Academic literature on the topic 'BTBR mice'

博士<br>國防醫學院<br>醫學科學研究所<br>107<br>As opposed to Gitelman syndrome (GS) caused by loss-of-function mutation in the Na+-Cl- cotransporter (NCC), pseudohypoaldosteronism type II (PHAII) is notable for an enhanced NCC function. PHAII is an autosomal dominant renal tubular disorder characterized by thiazide-correctable salt-sensitive hypertension with low renin activity, hyperkalemic metabolic acidosis and hypercalciuria. Despite no identified NCC mutation in PHAII, genetic defects in the well-known with-no-lysine [K] (WNK) kinases WNK1 and WNK4 (minority) and two newly-discovered Kelch-like 3 (KLHL3, majority) or Cullin3 (Cul3) are most responsible for inherited PHAII. Our laboratory and other research groups have clearly demonstrated that the constitutive activation of WNK kinases with downstream phosphorylation of the Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response kinase 1 (OSR1) which in turn stimulate NCC and NKCC [WNKs-SPAK/OSR1-N(K)CC cascade] has contributed to be the pathogenesis of PHAII. Both of KLHL3 and Cul3 regulated WNKs-SPAK/OSR1-N(K)CC cascade by promoting degradation of WNK1/4. Arterial hypertension is among the leading global risks for premature mortality and mortality. Increased renal salt reabsorption was thought to be as an important culprit for hypertension, thus exploring unknown molecular mechanisms of regulation of iron homeostasis will enhance the understanding of the fundamental biology and to provide a new vision for antihypertensive treatment. In view of this, we generated and analyzed Klhl3 knock-in (KI) mice carrying a missense M131V mutation in the BTB domain (corresponding to human KLHL3 M78V mutation). KLHL3M131V/+ KI mice exhibited typical PHAII phenotypes including an exaggerated diuretic response to hydrochlorothiazide. Their kidney tissues showed an unchanged KLHL3, decreased cullin3 (Cul3), and increased WNK1 and WNK4 expression along with an enhanced downstream SPAK/OSR1-N(K)CC phosphorylation. In microdissected renal tubules, Klhl3M131V/+ KI mice expressed high levels of Wnk4 mRNA and large Wnk4 to Wnk1 mRNA ratio in the distal nephron. In vitro co-immunoprecipitation (Co-IP) showed the KLHL3 BTB domain mutation retained intact interaction with WNKs but reduced binding to Cul3, thus leading to the increased abundance of total WNKs. Next, we generated Wnk4 -/- mice by targeting disruption from the promoter to exon 2 of Wnk4 and further crossed them with Klhl3M131V/+ KI mice to elucidate the issue about which WNKs plays a dominant role in KLHL3 mutation-causing PHAII. Wnk4-/- mice exhibited GS-like features and their kidney tissues showed an absence of WNK4 protein expression with compensatively increased WNK1 expression. The expressions of OSR1 and NKCC2 were increased, but the levels of SPAK and NCC were obviously decreased. Notably, Klhl3M131V/M131V．Wnk4-/- double transgenic mice still exhibited the overwhelming GS-like phenotypes with remarkably decreased SPAK-NCC phosphorylation despite an increased WNK1 expression compared to WT mice. In summary, my thesis utilized the technique of genetic mouse model to clarify the underlying pathophysiological mechanism of PHAII. Klhl3M131V/+ KI mice featured the typical phenotypes of PHAII via a defective BTB domain impaired binding to Cul3, resulting in a reduced Cul3 expression and consequently increased WNK1/4 levels. Klhl3 M131V mutation failed to rescue the GS-like phenotypes in Wnk4-/- mice despite increased expression of WNK1, confirming WNK4 kinase is a dominant upstream WNKs in regulation of the N(K)CC pathway on KLHL3 mutation-causing PHAII background. New insights into the role of WNK4 could contribute significantly to the effort to develop novel therapeutics targeted to PHAII.