Hypercalciuria

Genetic and environmental basis

Pascal HouillierParis-Descartes University

Hypercalciuria is a risk factor for calcium nephrolithiasis

N Lerolle et al, Am J Med, 2002

Thiazide diuretics decreases the

recurrence of stone

FL Coe et al, Kidney Int, 1988

SHOK syndrome

• Stroke• Hypertension• Osteoporosis• Kidney

Primary mechanisms resulting in hypercalciuria

ECFCa

ECFCa

ECFCa

Hypercalciuria Hypercalciuria Hypercalciuria

Primary disorderRenal leak

Primary disorderBone resorption

Primary disorderIntestine hyperabsorption

Idiopathic (genetic) hypercalciuria

• Familial inheritance

• Heavy influence of environmental (dietary) factors

• Complex pathophysiology

Low Ca excretion

High Ca excretion

Gene1Gene2 Gene3 Gene4 Gene5Gene1

Influence of environmental factors

Low Ca excretion

High Ca excretion

High Na intake increases urinary Ca excretion

High Na intake

Increased ECF volume

Decreased proximalNa and Ca

absorptions

J Lemann, Jr, 1992

Thiazides reduce urinary calcium excretion through a decrease in ECF volume

T Nijenhuis et al, JCI, 2005

7 male patients with Dent syndrome (CLNC5 defect)

A. Blanchard, unpublished results.

Ncc inactivation is associated with an increased bone mineral density

Humans Mice

L. Nicolet-Barousse et al, JBMR, 2005

High dietary protein intake increases

urinary Ca excretion

ECFCa

Hypercalciuria

Increased bone resorption

Decreased tubular Ca reabsorption

Increased animal protein intake :Increased acid load

J Lemann, Jr, 1992

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

UC

aV

,µ

mol/m

in

Filt

ere

d load

of

Ca,

µm

ol/m

in

Acid load

Metabolic acidosis induces a negative calcium balance

Sebastian et al, N Eng J Med, 1994J Lemann et al, J Clin Invest, 1966

Carbohydrates induce an increase in urinary calcium excretion

J. Lemann Jr et al, N Engl J Med, 1969

Pathophysiology of human idiopathic hypercalciuria

ECFCa

Hypercalciuria

Increased intestinal Ca absorption

Increased Ca release(especially on a low Ca diet)

Decreased renal tubular Ca reabsorption

Primary or secondary disorders ?

Pathophysiology of rat idiopathic hypercalciuria

ECFCa

Hypercalciuria

D. Bushinsky et al, Semin Nephrol, 1996S. Tsuruoka et al, Kidney Int, 1997

Role of vitamin D receptor in intestinal epithelial cells

In vitro rat duodenal calcium transportFlux N males IH males N females IH femalesJms 51 ± 12 264 ± 27 29 ± 9 258 ± 40Jsm 11 ± 12 19 ± 2 14 ± 2 23 ± 2Jnet 40 ± 11 245 ± 28 14 ± 8 235 ± 40

From Li, 1993

VDR-binding sites in intestine and kidney from normal and IHrats

Group N max KdIntestine

Normal Males 243 ± 42 0.33 ± 0.01IH males 536 ± 74 0.49 ± 0.01Kidney

Normal males 34 ± 4 0.27 ± 0.16IH males 87 ± 4 0.45 ± 0.28

From Li, 1993

0

5

10

15

Uri

nar

y ca

lciu

m e

xcre

tio

n (

mm

ol/d

)

0 5 10 15

Net intestinal calcium absorption (mmol/d)From Coe, 1991

Role of kidney in idiopathic hypercalciuria

0

50

100

150

0

1

2

3

4

Filt

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Uri

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ca

lciu

m e

xc

reti

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(µ

mo

l/min

)

Controls (n=9)

Hypercalciuric patients (n=34)

Controls (n=9)

Hypercalciuric patients (n=34)

P < 0.02

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

Acute response to hydrochlorothiazide

0

0.05

0.1

0.15

0

0.005

0.01

0.015

0.02

0.025

Incr

ease

in u

rin

ary

calc

ium

exc

reti

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(m

mo

l/mm

ol c

reat

inin

e)

Incr

ease

in u

rin

ary

calc

ium

ex

cret

ion

(m

mo

l/l G

F)

Controls (n=20)

Fasting hypercalciuria

(n= 20)

Controls (n=9)

Renal hypercalciuria

(n= 20)

AB

From Sutton, 1980 and Sakhaee, 1985

Calciuric response to an acute acid load

-2

0

2

4

6

8

Mea

n in

crea

se in

cal

ciu

m e

xcre

tio

n

(µm

ol/m

in)

0 1 2 3 4 5 6 7

Baseline Calcium excretion (µmol/min)

From Houillier, 1996

Calciuric response to furosemide

0

2

4

6

8U

rin

ary

calc

ium

ex

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Furo 0 + 0 +

Control rats Hypercalciuric ratsFrom Tsuruoka, 1997

CLC-5OCRLATP7BFAHG6PCNPT2NHERF-1AKr1b1CAII

TRPV5VDRCalbindin-D28k

WNK1-4T Kallikrein

NKCC2ROMKCLC-KbBarttinCaSRPCLN-1

ATP6V1B1ATP6V0V4SLC4A1 (AE1)SCNN1B and G(ENaC ß and subunits)

Kidney as the primary defect : monogenic disease in humans and/or mice

TRPV5 (ECaC 1)

Copyright ©2000 American Physiological Society

Hoenderop, J. G. J. et al. Am J Physiol Renal Physiol 278: F352-F360 2000

Fig. 1.TRPV5 (ECaC) is the gatekeeper for Ca absorption in the distal tubule

Phenotype of Trpv5 -/- mice

J. Hoenderop et al, J Clin Invest, 2003

Phenotype of Trpv5 -/- mice

Decreased distal tubular Ca reabsorption

Adaptive increase in intestinalCa absorption

CaSR

CaSR and model of ion transport in the TAL

Na

2Cl

K

K

Ca, Mg

-

-+

Ca

CaSR

Lumen Cell Interstitium

Cl

PCLN-1

-

PTH

CaSR +/+

CaSR +/-

CaSR -/-

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-/-

Copyright ©2003 American Society for Clinical Investigation

Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037

Copyright ©2003 American Society for Clinical Investigation

Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037

Copyright ©2003 American Society for Clinical Investigation

Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037

CaSR +/+Gcm2+/+

CaSR -/-Gcm2+/+

CaSR +/+Gcm2-/-

CaSR -/-Gcm2-/-

CaSR +/-Gcm2+/+

CaSR +/-Gcm2-/-

Copyright ©2003 American Society for Clinical Investigation

Tu, Q. et al. J. Clin. Invest. 2003;111:1029-1037

Gain-of-function mutations in CASR gene induce a renal leak of calcium

Yamamoto et al, JCEM, 2000

Table 1. Clinical laboratory data of patient 1

7 yr 9 yr Normal Values--------------------------------------- --------------- ----------- ---

Plasma

potassium 2.5 to 3.4 2.9 to 3.0 3.5 to 4.7 mmol/L

calcium 1.5 to 1.8 1.7 to 2.16 2.25 to 2.65 mmol/L

magnesium 0.5 to 0.63 0.59 to 0.67 0.75 to 0.96 mmol/L

bicarbonate 27 to 32 30.2 to 30.4 23 to 28 mmol/L

creatinine 50 60 30 to 90 µmol/L

renin 158 113 6.5 to 80 pg/ml

aldosterone ND 56 4 to 40 ng/dl

PTH 6 Undetectable 10 to 65 pg /ml

Urine

calcium 2.4 to 2.6c 1.44 to 1.7 <0.3 mmol/mmol of creatininemagnesium 0.8 1.8 to 2 <0.4 mmol/mmol of creatininesodium 53 10.7 <1.6 mmol/mmol of creatininefractional excretion of16.5 to 26.7e 17.5 to 21.6e2.15 to 15.9 %K (FEK)osmolality after nasalND 460 >800 mOsm/kgdDAVP

7 yr 9 yr Normal Values

Plasmapotassium 2.5 to 3.4 2.9 to 3.0 3.5 to 4.7 mmol/L

calcium 1.5 to 1.8 1.7 to 2.16 2.25 to 2.65 mmol/L

magnesium 0.5 to 0.63 0.59 to 0.67 0.75 to 0.96 mmol/L

bicarbonate 27 to 32 30.2 to 30.4 23 to 28 mmol/L

creatinine 50 60 30 to 90 µmol/L

renin 158 113 6.5 to 80 pg/ml

aldosterone ND 56 4 to 40 ng/dl

PTH 6 Undetectable 10 to 65 pg /ml

Urine

calcium 2.4 to 2.6c 1.44 to 1.7 <0.3 mmol/mmol of creatinine

magnesium 0,8 1.8 to 2 <0.4 mmol/mmol of creatinine

sodium 53 10,7 <1.6 mmol/mmol of creatinine

fractional excretion of 16.5 to 26.7e 17.5 to 21.6e2.15 to 15.9 %

K (FEK)

osmolality after nasal ND 460 >800 mOsm/kgdDAVP

Expression hétérologue du CaSR

Vargas-Poussou, JASN, 2002

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

Paracellin-1 (Claudin 16) and hypercalciuria

Tubular phenotype of patients with loss-of-function in PCLN-1 gene

HHFControls

0

5

10

15

20

UV/GFRmmol/l GF

Na +

0

0,1

Ca ++

baseline furo0

0,1

0,2

Mg ++

0

10

Cl -

20

baseline furo baseline furo

baseline furo

Na+K+2 Cl-

3 Na+

2 K+

Cl-K+

Ca++

Mg++

Na+K+2 Cl-

3 Na+

2 K+

Cl-K+

?

+ -

Paracellin-1

A. Blanchard et al, Kidney Int, 2002

CLC5 and hypercalciuria

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

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)

Syndrome Maladie deDent

Protéinurie deBPM avecnéphrocalcinose

Néphrolithiaserécessive liée àl'X

Rachitismehypophosphatémique récessif lié à l'X

Rachitisme + +Protéinurie + + + +Perte rénale dephosphate

+ + +

Hypercalciurie + + + +Lithiase + +Néphrocalcinose + + + +Insuffisancerénale

+ +

Prédominancemasculine

+ + + +

Mutation du gèneCLCN-5

+ + + +

Xp11.22Xp22.1 Xq22 Xq28

Rachitisme hypophosphatémique

Maladie de Dent

Diabète insipide

néphrogénique

Syndrome d'Alport

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

CLC-5

CLC-5CLC-5

Colocalisation avec Rab4 et H+-ATPase

A-ClC-5B-H+ATPaseC=A+B

D-CLC-5E-2microglob.F=D+E

G-CLC-5H- 2microglobI=G+H 13min.

ME CLC-5

Localisation de CLC-5 dans le tubule proximal

ß2 microglobLactoglobuline

lactoglobuline

FITC-dextran

CLC-5

horseradish peroxydase CLC-5

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

CLC-5Mégaline CLC-5+mégaline

mégaline mégaline + CLC-5ClC-5

NaPi 2+/+ -/- -/-

+/- +/-

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+/-.

S1

Apex

H+ Cl-

Fonction de CLC-5

PHYSIOPATHOLOGIERôle de ClC5 dans le diabète phosphaté

Piwon Nils, Nature, 2000, vol 408, 369-373.

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 filtrée

[PTH] nle

[PTH]>

Nle« hyperparathyroidisme »

luminal (S3)

Tissue kallikrein and hypercalciuria

Tissue kallikrein and TRPV5 are coexpressed in the renal tubule

Copyright ©2001 by the National Academy of Sciences

Meneton, Pierre et al. (2001) Proc. Natl. Acad. Sci. USA 98, 2634-2639

FemellesC57Bl6/J

MalesC57Bl6/J

Femelles129Sv

Tissue kallikrein gene expression is controlled by calcium intake

Expression des transcrits des transporteurs

Mécanisme d’action de la TK

D. Gkika et al, EMBO J, 2006

L’effet de la TK est dépendant de la PKC

Effet des mutations des sites consensus de la PKC

La TK stabilise TRPV5 à la membrane

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 : – > 5 genes, no gene encoding a Ca transporter

• Kidney as the primary defect : – > 18 genes, only one gene encoding a Ca

transporter (PCLN-1)

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

regionsR. Hoopes et al, J.A.S.N., 2003

Conclusion• Hypercalciuria is a complex trait, and its expression

depends on both– Environmental factors– Genetic factors

Modification of dietary factors is efficient but not specificContinuing efforts are warranted

- detailed proximal phenotype definition- study of monogenic causes of hypercalciuria- identification of loci linked to idiopathic hypercalciuria

• Georges Pompidou 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

Pathophysiology of human idiopathic hypercalciuria

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mo

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Controls (n=9)

Hypercalciuric patients (n=34)

Controls (n=9)

Hypercalciuric patients (n=34)

P < 0.02

0

2.5

5

7.5

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ControlsIdiopathic

hypercalciuria

Diet Ca25.4 mmol/d

Diet Ca23.6 mmol/d

Adapted from Lemann, 1992

ECFCa

HypercalciuriaAdapted from Houillier, 1996

Monogenic renal hypercalciuria : clues to the genetics of idiopathic

hypercalciuria ?

• Trpv5 (ECaC1)• CaSR• Paracellin-1 (Claudin 16)