{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/289398"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/289398","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Gitelman & Gordon: Mirror image syndromes reveal the roles of WNKs in blood pressure homeostasis and novel anti-hypertensive targets","abstract":"Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy.","abstract_html":"Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3<span class=\"etd-inline-math\"><sup>WT/\\Delta403-459</sup></span> mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK<span class=\"etd-inline-math\"><sup>L502A/L502A</sup></span> knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3<span class=\"etd-inline-math\"><sup>\\Delta403-459</sup></span> proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3<span class=\"etd-inline-math\"><sup>\\Delta</sup></span><span class=\"etd-inline-math\"><sup>ex9</sup></span>PHAII. In mice, homozygous MO25<span class=\"etd-inline-math\">&alpha;</span> knock-out proved embryonically lethal, while homozygous MO25<span class=\"etd-inline-math\">&beta;</span> knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK<span class=\"etd-inline-math\"><sup>L502A</sup></span> protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK<span class=\"etd-inline-math\"><sup>L502A/L502A</sup></span> mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy.","abstract_has_math":true,"creators":["Siew, Keith"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["O'Shaughnessy, Kevin"],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-02-20","date_published":"2018-02-20","updated_at":"2026-07-22T22:23:54Z","subjects":["Blood Pressure","hypertension","WNK","SPAK","CUL3","Gitelman Syndrome","Gordon Syndrome","PHAII","PHA2E","FHHt","Familial Hyperkalaemia Hypertension","Pseudohypoaldosteronism Type II","MO25","CAB39","electrolytes","homeostasis","Cullin-3","renal","cardiovascular","drug targets","mouse models"],"languages":["en"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/e1e03f49-0ea7-43de-a7d5-75857d10ab94/download","https://creativecommons.org/licenses/by/4.0/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000265025095","0000000214767566"],"render_values":[{"text":"0000-0002-6502-5095","href":"https://orcid.org/0000-0002-6502-5095","code":true},{"text":"0000-0002-1476-7566","href":"https://orcid.org/0000-0002-1476-7566","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.36646","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["O'Shaughnessy, Kevin"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["British Heart Foundation"]},{"key":"dc:creator","label":"Author","values":["Siew, Keith"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0000000265025095","0000000214767566"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2018-02-20"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/289398"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Blood Pressure","hypertension","WNK","SPAK","CUL3","Gitelman Syndrome","Gordon Syndrome","PHAII","PHA2E","FHHt","Familial Hyperkalaemia Hypertension","Pseudohypoaldosteronism Type II","MO25","CAB39","electrolytes","homeostasis","Cullin-3","renal","cardiovascular","drug targets","mouse models"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/e1e03f49-0ea7-43de-a7d5-75857d10ab94/download","https://creativecommons.org/licenses/by/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.36646"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/69c35e24-97be-41ed-b219-b788c0da2c67/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["87eda9de84448d1f82354d60eee3eb5f","459c618105ce63babcc59379733a34f1"]},{"key":"dc:title","label":"Title","values":["Gitelman & Gordon: Mirror image syndromes reveal the roles of WNKs in blood pressure homeostasis and novel anti-hypertensive targets"]}]}],"canonical_facts":{"dc:contributor.advisor":["O'Shaughnessy, Kevin"],"dc:contributor.sponsor":["British Heart Foundation"],"dc:creator":["Siew, Keith"],"dc:creator.authoridentifier":["0000000265025095","0000000214767566"],"dc:date.issued":["2018-02-20"],"dc:description.abstract":["Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy."],"dc:format.checksum.md5":["87eda9de84448d1f82354d60eee3eb5f","459c618105ce63babcc59379733a34f1"],"dc:identifier.doi":["10.17863/CAM.36646"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/69c35e24-97be-41ed-b219-b788c0da2c67/download"],"dc:language":["en"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/289398"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/e1e03f49-0ea7-43de-a7d5-75857d10ab94/download","https://creativecommons.org/licenses/by/4.0/"],"dc:subject":["Blood Pressure","hypertension","WNK","SPAK","CUL3","Gitelman Syndrome","Gordon Syndrome","PHAII","PHA2E","FHHt","Familial Hyperkalaemia Hypertension","Pseudohypoaldosteronism Type II","MO25","CAB39","electrolytes","homeostasis","Cullin-3","renal","cardiovascular","drug targets","mouse models"],"dc:title":["Gitelman & Gordon: Mirror image syndromes reveal the roles of WNKs in blood pressure homeostasis and novel anti-hypertensive targets"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:23:54Z"}