Culturing Primary Rat Inner Medullary Collecting Duct Cells

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Preparation

1. Preparing the culture dishes
   1. Thaw Collagen Type IV O/N at 4 °C and dissolve it in 0.1% sterile acetic acid. The volume to use depends on the type and number of dishes in which the cells are to be seeded. Use 2 μg/cm².
   2. Incubate dishes at least for 1 hr at RT and wash twice with distilled water (A. tridest).
   3. Allow the dishes to dry properly.
2. Supplementation of medium
   1. Increase the glucose level up to 4.5 g/L by adding 1.75 g glucose to 500 ml medium. To adjust the medium to 600 mosmol, add 100 mM NaCl and 100 mM urea, to increase the osmolarity by 200 mosmol....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagen Type IV Mouse	BD Biosciences	356233
Hyaluronidase	SIGMA	H6254
Collagenase type CLS-II	Biochrom AG	C2-22
DMEM + GlutMAX	Invitrogen GIBCO	21885108
Nystatin	SIGMA	N4014
Ultroser G	Cytogen	15950-017
Non essential amino acids (NEA)	Biochrom AG	K0293
Gentamicin	Invitrogen GIBCO	15710
DBcAMP	BIOLOG	D009