EFFECT OF RAPAMYCIN ON MORPHOLOGICAL AND FUNCTIONAL PARAMETERS IN THE KIDNEY OF THE RABBIT (1995)

Rapamycin (RAPA)* is a macrolide antibiotic produced by Streptomyces hydroscopicus (1,2). The drug, initially isolated as an antifungal agent, has been shown to possess immunosuppressive activity with potency similar to FK506 and up to 100-fold greater than CsA (3). RAPA has shown considerable promise as an immunosuppressive agent in the prolongation of allograft survival (4). Its efficacy alone or in combination with CsA has been studied in vivo in a number of animal models (3–7). In addition to assessing efficacy, one must also address the question of potential adverse side effects induced by an immunosuppressant. Few studies have examined this issue, particularly in the case of renal dysfunction, which is one of the major complications of immunosuppressive therapy with CsA. In this regard, studies to date have been limited to the rat (3, 8, 9), which, when administered RAPA for 14 days, did not demonstrate changes in urine output, plasma creatinine, or creatinine clearance. Furthermore, histological examination of the kidneys failed to reveal any significant changes. To determine more definitively the potential renal side effects of RAPA, one should utilize more than a single species of animal. In view of this, the present study was designed to examine RAPA-induced renal side effects in the rabbit, an animal model chosen because of previous demonstration of its reliability (10, 11) in assessing CsA-induced, renal structural and functional changes similar to those seen in man. Twenty age-matched New Zealand White rabbits (2.5–3.0 kg) were housed in individual cages and randomly assigned to 4 groups of 5 animals each. The animals were fed Prolab rabbit formula (Agway Inc., Syracuse, NY) containing all essential vitamins and minerals, and water ad libitum for the duration of the study. The rabbits received RAPA (2.0 or 0.5 mg/kg), vehicle (1% w/w polysorbate 80 in phospho-50 PG/water), or saline orally daily for 60 days. The dosage levels of RAPA were selected to achieve drug concentrations in whole blood that result in prolongation of graft survival based on our previous work assessing the efficacy of the drug in a rabbit heterotropic heart transplant model (7). Animals were bled via the marginal ear vein on a weekly basis for determination of RAPA whole blood levels by high pressure chromatography, as described previously (12). At the end of the study period, blood pressure, glomerular filtration rate (GFR), renal blood flow (RBF), and serum creatinine levels were determined. To rule out opportunistic, pathogen-induced structural/functional changes in the kidney in an immune-suppressed host, as reported previously (11), sera were obtained for subsequent serological analysis (Institut Armand-Frappier, Laval, Quebec, Canada). Animals were anesthetized using an intramuscular injection of atrevet (1 mg/kg), xylazine (3 mg/kg), and ketamine (50 mg/kg). Polyethylene catheters were inserted into the common carotid (PE 60). Continuous blood pressure monitoring via the femoral artery was performed using a Stather blood pressure transducer (model P230C) attached to a polygraph recorder (Gross Instruments Co., Quincy, MA). For GFR determinations, a solution of normal saline containing 1.5% (w/v) inulin was administered via the femoral vein as a bolus of 2 ml over 2 min, followed by continuous infusion of 1.0 ml/min using a Harvard apparatus infusion/withdrawal pump. After a 30-min equalization period, 2 15-min urine collections were made, followed by a 2-ml collection of arterial blood via the femoral artery. Inulin concentrations in plasma and urine were determined by the anthrone colori-metric assay as described previously (10, 11). RBF was determined using 15-μm microspheres labeled with 85Sr (3 M Co., Inc., St. Paul, MN), injected, and analyzed according to the method of Bartrum et al. (13). Briefly, a 1-ml volume of the microsphere preparation sonicated in Tween 80 to prevent aggregation was injected into the left ventricle via the indwelling carotid catheter and blood was drawn simultaneously from the femoral artery and placed into counting tubes. One kidney per animal was then removed, weighed, and divided into several counting tubes so that the tissue was not more than 2 cm from the bottom of the tube. Tubes containing blood were centrifuged at low speed for 5 min to sediment the microspheres. The tubes containing blood and tissue were then counted in a gamma-counter. The counts for all tissue tubes were summed to give the total organ count. Serum creatinine concentration was determined by the alkaline picrate method (14) on a Beckman Astra analyzer (Beckman Instruments, Brea, CA). For morphological study, harvesting and processing of the remaining kidney for light microscopic assessment were carried out as reported previously (70). Kidneys were processed, and sections were stained with hematoxylin and eosin and scored semiquantitatively on a scale of 0–4+ (absent, minimal, mild, moderate, or severe) for tubule atrophy and interstitial fibrosis, as described previously (10). Additional sections were stained with periodic acid-Schiff stain for the detection of arteriopathy. Tissue sections were examined without foreknowledge of their source. Statistical assessment of the data obtained was carried out using analysis of variance, and Duncan's multiple-range test and chi-square analyses, where appropriate. A P-value less than 0.05 was considered to be statistically significant.