Gain-of-function factor H-related 5 protein impairs glomerular complement regulation resulting in kidney damage.

Genetic variation within the factor H-related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury.

O- and N-Glycosylation of Serum Immunoglobulin A is Associated with IgA Nephropathy and Glomerular Function.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and is a leading cause of renal failure. The disease mechanisms are not completely understood, but a higher abundance of galactose-deficient IgA is recognized to play a crucial role in IgAN pathogenesis. Although both types of human IgA (IgA1 and IgA2) have several N-glycans as post-translational modification, only IgA1 features extensive hinge-region O-glycosylation. IgA1 galactose deficiency on the O-glycans is commonly detected by a lectin-based method. To date, limited detail is known about IgA O- and N-glycosylation in IgAN. METHODS: To gain insights into the complex O- and N-glycosylation of serum IgA1 and IgA2 in IgAN, we used liquid chromatography-mass spectrometry (LC-MS) for the analysis of tryptic glycopeptides of serum IgA from 83 patients with IgAN and 244 age- and sex-matched healthy controls. RESULTS: Multiple structural features of N-glycosylation of IgA1 and IgA2 were associated with IgAN and glomerular function in our cross-sectional study. These features included differences in galactosylation, sialylation, bisection, fucosylation, and N-glycan complexity. Moreover, IgA1 O-glycan sialylation was associated with both the disease and glomerular function. Finally, glycopeptides were a better predictor of IgAN and glomerular function than galactose-deficient IgA1 levels measured by lectin-based ELISA. CONCLUSIONS: Our high-resolution data suggest that IgA O- and N-glycopeptides are promising targets for future investigations on the pathophysiology of IgAN and as potential noninvasive biomarkers for disease prediction and deteriorating kidney function.

Circulating complement factor H-related protein 5 levels contribute to development and progression of IgA nephropathy.

IgA nephropathy (IgAN) is a disease associated with activation of the complement system. But the factors influencing complement activation in IgAN are not fully understood. Complement factor H (FH) is an essential negative regulator of complement C3 activation. Complement factor H-related protein (FHR)-5 shares high sequence similarity with factor H. However, unlike factor H, on binding to activated C3 it enables further activation to proceed. Previously, we reported the contribution of rare variants of the CFHR5 gene to IgAN susceptibility. Here we compared circulating levels of FHR-5 in 1126 patients with IgAN and regular follow-up with those of 153 unrelated healthy individuals to explore the relationship of FHR-5 levels with IgAN development and progression. Circulating FHR-5 levels were significantly elevated in patients with IgAN compared to healthy individuals (median 4.55 [interquartile range 3.58 to 5.85] µg/ml vs 3.19 [interquartile range 2.55 to 3.92] µg/ml). Higher circulating FHR-5 levels were associated with a lower estimated glomerular filtration rate, hypertension, and severe Oxford-T and Oxford-C scores. High FHR-5 levels were independently and significantly associated with a risk of developing either a 30% decline in the estimated glomerular filtration rate or end-stage renal disease (hazard ratio, per standard deviation increment of natural square root transformed FHR-5 of 1.226; 95% confidence interval: 1.106-1.359). Thus, the circulating FHR-5 level is an independent risk factor for IgAN progression.

IgA1 Glycosylation Is Heritable in Healthy Twins.

IgA nephropathy (IgAN) is the most common form of primary GN and an important cause of kidney failure. Characteristically, patients with IgAN have increased serum levels of undergalactosylated IgA1 (gd-IgA1). To assess the degree to which serum gd-IgA1 levels are genetically determined in healthy individuals, we determined serum IgA and gd-IgA1 levels by ELISA in a sample of 148 healthy female twins, including 27 monozygotic and 47 dizygotic pairs. Using the classic twin model, we found the heritability of serum gd-IgA1 and IgA levels to be 80% (95% confidence interval, 66% to 89%) and 46% (95% confidence interval, 15% to 69%), respectively. These data indicate that serum gd-IgA1 levels are highly heritable. Elucidating the genetic basis of this heritability will be important in understanding the pathogenesis of IgAN.

Circulating complement factor H-related proteins 1 and 5 correlate with disease activity in IgA nephropathy.

IgA nephropathy (IgAN) is a common cause of chronic kidney disease and end-stage renal failure, especially in young people. Due to a wide range of clinical outcomes and difficulty in predicting response to immunosuppression, we need to understand why and identify which patients with IgAN will develop progressive renal impairment. A deletion polymorphism affecting the genes encoding the complement factor H-related protein (FHR)-1 and FHR-3 is robustly associated with protection against IgAN. Some FHR proteins, including FHR-1 and FHR-5, antagonize the ability of complement factor H (fH), the major negative regulator of the complement alternative pathway, to inhibit complement activation on surfaces, a process termed fH deregulation. From a large cohort of patients, we demonstrated that plasma FHR-1 and the FHR-1/fH ratio were elevated in IgAN and associated with progressive disease. Plasma FHR-1 negatively correlated with eGFR but remained elevated in patients with IgAN with normal eGFR. Serum FHR5 was slightly elevated in IgAN but did not correlate with eGFR. Neither FHR5 levels nor the FHR-5/fH ratio was associated with progressive disease. However, higher serum FHR-5 levels were associated with a lack of response to immunosuppression, the presence of endocapillary hypercellularity, and histology scores of disease severity (the Oxford Classification MEST score). Thus, FHR-1 and FHR-5 have a role in IgAN disease progression.

Association of Histologic Parameters with Outcome in C3 Glomerulopathy and Idiopathic Immunoglobulin-Associated Membranoproliferative Glomerulonephritis.

BACKGROUND AND OBJECTIVES: C3 glomerulopathy and idiopathic Ig-associated membranoproliferative GN are kidney diseases characterized by abnormal glomerular complement C3 deposition. These conditions are heterogeneous in outcome, but approximately 50% of patients develop kidney failure within 10 years. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: To improve identification of patients with poor prognosis, we performed a detailed analysis of percutaneous kidney biopsies in a large cohort of patients. Using a validated histologic scoring system, we analyzed 156 native diagnostic kidney biopsies from a retrospective cohort of 123 patients with C3 glomerulopathy and 33 patients with Ig-associated membranoproliferative GN. We used linear regression, survival analysis, and Cox proportional hazards models to assess the relationship between histologic and clinical parameters with outcome. RESULTS: Frequent biopsy features were mesangial expansion and hypercellularity, glomerular basement membrane double contours, and endocapillary hypercellularity. Multivariable analysis showed negative associations between eGFR and crescents, interstitial inflammation, and interstitial fibrosis/tubular atrophy. Proteinuria positively associated with endocapillary hypercellularity and glomerular basement membrane double contours. Analysis of second native biopsies did not demonstrate associations between immunosuppression treatment and improvement in histology. Using a composite outcome, risk of progression to kidney failure associated with eGFR and proteinuria at the time of biopsy, cellular/fibrocellular crescents, segmental sclerosis, and interstitial fibrosis/tubular atrophy scores. CONCLUSIONS: Our detailed assessment of kidney biopsy data indicated that cellular/fibrocellular crescents and interstitial fibrosis/tubular atrophy scores were significant determinants of deterioration in kidney function.

Lectin microarray profiling of metastatic breast cancers.

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 µm section of a primary breast tumor or <1 µL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.

Glomerular Complement Factor H-Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment.

INTRODUCTION: Therapeutic agents that target complement are increasingly available for glomerular diseases. However, the mechanisms linking glomerular complement deposition with inflammation and damage are incompletely understood. Complement factor H-related protein 5 (FHR5) interacts with complement C3 and is considered to promote activation. Circulating and glomerular FHR5 associates with IgA nephropathy and abnormal FHR5 associates with familial C3 glomerulopathy (C3G). We characterized glomerular FHR5 staining in C3G and assessed its relationships with histological features of glomerular injury and clinical outcome. METHODS: We developed FHR5 staining protocols for formalin-fixed paraffin-embedded (FFPE) renal tissue and applied them to surplus biopsy sections from a C3G cohort. RESULTS: Glomerular FHR5 was highly prevalent in native and transplant C3G and correlated with glomerular C3 and C5b-9 staining. Glomerular FHR5 staining correlated negatively with estimated glomerular filtration rate (eGFR) (P = 0.04, difference of medians 19.7 ml/min per 1.73 m2; 95% confidence interval [CI] 1.1-43.0) and positively with a membranoproliferative glomerulonephritis pattern at diagnostic biopsy (odds ratio 18; 95% CI 1.6-201; P = 0.049). Glomerular FHR5 staining intensity positively correlated with glomerular complement C3b/iC3b/C3c (Pearson's correlation coefficient [R] = 0.59; P = 0.0008), C3dg (R = 0.47; P = 0.02) and C5b9 (R = 0.44, P = 0.02). CONCLUSIONS: Glomerular FHR5 is highly prevalent in C3G, interacts with glomerular C3, and is associated with markers of disease severity. Glomerular FHR5 likely exacerbates complement-mediated glomerular damage in C3G and its interaction with glomerular complement might be exploited to target complement therapeutic agents.

Serum IgA1 shows increased levels of α2,6-linked sialic acid in breast cancer.

The lectin Helix pomatia agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum samples from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary N-linked glycans on IgA1 from BCa patients (p < 0.0001: non-core fucosylated; p = 0.0345: core fucosylated) and increased asialo-Thomsen-Friedenreich antigen (TF) and disialo-TF antigens in the O-linked glycan preparations from IgA1 of cancer patients compared with healthy control individuals. An increase in Sambucus nigra binding was observed, suggestive of increased α2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases (χ 2 13.359; n = 114; p = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue samples showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication.

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.

INTRODUCTION: IgA nephropathy (IgAN) is characterized by glomerular deposition of galactose-deficient IgA1 and complement proteins and leads to renal impairment. Complement deposition through the alternative and lectin activation pathways is associated with renal injury. METHODS: To elucidate the contribution of the lectin pathway to IgAN, we measured the 11 plasma lectin pathway components in a well-characterized cohort of patients with IgAN. RESULTS: M-ficolin, L-ficolin, mannan-binding lectin (MBL)-associated serine protease (MASP)-1 and MBL-associated protein (MAp) 19 were increased, whereas plasma MASP-3 levels were decreased in patients with IgAN compared with healthy controls. Progressive disease was associated with low plasma MASP-3 levels and increased glomerular staining for C3b/iC3b/C3c, C3d, C4d, C5b-9, and factor H-related protein 5 (FHR5). Glomerular FHR5 deposition positively correlated with glomerular C3b/iC3b/C3c, C3d, and C5b-9 deposition, but not with glomerular C4d. These observations, together with the finding that glomerular factor H (fH) deposition was reduced in progressive disease, are consistent with a role for fH deregulation by FHR5 in renal injury in IgAN. CONCLUSION: Our data indicate that circulating MASP-3 levels could be used as a biomarker of disease severity in IgAN and that glomerular staining for FHR5 could both indicate alternative complement pathway activation and be a tissue marker of disease severity.

Molecular interactions in cancer cell metastasis.

Metastasis, the process by which cancer cells leave the primary tumour, disseminate and form secondary tumours at anatomically distant sites, is a serious clinical problem as it is disseminated disease, which is often impossible to eradicate successfully, that causes the death of most cancer patients. Metastasis results from a complex molecular cascade comprising many steps, all of which are interconnected through a series of adhesive interactions and invasive processes as well as responses to chemotactic stimuli. In spite of its clinical significance, it remains incompletely understood. This review provides an overview of some of the molecular interactions that are critical to metastasis. It summarises the principle molecular players in the major steps of the metastatic cascade. These are: (1) tumour angiogenesis, (2) disaggregation of tumour cells from the primary tumour mass, mediated by cadherins and catenins, (3) invasion of, and migration through, the basement membrane (BM) and extracellular matrix (ECM) surrounding the tumour epithelium, and subsequent invasion of the BM of the endothelium of local blood vessels. This is mediated through integrins and proteases, including urokinase form of plasminogen activator (uPA), matrix metalloproteinases (MMPs) and cathepsins, (4) intravasation of the tumour cells into the blood vessels prior to hematogeneous dissemination to distant sites, (5) adhesion of the circulating tumour cells to the endothelial cell lining at the capillary bed of the target organ site. This occurs through adhesive interactions between cancer cells and endothelial cells involving selectins, integrins and members of the immunoglobulin superfamily (IgSF), (6) invasion of the tumour cells through the endothelial cell layer and surrounding BM (extravasation) and target organ tissue and (7) the development of secondary tumour foci at the target organ site.