cotransport,electrogenic

Na-Pi cotransport,electrogenic

Substrate Pi, organic anions Pi Pi Pi

Affinity for Pi ;1.0 mM 0.1–0.2 mM 0.05 mM 0.025 mMAffinity for Na1 50–60 mM 50–70 mM 33 mM 40–50 mMNa1-Pi coupling .1 3 3 3pH dependence Stimulated at high pH “Decreased” at high pH “Decreased” at high pHTissue expression

(mRNA protein)Kidney cortex/PT, liver, brain Kidney cortex/PT Small intestine, lung,

and other tissuesUbiquitous

Regulated by PTH/Pi diet No PTH and Pi diet Pi diet (Pi diet)

PT, parathyroid; PTH, parathyroid hormone.


observations). Studies on brush-border membranes pro-vided evidence for a higher expression in “deep” jux-tamedullary compared with superficial nephrons (97).

On the basis of hydropathy predictions, the type INa-Pi cotransporter protein may contain six to eight trans-membrane regions (Fig. 2); it contains three N-glycosyla-tion motifs of which some are used as indicated by im-munoblotting studies with isolated brush-bordermembrane vesicles and by in vitro translation experi-ments (416; for review, see Refs. 285, 284, 414).

The induction of increased Na-Pi cotransport activityafter injection into X. laevis oocytes was the basis for theexpression cloning of the cDNA encoding the type I Na-Pi

cotransporter cDNA (416). Stable transfection of type Itransporter cDNA into Madin-Darby canine kidney(MDCK) and LLC-PK1 cells resulted also in an increasedcellular uptake of Pi (325). Na1-dependent Pi uptake,induced after expression of the type I transporter in oo-cytes, has been extensively characterized (50, 56, 276, 297,416). The apparent Km for Pi was ;0.3 mM for expressionof the human and ;1 mM for the rabbit type I Na-Pi

cotransporter. The apparent Km value for Na1 interactionwas ;50 mM,with a Hill coefficient exceeding unity. Fur-thermore, no pH dependence of type I transporter-medi-ated Na1-dependent Pi uptake could be observed in oo-cytes. In electrophysiological studies in oocytes, evidencewas obtained that the type I transporter protein might bemultifunctional, since evidence for anion channel func-tion with permeability for chloride and different organicanions was obtained (50, 57). In the oocyte experiments itwas observed that the induction of chloride conductionby expression of the type I transporter cDNA was timeand dose dependent, in contrast to Na1-dependent Pi

uptake, which was maximally increased at low doses ofinjected cRNA and after short time periods of expression(50). This could suggest that the type I transporter proteinmay modulate an intrinsic “oocyte” Na-Pi uptake activity,present not only in oocytes, and that the type I transporter

protein may or may not be a Na-Pi cotransporter itself butrather an anion channel protein with expression in renalbrush-border membrane. Its role in proximal tubular se-cretion of anions (e.g., organic anions, xenobiotics) needsto be determined. Certainly, the above-described charac-teristics of type I transporter-induced Na1-dependent Pi

uptake does not resemble the characteristics of Na1-dependent Pi uptake in brush-border membrane vesicles(e.g., Ref. 14; for review, see Refs. 138, 283). Therefore,the type I transporter is not a major player in mediating orcontrolling brush-border membrane Na-Pi cotransport.

Yabuuchi et al. (426) have studied in more detail theanion conductive properties of the type I Na-Pi cotrans-porter (human Npt 1). In oocytes, benzylpenicillin, b-lac-tam antibiotics, probenecid, foscarnet, and melavonicacid were transport substrates. In the hepatocytes, Npt 1

was located on the sinusoidal membrane (426).To establish the physiological role of NPT 1 in above

anion secretion (as well as in renal Pi handling), genedeletion experiments are required (H. S. Tenenhouse andI. Soummounou, personal communication).

B. Type II Na-Pi Cotransporter

The cDNA encoding the type II (type IIa) Na-Pi co-transporter was identified by expression cloning in X.

laevis oocytes, from rat and human kidney cortex librar-ies, respectively (260). Homology-based approaches thenled to the identification of type II-related transporters inkidneys from different species including flounder andzebrafish, in opossum kidney cells (OK cells), and in abovine epithelial cell line (NBL-1; Refs. 87, 88, 163, 168,219, 294a, 366, 405, 417; see also Fig. 3). A type II-relatedNa-Pi cotransporter was identified in apical membranes ofmammalian small intestine and type II pneumocytes (121,175, 396) and has been designated type IIb Na-Pi cotrans-porter (Table 1). The regions with highest homology be-

FIG. 3. Proximal tubular expres-sion of the type IIa Na-Pi cotrans-porter protein (left) and mRNA(right). The protein shows a luminaland some distinct intracellular local-ization. The mRNA is also exclusivelylocated in the proximal tubules. P,proximal tubules; G, glomeruli; D, dis-tal tubules. For comparison, see alsoFigures 11 and 12. For details, seetext and references given in the text.


tween type IIa and type IIb transporters are in transmem-brane domains, and regions with no or little homology areat the cytoplasmic NH2 and COOH termini (Fig. 4; Ref.175).

Werner et al. (414) compared the sequences of thetype II Na-Pi cotransporters (Fig. 5) and three “families”were identified. Interestingly, the type IIa transporter ispreferentially expressed in kidney, with a proximal tubu-lar apical location (see sect. VB1). The type IIb transportercan have multiple locations; in mammals, it is expressedin the small intestine, type II pneumocytes, and othertissues, whereas in nonmammalian vertebrates it can beeither in the kidney and/or small intestine (175, 190, 219,

294a, 396). A type IIa Na-Pi cotransporter appears to beexpressed also in osteoclasts and may play a role in boneresorption (145). A type II Na-Pi cotransporter seems alsoto be expressed in brain, where the function is not yetestablished (177). Finally, type II related proteins ap-peared very early in evolution, and related genes werefound in Vibrio cholerae and C. elegans (see Fig. 5; forreview, see Ref. 414).

1. Chromosomal location/genomic organization

The human type IIa cotransporter gene (NPT 2) mapsto chromosome 5q35 (Fig. 6; Refs. 222, 223, 269, 277) and

FIG. 4. Sequence comparisons between type IIa and type IIb Na-Pi cotransporters. Predicted transmembrane areasare given by black bars above the sequences and show high sequence homologies. For further discussion, see text andincluded references. [Adapted from Hilfiker et al. (175).]


the murine (Npt 2) gene to chromosome 13B (437). Thehuman type IIb Na-Pi cotransporter maps to chromosome4p15–16 (418a).

The genomic structure of NPT 2 (human IIa) and Npt

2 (murine IIa) has been determined; they are ;16 kb inlength and consist of 13 exons and 12 introns (Fig. 6; Refs.162, 379). In the promoter region of the human, murineand OK cell NPT 2/Npt 2 gene, a TATA box is present 31bp upstream from the transcription start sites. A GCAATelement and several AP-1 sites may control promoteractivity (162, 379). The NPT 2/Npt 2 promoter is activeonly in a proximal tubular environment, i.e., in OK cells(162, 174, 176). 59-Flanking sequences of the OK cell typeII Na-Pi cotransporter gene contain elements mediatingtranscriptional control under different bicarbonate/car-bon dioxide tensions (194). For the rat Npt 2 promoter, animportant role of repeating AP-2 consensus sites in regu-

lating cell-specific expression was documented (359). Inreporter gene studies, no physiological regulation (e.g., bylow-Pi medium, PTH, thyroid hormones, and growth fac-tors) was observed using a short promoter (327 bp) frag-ment (174, 176). However, in COS-7 cells expressing thehuman vitamin D receptor, a vitamin D response elementwas observed at ;2 kb upstream from the transcriptioninitiation site of the NPT 2/Npt 2 gene (380). Furthermore,regulatory sequences within the NPT 2 gene, ;1 kb up-stream of the transcription start site, were identified asbinding sites to nuclear proteins upregulated in kidneys ofweaning mice fed a low-Pi diet (212a). The correspondingDANN-binding protein could be identified; it correspondsto a known transcription factor (TFE3) that activatestranscription through the mE3 site of the immunoglobinheavy chain enhancer (212a). The mRNA encoding TFE3was found to be significantly increased in kidney tissuesof weaning mice fed a low-Pi diet (212a).

2. Tissue-specific expression

In situ hybridization of renal sections (Fig. 3) andnephron microdissection, followed by RT-PCR, docu-mented that type IIa mRNA expression is restricted to thekidney proximal tubule (87, 91, 348, 388). Therefore, inmouse kidney, the type IIa is by far the most abundant ofknown Na-Pi cotransporters (388). Type IIa Na-Pi cotrans-porter protein is found in the brush-border membrane ofproximal tubules (see Fig. 3; Refs. 91, 348). Inter- andintranephron distribution type of IIa Na-Pi cotransporterhighly depends on the physiological requirements withinoverall Pi homeostasis, (see Figs. 3 and 11; Refs. 91, 210,243, 348; for review, see Refs. 242, 284–287). The type IIaNa-Pi cotransporter is also expressed in OK cells but notin other renal cell lines (310, 311, 366, 386, 424; and J.Forgo, G. Strange, J. Biber, and H. Murer, unpublishedobservations). Recently, a type IIa transporter protein-related immunoreactivity was observed in membranefractions isolated from nontransformed immortalizedmice kidney cortex epithelial cells (71). There is no evi-dence that the type IIa Na-Pi cotransporter is expressed inprimary renal proximal tubular epithelial cell cultures(Forgo et al., unpublished observations). Its expression inOK cells is the basis for the use of this cell line as an invitro model for the study of cellular mechanisms involvedin regulation of type IIa Na-Pi cotransport activity (seeRefs. 192–194, 234–236, 263, 307–311; for review, see Refs.283–288).

The related type IIb Na-Pi cotransporter is found inthe apical membrane of upper small intestinal enterocytesand type II pneumocytes (see Refs. 175, 396); type IIbtranscripts have been found in a variety of other tissues(175).

FIG. 5. Evolutionary tree of type II Na-Pi cotransporters identified indifferent species. The database accession numbers are as follows:mouse kidney, L33878 and U22465; rat kidney, L13257; human kidney,L13258; sheep, AJ001385; rabbit kidney, U20793; opossum kidney cells,L26308; mouse intestine, AF081499; human intestine, AF111856; bovinekidney cells, X81699; chicken kidney (A. Werner, unpublished data);carp kidney (Werner, unpublished data); zebrafish kidney (Werner, un-published data), flounder kidney and intestine, U13963; Xenopus intes-tine, L78836; zebrafish intestine (Werner, unpublished data); Vibrio

cholerae, AJ010968; Caenorhabditis elegans, AF095787. A type II Na-Pi

cotransporter was also identified from sheep kidney (S. P. Shirazi-Beechey, unpublished data; accession number AJ001385). [Dendrogramis extended from that published by Werner et al. (414) and given to uscourtesy of Andreas Werner.]


3. Structural aspects

Hydropathy analysis predicted eight transmembranesegments for the type IIa cotransporter protein (Fig. 2;Ref. 260; for review, see Refs. 284, 285). This membranetopology was supported by several experimental findings:1) insertion of FLAG epitopes and accessibility of theepitope to antibodies (231); 2) lack of accessibility ofantibodies directed against COOH- and NH2-specificamino acid sequences (231); 3) identification of two gly-cosylation sites in the second “suggested” extracellularloop (166); and 4) accessibility to membrane-impermeantsulfhydryl reagents after insertion of cysteine residues atspecific sites of the protein (228, 229). Two regions maypenetrate partially into the lipid bilayer (Fig. 7).

Type IIa Na-Pi cotransporters contain numerous poten-tial phosphorylation sites for protein kinase C and casein IIkinases (165, 260). The role of these sites in physiologicalcontrol of transport activity is not clear (see sect. VC4).

On immunoblots of brush-border membrane proteinsperformed under nonreducing conditions, the type IIaNa-Pi cotransporter shows an apparent molecular mass of80–90 kDa; under reducing conditions two bands of;45–50 kDa appear (39, 48, 91, 425). The latter suggeststhat the transporter might be proteolytically cleaved be-tween the two glycosylation sites at positions N298 andN328 (39, 48, 228, 229, 305). It is not known whether thisproteolytic cleavage occurs in situ or whether it is exper-imentally induced. Site-directed mutagenesis studies doc-

umented an S-S bridge in the second extracellular loop(Fig. 7; Refs. 228, 229). It is of interest that separateoocyte injections of cRNA encoding NH2- and COOH-terminal fragments of the flounder type IIb Na-Pi cotrans-porter resulted in induction of Pi uptake activity only ifboth “parts” of the proteins were “present” (220).

The question of a multimeric structure of the type IIacotransporter has been addressed mainly in radiation in-activation studies (33, 98, 195). The size of the functionalunit of brush-border membrane Na-Pi cotransport (mostlytype IIa Na-Pi cotransporter mediated) was found to bebetween 170 and 200 kDa, suggesting a multimeric struc-ture. Recent experiments in oocytes expressing wild-typeand mutant (inactivatable, cysteine insertion) type IIaNa-Pi cotransporters suggested that each individual wild-type cotransporter molecule within an assumed homo-multimeric complex is functional (220a). The apparenthigh functional molecular mass observed in brush-bordermembranes could also be due to a heteromultimeric com-plex (see below). The experiments in different heterolo-gous expression systems (e.g., in Sf9 cells, Refs. 134, 135;in MDCK cells and LLC-PK1 cells, Refs. 325, 326; and inoocytes, Ref. 260) suggest that an unknown additionalprotein within the functional complex is not an obligatoryrequirement for the type IIa Na-Pi cotransporter-mediatedPi uptake activity or, rather unlikely, is present as anintrinsic protein (to serve as a transporter subunit) indifferent expression systems.

FIG. 6. Chromosomal location and genomic organization of human type IIa Na-Pi cotransporter. The gene is localizedon chromosome 5 (5q35) and has 12 introns and 13 exons. The position of the introns and their size is given with respectto the transcription initiation site. For further discussion, see text and included references. [Adapted from Hartmann etal. (162) and Kos et al. (222).]


Tatsumi et al. (381) have identified type IIa Na-Pi

cotransporter-related cDNA (named NaPi-2a, NaPi-2b,and NaPi-2g). The NaPi-2a-encoded protein (355 aminoacids) has a high homology to the NH2-terminal half of thetype IIa cotransporter, NaPi-2b encodes for 327 aminoacids identical to the NH2-terminal part of type IIa co-transporter with a completely different 146-amino acidCOOH-terminal end, and NaPi-2g encodes a 268-aminoacid protein from the COOH-terminal end of the molecule(381). It seems that the related mRNA are formed byalternative splicing of the type IIa cotransporter gene(381). Isoform specific mRNA were found on Northernblots of rat kidney cortex mRNA. With the use of a full-length type IIa Na-Pi cotransporter cDNA probe, the ma-jor transcript detected was ;2.6 kb (260, 381). Additional

bands (9.5, 4.6, and 1.2 kb) were seen, although in ourexperience, these bands are not abundant (260, 381). TheNaPi-2a probe hybridizes with transcripts of 9.5 and 4.6kb, the NaPi-2b probe with a transcript of 1.2 kb, and theNaPi-2g probe with transcripts of 9.5 and 2.6 kb (381). InWestern blots, with the use of NH2- or COOH-terminaltype IIa Na-Pi cotransporter specific antibodies, proteinsof 45, 40, and 37 kDa were observed, corresponding ap-proximatively to the size of the in vitro translated proteins(NaPi-2a, NaPi-2b, NaPi-2g; Ref. 381). The full-length typeIIa Na-Pi cotransporter protein is recognized in Westernblots from brush-border membrane as a 80- to 90-kDaprotein in its glycosylated form (87). In our hands, thelower molecular mass bands are not detected in the ab-sence of reducing agents (see Refs. 39, 91). Because they

FIG. 7. Secondary structure (membrane topology) of type IIa Na-Pi cotransporter (rat, NaPi-2). The model is derivedfrom hydropathy predictions (Fig. 2; Ref. 260) and is experimentally supported by studies on N-glycosylation (166),accessibilities of specific antibodies to either the NH2 or COOH terminus (231), FLAG-epitope insertion (231), andcysteine insertions/deletions (228, 229). For further details, see text and included references.


are only visible under reducing conditions, type 2a, 2b,and/or 2g related proteins might be linked to the full sizetype IIa Na-Pi cotransporter via S-S bridges. Alternatively,the possibility exists that the smaller proteins, apparentafter reduction of S-S bridges, are a product of proteo-lytic cleavage of the full size type IIa Na-Pi cotrans-porter protein (see above). Based on coexpression ex-periments in oocytes, Tatsumi et al. (381) postulatedthat the smaller isoforms might regulate, in a dominantnegative manner, the function of the type IIa Na-Pi

cotransporter protein. However, this interpretation re-quires further studies to, for example, document thecoexistence, within a “heterologous” complex, of thedifferent proteins at the brush-border membrane. Fur-thermore, quantitative aspects are crucial, since in ourexperience NaPi-2a, -2b, and -2g can only be present inrather small amounts relative to the full size type IIacotransporter. Thus the role of the small type IIa Na-Pi

cotransporter-related proteins in brush-border mem-brane Na-Pi cotransport in vivo is not clear.

An antisense type IIb Na-Pi cotransporter transcriptwas detected in different nonmammalian tissues. It waspostulated that it might be involved in the control ofcotransporter protein expression (physiological control;tissue specificity; Ref. 184).

When expressed in X. laevis oocytes, the type IIaNa-Pi cotransporter mediates Na-Pi cotransport activitywith functional characteristics identical to those ob-served in isolated brush-border vesicles (87, 163, 260, 366,405). A 3:1 stoichiometry (Na1:Pi) is the basis for itsmembrane potential sensitivity (electrogenicity; e.g., Refs.59, 123). As discussed in section V, the transport charac-teristics and kinetic behavior of the type IIa transporterhave been studied in great detail. Similar transport char-acteristics were also observed in different other heterol-ogous expression systems such as insect Sf9 cells, fibro-blasts, and MDCK cells (134, 135, 326, 395).

C. Type III Na-Pi Cotransporter

Surprisingly, the receptor for gibbon ape leukemiavirus (Glvr-1) and the receptor for the mouse ampho-tropic retrovirus (Ram-1) have been shown to mediateNa-Pi cotransport activity after their expression in X.

laevis oocytes (201, 202, 302). The transporter proteinshave been named PiT-1 and PiT-2 and are now classifiedas type III Na-Pi cotransporters (Table 1).

Expression of type III Na-Pi cotransporters seems tobe ubiquitous, and related mRNA have been identified inkidney, parathyroid glands, bone, liver, lung, striated mus-cle, heart, and brain (Table 1; Refs. 84, 201, 202, 302, 303,362, 382). In mouse kidney, transcripts of type III cotrans-porters are found throughout the different structures(362, 388). Immunofluorescence studies showed in the

proximal tubule a basolateral location (C. Silve, personalcommunication). Based on mRNA levels, type III Na-Pi

cotransporters are two orders of magnitude less abundantthan type IIa transporters (388). Its role in the proximaltubule seems not to be in transcellular Pi transport butrather in cell Pi uptake if luminal Pi entry is insufficient forcell metabolic functions. Type III transporter expressionseems not to be altered by PTH (386).

The type III transporters show some homology to aNeurospora crassa gene (Pho-41) involved in transmem-brane Pi movements (302). Hydropathy analysis suggests10 transmembrane regions (Fig. 2; Refs. 201, 202).

PiT-1- and PiT-2-mediated Na-Pi cotransport has beenstudied by expression in X. laevis oocytes or in fibroblasttransfection (201, 202, 302). Transport is characterized bya Km for Pi in the order of 20–30 mM and a Km for Na of40–50 mM. pH dependence of type III Na-Pi cotransporteris opposite to the type IIa cotransporter, i.e., decreasedactivity by increasing pH. Similar to the type IIa, typeIII-mediated transport of Pi is electrogenic with a netinflux of a positive charge during the transport cycle,suggesting also a 3:1 stoichiometry .

V. TYPE IIA SODIUM-PHOSPHATE

COTRANSPORTER: THE KEY PLAYER

IN BRUSH-BORDER MEMBRANE

PHOSPHATE FLUX

The tissue expression, the relative renal abundance,and overall transport characteristics of type I, II (IIa), andIII Na-Pi cotransporters suggest that the type IIa trans-porter plays a key role in brush-border membrane Pi flux.As discussed in this section, changes in expression of thetype IIa Na-Pi cotransporter protein parallel alterations inproximal tubular Pi handling, documenting its physiolog-ical importance (for review, see Refs. 242, 284–289). Inaddition, experiments on molecular (genetic) suppressionof the type IIa Na-Pi cotransporter support its role inmediating brush-border membrane Na-Pi cotransport. 1)Intravenous injection of specific antisense oligonucleo-tides led to reduced brush-border membrane Na-Pi co-transport activity that was associated with a decrease intype IIa cotransporter protein (300). 2) Disruption of thetype IIa Na-Pi cotransporter gene (Npt 2) in mice led to an;70% reduction in brush-border Na-Pi cotransport rateand complete loss of the protein (25, 178; see also below).The molecular basis for the remaining brush-border mem-brane Na-Pi cotransport after Npt 2 gene disruption isunclear. Either the type I transporter protein or anothernot yet identified Na-Pi cotransporter could account forresidual transport activity. 3) Injection of type IIa anti-sense oligonucleotides in oocytes completely inhibitedNa-Pi cotransport mediated by kidney cortex mRNA, con-firming its major role in brush-border membrane Na-Pi

cotransport (275, 276, 389, 415).


A. Transport Characteristics

As already indicated, the transport characteristics ofthe type IIa cotransporter heterologously expressed indifferent cellular systems (mainly X. laevis oocytes) re-sembles closely those of Na-Pi cotransport activity ob-served in isolated brush-border membranes (e.g., Refs. 87,88, 163, 168, 219, 260, 366, 405, 417). In particular, in allexpression systems studied thus far, type IIa-mediatedNa-Pi cotransport activity increased with increasing me-dia pH values, a “signature” for proximal tubular brush-border membrane Na-Pi cotransport (e.g., Refs. 14, 260).

The simplest experimental technique to analyze thetransport characteristics of a Na-substrate cotransporteris by studying Na1 gradient-driven tracer substrate influxunder different conditions. For the type IIa cotransporter,this has been done already in 1976 in isolated rat brush-border membrane vesicles (179); obviously, it was thennot known that the brush-border Na-Pi cotransport activ-ity is mostly associated with the type IIa cotransporterprotein (25, 260). The studies with isolated membranevesicles provided, however, significant insights into themechanism/kinetic of brush-border membrane Na-Pi co-transport (e.g., Refs. 14, 34, 35, 55, 80, 352). A detailedkinetic characterization of type IIa-mediated Na-Pi co-transport activity was performed after its expression in X.

laevis oocytes (e.g., Ref. 260). The first characterization,performed using tracer techniques, suggested a Na1:Pi

stoichiometry exceeding unity (see sect. VA1; Ref. 260).These data and the evidence for electrogenicity of Na-Pi

cotransport across the brush-border membrane from mi-croperfusion experiments in vivo (133) and from studieswith isolated vesicles (35, 55) were the rationale for anelectrophysiological characterization of the type IIa Na-Pi

cotransporter after its expression in oocytes. The electro-physiological studies, performed under steady-state con-ditions, complemented the tracer uptake study, whereaspre-steady-state measurements provided new insightsinto individual steps within the transport cycle (see be-low).

1. Steady-state electrophysiological characteristics

Under voltage-clamp conditions, superfusion of oo-cytes expressing the rat type IIa cotransporter with 1 mMPi in the presence of Na1 (100 mM) elicits an inwardcurrent, the magnitude of which depends on the holdingpotential (Fig. 8, A and B; Refs. 56, 58, 59, 122, 123, 125,126, 407, 408). This observation indicates that a Pi-in-duced inward movement of positive charge(s) occurs dur-ing the transport cycle. At a given membrane potential,dose-response relationships can then be obtained for bothNa1 and Pi. Furthermore, the interdependence of theapparent affinity for either Na1 or Pi and/or the appliedmembrane potential was studied (e.g., Ref. 122). For Pi, a

hyperbolic saturation curve is observed, whereas for Na1,the saturation curve is sigmoidal (Fig. 8, C and D). At 100mM Na1, the apparent Km for Pi interaction is ;0.1 mMand shows little dependency on the holding potential (Fig.8E). At 1 mM Pi, the apparent Km for Na1 interaction is;50 mM (Fig. 8F). The concentration dependence of Pi-induced current depends on the external Na1 concentra-tion with a dual effect: increasing Na1 leads to a decreasein the apparent Km for Pi and to an increase in theapparent Vmax (Fig. 8C). On the other hand, increasing Pi

also leads to an increase in affinity for Na1 (Fig. 8D). Atthe lower Na1 concentrations, the apparent Km for Pi

interaction shows a marked dependence on the holdingmembrane potential (Fig. 8E); this is not observed for theKm for Na1 interaction (Fig. 8F). Finally, these saturationexperiments provide some information with respect tothe stoichiometry (Pi :Na1). Hill coefficients calculated onthe basis of the Pi saturation curves were always close to1, whereas those calculated for Na1 saturation were al-ways close to 3 (e.g., Refs. 59, 122 123). A 3:1 stoichiom-etry explains the positive inward current (59, 122, 123).The stoichiometry (Na1:Pi) has been determined moredirectly by simultaneous measurements of substrate fluxand charge movement under voltage-clamp conditions inthe same oocytes (123). It was found that translocation ofa positive charge into the oocyte is associated with thetransfer of 1 Pi and 3 Na1. These experiments also pro-vided evidence for the preferential transport of divalent Pi

anions (123).The antiviral agent foscarnet (phosphonoformic acid,

PFA) is a known competitive inhibitor of brush-bordermembrane Na-Pi cotransport (e.g., Refs. 10, 205, 256, 375,404). In electrophysiological studies, PFA inhibited Pi-induced inward currents but did not elicit PFA-inducedcurrents (59, 122). Thus PFA interferes with Pi binding butis not a transported substrate. In addition, arsenate is acompetitive inhibitor of brush-border membrane and oo-cyte type IIa Na-Pi cotransporter-mediated Pi uptake(179). In contrast to PFA, arsenate induces inward cur-rents and is thus a transported substrate (e.g., Ref. 163).Recently, a “slippage current” associated with the transferof the partially loaded type IIa cotransporter was identi-fied (only with Na1, see below; Fig. 9; Ref. 122). Thiscurrent was blocked by PFA and showed a dose depen-dence suggesting interaction with only one Na1. The slip-page current accounts for ;10% of maximally inducedcurrent of the fully loaded carrier (122). Although thisslippage current is of little functional significance, it isimportant in our understanding of the transporter cycle(see sect. VA3).

2. Pre-steady-state electrophysiological characteristics

Pre-steady-state relaxation’s resulting from the applica-tion of voltage steps to the voltage-clamped cell have been


first reported for the cloned Na1-glucose cotransporter(SGLT-1; Refs. 45, 75, 167) expressed in oocytes and subse-quently for many other Na1-solute cotransport systems (e.g.,Ref. 117). They permit an identification of partial reactionswithin the transport cycle. Such measurements were alsoperformed with the rat (types IIa and IIb) and flounderisoforms (type IIb) of the type II Na-Pi cotransporters ex-pressed in oocytes (122, 124, 125, 126). Figure 9 provides anexample from a study on the rat type IIa cotransporter (122).Application of a voltage step in the presence of 96 mM Na1,and in the presence or absence of saturating Pi, leads to

current transients that are primarily due to charging oocytemembrane capacitance (Fig. 9A). Recording at a higher gainresults in a slower relaxation to the steady state in theabsence of Pi (Fig. 9B). Subtraction of the curves obtained inthe presence/absence of Pi shows transporter cycle-depen-dent relaxation currents (Fig. 9C), whose magnitude couldbe directly related to the magnitude of Pi-induced steady-state currents in oocytes expressing different amounts ofcotransporters at their surface (122). Furthermore, the Pi-induced effects on the pre-steady-state relaxation shows thesame saturation characteristics (apparent Km) as that ob-

FIG. 8. Electrogenic behavior of type IIa Na-Pi cotransporter (rat, NaPi-2). A: basic scheme for the two-electrodevoltage-clamp system as used in oocytes expressing the transporter. Vc, current potential; Im, membrane current. B: basicexperimental recording. When Pi is suppressed in the presence of Na1, an inward current is recorded; its amplitudedepends on the transmembrane holding potential (steady-state current recordings). Vh, holding potential. C: dose-response data obtained at 250 mV (holding potential) by altering Pi concentrations in the superfusate containing either96 mM Na1 (■) or 50 mM Na1 (h), resulting in Michaelis constant (Km) values for Pi interaction (see E). Ip, pipettecurrent. D: dose-response data obtained at 250 mV (holding potential) by altering Na1 concentration at either 1.0 mM Pi (■)or 0.1 mM Pi (h), resulting in Km values for Na1 interaction (see F). E: dependence of Km values for Pi interaction on differentholding potentials (see C). F: dependence of Km values for Na1 interaction on different holding potentials (see D).


served for Pi interaction in steady-state measurements (122).These current transients are consistent with the transloca-tion of charged entities within the transmembrane electricfield. The voltage dependence of the time constants of re-laxation and equivalent charge associated with the relax-ation can be obtained from measurements after voltagejumps of different magnitudes from the same holding poten-tial. With the Boltzmann equation, the transporter number,turnover number, and the apparent valency for the chargemovement can be calculated, taking also into account Pi-induced steady-state currents obtained in the same oocytesas the pre-steady-state measurements. A value of 25 s21 wasestimated for the turnover of the rat type IIa Na-Pi cotrans-porter at 2100 mV, in agreement with that measured forother Na-solute transporters (e.g., Refs. 45, 75, 117, 167); theapparent valency of the charge translocated within the re-laxation cycle is 21 (122).

3. Kinetic scheme

Above steady-state measurements (electrophysiolog-ical and tracer studies) as well as electrophysiological

pre-steady-state measurements led to the formulation of akinetic scheme as shown in Figure 10 (122). The emptycarrier has a valency of 21 and can interact at the extra-cellular surface with one Na1. Translocation of the emptycarrier as well as interaction with one Na1 are voltage-dependent partial reactions within the transporter cycle.The carrier loaded with only 1 Na1 can translocate (“slip-page”); this is an electroneutral process. The Na1 inter-action then allows interaction with Pi (or with the inhib-itor PFA) at the extracellular surface. Finally, the fullyloaded carrier is formed by interaction with two addi-tional Na1. Translocation of the fully loaded carrier isagain an electroneutral process. We have no informationfor distinct steps occurring at the cytoplasmic surface butassume a transition from the fully loaded to the emptystate (mirror symmetry). A net inward movement of onepositive charge occurs per transport cycle, due to thereorientation of the charged (21) empty carrier. Althoughthe carrier might interact with mono- or divalent Pi, apreferential transport of divalent Pi prevails under mostphysiological and experimental conditions.

FIG. 9. Pre-steady-state charge movements related to type IIa Na-Pi cotransporter (rat NaPi-2). A: voltage stepbetween 2100 to 0 mV applied to oocyte and corresponding membrane currents superimposed for 96 mM Na1 with andwithout 1 mM Pi. The small displacement in the baseline preceding the step represents the steady-state induced currentat 2100 mV. B: magnified view of current records in A showing a clear difference in the relaxations depending on thepresence or absence of substrate. Thin trace is for 1 mM Pi; bold trace is for 0 mM Pi. C: difference between records inB, showing the relaxations superimposed on the Pi-induced steady-state currents. Note that the rate of relaxation for theforward step (2100 to 0 mV) is faster than the return relaxation, indicating that the relaxation time constant dependson the target potential that is a characteristic of voltage-dependent charge movements. The area under each relaxation,determined by numerical integration, is a measure of the apparent charge translocated and should be equal for thedepolarizing and hyperpolarizing steps. For further discussion, see text and included references.


4. pH dependence of transport

Proximal tubular brush-border membrane Na-Pi

cotransport is increased by increasing pH (e.g., Refs.14, 52, 55, 80, 327, 328, 334, 352). Studies with brush-border membrane vesicles provided evidence that thisphenomenon is to a significant extent explained by acompetition of protons with sodium for an interactionwith the carrier (14). Preferential transport of divalentPi also contributes to the observed pH dependence(352). Steady-state electrophysiological measurementsalso suggested a competitive interaction of H1 with theNa1-binding site(s) (59). Pre-steady-state measure-ments provided evidence for an additional direct effectof H1 on the carrier, on the reorientation of the emptytransporter (124). As indicated in Figure 10, the pHdependence of the carrier includes a kinetic effect onthe reorientation of the free carrier as well as compe-tition for Na1 binding. Thus the pH dependence of typeIIa Na-Pi cotransport activity is in part but cannot befully explained by preferential transport of divalent Pi.More recent studies indicated that this pH dependenceis determined by basic amino acid residues in the thirdextracellular loop (96).

B. Altered Expression as the Basis for Altered

Pi Reabsorption

With the cloning of the type IIa Na-Pi cotransporter(Npt 2), the key player in brush-border membrane Na-Pi

cotransport (see above and below), the tools were avail-able for a more detailed analysis of cellular/molecularmechanisms involved in physiological/pathophysiologicalalterations of proximal tubular Na-Pi cotransport.

The Npt 2 knockout mice (25, 178) documentedclearly the importance of the type IIa Na-Pi cotransporterin renal Pi handling and in the overall maintenance of Pi

homeostasis. In Npt 2 knockout mice, other Na-Pi co-transporters (e.g., Npt 1, Glvr-1, and Ram-1) are notupregulated (178). Furthermore, renal handling of Pi is inNpt 2 knockout mice unaffected by PTH and Pi diet (178;N.-X. Zhao and H. S. Tenenhouse, personal communica-tion). In addition to the renal defects, Npt 2 knockoutmice have intrinsic skeletal abnormalities most likely re-lated to the lack of type IIa Na-Pi cotransporter in oste-oclasts (145a).

Alterations in proximal tubular Na-Pi cotransport ac-tivity after induction of altered renal Pi handling in theintact organism or in the OK cell tissue-culture model arealways associated with an altered apical membrane ex-pression of the type II Na-Pi cotransporter protein (fastingas an exception). In the following sections, we might firstdescribe situations of altered expression and discuss thencellular mechanisms leading to altered brush-border ex-pression.

1. Ontogeny/aging

Pi reabsorption in the kidney shows a strong ontoge-netic/developmental as well as age-dependent behavior

FIG. 10. Kinetic models for type IIa Na-Pi cotransporter. A: model derived from studies on rat renal brush-bordermembrane vesicles. [From Gmaj and Murer (138). This model was published in Physiological Reviews in 1986.] B: anordered kinetic model for type IIa Na-Pi cotransporter. This scheme shows the partial reactions that have been identifiedby the steady-state and pre-steady-state analyses (see Figs. 8 and 9). The shaded reactions represent the two voltage-dependent steps that contribute to the pre-steady-state charge movements. Substrate concentrations are assumed tomodify the binding rate constants only. Protons modulate the voltage-dependent rate constant associated with the emptycarrier as well as the final Na1-binding step, whereas phosphonoformic acid (PFA) most likely competes directly withPi binding. The binding/debinding steps on the cytosolic side have not yet been characterized. For further details, see textand included references (see also Refs. 122, 229).


(e.g., Refs. 20, 66, 160, 161, 383). Age dependence was alsoobserved at the level of the type IIa Na-Pi cotransporterprotein expression (365). In kidneys of newborn rats,expression of the type IIa Na-Pi cotransporter was ob-served in differentiated juxtamedullary and intermediatenephrons only and was absent in nephron “Anlagen” inthe outer cortex (S-shaped bodies; Ref. 397). After com-pletion of nephron formation, during suckling, expressionof the transporter was similarly high in the brush-bordermembrane of all nephron generations. In weaning, theexpression pattern resembled that in adults (238, 397),i.e., type IIa abundance decreased in the brush-bordermembrane of superficial and midcortical nephrons. Theimmunohistochemical data suggest that, as soon asnephrogenesis is completed, the type IIa transporter inthe kidney is functional. As indicated above, in weaningmice, a specific transcription factor (TFE3) might contrib-ute to the expression of the Npt 2 gene, especially in lowPi conditions (212a).

A specific type IIa-related Na-Pi cotransporter proteinwas postulated to account for high Pi transport rates inweaning rats (363, 364). Evidence for this postulate wasobtained by antisense experiments and transport expres-sion in X. laevis oocytes. When mRNA isolated fromkidney cortex of rapidly growing rats was treated withtype IIa transporter antisense oligonucleotides, or de-pleted of type IIa specific mRNA by a subtractive hybrid-ization procedure, Na1-dependent Pi uptake was still de-tected in injected oocytes. The type IIa transporter-depleted mRNA contained a mRNA species that has somesequence homology to the type IIa transporter encodingmessage. This interesting observation was not followedup, and at present, the identity of this growth-relatedtransporter is not clear and/or remains hypothetical.

Tubular Pi reabsorption decreases during aging ashas been indicated by metabolic balance studies, clear-ance studies, and studies with isolated vesicles (66, 160,161, 214). This decrease is due to a reduction in the Vmax

without a change in the apparent Km for Pi of brush-border membrane Na-Pi cotransport. The type IIa Na-Pi

cotransporter brush-border membrane protein mRNAabundance decreased approximatively twofold when3-mo-old rats are compared with 12- to 16-mo-old rats, inparallel to the decrease in brush-border membrane Na-Pi

cotransport activity (365).

2. Regulatory control

As already indicated, physiological (and pathophysi-ological) alterations in renal handling of Pi are related toan altered brush-border content of the type IIa Na-Pi

cotransporter protein.This was observed for altered brush-border mem-

brane Na-Pi cotransport activity as observed in responseto altered dietary Pi intake (e.g., Refs. 48, 53, 61, 76, 78,

204, 243, 245, 246, 348, 370–372, 387, 405, 415, 434; forreview, see Ref. 283). An increase in brush-border mem-brane Na-Pi cotransporter activity in response to a low-Pi

diet correlated with an increase in type IIa transporterprotein in Western blots and in immunohistochemistry(e.g., Refs. 48, 243, 258). The histochemical analysis sug-gests a “recruitment” phenomenon (Fig. 11; Ref. 348). Inanimals fed a high (or normal)-Pi diet, expression oftransporter is mostly in deep (juxtamedullary) nephrons,and in animals fed a low-Pi diet, the transporter is alsohighly expressed in superficial nephrons. The diet-in-duced changes are observed at the functional and proteinlevels within the first 2 h, but feeding a low-Pi diet forprolonged time periods also leads to a change in type IIaNa-Pi cotransporter mRNA that is not observed after shorttime periods. Refeeding high-Pi diets to animals adaptedchronically to low-Pi diet results in a reversal of thisphenomenon, first a decrease in brush-border expressionof the transporter protein without a decrease in specificmRNA content (243; see also Refs. 27, 199, 376, 387, 415).The type I Na-Pi cotransporter does not show such alter-ations (e.g., Refs. 48, 405). Similar findings could also beobtained in OK cells, i.e., an increase in type IIa proteincontent in response to a low-Pi media occurred withinhours, in parallel with an increase in Na-Pi cotransportactivity (e.g., Refs. 266, 309). The findings on the responseof OK cell-specific type IIa transporter mRNA to low-Pi

diet are controversial (266, 309, 353, 354). In our labora-tory, the low-Pi diet-induced changes in specific mRNAcontent were rather small, and transport adaptation wasnot prevented by actinomycin D (41, 266, 309). Further-more, the adaptive phenomena were also observed in OKcells with the transfected rat type IIa cotransporter on theprotein but not mRNA level (309).

An altered PTH status in the animal is associatedwith an altered brush-border expression of the type IIaNa-Pi cotransporter as analyzed by Western blots of iso-lated brush-border membrane vesicles or by immunohis-tochemical staining on kidney sections (210). Injection ofPTH in rats or mice leads within minutes to a reduction inbrush-border membrane transporter content without aconcomitant loss of other brush-border proteins, e.g., theNa-sulfate cotransporter protein (Fig. 12; Refs. 210, 257,259; see also Ref. 265). A prolonged increase in PTH canalso lead to a decrease in type II Na-Pi cotransportermRNA content (210). Also in OK cells, PTH leads to adecrease in the apical expression of the intrinsic andtransfected rat type IIa Na-Pi cotransporters (235, 308–311).

A role for insulin has been postulated for the adaptiveresponse of the proximal tubule to changes in dietary Pi

intake (247). In vitro and in vivo experiments providedevidence that insulin stimulates brush-border membraneNa-Pi cotransport. Streptozotocin-induced diabetes is as-sociated with a reduced proximal tubular Pi reabsorption,


a decreased type I Na-Pi cotransporter content, and nochange in either type IIa or III Na-Pi cotransporter content(247). It is, however, questionable whether phosphaturiain diabetes can be explained by a reduced type I Na-Pi

cotransporter expression. Phosphaturia could rather beexplained by an inhibition of type IIa Na-Pi cotransportactivity related to a competition for driving forces (in-crease in glucose reabsorption; see Refs. 22, 392). Instreptozotocin-induced diabetes, the adaptive response(dietary intake) of the type IIa Na-Pi cotransporter wasprevented (1, 7, 358).

Thyroid hormone (3,39,5-triiodothyronine; T3) in-creases proximal tubular brush-border membrane andOK cell apical Na-Pi cotransport (e.g., Refs. 31, 107, 118,213, 433); a parallel increase in PFA binding sites sug-

gested an increased brush-border membrane content oftransporters (433, 434). Euzet et al. (119) have shownan important role for T3 in the maturation of the typeIIa Na-Pi cotransporter (119). It was shown that phys-iological doses of T3 lead in rats to an increase inbrush-border membrane Na-Pi cotransport activity,type IIa transporter protein content, and specific mRNAcontent (6). The stimulatory effect of T3 is less evidentin aging animals (6). Nuclear run-on experiments pro-vided evidence that these effects are due to an in-creased transcription of the transporter gene (6). In OKcells, the stimulatory effect of T3 was completely pre-vented by the transcriptional inhibitor actinomycin D(367).

IGF-I directly stimulated OK cell Na-Pi cotransport

FIG. 11. Effect of dietary Pi intake on type IIa Na-Pi cotransporter (rat, NaPi-2) mRNA (A–C) and protein (D–I). Left:animals fed chronically a high-Pi diet (1.2% Pi) and switched for different time periods to low-Pi diets (0.1% Pi). Right:animals fed chronically a low-Pi diet (0.1% Pi) and switched for different time periods to high-Pi diets. Magnifications:A–F, 345; G–I, 31,200. Bars: A–F, 200 mm; G–I, 5 mm. For further details, see text and included references. [Adaptedfrom Ritthaler et al. (348).]


associated with an increase in specific type IIa Na-Pi

cotransporter content, an effect most likely involving ac-tivation of tyrosine kinase activity (193).

EGF caused a time- and dose-dependent decrease inOK cell Na-Pi cotransport rate, an effect associated with adecrease in type IIa Na-Pi cotransporter mRNA abun-dance (15). This observation is in agreement with findingson weaned and suckling rats but in disagreement withobservations on isolated perfused rabbit proximal tubuleswhere EGF stimulates Na-Pi cotransport (17, 336).

Glucocorticoid administration to rats and neonatalrabbits decreased rat brush-border membrane Na-Pi co-transport activity and kidney cortex type IIa transportercontent (16, 244, 254, 320). Also in OK cells glucocorti-coids led to a reduction in Na-Pi cotransport activity andassociated transporter protein content (192; see also Ref.156a).

Fasting-induced phosphaturia leads to decreased ex-pression of type I Na-Pi cotransporter mRNA but to nochange in type IIa mRNA content (247). However, it is

unlikely that a decreased type I Na-Pi cotransporter con-tent accounts for the phosphaturia observed in fastingconditions (28). As indicated above, the type IIa Na-Pi

cotransporter determines overall renal Pi handling.Acid-base changes also induce alterations in the ex-

pression of the type IIa Na-Pi cotransporter. Chronic met-abolic acidosis in rats significantly decreased brush-bor-der membrane Na-Pi cotransport activity, associated witha decrease in type IIa cotransporter protein and mRNAcontent (11). At the onset of acute (6 h) metabolic acido-sis, changes in transport activity and brush-border trans-porter protein content were not paralleled by a change incortical tissue type IIa Na-Pi cotransporter mRNA or pro-tein (11). This may indicate that in acute situationschanges in membrane trafficking of the type IIa Na-Pi

cotransporter (see below) contribute to the acid-inducedchanges. Interestingly, in OK cells, we have found anopposite effect of exposure to an acid medium (192, 194).Transport activity, transporter protein, and mRNA con-tent were all increased; the increase was, however, pre-

FIG. 12. Parathyroid hormone(PTH)-dependent retrieval of type IIaNa-Pi cotransporters from the brush-border membrane. Rats with removedparathyroid glands (PTX) were in-jected with PTH, and kidney sliceswere then stained for the type IIaNa-Pi cotransporter (NaPi-2) or forthe Na-SO4 cotransporter (NaSi-1;Ref. 265). It was seen that the type IIaNa-Pi cotransporter is removed fromthe brush-border membrane in re-sponse to PTH administration. Atearly time periods (35 min), a subapi-cal appearance of the transporter isvisible. The Na-SO4 cotransporter isnot removed from the brush-bordermembrane in response to PTH admin-istration. For further reading, see textand included references.


vented or reversed by adding glucocorticoids (192). Thisindicates that the above observations in rat might be theresult of acid base-induced changes in glucocorticoid lev-els rather than of direct cellular effects (see also Refs. 47,127).

3. Acquired alterations

Proximal tubular brush-border membrane Na-Pi co-transport is sensitive to intoxication by heavy metals (5,141, 255, 321). A direct acute inhibitory effect of Hg21,Pb21, and Cd21 has been shown in X. laevis oocytesexpressing the type IIa Na-Pi cotransporter (408). Hg21

and Cd21 effects were mostly on Vmax effects, but Pb21

also increased the apparent Km for Pi (408). It will be ofinterest to determine whether the described inhibitoryeffect can be assigned to specific sulfhydryl groups, whichwould then also explain the observations in brush-bordermembranes (Refs. 255, 321; e.g., also in studies with cis-platin, Ref. 90). In vivo intoxication of rats with cadmiumreduced brush-border membrane Na-Pi cotransport rateaccompanied by a loss of the type IIa Na-Pi cotransporterprotein (169).

4. Genetic abnormalities

Among the different genetically determined alter-ations in renal Pi handling, only X-linked defects havebeen characterized at the level of brush-border membranePi transporters. Studies on the murine Hyp and Gy ho-mologs of the human disease X-linked hypophosphatemiahave identified a specific reduction in brush-border mem-brane Na-Pi cotransport activity that was associated witha reduction in type IIa Na-Pi cotransporter protein andspecific mRNA content, thereby accounting for the Pi leakin affected mice (27, 384, 385, 387, 389, 393; see also Refs.87–89). Because the Npt 2 gene does not map to theX-chromosome (see above and Refs. 222, 223, 437), adefect in a gene on the X-chromosome must influence therenal brush-border expression of the type IIa Na-Pi co-transporter (for review, see Ref. 384). This mutant genehas been identified in affected humans (106, 110, 112, 185)by linkage/positional cloning and subsequently in Hyp

and Gy mice (26, 373); it was designated PHEX/Phex

(formerly PEX/Pex) to signify phosphate regulating genewith homology to endopeptidases on the X-chromosome.The PHEX/Phex protein is not expressed in the kidney butrather in bones (26). On the basis of its homology to amembrane-bound endopeptidases, it is postulated thatPHEX/Phex is involved in the processing of humoral fac-tor(s) (e.g., phosphatonin and stanniocalcin 1/2; see alsoRefs. 72, 105, 111, 189, 224, 225, 272) which regulate renalPi handling by altering the type IIa Na-Pi cotransporterexpression, and Pi metabolism in general (for review, seeRefs. 110, 112, 295, 384). In Hyp mice, a decreased tran-scription rate of the type IIa Na-Pi cotransporter gene was

observed (89). In Hyp and Gy mice, different gene dele-tions were identified: a 39-deletion in Hyp and a 59-dele-tion in Gy (26, 273).

C. Cellular Mechanisms in the Control of Type II

Na-Pi Cotransporter Expression

As discussed in section VB, changes in renal Pi han-dling are attributable, for the most part, to altered brush-border membrane expression of the type IIa Na-Pi co-transporter. It is apparent that alterations in the type IIaNa-Pi cotransporter expression occur independently oftranscriptionally regulated changes in mRNA, perhapswith the exception of T3 (6, 367), 1,25(OH)2D3-mediatedeffects (8, 380), or prolonged feeding of low-Pi diets inweaning mice (212a). If changes in type IIa Na-Pi cotrans-porter mRNA are observed, they are either rather small orthey occur only after prolonged stimulation (e.g., Refs.210, 243), i.e., after changes in the specific transporterprotein content, suggesting that changes in mRNA repre-sent a phenomenon secondary to the primary event, i.e.,downregulation or upregulation of brush-border expres-sion of type IIa Na-Pi cotransporter protein. In agreementwith this apparent lack of transcriptional control mecha-nisms are the negative observations with promoter/re-porter gene studies in OK cells (e.g., Refs. 174, 176),where with the above-mentioned exceptions of alter-ations in 1,25(OH)2D3 (380), of the low-Pi diet-dependentincrease in TFE3 in weaning mice (212a), or in ambientbicarbonate/carbon dioxide tension (194) of the media, noactivation of the NPT 2/Npt 2 promoter could be detected.Therefore, it is appropriate to restrict the discussion pri-marily to mechanisms leading to altered membrane ex-pression, i.e., mainly to membrane retrieval and mem-brane insertion of the type IIa Na-Pi cotransporter (forreview, see Refs. 286, 288). The crucial role of “membranetrafficking” in the control of brush-border membrane ex-pression of the type IIa Na-Pi cotransporter offers thepotential for physiological/pathophysiological control ofNa-Pi cotransport involving alterations in the participatingcomplex machinery. In this way, for example, an endoso-mal chloride channel defect in Dent’s disease may affectmembrane insertion and thus lead to phosphaturia (252,253, 391).

1. Membrane retrieval

The phenomenon of membrane retrieval of the trans-porter can be best studied in PTH-induced or high-Pi

diet-induced downregulation of brush-border membraneNa-Pi cotransport activity and type IIa Na-Pi cotransporterprotein abundance (see Figs. 11 and 12 and Refs. 208, 210,257, 259; see also Refs. 180, 209, 211, 306). The data fromimmunohistochemical (immunogold) studies on PTH-in-duced internalization are summarized in Figure 13 (398).


In rats, infusion of PTH or feeding high-Pi diets leads to atransient accumulation of type IIa Na-Pi cotransporters inthe so-called subapical vacuolar apparatus (83, 164). In-ternalization can occur at intermicrovillar clefts via clath-rin and adapter protein (AP-2) containing membrane car-gos (398). The same endocytic structures can be filled byhorseradish peroxidase injected into the animals beforePTH treatment (398). Thus type IIa Na-Pi cotransportersare internalized via the same pathway as soluble proteins.Evidence from experiments with cholchicine-pretreatedrats indicates that there might be an additional “un-known” internalization step, separate from the intermi-crovillar clefts (259; Fig. 13). Morphological and biochem-ical data suggest that internalized type IIa Na-Pi

cotransporters are directed to the lysosomes for degrada-tion (212, 311, 398). Although recycling of transportersback to the membrane cannot be entirely excluded, underthe experimental conditions applied, no evidence for sucha reutilization of internalized transporters could be ob-tained in studies on OK cells (262, 311). Surface biotiny-lation experiments performed in OK cells provided directevidence that the increase in intracellularly located trans-porters after PTH treatment is related to a decrease of

transporters at the cell surface (191). An alternate mech-anism that would lead to reduced transporter expressionat the surface would have been degradation at the mem-brane site and/or a reduced insertion rate (see sect. VC2)to account for intracellular accumulation. Obviously, thisphenomena of downregulation requires a complex ma-chinery that provides specificity for the transporter mol-ecule and specific signaling events. These issues are dis-cussed in the following sections.

2. Membrane insertion

This phenomenon can be best analyzed in ratsadapted to high-Pi diet and fed “acutely” a low-Pi diet (e.g.,Fig. 11; e.g., Refs. 243, 258). The increase in brush-bordertype IIa Na-Pi cotransporter content is observed within1–2 h and can also be observed in OK cells after exposureto media with adjusted Pi contents (309). This rapid in-crease occurs independently of any change in specifictype IIa Na-Pi cotransporter mRNA content (243, 309). InOK cells, this adaptive response could be prevented byblocking protein synthesis at the translational level (e.g.,Ref. 41). In contrast, in animal models, pretreatment with

FIG. 13. “Mechanisms” of type IIa Na-Pi cotransporter internalization. A and B: double labeling of Na/Pi-2 (rattransporter) and horseradish peroxidase (HRP; fluid-phase marker) by the immunogold labeling technique on ultrathincryosection of rat kidneys after HRP and parathyroid hormone (PTH) injection. Gold particles (12 nm) bound to NaPi-2are located at the microvillar membrane (MV) and at intermicrovillar clefts (arrows). NaPi-2 and HRP (gold particles 6nm) are colocalized in the clefts (arrows) and in subapical vesicles of the vacuolar apparatus (A and B). Magnifications:A, 3;23,000; B, 3;33,000. Bars: ;1 mm. C: proposed pathway of trafficking of NaPi-2 cotransporter in proximal tubulecells. Newly synthezised NaPi-2 seems to travel to the brush-border membrane via the subacpical compartment (SAC;corresponding to the vacuolar apparatus). During downregulation of the protein in the brush-border membrane,internalized NaPi-2 seems to follow the same general endocytotic pathway like HRP via small endocytotic vesicles (SEV),subapical compartment (SAC), and large endosomal vesicles (LEV); finally, the proteins will be degraded in lysosomes(Ly). This pathway includes also the participation of clathrin and adaptor protein 2 (AP-2). For further details, see textand included references. The Na/Pi cotransporter may also be internalized via a non-clathrin-mediated pathway (dashedline; see text). [Adapted from Traebert et al. (398).]


cycloheximide did not prevent upregulation of brush-bor-der membrane type IIa Na-Pi cotransport activity andprotein (242, 245, 246, 258). However, it has not beenshown whether protein synthesis is indeed blocked inproximal tubular cells after intraperitoneal injection ofcycloheximide. Upregulation of Na-Pi cotransporter activ-ity and type IIa transporter protein content is obviouslyalso observed after recovery from PTH inhibition. In OKcells it was found that the recovery of Na-Pi cotransportactivity and type II Na-Pi transporter protein is entirelydependent on protein synthesis (262, 311). In chronicparathyroidectomized rats, a specific upregulation of typeIIa Na-Pi cotransporter protein was also observed in theabsence of changes in specific mRNA content (210).

3. Involvement of microtubules

In contrast to the known dependence of the endocy-tosis of soluble proteins on an intact microtubular net-work (115), the internalization of type IIa Na-Pi cotrans-porters in response to either PTH or acute high-Pi dietfeeding was not impaired (258, 259) by agents disturbingmicrotubules. Similar observations were made in OK cells(158). In contrast, the intracellular routing of the trans-porter to the lysosomes depends on an intact microtubu-lar network (258, 259). Membrane insertion of the trans-porter, observed after acute administration of a low-Pi

diet, requires an intact microtubular network (258), inagreement with the important role of microtubules forapical routing of post-Golgi vesicles and for the mainte-nance of the dense tubular subapical network (115, 270).It is of interest that PTH and altered medium Pi contentwere found to have profound effects on the cytoskeletonof cultured proximal tubular cells (139, 258, 259, 304).

4. Signaling pathways

A) CAMP AND DIACYLGLYCEROL/INOSITOL TRISPHOSPHATE (CAL-CIUM). cAMP- and diacylglycerol (DAG)/inositol trisphos-phate (IP3)-related signaling pathways in the regulation ofbrush-border membrane type II Na-Pi cotransporter activ-ity and expression have been studied in relation to PTH-dependent regulation of proximal tubular Pi reabsorption.PTH and PTH-related peptide receptors, respectively, arelocated in both apical and basolateral membranes of prox-imal tubular epithelial cells and of OK cells (e.g., Refs.68–70, 73, 85, 86, 186, 200, 215, 261–264, 331–333, 341, 342,346; for review, see Refs. 280, 283). The PTH receptor cansignal through activation of both adenylate cyclase andphospholipase C, generating the second messengerscAMP, IP3, a rise in intracellular Ca21, and DAG (e.g.,Refs. 2, 183, 271). The PTH-(1O34) analog activates bothsignaling pathways, whereas the PTH-(3O34) analog onlyactivates the cAMP-independent signaling pathway (e.g.,Refs. 85, 86, 235, 308). Although pharmacological activa-tion of either pathway separately leads to an inhibition of

apical Na-Pi cotransport activity associated with a de-crease in brush-border membrane type IIa Na-Pi cotrans-porter protein content (e.g., Refs. 235, 264, 308), thereseems to be an interdependence of the two regulatorypathways. In OK cells, abolition of the cAMP/protein ki-nase A pathway prevents PTH inhibition (267, 268, 357);similarly, inhibition of the DAG/protein kinase C pathwayalso reduces PTH inhibition of the Na-Pi cotransporter(330, 333). Furthermore, in OK cells, the maximal inhibi-tion induced by PTH-(3O34) is only ;50% of the maximalPTH-(1O34)-induced inhibition (85, 86, 235, 308).

Although most observations on PTH-dependent sig-naling mechanisms were derived from studies with OKcells, similar mechanisms may be operative in the proxi-mal tubule; PTH-(3O34) similar to PTH-(1O34) leads to areduction in apical type IIa Na-Pi cotransporter content inintact rats and in vitro perfused mice proximal tubules(399). Furthermore, the experiments with the isolatedperfused tubules showed that PTH-(1O34) leads to areduction in brush-border transporter content after per-fusion either through the lumen or basolateral side,whereas PTH-(3O34) only induces this reduction afterapical perfusion only (399). It is of interest that earlierstudies on OK cells have also provided evidence for asym-metrical signaling mechanisms in PTH control of apicalNa-Pi cotransport (341, 342). The experiments with iso-lated perfused tubules also documented the involvementof protein kinases A and C in internalization of the apicalNa-Pi cotransporter (399).

The events after second messenger generation lead-ing to inhibition and/or retrieval of the type IIa Na-Pi

cotransporter are not clear. Obviously, phosphorylationevents do play a role, but there is no direct evidence thataltered phosphorylation of the type IIa Na-Pi cotrans-porter protein is essential in the regulation (42, 152, 182).Recently, in studies in OK cells, it has been shown that thetype IIa transporter is a phosphoprotein. However, PTH-induced alterations in the type IIa Na-Pi cotransporterphosphorylation are difficult to document (M. Jankowski,H. Hiefiker, J. Biber, and H. Murer, unpublished observa-tions). Because there are several potential phosphoryla-tion sites within the protein sequence, changes at individ-ual sites need to be analyzed. It is of interest that in X.

laevis oocytes, activation of protein kinase C leads to aninternalization of the transporter and to an associatedinhibition of transport (125). This inhibition/internaliza-tion is not prevented after mutagenesis of known kinaseconsensus sites, indicating that at least in this experimen-tal model, the phosphorylation consensus sites are notinvolved in this regulation (165). Finally, it is reasonableto assume that PTH-induced regulatory phosphorylationevents are not at the level of the transporter but rather atinteracting proteins required for its regulation (see sect.VC4D). In this respect it is of interest that regulation byPTH and/or pharmacological activation of protein kinases


A/C requires the correct cellular context; transfectionexperiments showed that only the OK cells provide thecorrect cellular environment for this regulation of thetype IIa Na-Pi cotransporter (Z. Karim-Jiminez, N. Her-nando, H. Murer, and J. Biber, unpublished data). Suchdependence on an interacting protein has also beenshown for brush-border membrane Na/H exchange(NHE-3) and its kinase A-induced inhibition (412, 413,428; see also Ref. 293).

Extracellular cAMP (intratubular) is known to inhibitproximal tubular and OK cell apical Na-Pi cotransport.This effect can be related to specific receptor interactionsand intracellular signaling or more likely to luminal hy-drolysis of cAMP followed by uptake of adenosine, whichmight after incorporation into ATP be the source foradditional intracellular cAMP (130, 131, 132; see also Refs.274, 361). A role of intratubular cAMP, generated in theliver after stimulation by glucagon, has been suggested forin vivo control of Pi reabsorption (3). It is unclear at thismoment how these observations and interpretations canbe reconciled with the stimulatory effect of adenosine ontubular Pi reabsorption in rats (312).

Alterations in cytoplasmic calcium concentrationsper se are not sufficient/important in PTH-induced signal-ing of OK cell apical Na-Pi cotransport; this has beendocumented using Ca21-clamping protocols (332). Saxenaand Allon (353) have postulated that an increase insteady-state cytosolic calcium plays a role in “chronic”adaptation of OK cell Na-Pi cotransport to low-Pi media.

B) PI ADAPTATION (PI SENSING). As discussed above,brush-border membrane type IIa Na-Pi cotransporter pro-tein content, and thus proximal tubular Pi reabsorption,responds within hours to alterations in dietary Pi intake(242, 243, 258). This phenomenon is also observed in OKcells, induced by alteration in media Pi content (41, 309).In the animal models the adaptive phenomena might be inpart related to alterations in different humoral factors andlocal auto/paracrine factors, although PTH can be ex-cluded as a major factor (see above; for review, see Ref.37). In this regard, it is interesting that intravenous injec-tions of Pi are sufficient to induce alterations in brush-border membrane Na-Pi cotransport rate in thyroparathy-roidectomized rats (76; for review, see Refs. 37, 46, 283).Because alterations in dietary Pi intake are paralleled byrapid changes in plasma Pi concentration and thus offiltered Pi load (61, 316), the question of direct Pi sensingby the cells has to be considered, as indicated by theexperiments on OK cells. Experiments with OK cells pro-vided an interesting clue on the potential mechanisms inPi sensing by the proximal tubular cell. In cells grown onpermeant filters, depletion of Pi at the apical site wassufficient to provoke an adaptive increase of apical Na-Pi

cotransport rate, whereas removal of Pi only from thebasolateral cell surface was without effect (339, 340).Thus there is the possibility of a Pi sensing mechanism at

the apical surface. Because the Ca21 sensor (Ca21 recep-tor CaR) is expressed at the apical cell surface of proxi-mal tubular cells, it may “indirectly” be involved in tubularPi sensing, because changes in Pi and Ca21 concentra-tions are interdependent (51, 347). Another possibility isthat the rate of Pi entry at the apical cell surface contrib-utes to Pi sensing. It was observed that inhibition of OKcell Na-Pi cotransport after prolonged exposure to PFAresults after PFA removal in an “adapted” state of Na-dependent Pi uptake (J. Forgo and H. Murer, unpublishedobservations). Similarly, reduced Pi entry in response toPTH inhibition could provide a signal for the preferredresynthesis of the type IIa cotransporter resulting in re-covery of Na-Pi cotransport rates after removal of PTH. Inthis case, the higher transport rates after recovery wouldthen serve as feedback to again slow down the rate oftransporter resynthesis. Finally, the adaptive response ofNa-Pi cotransport rate to low Pi intake (or low-Pi media)may also involve a more general signaling that is relatedto changes in intracellular Pi metabolism after lowering ofapical (and/or basolateral) Pi (20, 102; see also Ref. 38). Inaddition, alterations in cytosolic Ca21 concentrationswere postulated to be part of the Pi-sensing mechanisms(353).

C) UBIQUITINYLATION. Membrane retrieval is involved inregulation of the type IIa Na-Pi cotransporter. Membraneretrieval of a variety of membrane proteins, includingmembrane receptors and perhaps also transporter pro-teins, e.g., the epithelial Na1 channel, can depend onubiquitinylation, which can be a signal for endocytosisfollowed by lysosomal and/or proteosomal degradation(368, 369; see also Ref. 173). In OK cells, lysosomal deg-radation, but not proteosomal degradation of the type IINa-Pi cotransporter protein, is involved in its PTH-depen-dent downregulation (311). Proteosomal (and lysosomal)degradation seems also to be involved in the basic turn-over of the OK cell type IIa cotransporter (311). Ubiquiti-nylation of the transporter itself or of interacting proteinscan be involved in membrane retrieval and/or targetingthe type IIa transporter to the degradation machinery.

D) INTRACRINE REGULATION. An intracrine regulationpathway was postulated to control apical expression oftype IIa Na-Pi cotransporter (107; see also Refs. 29, 30,207, 427). Different metabolic and/or hormonal stimulimay lead to the synthesis of cyclic ADP-ribose, which inturn may initiate an intracellular regulatory cascade, withthe release of Ca21, activation of a Ca21/calmodulin-de-pendent protein kinase, and changes in the turnover ofthe transporter at the apical membrane (107).

E) PHOSPHATIDYLINOSITIDE 3-KINASE. Phosphatidylinosit-ide 3-kinase (PI 3-kinase) seems to be involved in variousmembrane trafficking processes. In OK cells, inhibition ofPI 3-kinase leads to a reduction of endocytosis of albumin(54). However, endocytosis of type IIa Na-Pi cotrans-porter in OK cells and its degradation by PTH are not


prevented by inhibition of PI 3-kinase (307). On the otherhand, inhibition of PI 3-kinase retards the recovery fromPTH inhibition and reduces transport activity and trans-porter protein content in control cells (307). Thus PTH-induced endocytosis of the type IIa Na-Pi cotransportoccurs via a different pathway than albumin endocytosis.PI 3-kinase activity may be involved in the resynthesis/reinsertion of apically located transporters.

F) TYROSINE KINASE ACTIVITY. In OK cells, vanadate, apotent inhibitor of protein tyrosine phosphatases, mim-icks the stimulatory effect of IGF-I on Na-Pi cotransportactivity (63, 65). Similarly, IGF-I and vanadate increasethe membrane abundance of type IIa Na-Pi cotransporterprotein content (193). The effects on transporter proteincontent could not be prevented by actinomycin D andcycloheximide (193). The latter observation led to theconclusion that tyrosine kinase-dependent mechanismsmay control the membrane stability (i.e., reduce the re-trieval rate) of the type IIa Na-Pi cotransporter (193).Tyrosine kinase activity may also be involved in TGF-aand EGF effects on apical Na-Pi cotransport activity (15,314). However, the participation of phospholipase C/pro-tein kinase C has also been proposed for the TGF-a-and/or EGF-mediated effects (335–337).

5. Endocytic motifs

Tyrosine based and dileucine signals within the typeIIa Na-Pi cotransporter sequence might be important mo-tifs for the endocytic removal and/or membrane targeting,in a manner similar to some “model” proteins in polarizedepithelial cells (270). In mutagenesis experiments on thetype IIa Na-Pi cotransporter, various tyrosine-based mo-tifs and dileucine motifs located at predicted intracellularsites were found not to be essential for internalization inoocytes and transfected OK cells (170–172). Because thetype IIb transporter (intestinal isoform) transfected in OKcells is also apically targeted but not internalized by PTH(171, 197, 198), endocytic motifs/domains can be identi-fied on studies of type IIa/IIb chimeras; these studiessuggested a critical role of a dibasic motif located in thelast intracellular loop (198; see Fig. 7).

6. Lipids

Alterations in brush-border membrane lipid compo-sition have been associated with altered brush-bordermembrane Na-Pi cotransport (e.g., Refs. 240, 241, 246, 279,406). Changes in membrane fluidity and a role of a sphin-gomyelin-ceramide regulatory pathways were postulated(157, 221, 237, 251, 435). Chronic adaptation to low-Pi dietwas reported to alter cholesterol and glycosphingolipidcontent and thus membrane fluidity (240, 241). However,alterations in Na-Pi cotransport induced by acute changesin low-Pi diets occur before the changes in brush-borderlipid composition and thus are not essential for the adap-

tive response (245, 246). In rat studies, evidence wasprovided that the dexamethasone-induced inhibition ofbrush-border membrane Na-Pi cotransport is associatedwith a decrease in transporter protein content and anincrease in glucosylceramide (244). Taken together, itseems unlikely that transport alterations are directly re-lated to changes in membrane fluidity, but alterations inlipid composition, e.g., in microdomains such as glycolip-id-cholesterol rafts, may facilitate internalization of thetype IIa Na-Pi cotransporter.

Recently, it was shown that different inhibitors ofP-glycoprotein functions stimulate proximal tubular Pi

reabsorption related to an increased expression of typeIIa Na-Pi cotransporter protein. It is speculated that thiseffect is associated with drug-induced changes in thesphyngomyeline-ceramide pathway and associatedchanges in the membrane traffic of the transporter protein(D. Prie, S. Couette, I. Fernandes, C. Sieve, and G. Fried-lander, personal communication).

7. Reduction of S-S bridge

In Western blots of isolated brush-border mem-branes, a significant proportion of the type IIa cotrans-porter appears to be cleaved under reducing conditions,suggesting that the transporter is stabilized by a S-Sbridge (e.g., Refs. 39, 48, 91, 229, 305, 425). The degree oftransport inhibition under reducing conditions correlatedwith the appearance of cleaved moieties (425). Internal-ization and cleavage of the transporter can also be in-duced in X. laevis oocytes by a treatment with reducingagents (230). On the basis of these findings, we suggestthat a reduction of S-S bridge within the type IIa Na-Pi

cotransporter may contribute to its basal turnover andperhaps regulation. Reduction of the S-S bridge within thesecond extracellular loop (Fig. 7) may destabilize thesecondary structure of the type IIa transporter proteinfollowed by a degradation at the cell surface or afterinternalization. Reducing agents might enter the proximaltubular lumen by the secretory mechanism for organicanions.

8. Interacting proteins

Type IIa Na-Pi cotransporters are located amongmany other membrane proteins along the microvilli. How-ever, in physiological regulation they are specifically in-ternalized at intermicrovillar clefts (210, 257, 259, 398).Conceptionally there should be “anchoring” mechanismskeeping proteins within the microvilli “en place.” In reg-ulation, the interaction between specific anchor (interact-ing) proteins and specific membrane proteins (e.g., thetype IIa Na-Pi cotransporter) could be controlled, permit-ting via lateral mobility the protein to enter into cleftswhere it is removed. Alternatively, unrestricted lateralmobility could allow the transporter protein to enter al-


ways into the cleft region and transporters to be removedwould then be posttranscriptionally modified (e.g., phos-phorylation, ubiquitinylation; see above) providing a sig-nal for membrane retrieval of the transporters to bedownregulated. Also, this latter mechanism would requirespecific proteins to recognize (“interact” with) the trans-porter protein. PDZ-domain containing proteins havebeen described to interact with the brush-border mem-brane Na/H exchange NHE-3 and to be required for itskinase-mediated regulation (293, 412, 413, 428); such reg-ulation also involves the internalization of the NHE-3exchanger. Interestingly, we recently identified a PDZ-domain containing protein (diphor-1) that colocalizedwith the type IIa Na-Pi cotransporter in brush borders andinteracting with the COOH-terminal cytosolic portion ofthe molecule (93, 418, 418a). When coexpressed in oo-cytes with the type IIa Na-Pi cotransporter protein, adiphor-1-induced specific increase in Na-Pi cotransportactivity was observed without a stimulatory effect onNa-SO4 cotransport after coexpression with a cloned Na-SO4 cotransporter (93). Interacting proteins might be re-quired for brush-border expression of type II Na-Pi co-transporter and/or for its regulation. However, the preciserole of above-mentioned PDZ-domain containing proteins(e.g., diphor-1) in controlling the apical expression of thetransporter is not yet established.

VI. SUMMARY AND OUTLOOK

The type IIa Na-Pi cotransporter located in the renalproximal tubular brush-border membrane is the keyplayer in renal Pi reabsorption and thus also in overall Pi

homeostasis. Physiological regulation of the type IIa Na-Pi

cotransporter is responsible for altered renal Pi handlingand is mostly directly related to its altered brush-borderexpression. The latter involves complex membrane re-trieval and reinsertion mechanisms. Its expression canalso be abnormally regulated in different disease states(e.g., in X-linked hypophosphatemia). Transport activitymediated by the type IIa transporters in the brush-bordermembrane can also be controlled by changes in intratu-bular/intracellular pH, in transmembrane potential differ-ence, and posttranslational modification (e.g., proteinphosphorylation, reduction of S-S bridge). Finally, thetype IIa Na-Pi cotransporter gene might also be directlyaffected in autosomal disorders in renal Pi handling.

With the cloning of the type IIa cotransporter, toolsand information (gene location and structure) becomeavailable to dissect physiological regulation and patho-physiological alterations at the molecular level. In thefuture, studies on the structure/function relationship willprovide new insights into the mechanisms of cotrans-porter regulation/function. Several novel approaches tounderstand the mechanisms determining the specific re-

trieval of the transporter and the specific steps within thetransport cycle were dicussed. Finally, for an understand-ing of renal Pi handling at a molecular level, the in vitromolecular studies (e.g., in oocytes) have to be reinte-grated into the function of the entire cell, nephron, organ,and animal. Tissue-culture experiments, studies on iso-lated perfused tubules, and experiments on animals (ratsand/or mice; normal and/or genetically modified) are es-sential to develop a more detailed understanding of renalPi handling.

This review is devoted to many co-workers and collabora-tors contributing to this work over the last 10 years. The excel-lent secretarial help of Denise Neukom-Rossi and the excellentart work done by Christian Gasser are gratefully ackowledged.We also specifically acknowledge the excellent collaborationswith the laboratories of Dr. M. Levi (Dallas), Dr. H. S. Tenen-house (Montreal), Dr. S. Kempson (Indianapolis), and Dr. B.Kaissling (Zurich). A special thank goes to H. S. Tenenhouse andM. Levi for critical reading and correction of the manuscript.

This review is in memory of our dear colleague, Thomas P.Dousa (10/01/99), a leading investigator in the field of renalphosphate handling.

Major financial support by the Swiss National ScienceFoundation is gratefully acknowledged.

Address for reprint requests and other correspondence: H.Murer, Institute of Physiology, Univ. of Zurich, Winterthur-erstrasse 190, CH-8057 Zurich, Switzerland (E-mail:[email protected]).

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Proximal Tubular Phosphate Reabsorption: Molecular Mechanisms

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