A NIMAL PROTOCOLS AND PROCEDURES

Animals. All animal protocols were approved by the University of Colorado Committee on Animal Research, and animal care was in accordance with the National Institutes of Health guidelines for ethical animal research (NIH publication No. 80-123).

All animals were housed in cages in a temperature and light controlled environment with free access to tap water and food ad libitum. All animal experiments and dosing were performed after at least two weeks of acclimatization.

4.5.1 Animal model for the quality control of conservation solutions

All surgical procedures were supervised or carried out by a board-certified surgeon, Volker Schmitz, MD. Experimental Groups. Ten- to fourteen-week old inbred male Lewis rats, weighing 300 to 350 were obtained from Charles River Laboratories (Wilmington, MA). The following groups were studied:

(I) Kidney transplantation after donor kidneys were flushed with 20mL of ice-cold HTK (Custodiol Köhler, Chemie GmbH, Alsbach, Germany), reflushed with 10mL UW (ViaSpan, DuPont Pharmaceuticals, Wilmington, DE) 30 minutes after explantation and stored in UW solution at 4º Celsius for 16 hours,

(II) donor kidney flushed with UW (20mL) and stored in UW for 16 hours, (III) donor kidney flushed with HTK (20mL) and stored in HTK for 16 hours;

(IV-VI) same preservation solution protocol as (I) to (III) but cold storage time extended to 24 hours.

4.5.2 Animal models: Model A-1 and A-2 (non-transplant, IS treatment)

Model A-1. Non-transplant model for the evaluation of drug nephrotoxicity of cyclosporine and sirolimus alone and in combination. Fifty-four male Wistar rats were randomly assigned to nine treatment groups (n=6/group):

(I) vehicle controls (skim milk for 6 and 28 days) (II) cyclosporine 10mg/kg/day for 6 days

(III) cyclosporine 25mg/kg/day for 6 days (IV) sirolimus 1mg/kg/day for 6 days

(V) cyclosporine 10mg/kg/day + sirolimus 1mg/kg/day for 6 days (VI) cyclosporine 25mg/kg/day + sirolimus 1mg/kg/day for 6 days (VII) cyclosporine 10mg/kg/day for 28 days

(VIII) sirolimus 1mg/kg/day for 28 days

(IX) cyclosporine 10mg/kg/day + sirolimus 1mg/kg/day for 28 days

Study drugs were administered by oral gavage. All drugs were the commercial formulation as approved by the US Federal Drug Administration for clinical use and were diluted in skim milk as appropriate. The volume of the oral gavage did never exceed 1 mL. All doses of the study drugs were based on systematic dose finding studies during the development of the rat model in order to achieve drug blood concentrations similar to those in patients. Twenty-five mg/kg/day cyclosporine was not included when long-term treatment effects were studied since a pilot study had shown that this dose in combination with 1mg/kg/day sirolimus was associated with a significant mortality of more than 50%.

Before the last day of treatment (6 or 28), rats were placed in metabolic cages for 24h-urine collections, then on the final day, two hours after receiving the final drug doses, animals were prepared for clearance measurements as described below (under Renal Function).

Model A-2. Non-transplant model for drug nephrotoxicity for tacrolimus and its combination with sirolimus. An additional twenty-four Wistar rats were randomly assigned to four treatment groups (n=6/group):

(I) vehicle controls (skim milk for 6/28 days) (X) tacrolimus1mg/kg/day for 6 days

Materials and Methods (XI) tacrolimus 1mg/kg/day + sirolimus 1mg/kg/day for 6 days

(XII) tacrolimus1mg/kg/day for 28 days

(XIII) tacrolimus 1mg/kg/day + sirolimus 1mg/kg/day for 6 days

Again, study drugs were administered by oral gavage. All drugs were the commercial formulation as approved by the US Federal Drug Administration for clinical use and were diluted in skim milk as appropriate. The volume of the oral gavage did never exceed 1 mL. As in Model A-1, all doses of the study drugs were chosen to yield drug blood concentrations similar to those in patients. These rats were also placed in metabolic cages for 24h-urine collections, before the last day of treatment (6 or 28). On the final day, two hours after receiving the final drug doses, animals were prepared for glomerular filtration rate measurements as described below (under Renal Function).

4.5.3 Animal models: Model B (transplanted rats, IS treatment)

Model B. Transplantation Model. Ten- to fourteen- week old inbred male Lewis rats, weighing 300 to 350 were obtained from Charles River Laboratories (Wilmington, MA).

Orthotopic rat kidney transplantation involved a donor animal that was sacrificed at the time of kidney harvest, and a recipient animal. Since inbred Lewis rats are genetically almost identical, organs can be transplanted without the risk of an immune reaction against the allograft and without the need for immunosuppression. Briefly, donor animals were anesthetized with isoflurane, and the left kidney was exposed through a midline incision. Following mobilization of the left kidney, the suprarenal aorta was clamped and an arterial flush catheter was placed in the abdominal aorta. After cutting the left renal vein, and pressure perfusion with UW solution, the aorta was cut above and below the renal artery to provide an aortic patch, and the kidney was removed and placed in either cold UW thereafter and kept at 4° C for 6 hours before transplantation. The recipient animal was anesthetized with isoflurane and placed on a warming surgical table for the procedure. Body temperature was monitored via a rectal probe. Through a midline incision, the left native kidney with its vessels was exposed. The aorta was clamped above and below the renal artery. A mini-bulldog clamp was placed on the renal vein adjacent to the vena cava. Renal vein, artery and ureter were cut and the kidney was removed. A longitudinal incision was applied to the recipient aorta, which matched the

length of the aortic patch on the donor kidney. For the renal artery, an end-to-side anastomosis between the donor vessels (using the aortic patch) and the recipient aorta was done (10-0 suture). The renal vein was anastomozed in an end-to-end fashion (8-0 suture). All anastomoses were completed with hand-sewn sutures. The donor ureter was anastomozed in an end-to-end fashion to the recipient's ureter as well using 6 to 8 single stitches (10-0 suture). After removal of the right recipient kidney and administration of 5 cc normal saline into the abdominal cavity, the abdomen was closed. Vascular anastomoses time was comparable in all groups (20±5 min). All animals were allowed to recover under a warming light thereafter. As aforementioned, the surgical procedures were carried out by Dr. V. Schmitz.

Experimental Groups: Thirty-six rats were assigned randomly to six groups, which were all treated for seven days. Groups II to VI all underwent unilateral orthotopic kidney transplantations (see below):

(I) group 1 (control) did not undergo kidney transplantation and received vehicle (skim milk) treatment only,

(II) transplantation but vehicle only,

(III) cyclosporine (25mg/kg/day) and sirolimus (1mg/kg/day), (IV) cyclosporine (10mg/kg/day) and sirolimus (1mg/kg/day), (V) sirolimus (1mg/kg/day) and

(VI) cyclosporine (10mg/kg/day).

The doses chosen in this study were based on the results of previous experiments that had shown adequate blood levels comparable to those found in human transplantations. 24 hours before the final day, after receiving dosing, rats were placed in metabolic cages for 24h urine collections. On day 7, four hours after receiving the final drug doses, animals were sacrificed. Plasma was analyzed for creatinine, blood urea nitrogen (BUN) in our hospital affiliated clinical laboratory using established and validated assays.