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

Eui Jung Park, Jung Suk Lim, Hyun Jun Jung, Eunjung Kim, Ki Hwan Han, Tae Hwan Kwon

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)F958-F971
JournalAmerican Journal of Physiology - Renal Physiology
Volume304
Issue number7
DOIs
StatePublished - Jun 10 2013
Externally publishedYes

Fingerprint

Collecting Kidney Tubules
Aquaporin 2
HSP70 Heat-Shock Proteins
5' Flanking Region
Phosphorylation
Colforsin
Luciferases
Cell Survival
Cell Membrane
Annexin A2
LLC-PK1 Cells
Biotinylation
Tropomyosin
Proteins
Luminescence
Vasopressins
Immunoblotting
Genetic Promoter Regions
Computer Simulation
Small Interfering RNA

Keywords

  • Aquaporin
  • Collecting duct
  • Heat shock protein
  • Phosphorylation
  • Vasopressin

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

The role of 70-kDa heat shock protein in dDAVP-induced AQP2 trafficking in kidney collecting duct cells. / Park, Eui Jung; Lim, Jung Suk; Jung, Hyun Jun; Kim, Eunjung; Han, Ki Hwan; Kwon, Tae Hwan.

In: American Journal of Physiology - Renal Physiology, Vol. 304, No. 7, 10.06.2013, p. F958-F971.

Research output: Contribution to journalArticle

Park, Eui Jung ; Lim, Jung Suk ; Jung, Hyun Jun ; Kim, Eunjung ; Han, Ki Hwan ; Kwon, Tae Hwan. / The role of 70-kDa heat shock protein in dDAVP-induced AQP2 trafficking in kidney collecting duct cells. In: American Journal of Physiology - Renal Physiology. 2013 ; Vol. 304, No. 7. pp. F958-F971.
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AU - Han, Ki Hwan

AU - Kwon, Tae Hwan

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AB - 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.

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