Glycogen Synthase Kinase-3 Modulates Hyperosmotic-Induced Urea Transporter A1 Relocation in the Inner Medullary Collecting Duct Cells

Abstract

Aim: Glycogen synthase kinase 3 (GSK3) regulates urine concentration by mediating the vasopressin-induced aquaporin 2 expression and water permeability, although it is unknown whether GSK3 also mediates the accumulation of the urea transporter A1 (UT-A1). The aim of this study is to investigate the effect of GSK3 on UT-A1 distribution. Methods: Mouse inner medullary collecting duct 3 cells were transfected with UT-A1-GFP construct. The stable transfected cells were cultured under hypertonic conditions, treated with GSK3 inhibitor lithium chloride, GSK3 activator, lysosome or proteasome inhibitor. The expression levels of UT-A1, GSK3, and phospho-GSK3 were analyzed using western blot. The interaction between UT-A1 and the Golgi apparatus was examined using confocal immunofluorescence microscope. The UT-A1 trafficking was examined using the biotinylation of surface membranes. Results: UT-A1 dissociated away from the Golgi apparatus and translocated to the plasma membrane under hypertonic-NaCl and NaCl plus urea stimulation. This movement was accompanied by the increased phosphorylation of GSK3 and its localization on the cellular membrane. Moreover, these results were duplicated by treating the cells with the GSK3 inhibitor, and by contrast, were partially reversed by the GSK3 activator. Treating cells with a lysosome or proteasome inhibitor failed to attenuate the effects of hypertonic stimulus, indicating that the loss of UT-A1 from the Golgi was not due to degradation. Conclusion: Our results suggest that GSK3 may in part modulate the hypertonic-induced intracellular UT-A1 redistribution and its accumulation on the plasma membrane, which may constitute another mechanism by which GSK3 modulates urine concentration.

Berberine ameliorates lipopolysaccharide‑induced inflammatory responses in mouse inner medullary collecting duct‑3 cells by downregulation of NF‑κB pathway

“…Collecting duct system of the kidney is a renal tubular segment and plays a role in the reabsorption of filtered water (~15%). A key feature of the collecting duct is water reabsorption through hormonal regulation in an autocrine/paracrine manner (32)(33)(34)(35)(36)(37)(38)(39)(40). For instance, vasopressin, also called as antidiuretic hormone (adH), affects osmotic water permeability and increases water reabsorption in IMCD cells (35).…”

“…A key feature of the collecting duct is water reabsorption through hormonal regulation in an autocrine/paracrine manner (32)(33)(34)(35)(36)(37)(38)(39)(40). For instance, vasopressin, also called as antidiuretic hormone (adH), affects osmotic water permeability and increases water reabsorption in IMCD cells (35). Hence, most reports of IMCD have been in the context of regulation of water homeostasis such as osmotic water, urea, and electrolyte permeability (35,(41)(42)(43)(44).…”

“…For instance, vasopressin, also called as antidiuretic hormone (adH), affects osmotic water permeability and increases water reabsorption in IMCD cells (35). Hence, most reports of IMCD have been in the context of regulation of water homeostasis such as osmotic water, urea, and electrolyte permeability (35,(41)(42)(43)(44). However, because many renal diseases are accompanied by inflammatory mediator production as well as water imbalance (45), it is very important to identify the underlying patho-physiology of inflammatory responses in renal diseases.…”