Interaction between GALNT12 and C1GALT1 Associates with Galactose-Deficient IgA1 and IgA Nephropathy.

BACKGROUND: Galactose-deficient IgA1 plays a key role in the pathogenesis of IgA nephropathy, the most common primary GN worldwide. Although serum levels of galactose-deficient IgA1 have a strong genetic component, the genetic link between this molecule and IgA nephropathy has not yet been clearly established. METHODS: To identify novel loci associated with galactose-deficient IgA1, we performed a quantitative genome-wide association study for serum galactose-deficient IgA1 levels, on the basis of two different genome-wide association study panels conducted in 1127 patients with IgA nephropathy. To test genetic associations with susceptibility to IgA nephropathy, we also enrolled 2352 patients with biopsy-diagnosed IgA nephropathy and 2632 healthy controls. Peripheral blood samples from 59 patients and 27 healthy controls were also collected for gene expression analysis. RESULTS: We discovered two loci, in C1GALT1 and GALNT12, that achieved genome-wide significance, explaining about 3.7% and 3.4% of variance in serum galactose-deficient IgA1 levels, respectively. We confirmed the previously reported association of C1GALT1 with serum galactose-deficient IgA1 levels, but with a different lead single-nucleotide polymorphism (rs10238682; ß=0.26, P=1.20×10-9); the locus we identified at GALNT12 (rs7856182; ß=0.73, P=2.38×10-9) was novel. Of more interest, we found that GALNT12 exhibits genetic interactions with C1GALT1 in both galactose-deficient IgA1 levels (P=1.40×10-2) and disease risk (P=6.55×10-3). GALNT12 mRNA expression in patients with IgA nephropathy was significantly lower compared with healthy controls. CONCLUSIONS: Our data identify GALNT12 as a novel gene associated with galactose-deficient IgA1 and suggest novel genetic interactions. These findings support a key role of genetically conferred dysregulation of galactose-deficient IgA1 in the development of IgA nephropathy.

Enhanced Bruton's tyrosine kinase activity in the kidney of patients with IgA nephropathy.

PURPOSE: Bruton's tyrosine kinase (BTK) is a vital biological molecule that contributes to immune regulation. Previous studies have showed that BTK can be detected in patients with lupus nephritis and rheumatoid arthritis. However, the role of BTK in IgA nephropathy (IgAN) has not yet been elucidated. The purpose of this research was to investigate the role of BTK activation in macrophages in IgAN. METHODS: Peripheral blood and renal tissue samples were collected from 63 patients with IgAN, and peritumoral normal tissues were collected from 20 patients after surgical resection of renal tumor for use as control. Additionally, 20 healthy volunteers were recruited as control. The levels of BTK, CD68, phosphorylated BTK (pBTK), phosphorylated NF-κB (p-NF-κB p65), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and monocyte chemotactic protein (MCP)-1 were measured by immunohistochemistry (IHC), real-time polymerase chain reaction (RT-PCR), western blotting, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to peritumoral normal tissues, the expression levels of CD68 and BTK were significantly increased in IgAN group (p < 0.001) and the differences between M0 and M1, E0 and E1, S0 and S1, T0 and T1-2, C0 and C1-2 were statistically significant in the updated Oxford Classification (p < 0.05). Also, CD68 and BTK were positively correlated with Katafuchi semi-quantitative glomerular and tubulointerstitial scores (r = 0.580, 0.637 and 0.442, 0.489, respectively, p < 0.05). The expression of BTK was significantly higher in C3b- and C4d-positive renal tissues of patients with IgAN (p < 0.05). In addition, BTK was positively correlated with 24-h urine protein, serum creatinine levels (r = 0.456 and 0.453, respectively, p < 0.001), and negatively correlated with serum albumin (r = 0.357, p < 0.05). The intensity of expression of pBTK and p-NF-κB p65 was observably increased in renal tissues and monocytes of patients with IgAN compared to the control group. The results of IHC, RT-PCR, and ELISA indicated that the levels of TNF-ɑ, IL-1ß, and MCP-1 were markedly increased in the IgAN group (p < 0.05). CONCLUSION: The results of this study indicate that activation of BTK in macrophages may play an important role in promoting the progression of renal inflammation in IgAN.

ST6Gal1 is up-regulated and associated with aberrant IgA1 glycosylation in IgA nephropathy: An integrated analysis of the transcriptome.

Galactose-deficient IgA1 (Gd-IgA1) plays a crucial role in the development of Immunoglobulin A nephropathy (IgAN), however, the underlying pathogenic mechanisms driving Gd-IgA1 production in B cells are not well understood. In this study, RNA-seq analysis identified 337 down-regulated and 405 up-regulated genes in B cells from 17 patients with IgAN and 6 healthy controls. Among them, ST6Gal1, which was associated with IgAN in a previous genome-wide association study (GWAS), was up-regulated in IgAN and significantly positive correlated with elevated Gd-IgA1. In addition, we identified increased plasma ST6Gal1 levels in 100 patients with IgAN, which were associated with higher levels of proteinuria, plasma IgA, Gd-IgA1 levels, greater degrees of systemic complement activation including C3a, Bb, C4d, MAC and a lower proportion classified as C2 grade (crescent proportion ≥25%). Interesting, in vitro, recombinant ST6Gal1 (rST6Gal1) exposure reduced the production of Gd-IgA1 in cultured peripheral blood mononuclear cells from IgAN patients. rST6Gal1 stimuli also increased expression of C1GALT1, which were well-known proportional to the decrease in galactose deficiency of IgA1. In conclusions, we identified increased plasma ST6Gal1 levels and the association of ST6Gal1 with disease severity of IgAN. Additionally, rST6Gal1 administration in vitro increased expression of C1GALT1 and reduced the production of Gd-IgA1.

Association analysis of DNA methyltransferases in IgA nephropathy.

DNA methylation, especially DNA methyltransferases (DNMTs), is involved in the pathogenesis of many autoimmune diseases through regulating immune function. This study aimed to explore the potential role of DNMTs in IgA nephropathy (IgAN). We evaluated mRNA expressions of DNMT1, DNMT3A, DNMT3B along with ß1,3-galactosyltransferase (C1GALT1) in peripheral blood mononuclear cells (PBMCs), and measured galactose-deficient IgA1 (Gd-IgA1) levels in plasma. The expression intensity of DNMT1 and DNMT3B in the renal specimen of IgAN patients were also detected. Results showed DNMT3B, not DNMT1 or DNMT3A, was notably increased in IgAN patients compared to controls and associated with pathologic types. However, DNMT1 and C1GALT1 were found positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with 24 h Urine protein in IgAN patients. No association was found between DNMT1 and Gd-IgA1. The expressions of DNMT3B and DNMT1 were barely observed in IgAN renal biopsy specimens. In conclusion, for the first time, we identified the relations of DNMTs and C1GALT1 to the clinical state and pathology of IgAN patients, which provide new clues for IgAN.

Indoleamine 2,3-Dioxygenase (IDO) Regulates Th17/Treg Immunity in Experimental IgA Nephropathy.

IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide. Current studies have shown that the Th17/Treg immune balance may be involved in the occurrence of IgAN, but the exact mechanism is still unclear. Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyses degradation of tryptophan (Trp) through the kynurenine (Kyn) pathway; it can control inflammation and immune response by inducing Trp starvation. IDO may be a key molecule in regulating the Th17/Treg immune balance. However, it is not clear whether IDO is involved in the IgAN disease occurrence by regulating the Th17/Treg immune balance. In this study, an IgAN mouse model was established. The mice were intraperitoneally inoculated with IDO inhibitor 1-MT or agonist ISS-ODN to observe whether the IDO signalling pathway participates in the occurrence and development of IgAN by regulating the Th17/Treg immune balance. The results showed that IDO inhibitor 1-MT significantly increased renal injury and glomerular IgA accumulation and up-regulated Th17/Treg and Th17-related cytokine expression in IgAN mice, while ISS-ODN significantly decreased renal injury and glomerular IgA accumulation, down-regulated Th17/Treg expression and inhibited Th17-related cytokine expression in IgAN mice. In conclusion, IDO was involved in the occurrence and progress of IgAN by regulating the Th17/ Treg balance.

p38 MAPK activity is associated with the histological degree of interstitial fibrosis in IgA nephropathy patients.

Activation of p38 mitogen-activated protein kinase (MAPK) is associated with tissue fibrosis, and inhibition of p38 MAPK can attenuate the progression of fibrosis. We aimed to investigate whether p38 MAPK activity in kidney tissue confirmed by immunohistochemical staining is associated with renal tubulointerstitial fibrosis in chronic kidney disease patients with IgA nephropathy. We collected kidney biopsy specimens from 341 IgA nephropathy patients and 15 control patients to identify the clinical and histopathological factors associated with kidney tubulointerstitial fibrosis and to find an association between kidney phosphorylated p38 immunoactivity and pathological grading. In addition, we aimed to investigate whether the anti-fibrotic effect of p38 MAPK inhibition can be identified by assessing the immunostaining intensity of phosphorylated p38 in kidney tissue. A renal tubulointerstitial fibrosis model was introduced using 7-week-old C57BL/6 mice subjected to unilateral ureteral obstruction (UUO). The p38 MAPK inhibitor SB-731445 was injected intraperitoneally every day for 7 days, and changes in renal fibrosis-associated markers were investigated. Assessment of kidney biopsy specimens from IgA nephropathy patients revealed that the degree of interstitial fibrosis was significantly associated with the tissue immunoactivity of phosphorylated p38. High-grade interstitial fibrosis was associated with a low glomerular filtration rate, high proteinuria, and high-grade histopathological changes, including tubular atrophy, interstitial inflammation, and glomerular sclerosis. In a mouse UUO model, renal protein expression of COL1 and phosphorylated p38 were significantly increased, and the protein expression of COL1 and phosphorylated p38 decreased in mice administered 10 mg/kg/day p38 MAPK inhibitor. We found that kidney interstitial fibrosis is associated with increased immunoactivity of phosphorylated p38 in a UUO mouse model and in human IgA nephropathy patients and that the anti-fibrotic effect of p38 MAPK inhibition can be confirmed using immunohistochemical staining for phosphorylated p38 in kidney tissue.

The Xanthine Oxidase Inhibitor Febuxostat Suppresses the Progression of IgA Nephropathy, Possibly via Its Anti-Inflammatory and Anti-Fibrotic Effects in the gddY Mouse Model.

Recent clinical studies have demonstrated the protective effect of xanthine oxidase (XO) inhibitors against chronic kidney diseases, although the underlying molecular mechanisms remain unclear. However, to date, neither clinical nor basic research has been carried out to elucidate the efficacy of XO inhibitor administration for IgA nephropathy. We thus investigated whether febuxostat, an XO inhibitor, exerts a protective effect against the development of IgA nephropathy, using gddY mice as an IgA nephropathy rodent model. Eight-week-old gddY mice were provided drinking water with (15 µg/mL) or without febuxostat for nine weeks and then subjected to experimentation. Elevated serum creatinine and degrees of glomerular sclerosis and fibrosis, judged by microscopic observations, were significantly milder in the febuxostat-treated than in the untreated gddY mice, while body weights and serum IgA concentrations did not differ between the two groups. In addition, elevated mRNA levels of inflammatory cytokines such as TNFα, MCP-1, IL-1ß, and IL-6, collagen isoforms and chemokines in the gddY mouse kidneys were clearly normalized by the administration of febuxostat. These data suggest a protective effect of XO inhibitors against the development of IgA nephropathy, possibly via suppression of inflammation and its resultant fibrotic changes, without affecting the serum IgA concentration.

Defective activation of the MAPK/ERK pathway, leading to PARP1 and DNMT1 dysregulation, is a common defect in IgA nephropathy and Henoch-Schönlein purpura.

Studies on IgA nephropathy (IgAN) have identified, through GWAS, linkage analysis, and pathway scanning, molecular defects in familial and sporadic IgAN patients. In our previous study, we identified a novel variant in the SPRY2 gene that segregates with the disease in one large family. The functional characterization of this variant led us to discover that the MAPK/ERK pathway was defective not only in this family, but also in two sporadic IgAN patients wild type for SPRY2. In the present study, we have deepened the molecular analysis of the MAPK/ERK pathway and extended our evaluation to a larger cohort of sporadic patients and to one additional family. We found that the ERK pathway is defective in IgAN patients and in patients affected by another IgA-mediated disorder, Henoch-Schönlein purpura (HSP). Furthermore, we found that two other proteins, PARP1 and DNMT1, respectively involved in DNA repair and in antibody class switching and methylation maintenance duties, were critically downregulated in IgAN and HSP patients. This study opens up the possibility that defective ERK activation, in some patients, leads to PARP1 and DNMT1 downregulation suggesting that IgAN could be the consequence of a dysregulated epigenetic maintenance leading to the upregulation of several genes. In particular, PARP1 could be used as a potential biomarker for the disease.

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy.

Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, Y1CS3, in the complementarity-determining region 3 of the heavy chain variable region compared with a Y1CA3 sequence in similar isotype-matched IgG from healthy controls. We previously found that the S3 residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the Y1C(A/V)3 amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S3 hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent ß-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.

Role of let-7b in the regulation of N-acetylgalactosaminyltransferase 2 in IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is characterized by aberrant O-glycosylation in the hinge region of IgA1. The early step in O-glycan formation is the attachment of N-acetylgalactosamine (GalNAc) to the serine/threonine of the hinge region; the process is catalysed by UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). In our previous work, the microarray analysis on peripheral blood mononuclear cells (PBMCs) identified an upregulated miRNA called let-7b. METHODS: To study the molecular mechanisms in which let-7b was involved, we performed a bioinformatic analysis to predict their target genes. To validate biologically let-7b targets, we performed transient transfection experiments ex vivo using PBMCs from an independent group of IgAN patients and healthy blood donors (HBDs). RESULTS: Bioinformatic analysis revealed that GALNT2 is the potential target of let-7b. We found this miRNA significantly upregulated in PBMCs of IgAN patients compared with HBDs. Then, we demonstrated in ex-vivo experiments that let-7b decreased GALNT2 levels in PBMCs of IgAN patients, whereas the loss of let-7b function in PBMCs of HBDs led to an increase of GALNT2 mRNA and its protein level. Finally, we found that upregulation of let-7b occurred also in B-lymphocytes from IgAN patients. CONCLUSIONS: Our results give novel additional information on the abnormal O-glycosylation process of IgA1 in IgAN patients. This study provides evidence for another important miRNA-based regulatory mechanism of the O-glycosylation process in which the deregulated expression of let-7b is associated with altered expression of GALNT2. This finding could be taken into consideration for new therapeutic approaches in IgAN because other serum glycosylated proteins do not display abnormal glycosylation.

ACE insertion/deletion polymorphism (rs1799752) modifies the renoprotective effect of renin-angiotensin system blockade in patients with IgA nephropathy.

INTRODUCTION: Little is known about genetic predictors that modify the renoprotective effect of renin-angiotensin system (RAS) blockade in IgA nephropathy (IgAN). MATERIALS AND METHODS: The present multicenter retrospective observational study examined effect modification between RAS blockade and three RAS-related gene polymorphisms in 237 IgAN patients, including ACE I/D (rs1799752), AT1R A1166C (rs5186) and AGT T704C (rs699). RESULTS: During 9.9 ± 4.2 years of observation, 63 patients progressed to a 50% increase in serum creatinine level. Only ACE I/D predicted the outcome (ACE DD vs ID/II, hazard ratio 1.86 (95% confidence interval 1.03, 3.33)) and modified the renoprotective effect of RAS blockade (p for interaction between ACE DD and RAS blockade = 0.087). RAS blockade suppressed progression in ACE DD patients but not in ID/II patients (ACE ID/II with RAS blockade as a reference; ID/II without RAS blockade 1.45 (0.72, 2.92); DD without RAS blockade 3.06 (1.39, 6.73); DD with RAS blockade 1.51 (0.54, 4.19)), which was ascertained in a model with the outcome of slope of estimated glomerular filtration rate (p = 0.045 for interaction). CONCLUSION: ACE I/D predicted the IgAN progression and the renoprotective effect of RAS blockade in IgAN patients whereas neither AT1R A1166C nor AGT T704C did.

N-acetylgalactosaminide α2,6-sialyltransferase II is a candidate enzyme for sialylation of galactose-deficient IgA1, the key autoantigen in IgA nephropathy.

BACKGROUND: Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a ß1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing. METHODS: We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry. RESULTS: ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide. CONCLUSIONS: Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.

Urokinase, urokinase receptor, and plasminogen activator inhibitor-1 expression on podocytes in immunoglobulin A glomerulonephritis.

BACKGROUND/AIMS: The purpose of this study was to investigate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor (PAI)-1 on podocytes in immunoglobulin A (IgA) glomerulonephritis (GN). METHODS: Renal biopsy specimens from 52 IgA GN patients were deparaffinized and subjected to immunohistochemical staining for uPA, PAI-1, and uPAR. The biopsies were classified into three groups according to the expression of uPA and uPAR on podocytes: uPA, uPAR, and a negative group. The prevalences of the variables of the Oxford classification for IgA GN were compared among the groups. RESULTS: On podocytes, uPA was positive in 11 cases and uPAR was positive in 38 cases; by contrast, PAI-1 was negative in all cases. Expression of both uPA and uPAR on podocytes was less frequently accompanied by tubulointerstitial fibrosis. CONCLUSIONS: Our results suggest a possible protective effect of podocyte uPA/uPAR expression against interstitial fibrosis.

Cytokines alter IgA1 O-glycosylation by dysregulating C1GalT1 and ST6GalNAc-II enzymes.

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by renal immunodeposits containing IgA1 with galactose-deficient O-glycans (Gd-IgA1). These immunodeposits originate from circulating immune complexes consisting of anti-glycan antibodies bound to Gd-IgA1. As clinical disease onset and activity of IgAN often coincide with mucosal infections and dysregulation of cytokines, we hypothesized that cytokines may affect IgA1 O-glycosylation. We used IgA1-secreting cells derived from the circulation of IgAN patients and healthy controls and assessed whether IgA1 O-glycosylation is altered by cytokines. Of the eight cytokines tested, only IL-6 and, to a lesser degree, IL-4 significantly increased galactose deficiency of IgA1; changes in IgA1 O-glycosylation were robust for the cells from IgAN patients. These cytokines reduced galactosylation of the O-glycan substrate directly via decreased expression of the galactosyltransferase C1GalT1 and, indirectly, via increased expression of the sialyltransferase ST6GalNAc-II, which prevents galactosylation by C1GalT1. These findings were confirmed by siRNA knockdown of the corresponding genes and by in vitro enzyme reactions. In summary, IL-6 and IL-4 accentuated galactose deficiency of IgA1 via coordinated modulation of key glycosyltransferases. These data provide a mechanism explaining increased immune-complex formation and disease exacerbation during mucosal infections in IgAN patients.

Glomerular angiotensin-converting enzyme 2 in pediatric IgA nephropathy.

BACKGROUND: Angiotensin-converting enzyme (ACE) 2 is a homolog of ACE and is thought to be a potent counter-regulator against ACE activity. However, the role of ACE2 has not been investigated in pediatric patients with IgA nephropathy (IgAN). This study was performed to examine the relationship between ACE2 expression and the development of pediatric IgAN. METHODS: We performed immunohistochemical analysis of ACE2 and ACE in 39 patients with pediatric IgAN and 14 patients with minor glomerular abnormalities, and elucidated the effects of various cytokines on ACE2 expression in cultured human mesangial cells. RESULTS: ACE2 expression levels in glomeruli and tubules were positively correlated with the mesangial hypercellularity score, while ACE expression levels in glomeruli and tubules are not. Multiple regression analysis showed that the mesangial hypercellularity score correlated with the ACE2 expression level in glomeruli and the urinary protein-creatinine ratio. In IgAN patients not treated with a renin-angiotensin system blocker, ACE2 expression levels in glomeruli were significantly increased compared to patients with minor glomerular abnormalities. IgAN patients treated with a renin-angiotensin system blocker did not show this increase in ACE2 expression. Furthermore, cultured human MC showed increased ACE2 mRNA and protein after treatment with IL-1ß, a pro-inflammatory cytokine in IgAN. In fact, glomerular expressions of IL-1ß were remarkably increased in patients with IgAN. CONCLUSION: These data indicate that ACE2 expression in glomeruli is associated with mesangial hypercellularity in early lesions of pediatric IgAN.

A meta-analysis of the association between angiotensin-converting enzyme insertion/deletion gene polymorphism and end-stage renal disease risk in IgA nephropathy patients.

BACKGROUND AND OBJECTIVE: The association between angiotensin-converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism and end-stage renal disease (ESRD) risk in IgA nephropathy (IgAN) patients is still controversial. A meta-analysis was performed to evaluate the association between ACE I/D gene polymorphism and ESRD susceptibility in IgAN patients. METHOD: A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic databases. RESULTS: Thirteen articles were identified for the analysis of the association between ACE I/D gene polymorphism and ESRD risk in IgAN patients. D allele and DD genotype were associated with ESRD susceptibility in IgAN patients for overall populations (p=0.01 and 0.003, respectively). In Asians, there was a markedly positive association between DD genotype and ESRD susceptibility (p=0.03). Furthermore, D allele and DD genotype were associated with ESRD susceptibility in Caucasians (p=0.02 and 0.03, respectively). However, II genotype might not play a protective role against ESRD onset for overall populations, Asians and Caucasians. CONCLUSION: DD homozygote is a significant genetic molecular marker for the onset of ESRD in IgAN patients.

Spleen tyrosine kinase is important in the production of proinflammatory cytokines and cell proliferation in human mesangial cells following stimulation with IgA1 isolated from IgA nephropathy patients.

IgA immune complexes are capable of inducing human mesangial cell (HMC) activation, resulting in release of proinflammatory and profibrogenic mediators. The subsequent inflammation, cellular proliferation, and synthesis of extracellular matrix lead to the progression of IgA nephropathy (IgAN). Spleen tyrosine kinase (SYK) is an intracellular protein tyrosine kinase involved in cell signaling downstream of immunoreceptors. In this study, we determined whether SYK is involved in the downstream signaling of IgA1 stimulation in HMC, leading to production of proinflammatory cytokines/chemokines and cell proliferation. Incubation of HMC with IgA1 purified from IgAN patients significantly increased the synthesis of MCP-1 in a dose-dependent manner. There was also significantly increased production of IL-6, IL-8, IFN-γ-inducible protein-10, RANTES, and platelet-derived growth factor-BB. Stimulation of HMC with heat-aggregated IgA1 purified from IgAN patients induced significantly increased HMC proliferation. Both pharmacological inhibition of SYK and knockdown of SYK by small interfering RNA significantly reduced the synthesis of these mediators and inhibited HMC proliferation. Moreover, positive immunostaining for total and phospho-SYK in glomeruli of kidney biopsies from IgAN patients strongly suggests the involvement of SYK in the pathogenesis of IgAN. To our knowledge, we demonstrate, for the first time, the involvement of SYK in the downstream signaling of IgA1 stimulation in HMC and in the pathogenesis of IgAN. Hence, SYK represents a potential therapeutic target for IgAN.

The IgA1 immune complex-mediated activation of the MAPK/ERK kinase pathway in mesangial cells is associated with glomerular damage in IgA nephropathy.

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has significant morbidity and mortality as 20-40% of patients progress to end-stage renal disease within 20 years of onset. In order to gain insight into the molecular mechanisms involved in the progression of IgAN, we systematically evaluated renal biopsies from such patients. This showed that the MAPK/ERK signaling pathway was activated in the mesangium of patients presenting with over 1 g/day proteinuria and elevated blood pressure, but absent in biopsy specimens of patients with IgAN and modest proteinuria (<1 g/day). ERK activation was not associated with elevated galactose-deficient IgA1 or IgG specific for galactose-deficient IgA1 in the serum. In human mesangial cells in vitro, ERK activation through mesangial IgA1 receptor (CD71) controlled pro-inflammatory cytokine secretion and was induced by large-molecular-mass IgA1-containing circulating immune complexes purified from patient sera. Moreover, IgA1-dependent ERK activation required renin-angiotensin system as its blockade was efficient in reducing proteinuria in those patients exhibiting substantial mesangial activation of ERK. Thus, ERK activation alters mesangial cell-podocyte crosstalk, leading to renal dysfunction in IgAN. Assessment of MAPK/ERK activation in diagnostic renal biopsies may predict the therapeutic efficacy of renin-angiotensin system blockers in IgAN.

Oxidative damages in tubular epithelial cells in IgA nephropathy: role of crosstalk between angiotensin II and aldosterone.

BACKGROUND: Inhibition of the renin-angiotensin-aldosterone system (RAAS) slows down the progression of chronic renal diseases (CKD) including IgA nephropathy (IgAN). Herein, we studied the pathogenetic roles of aldosterone (Aldo) in IgAN. METHODS: Human mesangial cells (HMC) was activated with polymeric IgA (pIgA) from IgAN patients and the effects on the expression of RAAS components and TGF-ß synthesis examined. To study the roles of RAAS in the glomerulotubular communication, proximal tubular epithelial cells (PTEC) was cultured with conditioned medium from pIgA-activated HMC with eplerenone or PD123319, the associated apoptotic event was measured by the generation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and reactive oxygen species (ROS). RESULTS: Polymeric IgA up-regulated the Aldo synthesis and aldosterone synthase expression by HMC. The release of TGF-ß by HMC was up-regulated synergistically by AngII and Aldo and this was inhibited by incubation of HMC with losartan plus eplerenone. Cultured PTEC express the mineralocorticoid receptor, but not synthesizing aldosterone. Apoptosis, demonstrated by cleaved PARP expression and caspase 3 activity, was induced in PTEC activated by conditioned medium prepared from HMC cultured with pIgA from IgAN patients. This apoptotic event was associated with increased generation of NADPH oxidase and ROS. Pre-incubation of PTEC with PD123319 and eplerenone achieved complete inhibition of PTEC apoptosis. CONCLUSIONS: Our data suggest that AngII and Aldo, released by pIgA activated HMC, served as mediators for inducing apoptosis of PTEC in glomerulo-tubular communications. Crosstalk between AngII and Aldo could participate in determining the tubular pathology of IgAN.