Maria Luisa Carnevali

Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Coexistence of Different Circulating Anti-Podocyte Antibodies in Membranous Nephropathy

BACKGROUND AND OBJECTIVES The discovery of different podocyte autoantibodies in membranous nephropathy (MN) raises questions about their pathogenetic and clinical meaning. This study sought to define antibody isotypes and correlations; to compare levels in MN, other glomerulonephritides, and controls; and to determine their association with clinical outcomes. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Serum IgG(1), IgG(3), and IgG(4) against aldose reductase (AR), SOD2, and α-enolase (αENO) were measured at diagnosis in 186 consecutive MN patients, in 96 proteinuric controls (36 with FSGS, and 60 with IgA nephropathy), and in 92 healthy people recruited in four Italian nephrology units. Anti-phospholipase A2 receptor (PLA2r) and anti-neutral endopeptidase (NEP) IgG(4) were titrated in the same specimens. Association with 1-year follow-up clinical parameters was studied in 120 patients. RESULTS IgG(4) was the most common isotype for all antibodies; IgG(1) and IgG(3) were nearly negligible. IgG(4) levels were positive in a significant proportion of MN patients (AR, 34%; SOD2, 28%; αENO, 43%). Antibody titers were higher in MN than in healthy and pathologic controls (P<0.005). Anti-NEP IgG(4) did not differ from normal controls (P=0.12). Anti-PLA2r IgG(4) was detected in 60% of patients and correlated with anti-AR, anti-SOD2, and anti-αENO IgG(4) (P<0.001). In MN patients negative for the whole antibody panel (20%), 1-year proteinuria was lower compared with patients with at least one antibody positivity (P<0.05). CONCLUSIONS Our data suggest that IgG(4) is the prevalent isotype for antibodies against cytoplasmic antigens of podocytes (AR, SOD2, αENO). Their levels were higher than in other proteinuric glomerulonephritides and in normal controls and were correlated with anti-PLA2r. Only baseline negativity for all known antibodies predicted lower 1-year proteinuria.

NGAL (Lcn2) monomer is associated with tubulointerstitial damage in chronic kidney disease

The rate of progression of chronic kidney disease (CKD) is difficult to predict using single measurements of serum creatinine or proteinuria. On the other hand, documented tubulointerstitial disease presages worsening CKD, but kidney biopsy is not practical for routine use and generally does not sample the tubulointerstitial compartment of the medulla. Perhaps a urine test that correlates with specific histological findings may serve as a surrogate for the kidney biopsy. Here we compared both immunoblot analysis (under non-reducing conditions) and a commercially available monomer immunoassays of Neutrophil Gelatinase Associated Lipocalin (NGAL) with pathological changes found in kidney biopsies, to determine whether specific histological characteristics associated with a specific NGAL species. We found that the urine of patients with advanced CKD contained NGAL monomers as well as higher molecular weight complexes containing NGAL, identified by MALDI-TOF/TOF mass spectroscopy. The NGAL monomer significantly correlated with glomerular filtration rate, interstitial fibrosis and tubular atrophy. Hence, specific assays of the NGAL monomer implicate histology associated with progressive, severe CKD.The type and the extent of tissue damage inform the prognosis of chronic kidney disease (CKD), but kidney biopsy is not a routine test. Urinary tests that correlate with specific histological findings might serve as surrogates for the kidney biopsy. We used immunoblots and ARCHITECT-NGAL assays to define the immunoreactivity of urinary neutrophil gelatinase-associated lipocalin (NGAL) in CKD, and we used mass spectroscopy to identify associated proteins. We analyzed kidney biopsies to determine whether specific pathological characteristics associated with the monomeric NGAL species. Advanced CKD urine contained the NGAL monomer as well as novel complexes of NGAL. When these species were separated, we found a significant correlation between the NGAL monomer and glomerular filtration rate (r=-0.53, P<0.001), interstitial fibrosis (mild vs. severe disease; mean 54 vs. 167 μg uNGAL/g Cr, P<0.01), and tubular atrophy (mild vs. severe disease; mean 54 vs. 164 μg uNGAL/g Cr, P<0.01). Monospecific assays of the NGAL monomer demonstrated a correlation with histology that typifies progressive, severe CKD.

Urinary NGAL Marks Cystic Disease in HIV-Associated Nephropathy

Nephrosis and a rapid decline in kidney function characterize HIV-associated nephropathy (HIVAN). Histologically, HIVAN is a collapsing focal segmental glomerulosclerosis with prominent tubular damage. We explored the expression of neutrophil gelatinase-associated lipocalin (NGAL), a marker of tubular injury, to determine whether this protein has the potential to aid in the noninvasive diagnosis of HIVAN. We found that expression of urinary NGAL was much higher in patients with biopsy-proven HIVAN than in HIV-positive and HIV-negative patients with other forms of chronic kidney disease. In the HIV-transgenic mouse model of HIVAN, NGAL mRNA was abundant in dilated, microcystic segments of the nephron. In contrast, urinary NGAL did not correlate with proteinuria in human or in mouse models. These data show that marked upregulation of NGAL accompanies HIVAN and support further study of uNGAL levels in large cohorts to aid in the noninvasive diagnosis of HIVAN and screen for HIVAN-related tubular damage.

Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo: α-Enolase and Annexin AI

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

Eotaxin/CCL11 in idiopathic retroperitoneal fibrosis

BACKGROUND Idiopathic retroperitoneal fibrosis (IRF) is a rare fibro-inflammatory disorder characterized by a periaortic tissue which often encases the ureters causing acute renal failure. IRF histology shows fibrosis and a chronic inflammatory infiltrate with frequent tissue eosinophilia. We assessed a panel of molecules promoting eosinophilia and fibrosis in IRF patients and performed an immunogenetic study. METHODS Serum levels of eotaxin/CCL11, regulated and normal T-cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-5, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) were measured using a multiplex assay in 24 newly diagnosed, untreated IRF patients and 14 healthy controls. Retroperitoneal biopsies (available in 8/24 patients) were histologically evaluated to assess eosinophil infiltration, whereas mast cells (MCs) were identified by immunohistochemical analysis for human tryptase. Immunohistochemistry for eotaxin/CCL11 and its receptor CCR3 was also performed. Six single nucleotide polymorphisms (SNPs) within the CCL11 gene (rs6505403, rs1860184, rs4795896, rs17735961, rs16969415 and rs17809012) were investigated in 142 IRF patients and 214 healthy controls. RESULTS Serum levels of eotaxin/CCL11 were higher in IRF patients than in controls (P = 0.009). Eotaxin/CCL11 drives tissue infiltration of eosinophils and MCs, which can promote fibrosis. Eosinophilic infiltration was prominent (>5 cells/hpf) in five (62.5%) cases, and abundant tryptase-positive MCs were found in all cases; notably, MCs were in a degranulating state. Immunohistochemistry showed that CCL11 was highly produced by infiltrating mononuclear cells and that its receptor CCR3 was expressed by infiltrating eosinophils, MCs, lymphocytes and fibroblasts. None of the tested CCL11 SNPs showed disease association, but the TTCCAT haplotype was significantly associated with IRF (P = 0.0005). CONCLUSIONS These findings suggest that the eotaxin/CCL11-CCR3 axis is active in IRF and may contribute to its pathogenesis; the TTCCAT haplotype within the CCL11 gene is significantly associated with IRF.

In vivo characterization of renal auto-antigens involved in human auto-immune diseases: the case of membranous glomerulonephritis.

Renal auto‐immune diseases represent a major source of morbidity in humans. For many years the knowledge on mechanisms of auto‐immunity involving the kidney has been uniquely based on animal models. However, these findings often could not be readily translated to humans owing to notably difference in antigen expression by human podocytes. One example is Heymann nephritis (HN), the experimental model of human membranous glomerulonephritis (MGN), which is obtained in rats by injecting antibodies against megalin, a protein that is not present in human glomeruli. Human studies could not be done in the past since sequencing required too much material exceeding what obtainable from tissue biopsies in vivo. Research is now on the way to identify auto‐antigens and isolate specific auto‐antibodies in humans. New technology developments based on tissue microdissection and proteomical analysis have facilitated the recent discoveries, allowing direct analysis of human tissue in vivo. Major advances on the pathogenesis of MGN, the prototype for the formation and glomerular deposition of auto‐antibodies, are now in progress. Two independent groups have, in fact, demonstrated the existence of specific IgG4 against phospholipase A2 receptor, aldose reductase and Mn‐superoxide dismutase in glomerular eluates and in plasma of a prominent part of patients with MGN, suggesting a major role of these proteins as auto‐antigens in human MGN. This review will focalize these aspects outlining the contribution of proteomics in most recent developments.

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 deposition 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 experimental 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 clusterin, 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 homologue 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 primary 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 (2DE) and western blot with liver extracts and with recombinant receptor-associated protein (RAP) domain as immobilized 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 represents 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 manipulation 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 assay. 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 recombinant 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 antibody=antigen ratio on the sensitivity of the assay. It seems relevant to stress that patients were enrolled both before (n 1⁄4 15) and after (n 1⁄4 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 podocytes did not correspond to LDL-r. They have been characterized 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 negativity of anti-LDL-r serum Ab was shown by two independent 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 approaches 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.

Immunological Basis of Membranous Glomerulonephritis

Primary membranous glomerulonephritis (MGN) is a major glomerular disease causing proteinuria in humans (Jones, 1957). It is the prototype of an autoimmune disease (Couser, et al., 1978) characterized by sub-epithelial immune deposits within glomeruli. Its pathogenesis remains still unknown. Immune deposits are formed by IgG4, their respective antigen and complement. The definition of the immune deposit architecture has been a main focus of the pathology research for years but advances were restricted, until recently, to animal models of the disease, in particular to Heymann nephritis (HN) (Heymann, et al., 1959; Heymann, et al., 1952; Van Damme, et al., 1978). Unfortunately, results from experimental HN cannot be readily exported to human MGN since the major antigen of immune deposits in rat is not present in human glomeruli. Therefore different podocyte antigens are involved in human MGN and their identification is a fundamental step in understanding human pathology. Technology problems, mainly concerning dissection of glomeruli and purification/characterization of glomerular antibodies from human biopsies, have limited the experimental approach in humans for years. In the last 5 years, human MGN has become the topic of renewed nephrologic research. Debiec et al. (Debiec, et al., 2002, 2004) first showed that neutral endopeptidase (NEP) emerges as podocyte antigen in a rare form of congenital MGN due to maternal NEP deficiency and alloimmunization during pregnancy. More recently, the existence of three new glomerular auto-antigens, i.e. phospholipase A2 receptor (PLA2R), aldose reductase (AR) and superoxide dismutase 2 (SOD2) have been proposed by independent groups (Beck, et al., 2009; Prunotto, et al., 2010). Technology evolution in the field of laser capture and proteomics allowed a direct experimental approach in humans, a crucial step for a direct analysis ‘in vivo’. It is now clear that IgG4 eluted from glomeruli of MGN patients recognize a panel of podocyte proteins