• 2018-07
  • 2018-10
  • 2018-11
  • diprenorphine cost Here we found that podocytes both in vitr


    Here we found that podocytes, both in vitro and in kidney-biopsy tissue sections, express CAII. Furthermore, IgG4 purified from the patient\'s serum elicits early intracellular acidification with profound effects on podocyte homeostasis as a likely consequence of the diprenorphine cost interaction of IgG4 autoantibodies with membrane CAII. The drop in cytosolic pH over time is even greater than the acidification elicited by a specific pharmacologic CAII inhibitor, indicating direct and potent inhibitory action of the patient\'s IgG4 on podocyte CAII that can behave as a functional pathogenic target. Notably, this interaction is reminiscent of the ability of non-complement-fixing IgG4 autoantibodies to interact with an antigen in terms of inhibiting its function, as has been shown for IgG4 to muscle-specific kinase in patients with myasthenia gravis [16,17] and suggested for anti-PLA2R diprenorphine cost in patients with idiopathic MN [18,19]. In this regard, in the case of our patient, the co-localization of CAII and IgG4 deposited in the glomeruli indicates that CAII is indeed present in the subepithelial immune deposits. This observation offers an important clue, which suggests that anti-CAII antibodies, which in our patient are restricted to the IgG4 subtype, could be responsible for initiating the disease. Relying on evidence of mitochondrial disruption after early acidification in response to injurious stimuli [20], we next analyzed the consequences of low pH on mitochondrial functions. Our data that the decrease of podocyte pH preceded mitochondrial alteration and fragmentation provide a link between IgG4-driven intracellular acidification and perturbation of mitochondrial dynamics. Mitochondrial dysfunction in IgG4-treated podocytes was associated with massive ROS production, which in turn promoted F-actin cytoskeletal alterations. In this regard, an important role for ROS in inducing cytoskeletal reorganization in injured podocytes has been documented [21]. Another major finding of the study consists in the externalization of the mitochondrial enzyme SOD2 in clusters in direct response to the patient\'s IgG4-CAII ligation in vitro. The causal role of IgG4 anti-CAII challenge on SOD2 externalization is provided by the combined evidence that incubating the cells with an anti-CAII antibody induced the mislocation of SOD2 on the podocyte surface and that the saturation of patient\'s serum antibodies with purified CAII prevented the SOD2 mobilization. This would indicate a direct effect of the patient\'s IgG4 anti-CAII in the activation of intracellular signaling that leads to SOD2 trafficking to the podocyte plasma membrane. The observed mitochondrial fragmentation along with the consequent altered membrane potential in response to anti-CAII IgG4 could affect the release of matrix proteins from failing mitochondria [22], including SOD2. The finding that prevention of mitochondrial depolarization attenuated IgG4-induced SOD2 externalization on the membrane of damaged podocytes points to mitochondrial dysfunction as the major cause of SOD2 mislocation after exposure to the patient\'s IgG4. Protein membrane trafficking in podocytes is regulated by cytoskeletal F-actin filaments [23] and cytoskeletal alterations have been associated with SOD2 mistargeting from spoiled mitochondria to the cell surface [24]. Here we suggest that the patient\'s IgG4-induced cytoskeletal F-actin corticalization could drag the released SOD2 toward the plasma membrane and influence its spatial patterning in podocytes. In line with our findings, a local enrichment of cortical actin filaments was found to regulate nanocluster formation of membrane proteins [25]. The role of cytoskeletal remodeling in SOD2 cluster formation rests on the evidence that H2O2, while promoting strong SOD2 externalization, did not induce the SOD2 cluster formation. This difference could conceivably be a consequence of the lower capacity of H2O2 to induce F-actin corticalization compared to IgG4. The consequent C3 deposition on the cell surface indicates that the externalized SOD2 is the target for the binding of the patient\'s IgG3, leading to complement activation. Since SOD2 becomes recognizable on the podocyte surface via a process that can be related to the patient\'s IgG4 and is induced with disease, SOD2 behaves like a neoantigen, as other intracellular proteins, which are not abundantly expressed in the normal glomerulus [19]. Given the absence of detectable circulating immune complexes in our patient and according to known disease models, these findings converged to suggest intraglomerular interaction between IgG3 and SOD2 further shaping the MN lesion in the presence of anti-CAII IgG4 (see scheme Fig. 6E).