Dent disease: A window into calcium and phosphate transport

Abstract This review examines calcium and phosphate transport in the kidney through the lens of the rare X‐linked genetic disorder Dent disease. Dent disease type 1 (DD1) is caused by mutations in the CLCN5 gene encoding ClC‐5, a Cl−/H+ antiporter localized to early endosomes of the proximal tubule (PT). Phenotypic features commonly include low molecular weight proteinuria (LMWP), hypercalciuria, focal global sclerosis and chronic kidney disease; calcium nephrolithiasis, nephrocalcinosis and hypophosphatemic rickets are less commonly observed. Although it is not surprising that abnormal endosomal function and recycling in the PT could result in LMWP, it is less clear how ClC‐5 dysfunction disturbs calcium and phosphate metabolism. It is known that the majority of calcium and phosphate transport occurs in PT cells, and PT endocytosis is essential for calcium and phosphorus reabsorption in this nephron segment. Evidence from ClC‐5 KO models suggests that ClC‐5 mediates parathormone endocytosis from tubular fluid. In addition, ClC‐5 dysfunction alters expression of the sodium/proton exchanger NHE3 on the PT apical surface thus altering transcellular sodium movement and hence paracellular calcium reabsorption. A potential role for NHE3 dysfunction in the DD1 phenotype has never been investigated, either in DD models or in patients with DD1, even though patients with DD1 exhibit renal sodium and potassium wasting, especially when exposed to even a low dose of thiazide diuretic. Thus, insights from the rare disease DD1 may inform possible underlying mechanisms for the phenotype of hypercalciuria and idiopathic calcium stones.


| ALTERED C ALCIUM -PHOS PHATE ME TABOLIS M IN DENT D IS E A S E
Most affected males are hypercalciuric, and this can be marked in young children. However, in teenagers and adults the hypercalciuria is typically comparable to that observed in routine calcium stone formers. 3 Nephrocalcinosis with onset in childhood is common, while nephrolithiasis (calcium oxalate and/or phosphate) occurs in only about 25%. Although hypercalciuria is common, urinary oxalate and citrate excretions are typically normal.
Detailed studies on calcium and phosphate metabolism in DD are few. 4

| LOCALIZATION AND FUNCTION OF CLC-5
ClC-5 is a 746-aminoacid protein with multiple membrane-spanning domains and intracellular N-and C-terminal domain.  belongs to the family of voltage-gated chloride channels, which is comprised of cell surface channels and intracellular Cl − /H + exchangers. 6 ClC-5 is highly expressed in the kidney, primarily in the proximal tubular cells (PTCs) of the S3 segment, in the ⍺-intercalated cells of the cortical collecting duct of mouse and rat kidney, but also in the cortical and medullary thick ascending limb of Henle's loop (mTAL). [7][8][9][10] In the PTCs, ClC-5 plays an important role during receptormediated endocytosis of albumin and LMW proteins that make it through the glomerulus and colocalizes with vacuolar H + -ATPase (V-ATPase). 11,12 There ClC-5 contributes to the acidic pH within the endosomes important for ligand:receptor dissociation and subsequent recycling of the receptor to the apical membrane and degradation of the ligand within endosomes 13,14 (Figure 1).
The prevalent view was that ClC-5 supports efficient acidification of endosomes either by providing a Cl − conductance to counterbalance the accumulation of positively charged H + pumped in by V-ATPase, either directly in parallel with V-ATPase by acting as a Cl − / H + exchanger. 15 Recently, however, experimental evidence indicates that the endosomal Cl − concentration might play a role in endocytosis independent of endosomal acidification, thus suggesting another possible mechanism by which ClC-5 dysfunction may cause disease and impair endocytosis. 7 ClC-5 may have additional functions since about 8% is located at the PTC plasma membrane. 16 ClC-5 plays a key role in the formation/function of the endocytic complex that includes the megalin/cubilin, the sodium-hydrogen antiporter 3 (NHE3) F I G U R E 1 Receptor-mediated endocytosis of LMW proteins in the proximal tubule. LMW proteins undergo receptor-mediated endocytosis involving megalin and cubilin. Receptors and ligands are internalized by the formation of endosomes, which are progressively acidified by V-ATPase. Chloride influx via ClC-5 facilitates acidification by maintaining electroneutrality of ion transport. Endosomal recycling brings receptors back to the apical surface. The absence or dysfunction of ClC-5 can potentially disrupt endosome cycling at three points (indicated by arrows): (1) reduced rate of receptor internalization; (2) disrupt progression of endosomes to lysosomes; and (3) disrupt recycling of endosomes to the cell surface and the V-ATPase. Evidence suggests that the large intracellular ClC-5 C-terminus plays a crucial function to mediate the assembly, stabilization and disassembly of the endocytic complex via protein-protein interactions. The Hryciw group demonstrated that the C-terminus of ClC-5 also binds the actin-depolymerizing protein cofilin. 17 When the nascent endosome forms, recruitment of cofilin by ClC-5 appears required to localize dissolution of the actin cytoskeleton, thereby allowing the endosome to pass into the cytoplasm. It has been demonstrated both in mouse models and in humans that loss of ClC-5 causes defective PTC trafficking of megalin and cubilin. [18][19][20] Thus, ClC-5 may serve two roles during receptor-mediated endocytosis: (a) vesicular acidification and receptor recycling; and (b) participation in the non-selective megalin-cubilin receptor complex at the apical membrane.
Protein endocytosis in the PTCs relies on active receptors that include not only megalin and cubilin, but also amnionless, disabled-2. 21 Megalin and cubilin bind many protein ligands from tubular fluid including the commonly measured LMW proteins β2-microglobulin, ⍺-1 microglobulin and retinol-binding protein (RBP). Whereas megalin binds certain ligands independently, it binds others, such as albumin, jointly with the coreceptor cubilin. Other megalin ligands play key roles in systemic phosphate and calcium metabolism including vitamin D-binding protein and PTH.
In the collecting duct, ClC-5 protein localizes to the ⍺-intercalated cells which are key for acid-base homeostasis. ClC-5 colocalizes with the V-ATPase in the apical and subapical vesicles. 12 Accordingly, ClC-5 might be important for insertion and recycling of these vesicles, and when ClC-5 function is lost, defective expression of V-ATPase may result causing impaired urinary acidification.
When ClC-5 expression was silenced by an antisense ClC-5 transfection in a collecting duct cell model (mIMCD-3), endocytosis was arrested, and calcium oxalate crystals agglomerated on the ClC-5 silenced cells, suggesting a possible physiological role for ClC-5 during crystal clearance in the collecting duct. Thus, loss of ClC-5 function in this segment may contribute to nephrocalcinosis risk. [22][23][24] The role of ClC-5 in mTAL remains circumstantial and speculative. 25 Studies suggest that endocytosis takes place in murine mTAL where V-ATPase is also present 26 albeit at a lesser extent than in PTCs, thus suggesting a possible role for ClC-5 during endocytosis and exocytosis processes in this nephron segment.

| LO C ALIZ ATI ON AND FUN C TI ON OF C ALCI UM AND PHOS PHATE TR AN S P ORTER S IN THE K IDNE Y
Approximately 80% of filtered phosphate is reabsorbed from the urine via proximal tubular transporters, mostly in juxtamedullary nephrons 27,28 (Figure 2). About 98% of filtered calcium is reabsorbed along the nephron, paracellularly in the PT and in the TAL of the loop of Henle and transcellularly in the distal convoluted tubule and connecting tubule ( Figure 3). No reabsorption occurs in the collecting duct; therefore, F I G U R E 2 Renal phosphate reabsorption in the proximal tubule. Phosphate is reabsorbed via three sodium phosphate cotransporters: NaPi2a, NaPi2c and PIT-2. In humans, NaPi2a and NaPi2c are believed to play the most important role in phosphate reabsorption. They are positioned at the apical membrane of renal proximal tubular cells and take advantage of an inward electrochemical gradient for sodium to move phosphate from the filtrate into the cell. The amount of phosphate reabsorbed is dependent on the abundance of the sodium phosphate cotransporters and variations in their number at the brush border membrane are a primary regulatory pathway for urinary phosphate excretion F I G U R E 3 Renal calcium reabsorption along the nephron. The majority of filtered calcium is reabsorbed in the proximal tubule (PT) and thick ascending limb (TAL), with the final highly regulated percentage in the distal convoluted tubule (DCT) and in the connecting tubule (CNT). In the PT, calcium is mainly reabsorbed paracellularly, partially driven by activity of the sodium/proton exchanger 3 (NHE3), which allows transcellular sodium entry at the apical brush border, while the Na-K-ATPase pumps sodium out of the cell at the basolateral side. In TAL, calcium is reabsorbed by specialized and controlled paracellular pathways involving claudin 16, 19 and 14. The driving force for calcium is produced by the combined action of the basolateral Na-K-ATPase, the Na-K-Cl cotransporter (NKCC2) and the outward rectifying ROMK channel on the apical membrane. In DCT-CNT, calcium enters the cell at the apical side through TRPV5 channels, binds intracellular calbindin-D-28k and exits the cell at the basolateral side by the Na-Ca exchanger (NCX1) and the Ca-ATPase PMCA4 29 any calcium delivered there is subject to precipitation depending most importantly upon the tubular fluid pH. 29 In the PT, the majority of calcium is reabsorbed by passive and hormone-independent paracellular transport. The claudin family of epithelial tight junction proteins are crucial for paracellular calcium permeability. Active calcium transport in the S3 segment accounts for up to 20%-30% of PT calcium flux, and however, the precise pathway of this transport is not well defined. Several studies detected apical PT calcium-sensing receptor (CaSR) expression, a cation-sensing G protein-coupled receptor also present in the parathyroid gland. 30,31 CaSR expression in the PT appears to be regulated by vitamin D, and activation of CaSR may in turn alter PT expression of the vitamin D receptor. Activation of the CaSR in the kidney has other effects on transport, including increased sodium reabsorption and proton secretion in the mouse PT.
Thus, the majority of calcium and phosphate transport localizes in PTCs where ClC-5 plays an important role in endocytosis. Do these endocytic processes in turn impact PT calcium and phosphate transport?

| Renal phosphate handling
NaPi2a is the predominant phosphate transporter in the PT. 27 Major physiological regulators of NaPi2a expression include PTH, dopamine and fibroblast growth factor-23 (FGF 23), each of which stimulates NaPi2a endocytic removal and degradation as well as decrease mRNA expression. 32 PTH regulates NaPi2a membrane expression by stimulating NaPi2a endocytosis ( Figure 4). This differs from regulation of many other transport proteins that requires modification of the protein itself. 33 NaPi2a, apical PTH receptors and PLCβ1 are organized in a macromolecular complex via the scaffolding protein Na + /H + exchanger regulatory factor (NHERF1). 33 Phosphorylation of NHERF1 is followed by dissociation of the NaPi2a/NHERF-1 complex, with NHERF1 remaining at the apical membrane, while NaPi2a is internalized. PTH-induced inactivation of NaPi2a is facilitated by megalin. 34 NHERF1 is necessary for PTH-induced internalization of NaPi2a via apical but not basolateral PTH receptors. 35 A similar mechanism has been proposed for FGF23 and dopamine regulation of NaPi2a expression.
A low-phosphate diet has the opposite effect, stimulating insertion of NaPi2a into the apical membrane and inhibiting endocytosis.
Because endocytosed cotransporters are degraded in lysosomes, recovery of NaPi2a to basal levels when PTH stimulation is removed depends on de novo synthesis. Thus, apical retention/removal of NaPi2a is a highly regulated process. 33 Since the scaffold protein NHERF1 plays an important role regulating NaPi2a apical trafficking, as either a chaperone, a scaffolding protein or both, 32 a link between ClC-5 and NHERF1 whereby ClC-5 influences PT apical NaPi2a expression seems possible.
NHERF scaffold proteins are also crucial for maintaining the macromolecular complex responsible for LMW proteins uptake at the PT brush borders 36 ( Figure 5). Evidence supports this series of events in animal models. 37,38 The presence of albumin increased levels of plasma membrane-associated NHERF2, and silencing NHERF2 led to significant reduction in apical membrane ClC-5, the number of actin clusters and albumin uptake. This in vivo data support a key role for ClC-5 nucleation of the endocytic complex, presumably by tethering of the complex via NHERF2 to the actin cytoskeleton. These data collectively suggest that NHERF2 is necessary for maintaining ClC-5 at the plasma membrane. However, the significance of these findings is still not clear, since NHERF1 or NHERF2 KO mice lacked LMWP, the hallmark of ClC-5 dysfunction. 39 Intriguingly, however, NHERF1 null mice manifest decreased apical membrane NaPi2a, hyperphosphaturia and hypercalciuria.
F I G U R E 4 PTH-induced endocytosis of NaPi2a. In the proximal tubule, PTH binds to apical and basolateral PTH receptors. Stimulation of either receptor is known to rapidly induce phosphaturia by decreasing apical phosphate transporter activity. Activation of phospholipase C (PLC) by apical PTH receptors leads to protein kinase C (PKC)-dependent stimulation of ERK1/2 kinases and internalization of NaPi2a. Basolateral PTH receptors are linked to adenylate cyclase (AC), protein kinase A (PKA) and ERK1/2. NaPi2a is internalized via clathrin-coated vesicles, transported to endosomes and targeted to lysosomes for degradation
These cells also contain 24-hydroxylase that converts 1,25(OH) 2 D to the biologically inactive metabolite calcitroic acid. 40 Together, these data strongly support a pivotal role for PT endocytosis in renal calcium and phosphorus handling.  (Table 1). However, targeted disruption of ClC-5 in the Jentsch model did not lead to hypercalciuria, kidney stones or nephrocalcinosis, while a similar disruption in the Guggino model did. 48 Mice in the Jentsch model produced slightly more acidic urines. Urinary phosphate excretion was increased in both models by about 50%. Hyperphosphaturia in the Jentsch model was associated with decreased apical NaPi2a expression, although principally at the brush border of the S1 PT segment and at subapical vesicles of other PT segments. This was surprising because defective NaPi2a endocytosis due to loss of ClC-5 function might be expected to result in increased plasma membrane presence. Indeed, observed changes in NaPi2a surface expression in these KO mice appeared ClC-5-independent since apical NaPi2a was not decreased in any PTs of chimeric female mice, while it was in all PTs of -/y male mice.
Depriving ClC-5 -/y mice of dietary Pi enhances plasma membrane NaPi2a expression to normal levels. When these KO mice were given PTH, NaPi2a was internalized but at a slower rate compared with WT mice. The reduced internalization of NaPi2a in response to PTH suggests that altered PTH delivery might lead to vesicular localization of NaPi2a in the S3 PT segment of KO mice. Indeed, whereas serum PTH is normal in KO mice, urinary PTH is increased by about 1.7-fold. Because megalin is down-regulated in these mice, luminal PTH levels are increased. The increased stimulation of apical PTH receptor in turn enhances internalization of NaPi2a in more distal segments of the PT.
In conclusion, the phosphaturia in KO animals may be a consequence of reduced endocytosis of filtered PTH.

| Calcium transport
The possibility that ClC-5 may participate in renal calcium transport was suggested by the finding that ClC-5 expression in the renal cortex is under PTH regulation and that renal ClC-5 expression inversely correlated with urinary calcium excretion. 46 The hypothesis that hypercalciuria in DD1 is intrinsic to kidney-mediated mechanisms is supported by the observation that hypercalciuria is absent after kidney transplantation. 3 The discrepancy between the two mouse models regarding mechanisms of hypercalciuria in DD1 might suggest another possibility, that is it might be caused by defective NHE3 expression at the apical PT. A link between sodium and calcium reabsorption is well known.
Thiazide diuretics that block the Na + -Cl − cotransporter (NCC) in the distal convoluted tubule have a hypocalciuric effect stimulating sodium and calcium reabsorption in the PT. 54,55 The effect of diuretics on renal tubular calcium transport has been recently reviewed by Alexander and Dimke, 56 and studies suggest the PT as the major site of calcium reabsorption strictly linked to sodium reabsorption. 57

| THE NA + /H + E XCHANG ER NHE3 AND C ALCI UM REG UL ATI ON AT THE PROXIMAL TUBULE
The Na + /H + exchanger (NHE) protein family contains at least nine isoforms divided into plasma membrane isoforms (NHE1-NHE5) and endomembrane isoforms (NHE6-NHE9). 58 The plasma membrane isoforms function to exchange extracellular Na + for intracellular H + .
In adult kidney, PT NHE3 is the predominant brush border Na + /H + exchanger ( Figure 7).
Recent studies highlight the importance of PT NHE3 for calcium reabsorption. NHE3 −/− KO mice manifest significant urinary calcium wasting. 59  From these studies, the importance of PT NHE3 regulation clearly emerges, not only on calcium but also on phosphate handling.

| CLC-5 KO MODEL S: WHAT C AN THE Y TELL US ON NHE3 REG UL ATI ON?
Sodium-hydrogen antiporter 3 is dysregulated in ClC-5 KO mice. In the Jentsch model, 46

| CON CLUS IONS
Observations in patients with DD1 and model systems highlight the importance of receptor-mediated endocytosis for PT function.
The PT plays a pivotal role in renal phosphate and calcium handling, exerted mainly through the regulation of ClC-5-dependent (PTH) endocytosis. In fact, reduced expression of megalin, by mediating the uptake of PTH, antagonizes the effect of increased PTH availability on the luminal PTH receptor. Thus, the delicate balance between PTH reabsorption and PTH receptor activation influences both NaPi2-mediated proximal tubular phosphate reabsorption and the highly regulated proximal tubular synthesis of vitamin D.
ClC-5-dependent PTH endocytosis and ClC-5-dependent exocytosis may regulate NHE3-mediated proximal tubular sodium reabsorption and consequently paracellular calcium reabsorption. The importance of NHE3 dysfunction in determining DD1 phenotype was not investigated in patients with DD1.
However, it could be hypothesized that the high rate of hypokalemia among adults patients with DD1 might be due to the increased Na + delivery into the distal tubules and the activation of the renin-angiotensin system secondary to hypovolaemia may result in metabolic alkalosis, with hypokalemia as a secondary phenomenon. Thus, NHE3 appears to play a crucial role in PT receptor-mediated endocytosis, suggesting that ClC-5-dependent NHE3 dysfunction might be an important factor in the cascade of events leading to the common DD1 phenotype that includes LMWP, hypercalciuria and hypophosphatemia. These observations relate to recent in vivo studies in hypercalciuric humans that implicate the PT as the site of abnormal calcium reabsorption. Thus, the insights from the rare disease DD1 may have implications for the more common phenotype of hypercalciuria and idiopathic calcium stones.

ACK N OWLED G EM ENTS
The authors gratefully acknowledge the support of the Rare Kidney

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
AF, LBL and JL wrote the manuscript, LG conceived and designed figures and contributed to the discussion on the review topics.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.