Toshinao Tsuge

Down-regulation of core 1 β1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1

Background. Patients with IgA nephropathy (IgAN) have an increased amount of abnormally O-glycosylated IgA1 in circulation, in glomerular deposits and produced by tissue cells in vitro. Although increased production of Th2 cytokines by peripheral blood lymphocytes and a functional abnormality of core 1 β1,3-galactosyltransferase (C1β3Gal-T) have been proposed as mechanisms underlying pathogenesis of IgAN, they are still obscure and are not connected. Methods. To clarify the effect of T-cell cytokines, we analysed the mRNA levels of C1β3Gal-T and its molecular chaperone Cosmc, C1β3Gal-T activity and subsequent O-glycosylation of IgA1 in a human B-cell line stimulated with these cytokines. The surface IgA1-positive human B-cell line was cultured with recombinant human IFN-γ, IL-2, IL-4 or IL-5. The production and glycosylation of IgA1 were determined by sandwich ELISA and enzyme-linked lectin binding assay, respectively. The mRNA levels of C1β3Gal-T and Cosmc were quantitatively measured by real-time PCR. C1β3Gal-T activity was analysed using high-performance liquid chromatography. Results. IgA1 production by IL-4-stimulated cells was significantly higher than controls or after IFN-γ or IL-5. The terminal glycosylation of secreted IgA1 was altered in response to IL-4. IL-4 stimulation significantly decreased the mRNA levels of both C1β3Gal-T and Cosmc and of C1β3Gal-T activity. IL-4 stimulation was clearly blocked by recombinant human IL-4 soluble receptor. Conclusions. It appears that Th2 cytokine IL-4 may play a key role in controlling glycosylation of the IgA1 hinge region.

Pioglitazone attenuates TGF-β1-induction of fibronectin synthesis and its splicing variant in human mesangial cells via activation of peroxisome proliferator-activated receptor (PPAR)γ

The peroxisome proliferator‐activated receptor (PPAR)γ is expressed not only in adipose tissue but also in macrophages/monocytes and plays important roles in acute/chronic inflammation. Transforming growth factor (TGF)‐β is a common pathogenic indicator of sclerosis because it induces the accumulation of extracellular matrix (ECM) in the glomerular mesangium of the kidney. Among components of the ECM, fibronectin (FN) is an acute reactant in inflammation, and isoforms of it produced by splicing of gene variants appear during abnormal conditions such as wound healing. In this study, we examined the effects of pioglitazone, a PPARγ agonist, on TGF‐β1‐induced FN synthesis in cultured mesangial cells using RT‐PCR and Western blot analysis. We also analyzed its splicing variant, extra domain (ED) A, containing FN (EDA+FN). TGF‐β1 enhanced the production of both FN and EDA+ FN and down‐regulated PPARγ expression. Pioglitazone reversed both these effects of TGF‐β1. These findings suggest that PPARγ activation by pioglitazone may affect the TGF‐β1‐induced FN accumulation observed in the glomerular mesangium in cases of glomerulosclerosis, although further in vivo experiments are needed to evaluate this inference.

Polymorphism in promoter region of Fcα receptor gene in patients with IgA nephropathy

Abstract. We have recently identified two novel polymorphisms (–114T/C and +56T/C relative to the major transcription start site) in the functional promoter region of the Fcα receptor (FcαR) gene. Since altered FcαR expression may be associated with IgA nephropathy, we examined these promoter polymorphisms in this disease. Patients with IgA nephropathy (n=90), patients with other primary glomerulonephritis (n=50), and healthy adults (n=83) were studied. Genotyping was performed by the polymerase chain reaction (PCR) followed by direct sequence analysis and was subsequently confirmed by PCR with mismatched primers followed by restriction fragment length polymorphism analysis. The study demonstrated that the frequency of the +56C allele in patients with IgA nephropathy (0.511) was significantly (P<0.01) higher than that in patients with other primary glomerulonephritis (0.350) and healthy adults (0.337). In addition, a significant increase in the frequency of the +56CC genotype was observed in patients with IgA nephropathy (27.8% vs 10.0% in other GN and vs 9.6% in healthy adults). In contrast, no significant difference in the frequencies of the +56CC genotype and +56C allele was observed between other primary glomerulonephritis patients and healthy adults. The frequency of the –114CC genotype in patients with IgA nephropathy was significantly increased compared with those in both control groups (15.6% vs 4.0% in other GN and vs 2.4% in healthy adults). Polymorphisms of the FcαR promoter region therefore appear to be associated with susceptibility to IgA nephropathy, suggesting the importance of the FcαR gene and its expression in this disease.

Effect of Mizoribine on Glomerulonephritis of Early-Stage IgA Nephropathy in ddY Mice

Immunopathological studies were performed to determine whether the glomerular injuries in ddY mice, a model for IgA nephropathy (Berger’s disease), are influenced by treatment with mizoribine, a new immunosuppressive agent. The ddY mice were treated with a low (0.05 mg/ml) or a high (0.1 mg/ml) dose of mizoribine for 35 weeks. Flow cytometry analysis showed that there was a marked decrease in the number of B cells and IgA-bearing B cells. In immunofluorescence, the deposition of IgA in the glomerular mesangial areas and capillary walls of the high-dose mizoribine-treated ddY mice was markedly decreased as compared with that of control ddY mice receiving drinking water. The glomerular mesangial expansion in the high-dose mizoribine-treated ddY mice was milder than that found in the control ddY mice. In 45-week-old ddY mice, the average number of intraglomerular cells in the high-dose and low-dose mizoribine-treated ddY mice was slightly lower than that in drinking water treated ddY mice. The levels of urinary protein excretion in the high-dose mizoribine-treated ddY mice were also lower than those in the low-dose mizoribine-treated or drinking water treated ddY mice. It appears that treatment of mizoribine might influence the proliferation of B cells, especially IgA-bearing B cells, and improve the glomerular IgA deposition and glomerular expansion in early-stage IgA nephropathy of ddY mice.

Monocyte chemoattractant protein (MCP)-1 production via functionally reconstituted Fcα receptor (CD89) on glomerular mesangial cells

AbstractBackground: Fc alpha receptor (FcαR; CD89) is the receptor for Fc portion of IgA in various cells, and displays various immunological responses on binding. It is important to analyze the mesangial functions via FcαR in the pathogenesis of IgA nephropathy. However, it is still controversial whether FcαR is expressed on mesangial cells. To assess biological functions of FcαR on the mesangial cells, we established mesangial transfectants that expressed FcαR with or without FcRγ chain that is a common signaling molecule of FcRs. The production of monocyte chemoattractant protein-1 (MCP-1) by mesangial cells is known to contribute to cellular infiltration into glomeruli and subsequent glomerular injuries. Methods: Murine mesangial cell lines (SV40 MES 13) were transfected with cDNA of the human FcαR. Furthermore, we co-transfected some of the FcαR transfectants with cDNA of human FcRγ chain. The tyrosine phosphorylation of the intra-mesangial proteins after FcαR cross-linking was examined by immunoprecipitation. MCP-1 production from each transfectant stimulated with heat aggregated IgA was determined by sandwich ELISA. Results: Two kinds of mesangial transfectants stably expressed human FcαR with or without FcRγ chain (FcαR+, FcαR+/γ+). Phosphorylation of FcRγ chain and syk kinase was detected in FcαR+ and FcαR+/γ+ cells, but not in untransfected cells. Aggregated IgA induced significantly higher MCP-1 production in FcαR+/γ+ than those in FcαR+ or untransfected control. Conclusions: Present study demonstrated that FcαR and FcRγ chain could be reconstituted in mesangial cells and mediated MCP-1 production by aggregated IgA in a dose-dependent manner. Current data would argue that FcαR can be activated in mesangial cells through their own machinery, although underlying mechanisms for FcαR induction in mesangial cells remain unclear.

One-year results of an open-label study on antiproteinuric effect of benidipine in elderly patients with chronic kidney disease.

BACKGROUND The long-term antiproteinuric effects of benidipine, a calcium channel blocker (CCB), have not been evaluated in detail in hypertensive patients with chronic kidney disease (CKD). METHODS Benidipine (4 mg/day) was administered to previously untreated hypertensive patients with CKD, or hypertensive patients with CKD not achieving target blood pressure (BP) despite taking an angiotensin II receptor blocker (ARB). The patients were followed up for 1 year. If target BP was not achieved by 2 weeks after the start of benidipine treatment, the dosage was increased to 8 mg/day. The urinary protein to creatinine (UP/cre) ratio was evaluated before and after benidipine treatment. RESULTS This study evaluated 65 hypertensive patients with CKD. BP (systolic/diastolic) decreased from 154 ± 19 / 91 ± 12 mm Hg before treatment to 134 ± 16 / 78 ± 10 mm Hg at 1 year after treatment (p<0.001). The UP/cre ratio decreased significantly from 2.21 ± 2.47 g/g creatinine (g/g cre) before treatment to 1.43 ± 2.21 g/g cre after treatment (p<0.001). In both the untreated and ARB-treated groups, the BP and UP/cre ratio decreased significantly at 1 year after treatment. The percentage change in the UP/cre ratio was significantly greater in patients aged 65 years or older than in those less than 65 years (79.1% vs. 48.7%, p=0.038). CONCLUSIONS Benidipine treatment reduced the UP/cre ratio in hypertensive patients with CKD, and the percentage decrease of the UP/cre ratio was greater in elderly patients, suggesting that benidipine may have more potent antiproteinuric effects in elderly hypertensive patients with CKD.

Dose-response to a jelly preparation of calcium polystyrene sulfonate in patients with hyperkalemia--changes in serum potassium levels with or without a RAAS inhibitor.

AIMS In this study, dose-response of the serum potassium-lowering effect of a calcium polystyrene sulfonate (PS) preparation was investigated. Changes in the serum potassium level were also examined with or without application of a RAAS inhibitor, which is said to increase the serum potassium level. SUBJECTS AND METHODS 23 patients diagnosed to have hyperkalemia associated with chronic renal failure were enrolled in this study. The study drug, a PS-Ca jelly preparation (Argamate jelly), was started at a daily dose of 1 preparation (5 g as PS-Ca), and the dose was increased by 1 preparation every month to finally reach 3 preparations per day. Blood samples were collected once a month and serum levels of creatinine and electrolytes were measured. RESULTS PS-Ca jelly decreased serum potassium levels in a dose-dependent manner. Decreases were 0.67 mEq/l at 5 g of PS-Ca/day, 1.06 mEq/l at 10 g/d, and 1.33 mEq/l at 15 g/d. Irrespective of the use of the RAAS inhibitor, serum potassium levels decreased significantly in a dose-dependent manner. Furthermore, no major change in serum creatinine levels occurred in subjects in which the RAAS inhibitor was used, although in subjects in which the RAAS inhibitor was not used, serum creatinine level tended to gradually increase. CONCLUSION Serum potassium levels were reduced in a dose-dependent manner by administration of 5-15 g/d of PS-Ca, and it appeared that together with control of serum potassium levels, renal function should be maintained by continuous administration of RAAS inhibitor.

Suppressive activity of pemirolast potassium, an antiallergic drug, on glomerulonephritis. Studies in glomerulonephritis model rats and in patients with chronic glomerulonephritis concurrently affected by allergic rhinitis.

BACKGROUND It is still difficult to manage chronic glomerulonephritis with corticosteroids because of safety concerns, especially for patients with mild symptoms and infants. Therefore, an alternative approach is greatly required. Pemirolast potassium (CAS 100299-08-9) is an antiallergic drug with high safety. METHODS Two glomerulonephritis rat models were prepared to examine the pharmacological actions of pemirolast potassium: one reversible model prepared with the anti-Thy-1 antibody, and another irreversible model by unilateral nephrectomy and with the anti-Thy-1 antibody. Pemirolast potassium was administered to 10 Japanese chronic glomerulonephritis patients concurrently affected by allergic rhinitis in order to examine its efficacy for mild proteinuria. RESULTS Pemirolast potassium 1 and 10 mg/kg markedly inhibited proteinuria in the reversible model. In the irreversible model, pemirolast potassium 3 mg/kg showed a significant decrease in the incidence of glomerulosclerosis. In chronic glomerulonephritis patients, pemirolast potassium, 10 mg twice daily, for 6 months, significantly reduced the severity of proteinuria. CONCLUSION Our research suggested the efficacy of pemirolast potassium in glomerulonephritis. A well-controlled study is considered necessary to validate pemirolast potassium as a therapeutic drug for glomerulonephritis.

Regulation of human FcαR gene expression

The Fc receptor for IgA (FccαR, CD89) is expressed exclusively on human phagocytic cells including monocytes/macrophages, neutrophils, and eosinophils, and is capable of triggering various IgA-mediated effector functions. Altered FcαR expression has been reported in several diseases such as IgA nephropathy (IgAN) and allergic diseases, suggesting a role for FcαR in pathogenesis of diseases. The description of a promoter that directs tissue- or cell type-specific transcription could offer new insights into the mechanisms underlying aberrant gene expression associated with diseases. Here we show that at least two transcription factors, C/EBPα and an Ets-like factor, are functionally important for myeloid-specific expression of the FcαR gene. In addition, the FcαR promoter could be transactivated by PU.l, an Ets protein family member which has been shown to regulate the promoters of many myeloid-specific genes. Furthermore, in the FcαR promoter region there are two novel functional polymorphisms (T to C transition) that are associated with IgAN. The information obtained in this study will facilitate further analyses of activation stimuli and transcription factors involved in FcαR-mediated immune system in various pathological situations of diseases.

Role of cytokine mediators in the pathogenesis of IgA nephropathy

Abstract Among the developmental and/or exacerbating factors of IgA nephropathy, cytokines have been found to play a role in the progression of the disease. We previously reported that the levels of urinary IL-6, monocyte chemoattractant protein (MCP)-1, and IL-8 in patients in the advanced stage of IgA nephropathy were significantly higher than those in patients in the mild stage of this disease. It appears that measurement of urinary cytokines and/or chemokines is useful in evaluating the degree of renal injuries in patients with IgA nephropathy. Although one of the most critical findings in patients with IgA nephropathy is glomerular mesangial deposition of IgA (mainly IgA1), it is still unknown whether the activation of mesangial cells is directly triggered by IgA per se. Fc alpha receptor (FcαR) is one of the receptors for binding of IgA in various cells, and displays various immunological responses on binding. It is important to analyze the mesangial functions in the production of MCP-1 via FcαR in the pathogenesis of IgA nephropathy. To assess biological functions of FcαR in the mesangial cells, we established mesangial transfectants which expressed the FcαR and FcRγ chains. Murine mesangial cell lines (SV40 MES 13) were transfected with cDNA of human FcαR. Furthermore, we cotransfected some of the FcαR transfectants with cDNA of the human FcRγchain, which is known as a common signaling molecule of Fc receptors. MCP-1 from each transfectant stimulated with heat aggregated IgA was assayed by sandwich enzyme-linked immunosorbent assay (ELISA). The secretion of MCP-1 was elicited in a dose-dependent manner by stimulation with aggregated IgA in FcαR with or without the FcRγ chain (Fγ/F cells). It is considered that glomerular mesangial cells might produce MCP-1 by cell-activation with FcαR cross-linking after incubation with aggregated IgA. It appears that the activation mechanism of mesangial cells was efficiently controlled by association with the FcRγ chain, and that FcαR with the FcRγ chain may contribute to mesangial response to IgA deposition.