Detection of Subclinical Rejection in Pediatric Kidney Transplantation: Current and Future Practices.

INTRODUCTION: The successes in the field of pediatric kidney transplantation over the past 60 years have been extraordinary. Year over year, there have been significant improvements in short-term graft survival. However, improvements in longer-term outcomes have been much less apparent. One important contributor has been the phenomenon of low-level rejection in the absence of clinical manifestations-so-called subclinical rejection (SCR). METHODS: Traditionally, rejection has been diagnosed by changes in clinical parameters, including but not limited to serum creatinine and proteinuria. This review examines the shortcomings of this approach, the effects of SCR on kidney allograft outcome, the benefits and drawbacks of surveillance biopsies to identify SCR, and new urine and blood biomarkers that define the presence or absence of SCR. RESULTS: Serum creatinine is an unreliable index of SCR. Surveillance biopsies are the method most utilized to detect SCR. However, these have significant drawbacks. New biomarkers show promise. These biomarkers include blood gene expression profiles and donor derived-cell free DNA; urine gene expression profiles; urinary cytokines, chemokines, and metabolomics; and other promising blood and urine tests. CONCLUSION: Specific emphasis is placed on studies carried out in pediatric kidney transplant recipients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03719339.

De novo lupus-like glomerulonephritis after pediatric non-kidney organ transplantation.

BACKGROUND: We propose a novel clinically significant finding, de novo lupus-like glomerulonephritis (DNLLGN), in patients with autoantibodies and kidney abnormalities in pediatric liver transplant (LT) and intestinal inclusive transplants (ITx). METHODS: We describe the clinical, serologic, and histopathologic presentation and kidney outcomes in eight patients from our center found to have DNLLGN on kidney biopsy. RESULTS: Pediatric recipients of non-kidney solid organ transplants developed an unusual de novo immune complex glomerulonephritis with morphologic similarity to lupus nephritis. Six had isolated LT (0.9% of all pediatric LT at our center) and two had ITx (2.1% of all ITx). Five (63%) presented with nephrotic syndrome. Five patients had autoantibodies. Patients underwent kidney biopsy at a mean of 11.5 years in LT and 2.8 years in ITx after the index transplant. Biopsies demonstrated changes similar to focal or diffuse active lupus. Follow-up eGFR at a mean of 6 years after biopsy showed a mean decrease of 30 ml/min/1.73 m2 in all patients (p = 0.11). CONCLUSIONS: DNLLGN has not been previously recognized in this clinical setting, yet 8 kidney biopsies from pediatric recipients of LT and ITx at our center in 25 years demonstrated this finding. DNLLGN appears to be an under-reported phenomenon of clinical significance. A higher resolution version of the Graphical abstract is available as Supplementary information.

Phase I study of single-dose pharmacokinetics and pharmacodynamics of belatacept in adolescent kidney transplant recipients.

Belatacept is an intravenously infused selective T cell costimulation blocker approved for preventing organ rejection in renal transplant recipients aged ≥18 years. This phase I trial examined the pharmacokinetics and pharmacodynamics (percentage CD86 receptor occupancy [%CD86RO]) of a single dose of belatacept (7.5 mg/kg) administered to kidney transplant recipients aged 12-17 years receiving a stable calcineurin inhibitor-based immunosuppressive regimen. Nine adolescents (mean age 15.1 years) who were seropositive for Epstein-Barr virus were enrolled; all completed the 6-month study. Pharmacokinetics suggested relatively low variability of exposure (coefficients of variation for maximum observed serum concentration [Cmax ] and area under the serum concentration-time curve from time zero extrapolated to infinity [AUC0-INF ] were 20% and 25%, respectively). Mean half-life (T1/2 ) occurred 7.2 days postinfusion. Belatacept total body clearance was 0.48 mL/h/kg, and volume of distribution at steady-state (Vss ) was low at 0.09 L/kg. Compared with historical data from healthy adult volunteers administered a single dose of belatacept 10 mg/kg and adult kidney transplant recipients administered multiple doses of belatacept 5 mg/kg, pharmacokinetic values for adolescents were similar, indicating consistency across adolescent and adult populations. Mean %CD86RO increased with increasing belatacept concentration, indicating a direct relationship between pharmacokinetics and pharmacodynamics. Four patients reported 7 serious adverse events; none was considered related to belatacept. These data will inform belatacept dose selection in future studies of adolescent kidney transplant recipients.

Bortezomib may stabilize pediatric renal transplant recipients with antibody-mediated rejection.

BACKGROUND: Current therapeutic strategies to effectively treat antibody-mediated rejection (AMR) are insufficient. Thus, we aimed to determine the benefit of a therapeutic protocol using bortezomib for refractory C4d + AMR in pediatric kidney transplant patients. METHODS: We examined seven patients with treatment-refractory C4d + AMR. Immunosuppression included antithymocyte globulin or anti-CD25 monoclonal antibody for induction therapy with maintenance corticosteroids, calcineurin inhibitor, and anti-metabolite. Estimated glomerular filtration rate (eGFR) calculated by the Schwartz equation, biopsy findings assessed by 2013 Banff criteria, and human leukocyte antigen (HLA) donor-specific antibodies (DSA) performed using the Luminex single antigen bead assay were monitored pre- and post- bortezomib therapy. RESULTS: Seven patients (86 % male, 86 % with ≥6/8 HLA mismatch, and 14 % with pre-formed DSA) age 5 to 19 (median 15) years developed refractory C4d + AMR between 1 and 145 (median 65) months post-transplantation. All patients tolerated bortezomib. One patient had allograft loss. Of the six patients with surviving grafts (86 %), mean pre-bortezomib eGFR was 42 ml/min/1.73 m(2) and the mean 1 year post-bortezomib eGFR was 53 ml/min/1.73 m(2). Five of seven (71 %) had improvement of histological findings of AMR, C4d staining, and/or acute cellular rejection. Reduction in HLA DSAs was more effective for class I than class II. CONCLUSIONS: Bortezomib appears safe and may correlate with stabilization of eGFR in pediatric kidney transplant patients with refractory C4d + AMR.

Sirolimus and tacrolimus coefficient of variation is associated with rejection, donor-specific antibodies, and nonadherence.

BACKGROUND: Immunosuppression medication nonadherence has been associated with donor-specific antibodies and treatment-refractory rejection. Drug-level monitoring is a practical direct marker for nonadherence, as variations indicate erratic ingestion of medication. We previously reported that high variability in tacrolimus trough levels determined by the percent coefficient of variation (CV %) and standard deviation (SD) were associated with biopsy-proven rejection. We hypothesized that the CV % and SD in patients on a sirolimus/low-dose tacrolimus regimen may associate with self-reported medication nonadherence, rejection and donor-specific antibodies. METHODS: In this pilot feasibility study, we studied 37 biopsies in 23 pediatric renal transplant patients on both sirolimus and tacrolimus immunosuppression; CV %, SD, de novo donor-specific antibodies, rejection, and self-reported adherence were examined. RESULTS: A cut-off sirolimus CV % of 25 maximized the percentage of biopsies correctly classified as rejection (32 of 37, or 86 %, p = 0.001). A cut-off tacrolimus CV % of 31 maximized the percentage of correctly classified biopsies (25 of 37, or 68 %, p = 0.09). Among patients with both high sirolimus and tacrolimus CV %, 67 % developed de novo donor-specific antibodies (p = 0.002) with a DQ predominance and 71 % reported nonadherence (p = 0.05). CONCLUSIONS: In pediatric renal transplantation, sirolimus and tacrolimus CV % is a potential tool for monitoring patients at risk for allograft rejection and donor-specific antibodies secondary to medication nonadherence.

Accelerated rejection, thrombosis, and graft failure with angiotensin II type 1 receptor antibodies.

BACKGROUND: Angiotensin II type 1 receptor antibodies (AT1R-Abs) have been implicated in renal transplant rejection and failure; however, the mechanism of allograft damage, patterns of clinical presentation, and response to desensitization of AT1R-Abs have not been clearly established. CASE DIAGNOSIS/TREATMENT: We present the case of a 7-year-old boy with preformed AT1R-Abs who developed accelerated vascular and cellular rejection and renal allograft thrombosis despite desensitization and treatment with angiotensin receptor blockade. Although an association between AT1R-Abs and microvascular occlusion has been previously described, we are the first to describe an association between AT1R-Abs and renal artery thrombosis, leading to devastating early allograft failure. CONCLUSIONS: This case highlights the risk of allograft thrombosis associated with AT1R-Abs and illustrates that previous treatments utilized for AT1R-Abs may not always be effective. Further studies are needed to better characterize the mechanisms of AT1R-Ab pathogenesis and to establish safe levels of AT1R-Abs both pre- and post-transplantation. Given the outcome of this patient and the evidence of pro-coagulatory effects of AT1R-Abs, we suggest that the presence of AT1R-Ab may be a risk factor for thrombosis. The role of treatment with anti-coagulation and novel immunomodulatory agents such as tocilizumab and bortezomib require further investigation.

Optimizing HLA matching in a highly sensitized pediatric patient using ABO-incompatible and paired exchange kidney transplantation.

BACKGROUND: Kidney transplantation is the treatment of choice for end-stage renal disease. However, since pediatric patients have long projected life-years, it is also optimal for them to get well-matched transplants to minimize long-term sensitization. In North America, pediatric kidney transplantation is largely dependent upon the use of deceased donor organs, making it challenging to identify timely, well-matched transplants. Pediatric recipients may have willing living donors who are either HLA- or ABO-incompatible (ABOi); therefore, one solution is to utilize ABOi transplants and paired exchange programs to enhance HLA matching and living donation. CASE-DIAGNOSIS/TREATMENT: We adopted this approach for a highly sensitized patient with cPRA 90%, who received a successful ABOi paired exchange transplant. The recipient received pre-transplant immunomodulation until an acceptable isohemagglutinin titer <1:8 was reached before transplantation. The patient was induced with anti-thymocyte globulin and maintained on steroid-based triple immunosuppression. Eighteen-month allograft function is excellent with an estimated glomerular filtration rate (eGFR) of 83.53 ml/min/1.73 m(2). The patient did not develop de novo donor-specific HLA antibodies or have any episodes of acute rejection CONCLUSIONS: This case highlights the safety and efficacy of using paired exchange in combination with ABOi transplants in pediatric kidney transplantation to optimize HLA matching, minimize wait times, and enhance allograft survival.

Mineral abnormalities and long-term graft function in pediatric renal transplant recipients: a role for FGF-23?

BACKGROUND: Although current guidelines recommend the evaluation of mineral and bone metabolism in patients with all stages of chronic kidney disease (CKD), the prevalence of altered mineral ion homeostasis in the pediatric posttransplant population is unknown. Moreover, the contribution of abnormal mineral ion metabolism to graft outcomes in this population has not been evaluated. METHODS: Serum calcium, phosphorus, 25(OH)vitamin D, 1,25(OH)(2)vitamin D, parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) levels were evaluated 4.9 ± 0.5 years after transplantation in 68 stable pediatric renal allograft recipients. Patients were subsequently followed for 2 years. RESULTS: At baseline, mean estimated glomerular filtration rate (GFR) was 60 ± 2 mL/min/1.73 m(2). Serum calcium and phosphorus values were within the reference interval. PTH values were elevated but did not differ by CKD stage. 25(OH)vitamin D levels were low in nearly half of all subjects. Tubular reabsorption of phosphate and 1,25(OH)(2)vitamin D values were lower, while FGF-23 and PTH values were higher in more advanced stages of CKD. Thirty percent of patients with FGF-23 values >110 RU/mL had a decrease in GFR of >50% (P < 0.05) and FGF-23 values predicted future episodes of rejection. CONCLUSIONS: Despite normal serum calcium and phosphorus levels in the majority of prevalent pediatric renal transplant recipients, abnormalities in PTH, 25(OH)vitamin D and FGF-23 are common. FGF-23 levels may be associated with increased risk for deterioration of kidney function and episodes of rejection.

Acute rejection associated with donor-specific anti-MICA antibody in a highly sensitized pediatric renal transplant recipient.

Allograft rejection in HLA identical transplant recipients and in patients without detectable donor-specific anti-HLA antibodies has lead to the identification of non-HLA antigens as targets of the alloimmune response. MICA antigen has been recognized as an important non-HLA target in renal transplantation. Recent studies have shown that anti-MICA antibodies are associated with acute renal allograft rejection and failure. Current cross match procedures using donor lymphocytes fail to detect MICA antibodies. Transplant candidates are not routinely tested for pre-sensitization to MICA antigens nor are transplant donors typed for MICA alleles. Optimal classification and treatment of acute rejection associated with MICA antibody remains unknown. In this case report, we are the first to describe the clinical course and treatment of donor-specific MICA antibody associated with both Banff type II A ACR and AMR in a highly sensitized pediatric renal re-transplant recipient. This case also emphasizes the importance of pre-transplant screening for donor-specific MICA antibody especially in highly sensitized renal transplant patients.

Trabecular bone volume and osteoprotegerin expression in uremic rats given high calcium.

Calcium (Ca)-containing phosphate binders have been recommended for the treatment of hyperphosphatemia in children with chronic kidney disease. To study the effects of high Ca levels on trabecular bone volume (BV) and osteoprotegerin (OPG) expression in uremic young rats, a model of marked overcorrection of secondary hyperparathyroidism was created by providing a diet of high Ca to 5/6 nephrectomized young rats (Nx-Ca) for 4 weeks. The results of chondrocyte proliferation and apoptosis, osteoclastic activity, OPG expression and BV were compared among intact rats given the control diet, intact rats given a high Ca diet and 5/6 nephrectomized rats given the control diet (Nx-Control) and the high Ca diet (Nx-Ca). Ionized Ca levels were higher and parathyroid hormone levels were lower in Nx-Ca rats than in the other groups. Final weight, final length and final tibial length of Nx-Ca rats were significantly less than those of the other groups, although the length gain did not differ among the groups. The hypertrophic zone width was markedly enlarged in Nx-Ca rats. Chondrocyte proliferation rates did not differ among the groups, whereas osteoclastic activity was decreased in Nx-Ca rats compared with the Nx-Control animals. The OPG expression and BV were increased in Nx-Ca rats compared with the Nx-Control rats. Increased BV should improve bone strength, whereas disturbance of osteoclastogenesis interferes with bone remodeling. Bone quality has yet to be determined in high Ca-fed uremic young rats.

An age-independent gene signature for monitoring acute rejection in kidney transplantation.

Acute rejection (AR) remains a significant problem that negatively impacts long-term renal allograft survival. Numerous therapies are used to prevent AR that differ by center and recipient age. This variability confounds diagnostic methods. Methods: To develop an age-independent gene signature for AR effective across a broad array of immunosuppressive regimens, we compiled kidney transplant biopsy (n=1091) and peripheral blood (n=392) gene expression profiles from 12 independent public datasets. After removing genes differentially expressed in pediatric and adult patients, we compared gene expression profiles from biopsy and peripheral blood samples of patients with AR to those who were stable (STA), using Mann-Whitney U Tests with validation in independent testing datasets. We confirmed this signature in pediatric and adult patients (42 AR and 47 STA) from our institutional biorepository. Results: We identified a novel age-independent gene network that identified AR from both kidney and blood samples. We developed a 90-probe set signature targeting 76 genes that differentiated AR from STA and found an 8 gene subset (DIP2C, ENOSF1, FBXO21, KCTD6, PDXDC1, REXO2, HLA-E, and RAB31) that was associated with AR. Conclusion: We used publicly available datasets to create a gene signature of AR that identified AR irrespective of immunosuppression regimen or recipient age. This study highlights a novel model to screen and validate biomarkers across multiple treatment regimens.

CD20+ lymphocytes in renal allografts are associated with poor graft survival in pediatric patients.

BACKGROUND: The presence of CD20+ lymphocyte renal allograft infiltrates has been associated with steroid-resistant rejection and poor graft survival. We quantified the number of CD20+ lymphocytes in renal allograft biopsies and correlated the results with graft survival. We also determined the relationships between CD20+ lymphocytes and acute cellular rejection versus antibody-mediated rejection. METHODS: We examined 45 biopsy samples from 31 pediatric patients biopsied for suspicion of rejection from November 2001 to November 2004. Immunohistochemical staining for CD20 and C4d was performed on all biopsies; CD20+ cell density per high-power field (hpf) was determined for each core. Patient graft status was followed postbiopsy and documented for graft survival or failure using the cutoff date of December 31, 2005. RESULTS: Patients with 2-10 and 11-100 CD20+ cells/hpf had worse graft survival in Kaplan-Meier analysis with a hazard ratio 4.56 (CI 1.07-19.35) two years postbiopsy compared to those with 0-1 cells/hpf (P = 0.02). The presence of CD20+ lymphocytes was significantly associated with acute cellular rejection (P = 0.0001) and not associated with antibody-mediated rejection (P = 0.16). Receiver-operating curve analysis confirmed > or =3 cells/hpf correlating with acute cellular rejection, yielding sensitivity 90% and specificity 76%. CONCLUSIONS: This study shows a significant 4.5-fold risk of graft failure at two years postbiopsy with presence of > or =2 CD20+ cells/hpf. Moreover, > or =3 CD20+ lymphocytes were highly associated with acute cellular rejection. They may be functioning as professional antigen-presenting cells in the graft. In steroid-refractory cellular rejections, therapies that target B cells may prolong graft survival.

Noncompliance with immunosuppressive medications in pediatric and adolescent patients receiving solid-organ transplants.

Advances in knowledge in transplantation have improved 1-year renal allograft survival in all age groups of pediatric patients. However, the results from many studies have shown that the long-term allograft survival is least successful in adolescent recipients. The major cause of late graft failure in adolescents can be contributed in large measure to medication noncompliance. Medication noncompliance in teenagers has been shown to be more than four times greater in adolescents than in adults. The teenage years are a time of transition from childhood to adulthood. Important tasks during this transition include the development of an autonomous identity that progresses to full independence. However, the cognitive skills and intellectual maturation of adolescents are still limited, and this is particularly true in adolescents with chronic diseases. They have difficulty with abstract thinking, particularly the conceptualization of future consequences of present actions. This leads to characteristic risk-taking behaviors, including noncompliance with medical treatments. This transition is more intricate for adolescents with chronic illness because of their physical limitations. There are a number of strategies that are helpful in mitigating noncompliance. Adolescents must be dealt with directly. Previous noncompliant behaviors need to be acknowledged and dealt with, because studies show that noncompliance is a "stable" personality attribute that persists over time. Efforts should be made to choose medications that have the least side effects. Psychological and psychiatric conditions such as posttraumatic stress disorder require early recognition, diagnosis, and treatment. It is necessary to build rapport with teenagers, and this should start before transplantation. A multidisciplinary approach with physicians, social workers, nurses, and transplant coordinators is an effective mean of enhancing compliance. These and other strategies outlined in this discussion will enable the adolescent to achieve good compliance rates and prevent graft loss.

A novel treatment regimen for BK viremia.

BACKGROUND: BK viremia, a prerequisite for BK virus nephropathy (BKVN), affects 5% to 16% of pediatric renal transplant recipients (PRTR). We evaluated the safety and efficacy of a novel approach to treating BK viremia using fluoroquinolones and leflunomide in PRTR. METHODS: We studied 230 PRTR at Mattel Children's Hospital, UCLA, who underwent renal transplantation between January 2003 and October 2010. Nineteen patients were found to have BK viremia. Ciprofloxacin was started when the BK viral load was greater than 625 copies/mL, and patients were switched to leflunomide if BK viral load did not decrease after 2 months of ciprofloxacin therapy. All patients underwent transplant kidney biopsy, and their estimated glomerular filtration rate (eGFR) and BK PCR was measured serially. The side effects of ciprofloxacin and leflunomide were recorded in each patient. RESULTS: There was a significant decrease in BK viral load in patients treated with ciprofloxacin and leflunomide (P<0.001) with only a small reduction in immunosuppression. BK viremia was associated with a significantly decreased eGFR (P<0.001), and treatment with ciprofloxacin and leflunomide was associated with improved eGFR (P<0.001). This approach resulted in a BKVN rate of only 1%. CONCLUSIONS: This analysis demonstrates for the first time that, used in a stepwise fashion, ciprofloxacin and leflunomide are effective and safe treatments for BK viremia in PRTR.

Monitoring nonadherence and acute rejection with variation in blood immunosuppressant levels in pediatric renal transplantation.

BACKGROUND: Acute rejection associated with medication nonadherence is a major cause of allograft loss in pediatric kidney transplant patients. There is currently no reliable method to detect medication nonadherence and prevent allograft rejection. METHODS: In 46 pediatric patients who underwent renal transplantation between 2002 and 2003, the variation of serum drug levels was studied as a potential objective tool to monitor medication nonadherence. Tacrolimus (TAC) and mycophenolic acid (MPA) trough levels were measured from 1 to 12 months posttransplant, and standard deviation (SD) and percent coefficient of variation (CV%) were calculated. Because SD increased as mean trough levels rose, CV% (CV%=SD/mean multiplied by 100%) was used to eliminate this confounding effect. RESULTS: Ten of 46 patients had biopsy-proven rejection. The median TAC CV% was 53.4% in patients with biopsy-proven rejection, which was significantly higher than 30% in those without rejection (P=0.005). Median MPA CV% was 51.9% in patients without rejection and 45.1% in patients with rejection (P=NS). High TAC CV% correlated with increased risk for rejection, whereas MPA CV% did not. CONCLUSION: The TAC CV% seems to be a useful and superior marker, compared with SD alone, for assessing medication nonadherence and the possibility of allograft rejection in pediatric renal transplantation.

Significance of intragraft CD138+ lymphocytes and p-S6RP in pediatric kidney transplant biopsies.

BACKGROUND: We have previously shown that intragraft CD20+ B cells are associated with acute cellular rejection (ACR) and allograft loss. Phosphorylation of S6 ribosomal protein, a downstream target of the PI3K/Akt/mTOR pathway, promotes growth and proliferation of cells and could identify metabolically active cells such as alloantibody secreting plasma cells. Because CD20+ lymphocytes can differentiate into CD138+ plasma cells, we aimed to identify functionally active plasma cells by using intragraft CD138 quantification and p-S6RP staining and correlate these results with allograft rejection, function, and survival. METHODS: We examined 46 renal transplant biopsies from 32 pediatric patients who were biopsied for clinical suspicion of rejection. Immunohistochemical staining for C4d, CD20, CD138, and p-S6RP was performed. Patient creatinine clearance and graft status was followed up postbiopsy. RESULTS: Patients with greater than or equal to six CD138+ cells/high power field (hpf) had worse graft survival with a hazard ratio of 3.4 (95% CI 1.3-9.2) 2 years postbiopsy compared with those with 0 to 5 cells/hpf (P=0.016). CD138+ cells were stained for p-S6RP, indicating functionally active plasma cells. They were associated with ACR (P=0.004) and deteriorating graft function (R=0.22, P=0.001). Intragraft CD20+ and CD138+ cells found together in ACR were associated with poorer graft survival than either marker alone, hazard ratio 1.5 (95% CI 1.1-2.2, P=0.01). CONCLUSIONS: A threshold of greater than or equal to six CD138+ metabolically active plasma cells per hpf, coexisting with CD20+ B cells, was associated with poor allograft function and survival. This may represent an additional antibody-mediated process present in the setting of ACR and could play an important role in characterization and treatment of transplant rejection.

Laparoscopic versus open renal procurement for pediatric recipients of living donor renal transplantation.

Despite reports demonstrating the safety of laparoscopic donor nephrectomy (LDN) for pediatric recipients of renal transplants, recent evidence has challenged using LDN for recipients 5 years of age or younger. We retrospectively reviewed the records of all pediatric recipients of living donor renal transplants from September 2000 through August 2004. We compared those who received allografts recovered by LDN (n = 34) with those recovered by open donor nephrectomy (ODN, n = 26). Outcomes of interest included operative complications, postoperative renal function, the incidence of delayed graft function or episodes of acute rejection and long-term graft function. Donor and recipient demographic data were similar for the LDN and ODN groups. Serum creatinine and calculated creatinine clearance were not significantly different between groups both in the early postoperative period and at long-term follow-up (p > 0.142). Rates of delayed graft function and acute rejection did not differ between groups. Among recipients aged 5 years old or younger stratified by donor technique (9 LDN, 5 ODN recipients), no difference was noted in graft outcomes both early and long-term (p > 0.079). At our center, pediatric LDN recipients have graft outcomes comparable to those of ODN recipients. At experienced centers, we recommend continued use of LDN for pediatric recipients of all ages.