Defining Nephritic Factors as Diverse Drivers of Systemic Complement Dysregulation in C3 Glomerulopathy.

Introduction: C3 glomerulopathy (C3G) is an ultrarare renal disease characterized by deposition of complement component C3 in the glomerular basement membrane (GBM). Rare and novel genetic variation in complement genes and autoantibodies to complement proteins are commonly identified in the C3G population and thought to drive the underlying complement dysregulation that results in renal damage. However, disease heterogeneity and rarity make accurately defining characteristics of the C3G population difficult. Methods: Here, we present a retrospective analysis of the Molecular Otolaryngology and Renal Research Laboratories C3G cohort. This study integrated complement biomarker testing and in vitro tests of autoantibody function to achieve the following 3 primary goals: (i) define disease profiles of C3G based on disease drivers, complement biomarkers, and age; (ii) determine the relationship between in vitro autoantibody tests and in vivo complement dysregulation; and (iii) evaluate the association between autoantibody function and disease progression. Results: The largest disease profiles of C3G included patients with autoantibodies to complement proteins (48%) and patients for whom no genetic and/or acquired drivers of disease could be identified (43%). The correlation between the stabilization of convertases by complement autoantibodies as measured by in vitro modified hemolytic assays and systemic biomarkers that reflect in vivo complement dysregulation was remarkably strong. In patients positive for autoantibodies, the degree of stabilization capacity predicted worse renal function. Conclusion: This study implicates complement autoantibodies as robust drivers of systemic complement dysregulation in approximately 50% of C3G but also highlights the need for continued discovery-based research to identify novel drivers of disease.

Renin and renin blockade have no role in complement activity.

Renin, an aspartate protease, regulates the renin-angiotensin system by cleaving its only known substrate angiotensinogen to angiotensin. Recent studies have suggested that renin may also cleave complement component C3 to activate complement or contribute to its dysregulation. Typically, C3 is cleaved by C3 convertase, a serine protease that uses the hydroxyl group of a serine residue as a nucleophile. Here, we provide seven lines of evidence to show that renin does not cleave C3. First, there is no association between renin plasma levels and C3 levels in patients with C3 Glomerulopathies (C3G) and atypical Hemolytic Uremic Syndrome (aHUS), implying that serum C3 consumption is not increased in the presence of high renin. Second, in vitro tests of C3 conversion to C3b do not detect differences when sera from patients with high renin levels are compared to sera from patients with normal/low renin levels. Third, aliskiren, a renin inhibitor, does not block abnormal complement activity introduced by nephritic factors in the fluid phase. Fourth, aliskiren does not block dysregulated complement activity on cell surfaces. Fifth, recombinant renin from different sources does not cleave C3 even after 24 hours of incubation at 37 °C. Sixth, direct spiking of recombinant renin into sera samples of patients with C3G and aHUS does not enhance complement activity in either the fluid phase or on cell surfaces. And seventh, molecular modeling and docking place C3 in the active site of renin in a position that is not consistent with a productive ground state complex for catalytic hydrolysis. Thus, our study does not support a role for renin in the activation of complement.

Complement Factor I Variants in Complement-Mediated Renal Diseases.

C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) are two rare diseases caused by dysregulated activity of the alternative pathway of complement secondary to the presence of genetic and/or acquired factors. Complement factor I (FI) is a serine protease that downregulates complement activity in the fluid phase and/or on cell surfaces in conjunction with one of its cofactors, factor H (FH), complement receptor 1 (CR1/CD35), C4 binding protein (C4BP) or membrane cofactor protein (MCP/CD46). Because altered FI activity is causally related to the pathogenesis of C3G and aHUS, we sought to test functional activity of select CFI missense variants in these two patient cohorts. We identified 65 patients (16, C3G; 48, aHUS; 1 with both) with at least one rare variant in CFI (defined as a MAF < 0.1%). Eight C3G and eleven aHUS patients also carried rare variants in either another complement gene, ADAMTS13 or THBD. We performed comprehensive complement analyses including biomarker profiling, pathway activity and autoantibody testing, and developed a novel FI functional assay, which we completed on 40 patients. Seventy-eight percent of rare CFI variants (31/40) were associated with FI protein levels below the 25th percentile; in 22 cases, FI levels were below the lower limit of normal (type 1 variants). Of the remaining nine variants, which associated with normal FI levels, two variants reduced FI activity (type 2 variants). No patients carried currently known autoantibodies (including FH autoantibodies and nephritic factors). We noted that while rare variants in CFI predispose to complement-mediated diseases, phenotypes are strongly contingent on the associated genetic background. As a general rule, in isolation, a rare CFI variant most frequently leads to aHUS, with the co-inheritance of a CD46 loss-of-function variant driving the onset of aHUS to the younger age group. In comparison, co-inheritance of a gain-of-function variant in C3 alters the phenotype to C3G. Defects in CFH (variants or fusion genes) are seen with both C3G and aHUS. This variability underscores the complexity and multifactorial nature of these two complement-mediated renal diseases.

Modeling C3 glomerulopathies: C3 convertase regulation on an extracellular matrix surface.

Introduction: C3 glomerulopathies (C3G) are ultra-rare complement-mediated diseases that lead to end-stage renal disease (ESRD) within 10 years of diagnosis in ~50% of patients. Overactivation of the alternative pathway (AP) of complement in the fluid phase and on the surface of the glomerular endothelial glycomatrix is the underlying cause of C3G. Although there are animal models for C3G that focus on genetic drivers of disease, in vivo studies of the impact of acquired drivers are not yet possible. Methods: Here we present an in vitro model of AP activation and regulation on a glycomatrix surface. We use an extracellular matrix substitute (MaxGel) as a base upon which we reconstitute AP C3 convertase. We validated this method using properdin and Factor H (FH) and then assessed the effects of genetic and acquired drivers of C3G on C3 convertase. Results: We show that C3 convertase readily forms on MaxGel and that this formation was positively regulated by properdin and negatively regulated by FH. Additionally, Factor B (FB) and FH mutants impaired complement regulation when compared to wild type counterparts. We also show the effects of C3 nephritic factors (C3Nefs) on convertase stability over time and provide evidence for a novel mechanism of C3Nef-mediated C3G pathogenesis. Discussion: We conclude that this ECM-based model of C3G offers a replicable method by which to evaluate the variable activity of the complement system in C3G, thereby offering an improved understanding of the different factors driving this disease process.

Functional characterization of 105 factor H variants associated with aHUS: lessons for variant classification.

Atypical hemolytic uremic syndrome (aHUS) is a life-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulation due to pathogenic variants in genes that encode complement components and regulators. Among these genes, the factor H (FH) gene, CFH, presents with the highest frequency (15% to 20%) of variants and is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remains challenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. To address this knowledge gap, we expressed and functionally characterized 105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign and, for each, we fully documented the nature of the pathogenicity. Twenty-six previously characterized FH variants were used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%) alter FH expression or function in vitro and, therefore, are proposed to be pathogenic. We show that rarity in control databases is not informative for variant classification, and we identify important limitations in applying prediction algorithms to FH variants. Based on structural and functional data, we suggest ways to circumvent these difficulties and, thereby, improve variant classification. Our work highlights the need for functional assays to interpret FH variants accurately if clinical care of patients with aHUS is to be individualized and optimized.

The transcriptional regulator GON4L is required for viability and hematopoiesis in mice.

The Gon4l gene encodes a putative transcriptional regulator implicated in the control of both cell differentiation and proliferation. Previously, we described a mutant mouse strain called Justy in which splicing of pre-mRNA generated from Gon4l is disrupted. This defect severely reduces, but does not abolish, GON4L protein expression and blocks the formation of early B-lineage progenitors, suggesting Gon4l is required for B-cell development in vertebrates. Yet, mutations that disable Gon4l in zebrafish impair several facets of embryogenesis that include the initiation of primitive hematopoiesis, arguing this gene is needed for multiple vertebrate developmental pathways. To better understand the importance of Gon4l in mammals, we created mice carrying an engineered version of Gon4l that can be completely inactivated by Cre-mediated recombination. Breeding mice heterozygous for the inactivated Gon4l allele failed to yield any homozygous-null offspring, indicating Gon4l is an essential gene in mammals. Consistent with this finding, as well previously published results, cell culture studies revealed that loss of Gon4l blocks cell proliferation and compromises viability, suggesting a fundamental role in the control of cell division and survival. Studies using mixed bone marrow chimeras confirmed Gon4l is required for B-cell development but also found it is needed to maintain definitive hematopoietic stem/progenitor cells that are the source of all hematopoietic cell lineages. Our findings reveal Gon4l is an essential gene in mammals that is required to form the entire hematopoietic system.

Mutation of complement factor B causing massive fluid-phase dysregulation of the alternative complement pathway can result in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome is an ultra-rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute kidney injury. Its pathogenesis is driven most frequently by dysregulated cell-surface control of the alternative pathway of complement secondary to inherited and/or acquired factors. Here we evaluated two unrelated patients with atypical hemolytic uremic syndrome. The first, a five-year-old Caucasian female, presented at 10 months with schistocytes, thrombocytopenia and kidney injury. The second, a 55-year-old Caucasian female, presented at age 31 following caesarean section for preeclampsia. Complement biomarker testing was remarkable for undetectable levels of C3 in both. Circulating levels of C5 and properdin were also low consistent with over-activity of the alternative and terminal pathways of complement. Genetic testing identified a heterozygous novel variant in CFB (c.1101 C>A, p.Ser367Arg) in both patients. Functional studies found strong fluid-phase C3 cleavage when normal and proband sera were mixed. Cell-surface C3b deposition was strongly positive when patient serum was supplemented with C3. In vitro control of C3 convertase activity could be restored with increased concentrations of factor H. Thus, CFB p.Ser367Arg is a gain-of-function pathogenic variant that leads to dysregulation of the alternative pathway in the fluid-phase and increased C3b deposition on cell surfaces. Our study highlights the complexities of complement-mediated diseases like atypical hemolytic uremic syndrome and illustrates the importance of functional studies at the variant level to gain insight into the disease phenotype.

Early B Cell Progenitors Deficient for GON4L Fail To Differentiate Due to a Block in Mitotic Cell Division.

B cell development in Justy mutant mice is blocked due to a precursor mRNA splicing defect that depletes the protein GON4-like (GON4L) in B cell progenitors. Genetic and biochemical studies have suggested that GON4L is a transcriptional regulator that coordinates cell division with differentiation, but its role in B cell development is unknown. To understand the function of GON4L, we characterized B cell differentiation, cell cycle control, and mitotic gene expression in GON4L-deficient B cell progenitors from Justy mice. We found that these cells established key aspects of the transcription factor network that guides B cell development and proliferation and rearranged the IgH gene locus. However, despite intact IL-7 signaling, GON4L-deficient pro-B cell stage precursors failed to undergo a characteristic IL-7-dependent proliferative burst. These cells also failed to upregulate genes required for mitotic division, including those encoding the G1/S cyclin D3 and E2F transcription factors and their targets. Additionally, GON4L-deficient B cell progenitors displayed defects in DNA synthesis and passage through the G1/S transition, contained fragmented DNA, and underwent apoptosis. These phenotypes were not suppressed by transgenic expression of prosurvival factors. However, transgenic expression of cyclin D3 or other regulators of the G1/S transition restored pro-B cell development from Justy progenitor cells, suggesting that GON4L acts at the beginning of the cell cycle. Together, our findings indicate that GON4L is essential for cell cycle progression and division during the early stages of B cell development.

NEDD4 ubiquitin ligase is a putative oncogene in endometrial cancer that activates IGF-1R/PI3K/Akt signaling.

OBJECTIVE: The PI3K/Akt pathway is frequently dysregulated in endometrial cancer, the most common gynecologic malignancy. Emerging evidence identifies the ubiquitin ligase NEDD4 as a key regulator of the PI3K/Akt pathway via activation of insulin-like growth factor-1 receptor (IGF-1R). Our objective was to understand the role of NEDD4 in endometrial cancer. METHODS: NEDD4 expression was assessed by immunohistochemistry in a tissue microarray with 77 endometrial lesions ranging from normal benign endometrium to tumor specimens of varying stage and grade. Studies were extended to a panel of eight endometrial cancer cell lines phenotypically representing the most common endometrial patient tumors. RESULTS: Immunohistochemistry demonstrated robust staining of NEDD4 in endometrial tumor specimens, with greater NEDD4 expression in the most aggressive tumors. Expression of NEDD4 was detected in a majority of endometrial cancer cell lines surveyed. Exogenous overexpression of murine Nedd4 in endometrial cancer cell lines with modest endogenous NEDD4 expression resulted in a significant increase in the rate of proliferation. Nedd4 overexpression also promoted an increase in cell surface localization of IGF-1R and activation of Akt. Inhibition of PI3K/Akt signaling reversed the enhanced cell growth in Nedd4-overexpressing endometrial cancer cells. In addition, the expression of NEDD4 in endometrial tumors positively correlated with the Akt downstream effector FoxM1. CONCLUSIONS: This study identifies NEDD4 as a putative oncogene in endometrial cancer that may augment activation of the IGF-1R/PI3K/Akt signaling pathway.

Genetic Deficiency of Mtdh Gene in Mice Causes Male Infertility via Impaired Spermatogenesis and Alterations in the Expression of Small Non-coding RNAs.

Increased expression of metadherin (MTDH, also known as AEG-1 and 3D3/LYRIC) has been associated with drug resistance, metastasis, and angiogenesis in a variety of cancers. However, the specific mechanisms through which MTDH is involved in these processes remain unclear. To uncover these mechanisms, we generated Mtdh knock-out mice via a targeted disruption of exon 3. Homozygous Mtdh knock-out mice are viable, but males are infertile. The homozygous male mice present with massive loss of spermatozoa as a consequence of meiotic failure. Accumulation of γ-H2AX in spermatocytes of homozygous Mtdh knock-out mice confirms an increase in unrepaired DNA breaks. We also examined expression of the DNA repair protein Rad18, which is regulated by MTDH at the post-transcriptional level. In testes from Mtdh exon 3-deficient mice, Rad18 foci were increased in the lumina of the seminiferous tubules. The Piwi-interacting RNA (piRNA)-interacting protein Mili was expressed at high levels in testes from Mtdh knock-out mice. Accordingly, genome-wide small RNA deep sequencing demonstrated altered expression of piRNAs in the testes of Mtdh knock-out mice as compared with wild type mice. In addition, we observed significantly reduced expression of microRNAs (miRNAs) including miR-16 and miR-19b, which are known to be significantly reduced in the semen of infertile men. In sum, our observations indicate a crucial role for MTDH in male fertility and the DNA repair mechanisms required for normal spermatogenesis.