Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus.

Sookkasem Khositseth,D Michael Payne,Komgrid Charngkaew,Panapat Uawithya,Trairak Pisitkun,Jason D Hoffert,Nattakan Thippamom,Robert A Fenton,Fahad Saeed,Shu-Hui Chen,Poorichaya Somparn

doi:10.1038/srep18311

Abstract

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

Highlights

When urine osmolality was measured in urine recovered from bladders of some of the potassium-deprived rats at 24 hours, lower urine osmolality was detected compared to the controls (Supplementary Fig. 1), suggesting that a urinary concentrating defect was present near the end of day 1 in those animals
Based on the ultrastructural analysis revealing the extensive presence of autophagic structures in inner medullary collecting duct (IMCD) cells of potassium deprived rats, we further investigated the potential role of autophagy in down-regulation of the proteins detected by proteomic analysis
In the present study, we devised a strategy to better understand the pathogenesis of this condition by (i) investigating changes that occur in the initial stage of hypokalemia-induced nephrogenic diabetes insipidus (NDI) and (ii) applying an unbiased discovery approach

Summary

Introduction

Potassium deprivation has been reported to induce urinary concentrating defects through alterations in abundances of various proteins, including NKCC2 in thick ascending limb cells as well as AQP2 and/or urea transporter proteins in inner medullary collecting duct cells[11,12,13,14]. While most studies of hypokalemia-induced NDI have focused on long-term effects (days to weeks) in the collecting duct, the onset of a urinary concentrating defect in potassium-deprived rats has been reported to be as early as 12–24 hours[13]. In an extension of our proteomic analysis, we employed both electron microscopy (EM) and immunofluorescence labeling of IMCD cells to identify the possible mechanism for the observed protein down-regulation Both of these analyses revealed that the decreased abundance of identified proteins was related to autophagic protein degradation.

Methods

Results

Conclusion