Complement in ANCA-associated glomerulonephritis.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) are found in pauci-immune necrotizing crescentic glomerulonephritis. In the past, the role of complement in ANCA-associated vasculitis (AAV) was assumed to be minimal. More recently, however, it was found that blocking the complement cascade in a mouse model of AAV reduces glomerular damage. Immune complex deposits have been found in biopsies from AAV patients. In this study, we questioned whether immune complex formation or deposition may result in complement activation in ANCA-associated glomerulonephritis. METHODS: ANCA-positive patients from the Limburg Renal Registry were included between 1979 and 2011. Renal histology was documented together with immunoglobulin and complement immunofluorescence. In addition, C3d, properdin, C4d and mannose-binding lectin (MBL) were stained. Electron microscopy was performed. Circulating immune complexes were determined in a subset of patients, as well as C3 allotypes. RESULTS: C3c was found in 78 of 187 renal biopsies (41.7%) divided over 32.3% of proteinase-3 (PR3)-AAV patients and 52.3% of myeloperoxidase (MPO)-AAV patients (P = 0.006), whereas C3d was found positive in 51.1% of PR3-AAV patients and 70.4% of MPO-AAV patients (P = 0.105). C4d was found positive in 70.8%, properdin in 38.7% and MBL in 30.4% of patients. Whereas C4d and MBL positivity was similar between the AGN groups, properdin was more common in biopsies classified as crescentic compared with biopsies classified as focal or mixed. Renal biopsies positive for C3d and/or properdin showed more cellular crescents and less normal glomeruli compared with biopsies negative for C3d and/or properdin (P < 0.05). In 3 out of 43 renal biopsies analysed by electron microscopy, small electron dense deposits were found. In 14 of 46 patients analysed, circulating immune complexes were detectable. No association between histological findings and C3 allotypes was found. CONCLUSIONS: In the majority of AAV patients, no immune complex deposits were found in their renal biopsies. C3d, C4d and C5b-9 staining, however, was found to be positive in a majority of analysed renal biopsies. Importantly, C3d and properdin staining was associated with cellular crescents. We hypothesize that local immune complexes are quickly degraded in AAV and therefore not visible by electron microscopy. Our findings are compatible with the hypothesis that complement activation in AAV occurs predominantly via alternative pathway activation.

DNA extraction from long-term stored urine.

BACKGROUND: Traditionally, for DNA analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly, this option is often not considered. To complete our DNA database in patients with ANCA-associated vasculitis, we aimed to extract DNA from stored urine. METHODS: Urine was stored at the time of kidney biopsy from patients included in our regional kidney biopsy database, who had given informed consent for further study. Urine was subsequently filtered, dialyzed, concentrated and freeze dried and finally resolubilized and centrifuged. DNA was extracted using the high pure PCR template preparation kit (Roche Diagnostics). Next, concentration and purity were determined by Nanodrop analysis and by Quant-iT analysis. RESULTS: One hundred and eighty-one patients with ANCA-associated vasculitis were included. Of 114 patients (63%), DNA was available. From 53 of the remaining 67 patients, stored urine was available. Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7 ng/µL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280). CONCLUSION: DNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4 °C or lower temperatures results in significant degradation of the DNA, making recovery of DNA more difficult with longer periods of storage. In the current study, we demonstrated that DNA could be retrieved from subsequently filtered, dialyzed, concentrated and freeze dried urine that was stored at room temperature. In addition, we demonstrated that this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.

Dendritic cells in renal biopsies of patients with ANCA-associated vasculitis.

BACKGROUND: Dendritic cells (DCs) maintain immune tolerance and are able to initiate immune responses. Their involvement in ANCA-associated vasculitis (AAV) is unknown. In this study, the participation of DC subsets is investigated in renal biopsies of AAV patients. METHOD: A total of 25 patients with biopsy-proven AAV and five healthy controls (HC) with normal renal histology were included. Renal biopsies were stained for mature (CD208), immature (CD209), plasmacytoid (CD303) and Langerhans (CD1a) DC subsets. Furthermore, T-cells were stained using a T-cell marker (CD3). The interstitial cellular infiltrate was graded semi-quantitatively from 0+ (= absence of cells) to 3+ (= numerous cells). Within the glomeruli, an absolute count was performed for positive cells. RESULTS: CD208+ and CD209+ cells were found within patients' glomeruli but not in HC (1 +/- 0.3 versus 0.08 +/- 0.1 cells/glom; 2 +/- 0.3 versus 0.1 +/- 0.07 cells/glom). An average of 0.3 +/- 0.1 cell/glom expressed CD3 in patients while few cells were found in HC (0.1 +/- 0.7 cell/glom). Focal interstitial cellular infiltrates were observed in patients' biopsies but not in HC. Interstitial infiltration with CD3+ and CD209+ cells was assessed at an average of 1+, but some glomeruli and tubuli were surrounded by CD3+ and CD209+ cells forming clusters. Serial sections revealed that CD209+ cells were present in CD3+ rich areas. CONCLUSION: Both mature and immature glomerular DCs are found in renal biopsies of patients with AAV. Immature DCs cluster with T-cells in interstitial infiltrates in these biopsies. Since DCs form aggregates in T-cell areas, we hypothesize that these cells interact with each other and are involved in lymphoid neogenesis.

Signs and symptoms of thin basement membrane nephropathy: a prospective regional study on primary glomerular disease-The Limburg Renal Registry.

BACKGROUND: To chart the epidemiology of primary glomerular disease by means of a prospective regional study in the southern part of The Netherlands. METHODS: Experienced renal technicians collected renal biopsies, blood, and 24-hour urine samples at the bed site in each of the participating hospitals. The material was processed and analyzed at the University Hospital Maastricht. Analysis included light microscopy, immunohistochemistry, and electron microscopy of the biopsies as well as serologic and chemical analysis. RESULTS: Primary IgA nephropathy (IgAN), membranous glomerulopathy, antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis and thin basement membrane nephropathy (TBMN) are the most common primary glomerular diseases in this order of sequence. Our data show the clinical and histologic phenotype of TBMN to be diverse: the vast majority of TBMN has chronic microscopic hematuria, frequently associated with hypertension in late middle age; about 15% of TBMN has in addition substantial proteinuria which is associated in the majority of cases with the lesions of focal segmental glomerulosclerosis (FSGS). In 5% of TBMN a nephrotic syndrome is observed, occasionally associated with FSGS tip lesions. CONCLUSION: These results support the notion that TBMN is a disease of genetic heterogeneity; it is not a benign renal condition in a substantial number of patients, particularly those in late middle age.