Published: June 21st, 2013
ERRATUM NOTICEImportant: There has been an erratum issued for this article. Read more …
Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.
Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.
In renal collecting duct principal cells, arginine-vasopressin (AVP) controls water reabsorption by stimulating the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane. AVP binds to the G protein-coupled vasopressin type-2 receptor (V2R) stimulating adenylyl cyclase and thereby cAMP formation. Initiation of this signaling cascade leads to activation of protein kinase A (PKA). PKA phosphorylates AQP2 at serine 256 (S256), which is the key trigger for its redistribution from intracellular vesicles into the plasma membrane. The membrane insertion facilitates water reabsorption along an osmotic gradient and fine-tunes body water homeostasis.
The successful cultivation of primary rat IMCD cells will result in a confluent monolayer 6-8 days after seeding (Figure 2). Per 60 mm culture dish there are approximately 6 x 106 cells. The cells tightly adhere to the culture dishes, as these were coated with collagen type IV, a basement membrane component18. Therefore, IMCD cells will not detach even during several thorough washing procedures. Up to 80% of the cultured cells express endogenously V2R and AQP2. These are the princip.......
We present a detailed protocol for the preparation and culturing of primary rat IMCD cells. The approach yields up to 21 cm2 of cells from one rat20. The experiment requires standard cell culture equipment and can be carried out by a single person within approximately 6 hr. Therefore, this approach is suitable as a standard laboratory method.
Primary rat IMCD cells can be seeded in culture dishes of different size, ranging from 96 well plates to 60 mm dishes. However, for.......
|Collagen Type IV Mouse
|Collagenase type CLS-II
|DMEM + GlutMAX
|Non essential amino acids (NEA)
ErratumErratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells
A correction was made to Culturing Primary Rat Inner Medullary Collecting Duct Cells. There was an error with an author's name. The author's last name had a typo and was corrected to:
Copyright © 2024 MyJoVE Corporation. All rights reserved