Masayuki Endoh

In situ hybridization of interleukin 6 in diabetic nephropathy

Increased mesangial expansion is one of the most characteristic histological changes in diabetic nephropathy (DN). Although the pathogenesis of DN remains unclear, recent studies associate interleukin (IL) 6 with mesangial proliferative glomerulonephritis. To elucidate the expression and localization of IL-6 mRNA in renal tissues of patients with DN, a high-resolution in situ hybridization using digoxigenin-labeled oligonucleotide was performed. Patients were divided into three groups based on light microscopy findings: mild (group 1), moderate (group 2), and severe (group 3) mesangial expansion. The relationship between the expression of IL-6 mRNA and the degree of glomerular mesangial expansion in DN was examined. Individual cells positive for IL-6 mRNA were observed in glomeruli. These cells were mesangial cells, glomerular epithelial cells, and Bowmans capsule. The signal intensity was strongest in tissues from group 2 but was weak in those from groups 1 and 3. Most cells in the area of mesangial proliferation were strongly stained for IL-6 mRNA, and few positive cells were found in the Kimmelstiel-Wilson nodular lesion. In the interstitium, some tubules, particularly atrophic tubules, and some infiltrating cells were positively stained for IL-6 mRNA. The interstitial expression of IL-6 mRNA correlated significantly with the degree of interstitial injury and was remarkable in tissues from groups 2 and 3. We conclude that IL-6 mRNA is expressed by glomerular resident cells and interstitial cells in the renal tissue of patients with DN and that its expression may be associated with mesangial proliferation and may be involved in the tissue injury of DN.

A scoring system to predict renal outcome in IgA nephropathy: a nationwide 10-year prospective cohort study

Background. Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis, and a substantial number of patients succumb to end-stage renal disease (ESRD). However, prediction of the renal outcome in individual patients remains difficult. We have already published a scoring system using the data in a prospective cohort of IgAN patients followed up from 1995 to 2002. Methods. The cohort was further followed up until 2005 in 97 clinical units in Japan. The data from 2283 patients were analysed by Cox regression to determine the predictors of ESRD in IgAN, and their β-coefficients were converted into scores to estimate ESRD risk within 10 years. Results. During the follow-up (median, 87 months), 252 patients developed ESRD. Male sex, age less than 30 years, family histories of chronic renal failure and chronic glomerulonephritis, hypertension, proteinuria, mild haematuria, hypoalbuminaemia, low glomerular filtration rate and a high histological grade at initial renal biopsy were associated with the risk of ESRD in the multivariable analysis. A scoring system was framed to estimate the 10-year ESRD risk using eight variables significant in both univariable and multivariable models. This prognostic score accurately classified patients by risk: patients with estimates of 0–4.9, 5.0–19.9, 20.0–49.9 and 50.0–100% had an observed incidence of 1.7, 8.3, 36.7 and 85.5%, respectively. The corresponding area under the receiver-operating characteristic curve was 0.942 (95% confidence interval, 0.925–0.958). Conclusion. This validated scoring system to quantitatively estimate ESRD risk during the 10-year follow-up of IgAN patients will serve as a useful prognostic tool in clinical practice.

Phenotypic Characterization of Cytokine Expression in Patients With IgA Nephropathy

To identify the cytokines that play a relevant role in the pathogenesis of IgA nephropathy, we analyzed and compared the gene expression of proinflammatory cytokines, immuno-regulatory cytokines, and growth factors in peripheral blood mononuclear cells (PBMC). Expression of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF-α, and PDGF was examined in 28 patients with IgA nephropathy (IgAN), 20 patients with non-IgA mesangial proliferative glomerulo-nephritis (mesPGN), and 19 healthy controls. Compared with healthy controls, a significant number of IgAN and mesPGN patients showed increased expression of IL-1β, IL-4, IL-10, IL-12, and IFN-γ. The cytokine profile of renal tissue of 10 IgAN and 5 mesPGN biopsies was simultaneously analyzed and compared with that of PBMC. The proinflammatory IL-1α and growth factor PDGF-B were expressed more in renal tissues than in PBMC. Furthermore, the renal profile of IL-α, IFN-γ, and TNF-α expression was associated with the expression of IFN-γ PBMC. The serum level of IFN-γ of IgAN correlated significantly (P = 0.0003) with that of IL-12, suggesting a potential role for cross-stimulation. More importantly, expression of IFN-γ in PBMC and the elevated serum level correlated with the decline in glomerular filtration rate (P = 0.0012) and severity of renal histopathologic grade. To elucidate the role of leukocytes in renal cytokine expression, surface markers of T cells (CD3), monocytes (CD14), natural killer cells (CD16), and B cells (CD19) were also examined in renal tissues. The prominent renal expression of CD3, CD14, and CD16 implicates the leukocytes as the major source of proinflammatory cytokines in IgAN. Collectively, these findings indicate that IFN-γ plays a prominent role in an interactive network of cytokines that contribute to the pathogenesis and progression of IgA nephropathy.

A histologic classification of IgA nephropathy for predicting long-term prognosis: emphasis on end-stage renal disease

BACKGROUND A multicenter case-control study on IgA nephropathy (IgAN) was conducted to develop an evidence-based clinicopathologic classification of IgAN for predicting long-term renal outcome. METHODS Two hundred and eighty-seven patients including those with isolated hematuria or very mild proteinuria were enrolled. During a median follow-up of 9.3 years after biopsy, 49 patients (17%) progressed to end stage renal disease (ESRD). The associations between pathological variables and the need for chronic dialysis was examined by multivariate logistic regression analysis separately in patients who required dialysis earlier than 5 years (Early Progressors) and those who required dialysis within 5 to 10 years (Late Progressors) after biopsy. RESULTS Independent pathological variables predicting progression to ESRD were global sclerosis, segmental sclerosis and fibrous crescents for Early Progressors, and global sclerosis and cellular/fibrocellular crescents for Late Progressors. Four histological grades, HG 1, HG 2, HG 3 and HG 4, were established corresponding to <25%, 25-49%, 50-74% and =75% of glomeruli exhibiting cellular or fibrocellular crescents, global sclerosis, segmental sclerosis or fibrous crescents. Eleven (7%) patients in HG 1, 12 (16%) in HG 2, 13 (31%) in HG 3 and 13 (68%) in HG 4 progressed to ESRD. Multivariate logistic analysis revealed that the risk of progression to ESRD was significantly higher in HG 2, 3 and 4 than in HG 1 (odds ratio, 2.4, 5.7 and 27.6 vs. 1.0). CONCLUSIONS Our evidence-based histologic classification can identify the magnitude of the risk of progression to ESRD and is useful for predicting long-term renal outcome in IgAN.

Detection of immune complexes in polymorphonuclear leukocytes by double immunofluorescence in patients with IgA nephropathy.

Abstract The amounts of cytoplasmic inclusion bodies in peripheral blood polymorphonuclear cells (PMN) were determined in patients with IgA nephropathy and other glomerular diseases to elucidate whether or not IgA-dominant immune complexes were phagocytized by PMN in these patients. Fifteen patients with IgA nephropathy, 8 patients with other glomerular diseases, and 10 healthy adults were examined. The amounts of cytoplasmic inclusion bodies were measured by a double immunofluorescence technique. It was demonstrated that the percentages of IgA with C3 and IgA without C3 cytoplasmic inclusion bodies were significantly increased in patients with IgA nephropathy compared with those obtained in other glomerular diseases and healthy adults. There was a significant correlation between the levels of serum IgA and the percentage of IgA with C3 cytoplasmic inclusion bodies in PMN from patients with IgA nephropathy. It was suggested that IgA-dominant immune complexes are phagocytized by peripheral blood PMN in patients with IgA nephropathy.

Glomerular FcαR expression and disease activity in IgA nephropathy

In this study, we examined the receptors for the Fc portion of immunoglobulin A (IgA) (Fc alphaR) in the glomeruli as well as circulating polymorphonuclear leukocytes and monocytes at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay and at the protein level by an immunohistochemistry/flow cytometry technique using a specific anti-Fc alphaR monoclonal antibody (My 43). Glomeruli were isolated from biopsy specimens of renal tissues from IgA nephropathy (IgAN; 20 cases) and non-IgA mesangial proliferative glomerulonephritis (PGN; 13 cases) patients, and from normal renal tissue specimens obtained from kidneys removed because of malignancies (five cases) applying the microdissection method. There was a relative increase in Fc alphaR in the circulating phagocytes from IgAN patients compared with those from PGN and healthy controls. Fc alphaR expression was present in approximately 40% of glomeruli samples from IgAN patients at the message levels. Fc alphaR-positive specimens were also strongly positive for expression of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 mRNA. Specimens from PGN patients and healthy controls did not show any detectable Fc alphaR message. Serum IgA levels and severity of hematuria were significantly higher in patients with positive Fc alphaR expression. A message for Fc alphaR was detected in the tissues that were more damaged histologically. Our data suggest that there is some in vivo induction of glomerular Fc alphaR expression, possibly mediated by a synergistic stimulus from IgA and inflammatory cytokines, and the expressed receptor is likely to be involved in the disease process of IgAN.

A Novel Phosphoinositide 3-Kinase-dependent Pathway for Angiotensin II/AT-1 Receptor-mediated Induction of Collagen Synthesis in MES-13 Mesangial Cells

Chronic activation of the angiotensin II (ANG II) type 1 receptor (AT-1R) is critical in the development of chronic kidney disease. ANG II activates mesangial cells (MCs) and stimulates the synthesis of extracellular matrix components. To determine the molecular mechanisms underlying the induction of MC collagen, a mouse mesangial cell line MES-13 was employed. ANG II treatment induced an increase in collagen synthesis, which was abrogated by co-treatment with losartan (an AT-1R antagonist), wortmannin (a phosphoinositide 3-kinase (PI3K) inhibitor), an Akt inhibitor, and stable transfection of dominant negative-Akt1. ANG II induced a significant increase in PI3K activity, which was abolished by co-treatment with losartan or 2′,5′-dideoxyadenosine (2′,5′-DOA, an adenylyl cyclase inhibitor) but not by PD123319 (an AT-2R antagonist) or H89 (a protein kinase A (PKA) inhibitor). The Epac (exchange protein directly activated by cAMP)-specific cAMP analog, 8-pHPT-2′-O-Me-cAMP, significantly increased PI3K activity, whereas a PKA-specific analog, 6-benzoyladenosine-cAMP, showed no effect. The ANG II-induced increase in PI3K activity was also blocked by co-treatment with PP2, an Src inhibitor, or AG1478, an epidermal growth factor receptor (EGFR) antagonist. ANG II induced phosphorylation of Akt and p70S6K and EGFR, which was abrogated by knockdown of c-Src by small interference RNA. Knockdown of Src also effectively abolished ANG II-induced collagen synthesis. Conversely, stable transfection of a constitutively active Src mutant enhanced basal PI3K activity and collagen production, which was abrogated by AG1478 but not by 2′,5′-DOA. Moreover, acute treatment with ANG II significantly increased Src activity, which was abrogated with co-treatment of 2′,5′-DOA. Taken together, these results suggest that ANG II induces collagen synthesis in MCs by activating the ANG II/AT-1R-EGFR-PI3K pathway. This transactivation is dependent on cAMP/Epac but not on PKA. Src kinase plays a pivotal role in this signaling pathway between cAMP and EGFR. This is the first demonstration that an AT1R-PI3K/Akt crosstalk, along with transactivation of EGFR, mediates ANG II-induced collagen synthesis in MCs.

Profiling the adult human liver transcriptome: analysis by cDNA array hybridization

BACKGROUND/AIMS A comprehensive profile of genes expressed at the mRNA level (transcriptome) in human liver tissue is important for elucidating the pathogenesis and treatment of hepatic diseases. The recent development of cDNA array hybridization allows the parallel monitoring of thousands of genes expressed in a single organ. METHODS High-density microarrays containing 4043 known and unique human cDNA gene targets were used to quantitatively analyze the expression of genes in human livers. Expressed gene transcripts were classified by function and listed with information of their chromosomal positions. Computational analysis was used to cluster genes according to similarity in pattern of gene expression. RESULTS A total of 2418 unique gene transcripts were detected in five liver specimens. Through relational database analysis, we determined 1212 genes that were commonly expressed in 4 of the five liver specimens. Furthermore, analysis of the total 2418 expressed genes by self-organizing maps and hierarchical clustering unexpectedly revealed a genomic acute phase response in two of the liver specimens. CONCLUSIONS These findings represent a comprehensive preliminary molecular index of genes transcribed in the adult human liver. The information may serve as a resource for speeding up the discovery of genes underlying human hepatic diseases.

Risk stratification for progression of IgA nephropathy using a decision tree induction algorithm

Background. Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis, and many patients are at risk of at least slow progression. However, prediction of the renal outcome in individual patients remains difficult. Methods. To develop a practical and user-friendly scheme for risk stratification of IgAN patients, data were extracted from a prospective cohort study conducted in 97 clinical units in Japan from 1995. Specifically, we examined deterioration in renal function, defined as doubling of serum creatinine, within 10 years of follow-up in 790 adult IgAN patients without substantial renal dysfunction at baseline using a decision tree induction algorithm. Results. Recursive partitioning indicated that the best single predictor of renal deterioration was severe proteinuria on urine dipstick testing, followed by hypoalbuminaemia and the presence of mild haematuria for patients with and without severe proteinuria, respectively. Serum total protein levels, diastolic blood pressure and histological grade were placed in the third tier of the decision tree model. With these six variables, patients can be readily stratified into seven risk groups whose incidence of renal deterioration within 10-year follow-up ranges from 1.0% to 51.4%. Logistic regression also identified severe proteinuria, hypoalbuminaemia and mild haematuria as significant predictors of deterioration. Areas under the receiver-operating characteristic curve for the prediction were comparable between the decision tree model and the logistic regression model [0.830 (95% confidence interval, 0.777–0.883) versus 0.808 (95% confidence interval, 0.754–0.861)]. Conclusion. Risk of substantial renal deterioration in IgAN patients can be validly estimated using six predictors obtained from clinical routine.

Altered production of IgE and IgA induced by IL-4 in peripheral blood mononuclear cells from patients with IgA nephropathy.

In order to elucidate the factors responsible for altered immunoglobulin production in patients with IgA nephropathy (IgAN). the in vitro effects oHL‐4 and intcrferon‐gamma (IFN‐γ) on the synthesis of IgE and IgA by peripheral blood mononuclear cells (PBMC) were studied. Spontaneous IgE and IgA synthesis by PBMC was significantly increased in patients with IgA nephropathy compared with controls. The maximum amounts of IgA and IgE synthesis by PBMC after stimulation with IL‐4 were almost the same both in patients with IgAN and in controls. The enhancement rate of IL‐4‐induccd IgE and IgA synthesis was significantly lower in IgAN than in the controls, suggesting in vivo preactivation of PBMC in IgAN patients. IFN‐γ suppressed IgA and IgE synthesis by PBMC from IgAN patients as well as controls. However, the suppressive effect on IgE synthesis was less prominent in patients with IgAN. These results suggested that altered IL‐4 action might be involved in the development of IgA ncphropathy.