Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Complement analyses

Article Snippet: Primary antibodies were polyclonal rabbit anti-C3c (#A0062; Dako, Glostrup, Denmark); polyclonal rabbit anti-C3d (#136916; Abcam, Cambridge, UK) that recognizes C3dg; polyclonal rabbit anti-properdin (#2097; Biorbyt, Cambridge, UK); monoclonal mouse anti-C5b9 (#M0777; Dako); polyclonal rabbit anti-C4d (#107-01; BD Biotech, Franklin Lakes, NJ); monoclonal rabbit anti-C1q (#A0136; Dako); monoclonal mouse anti-factor H, OX-24 (#118820; Abcam); monoclonal mouse anti-FHR1 (#3078-M01; Abnova, Taipei, Taiwan); polyclonal rabbit anti-FHR5 (#81494-D01P; Abnova); and monoclonal mouse anti-CD68 (#M0876; Dako).

Techniques: Diagnostic Assay, Variant Assay

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Glomerular FHR5 staining in C3 glomerulopathy. (a) Glomerular staining intensity in native and transplant biopsies. FHR5 was the most frequent protein at 3+ intensity and detected in all of the transplant biopsies. (b) Representative images of complement staining in the native kidney. For each set of images, the biopsy indication, the appearances of the glomeruli by light microscopy, and the estimated glomerular filtration rate (eGFR) and proteinuria at the time of biopsy are indicated. *The cases represented by the fourth and fifth rows of images are from the same family. (c) Representative images of complement staining in transplant kidneys. For each set of images, the biopsy indication (either protocol or due to clinical changes; i.e., indication biopsy), time posttransplant together with the eGFR, and proteinuria at the time of biopsy are indicated. The histology including the presence or absence of rejection and the histology of subsequent biopsies is also shown. C3G, C3 glomerulopathy; C3GN, C3 glomerulonephritis; EH, endocapillary hypercellularity; fH, factor H; FHR1, factor H–related protein 1; FHR5, factor H–related protein 5; FHR5 mutation, the mutation described in CFHR5 nephropathy ; MPGN, membranoproliferative glomerulonephritis; NS, nephrotic syndrome; UPCR, urinary protein:creatinine ratio. Original magnification ×400. Bars = 100 μm.

Article Snippet: Primary antibodies were polyclonal rabbit anti-C3c (#A0062; Dako, Glostrup, Denmark); polyclonal rabbit anti-C3d (#136916; Abcam, Cambridge, UK) that recognizes C3dg; polyclonal rabbit anti-properdin (#2097; Biorbyt, Cambridge, UK); monoclonal mouse anti-C5b9 (#M0777; Dako); polyclonal rabbit anti-C4d (#107-01; BD Biotech, Franklin Lakes, NJ); monoclonal rabbit anti-C1q (#A0136; Dako); monoclonal mouse anti-factor H, OX-24 (#118820; Abcam); monoclonal mouse anti-FHR1 (#3078-M01; Abnova, Taipei, Taiwan); polyclonal rabbit anti-FHR5 (#81494-D01P; Abnova); and monoclonal mouse anti-CD68 (#M0876; Dako).

Techniques: Staining, Light Microscopy, Filtration, Mutagenesis

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Sequential glomerular FHR5 staining in a single case of C3 glomerulopathy transplant recurrence. Images and information from the same biopsy are organized in columns. A protocol surveillance biopsy performed 3 months after transplantation (Transplant biopsy 1) showed recurrent C3 glomerulonephritis (C3GN) with mild endocapillary hypercellularity (EH). Glomerular factor H–related protein 5 (FHR5), C5b9, C3b/iC3b/C3c, and C3dg were detected. The estimated glomerular filtration rate (eGFR) was stable and there was no significant proteinuria. Approximately 6 months posttransplant, the patient developed proteinuria and a fall in eGFR. Biopsy showed crescentic C3GN and increased glomerular CD68-positive cells (Transplant biopsy 2). Glomerular FHR5, C5b9, C3b/iC3b/C3c, and C3dg were detected but at increased staining intensity compared with the first transplant biopsy. The eGFR and proteinuria improved with eculizumab and prednisolone treatment. After 4 months of eculizumab treatment, renal biopsy (Transplant biopsy 3) showed resolution of glomerular CD68 staining, but glomerular staining for FHR5, C5b9, C3b/iC3b/C3c, and C3dg remained unchanged. After a further 3 months, proteinuria increased and biopsy showed crescentic C3GN and the recurrence of glomerular CD68-positive cells (Transplant biopsy 4). Proteinuria improved with re-introduction of eculizumab. UPCR, urine protein:creatinine ratio. Original magnification ×200 or ×400. Bars = 100 μm.

Article Snippet: Primary antibodies were polyclonal rabbit anti-C3c (#A0062; Dako, Glostrup, Denmark); polyclonal rabbit anti-C3d (#136916; Abcam, Cambridge, UK) that recognizes C3dg; polyclonal rabbit anti-properdin (#2097; Biorbyt, Cambridge, UK); monoclonal mouse anti-C5b9 (#M0777; Dako); polyclonal rabbit anti-C4d (#107-01; BD Biotech, Franklin Lakes, NJ); monoclonal rabbit anti-C1q (#A0136; Dako); monoclonal mouse anti-factor H, OX-24 (#118820; Abcam); monoclonal mouse anti-FHR1 (#3078-M01; Abnova, Taipei, Taiwan); polyclonal rabbit anti-FHR5 (#81494-D01P; Abnova); and monoclonal mouse anti-CD68 (#M0876; Dako).

Techniques: Staining, Transplantation Assay, Filtration

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Glomerular factor H–related protein 5 (FHR5) colocalizes with C3 in C3 glomerulopathy. (a) Glomerular FHR5 staining intensity positively correlated with the staining intensity of C3b/iC3b/C3c, C3dg, and C5b9. Both glomerular C3b/iC3b/C3c and C3dg positively correlated with glomerular C5b9. R values are calculated from Pearson’s correlation coefficient and P values are adjusted for multiple comparisons. (b) Representative images of combined immunofluorescence staining in three C3G cases for glomerular FHR5 with either C3b/iC3b/C3c or C3dg, and C3b/iC3b/C3c with C3dg. Renal biopsies in all 3 cases showed C3-dominant membranoproliferative glomerulonephritis, and the biopsy indications together with the urine protein:creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), and serum C3 levels at the time of biopsy are listed. The staining patterns for C3b/iC3b/C3c and C3dg (right-hand column of images) showed areas of colocalization (arrows), areas of C3b/iC3b/C3c alone (stars), and areas of C3dg alone (triangles). The staining pattern for C3b/iC3b/C3c and FHR5 (left-hand column of images) showed areas of colocalization (arrows), areas of C3b/iC3b/C3c alone (stars), and areas of FHR5 alone (triangles). The staining pattern for C3dg and FHR5 (middle column of images) showed areas of colocalization, particularly along capillary walls in cases 2 and 3 (arrows) and areas of FHR5 alone in cases 1 and 2 (triangles). Notably, we did not detect areas of C3dg without FHR5 staining. (c) FHR5, C3b/iC3b/C3c, and C3dg glomerular locations correlate in C3G. We calculated the correlation of glomerular antigen locations in all available glomeruli from the three C3G cases (c). The Pearson’s correlation coefficient for all glomeruli from cases 1 (circles), 2 (squares), and 3 (triangles), and the median values of the correlations for each case (horizontal lines) are shown. The median values of the correlation coefficients ( r ) for each case were as follows: C3b/iC3b/C3c with FHR5: 0.73 (case 1), 0.76 (case 2), and 0.68 (case 3); FHR5 with C3dg: 0.71 (case 1), 0.90 (case 2), and 0.5 (case 3); and C3b/iC3b/C3c with FHR5: 0.68 (case 1), 0.81 (case 2), and 0.58 (case 3). MMF, mycophenolate mofetil. Original magnification ×400. Bars = 100 μm.

Article Snippet: Primary antibodies were polyclonal rabbit anti-C3c (#A0062; Dako, Glostrup, Denmark); polyclonal rabbit anti-C3d (#136916; Abcam, Cambridge, UK) that recognizes C3dg; polyclonal rabbit anti-properdin (#2097; Biorbyt, Cambridge, UK); monoclonal mouse anti-C5b9 (#M0777; Dako); polyclonal rabbit anti-C4d (#107-01; BD Biotech, Franklin Lakes, NJ); monoclonal rabbit anti-C1q (#A0136; Dako); monoclonal mouse anti-factor H, OX-24 (#118820; Abcam); monoclonal mouse anti-FHR1 (#3078-M01; Abnova, Taipei, Taiwan); polyclonal rabbit anti-FHR5 (#81494-D01P; Abnova); and monoclonal mouse anti-CD68 (#M0876; Dako).

Techniques: Staining, Immunofluorescence, Filtration

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Glomerular factor H–related protein 1 (FHR5) and C5b9 staining intensity associated with lower estimated glomerular filtration rate (eGFR) at biopsy in C3 glomerulopathy. The eGFR at the time of biopsy was significantly lower in biopsies that had maximal staining intensities for FHR5 ( P = 0.04, difference of medians 19.7 ml/min per 1.73 m 2 ; 95% confidence interval [CI] 1.1–43.0) and C5b-9 ( P = 0.03, difference of medians 14.86 ml/min per 1.73 m 2 ; 95% CI 3.8–46.6). This was not seen for glomerular factor H–related protein 1 (FHR1), C3b/iC3b/C3c, C3dg, and C4d. P values are derived from Mann-Whitney U tests.

Article Snippet: Primary antibodies were polyclonal rabbit anti-C3c (#A0062; Dako, Glostrup, Denmark); polyclonal rabbit anti-C3d (#136916; Abcam, Cambridge, UK) that recognizes C3dg; polyclonal rabbit anti-properdin (#2097; Biorbyt, Cambridge, UK); monoclonal mouse anti-C5b9 (#M0777; Dako); polyclonal rabbit anti-C4d (#107-01; BD Biotech, Franklin Lakes, NJ); monoclonal rabbit anti-C1q (#A0136; Dako); monoclonal mouse anti-factor H, OX-24 (#118820; Abcam); monoclonal mouse anti-FHR1 (#3078-M01; Abnova, Taipei, Taiwan); polyclonal rabbit anti-FHR5 (#81494-D01P; Abnova); and monoclonal mouse anti-CD68 (#M0876; Dako).

Techniques: Staining, Filtration, Derivative Assay, MANN-WHITNEY

Journal: Kidney International Reports

Article Title: Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

doi: 10.1016/j.ekir.2019.06.008

Figure Lengend Snippet: Tubulo-interstitial staining for FHR5 in C3 glomerulopathy. (a) Representative images of staining for factor H–related protein 5 (FHR5), factor H–related protein 1 (FHR1), factor H (fH), properdin, and C3b/iC3b/C3c. No tubulo-interstitial staining for FHR5 or fH was evident but there was strong tubulo-interstitial staining for both properdin and FHR1. (b) Tubulo-interstitial staining for properdin was also seen in other renal diseases (thin basement membrane disease [TBM], lupus nephritis [LN], and membranous nephropathy). Tubulo-interstitial staining for FHR1 was demonstrable in biopsies from patients with TBM and IgA nephropathy. Tubulo-interstitial FHR1 staining in TBM was still detectable but less intense when the proteinase XXIV enzyme was used instead of pressure cooker antigen retrieval. Glomerular and tubulo-interstitial FHR1 was absent in renal tissue from a patient with IgA nephropathy and FHR1 deficiency. C3G, C3 glomerulopathy; LN IV(A/C), lupus nephritis class 4, active and chronic. Original magnification ×200. Bars = 100 μm.

Article Snippet: Primary antibodies were polyclonal rabbit anti-C3c (#A0062; Dako, Glostrup, Denmark); polyclonal rabbit anti-C3d (#136916; Abcam, Cambridge, UK) that recognizes C3dg; polyclonal rabbit anti-properdin (#2097; Biorbyt, Cambridge, UK); monoclonal mouse anti-C5b9 (#M0777; Dako); polyclonal rabbit anti-C4d (#107-01; BD Biotech, Franklin Lakes, NJ); monoclonal rabbit anti-C1q (#A0136; Dako); monoclonal mouse anti-factor H, OX-24 (#118820; Abcam); monoclonal mouse anti-FHR1 (#3078-M01; Abnova, Taipei, Taiwan); polyclonal rabbit anti-FHR5 (#81494-D01P; Abnova); and monoclonal mouse anti-CD68 (#M0876; Dako).

Techniques: Staining

Journal: Kidney International Reports

Article Title: Progressive IgA Nephropathy Is Associated With Low Circulating Mannan-Binding Lectin–Associated Serine Protease-3 (MASP-3) and Increased Glomerular Factor H–Related Protein-5 (FHR5) Deposition

doi: 10.1016/j.ekir.2017.11.015

Figure Lengend Snippet: Glomerular staining for alternative pathway regulators in IgA nephropathy (IgAN). (a) Representative images for complement factor H (fH), and factor H–related proteins 1 (FHR1) and 5 (FHR5). The top row represents positive staining (+) and the bottom row represents negative staining (−). Original magnification ×400. Bar = 100 μm. (b) The proportion of cases with positive (black) versus absent/minimal (gray) glomerular staining in stable and progressive IgAN. P values derived from the Fisher exact test.

Article Snippet: Immunohistochemistry protocols were optimized ( ) for formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rabbit polyclonal anti-human C4d (DB Biotech, Kosice, Slovakia), mouse monoclonal anti-human factor H (OX-24; Abcam, Cambridge, UK), rabbit polyclonal anti-human C3d (Abcam), mouse monoclonal anti-human C5b9 (Dako), mouse monoclonal anti-human FHR1 (Abnova, Taipei, Taiwan), and rabbit polyclonal anti-human FHR5 (Abnova).

Techniques: Staining, Negative Staining, Derivative Assay

Journal: Kidney International Reports

Article Title: Progressive IgA Nephropathy Is Associated With Low Circulating Mannan-Binding Lectin–Associated Serine Protease-3 (MASP-3) and Increased Glomerular Factor H–Related Protein-5 (FHR5) Deposition

doi: 10.1016/j.ekir.2017.11.015

Figure Lengend Snippet: Complement glomerular deposition in IgA nephropathy (IgAN). Glomerular staining intensity scores from surplus native renal biopsy tissue from patients with either stable or progressive IgAN. Each row represents information from a single patient. Staining intensity was scored: 0, absent; 0.5, minimal; 1, mild; 2, moderate; 3, strong. Filled cells indicate insufficient renal tissue to perform staining. Mannan-binding lectin (MBL) deficiency was defined as a plasma level of less than 100 ng/ml. CFHR3-1 , complement factor H–related 3 and 1 genes; fH, complement factor H; FHR1, factor H–related protein 1; FHR5, factor H–related protein 5.

Article Snippet: Immunohistochemistry protocols were optimized ( ) for formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rabbit polyclonal anti-human C4d (DB Biotech, Kosice, Slovakia), mouse monoclonal anti-human factor H (OX-24; Abcam, Cambridge, UK), rabbit polyclonal anti-human C3d (Abcam), mouse monoclonal anti-human C5b9 (Dako), mouse monoclonal anti-human FHR1 (Abnova, Taipei, Taiwan), and rabbit polyclonal anti-human FHR5 (Abnova).

Techniques: Staining, Binding Assay

Journal: Kidney International Reports

Article Title: Progressive IgA Nephropathy Is Associated With Low Circulating Mannan-Binding Lectin–Associated Serine Protease-3 (MASP-3) and Increased Glomerular Factor H–Related Protein-5 (FHR5) Deposition

doi: 10.1016/j.ekir.2017.11.015

Figure Lengend Snippet: Complement and IgA nephropathy. (a) Schematic diagram depicting lectin and alternative pathway complement activation. Lectin pathway activation is triggered by the binding of pattern-recognition molecules (PRMs) to carbohydrate or acetyl molecular patterns. Alternative pathway activation happens through the spontaneous, constant generation of reactive forms of C3. Both pathways result in the generation of C3b. C3b can be proteolytically cleaved to iC3b and C3d by complement factor I in the presence of cofactors, such as complement factor H. Similarly, C4b produced during lectin pathway activation can be cleaved to C4d. C3b generation can be rapidly amplified through an amplification loop. This results in the generation of large amounts of the opsonin C3b and can trigger complement C5 activation. This leads to the generation of the anaphylatoxin C5a, and the membrane attack complex (C5b-9) through the terminal pathway. fH, factor H; fI, factor I; MAp, MBL-associated protein; MASP, MBL-associated serine protease; MBL, mannose-binding lectin. (b) Complement proteins and severity of IgA nephropathy. Within plasma, increased levels of FHR1, , FHR5, and the FHR1:fH ratio , associate with progressive IgAN. Conversely, we found that reduced levels of MASP-3 associated with progressive disease. Within glomeruli, we replicated the association between increased C4d , , and C3b/iC3b/C3c with IgAN severity, and we showed that increased glomerular C3d, C5b-9, and FHR5 associated with progressive disease. The presence of FHR1 in the absence of fH was also more frequently seen in patients with progressive disease. FHR1, factor H–related protein 1; FHR5, factor H–related protein 5. (c) Hypothetical depiction of glomerular complement activation in IgA nephropathy. Galactose-deficient IgA1 (gd-IgA1) activates the lectin and alternative complement pathways in IgAN. Glomerular complement deposition is enhanced in progressive disease. Glomerular complement activation is influenced by FHR5 and the FHR1-fH ratio and associated with changes in circulating MASP-3 levels. Changes in FHR1, FHR5, and fH influence complement activation through the alternative pathway. fH negatively regulates activation, whereas FHR1 and FHR5 promote activation through antagonizing fH (“fH de-regulation”). The mechanism through which circulating MASP-3 levels fall in progressive disease are not understood but are presumed to be linked to lectin pathway activation. Red text highlights proteins demonstrated to associate with progressive IgAN and larger boxes indicate more deposition.

Article Snippet: Immunohistochemistry protocols were optimized ( ) for formalin-fixed paraffin-embedded renal biopsy tissue with the following antibodies: rabbit polyclonal anti-human C3c (Dako, Glostrup, Denmark), rabbit polyclonal anti-human C4d (DB Biotech, Kosice, Slovakia), mouse monoclonal anti-human factor H (OX-24; Abcam, Cambridge, UK), rabbit polyclonal anti-human C3d (Abcam), mouse monoclonal anti-human C5b9 (Dako), mouse monoclonal anti-human FHR1 (Abnova, Taipei, Taiwan), and rabbit polyclonal anti-human FHR5 (Abnova).

Techniques: Activation Assay, Binding Assay, Produced, Amplification

Journal: Human immunology

Article Title: Complement factor H gene ( CFH ) polymorphisms C-257T, G257A and haplotypes are associated with protection against severe dengue phenotype, possible related with high CFH expression

doi: 10.1016/j.humimm.2013.05.005

Figure Lengend Snippet: The promoter C-257T variant locus and CFH gene expression. Complement factor H plasma levels in CC and CT/TT genotype patients, during dengue convalescence phase (p = 0.005).

Article Snippet: After washing, the plates were incubated for 1 h at 37 °C with mouse monoclonal antibody anti-human factor H (Abcam) followed by washing and one hour incubation at 37 °C with goat anti-mouse IgG-HRP (Jackson ImmunoResearch).

Techniques: Variant Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Factor H and Properdin Recognize Different Epitopes on Renal Tubular Epithelial Heparan Sulfate

doi: 10.1074/jbc.M112.380386

Figure Lengend Snippet: TABLE 1

Article Snippet: After washing in PBS with 0.05% Tween 20, the wells were blocked with 3% skimmed milk powder in PBS for 1 h. In a separate microtiter plate, factor H (2 μg/ml diluted in PBS) was incubated with a dilution range of different HS-like heparinoids (see below) for 30 min, then transferred to the ELISA plate washed wells, and incubated for 1 h. The wells were washed again, and monoclonal mouse anti-human factor H antibody (2 μg/ml; Abcam, Cambridge, UK) diluted in PBS was added for 1 h. Secondary antibody was added after a washing step (HRP-labeled goat anti-mouse immunoglobulins, 1:250; DAKO, Glostrup, Denmark).

Techniques: Inhibition, Binding Assay, Derivative Assay, Modification