Quick isolation of rat medullary thick ascending limbs

Publisher Website
Google Scholar
Abstract
This paper describes a rapid and simple method for isolation of medullary thick ascending limbs (MTAL) from rat kidney. The technique takes advantage of the fact that MTAL represents a high fraction of the inner stripe (IS) tissue in the outer medulla, and that this nephron segment is more resistant than others to mechanical and enzymatic disruption. Special attention was given in the design of each step of the isolation procedure in order to improve purity and yield of the preparation. Major steps are the following: 1) careful dissection of the IS; 2) cutting IS tissue into small pieces of regular size (≈ 1 mm3); 3) mild and brief enzymatic hydrolysis in a 65 U/ml collagenase solution; 4) separation of long MTAL segments from other tubule fragments and cells, and washing of the collagenase solution, on a nylon sieve (100 μm opening). This technique does not require lengthy centrifugations and provides about 6 mg fresh tisue (=1 mg protein) from two rat kidneys in 2 h. Light microscopy and transmission electron microscopy show a good purity (at least 95%) and good preservation of TAL ultrastructural morphology. Adenylate cyclase responsiveness to arginine-vasopressin (AVP), glucagon (GLU) and salmon calcitonin (SCT) of the MTAL suspension is similar to that reported for single microdissected rat MTAL. Viability of the MTALs was demonstrated by the ability to accumulate cyclic AMP in presence of AVP, GLU, SCT and forskolin. Normal oxygen consumption was 45.1±2.4 (SEM) μl·mg protein−1·h−1 (n=8). It was not enhanced with addition of succinate, indicating that integrity of cellular membrane is preserved. It was decreased by half in presence of either furosemide or ouabain. This indicates that electrolyte transport is maintained in the MTAL tubule suspension. This technique represents an easy and quick way in obtaining milligrams of medullary thick ascending limb from small laboratory animals, with a high purity and viability, and maintenance of cell polarity.