Study design
This is a prospective multicenter paired study including three kidney transplant institutions (The Third Affiliated Hospital of Sun Yat-sen University, Zhujiang Hospital of Southern Medical University, and The Second Affiliated Hospital of Guangzhou Traditional Chinese Medicine University). The study protocol was approved by the three institutions’ Ethics Committee of Human Study, which was established according to the Operational Guidelines for Ethics Committees that Review Biomedical Research developed by the World Health Organization (WHO) [15].
No organs from executed prisoners were used in the study, and the kidneys from all donors were procured in accordance with the WHO principles, Declaration of Helsinki, and Istanbul Declaration [16, 17].
Donors were selected based on confirmed patient identity, age ≤ 65 years, no history of kidney disease, drug abuse or uncontrollable psychotic symptoms, no active infection including HIV, bacteria or fungus, no history of uncontrolled hypertension, diabetes mellitus with complications, no history of malignant melanoma, metastatic or incurable malignancy [18].
Organ donation and recovery was facilitated by organ procurement organizations in the three kidney transplant institutions, which were established by the National Health and Family Planning Commission of China. Before procurement, written informed consent was obtained from the donor’s immediate family, who agreed to withdraw life support and donate the kidney. The obtained consent was provided to the Organ Donation Committee, which supervised the donation process. Organ procurement and management was strictly processed according to the national guidelines for donation after cardiac death in China [18].
Participants
Based on preliminary studies [3, 19], 15 patients per arm should be required to achieve a power of 90% with a two-sided significance level of P < 0.05. To account for possible dropouts, the target number of patients was, therefore, set at 20 per arm. In the pilot study, 42 participants who received graft donations from the same donors from January 2016 to December 2016 were recruited and divided into two groups randomly. The participants were assigned to either the UC-MSCs treatment group or the control group in a 1:1 ratio using a block randomization method. A randomization list has been pregenerated. The participants were blinded to the treatment group throughout the study. The trial group simultaneously received UC-MSCs via the peripheral vein before operation and via the renal artery during operation. All the participants were recruited from the three transplant units. Written informed consent to participate in this study was obtained from the participants (Fig. 1).
Endpoints
The primary endpoints of this study included DGF in 1 week post transplantation, biopsy proven acute rejection (BPAR) in 1 year. The secondary endpoints were severe opportunistic infections related with opportunistic infection as well as pulmonary and urinary tract infection, and other donor specific immune response in 1 year postoperatively.
Immunosuppressive regimen
Antithymocyte globulin (50 mg/day) and methylprednisolone (500 mg/day) were continuously administered intravenously during the first 3 postoperative days as induction therapy. Maintenance immunosuppressive regimens consisted of a calcineurin inhibitor, mycophenolate mofetil, and prednisone. Mycophenolate mofetil, which was initiated immediately after transplantation, was maintained at a daily dose of 1.0–1.5 g. Tacrolimus or cyclosporine was started on days 2–4 at 0.1–0.15 or 6–8 mg/kg/day, respectively, based on the level of recovery of renal graft function. The immunosuppressive regimens were adjusted to achieve the target therapeutic trough levels in the peripheral blood (5–8 ng/ml for tacrolimus, and 130–150 ng/ml for cyclosporine). Oral administration of prednisone, which was initiated at 30 mg/day on day 4 following transplantation, was reduced by 5 mg/week until a maintenance dose of 10–15 mg/day.
Umbilical cord blood units
The UC-MSCs used in this study were isolated after birth, with written consent from the parents, and a total volume of 100–120 ml was harvested at passages 4–7. The processing and expansion of the cells took place at the Good Manufacturing Practice Stem Cell Laboratory Facility of Sun Yat-sen University, as previously described [19]. Characterization of the final product, which expressed CD90, CD73, CD105, CD44, and CD166, was determined by flow cytometric analysis. Before infusion, the UC-MSCs were subjected to aerobic, anaerobic, and fungal cultures and tested for mycoplasma infection; subsequently, their sterility was confirmed.
Procedures
A stem-cell dose of 2 × 106 UC-MSCs per kilogram body weight was administered for 30 min via the peripheral vein before renal transplantation. Vital signs were monitored continuously during the infusion. The dose of UC-MSCs administered via the renal artery during the surgical procedure was 5 × 106 cells. Before releasing the renal artery, the targeted UC-MSCs were administered into the kidney and maintained for 10 min (Fig. 1).
Clinical assessments and data collection
Participants were observed during the infusion and monitored for infusion reactions before operation. Additional adverse events (AEs) were identified through interviews with the participants during UC-MSC administration. For renal artery administration, renal perfusion after releasing the allograft artery was observed, and we assessed whether the renal capsule was full and plump and the renal cortex had ischemic areas. Additionally, urine volume from the release of the allograft vessels was also observed. After hospital discharge, AEs were identified through clinic visits or phone interviews with the participants at 3, 9, and 12 months after infusion.
Baseline clinical demographics of the donors (age, gender, body mass index, medical history, type of allograft, infection status, serum creatinine, causes of death, time of ICU, urine volume per day, creatinine level at organ procurement, time of warm and cold ischemia, vasoactive drugs, and cardiopulmonary resuscitation, CMV infection status) were documented. For the recipients, the clinical data included age, gender, medical history, current medication, blood type, previous blood transfusions, panel-reactive antibody (PRA), infection status, physical and laboratory examinations, and dialysis methods and duration. Other specifications, such as the number of human leukocyte antigen (HLA) mismatching between donor and recipient, complement dependent cytotoxicity, time-zero biopsy of the donated kidney, were also collected. All immunosuppressive agents and other drugs used and dosages administered were recorded during the study. The Modification of Diet in Renal Disease 2006 formulae were used to calculate the estimated glomerular filtration rate (eGFR) [20].
Serum creatinine and blood urea nitrogen levels, and urine volume per day were recorded to evaluate the renal function. Renal color ultrasound was performed postoperatively. In our study, DGF was defined as the use of dialysis in the first postoperative week, or failure of serum creatinine to decrease by 10% in the first 48 h following transplantation [21]. When acute rejection is suspected clinically, renal allograft biopsy was performed and classified according to Banff 2013 classification [22]. Incidences of pulmonary and urinary tract infections as well as other opportunistic infections such as CMV infection were monitored after the operation. Additionally, laboratory examinations, such as routine blood test, routine urine test, liver and renal function, and concentration of CNI, were performed once a week for 3 months after the operation and, thereafter, once every 2 weeks for 3–6 months and once a month for 6–12 months.
Statistical methods
Differences in clinical characteristics of the participants were examined with paired t test for continuous variables and McNemar’s test for discrete variables. Kaplan–Meier curves were plotted to depict graft and recipient survivals, and a curve comparison was performed between the two groups using the log-rank test. All analyses were performed using the Statistical Package for Social Science 21.0 for Windows (IBM Corp., Armonk, NY). P value < 0.05 was considered statistically significant.