C3 glomerulopathy-associated CFHR1 mutation alters FHR oligomerization and complement regulation

Tortajada, Agustín et alt.C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H-related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo- and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G. Copyright © 2013, American Society for Clinical Investigation.Work in this report was funded by the Spanish Ministerio de Economía y Competitividad (SAF2011-26583), the Fundación Renal Iñigo Alvarez de Toledo and the 7FP European Union project EURenOmics to S. Rodríguez de Córdoba; the “Ramón Areces” Foundation, the Spanish Ministerio de Economía y Competitividad (SAF2011-22988), and the “Red Temática de Investigación Cooperativa en Cáncer(RTICC)” (RD06/0020/1001) to O. Llorca; the Spanish “Ministerio de Economía y Competitividad” (PI0900268) to P. Sánchez-Corral; the MRC UK (G0701298) to C.L. Harris; and the Spanish “Ministerio de Economía y Competitividad” (PS09/00122) to M. López Trascasa. In addition, this work was supported by a grant from the Autonomous Region of Madrid (S2010/BMD-2316) to S. Rodríguez de Córdoba, O. Llorca, M. López Trascasa, and P. Sánchez-Corral. M.C. Pickering is a Wellcome Trust Senior Fellow in Clinical Science (WT098476MA). H. Yébenes was supported by a postdoctoral contract by the RTICC, and C. Abarrategui-Garrido was supported by the “Fundación de Investigación Biomédica del Hospital Universitario La Paz.”Peer reviewe

Molecular basis of factor H R1210C association with ocular and renal diseases

27 p.-3 fig.-1 tab.-3 fig. compl.The complement factor H (FH) mutation R1210C, which was described in association with atypical hemolytic uremic syndrome (aHUS), also confers high risk of age-related macular degeneration (AMD) and associates with C3 glomerulopathy (C3G). To reveal the molecular basis of these associations and to provide insight into what determines the disease phenotype in FH-R1210C carriers, we identified FH-R1210C carriers in our aHUS, C3G, and AMD cohorts. Disease status, determined in patients and relatives, revealed an absence of AMD phenotypes in the aHUS cohort and, vice versa, a lack of renal disease in the AMD cohort. These findings were consistent with differences in the R1210C-independent overall risk for aHUS and AMD between mutation carriers developing one pathology or the other. R1210C is an unusual mutation that generates covalent complexes between FH and HSA. Using purified FH proteins and surface plasmon resonance analyses, we demonstrated that formation of these FH-HSA complexes impairs accessibility to all FH functional domains. These data suggest that R1210C is a unique C-terminal FH mutation that behaves as a partial FH deficiency, predisposing individuals to diverse pathologies with distinct underlying pathogenic mechanisms; the final disease outcome is then determined by R1210C-independent genetic risk factors.This work was supported by The Spanish "Ministerio de Economía y Competitividad" (SAF2011-26583, PI11/00898 and RETICS RD12/0034) , The Fundación Renal Íñigo Álvarez de Toledo, the Seventh Framework Programme European Union Project EURenOmics (305608) and the Autonomous Region of Madrid S2010/BMD-2316)Peer reviewe

Hemolysis in eculizumab-treated PNH patients

35 p.-3 fig.-4 tab. Subías, Marta et al.Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n=12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administration.S.RdeC is supported by the Spanish “Ministerio de Economía y Competitividad” (SAF2011-26583) and the Autonomous Region of Madrid (S2010/BMD-2316).Peer reviewe

In vivo biological activity of rocket extracts (Eruca vesicaria subsp. sativa (Miller) Thell) and sulforaphane

Eruca is thought to be an excellent source of antioxidants like phenolic compounds, carotenoids, glucosinolates and their degradation products, such as isothiocyanates. Sulforaphane is one of the most potent indirect antioxidants of Eruca isolated until the date. In this work we investigate: (i) the safety and DNA protective activity of Eruca extracts and sulforaphane (under and without oxidative stress) in Drosophila melanogaster; and (ii) the influence on D. melanogaster life span treated with Eruca extracts and sulforaphane. Our results showed that among the four concentrations of Eruca extracts tested (from 0.625 to 5. mg/ml), intermediate concentrations of the Es2 accession (1.25 and 2.5. mg/ml) exhibited no genotoxic activity, as well as antigenotoxic activity (inhibition rate of 0.2-0.6) and the lowest concentration of Es2 and Es4 accessions (0.625. mg/ml) also enhanced the health span portion of the live span curves. Sulforaphane presented a high antigenotoxic activity in the SMART test of D. melanogaster and intermediate concentrations of this compound (3.75. μM) enhanced average healthspan. The results of this study indicate the presence of potent antigenotoxic factors in rocket, which are being explored further for their mechanism of action. © 2012 Elsevier Ltd.The authors thank to the Consejería de Innovación, Ciencia y Empresa (Junta de Andalucía, Spain) for funding the Project P06-AGR-02230.Peer Reviewe

Cytotoxic and genotoxic effects of metal(oid)s bioactivated in rocket leaves (Eruca vesicaria subsp. sativa Miller)

Rocket is an important source of essential elements. However, it may also accumulate toxic elements such as metal(oids). The objectives of the present work were (i) to study the uptake of arsenic, lead, cadmium and zinc in rocket grown in contaminated soils, (ii) to establish the genotoxic and cytotoxic activities of this vegetable material, and (iii) to study the modulator role of the glucosinolate and metal contents in the genotoxic/cytotoxic activities. Lead, cadmium and zinc leaf concentrations in our study were over the concentrations allowed by the statutory limit set for metal(oid) contents in vegetables. The accessions were non genotoxic at the different concentrations studied, although one of the accessions showed the highest mutation rates doubling those of negative control. The cytotoxicity assays with HL60 human leukaemia cells showed that the tumouricide activities of rocket leaves decreased with the increasing of metal(oid) concentrations and also with the decreasing of glucosinolate concentrations in their tissues. An interaction between metal(oid)s and glucosinolate degradation products contained in rocket leaves is suggested as the main modulator agents of the biological activity of the plants grown in metal-contaminated soils. © 2013.The authors thank to the Department for Innovation, Science and Business of the Regional Autonomous Government of Andalusia for funding the Project P06-AGR-02230.Peer Reviewe