The classical pathway triggers pathogenic complement activation in membranous nephropathy.

Membranous nephropathy (MN) is an antibody-mediated autoimmune disease characterized by glomerular immune complexes containing complement components. However, both the initiation pathways and the pathogenic significance of complement activation in MN are poorly understood. Here, we show that components from all three complement pathways (alternative, classical and lectin) are found in renal biopsies from patients with MN. Proximity ligation assays to directly visualize complement assembly in the tissue reveal dominant activation via the classical pathway, with a close correlation to the degree of glomerular C1q-binding IgG subclasses. In an antigen-specific autoimmune mouse model of MN, glomerular damage and proteinuria are reduced in complement-deficient mice compared with wild-type littermates. Severe disease with progressive ascites, accompanied by extensive loss of the integral podocyte slit diaphragm proteins, nephrin and neph1, only occur in wild-type animals. Finally, targeted silencing of C3 using RNA interference after the onset of proteinuria significantly attenuates disease. Our study shows that, in MN, complement is primarily activated via the classical pathway and targeting complement components such as C3 may represent a promising therapeutic strategy.

An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation.

Developmental neurotoxicity (DNT) of chemicals poses a serious threat to human health worldwide. Current in vivo test methods for assessing DNT require the use of high numbers of laboratory animals. Most alternative in vitro testing methods monitor rather simple toxicological endpoints, whereas the formation of a functional brain requires precisely timed navigation of axons within a complex tissue environment. We address this complexity by monitoring defects in axonal navigation of pioneer axons of intact locust embryos after exposure to chemicals. Embryos develop in serum-free culture with test chemicals, followed by immunolabeling of pioneer neurons. Defects in axon elongation of pioneer axons are quantified in concentration-response curves and compared to the general viability of the embryo, as measured by a resazurin assay. We show that selected chemical compounds interfering with calcium signaling, the cytoskeletal organization, and the reference developmental neurotoxicant rotenone, can be classified as DNT positive. The pesticide rotenone inhibits pioneer neuron elongation with a lower IC50 than the viability assay. The rho kinase inhibitor Y27632 can partially rescue outgrowth inhibition, supporting the classification of rotenone as a specific DNT positive compound. Since mechanisms of axonal guidance, such as growth cone navigation along molecular semaphorin gradients are conserved between locust and mammalian nervous systems, we will further explore the potential of this invertebrate preparation as an assay for testing the DNT potential of chemicals in humans.