Abstract

Lithium chloride (LiCl) is a widely used drug for the treatment of bipolar disorders, but as a side effect, 40% of the patients develop diabetes insipidus. LiCl affects the activity of the glycogen synthase kinase 3 (GSK3), and mice deficient for GSK3β showed a reduction in the urine concentration capability. The cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct cells to analyze the underlying mechanisms. LiCl and the inhibitor of GSK3 (SB216763) induced a decrease in the aquaporin-2 (Aqp2) protein level. LiCl induced downregulation of Aqp2 mRNA expression while SB216763 had no effect and TWS119 led to increase in expression. The inhibition of the lysosomal activity with bafilomycin or chloroquine prevented both LiCl- and SB216763-mediated downregulation of Aqp2 protein expression. Bafilomycin and chloroquine induced the accumulation of Aqp2 in lysosomal structures, which was prevented in cells treated with dibutyryl cyclic adenosine monophosphate (dbcAMP), which led to phosphorylation and membrane localization of Aqp2. Downregulation of Aqp2 was also evident when LiCl was applied together with dbcAMP, and dbcAMP prevented the SB216763-induced downregulation. We showed that LiCl and SB216763 induce downregulation of Aqp2 via different mechanisms. While LiCl also affected the mRNA level, SB216763 induced lysosmal degradation. Specific GSK3β inhibition had an opposite effect, indicating a more complex regulatory mechanism.

Highlights

  • In the renal collecting duct, the final urine concentration is predominantly regulated by the action of the antidiuretic hormone vasopressin (AVP), which is secreted by the neuronal lobule of the pituitary gland [1]

  • ANOVA analysis with Tukey post-test to compare the AQP2 columns, * indicates statistically significant differences compared to untreated cells (p ≤ 0.05); n = 3

  • To test the cellular mechanisms that lead to downregulation of Aqp2 mRNA induced by LiCl and SB216763, we examined the expression of proteins and genes that are used as clear target genes of tonicity-responsive enhancer binding protein (TonEBP), like BGT-1 and aldose reductase (AR) [25]

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Summary

Introduction

In the renal collecting duct, the final urine concentration is predominantly regulated by the action of the antidiuretic hormone vasopressin (AVP), which is secreted by the neuronal lobule of the pituitary gland [1]. The binding of AVP to the vasopressin type 2 receptor (V2 R) at the basolateral membrane of the collecting duct’s principal cells leads to, via an activation of a Gs -protein, the stimulation of the adenylyl cyclase and an increase of the cyclic adenosine monophosphate (cAMP) level [2] This activates the protein kinase A (PKA), which in turn phosphorylates the water channel aquaporin-2. Water can enter the cell along an osmotic gradient and leaves the cell on the basolateral site facilitated by aquaporin-3 (Aqp3) and aquaporin-4 (Aqp4) [4] Defects in this pathway often lead to a urine concentration deficiency, a disorder called diabetes insipidus (DI) [5]. This disorder can be inherited, either when specific mutations lead to a defect in

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