Thirsting/Dehydration Protocol
Objective: To examine the effects of water deprivation on aquaporin-2 (AQP2) expression and urinary osmolality in lithium-treated rats
Gather these items before starting the experiment. Check off items as you prepare.
Equipment6
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Materials1
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Protocol Steps
Lithium treatment
Rats were treated with lithium for 10 to 25 days prior to thirsting protocol
Note: This treatment precedes the thirsting protocol and results in reduced AQP2 expression
View evidence from paper
“lithium treatment reduced AQP2 expression dramatically, to 31 +/- 8% after 10 d and to 4 +/- 1% after 25 d”
Water deprivation (thirsting)
Rats were subjected to water deprivation while continuing lithium treatment
Note: Thirsting was performed in the continued presence of lithium treatment
View evidence from paper
“2 d of thirsting or 7 d of dDAVP treatment, in the continued presence of lithium, increased AQP2 expression by six- and threefold”
Kidney tissue collection
One kidney from each rat was used for membrane preparation from inner medulla; contralateral kidney was fixed for immunofluorescence and immunoelectronmicroscopy
Note: Tissues were collected after the thirsting period
View evidence from paper
“Membranes were prepared from inner medulla of one kidney from each rat, while the contralateral one was fixed for immunofluorescence and immunoelectronmicroscopy”
Immunoblotting analysis
Membrane samples were analyzed using immunoblotting to measure AQP2 expression levels
Note: Results showed sixfold increase in AQP2 expression after thirsting
View evidence from paper
“Immunoblotting revealed that lithium treatment reduced AQP2 expression dramatically”
Immunofluorescence analysis
Fixed kidney tissue was analyzed using immunofluorescence to visualize AQP2 localization
Note: Thirsting increased AQP2 immunolabeling mainly of vesicles
View evidence from paper
“Immunofluorescence and immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression”
Immunogold quantitation
Quantitative analysis of AQP2 particles per micrometer squared using immunogold labeling
Note: Measured AQP2 expression changes from control to lithium-treated conditions
View evidence from paper
“Immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression (from 11.2 +/- 1.0 to 1.1 +/- 0.2 particles/microns 2)”
Urinary osmolality measurement
Urine was collected and analyzed for osmolality changes following thirsting
Note: Increased urinary osmolality was observed coincident with increased AQP2 expression
View evidence from paper
“coincident with increased urinary osmolality”