1. Determinants of Serum Phosphate

The story of parathyroid hormone (PTH) and renal phosphate handling is an old one, and yet an incomplete one. Decades of research dating back to the 1920s detail the effects of PTH on proximal tubular reabsorption of the filtered load of phosphate [1]. PTH is one of the major phosphaturic hormones, and as such, plays a critical role in regulating total body phosphate homeostasis [2], the maintenance of which being crucial for cardiovascular health [3,4,5]. Deviations in serum phosphate can produce severe pathological conditions. Normal clinical values for serum phosphate have been defined as 3.4 to 4.5 mg/dL [6]. Even mild hyperphosphatemia with serum values of 5.5 mg/dL or greater correlate with a statistically significant increase in the risk of death from cardiovascular and renal diseases [7]. As a crucia l biological mineral, phosphate and the processes underlying its homeostasis remain significant areas of research.

Total body phosphate homeostasis is tightly maintained through a complex axis involving the intestines, kidneys, and bones. While the minimum daily dietary requirements for phosphate are approximately 20 mmol/day [8], the Western diet typically contains a much higher amount than stipulated by metabolic needs. In adults, serum phosphate levels result from a combination of dietary intake, bone modeling, and kidney excretion rates, the last of which is the most crucial determinant of serum phosphate. Dietary phosphate can be obtained from all natural foods, with particular abundance in protein-rich food as well as cereal grains [9]. Phosphate obtained from animal sources is more bioavailable and more readily absorbed than phosphate obtained from plant sources [10]. In more recent years, dietary loads of phosphate have increased through the heavy reliance of the food industry on phosphate-rich food additives to preserve food shelf life [10]. In the steady state, however, the amount of phosphate absorbed from the diet is balanced by the amount of phosphate excreted by the kidney.

In the gut, absorption of dietary phosphate into the circulation occurs through paracellular pathways and through transcellular pathways mediated primarily by the type IIb sodium-phosphate cotransporter (Npt2b), expressed in the apical membrane of the duodenum and early jejunum [11]. Npt2b expression is regulated by several factors, including dietary phosphate, 1,25-dihydroxyvitamin D, and fibroblast growth factor 23 (FGF23). A low phosphate diet induces increased expression of Npt2b, whereas a high phosphate diet decreases Npt2b expression [12]. 1,25-dihydroxyvitamin D has well been established as a positive regulator of Npt2b expression through post-transcriptional mechanisms [13,14,15,16]. In accordance with these findings, Kaneko et al. [17]found that mice lacking the Vitamin D receptor displayed decreased intestinal phosphate transport activity and Npt2b expression. FGF23, on the other hand, decreases intestinal absorption of phosphate through its inhibition of Vitamin D synthesis [16]. Together, these studies suggest that intestinal phosphate absorption undergoes more regulation than previously thought, and will perhaps lead to a clearer view on the role of Npt2b in maintaining total phosphate homeostasis.

In addition to the contribution of the gut to phosphate homeostasis, bone also plays a major role in phosphate homeostasis, as the hydroxyapatite matrix serves as a critical pool of phosphate and calcium reserves. Adult bones undergo constitutive remodeling, with phosphate and calcium cycling into and out of the bones and circulation as osteoclastic bone resorption is countered by osteoblastic bone formation [18]. The flux of phosphate into and out of bone is normally balanced in the adult, and thus serum phosphate levels are mostly determined by the rate of renal excretion.

In the kidney, approximately one-fifth of renal plasma flow is filtered into Bowman’s space. This glomerular ultrafiltrate then enters the nephron, wherein water and solutes are either reclaimed back into the circulation, or pass through to be excreted in the final urine. A small solute, phosphate is freely filtered at the glomerulus, and its reabsorption within the nephron occurs almost exclusively in the proximal tubule [19]. There is no evidence for significant phosphate secretion within any segment of the nephron, and thus rates of renal phosphate excretion are dependent on the rate of phosphate reabsorption from the ultrafiltrate of the glomerulus. The proximal tubular transport maximum for phosphate reabsorption is directly related to the abundance of phosphate transporters expressed within the apical membrane. Once the transport maximum is reached, any excess filtered phosphate will end up in the final urine. Phosphate is reclaimed from the ultrafiltrate through secondary-active transport coupled to the movement of sodium down its concentration gradient, which allows for entry of phosphate from the tubule lumen into the tubular epithelial cell. Reabsorption of phosphate back into the bloodstream is achieved by phosphate exiting the cell across the basolateral membrane through unknown mechanisms.

2. Contribution of Phosphate Transporters to Renal Phosphate Reabsorption

Mammalian phosphate transport is accomplished by two families of secondary-active sodium-coupled phosphate transporters: SLC20 and SLC34. The SLC20 family is comprised of PiT-1 (SLC20A1) and PiT-2 (SLC20A2). PiT-1 and PiT-2 mRNAs are ubiquitously expressed in both murine and human tissue, and they are thought to serve a housekeeping function of providing phosphate for intracellular metabolic needs [20,21]. Within the kidney, although expression of both PiT-1 and PiT-2 has been found, only PiT-2 has been identified in the apical membrane of the proximal tubule [22].

The SLC34 family of phosphate transporters is comprised of type II sodium-coupled phosphate transporters. Two members of the SLC34 family, Npt2a (SLC34A1) and Npt2c (SLC34A3), are expressed in the kidney in the apical membrane of the proximal tubule. Npt2a is an electrogenic transporter, cotransporting three sodium ions and one divalent phosphate ion into the cell (3 Na⁺:1 HPO₄^2-), whereas Npt2c is electroneutral, coupling the entry of two sodium ions and one divalent phosphate ion (2 Na⁺:1 HPO₄^2-) [23]. Npt2a and Npt2c are the primary mediators of proximal tubule reclamation of filtered phosphate, with PiT-2 contributing only a small fraction to overall phosphate reabsorption [24,25,26]. In murine tissue, Npt2a is expressed throughout the S1, S2, and S3 segments of the proximal tubule, with higher expression in juxtaglomerular versus superficial tubules [27,28], while Npt2c is expressed only in the S1 and S2 segments of superficial and midcortical proximal tubules [29]. Both Npt2a and Npt2c exhibit the highest expression in the S1 segments. Npt2a and Npt2c expression also vary with age, with rat Npt2c expression levels peaking during the weaning phase, whereas Npt2a expression is maintained throughout the murine lifespan, declining only somewhat with age [29,30]. Expression of Npt2a and Npt2c is also dependent on the dietary load of phosphate, with high phosphate diets inducing decreased expression of the transporters and low phosphate diets inducing increased expression [22,31,32].

In rodents, the type IIa sodium-phosphate cotransporter (Npt2a) has been identified as the transporter responsible for the majority of phosphate reabsorption, accounting for up to 80% of brush-border phosphate transport [33,34]. Studies in mice support a preeminent role for Npt2a in contributing to overall phosphate homeostasis, as Npt2a knockout mice not only display significant phosphate wasting, but they also develop hypercalciuria with tubular and interstitial calcium-phosphate deposits, and display developmental skeletal abnormalities [2,35,36]. Kidney-specific ablation of the Npt2c gene (Slc34a3) in mice does not elicit a hypophosphatemic phenotype, with no apparent disturbances in calcium or phosphate homeostasis [37]. However, global deletion of Npt2c in mice disrupts calcium homeostasis, with Npt2c^-/- mice exhibiting hypercalcemia and hypercalciuria, accompanied by elevated serum 1,25-dihydroxyvitamin D and decreased serum FGF23 levels [38]. Notably, the global Npt2c-null mice did not di splay hypophosphatemia or hyperphosphaturia. Thus, Npt2c appears to play an important role in maintaining systemic calcium homeostasis through its effect on FGF23 and vitamin D metabolism, while its importance in maintaining phosphate homeostasis is still subject to some debate.

The contribution of the renal phosphate transporters to overall phosphate homeostasis may be species-specific, however. Clinical syndromes associated with Npt2a and Npt2c gene mutations suggest that Npt2c expression and function may be more important for total phosphate homeostasis than Npt2a in humans. Loss-of-function mutations in the human Npt2c gene have been identified as the cause of hereditary hypophosphatemic rickets with hypercalciuria and accompanying nephrolithiasis renal phosphate wasting [39,40]. In contrast, human Npt2a mutations were found to be associated with a considerably milder phenotype of osteoporosis and nephrolithiasis[25,41]. However, recent clinical reports suggest a more significant role for Npt2a expression and function in human phosphate homeostasis than previously thought. Magen et al. [42]reported a clinical case of autosomal recessive hypophosphatemic rickets with renal Fanconi’s Syndrome secondary to an Npt2a loss-of-function genetic mutation. Likewise, Rajagopal et al. [43] and Kenny et al. [44] both reported case studies of mutations in the Npt2a gene that resulted in phenotypes of renal phosphate wasting with resultant hypercalcemia, hypercalciuria, and hypophosphatemia. Additionally, impaired expression of Npt2a is associated with dysregulated serum calcium homeostasis as a result of its impact on serum phosphate levels. In characterizing the genetic causes of Idiopathic Infantile Hypercalciuria (IIH), Schlingmann et al. [45] identified a mutation in the SLC34A1 gene that leads to impaired Npt2a trafficking with a subsequent loss of transport activity. In patients with this mutation, phosphate wasting is associated with the inappropriate production of 1,25-dihydroxyvitamin D, resulting in the IIH phenotype of hypercalciuria and nephrocalcinosis. In addition to these studies demonstrating the significance of Npt2a in the maintenance of phosphate homeostasis in humans, further supportive evidence comes from a genome-wide association study (GWAS) reported by Kestenbaum et al. [46], which revealed that polymorphisms at a locus near the SLC34A1 gene—the gene that encodes Npt2a—are a significant determinant of serum phosphorus. While these clinical reports suggest that the contribution of specific renal phosphate transporters to overall phosphate homeostasis may be more nuanced than previously recognized, they provide evidence and support for Npt2a as an important determinant of serum phosphate, as well as a critical target of regulators of renal phosphate reabsorption.

3. Regulation of Serum Phosphate by PTH

The maintenance of serum phosphate is dependent on the rate of its reabsorption in the kidney, which is subject to strict regulation by a host of factors, including diet and hormones. Npt2a expression and function are determined by physiologic regulators of serum phosphorus, including parathyroid hormone (PTH), one of the major phosphaturic hormones [2]. PTH is an 84-amino acid peptide hormone that is released from the parathyroid glands in response to either decreased serum Ca²⁺ or increased serum P_i [47]. In bone, PTH stimulates osteoclastic activity indirectly through its effect on osteoblast secretion of paracrine factors [9,48]. Bone resorption as a result of PTH stimulation releases Ca²⁺ and P_i into the circulation. In the proximal tubule, PTH upregulates 1α-hydroxylase and decrease 24-hydroxylase expression, which enhances production of 1,25-dihydroxyvitamin D, promoting increased calcium absorption in the gut as well as increased phosphate absorption [16,49]. In the process of releasing calcium from bone, phosphate stores also become liberated. To excrete the excess phosphate and preserve the newly restored serum calcium level, PTH exerts a phosphaturic effect in the kidney to decrease the reabsorption of phosphate from the plasma ultrafiltrate, while simultaneously increasing calcium reabsorption in the distal tubule to sustain the elevation in serum calcium [8,50]. As serum PTH levels increase, the renal transport maximum for phosphate decreases [51], which is accomplished through the downregulation of both of the major renal phosphate transporters, Npt2a and Npt2c [52].

While the actions of PTH on proximal tubular phosphate reabsorption are direct and relatively straight-forward, the actions of vitamin D on phosphate reabsorption, and Npt2a in particular, are complex. Acutely, vitamin D appears to stimulate phosphate reabsorption by increasing expression of Npt2a through upregulation of Npt2a promoter activity in a cell-specific manner, although this action appears to have a weak direct influence on phosphate transport [53]. Rather, vitamin D appears to play a more important role in overall phosphate homeostasis by modulating the FGF23/Klotho/PTH bone-endocrine axis [39]. PTH stimulates the formation of active vitamin D, which, in a negative feedback loop, suppresses further PTH release from the parathyroid glands [54,55]. Vitamin D also increases FGF23 expression, which decreases renal 1α-hydroxylase expression in addition to promoting phosphaturia [56]. Despite vitamin D’s direct and indirect actions in the proximal tubule to increase apical membrane phosphate transport, chronic exposure to vitamin D has actually been shown to decrease Npt2a expression synergistically with PTH, likely in response to increased phosphate release from increased bone turnover [57]. Given the complexities of the downstream responses to vitamin D, further work is required to more definitively determine the direct versus indirect actions of vitamin D on phosphate homeostasis over time.

4. Npt2a Expression in the Apical Membrane

The rate of phosphate reabsorption in the proximal tubule is directly related to the relative abundance and stability of Npt2a in the apical membrane. The expression of Npt2a in the brush border membrane is heavily reliant on its interaction with the scaffolding protein NHERF-1 (sodium-hydrogen exchanger regulatory factor 1). As its name implies, NHERF-1 was originally identified for its role in the regulation of NHE3, but has since been identified as a protein with a more global function than just the regulation of sodium-hydrogen exchange [58,59]. Of the population of Npt2a present in the apical membrane of the proximal tubule, approximately 35−50% is bound to NHERF-1, and this interaction stabilizes Npt2a in the membrane and prolongs its retention there [60]. NHERF-1 contains two PDZ domains—a conserved motif among scaffolding proteins that is crucial for its interaction with other proteins. Mutation analyses have shown the PDZ-1 domain of NHERF-1 to be critical not only for NHERF1-Npt2a associations, but also for regulated membrane expression, serving as a regulatory target for several hormones, including PTH. NHERF-1 assembles a complex of proteins involved in PTH receptor (PTHR1) signaling, including PTHR1, PLCβ, AKAP79/150, and actin [61,62,63], and stabilizes the apical expression of those complexes through its interaction with the cytoskeleton [64]. Accordingly, NHERF-1 is also required for maximal PTH-responsive inhibition of phosphate uptake [65]. In addition to the assembly of the PTH receptor complex, however, NHERF-1 also augments phosphate uptake inhibition by 8-Br-cAMP, PMA, and forskolin (activating PKA, PKC, and adenylyl cyclase, respectively), indicating a role for NHERF-1 in PTH receptor signaling beyond the assembly of the PTHR1 complex [65].

PTH-stimulated endocytosis of Npt2a occurs through the phosphorylation of a serine residue in the PDZ domain of NHERF-1 [60], which induces the rapid dissociation of Npt2a from NHERF-1. Maximal disassociation occurs 30 to 40 minutes following PTH stimulation [66]. Whereas Npt2a undergoes rapid endocytosis in response to PTH, NHERF-1 remains present in the membrane, as an important regulator for other ion transporters and protein complexes not involved in phosphate transport [66]. Following endocytosis to the endosomal compartment, Npt2a is targeted to lysosomes for degradation [67,68]. Re-insertion of Npt2a back into the apical membrane following removal of the PTH stimulus requires de novo synthesis, and thus takes several hours before new Npt2a begins to repopulate the apical membrane [69].

5. PTH Signaling Events in the Proximal Tubule

PTH signaling in the proximal tubule is elicited through its interaction with the PTH receptor type I (PTHR1). The PTH receptor is a member of the G protein-coupled receptor (GPCR) family, with seven transmembrane domains, an extracellular amino terminus, and an intracellular carboxyl terminus. The amino acid sequence of the PTH receptor is conserved across species, with PTHR1 cloned from rat osteoblasts displaying 78% conservation to the opossum kidney PTHR1, indicating an important systemic role for the receptor [70]. PTH receptor expression has been identified in a wide array of tissues, including the uterus, gut, ovaries, and liver, although expression of the PTHR1 in these tissues is thought to be more important for paracrine/autocrine signaling by the PTH-related peptide PTHrP, which also couples to the PTHR1 [71]. The highest expression of the PTHR1 within the body is found in the bone and kidney. Within the kidney, PTH receptors have been found both in the proximal and distal tubules, as well as the collecting duct [71,72].

Within the proximal tubule, PTH receptors are expressed in both the apical and basolateral membranes [73]. The PTH receptor is a member of the class II GPCR family, and possesses the ability to activate several intracellular signaling pathways. It has been shown to couple to several G proteins, including G_s and G_q, leading to the subsequent activation of the cAMP/PKA and PLC/PKC pathways, respectively [74]. The signaling pathways activated by PTH are complex and cell-specific, however, and influence more than just renal phosphate transport. Although the PKA and PKC pathways are the primary pathways activated in the proximal tubule, PTH receptors throughout the body differ in their structural requirements for signaling pathway activation, particularly when it comes to phospholipase activation. Proximal tubular PTH receptors, for example, are capable of activating different phospholipases from the ones activated by distal tubule PTH receptors [72]. PTH receptors even within the same cell may differ in their G protein-coupling capacities based on their distribution in the apical versus basolateral membrane. Early studies suggested that the PTH receptor in the proximal tubule coupled to distinct G proteins on the apical and basolateral membranes, with apical PTH1Rs thought to activate PKC exclusively, whereas basolateral PTH1Rs activated both PKA and PKC [75]. However, Npt2a-GST pull-down experiments by Khundmiri et al. [62]showed that Npt2a in the BBM of OK cells associates with AKAP79, PKA (both the catalytic and regulatory subunits), and the PTH receptor, and that AKAP-mediated activation of PKA was critical for downregulation of phosphate transport by PTH. While the aforementioned studies provide conflicting evidence for specific G protein coupling dependent on receptor localization, they nonetheless confirm the role of both G_s and G_q in mediating PTH signaling within the proximal tubule.

Although the predominant signaling pathways activated by PTH include the cAMP/PKA and PLC/PKC pathways [70], there are a number of other pathways that PTH has also been shown to activate, and these also appear to be tissue-specific. These include phospholipase A2 in osteoblasts and proximal tubules [76], phospholipase D in distal tubules [72], and the mitogen-activated protein kinase (MAPK) pathway in OK cells and mouse kidneys [77,78,79]. In addition to activating PKA, cAMP also activates another category of proteins known as exchange proteins directly activated by cAMP (Epac), which are guanine nucleotide exchange factors for the Ras GTPase family of proteins [80]. Epac1 and Epac2 are both expressed in the brush border of the proximal tubule [81], and are known regulators of several proximal tubule proteins, including NHE3, aquaporin-2, and the H⁺/K⁺-ATPase. However, no evidence currently exists for a role of Epac in regulating phosphate transport [80]. Of the pathways activated by PTH in the proximal tubule, PKA and PKC remain the most influential in mediating downstream phosphaturic effects of PTH signaling, and in that regard are the most highly studied.

5.1. PTH-induced PKA signaling

The PTHR1 is a potent stimulator of cAMP generation and PKA activity through its association with the G protein Gα_s. When activated by ligand-receptor interaction, Gα_s activates the closely associated adenylyl cyclase (AC), an enzyme that rapidly catalyzes the formation of cyclic AMP (cAMP) from ATP. Within the AC family of proteins, nine isoforms have been identified, and the effects of those isoforms are dependent on “signalosomes” recruited by each specific isoform [82]. As studied in the rat nephron, AC isoforms II, III, VI, VII, and IX are expressed in the proximal tubule, with II, III, and IX confirmed through IHC to be expressed only in the apical membrane (IHC data unavailable for VI and VII) [83]. NHERF-1 has also been found to be important not only in the formation of the apical membrane signalosomes, but also for modulating the amount of cAMP generated in response to PTH stimulation, and its presence is mandatory for cAMP inhibition of NHE3 [84,85].

Within 10 seconds of receptor binding, PTH induces a significant accumulation of cAMP [86], which in turn activates protein kinase A (PKA) by binding to the regulatory subunits, causing dissociation from and activation of the PKA catalytic subunits, thus resulting in phosphorylation of downstream targets. The cAMP signal is typically terminated through the actions of phosphodiesterases (PDEs) that hydrolyze cAMP to form 5’ AMP. In the case of PTH signaling, PTH also rapidly increases PDE activity in a dose-dependent and PKA-dependent manner, providing a negative-feedback mechanism for the termination of PTH-induced cAMP signaling [87].

The dynamics of PTH signaling vary during acute versus sustained signaling. AC is a membrane-bound protein, and the cAMP produced by it is thus produced at the cell membrane. However, this site of cAMP generation is short-lived, as the PTHR1 undergoes endocytosis shortly after stimulation by PTH. This series of events was originally thought to terminate receptor signaling, as association of β-arrestins with the internalized receptor would desensitize downstream signaling. However, more recent studies have demonstrated that, following endocytosis of the receptor, cAMP generation is sustained. Whereas the previous dogma held that cAMP accumulation occurred only at the cell membrane, studies by Ferrandon et al. [86]challenge this notion, when they showed that PTH(1-34) was endocytosed to early endosomal compartments, where it not only remained associated with the PTHR1, Gα_s, and AC, but continued to produce cAMP over a period of 20 minutes. In this manner PTH evokes sustained signal generation in the proximal tubule, even after removal of the original stimulus.

5.2. PTH-induced PKC signaling

While the PTHR1 is well known to activate cAMP-dependent pathways by coupling to G_s, the receptor also couples to G_q and is thus a potent stimulator of phospholipase C (PLC)-dependent signaling. Following the activation of PLC by G_q, PLC cleaves membrane phospholipids such as phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate (PIP₂), producing the two signaling molecules inositol triphosphate (IP₃) and diacylglycerol (DAG). Downstream activation of PKC occurs through two processes: DAG directly binds to and activates PKC, and IP₃ binds to ligand-gated calcium channels in the membrane of the endoplasmic reticulum. The subsequent rise in intracellular [Ca²⁺] produces activation of PKC in cooperation with DAG. In OK cells, PKC is maximally stimulated 60−90 seconds following activation of the PTHR1 by PTH [66]. In addition to this PLC-mediated activation of PKC, the PTHR1s expressed in human, mouse, and opossum kidneys are also capable of activating PKC through PLC-independent mechanisms [88].

PTH-induced signaling is heavily dependent on the interaction of the PTHR1 with accessory proteins. NHERF-1, required not only for apical membrane localization for Npt2a, is also crucial for the regulation of PTH receptor signaling and second messenger generation. In OKH cells, a clonal cell line of the OK WT cell that is deficient in NHERF-1, PTH produces robust levels of cAMP, but fails to stimulate phospholipase C and does not inhibit phosphate transport [65]. In cells that express PTHR1-NHERF-2 complexes, stimulation by PTH potently activates PLC signaling while inhibiting AC through the simultaneous activation of inhibitory G_i/o proteins [89]. PTH is thus capable of producing tissue-specific effects in accordance with the specific receptor-protein interactions present within the tissue membrane.

5.3. Modification of receptor coupling and signaling through PTH fragments

PTH is a peptide hormone, which in the mature, secreted form is 84 amino acids in length. The first 34 amino acids (1-34) are the biologically active portion of the hormone, responsible for full receptor coupling and subsequent G protein recruitment and activation [88]. Deletion of select amino acids within the peptide hormone results in an incomplete coupling of the ligand to the receptor, and a subsequent incomplete activation of downstream signaling pathways. Thus, modification of the amino acid composition of PTH yields analogues with selective PKA- or PKC-activating properties.

PTH(1-31) has been suggested as a relatively cAMP-selective PTHR1 agonist that is deficient for both PLC-dependent and -independent PKC signaling [88]. Treatment of proximal tubular cells with PTH(1-34) and PTH(1-31) produces a similar rise in cAMP, indicating that the carboxy-terminal deletion of residues (32-34) does not interfere with Gs activation [88]. PTH analogues truncated at the C-terminus further than the 31^st residue, however, fail to stimulate cAMP accumulation [72]. In contrast to G_s stimulation, the C-terminus (residues 32-34) was found to be critical for G_q signaling, as demonstrated by Jouishomme et al., who showed that the PTH(29-32) fragment is the smallest hormone fragment capable of stimulating PKC, and that the His32 residue is critical for this stimulation to occur [90]. Loss of the His32 residue thus prevents PTH from activating PKC. This property of His32-receptor coupling prevents PTH(1-31) from activating PKC, while preserving full adenylyl cyclase-stimulating capabilities.

Truncating PTH at the amino terminus by two amino acids yields the PTH fragment PTH(3-34). Whereas the carboxyl terminus is crucial for activation of PKC, the amino terminus is likewise crucial for the activation of PKA. PTH(3-34), devoid of the amino terminal residues necessary for G_s activation, selectively activates PKC through non-PLC-dependent mechanisms [91], albeit less effectively than the full-length analogue, and notably does not activate PKA [88]. Whereas stimulation of the proximal tubule by PTH(1-31) fails to result in an increase of intracellular calcium, stimulation with PTH(3-34) results in a partial increase, indicating a selective role for PTH(3-34) in the activation of PKC [72].

5.4. PTHR1 endocytosis and desensitization

The cessation of GPCR signaling is generally dependent on removal of the stimulus and receptor desensitization through endocytosis of the receptor from the plasma membrane. GPCR desensitization is a process that normally requires receptor phosphorylation and/or ubiquitination, promoting the association of β-arrestins that stop receptor signaling. Resensitization thus usually requires a process of dephosphorylation and removal of β-arrestin binding. In response to PTH stimulation, the PTHR1 carboxy-terminus becomes phosphorylated, promoting β-arrestin2 binding and receptor desensitization, followed by endocytosis to an internal compartment [92]. PTH induces a transient polyubiquitination of the PTHR1 by promoting coupled ubiquitination followed by deubiquitination through the rapid upregulation of the deubiquitinating enzyme USP2 [93]. Following receptor endocytosis and desensitization, GPCRs usually re-sensitize and recycle back to the plasma membrane, which for the PTHR1 occurs within 2h after PTH stimulation [93]. The PTHR1 is unique amongst GPCRs in that it does not require dephosphorylation or dissociation from β-arrestin in order to recycle back to the plasma membrane [94].

NHERF-1 association with the PTHR1 helps to prolong the expression of the PTHR1 in the plasma membrane and sustain PTHR1 signaling. Wang et al. found that NHERF-1 was crucial for preventing β-arrestin2 from interacting with PTHR1, as well as for maintaining the association of the PTHR1 with G_αs [92]. The presence of NHERF-1 in the apical membrane also protects against receptor desensitization by prolonging the retention of the PTHR1 in the plasma membrane [95]. While NHERF-1 has been shown to associate with the dopamine receptor D1 and the Na⁺/K⁺-ATPase in the basolateral membrane of the proximal tubule [96,97], direct association between basolateral NHERF-1 and PTHR1 has yet to be demonstrated. This potential difference in NHERF1-PTHR1 coupling may contribute to the differential signal generation of apical versus basolateral PTHR1s. In addition to NHERF-1 assisting PTHR1 signaling, the Golgi apparatus appears to play an important role in PTHR1 recycling, as internalized PTHR1 colocalizes with the Golgi, and disrupting the Golgi impairs PTHR1 recycling [98]. The PTHR1 has also been shown to traffic in and out of the nucleus, although the physiological function of this trafficking pattern is currently unknown [99].

6. Pathways Involved in Npt2a Protein Downregulation

The studies examining the relative contributions of the signaling pathways to the regulation of Npt2a by PTH have been inconclusive at best. Activation of both the PKA and PKC pathways has been shown to downregulate expression of Npt2a at the apical membrane in proximal tubule cells [60,100]. Several studies using cell-based and animal-based models have demonstrated that the two signaling pathways both contribute to PTH-mediated downregulation of Npt2a protein expression [26,65,101,102,103], and the relative contribution of each pathway is still under debate.

Several studies support a principal role for PKA signaling over PKC signaling in the sustained downregulation of Npt2a by PTH. In mice that lack adenylyl cyclase isoform 6 (AC6), PTH-induced endocytosis and subsequent lysosomal degradation of Npt2a is absent [104]. Through the use of PTH analogues with selective signaling properties, Nagai et al. showed that the acute down-regulation of phosphate transport in response to PTH involves primarily the cAMP/PKA pathway [101]. Genetic studies in Npt2c KO mice expressing WT or PLC-signaling-deficient PTHR1 showed that the hypophosphatemic response to PTH in PLC-signaling-deficient Npt2c KO mice was only slightly impaired, suggesting that chronic downregulation of Npt2a by PTH is primarily reliant on non-PLC signaling pathways [52]. Support for this conclusion is seen in patients with type Ia or type Ib pseudo-hypoparathyroidism, wherein a mutation in renal proximal tubule Gα_s results in proximal tubule-specific PTH-resistance, resulting in diminished phosphate excretion and hyperphosphatemia [105,106]. This clinical picture implicates a preeminent role for PKA in mediating downregulation of sodium-phosphate cotransport by PTH, as PLC/PKC pathway signaling, which is presumably unaffected by the mutation, cannot compensate for the loss of cAMP/PKA signaling.

Although several studies have implicated PKA as the primary mediator of the PTH phosphaturic response, other studies highlight the importance of PKC signaling over PKA signaling. Whereas Nagai et al. [101] used PTH analogues to demonstrate a dominant role for PKA signaling, Carpenter et al. [107]showed preservation of the phosphaturic effects of PTH in the absence of cAMP generation, through the use of PTH analogues with G_q-specific-activating properties. In mouse renal proximal tubule cells, inhibiting PKC completely blunted the inhibitory effect of PTH on phosphate transport, while inhibiting PKA did not affect PTH-stimulated downregulation of phosphate transport [108]. These studies by Cunningham et al. suggest that the inhibitory effect of cAMP on phosphate transport is reliant on downstream PKC activation, which would partially explain the phosphaturic effects of PKA, but does not address the mechanism for PTH-resistant hypophosphatemia in patients with pseudohypoparathyroidism. While Guo et al. found that PLC activation by PTH only contributes slightly to the hypophosphatemic response, they also found that sustained downregulation of Npt2a by PTH requires activation of a non-cAMP/PKA pathway downstream of the PTH receptor [52]. The differences between these studies may highlight species discrepancies in the contribution of PTH-stimulated signaling pathways to the regulation of phosphate transport by PTH.

PTH also activates several other signaling pathways in proximal tubule, but their roles, both as independent contributors to Npt2a regulation as well as adjunctive to PKA and PKC, in the regulation of Npt2a are not well understood. For instance, FGF23, another major phosphaturic factor, decreases both Npt2a protein and mRNA abundance through MAPK-dependent-signaling in the proximal tubule [109]. Although the MAPK pathway is critical for FGF23-induced hypophosphatemia, which is independent of PKA and PKC activation, the exact role of MAPK in PTH- and dopamine-mediated inhibition of phosphate transport is unclear [108]. However, Bacic et al. [79] showed that PTH-stimulated internalization of Npt2a in mouse kidney cortex could be blocked by the application of a MAPK inhibitor, and demonstrated that PKA-, PKC-, and PKG-mediated internalization of Npt2a are all dependent to some degree on MAPK signaling, suggesting that the three pathways converge on MAPK as a downstream signaling target of PTH. PI3 kinase and phospholipase A2 are additional signaling pathways known to be activated by PTH, but their role in regulating phosphate transport has not been thoroughly investigated. To date, the cAMP/PKA and PLC/PKC pathways are known to be the most influential on phosphate transport.

7. Evidence for a Role of PTH in the Regulation of Npt2a mRNA

The vast majority of studies that have examined the regulation of Npt2a by PTH have focused on regulation at the protein level. While it has been clearly demonstrated that apical protein expression of Npt2a is greatly diminished in response to PTH, few studies have examined whether regulation of the transporter also occurs at the mRNA level, and those that have studied the effect of PTH on Npt2a mRNA expression have been limited and inconclusive. Kilav et al. [110] were unable to demonstrate an effect of elevated PTH on Npt2a mRNA in a rat model of diet-induced hyperparathyroidism. In contrast to Kilav’s findings, three other laboratories have reported an effect of PTH on Npt2a mRNA. Kempson et al. [67]showed that acute PTH treatment in parathyroidectomized rats decreased Npt2a mRNA expression by 30%. Independently, Moe et al. [111] demonstrated that rats with hyperparathyroidism secondary to chronic kidney disease display a 50% reduction of levels of the Npt2a transcript. Friedlaender et al. [57] also reported modest inhibitory effects of PTH on Npt2a mRNA expression in PTH-infused rats, which was augmented further when administered alongside vitamin D.

Decreased mRNA expression of a gene can be produced by two general mechanisms: (1) decreased gene transcription, or (2) decreased mRNA stability. Work done by Hilfiker and colleagues suggests that the inhibitory effect of PTH on Npt2a mRNA may be due to the latter. In their studies on the Npt2a gene promoter, Hilfiker et al. showed that PTH has no inhibitory effect on Npt2a promoter function [112]. However, they did not examine Npt2a mRNA expression in response to PTH. Since PTH has been shown to have no effect on Npt2a gene promoter activity, changes in Npt2a mRNA levels in response to PTH most likely occur at the post-transcriptional level.

While some controversy existed as to whether PTH regulates Npt2a mRNA levels, previously published studies indicated that PTH is capable of regulating Npt2a beyond the protein level. Recently, we established that PTH induces rapid destabilization of Npt2a mRNA in the proximal tubule, and that this destabilization is sustained with chronic PTH [113]. While PTH is a known regulator of Npt2a protein degradation, we showed that PTH also regulates the rates of de novo synthesis through its effect on Npt2a mRNA levels in the cell. Through both animal and cell culture models, we established that PTH significantly and rapidly reduces Npt2a mRNA expression through post-transcriptional mechanisms, independent of Npt2a gene promoter activity.

Exploring further the mechanism behind the regulation of Npt2a mRNA by PTH, we found that the effect of PTH on Npt2a is dependent on both transcription and translation. When inhibiting transcription with actinomycin D, the reduction of Npt2a mRNA to the 50% level required 8.6 hours. PTH greatly reduced that time to just over 2 hours. Inhibiting transcription with actinomycin D while treating cells with PTH completely blocked the ability of PTH to destabilize the transcript. We achieved similar results when treating OK cells concomitantly with both PTH and cycloheximide, indicating that the PTH effect is also dependent on translation. The observation that this is a cycloheximide-sensitive mechanism indicates that enhanced protein synthesis, potentially of an RNA-binding protein, is vital for this destabilization process.

While this is the first report of protein synthesis potentially leading to altered Npt2a mRNA stability in response to PTH, previous studies have demonstrated that Npt2a mRNA stability is regulated in response to various stimuli through differential protein expression. In a study examining hypophosphatemia-induced increased Npt2a mRNA expression, Moz et al. [114]showed that a low phosphate diet in rats induces post-transcriptional stabilization of Npt2a mRNA. In their model of hypophosphatemia, a low phosphate diet induced increased production of renal cytosolic proteins, and that these proteins had a protective effect on the Npt2a mRNA by binding to both the 5’ untranslated region (UTR) and 3’ UTR. Their work indicates that there is a cis-acting instability element in the protein-binding region of the 3’ UTR of Npt2a mRNA [114,115], and that cytosolic proteins produced in response to hypophosphatemia bind to this region and stabilize the transcript. Glucocorticoids, known downregulators of sodium-phosphate cotransport in the proximal tubule, have also been shown to reduce both Npt2a protein and mRNA expression. Dexamethasone, a synthetic glucocorticoid, decreases Npt2a expression in a manner that is also dependent on both transcription and translation [116]. This study in conjunction with ours suggests the possibility that the characteristics of the specific proteins interacting with the 3’ UTR of Npt2a mRNA determine the fate of the transcript.

8. Summary

Our understanding of the mechanisms underlying phosphate homeostasis is still evolving. PTH potently induces phosphaturia through the rapid downregulation of both Npt2a and Npt2c. Downregulation of Npt2a by PTH is responsible for the majority of phosphate transport loss induced by PTH, and is also responsible for the sustained hypophosphatemic response evoked by chronic PTH. The current understanding of the mechanisms behind PTH-induced phosphaturia is outlined in Figure 1. Activation of the PTHR1 by PTH produces activation of the G_s/AC/cAMP/PKA and G_q/PLC/IP₃/Ca²⁺/PKC pathways. Both PKA and PKC promote endocytosis of Npt2a followed by lysosomal degradation, as well as mRNA decay. Our studies support the notion that changes in Npt2a mRNA stability evoked by PTH are due to increased protein synthesis, potentially involving increased expression of specific RNA-binding proteins. Based on these findings, we propose that PTH upregulates the expression of an RNA binding protein that enhances Npt2a mRNA degradation. Further studies are required to validate this hypothesis.

Figure 1. Mechanisms of PTH-Induced Phosphaturia in the Proximal Tubule. (A) In the absence of PTH, Npt2a mRNA is translated and trafficked to the apical membrane, where it is stabilized through its interaction with the regulatory factor NHERF-1. (B) In the presence of PTH, the PTHR1 activates Gs and Gq signaling pathways. Both PKA and PKC contribute to the phosphorylation of NHERF-1, causing the dissociation of Npt2a from NHERF-1 and reducing the stability of Npt2a expression in the apical membrane. Npt2a then undergoes clathrin-mediated endocytosis to an endosomal compartment, followed by lysosomal degradation; reinsertion of Npt2a back into the apical membrane following removal the PTH stimulus requires de novo synthesis. PKA and PKC activation causes rapid decay of Npt2a mRNA, thus decreasing de novo synthesis of the transporter and promoting sustained hypophosphatemia.

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Acknowledgments

The opinions expressed in this paper do not reflect the views of the Veteran’s Administration. The work was supported by a Veteran’s Affairs Merit Review and University of Louisville School of Medicine grant (EDL) and NIH 5R21-AG047474 to SJK .

Conflict of Interest

The authors have no conflicts to report in this paper.