Targeted Knockin of Constitutively Active SPAK (CA‐SPAK) in the Early Distal Convoluted Tubule (DCT1) Causes Hyperkalemic Hypertension

Familial Hyperkalemic Hypertension (FHHt) is caused by aberrant gain in WNK function, leading to elevate blood pressure via hyperactivation of the thiazide‐sensitive sodium chloride co‐transporter (NCC) in DCT. Altered potassium homeostasis (hyperkalemia) is also observed in FHHt and attributed to reduced ROMK mediated potassium secretion. Two mechanisms are thought to contribute to attenuated kaliuresis in FHHt: 1) Increased phospho‐activation of NCC via the WNK‐SPAK signaling cascade results in limited sodium availability for electrogenic potassium secretion in the connecting tubule and cortical collecting duct (CNT and CCD); 2) increased ROMK endocytosis induced via the WNK‐Erk5‐ARH pathway in the CNT and CCD. The relative contribution of these two pathways in limiting potassium secretion is unclear. Here, we explore if constitutive activation of SPAK (CA‐SPAK) in the DCT1 is sufficient to drive hyperphosphorylation of NCC, can phenocopy FHHt (hypertension and hyperkalemia). Knockin‐CA‐SPAK (CA‐SPAK KI) lox/lox mice were created by inserting a SPAK phosphomimetic mutant (T243E and S383D) between LoxP sites in the first exon of the SPAK gene. To drive DCT1‐specific expression of CA‐SPAK in the kidney, mice expressing Cre‐recombinase under the parvalbumin promoter were crossed with the CA‐SPAK KI lox/lox mice. The phenotypes of DCT‐specific CA‐SPAK KI were compared to matched wild‐type (WT) controls. As observed by immunofluorescent confocal microscopy, CA‐SPAK is specifically targeted to parvalbumin positive tubules in CA‐SPAK KI, consistent with DCT1‐specific expression. CA‐SPAK KI display significantly more total and phosphorylated NCC (T58) than WT even though the abundance of CA‐SPAK protein was slightly less than SPAK in WT, verifying the constitutively active nature of the knockin. Telemetric blood pressure (BP) measurements revealed CA‐SPAK KI are hypertensive despite low renin activity. The BP of KI mice is further elevated by a high salt diet, but is normalized to WT levels by hydrochlorothiazide (HCTZ). CA‐SPAK KI display metabolic acidosis, hyperkalemia, and a diminished capacity to secret potassium, which are all remediated by HCTZ. These results suggest increasing sodium delivery to the CNT and CCD is sufficient to ameliorate hyperkalemia. However, further investigation via confocal microscopy in conjunction with morphometric analysis indicates CA‐SPAK KI mice have a structural contraction in the CNT that is matched by an expansion of the DCT1, consistent with other FHHt animal models. Additionally, significant down‐regulation of ROMK is observed via western blot analysis in CA‐SPAK KI, which is nearly normalized to WT levels following 3 days of HCTZ. In summary: 1) DCT1 expression of CA‐SPAK KI phenocopies FHHt, underscoring the chief role NCC activation plays in the pathogenesis of the disease; 2) Normalization of ROMK expression and plasma potassium levels in CA‐SPAK KI following HCTZ treatment suggests an anti‐kaliuretic process may be activated in FHHt, but is inhibited during HCTZ treatment. Support or Funding Information DK093501, DK063049, DK54231