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

2. 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

3. 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

4. Case 1 - progress Age 2½ years Age 10½ years
phosphate supplement stopped. Serum PO4 maintained.
Age 10½ years
persistent proteinuria – P:CR mg/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

5. 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

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

7. 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

8. 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

9. Case 2 - progress Age 4½ years Age 13 years – 16 years
Cr 42 μmol/L; GFR 137mls/min/1.73m2
Persistent proteinuria P:CR mg/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)

10. 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

11. 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 mg/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

12. 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
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

13. 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)

14. 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.

15. 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): , February 1, 1996.
© 1996 Macmillan Magazines Ltd. Published by Nature Publishing Group. 3

16. 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

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. 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

23. 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

24. 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.

25. 19/32 families (60%) with CLCN5 mutations
13 (40%) without CLCN5 mutation
No clinical correlations

26. 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

27. 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.”

28. 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

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

30. 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%

31. 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

32. 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)

33. 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

34. 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