Kidney transplantation is the optimal treatment for end-stage kidney disease, but most grafts fail prematurely. Antibody-mediated rejection (AMR) accounts for >50% of graft loss. AMR is caused by antibodies against HLA and non-HLA antigens in two main renal compartments: glomeruli and tubulointerstitium. We hypothesized that compartment-specific proteome alterations may uncover the mechanisms of early antibody-mediated injury.
We performed laser-capture microdissection to isolate glomeruli and tubulointerstitium from FFPE kidney biopsies, and subjected unfractionated samples to label-free proteome analysis by LC-MS/MS on Q-Exactive-Plus mass spectrometer. Analyses were performed using MaxQuant, Perseus, IID and pathDIP. We compared 7 biopsies with AMR to 23 non-AMR biopsies with cellular rejection or acute tubular necrosis. Primary human glomerular microvascular endothelial cells (HGMEC) were studied in vitro.
We identified 2,026 proteins in glomeruli and 2,399 in tubulointerstitium (FDR=0.01). 120 proteins were differentially expressed in AMR vs. non-AMR glomeruli and 180 in the tubulointerstitium (p<0.05). Proteins involved in HLA-mediated antigen presentation were increased in AMR. Basement membrane and cytoskeletal proteins were significantly decreased in AMR. Reduced glomerular protein levels of LAMC1, NPHS1, and PTPRO in AMR were verified by immunostaining. Levels of basement membrane proteins correlated directly and significantly (R>0.7; p<0.05), suggesting co-regulation in AMR. Protein expression of CCT8 (cytoskeleton dynamics) and CALU (protein folding) correlated with histological features of AMR, namely glomerulitis and peritubular capillaritis (p=0.017). Protein-protein interaction and comprehensive pathway analysis of our glomerular protein signature revealed enrichment of inflammatory pathways, such as IL-8 signaling. Stimulation of HGMECs with anti-HLA class I antibody increased the secretion of IL-8 and MCP-1 cytokines(p<0.05).
Basement membranes are often remodeled in late chronic AMR and are the targets of non-HLA antibodies, suggesting that our findings may represent early, important alterations in AMR. Targeting early basement membrane remodeling in AMR may represent a new therapeutic opportunity.