**2.1.1 Megalin**

306 Basic and Clinical Endocrinology Up-to-Date

In this review, we focus on actions and interactions of various hormones and hormone-like substances on the regulation of apical membrane receptors and transporters that mediate three major functions of PTCs: 1) receptor-mediated protein endocytosis, 2) sodium reabsorption, and 3) phosphate reabsorption, which are particularly associated with the

**2. Hormonal actions and interactions for regulating protein endocytosis** 

Receptor-mediated endocytosis is a pivotal function of PTCs through which the cells reabsorb and metabolize proteins (and other substances) from glomerular filtrates (Saito, Sato et al. 2010). This reabsorption process is extremely efficient as urine is virtually proteinfree in humans, and it accounts for the essential conservation of nutrients, carrier-bound vitamins, and trace elements filtered by glomeruli. Impairment of the process results in a

The two major endocytic receptors expressed at the apical membranes of PTCs are megalin and cubilin (Saito, Sato et al. 2010) which act cooperatively in the uptake of glomerular-

Some hormones, such as dopamine, renalase, vitamin D3 and klotho, are synthesized in PTCs and

Fig. 2. Hormonal actions and interactions on the functions of receptors and transporters involved in receptor-mediated protein endocytosis and reabsorption of sodium and

AMN, amnionless; FGF23, fibroblast growth factor 23; GLP1, glucagon-like peptide 1; NaPi-IIa, sodiumdependent phosphate cotransporter type IIa; NaPi-IIc, sodium-dependent phosphate cotransporter type IIc; NHE3, Na+/H+ exchanger isoform 3; Pit-2, sodium-dependent phosphate symporter 2; PTC,

pathophysiology of CKD and the increased risk of CVD (Fig. 2).

loss of such substances and development of proteinuria.

**2.1 Endocytic receptors involved in protein reabsorption in PTCs** 

filtered proteins and mediate their metabolism in PTCs (Fig. 2).

others come from other organs. Renalase acts to metabolize dopamine.

phosphate in the apical membrane of PTCs.

proximal tubule cell; PTH, parathormone; SGLT2, sodium-glucose cotransporter 2

**in PTCs** 

Megalin is a large (~600 kDa) glycoprotein member of the low-density lipoprotein receptor family that is primarily expressed in clathrin-coated pits (Saito, Sato et al. 2010). Megalinligand complexes are internalized by invagination of clathrin-coated pits mediated by multiple adaptor proteins and motor molecules, forming endosomal vesicles. Acidification of the intravesicular lumen dissociates the ligands from megalin and they are transported to lysosomes for degradation or storage or are secreted into the cytosol for further processing or transport. Megalin is then recycled to the apical membranes through a recycling compartment. Megalin thus plays a critical role in reabsorption and metabolism of glomerular-filtered substances including albumin and low molecular weight proteins. Vitamin D-binding protein (DBP) is an endocytic ligand of megalin, and megalin knockout mice display decreased utilization of vitamin D for its activation in PTCs (Nykjaer et al. 1999; Leheste et al. 2003) (Fig. 3).

Decreased megalin expression in PTCs has been observed in the early diabetic stages of experimental animals (Tojo et al. 2001; Russo et al. 2007), and it has been suggested that the functions of megalin are also impaired in patients during the early stages of diabetic nephropathy, since low molecular weight proteinuria is frequently observed in patients at these stages (Pontuch et al. 1992; Hong et al. 2003). Therefore, the altered regulation of megalin expression and its functions must be primarily responsible for the early development of proteinuria/albuminuria in diabetic patients.

Megalin also interacts with various membrane receptors and transporters such as the cubilin/amnionless complex, sodium-hydrogen exchanger isoform 3 (NHE3) and sodiumdependent phosphate cotransporter type IIa (NaPi-IIa) as discussed in the following sections.

## **2.1.2 Cubilin**

Cubilin is a 460-kDa peripheral glycoprotein that lacks transmembrane and intracellular segments but is anchored to apical PTC membranes. It was originally identified as the receptor for intrinsic factor-vitamin B12 complex (Seetharam et al. 1988; Seetharam et al. 1997). Cubilin requires interaction with megalin to regulate its endocytic functions (Yammani, Seetharam, and Seetharam 2001; Kozyraki et al. 2001); however, it is bound more firmly by a protein called amnionless forming the complex CUBAM (Fyfe et al. 2004; Coudroy et al. 2005). Cubilin gene defects are the cause of hereditary megaloblastic anaemia 1 and Imerslund-Gräsbeck syndrome, also known as selective vitamin B12 malabsorption with proteinuria (Aminoff et al. 1999). Cubilin is also involved in the absorption of various protein ligands present in glomerular filtrates, including albumin, transferrin and DBP (Christensen, Verroust, and Nielsen 2009). A recent meta-analysis of genome-wide association studies identified a missense cubilin gene variant associated with levels of albuminuria in both the general population and in diabetic individuals (Boger et al. 2011).

#### **2.1.3 Amnionless**

Amnionless is a 38–50 kDa membrane protein with a single transmembrane domain that was initially identified as a component required for normal development of the trunk mesoderm derived from the middle streak (Kalantry et al. 2001). In addition, defects of the amnionless gene cause hereditary megaloblastic anaemia (Tanner et al. 2003).

Hormonal Actions and Interactions in Proximal Tubule Cells

**3.1.1 Sodium-hydrogen exchanger isoform 3** 

reviewed elsewhere (Bobulescu and Moe 2009).

diabetic patients (Bailey et al. 2010; List and Whaley 2011).

**3.1.3 Sodium-dependent phosphate cotransporters** 

**3.1.2 Sodium-glucose cotransporter-2** 

**sodium reabsorption in PTCs** 

**3.2.1 Angiotensin II** 

through the function of NHE3 (Fig. 2).

**in PTCs** 

Associated with the Development of Chronic Kidney Disease 309

Various transporters and channels are involved in sodium reabsorption in the apical and basolateral membranes of PTCs. Here, we focus on three types of apical transporters (Fig. 2).

Sodium-hydrogen exchanger isoform 3 (NHE3), the main NHE isoform of PTCs, mediates isotonic reabsorption of approximately two-thirds of filtered NaCl and water, the reabsorption of bicarbonate, and the secretion of ammonium (Bobulescu and Moe 2009). It also contributes to the reabsorption of filtered citrate, amino acids, and oligopeptides by providing H+ used by H+-coupled cotransporters. Indeed, enhanced NHE3 activity is assumed to be a factor for the increased sodium reabsorption and the development of hypertension in diabetes. NHE3 was reported to interact with megalin in PTC intermicrovillar clefts (Biemesderfer et al. 1999; Biemesderfer, DeGray, and Aronson 2001) and, following endocytosis with megalin, it is postulated to utilize the outward transvesicular sodium gradient of endocytic vesicles and early endosomes to drive the inward movement of H+ and endosomal acidification. This is important for dissociating reabsorbed ligand proteins from megalin both for further processing and megalin recycling to the cell surface. Hormonal regulation of NHE3 has been intensively investigated and

Glucose is freely filtered by glomeruli before being almost entirely reabsorbed into the circulation from PTCs. Reabsorption of the majority of glucose is achieved by sodiumglucose cotransporter-2 (SGLT2), which is present in the S1 segment of proximal tubules. Expression of SGLT2 is enhanced in the kidney of diabetic Zucker rats (Tabatabai et al. 2009). SGLT2 inhibitors currently in development are expected not only to treat hyperglycemia but also to have an effect on hypertension and glomerular hyperfiltration in

Sodium-dependent phosphate cotransporters reabsorb inorganic phosphate in a sodium-

**3.2 Hormones regulating the expression and function of the transporters involved in** 

Many hormones are involved in the regulation of sodium reabsorption in PTCs, particularly

Angiotensin II is important for increasing sodium uptake by PTCs. Intrarenal levels of angiotensin II are augmented in CKD and are also involved in the development of hypertension (Kobori et al. 2007; Navar et al. 2011). The action of angiotensin II on NHE3 has been intensively investigated, indicating biphagic dosage-dependent acute effects and chronic stimulatory effects on NHE3 (Bobulescu and Moe 2009). Classically, angiotensin II has been thought to act on its specific receptors in PTC plasma membranes by endocrine

dependent manner in PTCs. These will be discussed in detail in section 4.1.

**3.1 Major transporters involved in sodium reabsorption and regulated by hormones** 
