Living unrelated donor kidney transplantation – a fourteen-year experience

Background. In countries without a national organization for retrieval and distribution of organs of the deceased donors, problem of organ shortage is still not resolved. In order to increase the number of kidney transplantations we started with the program of living unrelated – spousal donors. The aim of this study was to compare treatment outcome and renal graft function in patients receiving the graft from spousal and those receiving ghe graft from living related donors. Method. We retrospectively identified 14 patients who received renal allograft from spousal donors between 1996 and 2009 (group I). The control group consisted of 14 patients who got graft from related donor retrieved from the database and matched than with respect to sex, age, kidney disease, immunological and viral pretransplant status, the initial method of the end stage renal disease treatment and ABO compatibility. In the follow-up period of 41 ± 38 months we recorded immunosuppressive therapy, surgical complications, episodes of acute rejection, CMV infection and graft function, assessed by serum creatinine levels at the beginning and in the end of the follow-up period. All patients had pretransplant negative cross-match. In ABO incompatible patients pretransplant isoagglutinine titer was zero. Results. The patients with a spousal donor had worse HLA matching. There were no significant differences between the groups in surgical, infective, immunological complications and graft function. Two patients from the group I returned to hemodialysis after 82 and 22 months due to serious comorbidities. Conclusion. In spite of the worse HLA matching, graft survival and function of renal grafts from spousal donors were as good as those retrieved from related donors.


Introduction
In spite of tremendous breakthrough in the field of organ transplantation, organ shortage remains as the main problem 1 .Two main sources of organs are living related and deceased donors, with better graft function and survival in the former group [2][3][4][5][6][7][8] .In countries like Serbia, without a national organization for retrieval and distribution of organs of the deceased, problem of organ shortage is much more pronounced.In attempt to increase the number of kidney transplantations we initiated high-risk transplant programs across positive cross-match, incompatible blood groups and from living unrelated -spousal donors.With these new transplant programs the total number of kidney transplantations in our unit increased by 15%.Transplantations from living unrelated -spousal donors contribute with 5%.The later situation is extremely delicate, because two emotionally and economically related members of the same family and their children are at possible risk at the same time 9 .The aim of this study was to investigate if there was any difference in the patients' and renal graft survival and function between recipients whose donors were unrelated -spouses compared to those whose donors were related.

Methods
This study was a retrospective matched pairs analysis of two groups.Both groups of kidney allograft recipients from spousal (the group I) and related donors (the control group) consisted of 14 patients transplanted in the period from 1996 to 2009.In 13 patients from the group I and 11 from the control group primary kidney disease was discovered in the advanced or end stage renal failure (ESRF) with laboratory and clinical presentation suggestive of chronic glomerulonephritis.One patient in each group had renal polycystic disease.They had bilateral nephrectomy prior to transplantation.One patient in the control group had IgA nephropathy and one lupus nephritis.The initial treatment of ESRF in 12 patients in the group I and 11 in the control group was in a period of one to four years hemodialysis, two patients in each group had preemptive kidney transplantation and one patient from the control group was one year on continuous ambulatory peritoneal dialysis (CAPD).Two patients from the group I and one from the control group had positive anti-hepatitis C virus (HCV) antibodies, while one from the group I was hepatitis B surface antigen (HBs Ag) and hepatitis B e antigen (HBe Ag) positive.The human leukocyte antigen system (HLA) matching between recipients and donors in the group I was from 10% to 62.5% (mean 39.2 ± 15%), and in the control group from 50% to 63% (mean 50.8 ± 3.3%).All the patients who got ABO compatible graft had negative pretransplant cross-match (one from the group I who had historically positive cross-match).The recipients of ABO incompatible grafts had pretransplant isoagglutinine titer zero and negative cross-match [10][11][12] .
The spousal donors aged from 33 to 60 years, mean 42.5 ± 7.5 years, and the related donors were significantly older, aged from 50 to 74 years, mean 60.14 ± 8.43 years (p < 0.01).
A potential donor and recipient were presented to the Ethical Committee of our institution if no exclusion criteria for transplant were met.After being accepted for the transplant program, the pretransplant evaluation was completed, and the couple was presented to the surgical team, which made a final decision about the operation.
All the patients got triple immunosuppressive protocol consisting of steroids, an antiproliferative drug (azathioprine, mycophenolate mofetil or mycophenolate sodium) and a calcineurine inhibitor (cyclosporine A or tacrolimus).Steroids were slowly tapered according to a standard protocol.The dose of antiproliferative drug was standardized, but also decreased during the follow-up.The calcineurine inhibitors' dose was adjusted depending on C0, C2 concentration and mini area under the curve of the first 4h (AUC 0-4).One patient with positive pretransplant cross-match was treated with selective IgG immunoabsorption (Imunosorba -Fresenius Medical Care), and became negative after the first procedure.The renal allograft recipients from ABO incompatible donor were pretreated with rituximab and plasmapheresis procedures [10][11][12] .The recipients with indexes near or over upper range in the mixed lymphocyte culture, beside standard triple immunosuppressive therapy received polyclonal (ATG-Fresenius -3 mg/kg daily for 4 days) or monoclonal (daclisumab -2 mg/kg, day 0 and 14) antibodies in the early postransplant period.
One patient received hyperimmune gamma globulin produced in our institution for the treatment of primary CVM infection 13 .
The duration of follow-up in both groups was between 3 and 132 months, mean 41.1 ± 38.2 months.
We analyzed the differences between the groups in early surgical complications, episodes of acute rejection, cytomegalovirus infection, graft and patients survival and graft function, as assessed by serum creatinine level at the time of discharge after the transplantation and at the end of the follow-up period.
HLA system of donors and recipients was determined serologically.All patients had negative pretransplant lymphocytotoxic cross-match (complement dependent cytotoxicity -CDC).Titers of ABO isoagglutinine were measured using the standard procedure described previously 14 .Blood groups and Rh factor were tested with blood grouping reagents ABO-Rh / Reverse grouping cassette (Ortho-Clinical Diagnostics, USA).The third generation of ELISA was performed for detection of serological markers for viral hepatitis.Polymerase chain reaction (PCR) Amplicor (Hoffman la Roche) was used for viral RNA and DNA detection.Cut-off levels for cytomegalovirus was 400 copies/mL and for hepatitis C virus 2 000 copies/mL.Concentrations of serum creatinine were determined colorimetrically.
According to the protocol in our institution, the diagnosis of early acute rejection (in the first 15 postransplant days) was based on a combination of clinical presentation, labora-tory findings and response to steroid pulse therapy.If there was no response to steroid therapy in three to five days the ultrasound-guided percutaneous graft biopsy was performed and tissue verification was obtained.
For data (frequencies) comparison between and within groups (inter-and intra-comparison) we used Kolmogorov-Smirnov test and ANOVA.The marked differences were significant at p < 0.05.

Results
The recipients in the groups were 8 males and 6 females, with the mean age of 49 ± 6.8 years in the group I and 47.5 ± 4.5 years in the control group (Table 1).
There were no significant differences between the groups according to sex, age, primary kidney disease, initial ESRF treatment, serology to hepatitis B virus (HBV) and hepatitis C virus (HCV), ABO compatibility and pretransplant cross match (Tables 1 and 2).As expected, a significant difference was in HLA matching, with worse matching of spousal grafts (Table 2).The spousal donors were not different significantly from the recipients in age, but related donors were significantly older (p < 0.01).
No significant difference was noticed between the groups in the duration of follow-up, surgical complications, episodes of acute rejection, cytomegalovirus (CMV) infection, graft outcome and patients survival.One patient from the group I had primary CMV infection as confirmed by CMV IgG D+/R-.Besides, six months of antiviral therapy  with Ganciclovir (iv and oral), in early postransplant period he received hyperimmune gamma globulin produced in our institution.
Four patients from the group 1 had surgical complications but only one patient needed reoperation within the first 24 hours due to bleeding from the renal vein suture.One hemathoma and two seromas resolved spontaneously.Late phlebothrombosis of the right femoral vein in one patient was successfully treated with low molecular weight heparin.
Three patients from the control group had seroma, but only one needed surgery.One patient had hematoma which reabsorbed.
These surgical complications had no long-term consequences on graft function, as the serum creatinine returned to normal range after healing in all patients.
In an early postransplant period (first 7 days), six patients from the group I and three from the control group had episodes of acute rejection.These rejections were successfully treated with methylprednisolone pulses in the dose of 5-7 mg/kg.Only one patient from the group I (with ABO incompatible renal graft) had three repeated episodes of acute rejection in the first 10 months.During the postransplant period in this patient we failed to achieve the balance between immunosuppressive therapy and numerous opportunistic bacterial and viral infections, hypersensitive and myelotoxic effects of immunosuppressive, antiviral and antibiotic therapy.After 22 months she restarted hemodialysis and two months later died due to agranulocytosis.
One patient from the group I was HBs Ag and HBe Ag positive with normal liver function at the time of transplantation, and was not started on preventive antiviral therapy with lamivudine after transplantation.Four years later, HBV restarted replication, which could not be controlled with antiviral therapy (lamivudine), and eventually developed all the laboratory and clinical signs of liver cirrhosis.At the same time we noticed a gradual increase in serum creatinine levels, then increasing proteinuria to the nephritic range, followed by poorly controlled hypertension and finally progressive renal failure to ESRD.Due to serious coagulation problem graft biopsy was not done.Almost seven years after transplantation (82 months) he restarted hemodialysis.
With the exception of the two patients mentioned above, the remaining patients had stable renal allograft function during the follow-up.Neither group showed significant difference in graft function, evaluated by serum creatinine level, at the beginning and at the end of the follow-up.Estimated graft function also was not different between the groups, neither at the beginning nor at the end of the followup (Table 3).

Discussion
Transplantation is the best possible treatment for the end-stage kidney disease 1 .The gap between waiting list and demands for organs for a transplant program increases yearly 1 .Originally, kidney transplantation was performed only from well-matched living donors.Later, many states developed national programs for retrieving and distribution of organs from deceased donors [1][2][3][4][5][6][7][8] .With the advances in pretransplant evaluation, immunosuppressive protocols and postransplant management, short-and long-term outcomes have improved 1 .Kidney transplantation offered to transplanted patient almost normal life with only few discomforts: renal graft lifelong immunosuppressive therapy and moderate life style modifications.
Organ shortage was the reason for many countries to start considering transplantation from marginal deceased and living unrelated donors.Most of these countries have a transplant program from spousal donors, but some of them developed a program from other unrelated donors: distant relatives, paired-exchange, living deceased exchange, altruistic donations, not directed and directed donors 8 .
In our institution, after an interview obtaining an informed consent and initial pretransplant evaluation, the Ethical Committee in each individual case gives a permission for the transplant program.Pretransplant evaluation is then completed and the couple is presented to the kidney transplant team, whose decision about medical indications for transplantation is final.Most of our spousal couples are deeply emotionally attached with their grown-up children.Their children show great interest in the transplant program for their parents with kidney disease.During the follow-up we noticed that the emotional and social connections between living donors and their recipients increased, and that they had positive influence on those who were in program for pretransplant evaluation and inspired the same couples to join the transplant program.
Similarly to the reports from other countries 2-8 our patients showed stable function during the whole follow-up period with the exception of two patients with significant pretransplant comorbidities.
The transplant program for spousal donors makes only 5% of total transplant program in our center.Similar percentage is found in other transplant centers worldwide.Some countries without a national program for transplantation from deceased donors formed an agency, which controls the program from living unrelated donors at the state level 5,15 .Only one third of these donors are spouses, while the rest are volunteers, in the most cases young adults reimbursed for donation.Both the short-and the long-term outcome of such transplantations is better than from deceased donor.Special situation is found in the countries with illegal living unrelated kidney transplant program.The boards in many international transplant organizations are considering how to control such program, because they are aware of the fact that it is almost impossible to cease it.

Conclusion
As a transplant center we developed different modalities of living related donor programs: preemptively, during hemodialysis and CAPD, from ABO compatible and incompatible living related or unrelated-spousal donor.From 1996 to 2009 we performed only 14 transplantations from unrelated donors.Pretransplant evaluation of these couples was more demanding due to more detailed psychosocial evaluation and administrative procedure.In this limited number of cases the outcome and function of transplanted kidneys from unrelated donor did not differ from related donors during the follow-up period.The only source which could increase significantly kidney transplant program is organization of a national program for retrieval and distribution of organs from deceased donors.

Table 1 Differences between the groups according to sex, age and primary kidney disease
*Kolmogorov -Smirnov test