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

Abstract

Background: The 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. Methods: Serine 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. Results: In-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. Conclusions: There 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.

A Urinary Proteomics Approach to Predict Treatment Response in Acute Antibody-Mediated Rejection

“…CPVL is a serine carboxypeptidase that is expressed in immune cells such as macrophages and monocytes and plays a role in phagocytic fragments digestion and antigen presentation [20]. It is also expressed in kidney tissue [21], and its presence in the urine of patients with acute rejection has been reported [22]. The specific role of this protein in acute graft rejection is still unknown.…”

Contribution of Proteomics in Transplantation: Identification of Injury and Rejection Markers

“…This study does stir the possibility that other enzymes may play critical roles in the rejection process. 62…”

“…This study does stir the possibility that other enzymes may play critical roles in the rejection process. 62 Perfusate has also been used to quantify and characterize the proteome to determine the quality of graft to reflect donor organ quality. For this study, the effect of warm ischemia time on the human donor kidney was compared between kidneys from DCD donors with controlled withdrawal or uncontrolled DCD donors after failed cardiopulmonary resuscitation and compared with kidneys from donation from brain-dead (DBD) donors that had relatively no warm ischemia.…”