Identification and Characterization of a New Autoimmune Protein in Membranous Nephropathy by Immunoscreening of a Renal cDNA Library

<div><p>Membranous Nephropathy (MN) represents a large amount of Nephrotic Syndromes in the adult population and its definitive diagnosis is currently carried out through biopsy. An autoimmune condition has been demonstrated in idiopathic MN (iMN) in which some kidney structures are targeted by patient autoantibodies. Some candidate antigens have been described and other likely involved target proteins responsible for the disease are not known yet. In this work our aim is to identify these proteins by screening a lambda-phage library with patients’ sera. We enrolled four groups of patients: two MN groups of 12 full iMN patients; one control group of 15 patients suffering from other renal diseases; one control group of 15 healthy individuals. A commercial cDNA phagemide library was screened using the above described sera, in order to detect positive signals due to antigen-antibody bond. We detected one phagemide clone expressing a protein which was shown to be targeted by the antibodies of the iMN sera only. Control sera were negative. The sequence analysis of cDNA matched the Synaptonemal Complex protein 65 (SC65) coding sequence. Further proteomic analyses were carried out to validate our results. We provide evidence of an involvement of SC65 protein as an autoimmune target in iMN. Considering the invasiveness and the resulting risk coming from renal biopsy, our ongoing aim is to set a procedure able to diagnose affected patients through a little- or non-invasive method such as blood sampling rather than biopsy.</p> </div

Clinical characteristics of patients.

<p>Clinical characteristics of patients.</p

Western blotting of SC65 in subcellular fractions of cultured differentiated podocytes.

<p><b>Cy</b>, cytosolic fraction. <b>Me</b>, membrane fraction. <b>Nu</b>, nuclear fraction. A band corresponding to SC65 is apparent in the cytosolic and membrane fractions.</p

Recombinant spot assay of each serum.

<p>Positively reacting sera are those showing the dark spot in the middle of the membrane, where the recombinant SC65 protein was spotted.</p

SC65 and IgG₄ staining of iMN renal tissue.

<p>The immunofluorescence image shows an iMN kidney glomerulus (and a particular of it) stained with monoclonal antibodies against SC65 (A, D) and IgG₄ (B, E). Pictures C and F result from the merge of A+B and D+E respectively. The costaining of SC65 and IgG₄ deposits in iMN specimens shows a polarized expression of IgG₄ in the subepithelial district adjacent to a cytoplasmic expression of SC65.</p

SC65 and Synaptopodin staining of normal and iMN renal tissue.

<p>The immunofluorescence image shows normal (A, B, C) and iMN kidneys (D, E, F) stained with monoclonal antibodies against SC65 (A, D) and synaptopodin (B, E). Pictures C and F result from the merge of A+B and D+E respectively. Costaining with synaptopodin show that SC65 and synaptopodin are co-expressed in the cytoplasm of podocyte cells.</p

Second selection of positive clone.

<p>Many plaques from pickup of first positive signal have developed on another plate. Exogenous proteins produced by single plaques have been adsorbed to this filter membrane, and tested for the immunoreactivity with iMN patient serum. The difference between positive and negative plaques is apparent.</p

Renal survival of patients according to the type of their variant.

<p>Renal survival of patients according to the type of their variant.</p

Table of genetic variants identified in the cohort.

<p>Table of genetic variants identified in the cohort.</p

Flow of information through the different phases of the selection of available studies.

<p>Flow of information through the different phases of the selection of available studies.</p