The role of 70-kDa heat shock protein in dDAVP-induced AQP2 trafficking in kidney collecting duct cells

It has been reported that several proteins [heat shock protein 70 (Hsp70 and Hsc70), annexin II, and tropomyosin 5b] interact with the Ser²⁵⁶ residue on the COOH terminus of aquaporin-2 (AQP2), where vasopressin-induced phosphorylation occurs for mediating AQP2 trafficking. However, it remains unknown whether these proteins, particularly Hsp70, play a role in AQP2 trafficking. Semiquantitative immunoblotting revealed that renal expression of AQP2 and Hsp70 was significantly increased in water-restricted or dDAVP-infused rats. In silico analysis of the 5′-flanking regions of AQP2, Hsp70-1, and Hsp70-2 genes revealed that transcriptional regulator binding elements associated with cAMP response were identified at both the Hsp70-1 and Hsp70-2 promoter regions, in addition to AQP2. Luciferase reporter assay demonstrated the significant increase of luminescence after dDAVP stimulation (10^-8 M, 6 h) in the LLC-PK1 cells transfected with luciferase vector containing 1 kb of the 5′-flanking region of Hsp70-2 gene. Hsp70-2 protein expression was also increased in mpkCCDc14 cells treated by dDAVP in a concentration-dependent manner. Cell surface biotinylation analysis demonstrated that forskolin (10^-5 M, 15 min)-induced AQP2 targeting to the apical plasma membrane was significantly attenuated in the mpkCCDc14 cells with Hsp70-2 knockdown. Moreover, forskolin-induced AQP2 phosphorylation (Ser²⁵⁶) was not significantly induced in the mpkCCDc14 cells with Hsp70-2 knockdown. In contrast, Hsp70-2 knockdown did not affect the dDAVP-induced AQP2 abundance. In addition, siRNA-directed knockdown of Hsp70 significantly decreased cell viability. The results suggest that Hsp70 is likely to play a role in AQP2 trafficking to the apical plasma membrane, partly through affecting AQP2 phosphorylation at Ser²⁵⁶ and cell viability.