David A Hughes Consultant Paediatric Nephrologist Dent Disease David A Hughes Consultant Paediatric Nephrologist

Case 1 - presentation Presented age 1½ years and referred age 2 years Polydipsia and polyuria Glycosuria Generalised aminoaciduria Nephrocalcinosis Ca:Creatinine – ‘normal’ ‘Normal’ acidaemia Low phosphate – supplemented Ix for cystinosis, galactosaemia and Wilson’s -ve

Case 1 - investigation Investigation at 2 years 3 months Ht 89cm (50-75th C) Wt 11.1 (3-10th C) BP 104/61 Urine: ++ blood, ++ protein, no glycosuria, pH 7 Creatinine 53; GFR 90mls/min/1.73m2; Alb 48g/L Ca:Cr 0.01mmol/mmol; TRP 47% Serum Ca, PO4, 1.25D3 and PTH normal P:CR 500mg/mmolCreat (normal <20) Normal acidaemia HCO3 25mmol/L Nephrocalcinosis; no bony rachitic changes Urine AAs – sample too dilute

Case 1 - progress Age 2½ years Age 10½ years phosphate supplement stopped. Serum PO4 maintained. Age 10½ years persistent proteinuria – P:CR 150-240mg/mmolCr Microscopic haematuria trace - 2+ Ca:Cr 1.0mmol/mmol Creatinine 120μmol/L; GFR 67mls/min/1.73m2 β2-microglobulin raised 94.8mg/L (normal <0.3) Urinary AAs – high normal excretion Progressive medullary nephrocalcinosis

Case 1 – genetic test Proband Mother Sister Nucleotide deletion in exon 3 causing frameshift mutation (c.181delA) in the CLCN5 gene consistent with diagnosis of X-linked Dent Disease Mother carrier status confirmed genetically with known mutation Minor nephrocalcinosis on US noted Sister confirmed not carrier No previous FH suggesting X-linked renal disease

Case 1 - current CKD 3 2ry hyperparathyroidism eGFR 50mls/min/1.73m2 2ry hyperparathyroidism controlled on alfacalcidol Proteinuria 2+ and haematuria 2+ P:CR 120 - 150mg/mmolCr on enalapril 5mg daily BP 96/59 Growth: Ht 152.5cm (2-9th C) Wt 37.5 (2nd C)

Case 2 - presentation Age 3 years old Balanitis and persistent proteinuria Proteinuria 4+; haematuria 2+ FH maternal grandfather and great uncle died age 30s from renal disease

Case 2 - investigation Ht 99.2cm (50-75th C) Wt 15.4kg (50th C) BP 100/60 Proteinuria 4+; haematuria 2+ P:CR 300mg/mmolCr Cr 42 μmol/L; Albumin 41g/L Normal acidaemia HCO3 21mmol/L Normal C3/C4; ANA –ve Normal renal US - ?cyst

Case 2 - progress Age 4½ years Age 13 years – 16 years Cr 42 μmol/L; GFR 137mls/min/1.73m2 Persistent proteinuria P:CR 250 -350mg/mmolCr Normal renal US – no cyst Renal biopsy – normal LM appearance; -ve IF; ??splitting of BM on EM Normal audiometry and opthalmology Age 13 years – 16 years Persistent proteinuria P:CR 966→150mg/mmolCr; A:CR 63mg/mmolCr β2-microglobulin >51 and 85 mg/L (normal <0.3) Ca:Creat 0.22; 0.64 No aminoaciduria Cr 57 – 72 μmol/L; Albumin 42 – 48 g/L Normal renal US (14 years) On Enalapril 15mg daily (introduced age 7 years)

Case 2 – genetic test Proband Mother Sister nucleotide transition (C1834A) in exon 10 in the CLCN5 gene should be responsible for the phenotype Mother microscopic haematuria 3+; no proteinuria carrier status not tested Sister -ve for haematuria and proteinuria carrier status not yet tested FH consistent with X-linked renal disease

Case 2 - current CKD 2 PTH within normal limits eGFR 82mls/min/1.73m2 PTH within normal limits Proteinuria 2+ and haematuria 2+ P:CR 102 - 146mg/mmolCr on Enalapril 15mg daily Irbersartan 150mg daily BP 122/47 Growth: Ht 173.3cm (25-50th C) Wt 80.3 (97th C) Transfer to adult services

Dent('s) Disease 1964 – Dent and Friedmann. Arch. Dis. Child. 2 unrelated English boys. Rickets and renal tubular abnormalities Phenotype development on follow up Familial proximal renal tubular syndrome Low-molecular-weight proteinuria Hypercalciuria Nephrocalcinosis Metabolic bone disease Progressive renal failure Marked male predominance Wrong et al. QJM. 1994. Initial 2 cases reported by Dent, followed up by Wrong in 1994 describing phenotype progression in these 2 families and a further 3 families. Female carriers asymptomatic, but all had low molecular weight proteinuria. About half had hypercalciuria. About one-third of affected males had rickets

Dent Disease 4 disorders of hereditary hypercalciuric nephrolithiasis Dent’s Disease X-linked nephrolithiasis (XLN) X-linked recessive hypophosphataemic rickets (XLHR) Idiopathic low molecular weight proteinuria of Japanese children (JILMWP)

Molecular Genetics of Dent Disease 11 kindreds Mutations in the CLCN5 gene on chromosome Xp11.22 Gene codes for chloride channel CLC-5 Lloyd et al. Nature, 1996. 19 of 32 families (60%) with CLCN5 mutations Total of 70 mutations in 90 families recorded Hoopes et al. Kidney International. 2004 Lloyd and colleagues in a letter to Nature describe CLCN5 gene mutations found in 11 kindreds (families) with 3 different inherited kidney stone disease diagnoses: Dent’s Disease X-linked nephrolithiasis (XLN) X-linked recessive hypophosphataemic rickets (XLHR) There were different mutations in all 11 kindreds encompassing the entire gene CLCN5 which codes for a chloride channel CLC-5. Mutations were 3 nonsense; 4 missense; two donor splice site mutations; one intragenic deletion and one microdeletion. I 2004 Hoopes and colleagues identified CLCN5 mutations in 19 unrelated males of 32 studied – 60% of the investigated group.

Schematic representation of a predicted topology of CLC-5 Figure 1 . Schematic representation of a predicted topology of CLC-5, based on the reported DNA sequence, to illustrate the mutations associated with X-linked hypercalciuric nephrolithiasis. The mutations found in 11 families with X-linked hypercalciuric nephrolithiasis Mutations found in families with 3 different inherited kidney stone disease diagnoses: Dent’s Disease X-linked nephrolithiasis (XLN) X-linked recessive hypophosphataemic rickets (XLHR) A common molecular basis for three inherited kidney stone diseases. Lloyd, Sarah; Pearce, Simon; Fisher, Simon; Steinmeyer, Klaus; Schwappach, Blanche; Scheinman, Steven; Harding, Brian; Bolino, Alessandra; Devoto, Marcella; Goodyer, Paul; Rigden, Susan; Wrong, Oliver; Jentsch, Thomas; Craig, Ian; Thakker, Rajesh Nature. 379(6564):445-449, February 1, 1996. © 1996 Macmillan Magazines Ltd. Published by Nature Publishing Group. 3

CLCN5 molecular function Sited in epithelial cells proximal renal tubule medullary Thick Ascending Limb of Henle’s loops collecting duct intercalated cells Codes the renal chloride channel CLC-5 Acidification of endosomes Solute reabsorption function Membrane recycling in proximal tubule Megalin-cubilin endocytic pathway

Filtered proteins may be absorbed to receptors, including megalin, on the apical surface of proximal tubular cells (site A). Endosomes form from this membrane (site B), and the first step in protein degradation requires acidification of the endosomal lumen through activity of the H+-ATPase as well as entry of chloride through the CLC-5 channel (site C). Reabsorbed proteins are further degraded in the lysosomal pathway (site D), while megalin and other membrane proteins are recycled to the apical surface. In patients with Dent’s disease mutations inactivate the CLC-5 chloride channel and impair endosomal function. Reabsorption of glucose, phosphate and amino acids are sodium-dependent processes involving specific apical membrane transporters (site E). Reabsorption of one or more of these solutes is often also impaired in Dent’s disease. The proximal tubule cell is also a site of production of 1.25 diOH-VitD3, which occurs through the 1-alpha-hydroxylase in mitochondria.

Endosomes formed from the apical surface of the proximal tubular cell bring in filtered proteins that are subsequently degraded. The process involves acidification of the endosomal lumen by the proton-ATPase. The chloride channel allows chloride to enter the vesicle to neutralise the charge generated by the proton pump, and this promotes maximal acidification. This may be the case in the early phase of endosomal creation from cell membranes. Other work (see below) suggests the CLC-5 acts as a proton chloride antiporter which would appear contrary to the proposal here – actually reducing – but perhaps more accurately “regulating” the internal endosomal pH.

Regulation and maintenance of organelle pH Protons are pumped into the organelle by V-ATPase and can leave by the proton-chloride exchanger CLC-5. Sodium entry is possible by the Na/K-ATPase and Na/H exchanger. A non-selective cation (Ca, Na, K) channel, mucolipin 1, controls the transport of cations and switches off at low pH (data not shown on diagram). The V-ATPase which acts as an antiporter for protons to leave by the proton-chloride exchanger CLC-5 would seem to act contrary to the theory of endosomal acidification internally maximised. This may true of the early phase endosome, in the absence of a V-ATPase when the endosome swaps intracellular organelle chloride for protons. CLC-5 chloride-proton exchanger adapted from Weisz in Ludwig et al. NDT 2006.

CLCN5 knock out mice Reduces expression of CLC-5 in KO mice Phenotype exhibited LMWP, glycosuria, aminoaciduria, polyuria, renal phosphate wasting Hypercalciuria, nephrocalcinosis, progressive renal failure Reduced amount of cell surface receptors megalin and cubilin Proximal tubular cell protein uptake function In CLC-5 KO mice there is selective loss of megalin and cubulin on the epithelial brush border of tubules Striking deficiency of urinary megalin in Dent’s 1 patients. Megalin deficiency causes LMWP – megalin recycling slowed and degradation of endocytosed proteins is also impaired. Not only reduced LMWP uptake but also albumin reabsorption.

Megalin and cubilin handling in the proximal tubular cells. Ligands bind to megalin/cubilin Megalin/cubilin receptors recycle to membrane Ligands released from receptor by low pH in endosome Ludwig et al. NDT 2006.

Events in the proximal tubule after glomerular filtration under normal physiological conditions and after glomerular damage. a During normal physiological conditions, all filtered proteins are efficiently internalized by the receptor complex megalin/cubilin/amnionless (AMN), resulting in a virtually protein-devoid urine. Proteins are degraded in lysosomes, and substances such as vitamins are transported basally for reuse. b During glomerular damage, filtration of low molecular weight proteins increases and larger proteins start to penetrate the glomerular barrier. Cells in the proximal tubule are thereby exposed to more, and new, proteins that compete for receptor-binding sites, eventually resulting in proteinuria. Further, in the cell, lysosomal degradation is unable to handle the increased amount of internalized protein, resulting in protein-clotted lysosomes

Megalin binds a variety of filtered molecules >50 ligands identified Cubilin - multiple binding domains 15 ligands identified peripheral membrane protein Amnionless probably assists cubilin in endocytosis Megalin, cubilin, and amnionless (AMN) presenting known domains and motifs. The three receptors colocalize in the renal proximal convoluted tubule (PCT), where they cooperate in ultrafiltrate clearance. Megalin binds a variety of filtered molecules (>50 ligands have been identified) through its complement type repeats and is able to mediate endocytosis via NPXY motifs in the cytoplasmic tail. Cubilin, on the other hand, includes multiple binding domains (CUB domains), but only around 15 ligands have been identified. Cubilin is a peripheral membrane protein and is thereby dependent on megalin and/or AMN to assure internalization of its ligands. AMN contains an NPXY and probably assists cubilin in endocytosis as well as in transport during synthesis

Biochemical consequences Proximal tubule Proteinuria – LMWP and albuminuria Urinary peptides unfiltered Prolactin, insulin, angiotensin II. Higher urinary PTH down-regulates Na-Phosphate co transporter AA and glucose loss related to poor membrane transporter recycling TAL of loop of Henle Role in hypercalciuria here? Impaired concentrating capacity? Collecting duct intercalated cells A nucleus for crystal agglomeration? Megalin deficiency causes LMWP – megalin recycling slowed and degradation of endocytosed proteins is also impaired. Not only reduced LMWP uptake but also albumin reabsorption.

19/32 families (60%) with CLCN5 mutations 13 (40%) without CLCN5 mutation No clinical correlations

Dent Diseases Dent Disease 2 5 of 13 families with Dent Disease and no CLCN5 mutation Mutation in the OCRL1 gene on chromosome Xq26 Features of (OculoCerebroRenal) Lowe’s syndrome absent Cataract Renal tubular Acidosis Mental retardation

Segregation of the R301C mutation in family 24 Segregation of the R301C mutation in family 24. The pedigree is shown, with affected subjects identified as blackened symbols. Digestion of the 298-bp exon 11 amplicons with the restriction endonuclease HhaI in normal subjects results in two products of 158 bp and 140 bp (subjects IV:1, II:3, II:4, and III:8). The mutated sequence produces a single detectable digest of 298 bp (subjects IV:2, II:5, III:4, III:5, III:6, and III:7). Heterozygous females display all three products (subjects III:2, III:3, II:7, and II:8). The lane containing PhiX174/HaeIII molecular weight markers is indicated by “M.”

OCRL1 molecular function OCRL1 on chromosome Xq25-27 Codes PIP2 5-phosphatase of OCRL1 protein Impaired function increases cellular PIP2 involved in vesicle trafficking at the Golgi apparatus PIP2 has wide distribution Functional disturbance in Lowe’s may require additional gene defect

Clinical diagnosis of Dent disease Affected male LMWP (> x5 ULN) Hypercalciuria At least one of: Nephrocalcinosis Nephrolithiasis Haematuria Hypophosphatemia Renal insufficiency

Dent Dent 1 Dent 2 (+) Clinical abnormality LMWP (> x5 ULN) 100% Wrong 1994 Dent 1 Scheinmann 1999 Hoopes 2004 Cho 2008 Dent 2 (+) n = 15 n = 19 n = 9 Hoopes (n = 12) Cho (n = 2) LMWP (> x5 ULN) 100% Hypercalciuria 92% 95% Nephrocalcinosis 73% 74% 89% 56% 64% 1/2 Nephrolithiasis 53% 49% 29% 8% Haematuria 94% 84% 67% 2/2 Hypophosphatemia 50% 58% 0/2 Renal rickets 40% 30% 38% 0/9 27% Renal insufficiency 26% 33% Concentrating defect 81% Aminoaciduria 76% 75% Glycosuria 54% 39% 11% 36% Acidification defect 17% Hypokalaemia 35%

Phenotype Dent 1 Dent 2 OCRL Case1 Case 2 LMWP + ++ Hypercalciuria +/- Nephrocalcinosis/ Nephrolithiasis + (56%) + (50%) + (43%) Haematuria + (67%) + (57%) Hypophosphatemia + (86%) Renal rickets Renal insufficiency + (adult) +(adult) + (10-30) Fanconi features phosphaturia aminoaciduria glycosuria proximal RTA

Phenotype Dent 1 Dent 2 OCRL Case1 Case 2 Lowe’s features cataract - ++ mental retardation +/- Other features Muscle enzymes (CK/LDH) mild ↑ ↑↑ ND Hyperuricosuria Growth retardation + ‘x-linked’ FH of CKD/ stones etc Urogenital anomalies (cryptorchidism)

Genotype and molecular consequences Case Dent 1 Dent 2 OCRL 1 2 Exon location 5-7 Frameshift and splice defects 9-15 (missense) 8-23 (all types) 3 10 Protein coded CLC-5 PiP2-5-phosphatase Tissue location PT cells; TAL of loop of Henle; Collecting duct cells Ubiquitous. Eyes, kidney, brain Molecular role Endosomal trafficking Golgi complex trafficking and actin dynamics End point effect Megalin and cubilin recycling with reduced PT reuptake of proteins and ligands

Treatment strategies Hypercalciuria Bone disease – hypophosphataemia Dietary sodium restriction Thiazide diuretics Bone disease – hypophosphataemia Phosphate supplements Vitamin D supplements Renal protection – anti-proteinuric agents ACEi and/or ARBs Dialysis and transplantation Genetic counselling in families