Detection of Acute Tubulointerstitial Rejection by Proteomic Analysis of Urinary Samples in Renal Transplant Recipients

This study investigates proteomic analysis of urinary samples as a non-invasive method to detect acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to analyze urinary samples in 19 patients with different grades of subclinical or clinical acute rejection (BANFF Ia to IIb), 10 patients with urinary tract infection and 29 patients without evidence of rejection or infection. A distinct urinary polypeptide pattern identified 16 out of 17 cases of acute tubolointerstitial rejection, but was absent in two cases of vascular rejection. Urinary tract infection resulted in a different polypeptide pattern that allowed to differentiate between infection and acute rejection in all cases. Potentially confounding variables such as acute tubular lesions, tubular atrophy, tubulointerstitial fibrosis, calcineurin inhibitor toxicity, proteinuria, hematuria, allograft function and different immunosuppressive regimens did not interfere with test results. Blinded analysis of samples with and without rejection showed correct diagnosis by CE-MS in the majority of cases. Detection of acute rejection by CE-MS offers a promising non-invasive tool for the surveillance of renal allograft recipients. Further investigation is needed to establish polypeptide patterns in vascular rejection and to explore whether changes in the urinary proteome occur before the onset of histologically discernible rejection.     

Acute Renal Transplant Rejection Possibly Related to Herbal Medications

Use of herbal and alternative medications in the United States is increasing. Many of these medications have unknown mechanisms of actions, and possible metabolic interactions with prescribed medications. We report a case of late acute rejection after exposure to two popular herbal medications.     

Characterization of Urinary Peptide Biomarkers of Acute Rejection in Renal Allografts

We previously demonstrated that 4.7 kDa and 4.4 kDa peptides are useful in diagnosing acute rejection in renal transplant recipients. The aim of this study was to characterize these polypeptides and assess their potential as biomarkers. The polypeptides were identified as human beta-Defensin-1 (4.7 kDa) and alpha-1-antichymotrypsin (4.4 kDa), by tandem mass spectrometry and ProteinChip immunoassay. The urinary abundance of both polypeptides, assessed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), revealed a reduction in beta-Defensin-1 while alpha-1-antichymotrypsin increased in patients with rejection (p < 0.05) compared with clinically stable transplants. The area under the curve (AUC) for the receiver operator characteristic (ROC) curve for the diagnosis of rejection for the ratio of both peptides combined was 0.912. Longitudinal analysis confirmed a reduction in beta-Defensin-1 with a reciprocal increase in alpha-1-antichymotrypsin as rejection developed. The difference in urinary beta-Defensin-1 levels quantified by radioimmunoassay was 176.8 +/- 122.3 pg/mL in stable patients compared with 83.2 +/- 52.2 pg/mL in patients with acute rejection, with an ROC AUC of 0.749 (p < 0.01). Immunohistochemistry (IHC) confirmed reduced beta-Defensin-1 expression in the renal parenchyma of patients experiencing acute rejection. In conclusion, the ratio of beta-Defensin-1 and alpha-1-antichymotrypsin excretion in the urine is a novel, potentially useful candidate biomarkers of acute rejection.     

Does Mycophenolate Mofetil Decrease the Recurrent Acute Rejection in Renal Transplant Recipients

Purpose: Mycophenolate mofetil (MMF) has emerged as a valuable immunosuppression in renal transplant patients. However, it is expensive and cannot be used routinely in our population. Material and methods: In a retrospective study, 60 renal transplant patients on MMF based triple immunosuppression were analysed. The indication for MMF was as rescue therapy after treatment of acute vascular rejection (Banff type-4, grade IIA, IIB and III) in all patients. However, 20 such patients also had associated chronic liver diseases. The patients were given 1.5–2.0 g MMF in two divided doses at least for 6 months, depending upon the tolerability, adverse effects and affordability, and followed-up at least for 1 year. The control group consisted of 60 cases of acute vascular rejection (Banff type-4, grade IIA, IIB and III) who were placed on cyclosporine, azathioprine and steroid based maintenance immunosuppressive regimen in same time frame. Results: The incidence recurrent acute rejections in MMF group was 18% and 42% in control group (P < 0.005). The serum transaminases in all patients of the liver diseases became normal in 3–6 months. The incidence of opportunistic infections in MMF and control group were 22% and 11% respectively (P < 0.05). The MMF based regimen was two times more expensive. The 1 year patient and graft survivals between two groups were not statistically significantly different. Conclusion: The MMF based regimen significantly decreases the recurrent acute rejections. However, it is expensive and cannot be used routinely in all patients in Indian scenario     

Acute Humoral Rejection in Hepatitis C-Infected Renal Transplant Recipients Receiving Antiviral Therapy

The use of interferon-alpha (IFN) in hepatitis C (HCV)-infected renal recipients has been associated with acute rejection and graft loss. We reviewed our recent experience in HCV (+) renal recipients treated with antiviral therapy for biopsy proven chronic hepatitis C. Twelve HCV (+) recipients who recently received antiviral therapy were analyzed. Post-treatment sera were tested for donor-specific human leukocyte antigen (HLA) antibodies (DSA). Within 6 months of initiating antiviral therapy, two of 12 patients (17%) developed acute rejection, which was characterized as acute humoral rejection (de novo DSA in serum and C4d deposits in peritubular capillaries). Both progressed to graft failure. Nine of the remaining 10 patients tested did not have DSA. The use of IFN was associated with severe acute humoral rejection (C4d +, DSA +). The recognition of IFN-associated acute humoral rejection in this series may explain the high rate of graft loss reported previously in renal recipients receiving IFN.     

MP84 Expression in the Urine Specimens of Acute Rejection Renal Transplant Recipients

MP84 is a novel protein synthesized in response to all cytokines. This antigen is expressed only in stimulated mesangial cells and decreased kidney sections, but not in the normal kidney sections (1,2). This study was performed to determine the excretion of MP84 in the urine of renal transplant recipients with acute rejection. Six persons with renal transplant acute rejection and 10 healthy persons were included. Two urine specimens from each person were collected. Dot-blot assay was performed. It was shown that 12 urine specimens from 6 persons with acute rejection revealed MP84 in the matrix dot-blot assay while there was no staining for MP84 in the urine specimens of healthy persons. This could be due to the immunological alteration during the acute rejection which could lead to autocrine and paracrine secretion of growth factors and then the excretion of MP84 in the urine. The mechanism of MP84 secretion is not clear.     

Urinary MicroRNA as Biomarker in Renal Transplantation

Urine represents a noninvasive source in which proteins and nucleic acids can be assessed. Such analytes may function as biomarkers to monitor kidney graft pathology at every desired frequency, thereby providing a time window to prevent graft damage by therapeutic intervention. Recently, several proteins have been measured in urine as markers of graft injury. However, the specificity is limited, and measuring urinary proteins generally lacks the potential to predict early kidney graft damage. Currently, urinary mRNA and microRNA are being investigated to evaluate the prognostic value of changes in gene expression during the initial stages of graft damage. At such time point, a change in treatment regimen and dosage is expected to have maximum potency to minimize future decline in graft function. Both mRNA and microRNAs have shown promising results in both detection and prediction of graft injury. An advantage of microRNAs compared to mRNA molecules is their stability, a characteristic that is beneficial when working with urine samples. In this review, we provide the current state of urinary biomarkers in renal transplantation, with a focus on urinary microRNA. In addition, we discuss the methods used to study urinary microRNA expression.     

Therapy Modalities for Antibody Mediated Rejection in Renal Transplant Patients

Introduction: The aim of our study was to determine the effectiveness of immunoglobulin, rituximab and plasmapheresis in renal transplant patients with antibody mediated rejection (AMR). Patients and Methods: Fourteen renal transplant patients with AMR were included in this study. The mean age of the patients was 33.9 ± 10.3 years and 10 (71.4%) of them were male. Lymphocyte cross match was negative for all patients and 10 (71.4%) of them were living donor transplants. Six patients were administered tacrolimus, three patients cyclosporine, two patients everolimus, and three patients sirolimus for immunosuppression. The patients with AMR were administered IVIG, rituximab and plasmapheresis. Results: Patient survival rate was 100%, graft survival rate after AMR was 50% in the first year and 33% in the 2nd and third years. AMR developed 31.9 ± 25.9 months after transplantation. Seven (50%) patients lost their grafts. Delayed graft function was observed in 28.6%, chronic allograft dysfunction in 78.5%, diabetes after transplantation in 14.3%, and cytomegalovirus infection in 7.1% of the patients. At the last follow-up, the mean blood creatinine was 3.1 ± 1.4, the mean proteinuria was 2300 (1300–3300) mg/day and the mean GFR was 34.5 ± 17.6 ml/min. C4d was positive in peritubullar capillaries in all patients, while neutrophil accumulation in peritubular and glomerular capillaries was observed in 8 patients. Chronic allograft vasculopathy was observed in 12 patients. Conclusion: AMR leads to progressive loss of renal function and has low graft survival. More effective treatment alternatives are needed for this clinical issue.     

Urinary miR‐210 as a Mediator of Acute T‐Cell Mediated Rejection in Renal Allograft Recipients

MicroRNAs (miRNAs) are small ribonucleotides regulating gene expression. Circulating miRNAs are remarkably stable in the blood. We tested whether miRNAs are also detectable in urine and may serve as new predictors of outcome in renal transplant patients with acute rejection. We profiled urinary miRNAs of stable transplant patients and transplant patients with acute rejection. The miR‐10a, miR‐10b and miR‐210 were strongly deregulated in urine of the patients with acute rejection. We confirmed these data in urine of a validation cohort of 62 patients with acute rejection, 19 control transplant patients without rejection and 13 stable transplant patients with urinary tract infection by quantitative RT‐PCR. The miR‐10b and miR‐210 were downregulated and miR‐10a upregulated in patients with acute rejection compared to controls. Only miR‐210 differed between patients with acute rejection when compared to stable transplant patients with urinary tract infection or transplant patients before/after rejection. Low miR‐210 levels were associated with higher decline in GFR 1 year after transplantation. Selected miRNAs are strongly altered in urine of the patients with acute renal allograft rejection. The miR‐210 levels identify patients with acute rejection and predict long‐term kidney function. Urinary miR‐210 may thus serve as a novel biomarker of acute kidney rejection.     

Acute Cellular Rejection or Cyclosporine A Nephrotoxicity? A Review of Transplant Renal Biopsies

Cyclosporine (CsA), a powerful immunosuppressive agent that increases graft survival in renal transplant recipients, is often nephrotoxic. The clinical distinction between acute rejection and CsA nephrotoxicity (NT) is a common challenge in the management of these patients. To seek a histologic distinction between acute rejection and CsA-NT, we reviewed the renal biopsies performed prior to initiation of therapy for rejection or nephrotoxicity in two groups of patients. Group 1 (ten patients) had criteria consistent with acute rejection and responded to steroid pulse therapy. Group 2 (15 patients) was treated for CsA-NT and responded to a decrease in the dose of CsA. We conclude that CsA-NT has no specific histologic features. A prominent interstitial mononuclear cell infiltrate as well as tubulitis are features of acute cellular rejection. These findings do not exclude the possibility that rejection and CsA-NT can co-exist in the same patient.     

Investigation of Urinary Angiotensinogen in Renal Transplant Recipients

Background

Recent studies have indicated that angiotensinogen (AGT) is also locally produced in the kidney and that urinary AGT is a marker of local renal renin-angiotensin system activation. Because urinary AGT levels are significantly higher in patients with chronic kidney disease (CKD) than in patients without CKD and correlate with urinary albumin and other levels, urinary AGT is increasingly recognized as a marker for CKD monitoring, prognosis, and treatment. In this study, we investigated urinary AGT levels in renal transplant recipients.

Methods

Among the patients who were treated as outpatients at the Department of Urology of Osaka City University Hospital from March 2012 to April 2013, 146 stable renal transplant recipients and 50 donors who gave informed consent were studied. Urinary AGT and creatinine (Cr) levels were measured. The urinary AGT-to-Cr ratio was calculated, and its correlation with clinical parameters was examined.

Results

The urinary AGT-to-Cr ratio of the renal transplant recipients was significantly higher than that of the renal transplant donors (P = .0143). Furthermore, the urinary AGT-to-Cr ratio had a significantly positive correlation with the urinary albumin-to-Cr ratio (ACR; r = 0.39, P < .0001), while on the other hand, it had a significantly negative correlation with estimated glomerular filtration rate (eGFR; r = −0.31, P = .0002). Multiple linear regression analysis of factors associated with eGFR showed that urinary AGT was a significant and independent factor after adjusting for age, sex, and ACR.

Conclusions

Our results indicated that urinary AGT levels were elevated in renal transplant recipients. In addition, urinary AGT significantly correlated with renal function and degree of albuminuria.     

Matching T-Cell Receptors Identified in Renal Biopsies and Urine at the Time of Acute Rejection in Pediatric Renal Transplant Patients

Urinary monitoring of kidney allograft function has been used for many years. More recently, molecular identification of cytotoxic T-cell products has been used as a diagnostic tool in acute rejection. Monitoring of T-cell infiltrates by analysis of the T-cell receptor (TcR) gene usage has been performed on biopsies with acute and chronic rejection, but not on urine samples. The aim of this study was to identify and compare TRBV gene usage assessing the CDR3 (Complementarity Determining Region 3) length distribution and sequence in urine and biopsies of pediatric renal allograft patients at the time of acute rejection and compare them with peripheral blood. We studied four pediatric renal transplant recipients with acute cellular rejection. We identified restricted and matched TRBV CDR3 spectratypes with overexpressed TRBV families and show identical, clonally expanded TRBV CDR3 sequences in all four patients present in the urine and renal allograft. We demonstrate that urinary monitoring can detect graft-infiltrating lymphocytes in acute rejection and may have a role in the monitoring of renal transplants.     

Proteomic-Based Identification of Cleaved Urinary β2-microglobulin as a Potential Marker for Acute Tubular Injury in Renal Allografts

Our aim is to develop noninvasive tests to monitor the renal allograft posttransplant. Previously, we have reported that an unbiased proteomic-based approach can detect urine protein peaks associated with acute tubulointerstitial renal allograft rejection. Identification of these proteins peaks by mass spectrometry demonstrated that they all derive from nontryptic cleaved forms of β2-microglobulin. In vitro experiments showed that cleavage of intact β2-microglobulin requires a urine pH < 6 and the presence of aspartic proteases. Patients with acute tubulointerstitial rejection had lower urine pH than stable transplants and healthy individuals. In addition, they had higher amounts of aspartic proteases and intact β2-microglobulin in urine. These factors ultimately lead to increased amounts of cleaved urinary β2-microglobulin. Cleaved β2-microglobulin as an indicator of acute tubular injury may become a useful tool for noninvasive monitoring of renal allografts.     

Sarcomatoid Renal Cell Carcinoma in a Renal Transplant Recipient

The incidence of transplanted kidneys derived from elderly donors is increasing because of the larger waiting lists and greater age of patients with end-stage renal failure. Compared with young donors, one of the problems is the heightened risk of neoplasm transmission. We report 2 cases of kidney recipients, both of whom developed a sarcomatoid renal cell carcinoma after receiving a kidney transplant from the same 68-year-old male donor, who did not show signs of a neoplasm on a previous abdominal ultrasound or a pretransplant biopsy.The first recipient was a 66-year-old woman who developed a kidney mass with several urologic obstructive complications, tumor dissemination, and death at 9 months after kidney transplantation. The second recipient was a 48-year-old asymptomatic man with normal renal function, who was studied after the results of the first recipient, revealing another renal tumor. Transplant nephrectomy was performed and a peritoneal implant was resected. The patient is alive without evidence of a neoplasm after 18 months.Herein we have discussed the mechanisms of neoplasm transmission in kidney transplantation and possible strategies for its prevention and treatment.