Abstract

Immunoglobulin A (IgA) nephropathy is the most prevalent form of glomerulonephritis worldwide. A renal biopsy is required for an accurate diagnosis, as no convenient biomarker is currently available. We developed a serological test based upon the observation that this nephropathy is characterized by undergalactosylated IgA1 in the circulation and in mesangial immune deposits. In the absence of galactose, the terminal saccharide of O-linked chains in the hinge region of IgA1 is terminal or sialylated N-acetylgalactosamine. A lectin from Helix aspersa, recognizing N-acetylgalactosamine, was used to develop an enzyme-linked immunosorbent assay that measures galactose-deficient IgA1 in serum. The median serum lectin-binding IgA1 level was significantly higher for 153 Caucasian adult patients with IgA nephropathy without progression to end-stage renal disease as compared with that for 150 healthy Caucasian adult controls. As the lectin-binding IgA1 levels for the controls were not normally distributed, the 90th percentile was used for determination of significant elevation. Using a value of 1076 U/ml as the upper limit of normal, 117 of the 153 patients with IgA nephropathy had an elevated serum lectin-binding IgA1 level. The sensitivity as a diagnostic test was 76.5%, with specificity 94%; the positive predictive value was 88.6% and the negative predictive value was 78.9%. We conclude that this lectin-binding assay may have potential as a noninvasive diagnostic test for IgA nephropathy. Immunoglobulin A (IgA) nephropathy is the most prevalent form of glomerulonephritis worldwide. A renal biopsy is required for an accurate diagnosis, as no convenient biomarker is currently available. We developed a serological test based upon the observation that this nephropathy is characterized by undergalactosylated IgA1 in the circulation and in mesangial immune deposits. In the absence of galactose, the terminal saccharide of O-linked chains in the hinge region of IgA1 is terminal or sialylated N-acetylgalactosamine. A lectin from Helix aspersa, recognizing N-acetylgalactosamine, was used to develop an enzyme-linked immunosorbent assay that measures galactose-deficient IgA1 in serum. The median serum lectin-binding IgA1 level was significantly higher for 153 Caucasian adult patients with IgA nephropathy without progression to end-stage renal disease as compared with that for 150 healthy Caucasian adult controls. As the lectin-binding IgA1 levels for the controls were not normally distributed, the 90th percentile was used for determination of significant elevation. Using a value of 1076 U/ml as the upper limit of normal, 117 of the 153 patients with IgA nephropathy had an elevated serum lectin-binding IgA1 level. The sensitivity as a diagnostic test was 76.5%, with specificity 94%; the positive predictive value was 88.6% and the negative predictive value was 78.9%. We conclude that this lectin-binding assay may have potential as a noninvasive diagnostic test for IgA nephropathy. Since its initial description in 1968,1.Berger J. Hinglais N. Les depots intercapillaires d′IgA-IgG (Intercapillary deposits of IgA-IgG).J Urol Nephrol. 1968; 74: 694-695PubMed Google Scholar immunoglobulin A (IgA) nephropathy (IgAN) has become recognized as the most common primary glomerulonephritis worldwide.2.Julian B.A. Waldo F.B. Rifai A. et al.IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States?.Am J Med. 1988; 84: 129-132Abstract Full Text PDF PubMed Scopus (233) Google Scholar IgAN is characterized by mesangial deposits of IgA1 with co-deposits of C3, and often also IgG or IgM or both.1.Berger J. Hinglais N. Les depots intercapillaires d′IgA-IgG (Intercapillary deposits of IgA-IgG).J Urol Nephrol. 1968; 74: 694-695PubMed Google Scholar, 3.Emancipator S.N. IgA nephropathy and Henoch–Schönlein syndrome.in: Jennette J.C. Olson J.L. Schwartz M.M. Silva F.G. Heptinstall's Pathology of the Kidney. Lippincott-Raven Publishers, Philadelphia1998: 479-539Google Scholar, 4.Conley M.E. Cooper M.D. Michael A.F. Selective deposition of immunoglobulin A1 in immunoglobulin A nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus.J Clin Invest. 1980; 66: 1432-1436Crossref PubMed Scopus (261) Google Scholar, 5.Julian B.A. Novak J. IgA nephropathy: an update.Current Opin Nephrol Hypertens. 2004; 13: 171-179Crossref PubMed Scopus (85) Google Scholar, 6.Novak J. Julian B.A. Tomana M. et al.Progress in molecular and genetic studies of IgA nephropathy.J Clin Immunol. 2001; 21: 310-327Crossref PubMed Scopus (99) Google Scholar Over 50% of IgAN patients exhibit increased serum levels of IgA and IgA-containing circulating immune complexes (CIC).7.Czerkinsky C. Koopman W.J. Jackson S. et al.Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies.J Clin Invest. 1986; 77: 1931-1938Crossref PubMed Scopus (167) Google Scholar, 8.Coppo R. Basolo B. Piccoli G. et al.IgA1 and IgA2 immune complexes in primary IgA nephropathy and Henoch–Schönlein nephritis.Clin Exp Immunol. 1984; 57: 583-590PubMed Google Scholar, 9.Schena F.P. Pastore A. Ludovico N. et al.Increased serum levels of IgA1-IgG immune complexes and anti-F(ab′)2 antibodies in patients with primary IgA nephropathy.Clin Exp Immunol. 1989; 77: 15-20PubMed Google Scholar The mesangial immune deposits likely originate from these CIC. We have found that these complexes contain aberrantly glycosylated polymeric IgA1 (galactose (Gal)-deficient in O-linked glycans).10.Tomana M. Novak J. Julian B.A. et al.Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J Clin Invest. 1999; 104: 73-81Crossref PubMed Scopus (392) Google Scholar, 11.Tomana M. Matousovic K. Julian B.A. et al.Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (279) Google Scholar, 12.Novak J. Vu H.L. Novak L. et al.Interactions of human mesangial cells with IgA and IgA-containing circulating immune complexes.Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13.Novak J. Tomana M. Matousovic K. et al.IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells.Kidney Int. 2005; 67: 504-513Abstract Full Text Full Text PDF PubMed Scopus (173) Google ScholarIn vitro studies have demonstrated that these CIC readily bind to mesangial cells and induce their proliferation.12.Novak J. Vu H.L. Novak L. et al.Interactions of human mesangial cells with IgA and IgA-containing circulating immune complexes.Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 13.Novak J. Tomana M. Matousovic K. et al.IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells.Kidney Int. 2005; 67: 504-513Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 14.Moura I.C. Arcos-Fajardo M. Sadaka C. et al.Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy.J Am Soc Nephrol. 2004; 15: 622-634Crossref PubMed Scopus (150) Google Scholar, 15.Coppo R. Amore A. Aberrant glycosylation in IgA nephropathy (IgAN).Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 16.Amore A. Cirina P. Conti G. et al.Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells.J Am Soc Nephrol. 2001; 12: 1862-1871PubMed Google Scholar Thus, aberrantly glycosylated polymeric IgA1 may be involved in the pathogenesis of the glomerular injury in IgAN. Indeed, elution of glomerular immune deposits from renal tissue of patients with IgAN has demonstrated substantial quantities of Gal-deficient IgA1.17.Allen A.C. Bailey E.M. Brenchley P.E.C. et al.Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar,18.Hiki Y. Odani H. Takahashi M. et al.Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy.Kidney Int. 2001; 59: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar Several laboratories have localized the aberrant glycosylation to the O-linked glycans in the hinge region of some IgA1 molecules.5.Julian B.A. Novak J. IgA nephropathy: an update.Current Opin Nephrol Hypertens. 2004; 13: 171-179Crossref PubMed Scopus (85) Google Scholar, 15.Coppo R. Amore A. Aberrant glycosylation in IgA nephropathy (IgAN).Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 19.Floege J. Feehally J. IgA nephropathy: recent developments.J Am Soc Nephrol. 2000; 11: 2395-2403PubMed Google Scholar, 20.Smith A.C. Feehally J. New insights into the pathogenesis of IgA nephropathy. Pathogenesis of IgA nephropathy.Springer Semin Immunopathol. 2003; 24: 477-493Crossref PubMed Scopus (30) Google Scholar, 21.Barratt J. Feehally J. IgA Nephropathy.J Am Soc Nephrol. 2005; 16: 2088-2097Crossref PubMed Scopus (397) Google Scholar In the absence of Gal, the terminal N-acetylgalactosamine (GalNAc) residues, sialylated GalNAc, or the hinge-region glycopeptides are recognized by naturally occurring IgG or IgA1 antibodies with anti-glycan or anti-hinge region peptide specificities, thus generating CIC.10.Tomana M. Novak J. Julian B.A. et al.Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J Clin Invest. 1999; 104: 73-81Crossref PubMed Scopus (392) Google Scholar Some of the resultant CIC escape normal clearance and deposit in the mesangium. These observations support the postulate that O-glycosylation abnormalities of IgA1 predispose to mesangial IgA deposition.5.Julian B.A. Novak J. IgA nephropathy: an update.Current Opin Nephrol Hypertens. 2004; 13: 171-179Crossref PubMed Scopus (85) Google Scholar, 12.Novak J. Vu H.L. Novak L. et al.Interactions of human mesangial cells with IgA and IgA-containing circulating immune complexes.Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 14.Moura I.C. Arcos-Fajardo M. Sadaka C. et al.Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy.J Am Soc Nephrol. 2004; 15: 622-634Crossref PubMed Scopus (150) Google Scholar, 15.Coppo R. Amore A. Aberrant glycosylation in IgA nephropathy (IgAN).Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21.Barratt J. Feehally J. IgA Nephropathy.J Am Soc Nephrol. 2005; 16: 2088-2097Crossref PubMed Scopus (397) Google Scholar, 22.van der Boog P.J. van Kooten C. van Seggelen A. et al.An increased polymeric IgA level is not a prognostic marker for progressive IgA nephropathy.Nephrol Dial Transplant. 2004; 19: 2487-2493Crossref PubMed Scopus (19) Google Scholar For the diagnosis of IgAN, a renal biopsy is required because there is no established serological marker. To avoid this invasive procedure, particularly in individuals with mild disease, we sought to develop a noninvasive test to support the diagnosis of IgAN using a GalNAc-specific lectin. Based on previous work by our group and others, the lectin from Helix aspersa (HAA) is well suited for detecting Gal-deficient IgA1 O-linked glycans.10.Tomana M. Novak J. Julian B.A. et al.Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J Clin Invest. 1999; 104: 73-81Crossref PubMed Scopus (392) Google Scholar, 11.Tomana M. Matousovic K. Julian B.A. et al.Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (279) Google Scholar, 15.Coppo R. Amore A. Aberrant glycosylation in IgA nephropathy (IgAN).Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 16.Amore A. Cirina P. Conti G. et al.Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells.J Am Soc Nephrol. 2001; 12: 1862-1871PubMed Google Scholar, 17.Allen A.C. Bailey E.M. Brenchley P.E.C. et al.Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients.Kidney Int. 2001; 60: 969-973Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 23.Allen A.C. Harper S.J. Feehally J. Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.Clin Exp Immunol. 1995; 100: 470-474Crossref PubMed Scopus (267) Google Scholar, 24.Carpenter G.H. Proctor G.B. Shori D.K. O-glycosylation of salivary IgA as determined by lectin analysis.Biochem Soc Trans. 1997; 25: S659Crossref PubMed Scopus (4) Google Scholar, 25.Hiki Y. Kokubo T. Iwase H. et al.Underglycosylation of IgA1 hinge plays a certain role for its glomerular deposition in IgA nephropathy.J Am Soc Nephrol. 1999; 10: 760-769PubMed Google Scholar, 26.Moore J.S. Kulhavy R. Tomana M. et al.Reactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteins.Mol Immunol. 2007; 44: 2598-2604Crossref PubMed Scopus (75) Google Scholar In this study, we show that a quantitative lectin-based enzyme-linked immunosorbent assay (ELISA) can be successfully used to measure serum levels of Gal-deficient IgA in sera of patients with IgAN. Median serum IgA concentration was 3 943 μg/ml (range 1 171–11 713 μg/ml) for 153 Caucasian IgAN patients who had not progressed to end-stage renal disease as compared with 2 843 μg/ml (range 886–8 185 μg/ml) for 150 healthy controls (P5 years after biopsy461.0930.2±11.913.3±7.01 974 (504–4 391)4 440 (2 037–9 148)425 (129–812)0.19 (0.06–19.3)60.7±38.5GFR90 ml/min/1.73 m2261.1727.5±9.53.2±4.91 466 (320–3 579)3 299 (1 409–7 422)373 (170–710)0.38 (0.06–2.53)105.0±16.3Normal urinalysis261.3630.0±10.311.8±8.92 030 (504–3 901)4 571 (2 037–7 350)417 (208–820)0.09 (0.06–0.19)45.5±16.2GFR, glomerular filtration rate; HAA, lectin from Helix aspersa; IgA, immunoglobulin A; UP/Cr ratio, urinary protein/creatinine ratio.a Estimated GFR was calculated using the MDRD formula (53). Open table in a new tab Table 2Diagnostic utility of serum IgA and HAA-IgA assaysControlPatientsSensitivitySpecificityPositive predictiveNegative predictiveYesNoYesNoIgA >4 761 μg/ml15135501030.327 (0.258, 0.405)0.900 (0.842, 0.938)0.769 (0.654, 0.855)0.567 (0.504, 0.629)HAA-IgA >1 076 U/ml15135116370.758 (0.685, 0.819)0.900 (0.842, 0.938)0.885 (0.820, 0.929)0.785 (0.718, 0.840)HAA-IgA >394 U/mg IgA1513594590.614 (0.535, 0.685)0.900 (0.842, 0.938)0.785 (0.628, 0.756)0.696 (0.628, 0.756)HAA, lectin from Helix aspersa; IgA, immunoglobulin A.The numbers in the brackets are the 95% confidence intervals. Open table in a new tab GFR, glomerular filtration rate; HAA, lectin from Helix aspersa; IgA, immunoglobulin A; UP/Cr ratio, urinary protein/creatinine ratio. HAA, lectin from Helix aspersa; IgA, immunoglobulin A. The numbers in the brackets are the 95% confidence intervals. Serum HAA-IgA levels for the patient and healthy control groups are shown in Figure 1a. The individual levels for the control group were not distributed normally with a marked skewing towards elevated levels. The median serum HAA-IgA level for the patients of 1 731 U/ml (range 320–8 317 U/ml) was significantly higher than the median of 615 U/ml (range 264–1 807 U/ml) for the control group (P<0.0001). The serum HAA-IgA level was higher than 1 076 U/ml (90th percentile for controls) for 117 of the 153 patients. These data indicate a sensitivity of 76.5% and a specificity of 90% with a positive predictive value of 88.6% and a negative predictive value of 78.9%. To illustrate the potential of this assay, the results are also presented as a receiver operating characteristic curve in Figure 1b. The area under the curve is 0.9021 with a standard error 0.0178, indicating that the true-positive rate was high and the false-positive rate was low. The 95% confidence interval was 0.8672–0.9370 (P<0.0001). Reproducibility of the HAA-IgA ELISA was high (repeated the assays had an r=0.970; P<0.0001). The serum HAA-IgA and IgA levels correlated significantly in IgAN patients (r2=0.571, P<0.0001) and healthy controls (r2=0.392, P<0.0001). The median value of the HAA-binding fraction of IgA was 445 U/mg IgA (range 129–879) for 153 Caucasian IgAN patients who had not progressed to end-stage renal disease as compared with 248 U/mg IgA (range 56–666) for 150 healthy controls (P<0.0001). Ninety-four patients had a HAA-binding fraction of IgA higher than 394 U/mg IgA (90th percentile for controls). These data indicate a sensitivity of 61.4% and specificity of 90% with a positive predictive value of 86.2% and a negative predictive value of 69.6%. Seven of 36 patients with normal HAA-IgA levels had an HAA-binding fraction of IgA above the 90th percentile for controls; among the 135 healthy controls with a HAA-IgA level below the cutoff point, 11 had a similarly increased value for HAA-binding fraction of IgA. To verify the ELISA data, selected serum samples from four IgAN patients and two healthy controls representing a wide range of HAA-IgA values were analyzed by Western blotting for total and Gal-deficient IgA (see Materials and methods). The band intensity was measured by densitometry and the ratio of HAA-IgA to IgA heavy chain was calculated. This value was plotted against HAA-binding fraction of IgA1 (U/mg IgA) determined by ELISA in the same sample. The data in Figure 2 are representative for the IgAN patients and healthy controls over the wide range of HAA-IgA values. The results from the two assays showed good correlation (r2=0.818, P=0.046). Serum HAA-IgA and IgA concentrations as well as values for the HAA-binding fraction of IgA (U/mg IgA) are shown for subgroups of patients in Table 2. There was no significant difference based upon gender. The median level for 26 patients with a normal urinalysis at the time of study was 2 030 Units/ml (88% with an elevated level). Levels of HAA-IgA for subjects with an estimated glomerular filtration rate (GFR) ≥90 ml/min/1.73 m2 were similar to those of patients with an estimated GFR <30 ml/min/1.73 m2. Furthermore, the HAA-IgA levels did not significantly differ between patients who had undergone renal biopsy within eight weeks of the blood sampling and patients whose blood was obtained more than five years after biopsy. Serum HAA-IgA levels did not significantly correlate with urinary protein/creatinine (UP/Cr) ratio (r=-0.128, P=0.211), estimated GFR (r=-0.147, P=0.069), or interval since biopsy (r=0.0844, P=0.300). Thus, the serum HAA-IgA level did not appear to be an indicator of disease activity in our cohort of Caucasian adults with IgAN. Renal biopsies from 34 patients, who had blood obtained within eight weeks of diagnostic biopsy, were graded according to the Haas system27.Haas M. Histologic subclassification of IgA nephropathy: a clinicopathologic study of 244 cases.Am J Kidney Dis. 1997; 29: 829-842Abstract Full Text PDF PubMed Scopus (397) Google Scholar by two investigators (WJC and BAJ) blinded to the clinical and laboratory data. Levels of HAA-IgA were above the cutoff value of healthy controls in five of eight (63%) patients with subclass 1, 2, and 3 histological features and in 20 of 26 (77%) patients with subclass 4 and 5 disease. In this study, we developed a quantitative assay to measure serum Gal-deficient IgA (HAA-IgA). Although the total IgA correlated with the HAA-IgA levels, only the serum HAA-IgA level differentiated IgAN patients from the healthy controls with high sensitivity (Table 2). This finding may be explained by a greater amount of Gal-deficient IgA recognized by naturally occurring anti-glycan antibodies, resulting in formation of CIC.10.Tomana M. Novak J. Julian B.A. et al.Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J Clin Invest. 1999; 104: 73-81Crossref PubMed Scopus (392) Google Scholar In our ELISA, serum IgA was captured by a polyclonal IgA heavy chain-specific antibody. As a result, both subclasses, IgA1 that accounts for about 84% of serum IgA28.Mestecky J. Russell M.W. IgA subclasses.Monogr Allergy. 1986; 19: 277-301PubMed Google Scholar,29.Mestecky J. Russell M.W. Jackson S. et al.The human IgA system: a reassessment.Clin Immunol Immunopathol. 1986; 40: 105-114Crossref PubMed Scopus (176) Google Scholar and IgA2, are captured. However, only IgA1 has O-linked glycans and, therefore, is the only IgA subclass that can bind to HAA, a GalNAc-specific lectin. Consequently, the HAA-IgA assay is a measure of serum Gal-deficient IgA1. Mesangial IgA deposition is incidentally encountered in asymptomatic individuals in frequencies that vary among studies, ranging from 2 to 16%.30.Waldherr R. Rambousek M. Duncker W.D. et al.Frequency of mesangial IgA deposits in non-selected autopsy series.Nephrol Dial Transplant. 1989; 4: 943-946PubMed Google Scholar, 31.Sinniah R. Occurence of mesangial IgA and IgM deposits in a control necropsy population.J Clin Pathol. 1983; 36: 276-279Crossref PubMed Scopus (88) Google Scholar, 32.Varis J. Rantala I. Pasternack A. et al.Immunoglobulin and complement deposition in glomeruli of 756 subjects who had committed suicide or met with a violent death.J Clin Pathol. 1993; 46: 607-610Crossref PubMed Scopus (60) Google Scholar, 33.Suzuki K. Honda K. Tanabe K. et al.Incidence of latent mesangial IgA deposition in renal allograft donors in Japan.Kidney Int. 2003; 63: 2286-2294Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 34.Rosenberg H.G. Martinez P.S. Vaccarezza A.S. et al.Morphological findings in 70 kidneys of living donors for renal transplant.Pathol Res Pract. 1990; 186: 619-624Crossref PubMed Scopus (18) Google Scholar For example, in a study in Japan, biopsies from 510 newly procured living-donor renal allografts taken at the time of transplantation showed mesangial IgA deposition in 82 (16.1%) of them.33.Suzuki K. Honda K. Tanabe K. et al.Incidence of latent mesangial IgA deposition in renal allograft donors in Japan.Kidney Int. 2003; 63: 2286-2294Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar By contrast, in a study of necropsy examinations of 756 Finish adults who had committed suicide or met with a violent death, mesangial IgA deposits were found in 18 (2.4%) of the individuals.32.Varis J. Rantala I. Pasternack A. et al.Immunoglobulin and complement deposition in glomeruli of 756 subjects who had committed suicide or met with a violent death.J Clin Pathol. 1993; 46: 607-610Crossref PubMed Scopus (60) Google Scholar However, these renal biopsies with IgA deposits rarely showed any significant pathological changes by light microscopy. Consequently, the clinical significance of these observations is not clear, but many otherwise normal healthy individuals may have mesangial deposits of IgA without clinical manifestation of nephritis or pathological changes of the glomeruli. It would have been interesting to have renal biopsy data on our cohort of healthy controls and to assess the frequency of IgA deposits with and without concurrent renal pathology by light microscopy. However, this could not have been carried out for ethical reasons. Several groups of investigators have shown significantly increased binding of GalNAc-specific lectins to serum IgA1 from patients with IgAN or Henoch–Schönlein purpura nephritis.10.Tomana M. Novak J. Julian B.A. et al.Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.J Clin Invest. 1999; 104: 73-81Crossref PubMed Scopus (392) Google Scholar, 11.Tomana M. Matousovic K. Julian B.A. et al.Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (279) Google Scholar, 23.Allen A.C. Harper S.J. Feehally J. Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.Clin Exp Immunol. 1995; 100: 470-474Crossref PubMed Scopus (267) Google Scholar, 35.Andre P.M. Le Pogamp P. Chevet D. Impairment of jacalin binding to serum IgA in IgA nephropathy.J Clin Lab Anal. 1990; 4: 115-119Crossref PubMed Scopus (98) Google Scholar, 36.Hiki Y. Horii A. Iwase H. et al.O-linked oligosaccharide on IgA1 hinge region in IgA nephropathy. Fundamental study for precise structure and possible role.Contrib Nephrol. 1995; 111: 73-84Crossref PubMed Google Scholar, 37.Mestecky J. Tomana M. Crowley-Nowick P.A. et al.Defective galactosylation and clearance of IgA1 molecules as a possible etiopathogenic factor in IgA nephropathy.Contrib Nephrol. 1993; 104: 172-182Crossref PubMed Google Scholar, 38.Allen A.C. Willis F.R. Beattie T.J. et al.Abnormal IgA glycosylation in Henoch–Schönlein purpura restricted to patients with clinical nephritis.Nephrol Dial Transplant. 1998; 13: 930-934Crossref PubMed Scopus (167) Google Scholar, 39.Linossier M.T. Palle S. Berthoux F. Different glycosylation profile of serum IgA1 in IgA nephropathy according to the glomerular basement membrane thickness: normal versus thin.Am J Kidney Dis. 2003; 41: 558-564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 40.Hashim O.H. Shuib A.S. Chua C.T. The interaction of selective plant lectins with neuraminidase-treated and untreated IgA1 from the sera of IgA nephropathy patients.Immunol Invest. 2001; 30: 21-31Crossref PubMed Scopus (18) Google Scholar Using HAA, Linossier et al.39.Linossier M.T. Palle S. Berthoux F. Different glycosylation profile of serum IgA1 in IgA nephropathy according to the glomerular basement membrane thickness: normal versus thin.Am J Kidney Dis. 2003; 41: 558-564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar showed increased binding for IgAN patients with normal thickness of the glomerular basement membrane on electron microscopy. Studies with other GalNAc-specific lectins, Vicia villosa lectin23.Allen A.C. Harper S.J. Feehally J. Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.Clin Exp Immunol. 1995; 100: 470-474Crossref PubMed Scopus (267) Google Scholar,38.Allen A.C. Willis F.R. Beattie T.J. et al.Abnormal IgA glycosylation in Henoch–Schönlein purpura restricted to patients with clinical nephritis.Nephrol Dial Transplant. 1998; 13: 930-934Crossref PubMed Scopus (167) Google Scholar and Glycine max lectin,40.Hashim O.H. Shuib A.S. Chua C.T. The interaction of selective plant lectins with neuraminidase-treated and untreated IgA1 from the sera of IgA nephropathy patients.Immunol Invest. 2001; 30: 21-31Crossref PubMed Scopus (18) Google Scholar showed increased serum Gal-deficient IgA1 in patients with Henoch–Schönlein purpura nephritis and IgAN, respectively. However, these results did not distinguish the patients from healthy controls; thus, the test would not be adequate to diagnose either disorder. Several candidates for a serological diagnostic marker of IgAN, such as increased levels of serum IgA or of IgA-containing complexes with fibronectin, IgG, or C3, have been previously examined.7.Czerkinsky C. Koopman W.J. Jackson S. et al.Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies.J Clin Invest. 1986; 77: 1931-1938Crossref PubMed Scopus (167) Google Scholar, 8.Coppo R. Basolo B. Piccoli G. et al.IgA1 and IgA2 immune complexes in primary IgA nephropathy and Henoch–Schönlein nephritis.Clin Exp Immunol. 1984; 57: 583-590PubMed Google Scholar, 9.Schena F.P. Pastore A. Ludovico N. et al.Increased serum levels of IgA1-IgG immune complexes and anti-F(ab′)2 antibodies in patients with primary IgA nephropathy