1. Human Hypertension caused by Mutations in WNK Kinases
Science Paper
Wilson et al 2001

2. Why is the study of Mendelian Forms of Hypertension important?
Hypertension affects 25% of most adult population and is a common risk factor for common causes of morbidity and mortality
Molecular Pathogenesis of common forms of hypertension is poorly understood
Studying rare Mendelian form of hypetension provide clues to pathways
Identify new drug targets and approaches to treatment

3. Features of PHAII/Gordons Syndrome
Autosomal Dominant
Hypertension; ↑ renal salt reabsorption
Hyperkalemia; ↓ renal K+ excretion
Metabolic acidosis; ↓ renal H+ secretion
These features suggest a defect in RENAL ELECTROLYTE HANDLING.
Treatment – Thiazide diuretics.
Autosomal Dominant – You just need one copy of the mutated gene to have the phenotype
Hypertension – high blood pressure
Hyperkalemia – increased Potassium levels in the blood
But with - Normal glomerular filtration
- Normal aldosterone secretion
Acidosis – increased acidity of body fluids
Thiazide diuretics – act by inhibiting salt reabsorption in the distal nephron.

4. Genetics of Affected Families
Linkage ↓
Genotype - (Look for mutations)
(Size of fragment)
PCR Sequencing
Southern blotting
Deletions Missense mutations

5. Genetics of Affected Families – K22
Genome wide scan shows linkage to Chromosome 12
PHA II Kindreds (affected have typical features of PHAII)
Null allele D12s94
3 Additional marker close to D12s94
PCR:
- using primers usually 42 kb apart in WT,
- only 600bp in affected individuals
confirm deletion and 41kb fragment in exon 1 of WNK1
Typical features of PHAII
Blood pressure > 140/90 mm Hg
Hyperkaleamia, Normal Glomerular filtration rate, Suppressed plama renin activity, Normal or elevated aldosterone levels, Hyperchloremia…
First found Linkage
Linkage to most telomeric 2cM Centimorgan of Chr 12p
Then
Additional markers in telomeric region
Genotyping – Affected members looked homozygotes
But the allele passed for affected parents to offspring violated mendelian transmission.
(Eg D12S94)
In all 4 informative matings (4 zeros), offspring inherited no allele from affected parent
Findings indicate null allele
Consistent with deletion within the disease linked choromosome segment

6. Genetics of affected families – K4
Null alleles at
STS45K
STS60K
PCR
Novel 24Kb product
Absent in WT
DNA sequencing
Deletion of 21Kb
NB – this deletion is located within the deletion shown in the K22 family.
Compare WNK 1 expression levels – 5x increase in WNK1 expression compared to unaffected individuals
Expression studies in K4 and WT show there I s 5 fold increase in WNK1 expression is affected individuals

7. Families with WNK 1 paralog mutations
Kindreds K13, K23, K11, K21
Paralogs of WNK1
↓ ↓ ↓
WNK WNK WNK4
Chr Chr X Chr 17
Additional PHAII loci have previously been mapped to Chromosome 1 and Chromosome 17 (Mansfield et al 1997)
4 missense mutations in WNK4 found in PHAII kindreds.
But what about families that did not have WNK1 mutations
Looked for WNK1 paralogs.
Paralogs are genes related by duplication within a genome.
Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.
Mutations found in highly conserved domain

8. Causes of PHAII No mutation in WNK 1 or WNK 4 Affected Families
Chromosome 12
WNK 1 mutation
Chromosome 17
WNK 4 mutation
Chromosome 1 ?
Deletion
Missense mutations
Genetic gain of function
41kb deletion Intron 1
21kb deletion Intron 1
a.a 565
Gln -> Glu
Exon 7
a.a 564
Asp -> Ala
Exon 7
a.a 562
Glu -> Lys
Exon 7
a.a 1185
Arg -> Cys
Exon 7
K22 K4
K13
K23
K11
K21

9. Expression of WNK 1 and 4 WNK 1 10kb transcript-kidney
Add expression in leukocytes
WNK 1
10kb transcript-kidney
12kb transcript- predominantly in heart and skeletal muscle
WNK 4
Approx 4.4kb expressed exclusively in kidney

10. Localisation WNK 1 WNK 4 WNK 4 WNK 1 Results not shown Anti WNK4-RED
Distal Convoluted Tubule Staining
Anti WNK4 -RED
Anti NCCT- GREEN
Distal Convoluted Tubule Staining
Anti WNK1-RED
Anti NCCT- GREEN
Renal Cortex Staining
Anti WNK1-RED
Anti AQP2- GREEN
Renal Cortex Staining
Anti WNK4-RED
Anti AQP2- GREEN
Fluorescent Microscopy
Results – Co-localisation of Anti WNK1 and Anti AQP2 shows WNK1 is found in the connecting tubule and collecting duct
Anti NCCT – GREEN – Marker of Distal Convoluted Tubule
Results – Co localisation of Anti WNK1 and anti NCCT
WNK1 – Present in cytoplasm
WNK4 – Present exclusively in intercellular junctions in DCT
- Present in cytoplasm in CCD
Results not shown
Anti WNK4-RED
ZO-1 GREEN – A tight junction complex
Results show that WNK is found in the intercellular junctions
Anti AQP2- GREEN- Marker of connecting tubule and collecting duct
Anti NCCT- GREEN- Marker of DCT

11. Summary
Mutations in WNK1 & WNK4 cause PHAII leads to a genetic gain of function mechanism.
Clustering of mutations in highly conserved domain suggest disruption at this site normally required for normal regulation.
Both kinases are found in the distal nephron which is well known to play a key role in electrolyte homeostasis, This process is disrupted in PHAII individuals.
Identification of upstream and downstream targets will help to understand WNK signalling pathways.
In DCT, salt reabsorption is mediated by NCCT
In CD, Na+ is reabsorbed by ENaC

12. WNK 4 Missense mutations
WNK1 in vitro
Prevents WNK4
interacting
with NCCT
WNK 4
Missense
mutations
NCCT │
↑overactivity
↑net sodium
Reabsorption
↑plasma volume
↑cardiac output
↑ BP
Hypertension
NCCT
In vitro WNK4
interacts with
NCCT and
Loses its ability to
suppress it
Hyperkaleamia
WNK4 directly
Interacts
with K+ channel
ROMK