Anne L. King

MicroRNA profiles in allograft tissues and paired urines associate with chronic allograft dysfunction with IF/TA

Despite the advances in immunosuppression, renal allograft attrition over time remains unabated due to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA). We aimed to evaluate microRNA (miRNA) signatures in CAD with IF/TA and appraise correlation with paired urine samples and potential utility in prospective evaluation of graft function. MiRNA signatures were established between CAD with IF/TA versus normal allografts by microarray. Validation of the microarray results and prospective evaluation of urine samples was performed using real‐time quantitative‐PCR (RT‐qPCR). Fifty‐six miRNAs were identified in samples with CAD‐IF/TA. Five miRNAs were selected for further validation based on array fold change, p‐value and in silico predicted mRNA targets. We confirmed the differential expression of these five miRNAs by RT‐qPCR using an independent set of samples. Differential expression was detected for miR‐142‐3p, miR‐204, miR‐107 and miR‐211 (p < 0.001) and miR‐32 (p < 0.05). Furthermore, differential expression of miR‐142‐3p (p < 0.01), miR‐204 (p < 0.01) and miR‐211 (p < 0.05) was also observed between patient groups in urine samples. A characteristic miRNA signature for IF/TA that correlates with paired urine samples was identified. These results support the potential use of miRNAs as noninvasive markers of IF/TA and for monitoring graft function.

Hepatitis C virus infection and kidney transplantation : Predictors of patient and graft survival

Background. The effect of hepatitis C virus (HCV) infection on patients undergoing kidney transplantation (KTx) is uncertain. This study aimed to evaluate the outcomes of our HCV+/end-stage renal disease (ESRD) patient population based on the therapeutic option including KTx or continuation in dialysis. Methods. KTx performed at Virginia Commonwealth University Hospital between January 2000 and December 2004 were tracked prospectively. Forty-three out of a total of 394 KTx patients included in the analysis were HCV+. A group of 52 contemporaneous HCV+/ESRD patients listed, but never transplanted, was also analyzed. HCV-negative transplanted patients were used as the control group. Results. Patient survival posttransplantation was 81.4% and 68.5% at 1 and 3 years in the HCV+ group, and 97.1% and 92.9% at 1 and 3 years in the HCV- group, respectively (P=0.001). Graft survival was 81.2% and 64.1% at 1 and 3 years in the HCV+ group, and 93.2% and 84.1% at 1 and 3 years posttransplantation in the HCV- group (P=0.01). Univariate analysis identified Knodell score as a predictor of mortality in HCV+ patients (P=0.04). Cox proportional hazards multivariate analysis identified deceased donor (P=0.02), previous kidney transplant (P=0.007), pretransplant diabetes (P=0.05), and Knodell Score (P=0.012) as predictors of patient mortality. Patient survival was superior in HCV+ patients undergoing KTx versus remaining on dialysis. Conclusions. Patients with ESRD/HCV+ benefit from KTx without achieving the excellent survival of HCV-/ESRD patients. Liver biopsy is a useful tool to identify advanced liver disease at pretransplantation time.

The urine microRNA profile may help monitor post-transplant renal graft function.

Non-invasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation and the longitudinal validation studies for non-invasive monitoring of graft function. Of 1,733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time-points post-kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early post-kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.

Calcineurin inhibitor-induced chronic nephrotoxicity in liver transplant patients is reversible using rapamycin as the primary immunosuppressive agent

Abstract: The purpose of this study was to determine whether calcineurin inhibitor (CNI)‐induced chronic nephrotoxicity in liver transplant patients is reversible by replacement of the CNI with rapamycin as the primary immunosuppressive agent. CNIs, while providing potent immunosuppression for liver transplant patients, exhibit nephrotoxicity as a major side‐effect. Whereas acute CNI‐induced nephrotoxicity is reversible by withdrawal of the CNI, chronic nephrotoxicity due to CNIs is a progressive process thought to be irreversible. Eight liver transplant patients with CNI‐induced chronic nephrotoxicity were converted to rapamycin as the primary immunosuppressive agent. The CNI was either discontinued (four patients) or the dosage lowered to maintain a subtherapeutic level (four patients). Renal function as assessed by serum creatinine was measured before and after conversion to rapamycin. Two patients progressed to dialysis dependence following conversion to rapamycin. These two patients had been on CNIs for a mean of 112 months (range 93–131 months) prior to conversion to rapamycin. Five patients experienced improvement in renal function. These patients had been on calcineurin inhibitors for a mean of 60 months (range 42–75 months) prior to conversion. One patient with chronic nephrolithiasis as a contributing factor to his renal dysfunction has progressed to dialysis dependence despite conversion to rapamycin following exposure to a CNI for 24 months. In the five patients with improved renal function, serum creatinine levels decreased significantly (2.4 ± 0.3 mg/dL to 1.5 ± 0.1 mg/dL, p < 0.05) by a mean of 7.2 months (range 5–10 months) after conversion from CNI to rapamycin‐based immunosuppression. Liver function remained stable after conversion to rapamycin. CNI‐induced chronic nephrotoxicity can be reversed upon withdrawal of the CNI. Rapamycin is an effective replacement agent as primary immunosuppressive therapy following withdrawal of CNIs in liver transplant patients with CNI‐induced chronic nephrotoxicity.

En bloc kidney transplantation from pediatric donors: comparable outcomes with living donor kidney transplantation.

Background. En bloc kidneys from pediatric donors have been considered suboptimal for transplantation to adult recipients and their outcomes have rarely been compared with living donor kidney transplantation (LDKT). Traditionally, there has been hesitancy in transplanting en bloc kidneys from donors weighing less than 10 kg due to high risk of technical complications. Methods. Retrospective chart reviews were performed to compare outcomes after pediatric en bloc (n=20, mean donor weight 11.4 kg), standard criteria deceased (n=249), and living donor (n=215) kidney transplantation in adult recipients at our center. The outcomes after en bloc transplantation from young donors weighing less than or equal to 10 kg were compared with those from 11 to 15 kg donors. Results. The 5-year graft survival after en bloc, standard deceased, and LDKT were 92%, 70%, and 88%, respectively (P=ns). There were no vascular complications, and urine leak was seen in 1 of 20 en bloc transplants. The 1-year serum creatinine of 1.1±0.2 mg/dL in recipients from less than or equal to 10 kg donors was comparable with 0.9±0.5 mg/dL in 11 to 15 kg group (P=ns). Conclusions. Excellent long-term outcome after pediatric en bloc kidney transplantation from donors weighing less than or equal to 15 kg are comparable with those after LDKT. By using meticulous surgical technique and judicious recipient selection criteria, technical graft losses can be minimized when using en bloc pediatric kidneys from donors weighing less than or equal to 10 kg. Use of pediatric en bloc kidneys should be encouraged continuously to address the problem of organ shortage.

Molecular pathways involved in loss of kidney graft function with tubular atrophy and interstitial fibrosis.

Loss of kidney graft function with tubular atrophy (TA) and interstitial fibrosis (IF) causes most kidney allograft losses. We aimed to identify the molecular pathways involved in IF/TA progression. Kidney biopsies from normal kidneys (n = 24), normal allografts (n = 6), and allografts with IF/TA (n = 17) were analyzed using high-density oligonucleotide microarray. Probe set level tests of hypotheses tests were conducted to identify genes with a significant trend in gene expression across the three groups using Jonckheere-Terpstra test for trend. Interaction networks and functional analysis were used. An unsupervised hierarchical clustering analysis showed that all the IF/TA samples were associated with high correlation. Gene ontology classified the differentially expressed genes as related to immune response, inflammation, and matrix deposition. Chemokines (CX), CX receptor (for example, CCL5 and CXCR4), interleukin, and interleukin receptor (for example, IL-8 and IL10RA) genes were overexpressed in IF/TA samples compared with normal allografts and normal kidneys. Genes involved in apoptosis (for example, CASP4 and CASP5) were importantly overexpressed in IF/TA. Genes related to angiogenesis (for example, ANGPTL3, ANGPT2, and VEGF) were downregulated in IF/TA. Genes related to matrix production-deposition were upregulated in IF/TA. A distinctive gene expression pattern was observed in IF/TA samples compared with normal allografts and normal kidneys. We were able to establish a trend in gene expression for genes involved in different pathways among the studied groups. The top-scored networks were related to immune response, inflammation, and cell-to-cell interaction, showing the importance of chronic inflammation in progressive graft deterioration.

Lethal graft-versus-host disease after simultaneous kidney-pancreas transplantation

BACKGROUND This case report is the first documentation of the occurrence and potential source of lethal graft-versus-host disease (GVHD) after simultaneous kidney-pancreas transplantation. The patient was a 27-year-old African-American male who received an ABO-compatible, five HLA antigen-mismatched kidney-pancreas transplant from a 17-year-old African-American female donor, who died after childbirth. METHODS Preoperative crossmatches using lymphocytotoxicity and flow cytometry were negative. The patient received four blood transfusions within 10 days of transplantation. Immunosuppression consisted of OKT3 induction, and then cyclosporine, azathioprine, and corticosteroids. RESULTS On postoperative day (POD) 9, the patient became febrile, and leukocytopenia and pancytopenia developed. Immunosuppression was reduced and granulocyte colony-stimulating factor was begun. Cultures were negative, interleukin 6 and interleukin 8 levels were elevated, and a cutaneous rash appeared on POD 18. A skin biopsy demonstrated dermatitis with focal epidermal necrosis consistent with GVHD. In an attempt to identify the source of GVHD, variable-number tandem repeat analysis fingerprinting was performed with DNA from donor splenocytes, from the skin biopsy, as well as from the patients buccal mucosa. The skin biopsy showed a mixed variable-number tandem repeat analysis type containing DNA fragments matching the recipient and donor. Blood donors were excluded as a source because they were serologically different from the organ donor. The patient developed liver abnormalities and died from multiorgan failure on POD 22. CONCLUSIONS We speculate that carryover of passenger donor lymphocytes within the transplanted organ were responsible for GVHD. Furthermore, donor traits such as sexual mismatching, African-American race, and alloimmune status may be important potential risk factors for GVHD.

Safe Conversion From Tacrolimus to Belatacept in High Immunologic Risk Kidney Transplant Recipients With Allograft Dysfunction.

There is no literature on the use of belatacept for sensitized patients or regrafts in kidney transplantation. We present our initial experience in high immunologic risk kidney transplant recipients who were converted from tacrolimus to belatacept for presumed acute calcineurin inhibitor (CNI) toxicity and/or interstitial fibrosis/tubular atrophy. Six (mean age = 40 years) patients were switched from tacrolimus to belatacept at a median of 4 months posttransplant. Renal function improved significantly from a peak mean estimated glomerular filtration rate (eGFR) of 23.8 ± 12.9 mL/min/1.73 m2 prior to the switch to an eGFR of 42 ± 12.5 mL/min/1.73 m2 (p = 0.03) at a mean follow‐up of 16.5 months postconversion. No new rejection episodes were diagnosed despite a prior history of rejection in 2/6 (33%) patients. Surveillance biopsies performed in 5/6 patients did not show subclinical rejection. No development of donor‐specific antibodies (DSA) was noted. In this preliminary investigation, we report improved kidney function without a concurrent increase in risk of rejection and DSA in six sensitized patients converted from tacrolimus to belatacept. Improvement in renal function was noted even in patients with chronic allograft fibrosis without evidence of acute CNI toxicity. Further studies with protocol biopsies are needed to ensure safety and wider applicability of this approach.

High prevalence of vitamin D deficiency in African American kidney transplant recipients.

Kidney transplant patients are at high risk for developing Vitamin D3 deficiency. The prevalence rates of 25(OH) Vitamin D3 deficiency and its association with parathyroid hormone (PTH) levels in African American kidney transplant recipients have not been examined. We measured 25(OH) Vitamin D3 and intact PTH concentrations in 38 African American transplant patients at our center in October 2006. We collected various laboratory data including serum creatinine, calcium, phosphate, alkaline phosphatase, and glomerular filtration rate. Vitamin D3 deficiency was present in 57.8% of the patients and 94.7% had insufficiency. Ten of 22 (45%) patients with chronic kidney disease stage 3 had intact PTH more than or equal to 70 pg/mL. On multivariate analysis, 25(OH) Vitamin D3 level was negatively correlated with intact PTH (P<0.01) and alkaline phosphatase level was positively associated with intact PTH levels (P<0.002). Vitamin D3 deficiency and insufficiency is present in most of the African American kidney transplant patients.

Gene Expression Changes Are Associated With Loss of Kidney Graft Function and Interstitial Fibrosis and Tubular Atrophy: Diagnosis Versus Prediction

Background. Loss of kidney graft function due to interstitial fibrosis (IF) and tubular atrophy (TA) is the most common cause of kidney allograft loss. Methods. One hundred one allograft tissues (26 samples with IF/TA, 17 normal allografts, and an independent biopsy group collected at 3 month [n=34] posttransplantation) underwent microarray analysis to identify early detection/diagnostic biomarkers of IF/TA. Profiling of 24 allograft biopsies collected at or after 9-month posttransplantation (range 9–18 months) was used for validation. Three-month posttransplantation biopsies were classified as IF/TA nonprogressors (group 1) or progressors (group 2) using graft function and histology at 9-month posttransplantation. Results. We identified 2223 differentially expressed probe sets between IF/TA and normal allograft biopsies using a Bonferroni correction. Genes up-regulated in IF/TA were primarily involved in pathways related to T-cell activation, natural killer cell-mediated cytotoxicity, and programmed cell death. A least absolute shrinkage and selection operator model was derived from the differentially expressed probe sets, resulting in a final model that included 10 probe sets and had 100% training set accuracy. The N-fold crossvalidated error was 2.4% (sensitivity 95.8% and specificity 100%). When 3-month biopsies were tested using the model, all the samples were classified as normal. However, evaluating gene expression of the 3-month biopsies and fitting a new penalized model, 100% sensitivity was observed in classifying the samples as group1 or 2. This model was evaluated in the sample set collected at or after 9-month posttransplantation. Conclusions. An IF/TA gene expression signature was identified, and it was useful for diagnosis but not prediction. However, gene expression profiles at 3 months might predict IF/TA progression.