Portal de Periódicos da CAPES

Cellular and Molecular Mechanisms of Renal Phosphate Transport

1997; Oxford University Press; Volume: 12; Issue: 2 Linguagem: Inglês

10.1359/jbmr.1997.12.2.159

1523-4681

Harriet S. Tenenhouse,

Renal and related cancers

The kidney is a major arbiter of extracellular phosphate (Pi( homeostasis and, as such, plays a key role in bone mineralization and growth. The bulk of filtered Pi is reabsorbed in the proximal tubule, with approximately 60% of the filtered load reclaimed in the proximal convoluted tubule and 15–20% in the proximal straight tubule. In addition, a small but variable portion (<10%( of filtered Pi is reabsorbed in more distal segments of the nephron. The overall capacity of the kidney to reabsorb Pi can be estimated by measuring the maximum tubular capacity (Tm( for Pi per unit volume of glomerular filtrate (TmPi/GFR(. In practice, this is achieved by measuring Pi and creatinine excretions and plasma concentrations during acute Pi infusions.1 Considerable effort has been devoted to the study of Pi transport in the proximal tubule, the principal site of its reabsorption (for reviews, see Refs. 2-8(. A wide variety of experimental systems, from the intact animal to vesicular membrane preparations, have been used to characterize proximal tubular Pi transport systems and to elucidate mechanisms involved in their regulation. These issues are briefly reviewed in the context of recent advances in the cloning of cDNAs encoding renal-specific Na+-Pi cotransporters and the application of these new tools to study the role of Na+-Pi cotransporter genes in Mendelian disorders of renal Pi reabsorption.9-13 Transepithelial Pi transport is essentially unidirectional and involves uptake across the brush border membrane (BBM(, translocation across the cell, and efflux at the basolateral membrane. Pi uptake at the apical cell surface is the rate-limiting step in the overall Pi reabsorptive process and the major site of its regulation.14 It is mediated by Na+-dependent Pi transporters that reside in the BBM and depend on the basolateral membrane-associated Na+,K+-ATPase to maintain the Na+ gradient that drives the transport process. Na+/Pi stoichiometries of both 2:1 and 3:1 have been documented. Given that divalent Pi (H2PO42−( is the predominant anion at physiological pH, the data suggest that Na+-Pi cotransport is both electroneutral and electrogenic. Direct evidence for electrogenic Na+-Pi cotransport has recently been obtained by expression studies in Xenopus laevis oocytes15 (see below(. Na+-Pi cotransport across the BBM is sensitive to changes in pH and is increased 10- to 20-fold when the pH is raised from 6 to 8.5. These findings reflect not only the preferential transport of divalent Pi but also the action of protons on the cotransporter per se. Indeed, it has been demonstrated that pH has a significant effect on the affinity of the cotransporter for Na+ ions.15, 16 At least two kinetically distinct Na+-Pi cotransport systems have been identified in the BBM: a high capacity, low affinity system in the proximal convoluted tubule in a position to reabsorb the bulk of filtered Pi; and a low capacity, high affinity system in the proximal convoluted and straight tubules in a position to reclaim residual Pi.17 This topological arrangement of Na+-Pi cotransport systems in series permits highly efficient reabsorption of Pi in the proximal tubule. There is little information on the translocation of Pi across the cell. Pi anions taken up by the cell rapidly equilibrate with intracellular inorganic and organic Pi pools. There is also a paucity of data regarding the mechanisms involved in the efflux of Pi at the basolateral cell surface. The latter appears to be a passive process that relies on the electrical gradient existing across the membrane and occurs via an anion exchange mechanism. Efforts to purify Na+-Pi cotransport proteins from isolated renal BBM preparations met with little success due to their low abundance and technical difficulties associated with their solubilization, reconstitution, and purification. Recently, however, cDNAs encoding renal-specific Na+-Pi cotransporters from several species have been identified, first by expression cloning in Xenopus laevis oocytes and then by homology screening.16, 18-26 Based on their nucleotide sequences, the cDNAs encode two classes of Na+-Pi cotransporters that share only 20% identity and have been designated NPT1 and NPT2 (Human Genome Mapping Workshop–approved symbols( (Table 1(. The NPT1 transporters are approximately 465 amino acids in length, and hydropathy analysis predicts seven to nine membrane spanning segments.18 The NPT2 transporters are comprised of approximately 635 amino acids and are predicted to span the membrane eight times.21 The NPT1 and NPT2 genes have been mapped to human chromosomes 6p2227 and 5q35,28 respectively, by fluorescence in situ hybridization. The human and murine NPT2/Npt2 genes have recently been cloned and characterized.29 The renal localization of NPT1 and NPT2 gene expression was determined by immunohistochemistry, in situ hybridization, and reverse transcriptase-polymerase chain reaction (RT/PCR( of RNA prepared from single microdissected nephron segments.30-32 These studies identified NPT1 and NPT2 transcripts in the proximal tubule and immunoreactive protein in the BBM of proximal tubular cells. However, while NPT1 is uniformly expressed in all segments of the proximal nephron, NPT2 expression is highest in the S1 segments of the proximal tubule. Functional studies of NPT1 and NPT2 have been conducted in cRNA-injected oocytes15, 18, 21, 33 and in cDNA-transfected renal cell lines.34, 35 Both NPT1 and NPT2 mediate high-affinity Na+-Pi cotransport. However, their pH profiles are significantly different, with that of NPT2 bearing closer resemblance to the pH dependence of Na+-Pi cotransport in renal BBM vesicles. Moreover, NPT1 appears to exhibit a broader substrate specificity than NPT2.36 NPT1 can induce a Cl− conductance that is inhibited by Cl− channel blockers and organic anions and, on this basis, it was suggested that NPT1 not only mediates BBM Na+-dependent Pi transport but also serves as an apical channel for Cl− transport and the excretion of anionic xenobiotics.36 The precise physiological role of NPT1 will thus require further study. Recent evidence has been obtained for the expression of two novel Pi transporters in the kidney. Both are cell surface viral receptors (gibbon ape leukemia virus [Glvr-1] and murine amphotropic virus [Ram-1]( that mediate high-affinity, electrogenic Na+-dependent Pi transport when expressed in oocytes and in mammalian cells.37 Although neither Glvr-1 nor Ram-1 show any sequence similarity to NPT1 or NPT2, they share 60% sequence identity with a putative Pi permease of Neurospera crassa.37 Since Glvr-1 and Ram-1 are widely expressed in mammalian tissues,37 they may serve as "housekeeping" Na+-Pi cotransporters. Additional studies are necessary to define the localization of Glvr-1 and Ram-1 in the kidney and to determine whether they play a significant role in renal Pi reabsorption. BBM Na+-Pi cotransport is a target for regulation by a variety of peptide and steroid hormones and growth factors.38 Because of the physiological importance of PTH in the regulation of renal Pi reabsorption, there is a vast literature devoted to this subject. PTH acts directly on proximal tubular cells and inhibits Na+-dependent Pi transport by mechanisms that are dependent on both the cAMP-protein kinase A and protein kinase C (PKC(-phosphoinositide signaling pathways.39 Both apical and basolateral PTH receptors mediate the action of PTH on Pi transport.40 PTH decreases the Vmax of high capacity, low affinity as well as low capacity, high affinity BBM Na+-Pi cotransport systems.41 The action of PTH on Na+-Pi cotransport is disrupted by agents that interfere with the endocytic pathway,42 and direct evidence for internalization of cell surface NPT2 protein in response to PTH was obtained by immunohistochemistry (Fig. 1(.43 Of interest is the demonstration that PKC-mediated inhibition of Na+-Pi cotransport is not prevented in Xenopus laevis oocytes injected with NPT2 cRNA in which PKC consensus phosphorylation sites were removed by site-directed mutagenesis.44 These data suggest that regulation of Na+-Pi cotransport by PTH does not involve phosphorylation of the predicted PKC consensus sites in NPT2. Mechanisms for regulation of NPT2 gene expression in the kidney. Dashed lines depict instances where direct evidence for the regulatory mechanism is not available. For discussion and references, see text. Several other hormones play a role in the regulation of proximal tubular Pi transport. Growth hormone, insulin-like growth factor-I, insulin, thyroid hormone, and 1,25-dihydroxyvitamin D3 all stimulate Pi reabsorption, while PTH-related peptide, calcitonin, atrial natriuretic factor, epidermal growth factor, transforming growth factor-α, and glucocorticoids inhibit renal Pi reclamation. While thyroid hormone elicits an increase in NPT2 mRNA abundance in opossum kidney cells,45 both dexamethasone46 and epidermal growth factor47 decrease NPT2 mRNA abundance in rat kidney cortex and opossum kidney cells, respectively (Fig. 1(. While the mechanism for the alterations in NPT2 mRNA has not been addressed, it is evident from other studies that thyroid hormone and glucocorticoids exert their effects on gene transcription through ligand-activated receptors that bind to response elements in the promoter region of target genes.48 Recent studies have implicated a novel hormonal system in the regulation of renal Pi handling. Stanniocalcin, an antihypercalcemic hormone that is produced by the corpuscles of Stannius in fishes and stimulates Pi reabsorption in the flounder proximal tubule,49 was discovered in humans.50 Stanniocalcin was identified in the distal convoluted and collecting tubules of human kidney by immunohistochemistry and shown to elicit a significant decrease in urinary Pi excretion when infused in the rat.50 These findings suggest that stanniocalcin may contribute to the overall maintenance of Pi homeostasis in mammals as well as fish.50 Dietary Pi intake is a major regulator of renal Pi handling. Pi deprivation elicits an increase in TmPi/GFR that is attributable to an adaptive increase in BBM Na+-Pi cotransport.51 The increase in Na+-Pi cotransport is independent of extrarenal factors. The signal for the adaptive response to Pi deprivation is not known. However, it has been suggested that a fall in the concentration of renal cell Pi plays a role in initiating the Pi transport response.52 The Vmax of both high capacity, low affinity and low capacity, high affinity Na+-Pi cotransport systems is increased by Pi restriction.53 The acute phase (2 h( of the adaptive response is associated with an increase in NPT2 protein but not NPT2 mRNA.54 In addition, the acute increase in Na+-Pi cotransport and NPT2 protein is rapidly reversed by a high Pi diet.54 Taken together, the data suggest that an exocytic/endocytic pathway is involved in mediating the acute response to Pi (Fig. 1(.54 In contrast, chronic Pi deprivation is associated with an increase in renal abundance of both NPT2 mRNA and protein.54 Studies in chronically Pi-starved opossum kidney cells demonstrated that the increase in NPT2 mRNA is associated with an increase in NPT2 mRNA stability rather than an increase in NPT2 gene transcription (Fig. 1(.55 While NPT1 gene expression is not markedly increased by Pi restriction,24 Glvr-1 and Ram-1 are regulated by extracellular Pi at both the mRNA and functional level.37 X-linked hypophosphatemia (XLH( and hereditary hypophosphatemic rickets with hypercalciuria (HHRH( are Mendelian disorders of Pi homeostasis characterized by rachitic bone disease, decreased growth rate and short stature, hypophosphatemia, and impaired renal Pi reabsorption.12 Features that distinguish XLH from HHRH are their mode of inheritance (X-linked dominant for XLH and autosomal dominant or recessive for HHRH( and the presence of an associated defect in renal vitamin D metabolism in XLH but not in HHRH.12 Studies in murine X-linked Hyp56 and Gy57 homologs of XLH have demonstrated that a specific defect in renal BBM Na+-Pi cotransport can account for the renal Pi leak and hypophosphatemia. Moreover, the decrease in BBM transport is associated with a corresponding decrease in the renal abundance of NPT2 protein and mRNA9-11 and, in Hyp mice, with a decrease in NPT2 gene transcription.58 The finding that NPT2 does not map to the X chromosome28 rules it out as a candidate gene for XLH (Hyp or Gy( and suggests that the X-linked gene at the XLH (Hyp or Gy( locus is involved in the regulation of renal NPT2 gene expression. The mutant gene in patients with XLH has recently been identified by positional cloning and was designated PEX to signify a Pi-regulating gene with homology to endopeptidases that maps to the X chromosome.59 The PEX gene contains significant homology to a family of endopeptidase genes that encode type II membrane glycoproteins and include neutral endopeptidase 24.11 (NEP(60 and endothelin converting enzyme-1 (ECE-1(.61 NEP is widely expressed and is involved in the proteolytic degradation and inactivation of various peptide mediators, including opioids, atrial natriuretic peptides, and endothelins, at the cell surface.60 In contrast, ECE-1 is expressed predominantly in vascular endothelium and functions in the proteolytic activation of endothelin from its inactive precursor peptide, big endothelin-1.61 Based on these findings, it is tempting to speculate that PEX is involved in the processing/inactivation of a peptide hormone that influences renal Pi handling and that loss of PEX function in XLH/Hyp/Gy results in either decreased production of a Pi conserving hormone or decreased clearance of a phosphaturic hormone. In either case, the net result would be the down-regulation of renal NPT2 gene expression. It remains to be established whether phosphotonin, the phosphaturic tumor-derived factor isolated from patients with tumor-induced osteomalacia62, 63 is a substrate for PEX and responsible for the renal Pi leak in XLHHyp/Gy. The molecular basis for the renal defect in Pi reabsorption in HHRH has not yet been addressed. Because of the absence of an associated disorder in vitamin D metabolism in HHRH and because Pi supplements alone are able to correct the bone lesions in HHRH, it has been suggested that HHRH arises from a primary renal defect in Pi transport.12 We have recently initiated studies to determine whether an NPT2 null mutation in mice will elicit the clinical features of HHRH. To this end, we have cloned the mouse NPT2 gene,29 constructed an NPT2 targeting vector, selected embryonic stem cell clones harboring one copy of the disrupted NPT2 gene, injected these into mouse blastocysts and obtained chimeric mice.64 The latter are being analyzed for germ line transmission and their progeny will be bred to homozygosity. It will thus be possible to define the precise contribution of NPT2 to the overall maintenance of Pi homeostasis and to establish whether other renal Pi transporters (e.g., NPT1, Glvr-1, and Ram-1( can compensate for the loss of NPT2 function. The NPT2 knockout mouse will also serve to delineate the impact of NPT2 gene expression on renal vitamin D metabolism, bone mineralization, and growth. At the same time, linkage analysis can be used to identify the mutant gene responsible for HHRH. Using polymorphic markers mapping to the 6p2219, 27 and 5q3528 chromosomal regions, it will be possible to include or exclude NPT1 and NPT2 as candidate genes. If these transporters are excluded, a genome search and positional cloning approach can be undertaken. Over the past 5 years, considerable progress has been made in identifying the molecular structure of two renal-specific, high affinity Na+-Pi cotransporters, NPT1 and NPT2, that reside in the BBM of proximal tubular cells where the bulk of filtered Pi is reabsorbed. The molecular mechanisms involved in the regulation of NPT2 gene expression by Pi availability, hormonal agents, and the X-linked Hyp and Gy mutations have been elucidated. However, no evidence for physiological regulation of NPT1 has been obtained. Further work is necessary to characterize the molecular entities involved in high capacity, low affinity BBM Na+-Pi cotransport, Pi efflux at the basolateral membrane, and the transepithelial transport of Pi in more distal segments of the nephron and to elucidate their contribution to the overall capacity of the kidney to reabsorb filtered Pi.