Long Term Regulation of Aquaporin-2 Expression in Vasopressin-responsive Renal Collecting Duct Principal Cells

Udo Hasler,David Mordasini,Marcelle Bens,Matthieu Bianchi,Françoise Cluzeaud,Martine Rousselot,Alain Vandewalle,Eric Féraille,Pierre-Yves Martin

doi:10.1074/jbc.m111880200

Abstract

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V(2) receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V(2) receptor agonist, produced the same effect as AVP, whereas the V(2) antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.

Highlights

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel
Endogenous AQP2 Expression of Untreated and AVP-treated Mouse Collecting Duct Principal Cells—The expression of AQP2 mRNA was analyzed by RNase protection assay (RPA) in confluent mpkCCDC14 cells
Compared with AVP alone, both of these compounds induced a similar rise in AQP2 protein levels, indicating that the action of AVP in mpkCCDC14 cells is mediated by cAMP. These results demonstrate, as observed in vivo, that AQP2 protein expression induced by AVP or dDAVP occurs via the occupancy of functional V2 receptors located in the basolateral membranes of mpkCCDC14 cells

Summary

Introduction

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. To establish further the involvement of V2 receptors in AVP-induced AQP2 expression, we assessed whether cAMP was able to mimic the AVP- or dDAVP-dependent increase of cellular AQP2 protein content by incubating cells 24 h with 51⁄710Ϫ6 M forskolin or with 10Ϫ3 8-bromo-cyclic AMP (Fig. 3D).

Results

Conclusion