1. Hypercalciuria Genetic and environmental basis Pascal Houillier
Paris-Descartes University

2. Hypercalciuria is a risk factor for calcium nephrolithiasis
Hypercalciuria is a well recognized risk factor for nephrolithiasis. This has been demonstrated in several studies. In one of them, reported here, we have studied members of families with idiopathic hypercalciuria. Some subjects had a quite normal urinary calcium excretion and others had a very high urinary calcium excretion, in excess of 0,15 mmol/kg/day. The first group served as the reference group with a relative risk of lithiasis equal to unity. In multivariate analysis, the risk of developing a calcium nephrolithiasis increased dose-dependently with the rate of urinary calcium excretion and became significantly higher than in the reference group for rates of urinary calcium excretion above 0,11 mmol/kg/day
N Lerolle et al, Am J Med, 2002

3. Thiazide diuretics decreases the recurrence of stone
In a very nice study by Coe and colleagues, in 1988, it was also shown that drugs that decrease urinary calcium excretion are able to dramatically influence the recurrence of stones. In this study, patients with recurrent stones have been studied before and after the beginning of a medical treatment with thiazides. The horizontal lines represent the duration of follow-up and each dot represent the time of a stone episode. By comparison between the left and the right part of the figure, it is apparent that the number of stone episode is lower after the beginning of thiazide treatment than before
FL Coe et al, Kidney Int, 1988

4. SHOK syndrome
Stroke
Hypertension
Osteoporosis
Kidney

5. Primary mechanisms resulting in hypercalciuria
Primary disorder
Renal leak
Primary disorder
Bone resorption
Primary disorder
Intestine hyperabsorption
ECF Ca
ECF Ca
ECF Ca
Hypercalciuria may result from several primary disorders
The primary disorder may be a renal leak of calcium that is a decrease in the ability of the renal tubule to reabsorb calcium. Under this condition, an adaptation of two other organs occur ; the intestinal absorption of calcium increases and the net bone release of calcium also increases.
The primary disorder may involve the bones with a primary increase in net calcium release. Under this condition, intestinal calcium absorption does not change or decreases but urinary calcium excretion obviously increases
Finally, the primary disorder may be an hyperabsorption of calcium from the gut ; the net bone calcium release is left unchanged but here again, the urinary calcium excretion increases
Hypercalciuria
Hypercalciuria
Hypercalciuria

6. Idiopathic (genetic) hypercalciuria
Familial inheritance
Heavy influence of environmental (dietary) factors
Complex pathophysiology
Idiopathic hypercalciuria is the most common condition in hypercalciuric patients. It has several characteristics that deserve to be mentionned
First, idiopathic hypercalciuria is not a diagnosis but rather the translation of our lack of knowledge
Second idiopathic hypercalciuria is frequently transmited within families
Third the expression of idiopathic hypercalciuria is highly influenced by environmental factors
Finally, idiopathic hypercalciuria has a complex pathophysiology.
I shall comment on these points now

7. Gene1 Gene1 Gene2 Gene3 Gene4 Gene5 Low Ca excretion High Ca excretion
On the top of the figure, you can see a pedigree of a typical french family in which hypercalciuria is transmitted. Hypercalciuric patients are represented as black symbol. As you can see, hypercalciuric patients are present in each generation and the transmission does not match a recessive pattern and hypercalciuria can be transmitted from male to male. Therefore, some authors have proposed that idiopathic hypercalciuria can be an autosomal dominant trait.
However, urinary calcium excretion is a continuous variable
Low Ca
excretion
High Ca
excretion

8. Influence of environmental factors
Low Ca
excretion
High Ca
excretion

9. High Na intake increases urinary Ca excretion
Increased ECF volume
Decreased proximal
Na and Ca
absorptions
J Lemann, Jr, 1992

10. Thiazides reduce urinary calcium excretion through a decrease in ECF volume
T Nijenhuis et al, JCI, 2005

11. 7 male patients with Dent syndrome (CLNC5 defect)
A. Blanchard, unpublished results.

12. Ncc inactivation is associated with an increased bone mineral density
Humans
Mice
L. Nicolet-Barousse et al, JBMR, 2005

13. High dietary protein intake increases urinary Ca excretion
Increased animal protein intake :
Increased acid load
Increased bone
resorption
ECF Ca
Decreased tubular
Ca reabsorption
J Lemann, Jr, 1992
Hypercalciuria

14. Metabolic acidosis induces an increase in urinary calcium excretion
Acute
Chronic
P. Houillier et al, Kidney Int, 1996
J. Lemann Jr et al, N Engl J Med, 1979
µmol/min
UCaV,
Filtered load
of Ca,
Acid load

15. Metabolic acidosis induces a negative calcium balance
J Lemann et al, J Clin Invest, 1966
Sebastian et al, N Eng J Med, 1994

16. Carbohydrates induce an increase in urinary calcium excretion
J. Lemann Jr et al, N Engl J Med, 1969

17. Pathophysiology of human idiopathic hypercalciuria
ECF Ca
Hypercalciuria
Increased intestinal
Ca absorption
Increased Ca release
(especially on a low Ca diet)
Decreased renal tubular
Ca reabsorption
Primary or secondary disorders ?

18. Pathophysiology of rat idiopathic hypercalciuria
ECF Ca
Hypercalciuria
D. Bushinsky et al, Semin Nephrol, 1996
S. Tsuruoka et al, Kidney Int, 1997

19. Role of vitamin D receptor in intestinal epithelial cells
From Li, 1993

20. From Li, 1993

21. From Coe, 1991

22. Role of kidney in idiopathic hypercalciuria

24. Factors decreasing renal tubular calcium reabsorption
Reduced PTH
NaCl intake (volume expansion)
Protein intake (metabolic acidosis)
Glucose, sucrose, ethanol
Phosphate restriction
Loop diuretics
Calcium, magnesium

25. Acute response to hydrochlorothiazide
From Sutton, 1980 and Sakhaee, 1985

26. Calciuric response to an acute acid load
From Houillier, 1996

27. Calciuric response to furosemide
From Tsuruoka, 1997

28. Kidney as the primary defect : monogenic disease in humans and/or mice
CLC-5
OCRL
ATP7B
FAH
G6PC
NPT2
NHERF-1
AKr1b1
CAII
TRPV5
VDR
Calbindin-D28k
WNK1-4
T Kallikrein
NKCC2
ROMK
CLC-Kb
Barttin
CaSR
PCLN-1
ATP6V1B1
ATP6V0V4
SLC4A1 (AE1)
SCNN1B and G
(ENaC ß and g
subunits)
Kidney as the primary defect : monogenic disease in humans and/or mice

29. TRPV5 (ECaC 1)

30. TRPV5 (ECaC) is the gatekeeper for Ca absorption in the distal tubule
Copyright ©2000 American Physiological Society
Hoenderop, J. G. J. et al. Am J Physiol Renal Physiol 278: F352-F
Fig. 1.

31. Phenotype of Trpv5 -/- mice
J. Hoenderop et al, J Clin Invest, 2003

32. Phenotype of Trpv5 -/- mice
Decreased distal tubular Ca
reabsorption
Adaptive increase in intestinal
Ca absorption

34. CaSR

35. CaSR and model of ion transport in the TALNa
2Cl K
Ca, Mg - + Ca
CaSR
Lumen
Cell
Interstitium Cl
PCLN-1
PTH

36. CaSR +/+
CaSR +/-
CaSR -/-

38. Croisement de souris mutées pour CaSR et de souris hypoparathyroïdiennes (Gcm2-/-)
I : normales ; II : CaSR -/-, Gcm2+/+; III : CaSR+/+, Gcm2-/- ; IV : CaSR-/-, Gcm2 -/- ;
V: CaSR+/-, Gcm2 +/+ ; VI : CaSR +/-,Gcm2-/-

39. Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037
Copyright ©2003 American Society for Clinical Investigation

40. Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037
Copyright ©2003 American Society for Clinical Investigation

41. CaSR +/+ CaSR -/- CaSR +/+ CaSR -/- CaSR +/- CaSR +/- Gcm2+/+ Gcm2+/+
Tu, Q. et al. J. Clin. Invest. 2003;111:
Copyright ©2003 American Society for Clinical Investigation

42. Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037
Copyright ©2003 American Society for Clinical Investigation

43. Gain-of-function mutations in CASR gene induce a renal leak of calcium
Yamamoto et al, JCEM, 2000

46. Expression hétérologue du CaSR
Vargas-Poussou, JASN, 2002

47. CaSR in idiopathic hypercalciuria
Petrucci et al, 2000:No significant linkage between CaSR variants and idiopathic hypercalciuria
Lerolle et al, 2002: No point mutation in CASR gene in families with idiopathic hypercalciuria
Vezzoli et al, 2002: higher urinary Ca excretion in patients bearing the R990G polymorphism (ARQ/AGQ or AGQ/AGQ)
Yao et al, 2005: GHS rats have a higher renal content in CaSR protein and mRNA

48. Paracellin-1 (Claudin 16) and hypercalciuria

52. Tubular phenotype of patients with loss-of-function in PCLN-1 gene
HHF
Controls 5 10 15 20
UV/GFR
mmol/l GF Na +
0,1 Ca ++
baseline
furo
0,2 Mg Cl -
Na+ K+
2 Cl-
3 Na+
2 K+
Cl-
Ca++
Mg++ ?
Paracellin-1
A. Blanchard et al, Kidney Int, 2002

53. CLC5 and hypercalciuria

54. Canaux chlore : 3 familles distinctes
"Cystic fibrosis transmembrane conductance regulator (CFTR)" Cl- channel
Extracellular-ligand gated (ELG), post synaptic Cl- channels
CLC family : voltage-gated Cl- channels
CLC-1 à CLC-7, CLC-Ka, CLC-Kb

55. Néphrolithiase hypercalciurique liée à l'X
Maladie de Dent (Grande-Bretagne)
Protéinurie de bas poids moléculaire avec hypercalciurie et néphrocalcinose (Japon)
Néphrolithiase récessive liée à l'X (Etats-Unis, Canada)
Rachitisme hypophosphatémique récessif lié à l'X (Italie, France)

58. Mutations du gène CLCN5 : perte de fonction du canal
Faux-sens
Non sens
Mutation d'un site d'épissage
Insertion
Délétion
Absence de parallélisme entre le phénotype et le génotype

59. Colocalisation avec Rab4 et H+-ATPase

60. A-ClC-5
B-H+ATPase
C=A+B
D-CLC-5
E-2microglob.
F=D+E
G-CLC-5
H- 2microglob
I=G+H 13min.
ME CLC-5
Localisation de CLC-5 dans le tubule proximal

61. ß2 microglob
Lactoglobuline
lactoglobuline
CLC-5
horseradish peroxydase
FITC-dextran
CLC-5

62. Mégaline
CLC-5
CLC-5+mégaline
Expression de la mégaline à la surface des cellules du tubule proximal en l’absence ou en présence de CLC-5

63. Physiopathologie diabète phosphaté
Diminution de l’expression apicale de NaPi-2 chez la souris CLC-5-/-
Pas de modification de l’expression de NaPi-2 à la surface des cellules CLC-5- chez la souris CLC-5+/-.
mégaline
ClC-5
mégaline + CLC-5
NaPi 2 S1
+/+
-/-
-/-
+/-
+/-

64. Fonction de CLC-5

65. Rôle de ClC5 dans le diabète phosphaté « hyperparathyroidisme »
PHYSIOPATHOLOGIE
Rôle de ClC5 dans le diabète phosphaté
Piwon Nils, Nature, 2000, vol 408,
Hypothèse : la diminution de l ’expression basale de NaPi-2 est liée à une augmentation de PTH. Elévation luminale et non basolatérale ([PTH] systémique Nle).
Diminution de l’endocytose
[PTH] nle
PTH
filtrée
[PTH]>
Nle
« hyperparathyroidisme »
luminal (S3)

66. Tissue kallikrein and hypercalciuria

67. Tissue kallikrein and TRPV5 are coexpressed in the renal tubule

68. Meneton, Pierre et al. (2001) Proc. Natl. Acad. Sci. USA 98, 2634-2639
Copyright ©2001 by the National Academy of Sciences

71. Femelles
C57Bl6/J

72. Males
C57Bl6/J

73. Femelles
129Sv

74. Tissue kallikrein gene expression is controlled by calcium intake

75. Expression des transcrits des transporteurs

76. Mécanisme d’action de la TK
D. Gkika et al, EMBO J, 2006

77. L’effet de la TK est dépendant de la PKC

79. Effet des mutations des sites consensus de la PKC

80. La TK stabilise TRPV5 à la membrane

82. Monogenic hypercalciuria : clues to the genetics of idiopathic hypercalciuria ?
Intestine as the primary defect :
> 5 genes, no gene encoding a Ca transporter
Bone as the primary defect :
Kidney as the primary defect :
> 18 genes, only one gene encoding a Ca transporter (PCLN-1)

83. Selective genotyping of F2 (GHS female x normocalciuric male WKY rats)
Genetics of idiopathic hypercalciuria : lessons from genetic hypercalciuric rats
Selective genotyping of F2 (GHS female x normocalciuric male WKY rats)
Linkage between hypercalciuria and chromosomal regions
Significant at D1Rat169
Suggestive to regions of Chr. 4, 7, 10, 14
No linkage with CaSR or VDR gene regions
R. Hoopes et al, J.A.S.N., 2003

84. Conclusion
Hypercalciuria is a complex trait, and its expression depends on both
Environmental factors
Genetic factors
Modification of dietary factors is efficient but not specific
Continuing efforts are warranted
- detailed proximal phenotype definition
- study of monogenic causes of hypercalciuria
- identification of loci linked to idiopathic hypercalciuria

87. Georges Pompidou Hospital Tenon Hospital
Pascal Houillier
Anne Blanchard
Marie Briet
Marc Froissart
Gérard Maruani
Laurence Nicolet
Tenon Hospital
Eric Rondeau
Pierre Ronco
Brigitte Lantz
Françoise Paillard
INSERM Unit
Nicolas Picard
Nijmegen University
Joost Hoenderop
Rene Bindels

88. Pathophysiology of human idiopathic hypercalciuria
ECF Ca
Hypercalciuria
Adapted from Lemann, 1992
Adapted from Houillier, 1996

90. Monogenic renal hypercalciuria : clues to the genetics of idiopathic hypercalciuria ?
Trpv5 (ECaC1)
CaSR
Paracellin-1 (Claudin 16)