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Molecular basis of distal Renal Tubular Acidosis
Rosa Vargas-PoussouDépartement de Génétique HEGP
Réseau de tubulopathies
Kidney and acid-base status
Kidney and acid-base status
Reabsorption of 80% of filtered bicarbonateGeneration of ammonia
NH3
Kidney and acid-base status
Reabsorption of 80% of filtered bicarbonateGeneration of ammonia
NH3
Reabsorption of10 – 15 % of bicarbonateAmmonia recycling
neration of ammonia
Kidney and acid-base status
Reabsorption of 80% of filtered bicarbonateGeneration of ammonia
NH3
Reabsorption of10 – 15 % of bicarbonateAmmonia recycling
neration of ammonia
Proton secretionAmmonia secretion
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
A-Intercalated cells
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
Vacuolar H+ ATPase
H+ ATPase
Vacuolar H+ ATPase Structure
C2A A
A
B1B1
B1
G3G3
H
E
D
F d2a4
e2 c’’c
Cytoplasm
Lumen
ATPADP+iP
H+
H+
V0
V1
Vacuolar H+ ATPase Structure
C2A A
A
B1B1
B1
G3G3
H
E
D
F d2a4
e2 c’’c
Cytoplasm
Lumen
ATPADP+iP
H+
H+
V0
V1
e2e2
V0d2
V0a4V0a4
V0a2
V1G3V1G3
V1C2
V1B1V1B1
Inner earKydney
Vacuolar H+ ATPase Structure
C2A A
A
B1B1
B1
G3G3
H
E
D
F d2a4
e2 c’’c
Cytoplasm
Lumen
ATPADP+iP
H+
H+
V0
V1
e2e2
V0d2
V0a4V0a4
V0a2
V1G3V1G3
V1C2
V1B1V1B1
Inner earKydney
V0d1V0cV0c’’V00a1V1HV1E1V1DV1c1V1B2V1A1
Ubiquitous
Vacuolar H+ ATPase Structure
C2A A
A
B1B1
B1
G3G3
H
E
D
F d2a4
e2 c’’c
Cytoplasm
Lumen
ATPADP+iP
H+
H+
V0
V1
e2e2
V0d2
V0a4V0a4
V0a2
V1G3V1G3
V1C2
V1B1V1B1
Inner earKydney
V0d1V0cV0c’’V00a1V1HV1E1V1DV1c1V1B2V1A1
Ubiquitous
Cellular physiology of the renal H+ATPase. Blake-Palmer, Katherine; Karet, Fiona.Current Opinion in Nephrology & Hypertension. 2009.
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
Vacuolar H+ ATPase
H+ ATPase
B1B1
B1
a4
ATPADP+iP
H+
H+
B1 Subunit – ATP6V1B1 gene
• Positional cloning - dRTA with deafness (Karet FE, 1999)
• 1 transcript• Protein: 513 amino acids
• Mutations affect function and/or pump assembly
• B1 KO mice (Finberg KE et al, PNAS 2005)
• B1 subunit increases the pump activity in response to external stimuli
2p13
a4 Subunit – ATP6V0A4 geneTAG ATG
• Positional cloning - dRTA with preserved hearing (Smith AN, 2000)
• dRTA with late onset sensorioneural hearing (Stover EH, 2002)
• dRTA with early sensorioneural hearing (Vargas-Poussou R, 2006)
• 3 additional transcripts• Protein: 840 amino acids• Mutation W520L:
dominant negative effect• a4 subunit interacts with
phosphophructokinase 1
7q33-34
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
AE1 anion exchanger, SLC4A1
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
AE1 anion exchanger, SLC4A1
H+ ATPase
AE1 – SLC4A1 gene
17q22
AE1 – SLC4A1 gene
17q22Band 3
AE1 – SLC4A1 gene
17q22Band 3kAE1
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
AE1 – SLC4A1 gene
17q22Band 3kAE1
Renal isoform
Blood group antigensSpherocytosis and Ovalocytosis
200285
488
518
663
701
773
850
Dominant dRTA Recessive dRTA
AE1 Mutations
G
S
S
V AV
R
Alper SL, Exp Physiol, 2006
AE1 Mutations
Spherocytosis and Ovalocytosis
Dominant dRTA
Recessive dRTA
Dominant dRTA
Heterozygous
Recessive dRTA
With hematologic abnormalities
Without hematologic abnormalities
AE1 Mutants and dRTA
Cordat E. et al. Traffic 2006 Devonald MA. et al. Nat Genet 2003
Model of compound heterozygous states of dRTA/SAO
Kittanakom S. et al. Biochem J 2008
Wt SAO Recessive dRTA SAO/dRTA
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
Carbonic Anhydrase type II
H+ ATPase
Carbonic Anhydrase II Deficiency syndromeOsteopetrosis with RTA and brain calcification
Autosomal recessive diseaseIncreased bone densityIntracerebral calcification, mental retardationGrowth failureFacial dysmorphismMixed RTA: proximal and distal
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
K+/Cl- cotransporter, KCC4
H+ ATPase
Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4.Boettger T et al. Nature 2002
knockout mice: Ur pH 7.3 0.1 wild type mice: Ur pH 6.4 0.1;n = 7 P < 0.001
CortexKcc4
PT
IC
Annual Reviews
Renal Ammonia Metabolism
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
RhCG
H+ ATPase
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
RhCG
H+ ATPase
S Biver et al. Nature 456, 339-343 (2008) doi:10.1038/nature07518
Impaired acid-stress handling in Rhcg−/− mice.
S Biver et al. Nature 456, 339-343 (2008) doi:10.1038/nature07518
Reduced NH3 permeability of microperfused collectingducts segments from Rhcg−/− acid-challenged mice.
Cl-
K+H+
CO2H2O
HCO3H+
HCO3-
ACII
H+
NH3NH3
NH4+
K+
Cl-
K+Na+
HCO3-
Cl-
Cl -Barttin
NH4+
NH3H+
AE1
KCC4
CLC-Kb
SLC26A7RhCG
Anion exchanger, SLC26A7
H+ ATPase
Slc26a7-null Mice
Xu J. et al. J Biol Chem 2009
Slc26a7-null Mice
Xu J. et al. J Biol Chem 2009
Isotonic
Hypertonic
Slc26a7-null Mice
Xu J. et al. J Biol Chem 2009
Distal Renal Tubular Acidosis
(n=109)
Recessive (n=84)
Dominant(n=25)
Consanguinity (n=31)
Complete(n=12)
Incomplete(n=10)
Haplotype Analysis SLC4A1 mutations(n=10)
No mutationin SLC4A1
Sporadic(n=53)
ATP6V0A4 mutations(n=16)
ATP6V1B1 mutations(n=12)
No mutations(n=3)
ATP6V0A4 mutations(n=19)
ATP6V1B1 mutations(n=23)
No mutation(n=11)
No mutation(n=1)
Distal Renal Tubular Acidosis
(n=109)
Recessive (n=84)
Dominant(n=25)
Consanguinity (n=31)
Complete(n=12)
Incomplete(n=10)
Haplotype Analysis SLC4A1 mutations(n=10)
No mutationin SLC4A1
Sporadic(n=53)
ATP6V0A4 mutations(n=16)
ATP6V1B1 mutations(n=12)
No mutations(n=3)
ATP6V0A4 mutations(n=19)
ATP6V1B1 mutations(n=23)
No mutation(n=11)
No mutation(n=1)
dRTA : global detection rate 77%Complete dRTA: detection rate 85%
ATG TAG
Ex1
c.1155dup, p.Ile386HisfsX56 (14)
17 mutations in 35 families(5 frameshift, 3 splice, 1 nonsens and
8 missense).
-1G>C (4)
Ex2 Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10 Ex11 Ex12 Ex13 Ex14
R394Q (3)P346R (2)G78RL81P(4)
-3C>G
A272V-2A>T
R157C584-6+1del
E195DfsX127
F365C
c.1356delPhe452fsX35
Q415X
ATPV1B1 Mutations
c.1260dupp.Ala421CysfsX21
c.270_273+4delp. Gln91CysfsX72
E161K
23 Homozygous7 Compound Heterozygous5 Heterozygous
ATG TAG
Ex1
c.1155dup, p.Ile386HisfsX56 (14)
17 mutations in 35 families(5 frameshift, 3 splice, 1 nonsens and
8 missense).
-1G>C (4)
Ex2 Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10 Ex11 Ex12 Ex13 Ex14
R394Q (3)P346R (2)G78RL81P(4)
-3C>G
A272V-2A>T
R157C584-6+1del
E195DfsX127
F365C
c.1356delPhe452fsX35
Q415X
ATPV1B1 Mutations
c.1260dupp.Ala421CysfsX21
c.270_273+4delp. Gln91CysfsX72
E161K
23 Homozygous7 Compound Heterozygous5 Heterozygous
ATP6VOA4 MutationsATG
Ex4
TAG
Ex2Ex1
+1G>A
Y129X(8) Q753XR770X(2)
33 mutations in 35 families.12 frameshift ,10 nonsens
6 missense, 4 splice1inframe
Ex3 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10 Ex11 Ex12 Ex13 Ex14 Ex15 Ex16 Ex17 Ex18 Ex19 Ex20 Ex21 Ex22 Ex23
c.188_9delp.Arg63AsnfsX12
E123X
-1G>A
D679YR6X
Y450X(2)
c.828_31dep.Thr277SerfsX25
c.1181_5delp.113fsX117(2)
R191X(3)
c.2137delp.Glu713SerfsX50
c.338delp.Asn113ThrfsX5
Q108X (2)
R243X(2)
c.709_11delp.Lys237del
+1G>A
R807Q
G820R
c.738_9del
W526R
c.1186dupp.Tyr396LeufsX34
c.1661delT
G175S
c.828_31delp.Thr277SerfsX2
c.201delp.Leu68TrpfsX10
R743L
+1G>Ac.1661delT
R807X
24 Homozygous8 Compound heterozygous3 Heterozygous
ATP6VOA4 MutationsATG
Ex4
TAG
Ex2Ex1
+1G>A
Y129X(8) Q753XR770X(2)
33 mutations in 35 families.12 frameshift ,10 nonsens
6 missense, 4 splice1inframe
Ex3 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10 Ex11 Ex12 Ex13 Ex14 Ex15 Ex16 Ex17 Ex18 Ex19 Ex20 Ex21 Ex22 Ex23
c.188_9delp.Arg63AsnfsX12
E123X
-1G>A
D679YR6X
Y450X(2)
c.828_31dep.Thr277SerfsX25
c.1181_5delp.113fsX117(2)
R191X(3)
c.2137delp.Glu713SerfsX50
c.338delp.Asn113ThrfsX5
Q108X (2)
R243X(2)
c.709_11delp.Lys237del
+1G>A
R807Q
G820R
c.738_9del
W526R
c.1186dupp.Tyr396LeufsX34
c.1661delT
G175S
c.828_31delp.Thr277SerfsX2
c.201delp.Leu68TrpfsX10
R743L
+1G>Ac.1661delT
R807X
24 Homozygous8 Compound heterozygous3 Heterozygous
Family 2(Algeria)
252
654
D7S2560D7S684D7S1824
65
10
65
13
65
10
116
13
65
13
664
Family 9(France/Algeria)
264
65
10
6510
264
1218
9613
656
65
13
65
13
65
13
Family 10(Algeria)
65
12
65
12
6512
65
12
665
1044
65
12
65
12
665
65
12
65
12
65
12
665
1044
Family 13(Algeria)
Family 34(Morocco)
654
658
Mutation Y129X – Founder effectFamily 5(Algeria)
D7S2560D7S684D7S1824
D7S2560D7S684D7S1824
D7S2560D7S684D7S1824
(Q753X/Y129X) (E123X/Y129X)
Vargas-Poussou R et al. JASN 2006
Family 38
�
1052
1054
1054
1052
D7S2560D7S684D7S1824
1059
1059
1059
1059
555
332
D7S2560D7S684D7S1824
Family 7
Mutation R770X - Malian families
1.661.390.2275%0.50.40.08Median0.040.270.0425%133232n =No MutB1a4*
* p = 0.0001
Nephrocalcinosisa4 Mutation 34/35B1 Mutation 27/32
7.337.307.2175%
7.277.287.15Median
7.237.197.1525%
82413n =
No MutB1a4
7.457.578.075%
7.07.157.5Median
6.97.07.025%
122821n =
No MutB1a4
14.2516.31475%
1413.811Median
10.2512825%
102927n =
No MutB1a4
* p = 0.01
4.23.53.4775%
3.72.83.05Median
2.92.62.625%
92724n =
No MutB1a4
*
Sensorioneural Hearing loss
AE1 Mutations
R589C (4)R589H (2)
Y904X
E90K
Renal isoform
S334_L342del
S525F G609R
SAO
8 mutations in 11 families 10 heterozygous,1 compound heterozygous (dRTA and ovalocytosis)
(1inframe deletion, 1 frameshift, 1 nonsense and 5 missense mutations)
A891PfX
HS
443075%27.517Median19.751625%811n =i dATRAE1
6.47.175%
66.9Median
5.56.7525%
79n =
i dATRAE1
* p = 0.0007
*
7.417.3475%
7.387.27Median
7.356.9525%
86n =
i dATRAE1
*
* p = 0.04
*
4.323.275%
3.93.2Median
3.323.125%
87n =
i dATRAE1
* p = 0.007
2820.2575%
2719.85Median
23.516.7525%
810n =
i dATRAE1
* p = 0.0004
*
Conclusions 1
Genes implicated in human dRTASLC4A1 (Dominant and Recessive)ATP6V1B1 and ATP6V0A4 (Recessive)ACII (Osteopetrosis with RTA)
Additional genes in miceKcc4Slc26a7Rhcg
Conclusions 2
Detection of loss of function mutations in 71 families with recessive and 10 families with dominant complete dRTA (detection rate of 85%).Similar frequency of a4 and B1 mutation in recessive dRTAOne patient with recessive dRTA and AE1 mutationPhenotypic variability of hearing loss More severe phenotype (age at diagnosis and metabolic acidosis) in patients with a4 mutations
Country? Access to medical care?a4 function in other nephron segments?
Conclusions 3
Detection of loss of function mutations in 11/12 families with dominant dRTA.No mutations were detected in 10 patients with incomplete dRTANo mutations were detected in 14 families with recessive dRTA
Supplementary genetic heterogeneity
MERCI
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