Abstract 068: Kelch-Like 3 Dephosphorylation by Calcineurin as a Mechanism Regulating Na-cl Cotransporter

Abstract

Tacrolimus, an inhibitor of the phosphatase calcineurin, is widely used as an immunosuppressive agent. However, its use is associated with hypertension and hyperkalemia. Although previous studies indicated that the activation of thiazide-sensitive, Na-Cl cotransporter (NCC) plays a key role in the pathogenesis, the identity of the direct target of calcineurin in kidney remained unclear. Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase that critically regulates NCC function. In pseudophyoaldoseronism type 2, the disease-causing mutations in KLHL3 impair binding and degradation of with-no-lysine 1 (WNK1) and WNK4, resulting in hypertension and hyperkalemia that are correctable by thiazide. Previously, we reported that phosphorylation of KLHL3 at serine 433 (KLHL3 S433-P ) by protein kinase C critically regulates KLHL3 function by abrogating the substrate-binding ability. However, phosphatases responsible for KLHL3 S433-P regulation were unknown. We here show that calcineurin acts as a phosphatase for KLHL3 S433-P . In in vitro phosphatase assay, we found that calcineurin suppressed KLHL3 S433-P levels (65% reduction from baseline). Consistently, the activation of calcineurin by ionomycin significantly reduced KLHL3 S433-P abundance in distal convoluted tubule cells and in HEK cells expressing KLHL3, an effect that was almost completely prevented by calcineurin knockdown. Moreover, we found that tacrolimus increased KLHL3 S433-P levels by 1.8 fold in mouse kidney, which was associated with the WNK accumulation and increased NCC activity. Finally, we found that tacrolimus attenuated KLHL3-mediated WNK4 ubiquitination and degradation, and this effect was abolished by non-phosphorylatable S433A substitution in KLHL3. These data identify Kelch-like 3 as a direct target of calcineurin, and provide evidence that KLHL3 is involved in the pathophysiology of tacrolimus-induced hypertension.