PS-BPB10-4: CREATION AND CHARACTERIZATION OF THE UBIQUITIN LIGASE KLHL3-S433A KNOCK-IN MICE

Abstract

Background and aim: Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that regulates blood pressure and electrolyte homeostasis. Mutation and inactivation of KLHL3 are known to cause hypertension and hyperkalemia mainly due to the impaired with-no-lysine (WNK) degradation and increased Na-Cl cotransporter (NCC) activity in the kidney. Previously, we have reported that angiotensin II (Shibata et al. PNAS 2014), potassium deficiency, and impaired glucose tolerance (Ishizawa et al. BBRC 2016 and JASN2019) can inactivate KLHL3 by altering phosphorylation at S433 in the Kelch-domain. We and others have also shown that decreased KLHL3 expression is also involved in the pathology of hypertension and hyperkalemia. The current study was designed to demonstrate the physiological importance of S433 in KLHL3 function in vivo in mice. Method: Using CRISPR/Cas9, S433 of KLHL3 was substituted to alanine in C57BL/6 mice (KLHL3-S433A Knock in mice: KI). Creation of KI was confirmed by DNA sequencing and RFLP analysis. Blood pressure was evaluated by telemetry, and blood gas was analyzed by iSTAT. Levels of KLHL3, WNK1/4, and NCC in the kidney were determined by Western blot analysis. Gene expression of KLHL3, WNK1 and WNK4 was evaluated by real time RT-PCR. Results: On a normal salt diet, blood pressure levels were similar between WT and KI; however, KI mice showed significant increase in response to a high salt diet compared with WT on the same diet. KI mice showed a significant increase in plasma potassium levels and a significant decrease in bicarbonate levels already at baseline, confirming that S433 in KLHL3 is also critical in regulating potassium and acid/base homeostasis. Urinalysis indicated that the increase in plasma potassium levels is mainly attributable to the decreased potassium excretion. Of interest, we found that total KLHL3 abundance was drastically reduced by Western blot analysis, which was accompanied by the increase in KS-WNK1 and WNK4 and an increase in NCC both. Real time RT-PCR showed that gene expression of Klhl3, as well as those of Wnk1 and Wnk4, was unchanged, indicating that S433A substitution promotes degradation of KLHL3, leading to hyperkalemia and increased salt sensitivity. Conclusion: The study confirmed the critical importance of S433 in KLHL3 in controlling the ubiquitin ligase function and electrolyte homeostasis in vivo. It also indicates that the modification of KLHL3-S433 influences the protein abundance without affecting gene transcription.