Clin Kidney J (2012) 5: 178–179 Research Article Patients with primary membranous nephropathy lack auto-antibodies against LDL receptor, the homologue of megalin in human glomeruli Primary membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. We know very little about the mechanism for the sub-epithelial immune dep- osition typical of MN [1], with the notable exception of a rare condition recently described by Debiec et al.[2] in which antibodies against neutral peptidase (NEP) are raised for alloimmunization during a first pregnancy and transferred to a second foetus determining neonatal MN. Most of our knowledge on immune deposition is derived instead from the characterization of Heymann nephritis (HN), an exper- imental rat model described in the early fifties [3] that is mediated by the deposition of anti-megalin antibodies in rat glomeruli where they co-localize with C5–9 and clus- terin, a natural ligand of megalin [4]. Since megalin is not present in human glomeruli, the findings obtained in rats cannot be completely exported to human MN and represent the missing point between HN and human MN. A recent work identified LDL receptor (LDL-r) as a megalin homo- logue in human glomeruli [5] raising the possibility that anti-LDL-r antibodies may be present in sera of patients with MN. The presence of LDL-r was then confirmed in pri- mary podocytes, human=murine podocyte cell lines and murine liver with specific antibodies and by MALDI-TOF (Figure 1A). Based on this background, we have screened sera of 38 patients (15 collected at the time of diagnosis, before the start of any therapy) for circulating anti-LDL-r antibodies utilizing two-dimensional electrophoresis (2D- E) and western blot with liver extracts and with recombi- nant receptor-associated protein (RAP) domain as immo- bilized antigens. In particular, the reason to utilize the RAP domain is that it is made up by a short sequence of 71 amino acids lacking glycosylation sites and actually repre- sents the accessible site for the binding of auto-antibodies. Both the intact protein in 2D-E and recombinant RAP (fusion complex with GST) in monodimensional-E were recognized by specific polyclonal antibodies at different dilutions, which rules out the possibility that chemical ma- nipulation alters the binding site of the protein (Figure 1A–C). MN sera were utilized at different dilutions (from 1:10 to 1:100) to exclude problems linked to sensitivity of the as- say. The sensitivity of the western blot technique to detect RAP was up to the pmole level (Figure 1C). Moreover, the assay was also tested by varying the dilution of anti-LDL-r antibodies (Figure 1D) in the presence of a constant amount of rRAP (13.4 pmol); the sensitivity of the assay was close to 1.2 mg=ml of the specific antibody under the condition used. In no case could circulating anti-LDL-r antibodies be detected in patients with MN (Figure 1B), and the same negative results were obtained utilizing re- combinant RAP domain as immobilized antigen (Figure 1E). In this case, several combinations of antigen and serum quantities were utilized to avoid any interference of the anti- body=antigen ratio on the sensitivity of the assay. It seems relevant to stress that patients were enrolled both before (n ¼ 15) and after (n ¼ 23) any therapy and therefore, even though this is a cross-sectional study, it gives insights unrelated to therapy. A final experiment tested with immunofluorescence the binding of a few sera to podocyte cell lines. In this case, MN sera indeed recognized that surface proteins on podo- cytes did not correspond to LDL-r. They have been char- acterized by proteomics and are currently under investigation (Ghiggeri, personal observation). Overall, our results suggest that LDL-r is not a target of an autoimmune response in human MN. Even though neg- ativity of anti-LDL-r serum Ab was shown by two independ- ent techniques (western blot and immunofluorescence) and by two different antigens (intact LDL-r and recombinant RAP) at different dilutions, the problem of sensitivity of the assay cannot be completely ruled out. Other technological ap- proaches should be considered. In consideration of the negative results presented here, also the extension of the analysis of autoimmunity to other podocyte components must be considered. Conflict of interest statement. None declared. 1 Laboratory on Pathophysiology of Uremia 2 Renal Child Foundation 3 Division of Nephrology, G. Gaslini Children Hospital, Genoa, Italy 4 Department of Clinical Medicine Nephrology and Health Sciences University of Parma, Parma, Italy 5 Chair of Nephrology University of Brescia and Division of Nephrology Montichiari Hospital, Brescia, Italy E-mail: labnefro@ospedale- gaslini.ge.it Maurizio Bruschi 1,2 Giovanni Candiano 1,3 Corrado Murtas 3,4 Marco Prunotto 1 Laura Santucci 1,2 Maria Luisa Carnevali 4 Francesco Scolari 5 Landino Allegri 4 Gian Marco Ghiggeri 1,3 References 1. Collins AB, Andres GA, McCluskey RT. Lack of evidence for a role of renal tubular antigen in human membranous glomerulonephritis. Nephron 1981; 27: 297–301 2. Debiec H, Guigonis V, Mougenot B et al. Antenatal membranous glomer- ulonephritis due to anti-neutral endopeptidase antibodies. N Engl J Med 2002; 346: 2053–2060 3. Heymann W, Hackel DB, Harwood S et al. Production of nephrotic syn- drome in rats by Freund’s adjuvants and rat kidney suspensions. Proc Soc Exp Biol Med 1959; 100: 660–664 4. Kerjaschki D, Horvat R, Binder S et al. Identification of a 400-kd protein in the brush borders of human kidney tubules that is similar to gp330, the nephritogenic antigen of rat Heymann nephritis. Am J Pathol 1987; 129: 183–191 5. Rastaldi MP, Candiano G, Musante L et al. Glomerular clusterin is associ- ated with PKC-alpha=beta regulation and good outcome of membranous glomerulonephritis in humans. Kidney Int 2006; 70: 477–485 doi: 10.1093/ckj/sfp002 Advance Access publication 31 January 2009 Ó The Author 2009. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org Downloaded from https://academic.oup.com/ckj/article-abstract/5/2/178/341502 by guest on 10 June 2020