Mass spectrometry analysis of IgA1 hinge region in patients with IgA nephropathy

BACKGROUND: Physicochemical alterations of the IgA molecule are supposed to play a pathogenetic role in IgA nephropathy (IgAN). The present study was carried out to analyze the structural variety of O-glycans on the IgA1 hinge region in IgAN. Sera from 9 IgAN patients and 9 healthy controls were individually examined to evaluate the IgA1 content and binding lectins (jacalin and Helix aspersa), using enzyme-linked immunosorbent assay (ELISA) techniques. The IgA1 from pooled sera were separated by affinity chromatography (jacalin), and the fragment containing the hinge region was prepared by pyridylethylation and trypsin treatment. The IgA fragments containing the hinge glycopeptide (33-mer hinge peptide core (HP) + O-glycans) were separated by jacalin affinity chromatography. Because we used jacalin, we only analyzed the Gal-3GalNAc residue containing IgA. The molecular weight (MW) of the IgA1 fragments was estimated using an ion trap mass spectrometer equipped with an electrospray ion source (ESI/MS). RESULTS: IgA1 concentration in pathological sera was higher than in the control serum (p<0.01). Compared with controls, serum IgA1 from IgAN patients showed significantly greater binding to the 2 lectins, jacalin (p<0.01) and Helix aspersa (HA, p<0.001), which are specific for O-linked Gal-beta1,3-GalNAc and GalNAc, respectively. Analyses of pooled sera showed that the number of O-glycosidic chains was comparable in IgAN and normal sera. With regards to the individual residues, we found that IgAN sera contained less sugar and galactose and sialic acid moieties than sera from control subjects, was reduced in IgAN sera, while terminal N-acetylgalactosamine levels were higher when compared with normal serum.CONCLUSIONS: Abnormalities of hinge region O-linked glycans were confirmed using advanced spectrometry technology. The pathogenetic implications for aggregation and defective removal of IgA1 are discussed.     

Immunohistochemical characterization of glomerular IgA deposits in IgA nephropathy

To characterize the IgA deposits found in glomeruli of IgA nephropathy, frozen sections of renal biopsy specimens from 191 consecutive patients with IgA nephropathy were examined by immunofluorescent microscopy. All 191 specimens were positive for IgA1 in glomeruli. IgA2 was detected in 3 out of these 191 specimens. Both kappa and lambda light chains were also detected in the glomeruli of all specimens. The presence of J chain was represented in 113 specimens after acid-urea pretreatment. Secretory component (SC) was detected in 13 out of the 191 specimens, but not in controls. Both J chain and SC were detected in 2 out of 3 specimens positive for IgA2 but not for IgM. These results suggest that IgA deposited in glomeruli in some patients with IgA nephropathy is perhaps mucosally derived IgA.     

The glomerular response to IgA deposition in IgA nephropathy

Compelling evidence points to a role for IgA receptors in the pathogenesis of IgA nephropathy. The soluble form of the type I IgA receptor (FcalphaRI or CD89) forms complexes with IgA that can be found in patients' serum and that initiate the disease in CD89 transgenic mice. A nonclassic IgA receptor, identified as the transferrin receptor (TfR), is highly expressed in patients' mesangium and colocalizes with IgA deposits. TfR preferentially binds polymeric IgA1 complexes, but not monomeric IgA1 or IgA2. The TfR-IgA1 interaction is dependent on carbohydrate moieties because hypoglycosylated IgA1 has superior binding to TfR than normally glycosylated IgA1. Polymeric IgA1 binding enhances mesangial cell TfR expression and results in cell proliferation and inflammatory and profibrogenic cytokine and chemokine production, suggesting a pivotal role in mesangial cell proliferation, matrix expansion, and recruitment of inflammatory cells. We propose that, as a second event, activation of the classic, FcRgamma-associated transmembrane FcalphaRI expressed on circulating myeloid leukocytes takes place. FcalphaRI/gamma2 cross-linking in human FcalphaRI transgenic animals promotes disease progression by enhancing leukocyte chemotaxis and cytokine production, and IgA immune complexes from IgA nephropathy patients induce FcalphaRI-dependent cell activation. This review therefore details the functional consequences of IgA/receptor interactions and discusses proposed mechanisms to explain the development and chronicity of the disease.     

Underglycosylation of IgA1 hinge plays a certain role for its glomerular deposition in IgA nephropathy

This study was performed to isolate and investigate the IgA1 that could accumulate in glomeruli (glomerulophilic IgA1). IgA1 was fractionated by the electric charge and the reactivity to Jacalin. Serum IgA1 of IgA nephropathy patients was separated and fractionated using a Jacalin column and subsequent ion-exchange chromatography. The fractions were divided into three groups of relatively cationic (C), neutral (N), and anionic (A). IgA1 was also divided into Jacalin low (L), intermediate (I), and high (H) affinity fractions by serial elution using 25, 100, and 800 mM galactose. The left kidneys of Wistar rats were perfused with 2, 5, or 10 mg of each group of IgA1. The rats were sacrificed 15 min, 30 min, 3 h, or 24 h after the perfusion. The accumulation of each IgA1 in the glomeruli was then observed by immunofluorescence. The IgA1 of the fractions N and H separated by the two methods was definitely accumulated in the rat glomeruli with a similar pattern. The electrophoresis revealed that the macromolecular IgA1 was increased in fraction H compared with other fractions. Therefore, Jacalin high-affinity IgA1(fraction H) was applied on a diethylaminoethyl column and divided into electrically cationic (HC), neutral (HN), and anionic (HA). Only the asialo-Galbeta1,3GalNAc chain was identified in the fraction HN IgA1 by gas-phase hydrazinolysis. Furthermore, the IgA1 fraction was strongly recognized by peanut agglutinin, Vicia Villosa lectins, and antisynthetic hinge peptide antibody. These results indicated that the IgA1 molecules having the underglycosylated hinge glycopeptide played a certain role in the glomerular accumulation of IgA1 in IgA nephropathy.     

Significance of IgA deposits on the glomerular capillary walls in IgA nephropathy

Based on immunofluorescence findings, 232 patients with IgA nephropathy were classified into two groups; one consisted of 88 patients (38%) with IgA deposits in the glomerular capillary walls together with the mesangial deposits (capillary type), and the other consisted of 144 patients (62%) with deposits confined to the mesangium (mesangial type). Electron microscopic findings revealed dense deposits on the capillary walls (subepithelial, 50%; intramembranous, 65%; and subendothelial, 24%) in 37 of 46 patients with capillary type and six of 47 with mesangial type (P less than .001). Crescent formation observed in greater than or equal to 10% of glomeruli was more frequently found in patients with the capillary type (30/88, 34%) than those with the mesangial type (9/144, 6%) (P less than .01), especially higher in those with subepithelial deposits (15/26, 57%). The capillary type patients showed heavier proteinuria (1.7 +/- 0.2 g/d) than the mesangial type patients (0.6 +/- 0.1 g/d) (P less than .05). Thirteen of the 14 patients in an acute exacerbation phase, manifested by an abrupt increase in urinary protein and development of macroscopic hematuria, showed capillary type IgA deposits. The ratio of patients with normal renal function in the fifth year after apparent onset was lower in the capillary type (74.0%) than in the mesangial type patients (96.9%) (P less than .05). These findings suggest that capillary IgA deposition is closely related to clinical and histologic activities of IgA nephropathy and is considered to be an important factor responsible for the progression of the disease, possibly through crescent formation.     

Ultrastructural alterations of glomerular anionic sites in IgA nephropathy

The ultrastructural alterations of glomerular anionic sites were studied in biopsy specimens from 34 patients with IgA nephropathy using polyethyleneimine (PEI). Prominent common findings in the glomeruli of the patients were few PEI particles in electron dense deposits in the mesangial and subepithelial area and marked reduction in glomerular anionic sites covered with deposits. The anionic sites of the glomerular basement membrane (GBM) and epithelial cell surface coat (ESC) appeared unaltered in the patients with hematuria and/or mild proteinuria. But in patients with proteinuria in the nephrotic range, focally discrete loss of anionic sites in the lamina rara externa (LRE) was seen and the number of anionic sites of the ESC were decreased with retraction of the foot processes. The anionic sites of the lamina rara interna showed much less change in these patients. Subepithelial deposits were often seen concomitantly with focal loss of anionic sites in the LRE at the site of the deposits, but subendothelial deposits had little influence on the anionic sites of the neighboring GBM. The anionic sites of GBM that showed focal thinning with small GBM projections were appreciably decreased in number, but those in split GBM were not decreased. These results suggest that either loss of the negative charge on the glomerular capillary wall associated with subepithelial immune deposition or morphological changes of the GBM contribute to the progression of proteinuria in IgA nephropathy.     

Pregnancy in IgA nephropathy, reflux nephropathy, and focal glomerular sclerosis

Fetal outcome was retrospectively studied in 217 pregnancies observed during the past two decades in 93 patients, 34 suffering from IgA nephropathy (IgAGN, 69 pregnancies), 53 from reflux nephropathy (RN, 137 pregnancies), and six from focal glomerular sclerosis (FGS, 10 pregnancies). Overall incidence of live births was 175 in 217 (81%). Fetal loss, corrected for induced abortions, was 10 in 66 (15%) in IgAGN, 18 in 129 (14%) in RN, and 2 in 10 in FGS. Renal failure and hypertension preexisting prior to conception or developing early in pregnancy were the most important factors associated with unsuccessful fetal outcome whereas urinary tract infection had limited effects in RN patients. Influence of pregnancy on the course of maternal renal disease was evaluated in the same groups of patients. An abnormally rapid deterioration of renal function was observed in three of the women with IgAGN and in one of the RN patients (with an additional case among 46 further female RN patients) but in none in the FGS group. All five women experiencing functional deterioration had a serum creatinine (SCr) level of greater than or equal to 200 mumol/L (2.3 mg/dL) and hypertension at conception. Hypertension in pregnancy was highly predictive of recurrence of hypertension in subsequent pregnancy and of the remote development of permanent hypertension in IgAGN patients. We conclude that when renal function is preserved, pregnancy is usually successful and no deleterious effects on maternal renal disease are to be expected in patients with IgAN, RN, and probably FGS.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgA nephropathy in children: significance of glomerular basement membrane deposition of IgA

Seventeen children with IgA nephropathy were grouped according to the absence (group I, n = 10) or presence (group II, n = 7) of glomerular basement membrane (GBM) deposition of IgA to determine whether GBM deposition of IgA correlated with laboratory or pathologic data at diagnosis or clinical status at follow-up. Children in group II had significantly (p less than 0.01) more proteinuria at diagnosis than children in group I. The percentage of glomeruli demonstrating crescent formation was significantly (p less than 0.05) higher in group II biopsies. Chronic changes of fibrous crescents, segmental sclerosis, global obsolescence, tubular atrophy, and interstitial fibrosis were also significantly (p less than 0.001) more common in group II biopsies. After a mean follow-up period of 2 years, all children in group II have persistent proteinuria of more than 1 g/24 h, and 3 of 5 have renal insufficiency (2 require dialysis). In contrast, 2 of 9 group I children have proteinuria exceeding 1 g/24 h, and only 1 has renal insufficiency. We conclude that, as compared to children with IgA localized to the mesangium, children with IgA nephropathy and GBM deposition of IgA have a higher urinary protein excretion at the time of diagnosis, more severe histologic alterations including a greater percentage of glomeruli demonstrating crescent formation, more chronic changes of segmental or global sclerosis, tubular atrophy, and interstitial fibrosis. Such children usually have persistent proteinuria and are more likely to develop progressive renal disease.     

Participation of complement components in glomerular deposition in IgA nephropathy

To clarify the role of complement components in glomerular deposition in IgA nephropathy, clinicopathological and immunohistological studies were performed on 299 patients (171 males and 128 females; age, 9-71 years). Glomerular IgA deposition with IgG and/or IgM was observed more frequently in patients with Clq and/or C4 than in those with only C3 deposition (P less than 0.001). Patients with glomerular deposition of Clq and/or C4 showed more severe proteinuria (1 g/24 hr less than), a lower glomerular filtration rate (GFR), a higher incidence of duplication of capillary walls and more severe proliferation of mesangial cells and an increase in mesangial matrix (P less than 0.05), as compared to those without both Clq and C4. Patients with glomerular C3 deposition had significantly lower serum CH50 levels at the time of renal biopsy (P less than 0.02) and a significantly higher incidence of sclerotic lesions (P less than 0.05). Patients with C3 deposition in the mesangium and peripheral capillaries had significantly higher serum IgA levels (P less than 0.02), a significantly higher incidence of adhesion (P less than 0.01), duplication and endocapillary proliferation (P less than 0.05) and a more severe increase in mesangial cells (P less than 0.01) than those with C3 deposition only in the mesangium. The above findings demonstrate that analysis of the complement system in glomeruli is important for the evaluation of glomerular damage, clinical findings and prognosis.     

Abnormalities of IgA1 production in IgA nephropathy

SUMMARY: IgA nephropathy (IgAN) is characterized by the mesangial deposition of polymeric IgA1 (plgA1). the original view that this plgA1 is derived from the mucosal immune system can no longer be sustained. Studies of duodenal mucosa and marrow indicate increased production of plgA1 in the marrow and decreased production in the mucosa. These changes are consistent with immunization studies showing exaggerated and prolonged plgA responses to systemic immunization, and reduced mucosal responses to mucosal neoantigens. However, the IgA1 and IgG systemic responses to mucosal antigen are increased in IgAN, a finding consistent with impairment in oral tolerance, the process by which systemic immune responses, to mucosal antigen challenge are normally suppressed. Both IgA1 production and the induction of oral tolerance are under T-cell control. T-cell populations involved in these processes include γδ T cells, Tr cells and T-helper (Th)3 cells; cytokines with a key role in the control of IgA production include interleukin (IL)-10 and transforming growth factor (TGF)-β. There is evidence of abnormal γδ T-cell V region usage in both mucosa and marrow in IgAN. Increased expression of relevant cytokines has also been reported in circulating T cells in IgAN. the increased O-glycosylation of circulating IgA1 in IgAN may also be further evidence of a shift in the production of mucosal-type plgA1 from the mucosa to marrow. These findings suggest that the specific lymphocyte homing mechanisms that normally maintain oral tolerance and control the site of IgA production require further study in IgAN.     

Antimouse laminin antibodies in IgA nephropathy and various glomerular diseases.

IgG, IgA and IgM class antibodies to mouse laminin and human fibronectin in sera from patients with various glomerular diseases (50 cases of IgA nephropathy, 5 cases of minimal-change nephrotic syndrome; 6 cases of membranous nephropathy, 5 cases of systemic lupus erythematosus, 2 cases of Henoch-Sch?nlein purpura, 3 cases of poststreptococcal nephritis and 4 cases of preeclampsia) and from 30 normal controls were tested using a solid-phase enzyme-linked immunosorbent assay method. IgA antimouse laminin antibody titers in sera from IgA nephropathy patients were significantly higher (p less than 0.05) than in controls. There were no statistical differences in IgA antimouse laminin antibody titers between patients with other glomerular diseases and normal controls. IgM antimouse laminin antibody was significantly raised (p less than 0.01) in sera from patients with preeclampsia. The reaction of mouse laminin with the IgA nephropathy and preeclampsia sera on each of the IgA and IgM assay systems was inhibited by the antigen at up to 5 micrograms/ml. However, it was not inhibited by anti-C3d, anti-C1q, anti-J chain and antisecretory component sera or saccharides. The reaction of mouse laminin with an exceptionally high-titer IgA antimouse laminin antibody serum from a normal control on the IgA assay system was clearly inhibited by 1 mM of melibiose, which contains alpha-galactosyl residues. The same concentration of melibiose, however, did not inhibit the reaction of mouse laminin with IgA nephropathy sera on the same assay system. Treatment of mouse laminin with alpha-galactosidase did not alter any binding from IgA nephropathy sera but binding was lost from an exceptionally high-titer normal control serum. There were no correlations between serum IgA level and IgA antimouse laminin antibody titer in sera from IgA nephropathy patients. Immunoblot techniques revealed the presence of antibody in sera from IgA nephropathy patients reacting with both subunits A and B of laminin, somewhat stronger with laminin A. None of the sera tested contained antifibronectin antibodies. These results indicate that the IgA antimouse laminin antibody is a specific antibody in IgA nephropathy and might play a role in the pathogenesis of the nephritis since mouse laminin and human mesangial laminin present a common epitope.     

Angiotensin converting enzyme inhibition improves glomerular size-selectivity in IgA nephropathy.

Clearances of uncharged dextrans of broad size distribution were used to evaluate the effects of a 30 day course of enalapril on glomerular barrier function in 10 patients with IgA nephropathy and proteinuria (from 1.4 to 5.6 g/day). Dextran clearance experiments were repeated three times: before enalapril therapy, after 30 days of enalapril and again 30 days after enalapril withdrawal. GFR, but not RPF, was significantly reduced by enalapril (basal 38.3 +/- 11.9, enalapril 30.2 +/- 12.6 ml/min/1.73 m2) and returned to basal values after enalapril withdrawal. Urinary protein excretion and fractional clearance of albumin were both significantly reduced by enalapril (basal 2.3 +/- 1.1 g/day and 102 +/- 90 x 10(-5), enalapril 1.2 +/- 0.6 g/day and 51 +/- 23 x 10(-5), respectively) and returned to basal values after enalapril withdrawal. Transglomerular passage of large dextrans (radii 54 to 62 A), but not of lower size (26 to 42 A) were significantly lowered by enalapril. When enalapril was withdrawn the dextran-sieving profile returned comparable to the baseline levels. A theoretical analysis of dextran-sieving profiles indicated that enalapril lowered the radius of largest membrane pores. This effect was independent from glomerular hemodynamic changes. We conclude that angiotensin converting enzyme inhibitors (CEI) in humans with IgA nephropathy reduces urinary protein excretion by a primary action on the intrinsic glomerular membrane properties enhancing barrier size-selective function. The hypofiltration associated with enalapril therapy in these patients, which was eliminated by its withdrawal, has to be taken into account as a possible undesired effect of CEI in long-term treatment.     

Contribution of macromolecular IgA1 to IgA abnormality in IgA nephropathy

To evaluate the contribution of macromolecular IgA₁ to IgA abnormality in childhood IgA nephropathy, serum samples from 29 healthy children and 26 patients with IgA nephropathy in different age-groups (7–9, 10–12, and 13–15 years) were each separated by sucrose density gradient ultracentrifugation and assayed for IgA₁ using an enzyme-linked immunosorbent assay. IgA₁ in fraction I (sedimentation coefficient >11.4s) was significantly greater in patients 7–15 years of age (median 36.3–57.0 mg/dl) than in the age-matched controls (median 8.8–10.4 mg/dl). IgA₁ in fraction II (11.4–9.3s) was significantly greater in patients 10–15 years of age (median 46.7–52.6 mg/dl) than in the controls (median 27.8–35.5 mg/dl), and IgA₁ in fraction III (<9.3s) was significantly greater in patients 13–15 years of age (median 156.9 mg/dl) than in the controls (median 120.7 mg/dl). The ratio of IgA₁ in fractions I–III was higher in the patients of each age-group (median 0.233–0.314) than in the controls (median 0.067–0.082), while the ratio of IgA₁ in fractions II–III was not significantly high in patients 7–12 years old (median 0.268 to 0.318) compared with the controls (median 0.182–0.264). Thus, IgA abnormality in childhood IgA nephropathy would be better represented by an increase in macromolecular IgA₁ of >11.4s than by an increase in IgA₁ in fractions of 11.4–9.3s or <9.3s. Received: 8 December 1999 / Revised: 2 March 2000 / Accepted: 2 May 2000     

Differences in glomerular leukocyte infiltration between IgA nephropathy and membranoproliferative glomerulonephritis

Background: An important aspect in glomerular nephritic processes is the enhanced influx of leukocytes into the glomerulus. Methods: To investigate the mechanisms of intraglomerular leukocyte infiltration in IgA nephropathy (IgA-N) and membranoproliferative glomerulonephritis type I (MPGN-I), we immunohistochemically examined the intraglomerular expression of leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18), macrophage-1 (Mac-1, CD11b/CD18) and intercellular adhesion molecule-1 (ICAM-1, CD54) together with glomerular deposition of C3c and fibrinogen. Results: In IgA-N (n=42), LFA-1⁺ cells were distributed mainly in glomeruli with intense expression of ICAM-1, and there was a positive correlation (P<0.001) between the number of LFA-1⁺ cells and the degree of ICAM-1 expression. Mac-1⁺ cells had no correlation with glomerular C3c deposition, but had a significant correlation with fibrinogen deposition (P<0.05). The number of LFA-1⁺ cells was significantly greater than of Mac-1⁺ cells (P<0.05). The number of LFA-1⁺ cells was strongly correlated with that of CD68⁺ cells (P<0.00001). In MPGN-I (n=43), on the contrary, Mac-1⁺ cells correlated only with C3c deposition (P<0.001), and they were observed mainly in peripheral loops of glomerular capillaries where C3c was deposited with a similar distribution. However, there was no relationship between LFA-1⁺ cells and ICAM-1 expression. The number of Mac-1⁺ cells was greater than that of LFA-1⁺ cells (P<0.0001), and most Mac-1⁺ cells were identical to CD15⁺ cells. Conclusion: These results indicate the possibility that different mechanisms may cause glomerular leukocyte infiltration in various forms of human glomerulonephritis. The LFA-1/ICCAM-1 pathway may play an important role in glomerular leukocyte infiltration in IgA-N, while the Mac-1/complement pathway may be important in MPGN-I. The former may promote mainly the infiltration of CD68⁺ cells, and the latter may promote that of CD15⁺ cells. In addition, Mac-1⁺ cells may act as fibrinogen and complement receptors in IgA-N and MPGN-I, respectively. Key words: adhesion molecules; complements; glomerular leukocytes infiltration; IgA nephropathy; membranoproliferative glomerulonephritis