Rat Renal Proximal Tubules, Hypoxia, lonomycin, and Calpain

The preparation of freshly isolated rat renal proximal tubules in suspension has been utilized by our laboratory for more than 10 yr and reported in numerous publications (1 –10 ). This method of tubule isolation by collagenase digestion and Percoll centrifugation has recently been used by us for the study of calpain activity in this tissue (11 –13 ). Briefly, the kidneys of two male Sprague-Dawley rats are perfused, and the cortices are digested with collagenase and hyaluronidase. After washing and filtering, proximal tubules are separated from other nephron segments by centrifugation on a Percoll gradient. The preparation has a purity of more than 95% proximal tubules. The tubule suspension is aliquotted into Erlenmeyer flasks and gassed with 95% O₂ 15% CO₂ . At this time, release of lactate dehydrogenase (LDH) activity into the incubation medium, which is used as an index of cell membrane integrity, consistently averages 10% or less of the total LDH activity of the whole preparation, and remains at this level under oxygenated conditions for the duration of atypical experiment (15–20 min). Hypoxia is achieved by gassing the suspension with 95% N₂ / 5% CO₂ , which results in a 2–3-fold increase in LDH release over 15 min. We have demonstrated that hypoxia results in a prelethal increase in free cytosolic Ca²⁺ in this model (14 ). Further, the Ca²⁺ ionophore, ionomycin, added to normoxic tubules, elicits a dose-dependent increase in free cytosolic Ca²⁺ (14 ), which permits the study of proximal tubules with a known free cytosolic [Ca²⁺ ]. Thus, hypoxia and ionomycin-induced proximal tubular injury provide excellent models in which to observe the effects of increased free cytosolic Ca²⁺ on membrane damage as well as the effects of cytoprotec tive agents on Ca²⁺ -mediated events. In this model of rat renal proximal tubules, we have demonstrated that calpain inhibitors attenuate the increase in calpain activity during both hypoxia and ionomycin treatment and protect against cell membrane injury (11 )