Recent advances in understanding the clinical and genetic heterogeneity of Dent's disease

Dent's disease 1 (OMIM 300009) is an X-linked proximal tubulopathy [ 1 ] first described in 1964 [ 2 ], with Fanconi syndrome, a consistent renal abnormality, and low-molecular-weight proteinuria (LMWP) being almost always present. Nephrocalcinosis and renal stone formation occur more frequently in Dent's disease 1 than in other forms of Fanconi syndrome. Various features of Dent's disease 1 predominate in different ethnic groups, and have been noted in earlier reports, resulting in several syndrome names of phenotypically similar disorders. These were referred to as X-linked recessive hypophosphataemic rickets (XLRH), X-linked recessive nephrolithiasis (XRN) and familial idiopathic LMWP in Japanese patients (JILMWP), often referred to as Dent's Japan disease [ 3 , 4 ]. All of these are now considered phenotypic variants of one unique entity, namely Dent's disease 1 [ 5 , 6 ]. Dent's disease 1 commonly presents in childhood or early adult life with symptoms of LMWP, hypercalciuria and nephrolithiasis/nephrocalcinosis, but less often with renal failure. LMWP is thought to be the only constant feature. However, in one patient, a ClC-5 mutation not associated with LMWP was detected [ 7 ]. Female carriers are usually asymptomatic (depending on lyonization) but often show LMWP and, more rarely, hypercalciuria [ 8 , 9 ]. Not all families and patients exhibit the same clinical features, although frequent thorough investigations have been rare. The most complete study is the report of Wrong et al . [ 1 ] ( Table 1 ). Not all patients had a decreased glomerular filtration rate (GFR). However, in all tested patients, GFR declined progressively with age. The frequency of reduced tubular phosphate reabsorption is difficult to judge, as decreased GFR has to be taken into account [ 10 ]. We calculated the tubular reabsorption from the data of Wrong et al . [ 1 ] ( Table 1 , Figure 1 ). Not all patients showed a decrease in phosphate reabsorption. In 14 patients, oral ammonium chloride was given to assess urinary acidification, and in seven patients it was found to be normal. Defective urinary acidification was related to the duration and severity of renal calcification. In four adequately tested children, urinary acidification was normal, thus indicating a secondary defect in the older patients. The cause of rickets in this series could not be adequately investigated [no measurements of parathyroid hormone (PTH) and vitamin D metabolites were available], but rickets was cured by pharmacological doses of vitamin D. In later reports [ 12 ], PTH was noted as normal or decreased and 1,25-dihydroxycholecalciferol (1,25-DHC) was elevated. A defect in ClC-5 influences the concentration of 1,25-DHC in an opposing way [ 13 ]. It has been hypothesized that 1-α-hydroxylase activity may be higher due to increased luminal PTH, while defective endocytosis leads to decreased availability of the precursor [ 14 ]. Hypophosphataemia also augments α-hydroxylase activity. Haematuria in Dent's disease 1 patients is probably related to nephrocalcinosis/stone formation. The CLCN5 gene, affected by mutations in Dent's disease 1 patients, was first identified in a family carrying a microdeletion [ 15 , 16 ]. The CLCN5 gene, encoding the voltage-gated chloride channel and chloride/proton exchanger (ClC-5) [ 17 , 18 ], spans ∼170 kb on chromosome Xp11.23/p11.22, comprises 17 exons and transcription initiates from at least four different start sites ( Figure 2 ). Transcripts (GenBank accession numbers X91906 and BK000969) including the untranslated exon 1a (start site 2) [ 19 ] or 1b (start site 4) [ 16 ] are spliced to exon 2 containing the start-ATG, whereas a third mRNA (arising from start site 3) comprises a larger exon 1b and retains intron 1 [ 20 ]. Two further transcripts (start site 1), due to alternative splicing of exon II, include exons I to IV [ 21 ]. Both these transcripts carry the start-ATG in exon III, thereby encoding a longer ClC-5 isoform consisting of 816 amino acids with an additional 70 amino acids at the intracellular amino terminus. Since these two mRNAs maintain the reading frame, the start-methionine of the shorter form (746 amino acids) resides at codon position 71. The longer variant, however, has only been detected at mRNA and not at the protein level [ 21 ]. To date, more than 80 distinct CLCN5 mutations, consisting of nonsense, missense, splice site and insertional and deletional mutations, have been reported in patients with Dent's disease 1 [ 3–5 , 16 , 22–45 ]. These data provide no evidence for a genotype–phenotype correlation, since various mutations were found to be associated with quite different clinical phenotypes ranging from ‘classic’ Dent's disease 1 to very slight urinary abnormalities, not only in unrelated patients but even within the same family [ 7 ]. Also, various groups provided evidence for genetic heterogeneity, in that, patients with typical features of Dent's disease 1, in whom no CLCN5 mutations could be detected, were encountered [ 21 , 24 , 29 , 39 , 43 ]. Recent investigations have revealed that defects in the OCRL1 gene (GenBank accession numbers NM001587 and NM000276) encoding a phospatidylinositol 4,5-bisphosphate 5-phosphatase [PtdIns(4,5)P 2 5-phosphatase; EC 3.1.3.36] are also responsible for a phenotype resembling Dent's disease 1 [ 46 ]. In the OMIM database, Dent's disease associated with OCRL1 mutations is now termed Dent's disease 2 (OMIM #300555). Mutations in the OCRL1 gene, located at Xq25 [ 47 ], were initially found to cause Lowe syndrome [ 48 ], a pleiotropic disease (OMIM 309000), affecting eyes, the nervous system and the kidney. OCRL1 mutations observed in Dent's disease 2 patients comprise insertion/deletion mutations, splice defects and missense mutations, located in various exons (E; E5: 259–262delTGTT, E7: 436-437insAA, E11: T901G, E14: A1385G) or intron 6 (intervening sequence 6: IVS6-2A->G). Due to frameshifts and premature stop codons, three of these defects were shown to cause absence of OCRL1 protein in Western blot analysis, whereas the two missense mutations give rise to reduced protein levels and diminished PtdIns(4,5)P 2 5-phosphatase activity [ 46 ]. Interestingly, in Lowe patients, these mutations have not been found to date, whereas mutations leading to a frameshift and/or premature stop codon have frequently been detected [ 49 ]. Thus the question remains: why, in these Dent's 2 cases, does the observed mutations not cause cataracts or metabolic acidosis. One obvious possibility is that this subset of patients carries variations in other gene(s) which provides a degree of cerebral and/or ocular protection. It is also worth mentioning that formal neuropsychological testing has not been reported for the majority of classic Dent's 1 patients, and it remains to be determined whether some might have subclinical neuropsychological problems. Genes other than CLCN5/OCRL1 have been investigated to explain the lack of mutations in patients with a Dent-like phenotype. First, CLCN4 , the gene encoding ClC-4 was analysed, since it is located on Xp22.3 and the mutations therein would consequently show an X-linked mode of inheritance. Moreover, ClC-4 (i) is also a member of the ClC-family of voltage-gated chloride channels and chloride/proton exchangers, (ii) gives rise to strong outwardly rectifying anion currents, (iii) exhibits chloride-coupled proton transport activity closely resembling those of ClC-5 [ 17 , 18 , 50 ], (iv) just as ClC-5, contributes to endosomal acidification by epithelial cells of the renal proximal tubule [ 51 ] and (v) could be co-immunoprecipitated with ClC-5, thereby indicating that both these proteins may interact in vivo [ 51 ]. As yet, however, no CLCN4 mutations have been found [ 46 , 52 ]. Hoopes et al . [ 46 ] also investigated the SLC9A6 gene, located at Xq26.3, which encodes the Na + /H + exchanger 6 (NHE6) [ 53 ]. This sodium–proton exchanger localizes to early recycling endosomes and is assumed to contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments [ 54 ]. No SLC9A6 mutation was observed in the 13 patients who met the strict criteria for Dent's disease [ 46 ]. By Northern analysis CLCN5 mRNA was almost exclusively detected in the kidney [ 15 ], but has also been found in aortic and coronary vascular smooth muscle cells and in aortic endothelial cells [ 55 ]. The RT–PCR analysis, however, revealed a wider ClC-5 distribution with CLCN5 mRNA expression of the various transcripts also detected in the liver, prostate, placenta, adrenals, leucocytes and the glial cells. Here, some tissues showed specific expression of only some of the transcripts and CLCN5 mRNA was undetectable in the samples from the skin, heart and the hippocampal tissue [ 21 ]. However, the role of ClC-5 in extrarenal tissues merits further investigation. In the kidney, ClC-5 was found to be predominantly expressed in the proximal tubule and α-intercalated cells of the distal nephron [ 56 , 57 ]. In the proximal tubule, ClC-5 mainly locates in intracellular subapical endosomes, where it is thought to participate in the process of endosomal acidification by dissipating the positive endosomal transmembrane voltage gradient generated by the action of the V-type proton ATPase (V-ATPase) [ 58 ]. Its presence in the medullary thick ascending limb of the Henle's loop has also been documented [ 57 ]. A small fraction has been localized at the cell surface [ 50 , 56 ]. However, we do not know whether ClC-5 plays a physiological role here, or whether it is only a by-product of recycling via the plasma membrane as suggested by Friedrich et al . [ 50 ]. Upon heterologous expression, either in Xenopus oocytes [ 50 , 59 ] or mammalian cells [ 50 , 60 ], the voltage-gated chloride channel, ClC-5, has been shown to elicit strong outwardly rectifying currents with an anion selectivity profile of nitrate > chloride >bromide > iodide [ 50 ]. Recently, it has been shown that ClC5, as ClC4, also shows chloride–proton antiport activity [ 17 , 18 ]. This will be discussed later. The selective flow of chloride ions is catalysed by a ClC-5 homodimer, with each channel subunit forming its own ion pore [ 61 , 62 ]. X-ray analysis revealed ClC-5 to comprise 18 helices (A–R; Figure 3 ) which show an internally repeated pattern (helices B–I and J–Q, respectively) forming two roughly repeated halves that span the membrane with opposite (antiparallel) orientations [ 61 ]. ClC-5 also harbours two carboxy-terminal intracellular CBS (cystathionine β-synthase) domains that form the so-called Bateman domain [ 63 ], an energy-sensing module that binds ATP necessary for the mediation of allosteric control [ 64 , 65 ]. A proline tyrosine (PY)-like motif ( Figure 3 ), located in the spacer region between the two CBS domains has been shown to be implicated in the internalization and ubiquitination of ClC-5 with mutant ClC-5 displaying a significant increase in surface expression [ 43 , 66 ]. Dysfunction of mutant ClC-5 may be caused by impaired dimerization, altered current kinetics, altered ion selectivity or defective intracellular trafficking. Functional consequences of naturally occurring CLCN5 nonsense and missense mutations were determined by heterologous expression in Xenopus oocytes. Here, all the tested nonsense mutations interfered with the CIC-5 function, in that all the recombinant ClC-5 mutants were shown to largely reduce or completely abolish ClC-5 chloride currents [ 5 , 29 , 34 , 43 , 67 ]. Functional properties established for several channels carrying an amino acid substitution, on the one hand, revealed complete loss of function, whereas other mutant constructs retained up to 50% wild-type activity [ 4 , 5 , 22 , 23 , 29 , 43 , 50 ], indicating that type and/or location of a mutation does not predict functional consequences. Defective trafficking of mutant ClC-5 was also in a renal tubular cell [ ]. of with reduced ClC-5 expression [ ] or of the CLCN5 gene in revealed phenotypic consequences displaying the renal tubular defects observed in Dent's disease 1 [ 58 , , ]. ClC-5 LMWP, and renal phosphate ClC-5 generated by different groups also hypercalciuria and with renal [ , ] but these features were in the established by et al . [ 58 ]. Moreover, some of these that hypercalciuria in the ClC-5 levels of of and renal [ ], than caused by increased [ ]. Dysfunction of ClC-5 in endosomes of ClC-5 further of Dent's disease Here, the internalization of the sodium–proton exchanger and the was indicating an impaired endocytosis from the membrane of proximal tubular cells [ 58 ]. a both these proteins from the membrane to intracellular probably due to a rise in luminal PTH ClC-5 show reduced of two cell surface and which are in the of proteins into the proximal tubular cells [ ]. This a selective loss at the a trafficking defect of the a pH from to is detected in early endosomes, endosomes and This acidification is generated and by + transport of The of + in a which may be by a of as chloride or by of + ). by Na + + the membrane and acidification ( Figure 4 ). for the role of ClC-5 in the acidification of endosomes is provided by several et al . [ ] that from ClC-5 a acidification rate and a level of and [ ] showed that + endosomal acidification of the proximal was diminished in the absence of et al . [ 80 ] found that endosomal chloride acidification in cells. The pH and chloride concentration in the proximal tubule cell from wild-type and was by et al . [ here, acidification and chloride was impaired in early endosomes but not in the endosomes and of ClC-5 by the of in a significant increase of the endosomal pH in cells [ 51 ] and it has also been shown that endocytosis in the proximal in wild-type but not in be by an of + ATPase [ ]. endosomes which have been and in of endosomal of the so-called with other early will in different expression of wild-type ClC-5 in Xenopus oocytes and cells outwardly rectifying chloride currents, which were or reduced by testing the ClC-5 observed in Dent's 1 patients [ 5 ]. The currents however, and it is whether similar be observed in vivo [ 17 , 80 ]. Two groups of that ClC-4 and ClC-5 as chloride/proton [ 17 , 18 ]. One pH to in the pH [ 17 ], while the other intracellular pH with [ 18 ]. This antiport would consequently to a loss of an + which to the that ClC-5 and [ 17 ] that these for ClC-5 a to acidification to a namely the one for endosomal It is that ClC-5 endosomes from the membrane by for luminal at that be similar to the concentration [ 18 ]. ClC-5 as the this might be in the absence of In these early endosomes, of has been shown to acidification and to which is consistent with the that have tubular proteinuria [ ]. The that 3–5 might be in the of intracellular is a more [ 17 ] that the would not into an A of the role of ClC-5 in intracellular compartments is and to proteins as as several and to and/or In the are from the by the endosomal pH and to the membrane [ ] ( Figure 5 ). In the ClC-5 a selective loss of and was observed at the [ ]. This the of urinary detected in Dent's 1 patients [ ]. As in the of LMWP [ ]. In ClC-5 not only does an of protein endocytosis of low-molecular-weight proteins occur due to the of the recycling of and to the but also an impaired of to the to an of 2 [ ]. As proteinuria in patients with Dent's disease 1 is frequently including ClC-5 will a role in the reabsorption of Defective of ClC-5 with a of may contribute to in patients with Dent's disease 1 [ ]. in the proximal tubule, may have on tubular The of urinary and proteins in Dent's disease 1 with the normal is [ , ] and and the [ ]. As binds and II, an increased concentration in the distal nephron may be with a on + ATPase activity [ , ]. The phosphate for the majority of the renal reabsorption of The is in the and via endocytosis by PTH, leading to No recycling As is in the reabsorption of PTH from the proximal tubular higher PTH availability was shown to be related to the of [ ]. A of the in the more distal of the proximal tubule is to this may be a reduced recycling of the as by in cells [ ]. The impaired reabsorption of amino acids and in the proximal tubular cells is due to the to the specific to the as initially assumed by et al . [ 34 ]. In this the functional role of ClC-5 in the medullary thick ascending limb of the Henle's loop and the cells of the tubule is [ ]. ClC-5 may a role in the recycling of on the site of the ascending A decreased presence of these contribute to the hypercalciuria and defective concentration as observed in syndrome [ ]. et [ ] on the role of ClC-5 in the of a et al . [ ] that phosphate and more to the ClC-5 as a for The that the of the membrane is altered or that a different of may occur and, most in a increase in plasma membrane a [ ]. et al . [ ] found that the expression of the + ATPase was in cells. However, this is difficult to with the normal observed in of Dent's 1 patients [ 1 ]. Recently, et al . [ ] reported that the loss of ClC-5 in the The that, to that in the kidney, ClC-5 is not for endocytosis in the its leads to a iodide associated with a of are to the of this ClC-5 and the with and to explain Dent's 1 patients do not a ClC-5 is in the small and of in the early endosomes associated with + where its role remains to be [ ]. has in hypercalciuria [ , ]. of renal are and will on the of disease from a small of patients revealed some normal show and were or completely was with as as [ 1 ]. These of and be at the of the of these Recent in ClC-5 give some for [ ] in that renal and the of renal the that was not completely on these early with be also be by In to the of renal and to a as as the of an might be as observed in patients with [ ]. in affected with either Dent's disease type 1 or is not only to the and for the patients, but also to those by who may be at for affected may recessive X-linked the Dent's being for the mutation due to a pattern of more in Dent's with evidence of genetic as by Hoopes et al . [ 39 , 46 ]. In series with patients the criteria of Dent's mutations in CLCN5 and OCRL1 were observed in 60 and of these patients, indicating at least one additional gene responsible for this phenotype in the patients. These several to Lowe syndrome, where cataracts are of the Dent's 2 patients, even an mutation resulting in the complete absence of PtdIns(4,5)P 2 showed this defect [ 46 ]. tubular a and often in Lowe syndrome [ 48 ] has not been observed in of the Dent's 2 to [ 46 ]. has been found in a Dent's 1 family with CLCN5 defect [ ]. is a further in Lowe syndrome, and was in three of the Dent's 2 patients [ 46 ]. This has clinical and the to a of the in patients with a Dent-like phenotype the features and of Lowe syndrome. some patients positive for OCRL1 mutations with the renal phenotype of Dent's disease 1 [ 46 ] is a further question that remains to be As with the features of Fanconi syndrome, is a between these The features the of X-linked Dent's disease provide no in to for either CLCN5 or OCRL1 both forms of Dent's as as Lowe syndrome, are by an impaired proximal tubular associated with LMWP, and and a renal is often observed [ 1 , , ]. and are features in both forms of Dent's disease but have also been observed in some Lowe patients [ ]. in muscle have often been found in Lowe patients [ ] and a level the of the normal was also observed in two patients with Dent's disease 2 [ 46 ]. is diminished in both Dent's and Lowe patients [ ]. Since this protein resides on the surface and in the endosomes of proximal tubular membrane recycling a defect to both the phenotypic observed in either Dent's 1 or OCRL1 Lowe a expression of a of detected in these also be in patients with Dent's disease 2 due to OCRL1 of . phosphate reabsorption in Dent's normal from [ patients with Dent's of Dent's disease to Wrong et al . ( [ 1 ] a No transport no one with renal failure. of Dent's disease to Wrong et al . ( [ 1 ] a No transport no one with renal failure. ( A ) of the CLCN5 and are by and [ 16 , 21 ]. be identified by and various start sites by that give rise to at least 5 mRNAs as in ( ). shows alternative splicing and exon 1b alternative transcription where intron 1 is in the longer from start site A of exon 2 in being in the two transcripts from start site of ClC-5 helices with the region shown and the region acids in helices and forming the chloride selectivity are by [ 61 , helices and are in the formation of the [ 61 , ]. of functional are in sequence [ 70 and forming the so-called Bateman domain [ 63 ] that binds ATP [ 65 internalization motif [ 66 ]. is from et al . [ 61 ] and et al . [ the additional 70 the alternative ClC-5 isoform [ 21 ] and with the of the et al . [ 43 ] with of and and maintenance of pH from [ ]. are into the by and by the chloride/proton exchanger is by Na + + ATPase and Na + /H + A , Na + , + ) channel, 1, the transport of and at pH [ ] not in the proximal tubular cells. to ( ) are from by the pH in the endosomes and the to the to [ ].