Urine Of Transplant Patients Research Articles

Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation

BackgroundThe pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection.MethodsSerine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine.ResultsIn-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group.ConclusionsThere appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

Comparison of Bacteria Isolated From Urinary Tract Infections in Patients on Transplant Versus Urologic Wards

Objective Urinary tract infections (UTI) are the most common hospital infections. Complications include sepsis and shock. Immunosuppressed transplant surgery patients may experience loss of the graft due to UTI. The purpose of this study was to determine the main microorganisms responsible for UTIs among patients of transplant wards compared to urologic wards. Additionally, drug susceptibility profiles of the most frequent microorganisms were analyzed. Materials and Methods We analyzed the results of positive urine cultures from patients on 2 transplant versus 1 urologic ward in 2010. Results The most common pathogen in urine samples from all 3 wards was Escherichia coli. Often, other Gram-negative bacilli of the genus Klebsiella spp, were cultured as well as Gram-positive cocci ( Enterococcus spp). Yeasts of the genus Candida were only found in urine of patients of transplant wards. The percentage of resistant bacteria was much higher among bacteria from transplant patients. Conclusions The high level of antimicrobial resistance of microorganisms isolated from the urine of transplant patients and the relatively high incidence of fungal infections, demand an especially quick, accurate microbiological diagnosis for this group of patients.

Anti-IL-2 receptor antibody decreases cytokine-induced apoptosis of human renal tubular epithelial cells (TEC)

Transplant rejection is mediated by T-cell activation which is modulated by interleukin-2 (IL-2) binding to IL-2R (CD25). Monoclonal anti-IL-2 receptor antibody is used in renal transplantation to reduce rejection. Interestingly, proximal tubular epithelial cells (TEC) express CD25, similar to T cells. We have demonstrated that IL-2 induces murine TEC apoptosis through down-regulation of the caspase-8 inhibitor protein c-FLIP. Anti-CD25 antibody may be useful clinically to limit renal injury, but this has not been tested in human TEC. Human PT-2 TEC were isolated and cloned from the urine of transplant patients. Apoptosis was determined by FACS with Annexin-V FITC. Protein expression was studied using western blot, and mRNA levels by quantitative real-time (PR-PCR). We demonstrated that the morphology of a human kidney cell line (PT-2) cloned from urine was consistent with proximal TEC and expresses alkaline phosphatase, cytokeratin, vimentin, CD13, CD26, and low levels of E-cadherin. Basal IL-2 receptor (CD25) was up-regulated by IL-2/IFN-γ stimulation, and cytokine exposure induced apoptosis in a dose-dependent manner. Apoptosis with IL-2/IFN-γ was associated with increased caspase-8 activity and decreased endogenous caspase-8 inhibitor c-FLIP mRNA and protein expression. IL-2/IFN-γ-induced apoptosis could be blocked by pre-treatment of PT-2 with anti IL-2R antibody (basiliximab) but not control IgG antibody. These data demonstrate for the first time in human TEC that IL-2 and IFN-γ can induce TEC apoptosis which can be blocked by CD25 blockade antibody. These data suggest that anti-CD25 mAb might similarly attenuate inflammation-induced TEC injury in vivo. Kidney-expressed CD25 may represent a clinically important new target for attenuating early inflammatory injury in donor kidneys and preserving renal function during anti-rejection therapy.

Quantitative Detection of Promoter Hypermethylation as a Biomarker of Acute Kidney Injury During Transplantation

Aberrant promoter hypermethylation, also known as epigenetics, is thought to be a promising biomarker approach to diagnose malignancies. Kidney repair after injury is a recapitulation of normal morphogenesis, with similarities to malignant transformation. We hypothesized that changes in urine epigenetics could be a biomarker approach during early kidney transplant injury and repair. We examined urine DNA for aberrant methylation of two gene promoters ( DAPK and CALCA) by quantitative methylation-specific polymerase chain reaction from 13 deceased and 10 living donor kidney transplant recipients on postoperative day 2 and 65 healthy controls. Results were compared with clinical outcomes and to results of the kidney biopsy. Transplant recipients were significantly more likely to have aberrant hypermethylation of the CALCA gene promoter in urine than healthy controls (100% vs 31%; P < .0001). There was increased CALCA hypermethylation in the urine of deceased versus living donor transplants (21.60 ± 12.5 vs 12.19 ± 4.7; P = .04). Furthermore, there was a trend toward increased aberrant hypermethylation of urine CALCA in patients with biopsy-proven acute tubular necrosis versus acute rejection and slow or prompt graft function (mean: 20.40 ± 6.9, 13.87 ± 6.49, 17.17 ± 13.4; P = .67). However, there was no difference of CALCA hypermethylation in urine of patients with delayed graft function versus those with slow or prompt graft function (16.9 ± 6.2 vs 18.5 ± 13.7, respectively; P = .5). There was no aberrant hypermethylation of DAPK in the urine of transplant patients. Urine epigenetics is a promising biomarker approach for acute ischemic injury in transplantation that merits future study.