Glomerular deposition of galactose-deficient IgA1-containing immune complexes via glomerular endothelial cell injuries.

BACKGROUND: Galactose-deficient immunoglobulin A1 (Gd-IgA1) plays a crucial role in the development of IgA nephropathy (IgAN). However, the pathological role of Gd-IgA1-containing immune complexes (ICs) and the mechanism of deposition in the mesangial region remain unclear. METHODS: To examine the deposition of Gd-IgA1-containing ICs in the mesangial region through glomerular endothelial cell injury, we evaluated the alteration of renal microvascular endothelial glycocalyx in nude mice injected with Gd-IgA1-IgG ICs. Human renal glomerular endothelial cells (HRGECs) were used to assess the potential capacity of Gd-IgA1-IgG ICs to activate endothelial cells. RESULTS: Nude mice injected with Gd-IgA1-containing ICs showed podocyte and endothelial cell injuries, with IgA, IgG and C3 depositions in glomerular capillaries and the mesangium. Moreover, albuminuria and hematuria were induced. Real-time glycocalyx imaging showed that renal microvascular glycocalyx was decreased immediately after injection of Gd-IgA1-containing ICs and then mesangial IgA deposition was increased. After coculture of Gd-IgA1-containing ICs with HRGECs, messenger RNA expression levels of endothelial adhesion molecules and proinflammatory mediators were upregulated significantly. CONCLUSION: Gd-IgA1-IgG ICs had a high affinity for glomerular endothelial cells, which resulted in glomerular filtration barrier dysfunction mediated by glycocalyx loss. Furthermore, Gd-IgA1-IgG ICs accelerated the production of adhesion factors and proinflammatory cytokines in glomerular endothelial cells. The glomerular endothelial cell injury induced by Gd-IgA1-containing ICs may enhance the permeability of Igs in the mesangial region and subsequent inflammatory responses in the pathogenesis of IgAN.

Usefulness of Dapagliflozin for Nephrotic Syndrome Secondary to Diabetic Kidney Disease.

An 81-year-old woman with a medical history of type 2 diabetes mellitus and diabetic nephropathy was admitted with a diagnosis of multiple cerebellar infarctions. Proteinuria and leg edema were observed on the day after admission and diagnosed as nephrotic syndrome. Furosemide and spironolactone were started but showed no diuretic effect, and the renal function deteriorated. These agents were then replaced with dapagliflozin, which resulted in a positive diuretic effect and subsequent improvement of hypoalbuminemia and renal dysfunction. This case report demonstrates the utility of dapagliflozin for nephrotic syndrome to achieve a positive diuretic effect and improve hypoalbuminemia without deteriorating the renal function.

Experimental evidence of pathogenic role of IgG autoantibodies in IgA nephropathy.

BACKGROUND: IgA nephropathy is thought to be an autoimmune disease wherein galactose-deficient IgA1 (Gd-IgA1) is recognized by IgG autoantibodies, resulting in formation and renal accumulation of nephritogenic immune complexes. Although this hypothesis is supported by recent findings that, in renal immunodeposits of IgA nephropathy patients, IgG is enriched for Gd-IgA1-specific autoantibodies, experimental proof is still lacking. METHODS: IgG isolated from sera of IgA nephropathy patients or produced as a recombinant IgG (rIgG) was mixed with human Gd-IgA1 to form immune complexes. IgG from healthy individuals served as a control. Nude and SCID mice were injected with human IgG and Gd-IgA1, in immune complexes or individually, and their presence in kidneys was ascertained by immunofluorescence. Pathologic changes in the glomeruli were evaluated by quantitative morphometry and exploratory transcriptomic profiling was performed by RNA-Seq. RESULTS: Immunodeficient mice injected with Gd-IgA1 mixed with IgG autoantibodies from patients with IgA nephropathy, but not Gd-IgA1 mixed with IgG from healthy individuals, displayed IgA, IgG, and mouse complement C3 glomerular deposits and mesangioproliferative glomerular injury with hematuria and proteinuria. Un-complexed Gd-IgA1 or IgG did not induce pathological changes. Moreover, Gd-IgA1-rIgG immune complexes injected into immunodeficient mice induced histopathological changes characteristic of human disease. Exploratory transcriptome profiling of mouse kidney tissues indicated that these immune complexes altered gene expression of multiple pathways, in concordance with the changes observed in kidney biopsies of patients with IgA nephropathy. CONCLUSIONS: This study provides the first in vivo evidence for a pathogenic role of IgG autoantibodies specific for Gd-IgA1 in the pathogenesis of IgA nephropathy.

Progressive Renal Dysfunction due to IgG4-Related Kidney Disease Refractory to Steroid Therapy: A Case Report.

Recently, as the number of case reports of IgG4-related kidney disease (IgG4-RKD) has increased, the histopathological features and clinical approach have been clarified. IgG4-RKD generally has a benign prognosis due to the efficacy of steroid therapy and rarely requires dialysis. Herein, we report a case of IgG4-RKD that presented with a subacute onset, advanced to end-stage kidney disease, and finally required maintenance hemodialysis despite steroid therapy. A 75-year-old man was admitted to our hospital for further evaluation of subacute renal failure. Diffuse enlargement of the kidney on computed tomography and increased urinary N-acetyl-ß-D-glucosaminidase and α1-microglobulin levels led us to suspect IgG4-RKD. Upon admission, the laboratory serological findings were as follows: creatinine 3.3 mg/dL, urea nitrogen 46.9 mg/dL, and IgG4 235 mg/dL. Urinalysis showed slight proteinuria without hematuria. Percutaneous renal needle biopsy showed diffuse infiltration of abundant lymphocytes and IgG4-positive plasma cells and storiform fibrosis, which is specific to IgG4-RKD, in the interstitium on light microscopy. Slight linear deposition of C3 was also observed in the tubules on immunofluorescence microscopy, with no electron-dense deposits. He was definitively diagnosed as having IgG4-RKD and started on prednisolone 0.6 mg/kg/day. However, the renal insufficiency continued to progress and hemodialysis was necessary. As the prednisolone was tapered, renal function did not improve and maintenance hemodialysis was started. In conclusion, this case indicates that the prognosis of IgG4-RKD is not necessarily benign and that further studies involving more patients are needed.

Serum galactose-deficient-IgA1 and IgG autoantibodies correlate in patients with IgA nephropathy.

IgA nephropathy is an autoimmune disease characterized by IgA1-containing glomerular immune deposits. We previously proposed a multi-hit pathogenesis model in which patients with IgA nephropathy have elevated levels of circulatory IgA1 with some O-glycans deficient in galactose (Gd-IgA1, autoantigen). Gd-IgA1 is recognized by anti-glycan IgG and/or IgA autoantibodies, resulting in formation of pathogenic immune complexes. Some of these immune complexes deposit in the kidney, activate mesangial cells, and incite glomerular injury leading to clinical presentation of IgA nephropathy. Several studies have demonstrated that elevated circulatory levels of either Gd-IgA1 or the corresponding autoantibodies predict progressive loss of renal clearance function. In this study we assessed a possible association between serum levels of Gd-IgA1 and IgG or IgA autoantibodies specific for Gd-IgA1 in serum samples from 135 patients with biopsy-proven IgA nephropathy, 76 patients with other renal diseases, and 106 healthy controls. Our analyses revealed a correlation between the concentrations of the autoantigen and the corresponding IgG autoantibodies in sera of patients with IgA nephropathy, but not of disease or healthy controls. Moreover, our data suggest that IgG is the predominant isotype of Gd-IgA1-specific autoantibodies in IgA nephropathy. This work highlights the importance of both initial hits in the pathogenesis of IgA nephropathy.

Impact of preoperative ultrasonography findings on the patency rate of vascular access in Japanese hemodialysis patients.

BACKGROUND: Although ultrasonography before a vascular access (VA) operation has become popular in recent years, benchmarks for the diameter or blood flow of arteries or veins are not defined in Japan. The objective of the present study is to analyze the relationship between preoperative US findings and the patency rate of VA in Japanese hemodialysis patients. METHODS: 139 patients with end stage kidney disease were enrolled in this study. They had been given primary radiocephalic arteriovenous fistula (AVF) from February 2009 to December 2010 at the Juntendo University Hospital and were followed up over 2 years. We defined the interval from the time of AVF creation until first access thrombosis or any intervention to maintain or restore blood flow as patency time (primary patency). We examined the correlation between the 2-year primary patency rate of VA and the diameter of the radial artery (RA), brachial artery (BA), or cephalic vein at an anastomosis presumptive region by US, the blood flow of RA or BA, as measured by US, age, gender, and primary kidney diseases. RESULTS: The average patency term was 448.6 ± 271.3 days, with the 1-year and 2-year patency rate as 64.0 and 51.2 %, respectively. The patency rate was significantly lower in elderly patients over the age of 75 and in patients with diabetes mellitus. US findings of 2.0 mm or less in the RA diameter also resulted in a noticeably low patency rate. A multivariate analysis indicated that those factors were risk factors for early VA failure. CONCLUSIONS: Preoperative US findings of the diameter of RA may involve the patency rate of VA, making it appears that an RA of 2.0 mm or more in diameter at an anastomosis region may be more effective for the improvement in the patency rate of VA.

Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.

BACKGROUND: Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). METHODS: Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. RESULTS: Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. CONCLUSION: This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease.

Diagnosis and activity assessment of immunoglobulin A nephropathy: current perspectives on noninvasive testing with aberrantly glycosylated immunoglobulin A-related biomarkers.

Immunoglobulin (Ig) A nephropathy (IgAN) is the most common form of glomerular disease worldwide and is associated with a poor prognosis. Thus, development of a curative treatment and strategies for early diagnosis and treatment are urgently needed. Pathological analysis of renal biopsy is the gold standard for the diagnosis and assessment of disease activity; however, immediate and frequent assessment based on biopsy specimens is difficult. Therefore, a simple and safe alternative is desirable. On the other hand, it is now widely accepted that multi-hit steps, including production of aberrantly glycosylated serum IgA1 (first hit), and IgG or IgA autoantibodies that recognize glycan containing epitopes on glycosylated serum IgA1 (second hit) and their subsequent immune complex formation (third hit) and glomerular deposition (fourth hit), are required for continued progression of IgAN. Although the prognostic and predictive values of several markers have been discussed elsewhere, we recently developed a highly sensitive and specific diagnostic method by measuring serum levels of glycosylated serum IgA1 and related IgA immune complex. In addition, we confirmed a significant correlation between serum levels of these essential effector molecules and disease activity after treatment, suggesting that each can be considered as a practical surrogate marker of therapeutic effects in this slowly progressive disease. Such a noninvasive diagnostic and activity assessment method using these disease-oriented specific biomarkers may be useful in the early diagnosis of and intervention in IgAN, with appropriate indication for treatment, and thus aid in the future development and dissemination of specific and curative treatments.

Serum under-O-glycosylated IgA1 level is not correlated with glomerular IgA deposition based upon heterogeneity in the composition of immune complexes in IgA nephropathy.

BACKGROUND: Although serum under-O-glycosylated IgA1 in IgA nephropathy (IgAN) patients may deposit more preferentially in glomeruli than heavily-O-glycosylated IgA1, the relationship between the glomerular IgA deposition level and the O-glycan profiles of serum IgA1 remains obscure. METHODS: Serum total under-O-glycosylated IgA1 levels were quantified in 32 IgAN patients by an enzyme-linked immunosorbent assay (ELISA) with Helix aspersa (HAA) lectin. Serum under-O-glycosylated polymeric IgA1 (pIgA1) was selectively measured by an original method using mouse Fcα/µ receptor (mFcα/µR) transfectant and flow cytometry (pIgA1 trap). The percentage area of IgA deposition in the whole glomeruli (Area-IgA) was quantified by image analysis on the immunofluorescence of biopsy specimens. Correlations were assessed between the Area-IgA and data from HAA-ELISA or pIgA1 trap. The relationships between clinical parameters and data from HAA-ELISA or pIgA1 trap were analyzed by data mining approach. RESULTS: While the under-O-glycosylated IgA1 levels in IgAN patients were significantly higher than those in healthy controls when measured (p<0.05), there was no significant difference in under-O-glycosylated pIgA1. There was neither a correlation observed between the data from HAA-ELISA and pIgA1 trap (r2=0.09) in the IgAN patients (r2=0.005) nor was there a linear correlation between Area-IgA and data from HAA-ELISA or the pIgA1 trap (r2=0.005, 0.03, respectively). Contour plots of clinical parameters versus data from HAA-ELISA and the pIgA1 trap revealed that patients with a high score in each clinical parameter concentrated in specific areas, showing that patients with specific O-glycan profiles of IgA1 have similar clinical parameters. A decision tree analysis suggested that dominant immune complexes in glomeruli were consisted of: 1) IgA1-IgG and complements, 2) pIgA1 and complements, and 3) monomeric IgA1-IgA or aggregated monomeric IgA1. CONCLUSIONS: Serum under-O-glycosylated IgA1 levels are not correlated with glomerular IgA deposition based upon heterogeneity in the composition of glomerular immune complexes in IgAN patients.

A panel of serum biomarkers differentiates IgA nephropathy from other renal diseases.

BACKGROUND AND OBJECTIVES: There is increasing evidence that galactose-deficient IgA1 (Gd-IgA1) and Gd-IgA1-containing immune complexes are important for the pathogenesis of IgA nephropathy (IgAN). In the present study, we assessed a novel noninvasive multi-biomarker approach in the diagnostic test for IgAN. MATERIALS AND METHODS: We compared serum levels of IgA, IgG, Gd-IgA1, Gd-IgA1-specific IgG and Gd-IgA1-specific IgA in 135 IgAN patients, 79 patients with non-IgAN chronic kidney disease (CKD) controls and 106 healthy controls. Serum was collected at the time of kidney biopsy from all IgAN and CKD patients. RESULTS: Each serum marker was significantly elevated in IgAN patients compared to CKD (P<0.001) and healthy controls (P<0.001). While 41% of IgAN patients had elevated serum Gd-IgA1 levels, 91% of these patients exhibited Gd-IgA1-specific IgG levels above the 90th percentile for healthy controls (sensitivity 89%, specificity 92%). Although up to 25% of CKD controls, particularly those with immune-mediated glomerular diseases including lupus nephritis, also had elevated serum levels of Gd-IgA1-specific IgG, most IgAN patients had elevated levels of Gd-IgA1-specific antibody of both isotypes. Serum levels of Gd-IgA1-specific IgG were associated with renal histological grading. Furthermore, there was a trend toward higher serum levels of Gd-IgA1-specific IgG in IgAN patients with at least moderate proteinuria (≥1.0 g/g), compared to patients with less proteinuria. CONCLUSIONS: Serum levels of Gd-IgA1-specific antibodies are elevated in most IgAN patients, and their assessment, together with serum levels of Gd-IgA1, improves the specificity of the assays. Our observations suggest that a panel of serum biomarkers may be helpful in differentiating IgAN from other glomerular diseases.

Changes in nephritogenic serum galactose-deficient IgA1 in IgA nephropathy following tonsillectomy and steroid therapy.

BACKGROUND: Recent studies have shown that galactose-deficient IgA1 (GdIgA1) has an important role in the pathogenesis of IgA nephropathy (IgAN). Although emerging data suggest that serum GdIgA1 can be a useful non-invasive IgAN biomarker, the localization of nephritogenic GdIgA1-producing B cells remains unclear. Recent clinical and experimental studies indicate that immune activation tonsillar toll-like receptor (TLR) 9 may be involved in the pathogenesis of IgAN. Here we assessed the possibility of GdIgA1 production in the palatine tonsils in IgAN patients. METHODS: We assessed changes in serum GdIgA1 levels in IgAN patients with clinical remission of hematuria and proteinuria following combined tonsillectomy and steroid pulse therapy. Further, the association between clinical outcome and tonsillar TLR9 expression was evaluated. RESULTS: Patients (n = 37) were divided into two groups according to therapy response. In one group, serum GdIgA1 levels decreased after tonsillectomy (59%) alone, whereas in the other group most levels only decreased after the addition of steroid pulse therapy to tonsillectomy (41%). The former group showed significantly higher tonsillar TLR9 expression and better improvement in hematuria immediately after tonsillectomy than the latter group. CONCLUSIONS: The present study indicates that the palatine tonsils are probably a major sites of GdIgA1-producing cells. However, in some patients these cells may propagate to other lymphoid organs, which may partially explain the different responses observed to tonsillectomy alone. These findings help to clarify some of the clinical observations in the management of IgAN, and may highlight future directions for research.

Serum levels of galactose-deficient immunoglobulin (Ig) A1 and related immune complex are associated with disease activity of IgA nephropathy.

BACKGROUND: The primary abnormal manifestation in immunoglobulin A nephropathy (IgAN) is recurring bouts of hematuria with or without proteinuria. Although immunohistochemical analysis of renal biopsy tissue remains the gold standard not only for diagnosis but also for evaluating the activity of IgAN, new sensitive and reasonably specific noninvasive tests are emerging to guide therapeutic strategy applicable to all stages of IgAN. The present study examined serum levels of galactose-deficient IgA1 (Gd-IgA1) and its immune complex (IgA/IgG-IC) as noninvasive markers for the disease activity. METHODS: We enrolled 50 IgAN patients (male 40 %, median age 37 years) showing complete or partial clinical remission after steroid pulse therapy with tonsillectomy (TSP) whose clinical data and serum could be followed up for 3-5 years. RESULTS: Cross-sectional analysis revealed that the degree of hematuria and proteinuria were significantly associated with levels of Gd-IgA1 and levels of IgA/IgG-IC. Longitudinal analysis further showed that from the group of 44 patients with heavy hematuria before TSP, 31 patients showed complete disappearance of hematuria (group A), but the remaining patients did not (group B). Although the levels of Gd-IgA1 and IgA/IgG-IC in the two groups before TSP were similar, percentage decrease of Gd-IgA1 and IgA/IgG-IC levels in group A was significantly higher than in group B. CONCLUSION: Disease activity of IgAN assessed by hematuria and proteinuria correlated with serum levels and changes of Gd-IgA1 and IgA/IgG-IC. These new noninvasive disease activity markers can be useful for future activity scoring system and guiding therapeutic approaches.

Autoantibodies targeting galactose-deficient IgA1 associate with progression of IgA nephropathy.

Mesangial and circulating IgA1 with aberrantly glycosylated hinge region O-glycans characterize IgA nephropathy (IgAN). Unlike healthy individuals, some IgA1 is galactose deficient in patients with IgAN, leaving terminal N-acetylgalactosamine residues in the hinge region exposed. Circulating autoantibodies that recognize such galactose-deficient IgA1 as an autoantigen, or the levels of the autoantigen itself, may allow prediction of disease progression. Here, we analyzed serum samples obtained at diagnosis for autoantigen and autoantibodies from 97 patients with IgAN selected from our prospective cohort according to their absolute renal risk for progression to dialysis or death (0, very low; 1, low; 2, high; 3, very high). We also analyzed samples from controls comprising 30 healthy volunteers and 30 patients with non-IgAN disease. The mean follow-up was 13.8 years. We found that mean serum levels of total autoantigen, normalized IgG autoantibody, and total IgA autoantibody were significantly higher in patients than in the combined controls (all P≤0.01). Furthermore, increasing levels correlated with worse clinical outcomes. In Cox regression and Kaplan-Meier analyses, IgG autoantibody levels ≥1.33 predicted dialysis or death (both P≤0.01). In conclusion, these data suggest that serum levels of IgG and IgA autoantibodies strongly associate with the progression of IgAN nephropathy.