*2.5. Intravenous and Oral Administration of Tofacitinib*

For oral and intravenous administration, pretreatment and surgical procedures were performed as previously reported [12]. For oral study, the rats were restricted from eating food overnight but water was freely accessible. The next day, after anesthesia with ketamine at a dose of 100 mg/kg, polyethylene 50 tubes (Clay Adams, Parsippany, NJ, USA) was cannulated into the carotid artery for blood collection. For intravenous study, polyethylene 50 tubes were cannulated into the carotid artery and jugular vein for blood collection and drug administration, respectively. After surgery, rats were allowed to rest for 2–3 h to recover from anesthesia and were free in the individual metabolic cage during the experiment.

For intravenous administration, tofacitinib (dissolved in 0.9% NaCl-injectable solution containing 0.5% β-cyclodextrin) was infused for 1 min at a dose of 10 mg/kg via the jugular veins of control (*n* = 6), G-ARF (*n* = 8), and C-ARF (*n* = 7) rats. Blood samples (110 µL) were collected through the carotid artery at 0 (prior to drug infusion), 1 (at the end of drug infusion), 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 600, and 720 min and were centrifuged at 8000 × *g* for 1 min. Plasma samples were collected and immediately stored in a −80 ◦C freezer until HPLC analysis of tofacitinib could be performed [22]. After collecting each blood sample, 0.3 mL of heparinized 0.9% NaCl-injectable solution (10 IU/mL) was immediately administered to the carotid artery to prevent blood clotting. At 24 h, the rat's abdomen was open and the entire gastrointestinal tract was removed, transferred to a beaker with 50 mL of methanol, and cut into small pieces. After mixing the contents in the beaker thoroughly, two 100 µL aliquots of supernatant were taken and stored at −80 ◦C until HPLC analysis of tofacitinib could be performed [22].

At 24 h after drug administration, urine samples were also collected. The metabolic cage was rinsed with 20 mL of distilled water, which was combined with the 24-h urine sample. The volume of combined urine sample was measured and two 100 µL aliquots of each combined sample were taken and stored in the −80 ◦C freezer until tofacitinib analysis by HPLC could be performed [22].

For oral administration, tofacitinib at a dose of 20 mg/kg was administered to control (*n* = 8), G-ARF (*n* = 6), and C-ARF (*n* = 8) rats. Blood samples (110 µL) were collected through the carotid artery at 0 (prior to drug administration), 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 600, and 720 min. At 24 h after drug administration, urine and gastrointestinal tract samples were collected and handled similarly to those in the intravenous study.
