Abstract: FR-PO627

Increased Phosphorylation of the Ubiquitin Ligase Kelch-Like 3 in the Kidney of Db/Db Mice

Session Information

Category: Diabetes

  • 501 Diabetes Mellitus and Obesity: Basic - Experimental

Authors

  • Shibata, Shigeru, Teikyo University School of Medicine, Tokyo, Japan
  • Ishizawa, Kenichi, Teikyo University School of Medicine, Tokyo, Japan
  • Nemoto, Yoshikazu, Teikyo University School of Medicine, Tokyo, Japan
  • Morimoto, Chikayuki, Teikyo University School of Medicine, Tokyo, Japan
  • Uchida, Shunya, Teikyo University School of Medicine, Tokyo, Japan
Background

Although clinical studies have shown that diabetic patients display salt-sensitive hypertension, its pathogenesis remains unclear. Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that regulates blood pressure by targeting With-No-Lyisne (WNK) kinases for degradation. Mutations and inactivation of KLHL3 cause hypertension resulting from increased Na-Cl cotransporter (NCC) activity in the kidney. Previously, we have reported that angiotensin II (Shibata et al. PNAS 2014) and potassium depletion (Ishizawa et al. BBRC 2016) inactivate KLHL3 by protein kinase C (PKC)-mediated phosphorylation at S433 in the Kelch-domain, thereby contributing to blood pressure elevation. In this study, we examined the possible involvement of KLHL3 in the diabetic kidney using a model of type 2 diabetes.

Methods

We examined the expression levels of total KLHL3 and KLHL3 phosphorylated at S433 (KLHL3S433-P) in the kidney of Db/+ and Db/Db mice. We also determined the levels of Cl transporters including NCC, Na-K-2Cl cotransporter NKCC2, and Cl/HCO3 exchanger pendrin in the membrane fraction of the kidney in this model.

Results

Western blotting revealed that KLHL3S433-P levels were significantly increased in the kidneys of Db/Db mice (2.2-fold increase versus Db/+ mice; P < 0.01), which was associated with the increased levels of WNK1/4. Moreover, NCC levels in the membrane fraction were significantly higher in Db/Db mice than Db/+ mice (2.3-fold increase, P < 0.01). Interestingly, NKCC2 was also increased, whereas pendrin was decreased in Db/Db mice. The decreased pendrin in this model was associated with the increased levels of inactive, phosphorylated form of mineralocorticoid receptor in the kidney. To determine the mechanism for the increased KLHL3S433-P levels, we evaluated PKC activity in the kidney. Of note, active, phosphorylated PKC was increased in Db/Db mice, explaining the KLHL3S433-P induction in this model.

Conclusion

These data indicate that the inactivation of KLHL3 is involved in the increased NCC activity in Db/Db mice, and suggest that KLHL3 is involved in the pathogenesis of salt-sensitive hypertension in type 2 diabetes.