Effects of hyperosmolality on expression of urea transporter A2 and aquaporin 2 in mouse medullary collecting duct cells

Abstract

In this study, the effects of hyperosmolality on the expression of urea transporter A2 (UTA2) and aquaporin 2 (AQP2) were investigated in transfected immortalized mouse medullary collecting duct (mIMCD3) cell line. AQP2-GFP-pCMV6 and UTA2-GFP-pCMV6 plasmids were stably transfected into mIMCD3 cells respectively. Transfected mIMCD3 and control cells were cultured in different hypertonic media, which were made by NaCl alone, urea alone, or an equiosmolar mixture of NaCl and urea. The mRNA and protein expression of AQP2 was elevated by the stimulation of NaCl alone, urea alone and NaCl plus urea in AQP2-mIMCD3 cells; whereas NaCl alone and NaCl plus urea rather than urea alone increased the mRNA and protein expression of UTA2 in UTA2-mIMCD3 cells, and all the expression presented an osmolality-dependent manner. Moreover, the mRNA and protein expression of UTA2 rather than AQP2 was found to be synergistically up-regulated by a combination of NaCl and urea in mIMCD3 cells. It is concluded that NaCl and urea synergistically induce the expression of UTA2 rather than AQP2 in mIMCD3 cells, and hyperosmolality probably mediates the expression of AQP2 and UTA2 through different mechanisms.

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

“…Previous studies have shown that acute stimulation using hypertonic NaCl or sucrose but not urea could regulate the distribution of UT-A1 [1,2]. Our study also showed that the distribution of UT-A1 could be induced by NaCl rather than urea alone.…”

“…Transfection of mIMCD 3 cells with UT-A1-GFP construct was performed using Lipofectamine plus according to the manufacturer's protocols [2]. Twenty-four hours after transfection and subsequently every 3-4 days, the cells were replenished with medium containing 400 μg/ml geneticin.…”

“…Cells were cultured in UT-A1-mIMCD 3 culture medium, referred to as iso medium containing 120 mmol/l NaCl and 300 mOsm/kg H 2 O osmolality [2]. Then, cells were treated with hyperosmolar solutions containing 550 or 800 mOsm/kg H 2 O for 1 h. Medium components used in acute adaptation were 250 mOsm/kg H 2 O (125 mmol/l) NaCl, 250 mOsm/kg H 2 O (250 mmol/l) urea, or 125 mOsm/kg H 2 O (62.5 mmol/l) NaCl plus 125 mOsm/kg H 2 O (125 mmol/l) urea, by adding the desired volume of solute stock solutions of 2.5 mol/l NaCl or 5 mol/l urea prepared in iso medium and filter sterilized.…”

“…The following antibodies were used: GFP, UT-A1, phospho-GSK3 or GAPDH. Western immunoblot assays were performed as previously described [2]. Band intensity was quantified with the ImageJ program (NIH, Bethesda, Md., USA).…”

“…This action, together with the vasopressin-regulated aquaporins (AQP), allows water reabsorption across the IMCD. The cells in the inner medulla are highly specialized and are effectors that not only concentrate the urine but also regulate the widely fluctuating NaCl and urea concentrations [1,2]. A number of studies have reported the accumulation of UT-A1 in the plasma membrane in response to hyperosmolar solutions containing NaCl or sucrose [1] and that hyperosmolality could increase urea permeability in perfused rat terminal IMCDs [3].…”