Abstract
Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.
Highlights
Proteinuria is a hallmark of glomerular filtration barrier injury in both native and transplanted kidneys and is an independent prognostic factor for decline in kidney function, cardiovascular disease, and death [1,2,3].Urine excretion of complement factors was first documented in relation to proteinuria in animal models of kidney transplantation more than 50 years ago [4]
The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation and apical membrane attack in kidney transplant recipients
Proteinuria is associated with a higher risk of kidney failure and changes in albuminuria predicts kidney outcome in chronic kidney disease (CKD) [5,6,7,8]
Summary
Proteinuria is a hallmark of glomerular filtration barrier injury in both native and transplanted kidneys and is an independent prognostic factor for decline in kidney function, cardiovascular disease, and death [1,2,3]. Urine excretion of complement factors (complementuria) was first documented in relation to proteinuria in animal models of kidney transplantation more than 50 years ago [4]. Experimental evidence suggests that one or several filtered proteins are directly causing progressive kidney injury [9,10,11]. The rate-limiting step in complement activation is C3 conversion, which can be quantified by measuring C3 split products C3c and C3dg [14, 15]. Downstream activation and membrane attack involves C5b-8 formation on a cell surface and the incorporation of 10 –
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