**4. Hormonal actions and interactions for regulating phosphate reabsorption in PTCs**

Phosphate uptake is an important function of PTCs which is regulated by the actions and interactions of various hormones and hormone-like substances, such as parathormone (PTH), fibroblast growth factor 23 (FGF23), klotho, glucocorticoids, vitamin D3, dopamine and so on (Figs. 2 and 3). Phosphate regulation is particularly associated with the development of cardiovascular disease and high mortality in patients with CKD, independently of the glomerular filtration rate (Kestenbaum et al. 2005). Therefore, the regulation of phosphate uptake in PTCs is clinically important.

#### **4.1 Transporters involved in PTC phosphate reabsorption that is hormonally regulated**

At least three types of sodium-dependent phosphate transporters have been identified in the brush border membrane of PTCs: NaPi-IIa, NaPi-IIc, and sodium-dependent phosphate symporter 2 (Pit-2) (Fig. 2). Renal reabsorption of inorganic phosphate is mainly (about 70%) mediated by NaPi-IIa in the adult kidney (Ohkido et al. 2003).

### **4.1.1 NaPi-IIa**

NaPi-IIa is a major phosphate transporter of PTCs, and changes in renal phosphate handling are primarily attributable to altered NaPi-IIa brush border membrane expression (Hernando et al. 2005). NaPi-IIa is expressed in the S1–S3 segment of superficial and juxtamedullary nephrons (Picard et al. 2010) and mediates the electrogenic transport of inorganic phosphate coupled to three sodium ions (Murer, Forster, and Biber 2004). NaPi-IIa is regulated by several hormones and other factors, as described in section 4.2. Adequate steady-state expression of NaPi-IIa and its inactivation by endocytosis and intracellular translocation require the presence of megalin (Bachmann et al. 2004), such that megalin dysfunction is likely to impair NaPi-IIa inactivation, inducing the increased reabsorption of phosphate (Fig. 3).

Hormonal Actions and Interactions in Proximal Tubule Cells

the progression of secondary hyperparathyroidism in CKD.

FGF23 has a direct action on PTCs (Huang and Moe 2011).

hyperparathyroidism in CKD (Kuro-o 2011).

**4.2.1 PTH** 

**4.2.2 FGF23** 

**4.2.3 Klotho** 

**4.2.4 Vitamin D3** 

Associated with the Development of Chronic Kidney Disease 313

PTH was first identified as an inactivator of NaPi-IIa through endocytic retrieval and degradation (Bacic et al. 2006). It was then found to be also associated with the endocytic regulation of NaPi-IIc (Segawa et al. 2007), although its acute effects differentially regulate these cotransporters including Pit-2 (Picard et al. 2010). The synthesis of PTH is increased in

FGF23 is a 32-kDa protein that is secreted mainly by bone osteocytes (Liu et al. 2006). Administration of FGF23 to mice decreases the renal expression of both NaPi-IIa and NaPi-IIc and induces hypophosphatemia predominantly via FGF receptor 1 (Gattineni et al. 2009). FGF23 also suppresses 1,25(OH)2D3 by inhibiting 1α-hydroxylase (CYP27B1), which converts 25-hydroxyvitamin D3 [25(OH)D3] to 1,25(OH)2D3, and by stimulating 24 hydroxylase (CYP24), which converts 1,25(OH)2D3 to inactive metabolites in PTCs (Shimada et al. 2004). Increased FGF23 appears to play a causative role in the abnormal renal phosphate handling and vitamin D3 metabolism of rats with CKD, leading to the development of secondary hyperparathyroidism and increased production of PTH (Hasegawa et al. 2010). The ternary complex of klotho-FGF23-FGF receptor in distal convoluted tubules (DCTs) has been thought to be a prerequisite for the exertion of FGF23 effects (Kurosu et al. 2006), and, indeed, initial FGF23-mediated signaling occurs in DCTs (Farrow et al. 2009). However, it remains undetermined how FGF23-mediated signaling in DCTs leads to the regulation of NaPi-IIa and NaPi-IIc in PTCs. It may be possible that

Klotho was first identified as an aging suppressor gene product (Kuro-o et al. 1997). It is a single-pass transmembrane protein expressed predominantly in the kidney, intensely in DCTs and to a lesser extent in PTCs and parathyroid gland (Kuro-o 2011). *Klotho*–/– and *Fgf23*–/– mice show very similar phenotypes such as premature aging and abnormal mineral metabolism (Kuro-o 2011). As mentioned above, klotho acts with FGF receptor as a coreceptor in the binding of FGF23 in DCTs for phosphate and vitamin D3 regulation (Kurosu et al. 2006). The Klotho-FGF23-FGF receptor complex in DCTs may produce putative paracrine factors on PTCs where phosphate transporters and enzymes for vitamin D3 regulation are located (Huang and Moe 2011). The extracellular domain of Klotho is also known to be cleaved from plasma membranes, secreted to the circulatoin and present in urine (Hu et al. 2011); this soluble klotho form may directly act on PTCs. Klotho was also found to act as a PTC-synthesized autocrine factor in the FGF23-independent inhibition of NaPi-IIa function by ß-glucuronidase-like enzymatic activity (Hu et al. 2011). Decreased Klotho expression in the kidney may be one of initial factors triggering dysregulation of various hormones involved in phosphate homeostasis and the development of secondary

As mentioned above, megalin and cubilin take up the 25(OH)D3/DBP complex from glomerular filtrates (Nykjaer et al. 1999; Nykjaer et al. 2001; Leheste et al. 2003). In PTCs, 25(OH)D3 is dissociated from DBP and converted by 1α-hydroxylase to the biologically active form 1,25(OH)2D3, which is released to peritubular capillaries. Therefore, dysfunction of these endocytic receptors is an important cause of deficiency of both 25(OH)D3 and

In CKD, renal expression of megalin and klotho is decreased, resulting in increased urinary phosphate excretion. Decreased megalin expression also causes decreased renal synthesis of 1,25(OH)2 D3. Synthesis of FGF23 in the bone is then stimulated, which also suppresses renal 1,25(OH)2 D3 synthesis, increasing the synthesis of PTH in the parathyroid.

Fig. 3. Presumed hormonal network involved in phosphate regulation in CKD

#### **4.1.2 NaPi-IIc**

NaPi-IIc is a growth-related transporter that is more active in the young and has a reduced role in adults (Segawa et al. 2002). NaPi-IIc transports inorganic phosphate in an electroneutral fashion and is highly pH dependent, exhibiting more transport at a more alkaline pH (Segawa et al. 2002). It is expressed in the S1 segment of PTCs (Picard et al. 2010) and mutations of the NaPi-IIc gene were found to cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal inorganic phosphate reabsorption (Bergwitz et al. 2006; Lorenz-Depiereux et al. 2006).

## **4.1.3 Pit-2**

Pit-2, expressed in S1 and weakly in S2 segments of superficial and juxtamedullary nephrons (Picard et al. 2010), mediates the electrogenic transport of inorganic phosphate (Ravera et al. 2007).

#### **4.2 Hormones regulating the expression and function of PTC transporters involved in phosphate reabsorption**

Several hormones and hormone-like substances are involved in the regulation of phosphate cotransporters. In particular, the actions and interactions of PTH, fibroblast growth factor 23 (FGF23), klotho and vitamin D3 are important in maintaining phosphate homeostasis and the development of CKD-mineral bone disease (MBD) (Fig. 2 and 3).

## **4.2.1 PTH**

312 Basic and Clinical Endocrinology Up-to-Date

In CKD, renal expression of megalin and klotho is decreased, resulting in increased urinary phosphate excretion. Decreased megalin expression also causes decreased renal synthesis of 1,25(OH)2 D3. Synthesis of FGF23 in the bone is then stimulated, which also suppresses renal 1,25(OH)2 D3 synthesis,

NaPi-IIc is a growth-related transporter that is more active in the young and has a reduced role in adults (Segawa et al. 2002). NaPi-IIc transports inorganic phosphate in an electroneutral fashion and is highly pH dependent, exhibiting more transport at a more alkaline pH (Segawa et al. 2002). It is expressed in the S1 segment of PTCs (Picard et al. 2010) and mutations of the NaPi-IIc gene were found to cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal inorganic phosphate

Pit-2, expressed in S1 and weakly in S2 segments of superficial and juxtamedullary nephrons (Picard et al. 2010), mediates the electrogenic transport of inorganic phosphate

**4.2 Hormones regulating the expression and function of PTC transporters involved in** 

Several hormones and hormone-like substances are involved in the regulation of phosphate cotransporters. In particular, the actions and interactions of PTH, fibroblast growth factor 23 (FGF23), klotho and vitamin D3 are important in maintaining phosphate homeostasis and

Fig. 3. Presumed hormonal network involved in phosphate regulation in CKD

reabsorption (Bergwitz et al. 2006; Lorenz-Depiereux et al. 2006).

the development of CKD-mineral bone disease (MBD) (Fig. 2 and 3).

increasing the synthesis of PTH in the parathyroid.

**4.1.2 NaPi-IIc** 

**4.1.3 Pit-2** 

(Ravera et al. 2007).

**phosphate reabsorption** 

PTH was first identified as an inactivator of NaPi-IIa through endocytic retrieval and degradation (Bacic et al. 2006). It was then found to be also associated with the endocytic regulation of NaPi-IIc (Segawa et al. 2007), although its acute effects differentially regulate these cotransporters including Pit-2 (Picard et al. 2010). The synthesis of PTH is increased in the progression of secondary hyperparathyroidism in CKD.
