Abstract 081: Pathogenic Role of Ubiquitin Ligase Klhl3 in Diabetic Nephropathy

Abstract

Background and Aim: 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 deficiency (Ishizawa et al. BBRC 2016) inactivate KLHL3 by protein kinase C (PKC)-mediated phosphorylation at S433 in the Kelch-domain, thereby contributing to increased blood pressure. Although clinical studies have shown that diabetic patients display salt-sensitive hypertension, its pathogenesis remains unclear. 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, KLHL3 phosphorylated at S433 (inactive form; KLHL3 S433-P ), and NCC in the kidney of Db/+ and Db/Db mice by Western blot. Distribution of KLHL3 S433-P and NCC was analyzed by immunofluorescent microscopy. In some experiments, bisindolylmaleimide (BIM; the PKC inhibitor) was administered intraperitoneally. Results: We found that KLHL3 S433-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 ) . Immunofluorescent study indicated that KLHL3 S433-P is increased in the distal convoluted tubules (where NCC is present). Of note, active, phosphorylated PKC was increased in the kidney of Db/Db mice (1.8-fold increase; P <0.01), explaining the KLHL3 S433-P induction in this model. To investigate the causal role of PKC and KLHL3 S433-P , we administered BIM to Db/Db mice. Importantly, KLHL3 S433-P levels were significantly decreased by BIM (29% decrease vs Db/Db mice; P < 0.05). Furthermore, the increased NCC at the plasma membrane in Db/Db mice was also ameliorated by the PKC inhibitor. Conclusion: These data indicate that the inactivation of KLHL3 is involved in the aberrant NCC activity in the kidney of Db/Db mice.