Mirjan M. van Timmeren

Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

BACKGROUND: Chronic transplant dysfunction is characterized by renal function decline and proteinuria. Kidney injury molecule (KIM)-1, a transmembrane tubular protein with unknown function, is undetectable in normal kidneys, but markedly induced after injury. Urinary KIM-1 excretion has been quantified as biomarker of renal damage. We prospectively studied whether urinary KIM-1 predicts graft loss, independent of renal function and proteinuria. METHODS: Renal transplant recipients (n=145) visiting our outpatient clinic between August 2001 and July 2003 collected 24-hour urine samples for assessment of baseline urinary KIM-1 excretion (microsphere-based Luminex technology), and were followed for graft loss. RESULTS: Recipients participated at a median (interquartile range) of 6.0 (2.5-12.0) years posttransplant in baseline measurements. Follow-up beyond baseline was 4.0 (3.2-4.5) years. Urinary KIM-1 excretion was 0.72 (0.42-1.37) ng per 24 hours. Occurrence of graft loss increased over tertiles of KIM-1 excretion: 3 (6.3%), 11 (22.4%), and 17 cases (35.4%; P=0.001), respectively. High KIM-1 excretion was associated with proteinuria, low creatinine clearance, and high donor age (all P<0.01). In multivariate Cox regression analyses, prediction of graft loss by KIM-1 appeared independent of creatinine clearance, proteinuria, and donor age. Hazard ratios (95% CI) for the second and third tertile of KIM-1 excretion were 3.6 (0.9-13.5) and 5.1 (1.5-17.8) in the final model. CONCLUSIONS: Urinary excretion of KIM-1 is an independent predictor of long-term graft loss and therefore a promising new biomarker in early prediction of graft loss.

Kim-1, a recently discovered membrane protein, is undetectable in normal kidneys but markedly induced in proximal tubules after ischemic and toxic injury. The function of Kim-1 is unclear, but it is implicated in damage/repair processes. The Kim-1 ectodomain is cleaved by metalloproteinases and detectable in urine. We studied Kim-1 in a nontoxic, nonischemic, model of tubulointerstitial damage caused by acute proteinuria. Uninephrectomized (NX) rats received daily (ip) injections of 2 g BSA (NX+BSA, n = 12) or saline (NX, n = 6) for 3 wk. Kidneys were stained for various damage markers by immunohistochemistry (IHC). Kim-1 mRNA (RT-PCR, in situ hybridization), protein (IHC, Western blotting), and urinary Kim-1 (Luminex) were determined. Spatial relations between Kim-1 and other damage markers were studied by double labeling IHC. NX+BSA rats developed massive proteinuria (1,217 +/- 313 vs. 18 +/- 2 mg/day in NX, P < 0.001) and significant renal damage. Kim-1 mRNA was upregulated eightfold in NX+BSA (ratio Kim-1/beta-actin, 4.08 +/- 2.56 vs. 0.52 +/- 0.64 in NX, P < 0.001) and localized to damaged tubules. Kim-1 protein expression was markedly induced in NX+BSA (2.46 +/- 1.19 vs. 0.39 +/- 0.10% staining/field in NX, P < 0.001). Urinary Kim-1 was significantly elevated in NX+BSA (921 +/- 592 vs. 87 +/- 164 pg/ml in NX, P < 0.001) and correlated with tissue Kim-1 expression (r = 0.66, P =0.02). Kim-1 protein was found at the apical membrane of dilated nephrons. Kim-1 expression was limited to areas with inflammation (MØ), fibrosis (alpha-smooth muscle actin), and tubular damage (osteopontin), and only occasionally with tubular dedifferentiation (vimentin). These results implicate involvement of Kim-1 in the pathogenesis of proteinuria-induced renal damage/repair. Urinary Kim-1 levels may serve as a marker of proteinuria-induced renal damage.

Kidney injury molecule-1 (KIM-1) is a marker for renal proximal tubular damage, the hallmark of virtually all proteinuric, toxic and ischaemic kidney diseases. KIM-1 has gained increasing interest because of its possible pathophysiological role in modulating tubular damage and repair. In this respect, it is interesting that the best biomarkers often turn out to be important in modulation of damage and some even become therapeutic targets. The emphasis of this review is on structural and biochemical aspects of KIM-1, its expression pattern and its pathophysiological role in renal disease. We also discuss the prognostic impact of KIM-1 in relation to urinary protein excretion. Glomerular (proteinuria) and interstitial markers (KIM-1) might have independent prognostic impact and so may provide independent treatment targets. Finally, the potential of KIM-1 as biomarker of renal damage, as a predictor of renal function decline and its perspectives for monitoring therapy response, are discussed.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (Pr3) are considered pathogenic in ANCA-associated necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. Modulation of ANCA IgG glycosylation may potentially reduce its pathogenicity by abolishing Fc receptor-mediated activation of leukocytes and complement. Here, we investigated whether IgG hydrolysis by the bacterial enzyme endoglycosidase S (EndoS) attenuates ANCA-mediated NCGN. In vitro, treatment of ANCA IgG with EndoS significantly attenuated ANCA-mediated neutrophil activation without affecting antigen-binding capacity. In a mouse model of anti-MPO IgG/LPS-induced NCGN, we induced disease with either unmodified or EndoS-treated (deglycosylated) anti-MPO IgG. In separate experiments, we administered EndoS systemically after disease induction with unmodified anti-MPO IgG. Pretreatment of anti-MPO IgG with EndoS reduced hematuria, leukocyturia, and albuminuria and attenuated both neutrophil influx and formation of glomerular crescents. After inducing disease with unmodified anti-MPO IgG, systemic treatment with EndoS reduced albuminuria and glomerular crescent formation when initiated after 3 but not 24 hours. In conclusion, IgG glycan hydrolysis by EndoS attenuates ANCA-induced neutrophil activation in vitro and prevents induction of anti-MPO IgG/LPS-mediated NCGN in vivo. Systemic treatment with EndoS early after disease induction attenuates the development of disease. Thus, modulation of IgG glycosylation is a promising strategy to interfere with ANCA-mediated inflammatory processes.