Background:
Chronic lung allograft dysfunction (CLAD) remains the leading cause of long-term mortality after lung transplant. The two main phenotypes of CLAD, bronchiolitis obliterans syndrome (BOS) and Restrictive allograft syndrome (RAS), are both characterized by chronic inflammation and fibrosis. Finding early markers of CLAD and identifying new therapeutic targets is a key goal to improve survival in this population. Angiotensin II (AngII), the main effector of the renin-angiotensin-aldosterone system (RAAS), is involved in fibrogenesis in the kidney equivalent of CLAD, interstitial fibrosis and tubular atrophy (IFTA). We have previously identified AngII-regulated proteins that are elevated in urine and kidneys of patients with IFTA. RAAS is involved in CLAD pathophysiology. We hypothesize that AngII-regulated proteins will be elevated in bronchoalveolar lavage (BAL) and lung tissue of CLAD patients, compared to stable controls.
Methods:
For the measurement of AngII-regulated proteins in BAL, we have developed parallel reaction monitoring (PRM) assays using Q-Exactive plus instrument. Two proteotypic peptides of each protein (TSP1, HMOX1, RHOB, BST1, GLNA, LAMB2, LYPA1) were selected and heavy isotope-labeled synthetic peptides were used for their absolute quantification. We performed immunohistochemical/immunofluorescence staining of TSP1 and AngII receptors in CLAD lungs and 1 control.
Results:
Using PRM assays, we detected 2 native peptides of TSP1, BST1, GLNA and LYPA1 protein and one of RHOB in a pool of BAL from controls and patients with CLAD. We constructed calibration curves and determined LOD and LOQ. In our preliminary analysis, AngII receptors and TSP1 were identified in characteristic lesions of CLAD.
Conclusion:
These preliminary data suggest that RAAS is expressed in CLAD lungs and that AngII-regulated proteins can be measured in BAL. To further these findings, we will quantify AngII-regulated proteins in BAL samples and CLAD lungs from a larger cohort of lung transplant patients.