Human Hypertension caused by Mutations in WNK Kinases

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.

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

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. • 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 ↓ ↓ ↓ WNK2 WNK3 WNK4 Chr9 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.

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

9. Expression of WNK 1 and 4 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 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 Results not shown Anti WNK4-RED ZO-1GREEN – 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.

12. 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