Ageing reduces in vitro thick ascending limb viability

The medullary thick ascending limb (mTAL) of Henle controls the renal cortico‐medullary osmotic gradient. Suspensions of mTALs are an in vitro model to study metabolism, signalling and physiology of this segment. In several cell types, ageing decreases in vitro viability. However, is unknown whether ageing affects mTAL in vitro viability. We hypothesize that ageing decreases tubular viability in mTALs suspensions. To study this, we generated mTAL suspensions by collagenase digestion with a purity of 97%. Then, we measured body weight (BW) in 5‐week old and 15‐week old Wistar‐Kyoto rats. Tubular viability was assessed by trypan blue exclusion assay and tubules counted by light microscopy at 40X. Experiments were done in parallel by two different operators. We found that BW of 5‐week old rats was 116 ± 3 g. In contrast, BW of 15‐week old rats was 289 ± 8 g (P < 0.01), as shown in previous reports. Then we measured tubular viability in mTALs suspensions. We found that in vitro mTALs suspension from 5‐week old rats had 87 ± 2 % of viable tubules. In contrast, in vitro mTAL suspensions from 15‐week old rats had only 65 ± 4 % (P < 0.01). Therefore, mTAL suspensions from younger rats have more viable tubuli than older rats when prepared by this technique. We conclude that ageing decreases in vitro mTALs viability. Therefore, care should be taken when interpreting results of experiments based on mTAL suspensions from rats of undefined age.