Choli Hartono

Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine

BACKGROUND Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation. METHODS We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status. RESULTS The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed. CONCLUSIONS Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.

CD103 mRNA levels in urinary cells predict acute rejection of renal allografts1

Background. CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. Methods. We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. Results. CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). Conclusion. CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.

SERINE PROTEINASE INHIBITOR-9, AN ENDOGENOUS BLOCKER OF GRANZYME B/PERFORIN LYTIC PATHWAY, IS HYPEREXPRESSED DURING ACUTE REJECTION OF RENAL ALLOGRAFTS

Background. Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1′ is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. Methods. We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. Results. The levels of PI-9 (P =0.001), granzyme B (P <0.0001), and perforin mRNAs (P <0.0001), but not the levels of 18S rRNA (P =0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P =0.01). Furthermore, PI-9 levels predicted subsequent graft function (r =0.43, P =0.01). Conclusions. PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.

Noninvasive Diagnosis of Acute Rejection of Renal Allografts

Purpose of reviewAcute rejection is an immune process that begins with the recognition of the allograft as nonself and ends in graft destruction. Histological features of the allograft biopsy are currently used for the differential diagnosis of allograft dysfunction. In view of the safety and the opportunity for repetitive sampling, development of noninvasive biomarkers of allograft status is an important objective in transplantation. Herein, we review some of the progress towards the development of noninvasive biomarkers of human allograft status. Recent findingsUrinary cell and peripheral blood cell mRNA profiles have been associated with acute rejection of human renal allografts. Emerging data support the idea that development of noninvasive biomarkers predictive of antibody-mediated rejection is feasible. The demonstration that intragraft microRNA expression predicts renal allograft status suggests that noninvasively ascertained microRNA profiles may be of value. SummaryWe are pleased with the progress to date, and anticipate clinical trials investigating the hypotheses that noninvasively ascertained mRNA profiles will minimize the need for invasive biopsy procedures, predict the development of acute rejection and chronic allograft nephropathy, facilitate preemptive therapy capable of preserving graft function, and facilitate personalization of immunosuppressive therapy for the allograft recipient.

Molecular signatures of urinary cells distinguish acute rejection of renal allografts from urinary tract infection

Acute rejection (AR) and urinary tract infection (UTI) continue to plague renal transplantation. We tested the hypotheses that UTI does not increase granzyme B mRNA levels in urinary cells, and that the levels distinguish AR from UTI. We measured the levels of granzyme B mRNA in 15 urine specimens from renal allograft recipients with UTI, 29 specimens from patients with AR but without UTI, and 14 specimens from patients without AR and without UTI. We also measured transcript levels in urine specimens from 41 nontransplant individuals, 11 with UTI and 30 without UTI. UTI did not increase granzyme B mRNA levels. Granzyme B mRNA levels were lower in renal allograft recipients with UTI compared with those with AR (P<0.0001). We conclude that bacterial UTI is unlikely to confound AR diagnosis made by measurement of granzyme B mRNA levels in urinary cells.

Independent Risk Factors for Urinary Tract Infection and for Subsequent Bacteremia or Acute Cellular Rejection: A Single Center Report of 1166 Kidney Allograft Recipients

Background Urinary tract infection (UTI) is a frequent, serious complication in kidney allograft recipients. Methods We reviewed the records of 1166 kidney allograft recipients who received their allografts at our institution between January 2005 and December 2010 and determined the incidence of UTI during the first 3 months after transplantation (early UTI). We used Cox proportional hazards models to determine the risk factors for early UTI and whether early UTI was an independent risk factor for subsequent bacteremia or acute cellular rejection (ACR). Results UTI, defined as 105 or more bacterial colony-forming units/mL urine, developed in 247 (21%) of the 1166 recipients. Independent risk factors for the first episode of UTI were female gender (hazard ratio [HR], 2.9; 95% confidence intervals [CI], 2.2–3.7; P<0.001), prolonged use of Foley catheter (HR, 3.9; 95% CI, 2.8–5.4; P <0.001), ureteral stent (HR, 1.4; 95% CI, 1.1–1.8; P=0.01), age (HR, 1.1; 95% CI, 1.0–1.2; P=0.03), and delayed graft function (HR, 1.4; 95% CI, 1.0–1.9; P=0.06). Trimethoprim/sulfamethoxazole prophylaxis was associated with a reduced risk of UTI (HR, 0.6; 95% CI, 0.3–0.9; P=0.02). UTI was an independent risk factor for subsequent bacteremia (HR, 2.4; 95% CI, 1.2–4.8; P=0.01). Untreated UTI, but not treated UTI, was associated with an increased risk of ACR (HR, 2.8; 95% CI, 1.3–6.2; P=0.01). Conclusions Female gender, prolonged use of Foley catheter, ureteral stent, age, and delayed graft function are independent risk factors for early UTI. UTI is independently associated with the development of bacteremia, and untreated UTI is associated with subsequent ACR.

Concurrent acute cellular rejection is an independent risk factor for renal allograft failure in patients with C4d-positive antibody-mediated rejection.

Background Identification of risk factors for renal allograft failure after an episode of acute antibody-mediated rejection (AMR) may help the outcome of this difficult-to-treat complication. Methods During December 2003 to February 2011, 833 kidney graft recipients underwent 1120 clinically indicated biopsies at our center. We reviewed the biopsy results and identified 87 biopsy specimens from 87 patients positive for the degradation product of complement component 4 (C4d) and acute AMR. We generated Kaplan-Meier survival curves and performed a multivariable analysis using the Cox proportional hazards regression model to identify risk factors for allograft failure after C4d+ acute AMR. Results Among the 87 patients, 26 had a diagnosis of acute AMR according to the Banff ’09 classification schema, 29 had acute AMR and chronic active AMR, 18 had acute AMR and acute T-cell mediated rejection (TCMR), and 14 had acute AMR, chronic active AMR, and acute TCMR. Kaplan-Meier survival estimates showed that concurrent acute TCMR (P=0.001, Mantel-Cox log-rank test), concurrent chronic active AMR (P=0.03), and time to biopsy (P=0.04) are associated with graft survival. The Cox proportional hazards regression analysis identified that concurrent acute TCMR (hazard ratio, 2.59 [95% confidence interval, 1.21–5.55]; P=0.01) and estimated glomerular filtration rate (hazard ratio, 0.65 [95% confidence interval, 0.48–0.88]; P=0.01) are independent risk factors for allograft loss. Concurrent chronic active AMR or time to biopsy was not associated with graft failure by the multivariable Cox analysis. Conclusions Our single-center study has elucidated that concurrent acute TCMR in kidney transplant recipients with C4d+ acute AMR is an independent risk factor for graft failure. Level of allograft function at the time of diagnosis was also an independent predictor of graft loss.

Urinary Cell mRNA Profiles and Differential Diagnosis of Acute Kidney Graft Dysfunction

Noninvasive tests to differentiate the basis for acute dysfunction of the kidney allograft are preferable to invasive allograft biopsies. We measured absolute levels of 26 prespecified mRNAs in urine samples collected from kidney graft recipients at the time of for-cause biopsy for acute allograft dysfunction and investigated whether differential diagnosis of acute graft dysfunction is feasible using urinary cell mRNA profiles. We profiled 52 urine samples from 52 patients with biopsy specimens indicating acute rejection (26 acute T cell-mediated rejection and 26 acute antibody-mediated rejection) and 32 urine samples from 32 patients with acute tubular injury without acute rejection. A stepwise quadratic discriminant analysis of mRNA measures identified a linear combination of mRNAs for CD3ε, CD105, TLR4, CD14, complement factor B, and vimentin that distinguishes acute rejection from acute tubular injury; 10-fold cross-validation of the six-gene signature yielded an estimate of the area under the curve of 0.92 (95% confidence interval, 0.86 to 0.98). In a decision analysis, the six-gene signature yielded the highest net benefit across a range of reasonable threshold probabilities for biopsy. Next, among patients diagnosed with acute rejection, a similar statistical approach identified a linear combination of mRNAs for CD3ε, CD105, CD14, CD46, and 18S rRNA that distinguishes T cell-mediated rejection from antibody-mediated rejection, with a cross-validated estimate of the area under the curve of 0.81 (95% confidence interval, 0.68 to 0.93). Incorporation of these urinary cell mRNA signatures in clinical decisions may reduce the number of biopsies in patients with acute dysfunction of the kidney allograft.

Pancreas transplantation considering the spectrum of body mass indices

Afaneh C, Rich B, Aull MJ, Hartono C, Kapur S, Leeser DB. Pancreas transplantation considering the spectrum of body mass indices. 
Clin Transplant 2011: 25: E520–E529.

Pancreas Transplantation: Does Age Increase Morbidity?

Introduction. Pancreas transplantation (PTx) is the only definitive intervention for type 1 diabetes. Medical advancements in diabetes care have led to an aging PTx candidate pool. We report our experience with patients ≥50 years of age undergoing PTx. Methods. We reviewed 136 consecutive PTx patients at our institution from 1996–2010; 17 were ≥50 years of age. We evaluated demographics, surgical complications, acute rejection (AR) rates, nonsurgical infections, and survival outcomes. Results. Demographic data was similar (P > .05) between groups, excluding age. The two groups had comparable major and minor surgical complication rates (P = .10 and P = .25, resp.). The older group had a lower 1-year and overall AR rate (P = .04 and P = .03, resp.). The incidence of non-surgical infections and overall patient and graft survival was similar between groups (P > .05). Conclusion. Older patients with type 1 diabetes are feasible candidates for PTx, as surgical morbidity, incidence of infections, and AR rates are low.