Source Paper
Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla.
D Marples, S Christensen, E I Christensen, P D Ottosen, S Nielsen
Journal of Clinical Investigation • 1995
Source Paper
D Marples, S Christensen, E I Christensen, P D Ottosen, S Nielsen
Journal of Clinical Investigation • 1995
Lithium, a widely used treatment for bipolar affective disorders, often causes nephrogenic diabetes insipidus. The effect of chronic lithium therapy on the expression of the vasopressin-regulated water channel Aquaporin-2 (AQP2) in rat kidney was examined. Membranes were prepared from inner medulla of one kidney from each rat, while the contralateral one was fixed for immunofluorescence and immunoelectronmicroscopy. Immunoblotting revealed that lithium treatment reduced AQP2 expression dramatically, to 31 +/- 8% after 10 d and to 4 +/- 1% after 25 d, coincident with development of severe polyuria. Immunofluorescence and immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression (from 11.2 +/- 1.0 to 1.1 +/- 0.2 particles/microns 2). The downregulation was only partly reversed by return to lithium-free diet for 1 wk (40 +/- 8% of control). Furthermore, immunoblotting and immunogold quantitation revealed that 2 d of thirsting or 7 d of dDAVP treatment, in the continued presence of lithium, increased AQP2 expression by six- and threefold, respectively, coincident with increased urinary osmolality. Thirsting increased AQP2 immunolabeling mainly of vesicles, whereas dDAVP caused accumulation of AQP2 predominantly in the subapical region and plasma membrane. Thus, lithium causes marked downregulation of AQP2 expression, only partially reversed by cessation of therapy, thirsting or dDAVP treatment, consistent with clinical observations of slow recovery from lithium-induced urinary concentrating defects.
Objective: Examine the effect of chronic lithium therapy on aquaporin-2 (AQP2) water channel expression in rat kidney medulla and the development of polyuria
Gather these items before starting the experiment. Check off items as you prepare.
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Begin chronic lithium therapy in rats to induce nephrogenic diabetes insipidus and examine effects on AQP2 expression
Note: Two timepoints examined: 10 days and 25 days of lithium treatment
“The effect of chronic lithium therapy on the expression of the vasopressin-regulated water channel Aquaporin-2 (AQP2) in rat kidney was examined”
Collect both kidneys from each rat - one for membrane preparation and one for immunofluorescence and immunoelectronmicroscopy analysis
Note: Bilateral sampling allows for parallel analysis of protein expression and tissue localization
“Membranes were prepared from inner medulla of one kidney from each rat, while the contralateral one was fixed for immunofluorescence and immunoelectronmicroscopy”
Isolate membranes from the inner medulla of one kidney for immunoblotting analysis
Note: Specific to inner medulla region
“Membranes were prepared from inner medulla of one kidney from each rat”
Conduct immunoblotting analysis to measure AQP2 expression levels in kidney membranes at different timepoints
Note: Measurements taken at 10 days and 25 days of lithium treatment
“Immunoblotting revealed that lithium treatment reduced AQP2 expression dramatically, to 31 +/- 8% after 10 d and to 4 +/- 1% after 25 d”
Conduct immunofluorescence microscopy on fixed kidney tissue to visualize and confirm AQP2 expression changes
Note: Performed on contralateral kidney fixed tissue
“Immunofluorescence and immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression (from 11.2 +/- 1.0 to 1.1 +/- 0.2 particles/microns 2)”
Conduct immunoelectronmicroscopy with immunogold quantitation to measure AQP2 particle density and subcellular localization
Note: Quantitative analysis of particle density per unit area
“Immunofluorescence and immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression (from 11.2 +/- 1.0 to 1.1 +/- 0.2 particles/microns 2)”
Discontinue lithium treatment and return rats to lithium-free diet to examine reversibility of AQP2 downregulation
Note: Partial reversal of AQP2 downregulation observed
“The downregulation was only partly reversed by return to lithium-free diet for 1 wk (40 +/- 8% of control)”
Subject rats to 2 days of water deprivation while continuing lithium treatment to examine AQP2 expression changes
Note: Increased AQP2 expression primarily in vesicles
“2 d of thirsting or 7 d of dDAVP treatment, in the continued presence of lithium, increased AQP2 expression by six- and threefold, respectively”
Administer dDAVP (vasopressin analog) for 7 days while continuing lithium treatment to examine AQP2 expression and localization
Note: Caused accumulation of AQP2 predominantly in subapical region and plasma membrane
“7 d of dDAVP treatment, in the continued presence of lithium, increased AQP2 expression by threefold, respectively, coincident with increased urinary osmolality”
Assess urinary osmolality as a functional measure of kidney concentrating ability in response to treatments
Note: Increased urinary osmolality observed with thirsting and dDAVP treatment
“coincident with increased urinary osmolality”
Determine subcellular distribution of AQP2 protein in response to different treatments using immunofluorescence and immunoelectronmicroscopy
Note: Thirsting increased AQP2 mainly in vesicles; dDAVP caused accumulation in subapical region and plasma membrane
“Thirsting increased AQP2 immunolabeling mainly of vesicles, whereas dDAVP caused accumulation of AQP2 predominantly in the subapical region and plasma membrane”
Bilateral kidney sampling performed - one kidney used for membrane preparation, contralateral kidney used for immunofluorescence and immunoelectronmicroscopy