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

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.

Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus

“…Similarly, an abolishment of autophagic degradation of AQP2 and recovery of normal phenotype were observed after normalization of serum potassium level in our previous study. 12 These findings support an important role for autophagy in the mechanism of hypercalcemia-induced NDI (Figure 11).…”

“…cAMP, cyclic adenosine monophosphate; CaSR, calcium-sensing receptor; PKA, protein kinase A. of AQP2 in autophagosomes prevents the development of NDI. For the multiple reasons previously stated in our hypokalemia study, 12 such specific inhibition in our animal model system would be highly problematic. One practical approach that we used to address this issue was a PTH withdrawal experiment.…”

“…Significantly, the results summarized above for hypercalcemia-induced NDI were qualitatively comparable to our results recently described for NDI induced by hypokalemia, in which activation of autophagy (especially of AQP2, cortactin, and other cell junction proteins) was also observed. 12 In our previous study of hypokalemic rats, the presence of damaged mitochondria and mitophagy in IMCD led us to hypothesize that intracellular K þ depletion may alter mitochondrial K þ balance (via mitochondrial K þ channels), leading to decreased adenosine triphosphate (ATP) production and increased generation of reactive oxygen species followed by mitophagy. 12 In contrast to hypokalemia, the early events in hypercalcemia-induced NDI did not include gross mitochondrial damage or mitophagy (as detected by electron microscopy analysis), and only a few mitochondrial membrane proteins were downregulated (as detected by proteomic analysis).…”

“…12 In our previous study of hypokalemic rats, the presence of damaged mitochondria and mitophagy in IMCD led us to hypothesize that intracellular K þ depletion may alter mitochondrial K þ balance (via mitochondrial K þ channels), leading to decreased adenosine triphosphate (ATP) production and increased generation of reactive oxygen species followed by mitophagy. 12 In contrast to hypokalemia, the early events in hypercalcemia-induced NDI did not include gross mitochondrial damage or mitophagy (as detected by electron microscopy analysis), and only a few mitochondrial membrane proteins were downregulated (as detected by proteomic analysis). In addition, immunofluorescence analysis for one of these downregulated mitochondrial membrane proteins, Atp5a, exhibited only limited colocalization with ATG12 (an autophagosome marker).…”

“…These findings support our recent study indicating a major role for autophagy as an early event in the development of NDI resulting from hypokalemia, another common electrolyte imbalance. 12…”