Cellular Immunity Delimits Adenoviral Gene Therapy Strategies for the Treatment of Neoplastic Diseases

Background: Adenoviral gene therapy is a promising new approach for the treatment of neoplastic diseases. To design rational clinical trials and distinguish the effects of therapeutic transgene expression from those caused by viral infection alone, the immune response to the vector must be understood. In these experiments, we further define cellular immunity to recombinant adenovirus.Methods: The immune response to hepatic adenoviral gene transfer was studied in infected mice by depleting T cells with an anti-CD3 antibody, measuring splenocyte cytokine production, determining the impact of transgene expression on inflammation, and assessing liver MHC protein expression.Results: The cellular immune response to recombinant adenovirus is (1) averted by T lymphocyte depletion, (2) marked by a T_H1 response with increased IL-2 production, (3) directed against both the transgene product and viral proteins, and (4) associated with increased hepatocyte MHC Class I expression.Conclusions: It is necessary to take into consideration the constraints imposed by the immunogenicity of recombinant adenovirus and its transient transgene expression in the clinical application of adenoviral gene transfer for the treatment of cancer.Presented at the 51st Annual Cancer Symposium of The Society of Surgical Oncology, San Diego, California, March 26–28, 1998.     

Overexpression of complement inhibitor Crry does not prevent cryoglobulin-associated membranoproliferative glomerulonephritis

BACKGROUND: Mice overexpressing thymic stromal lymphopoietin (TSLP) develop mixed cryoglobulinemia with renal disease closely resembling human cryoglobulin-associated membranoproliferative glomerulonephritis (MPGN), including glomerular deposits of immunoglobulins and complement. We assessed the effect of complement inhibition through overexpression of Crry (complement receptor-1 related gene/protein Y), which blocks the classic and alternative pathway of complement activation through inhibition of the C3 convertase, in cryoglobulinemia-associated immune complex glomerulonephritis. METHODS: TSLP transgenic mice were crossbred with animals overexpressing Crry. Mice were sacrificed after 50 days (females) or 120 days (males), and kidneys, blood, and urine were collected from seven mice of each experimental group (wild type, Crry transgenic, TSLP transgenic, and Crry/TSLP doubly transgenic). RESULTS: TSLP/Crry doubly transgenic animals demonstrated expected serum levels of Crry. Renal involvement, both in TSLP transgenic and TSLP/Crry doubly transgenic animals, was characterized by glomerular matrix expansion, macrophage influx, activation of mesangial cells, and deposition of immunoglobulins and complement. Overexpression of Crry did not result in significant improvement of renal pathology or laboratory findings. Expression of recombinant soluble Crry was confirmed by enzyme-linked immunosorbent assay (ELISA) in Crry transgenic animals. However, formation of the membrane attack complex C5b-9 as a marker of terminal active complement components and represented by glomerular C9 staining could not be inhibited in Crry transgenic TSLP mice. CONCLUSION: These results indicate that overexpression of Crry was not sufficient to prevent renal injury in TSLP transgenic mice. We suggest that the inhibitory capacity of Crry may be overwhelmed by chronic complement activation. Further studies need to address the role of complement in cryoglobulinemic glomerulonephritis before therapeutic complement inhibition can be attempted.     

Suppression of experimental crescentic glomerulonephritis by peroxisome proliferator-activated receptor (PPAR)γ activators

Background. It has been recently reported that peroxisome proliferator-activated receptors (PPARs)γ exist in various tissues and that they exibit anti-inflammatory effects. Methods. We investigated the effects of PPARγ activators on the development of crescentic glomerulonephritis. Crescentic glomerulonephritis was induced by the injection of rabbit anti-rat glomerular basement membrane antibody in WKY rats. Results. Administration of troglitazone suppressed urinary protein excretion and crescent formation as indicated by crescent scores. Pioglitazone, a PPARγ activator, mimicked the effect of troglitazone, but bezafibrate, a PPARα-activator, did not. Immunohistology revealed that troglitazone and pioglitazone inhibited the infiltration of ED-1-positive monocyte/macrophages and CD8-positive cells into glomeruli. Conclusions. In the present study, we demonstrated that PPARγ activators exert antinephritic effects by suppressing the recruitment of inflammatory cells via a PPARγ-dependent mechanism. Received: June 4, 2002 / Accepted: November 20, 2002 Acknowledgments This study was supported in part by Grants for Scientific Research from the Japanese Ministry of Education, Science, and Culture and by the U-Television Yamanashi (UTY) broadcasting company. Correspondence to:K. Haraguchi     

A case of atypical hemolytic uremic syndrome with a transient decrease in complement factor H

We report a case of sporadic atypical hemolytic uremic syndrome (HUS) with a transient decrease in complement factor H. Referred for hemolysis and azotemia without diarrhea prodrome, this 31-month-old boy showed a decreased complement 3 (C3) and complement factor H (FH) level. However, the factor H gene (HF1) mutation was missing. After the hemolysis was controlled with plasma infusion, the C3 and FH levels recovered. The patients renal function fully recovered and remained normal, and there was no recurrence of the HUS.     

Roles of connective tissue growth factor and prostanoids in early streptozotocin-induced diabetic rat kidney: the effect of aspirin treatment

Background. Connective tissue growth factor (CTGF) is a cysteine-rich growth factor induced by transforming growth factor-β (TGF-β) and is thought to play a critical role in TGF-β-stimulated extracellular matrix accumulation. To explore its involvement in early diabetic nephropathy, we investigated the time course of CTGF gene expression and its regulation in streptozotocin (STZ)-induced diabetic rat kidney. Methods. Northern blot analysis for CTGF, TGF-β, and fibronectin expression was performed in the glomeruli of STZ-induced diabetic rats from 3 days to 12 weeks after the induction of diabetes, together with histological examination. To investigate the role of prostanoids in this process, aspirin was administered in one group of diabetic rats. Furthermore, CTGF expression was analyzed in rat mesangial cells cultured under high-glucose conditions. Results. Glomerular expression of CTGF and TGF-β1 mRNA was coordinately upregulated as early as day 3, followed by fibronectin induction and mesangial matrix accumulation. Chronic aspirin treatment in diabetic rats significantly attenuated mesangial expansion, and effectively suppressed CTGF induction, as well as inhibiting the upregulation of TGF-β1 and fibronectin expression. In cultured mesangial cells, aspirin treatment abolished high glucose-stimulated CTGF upregulation. Conclusions. These results indicate that CTGF expressed in the glomeruli is upregulated in the early stage of STZ-induced diabetic nephropathy in rats, and could be a critical mediator of the development of diabetic glomerulosclerosis. In addition, the modulatory effects of aspirin during this process suggest a role of the cyclooxygenase pathway in the progression of diabetic nephropathy. Received: March 5, 2002 / Accepted: December 6, 2002 Acknowledgments We thank Ms. A. Wada, Ms. J. Nakamura, and Ms. Y. Oki for technical assistance, and Ms. S. Doi and Ms. A. Sonoda for secretarial assistance. This work was supported in part by research grants from the Japanese Ministry of Education, Science, Sports and Culture; the Japanese Ministry of Health and Welfare; “Research for the Future (RFTF)” of the Japan Society for the Promotion of Science; the Ono Foundation for Medical Research; the Smoking Research Foundation; and the Salt Science Research Foundation. Correspondence to:M. Mukoyama     

Mast cells and tubulointerstitial fibrosis in patients with ANCA-associated glomerulonephritis

Background. To elucidate the role of mast cells (MCs) in the pathogenesis of crescentic glomerulonephritis, we investigated the immunohistochemical localization of MCs in the kidney and the correlation between MC localization and tubulointerstitial lesions in biopsy specimens and serum stem cell factor (SCF) levels in patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis. Methods. Thirteen patients with ANCA-associated glomerulonephritis were enrolled in this study. Clinical parameters, such as serum creatinine and urinary protein excretion, were obtained from each patient at the time of biopsy. Paraffin-embedded sections were used for immunohistochemical staining, using the labelled streptavidin bio-tin (LSAB) method. Monoclonal antibodies to human tryptase, α-smooth-muscle actin, and CD68 were used as primary antibodies. Ten cortical interstitial fields were randomly selected at an original magnification of ×400 and assessed using a computer-assisted color image analyzer. Tubulointerstitial fibrosis was assessed as the percentage of the area stained with Masson trichrome in ten cortical interstitial fields. The measurement of serum SCF levels was performed by using an enzyme-linked immunoassay. Results. In all of the control subjects, a few tryptase-positive MCs were observed in the glomeruli and interstitium. In contrast, sparse MCs were observed in the interstitium, but not in the glomeruli of diseased kidneys. The number of interstitial MCs in the tubulointerstitial lesions was positively correlated with the degree of interstitial fibrosis. Moreover, a significant positive correlation was observed between the number of interstitial MCs and the serum SCF concentration (r = 0.85; P = 0.001). Conclusions. Our findings suggest that MC infiltration induced by SCF in interstitial tissues seems to be associated with tubulointerstitial fibrosis in human ANCA-associated glomerulonephritis. Received: May 31, 2002 / Accepted: November 8, 2002 Acknowledgments The authors thank Professor Hiroshi Toma of the Department of Urology, Tokyo Women's Medical University, for donating the control tissues. We wish to thank Mr. Shigeru Horita and Mr. Tsutomu Ishizuka for their special technical assistance. This study was supported in part by Grants for Progressive Renal Diseases from the Ministry of Health of Japan and a Grant-in-Aid from the Ministry of Health, Science, Sports, and Culture of Japan. Correspondence to:S. Otsubo     

腺病毒介导反义c-myc增强骨肉瘤细胞对顺铂化疗敏感性的实验研究

目的构建表达反义cmyc的重组腺病毒,探讨重组腺病毒介导反义cmyc转染的人骨肉瘤MG63细胞对顺铂化疗敏感性的影响。方法应用基因重组技术,将约720bp的人cmyccDNA反向克隆到腺病毒载体,经重组、扩增、病毒包装后构建表达反义cmyc的重组腺病毒(AdAscmyc),并在体外转染骨肉瘤MG63细胞,采用瑞士染色、吖啶橙染色、蛋白免疫印迹(WesternBlot)、细胞体外增殖抑制试验(MTT)、流式细胞仪(FCM)等观察细胞形态、检测cmyc蛋白表达、瘤细胞体外增殖抑制、凋亡及细胞周期,分析AdAscmyc体外转染的骨肉瘤MG63细胞对顺铂化疗敏感性。结果成功构建AdAscmyc,滴度可达2×109pfu/ml,体外转染MG63细胞48h后,可降低cmyc蛋白表达,并与浓度为2.0、5.0μg/ml的顺铂作用2h后,可抑制MG63细胞的体外增殖,抑制率分为33.4%、54.2%,与对照腺病毒(AdLacZ)转染组相比差异有统计学意义(P<0.05),FCM检测证实AdAscmyc的转染可诱导骨肉瘤细胞凋亡,且顺铂治疗后凋亡比例增加,细胞周期分析显示AdAscmyc转染的骨肉瘤细胞出现G2/M期阻滞。结论腺病毒介导反义cmyc能诱导骨肉瘤MG63细胞凋亡并增加MG63细胞对顺铂化疗敏感性。     

Anti-GBM glomerulonephritis in mice lacking IL-1β-converting enzyme (ICE)

Background. Interleukin-1 (IL-1) has been reported to play a major role in the initiation and progression of several glomerulonephritis, and inhibition of IL-1 by the blockade of IL-1β-converting enzyme (ICE) has been suggested to be an ideal therapeutic strategy. Methods. To examine the effect of ICE inhibition on glomerulonephritis, we examined the susceptibility of ICE-deficient mice (ICE−/−) to anti-glomerular base-ment membrane antibody-induced glomerulonephritis (antiGBMGN), which has been previously reported to be mediated by IL-1β. Results. After the injection of antiGBM antibody to ICE−/− and wild type mice, albuminuria rose progressively and both groups of mice died within 7–9 days. Laboratory analysis of proteinuria, serum creatinine, and glomerular histology revealed no significant difference between the two groups. To pursue the mechanism of this result, bone marrow-derived monocyte/macrophage lineage cells (Mo/Mq cells) were established from both groups and the potency of IL-1β production in response to lipopolysaccharide was examined. An enzyme-linked immunosorbent assay (ELISA) of IL-1β revealed that, Mo/Mq cells from ICE−/− mice secreted IL-1β in response to lipopolysaccharide, although to a lesser extent than the Mo/Mq cells from ICE+/+ mice, suggesting that other protease(s) may process proIL-1β to generate the mature form. In fact, as was seen in the wild type mice, serum from antiGBM-injected ICE−/− mice contained IL-1β. Conclusions. These data suggest a limited effectiveness of ICE inhibition as a therapeutic strategy for glomerulonephritis. Received: May 18, 1999 / Accepted: October 25, 1999     

Effect of p53 Gene Therapy Combined with CTLA4Ig Selective Immunosuppression on Prolonged Neointima Formation Reduction in a Rat Model

p53 to the injured rat carotid artery. The purpose of this study was to determine if the effect of p53 gene in reducing neointimal formation would still be present up to 8 weeks after arterial injury and whether it could be enhanced by adding immunosuppression. Cytotoxic T lymphocyte–associated antigen-4 Ig (CTLA4Ig), a novel immunosuppressive agent, is a recombinant soluble protein that blocks T cell–dependent immune response. Animals were divided into eight groups (n= 6 in each). In vivo gene transfer was used in isolated segments of balloon-injured rat carotid arteries. Genetically modified adenovirus encoding for wild-type p53 protein was applied at 8 × 10¹⁰ pfu/mL. Control rats received adenovirus null at the same concentration. A daily dose of 300 μg of CTLA4Ig was given intraperitoneally, either once, twice, or three times. Expression of p53 was determined by Western blot analysis. Neointimal formation was assessed at 4 or 8 weeks by harvesting carotid arteries and determining the intima/media (I/M) ratio cross-sectional area measurements. p53 expression was confirmed by Western blot analysis. We concluded that adenovirus-mediated p53 gene transfer significantly decreases the formation of neointima up to 8 weeks following rat carotid injury. However, there is loss of effectiveness on neointimal formation inhibition as time elapses. When CTLA4Ig is added, there is significant improvement in results, with sustained neointimal formation inhibition at 8 weeks after the procedure.     

Gene therapy for glomerulonephritis using bone marrow stem cells

We have proposed two novel therapeutic strategies for glomerulonephritis based on bone marrow transplantation technology. In the ex vivo differentiation system, bone marrow cells were differentiated ex vivo to express ligands of adhesion molecules and acquire the potential to be recruited to the inflamed site, and they were adenovirally transfected with a therapeutic gene followed by transfusion to affected subjects. These cells may deliver anti-inflammatory cytokines into inflamed glomeruli. In the in vivo differentiation system, bone marrow cells were genetically modified using a retrovirus and transplanted to the affected subjects before differentiation so that they might retain the potential for self-renewal as well as differentiation in vivo. Both systems were able to prove the therapeutic benefit in at least anti-glomerular basement membrane (anti-GBM) nephritis in mice. These strategies have several advantages over the previous glomerulus-targeted gene-delivery system, i.e., use of peripheral vessels for administration, and possibly no immunoreaction due to autologous transfer, and suggest that bone marrow-derived cells can be utilized for therapeutic intervention to treat glomerulonephritis. Received: June 4, 2002 / Accepted: September 17, 2002 Acknowledgments This work was supported by a grant from the Ministry of Education (Japan), Ministry of Human Health and Welfare (Japan), and the Sankyo Foundation of Life Science. Correspondence to:T. Yokoo     

Final common pathways of progression of renal diseases

Complement activation plays a critical role in the pathogenesis of many forms of glomerulonephritis. Studies utilizing cultured glomerular cells and animal models have clarified the important role of imbalance between complement activation and complement regulatory proteins in the pathogenesis of immune-mediated glomerular injury. Glomerular injury results not only from glomerulonephritis but occurs through a variety of mechanisms, including those associated with hypertension and diabetes mellitus. However, once glomerular damage reaches a certain threshold, progression of renal disease is consistent and irreversible. Pathological studies have revealed a close correlation between tubulointerstitial damage and poor prognosis, emphasizing the crucial role of tubulointerstitial injury in eventual kidney failure. One common mechanism that leads to renal failure via tubulointerstitial injury is massive proteinuria. Specific components of the filtered protein may be toxic to tubular cells. Among various harmful proteins filtered through the glomerular barrier are complement components, which are activated by ammonium resulting from excess delivery of protein to tubular cells. Another common mechanism is chronic ischemia in the tubulointerstitium. Tubulointerstitial damage results in the loss of peritubular capillaries and induces chronic hypoxia in this compartment, producing a vicious cycle. Therapeutic approaches that target these final common pathways may prove valuable in many kinds of renal disease. Received: June 17, 2002 / Accepted: September 17, 2002 Acknowledgments The author is very grateful to Dr. Nobutaka Hirokawa (University of Tokyo, Tokyo, Japan) for his mentorship in my research career, to Dr. Toshiro Fujita (University of Tokyo, Tokyo, Japan) for his warm and generous support since my return from Seattle, and to Dr. Kiyoshi Kurokawa (Tokai University, Isehara, Japan), who was the main reason for me to choose my career as a nephrologist. The author is also grateful to Drs. Richard J. Johnson (University of Washington, Seattle, WA, USA), Seiichi Matsuo (University of Nagoya, Nagoya, Japan), Kenjiro Kimura (St. Marianne University, Japan), Fujio Shimizu (Niigata University, Niigata, Japan), Reiko Inagi (Tokai University, Isehara, Japan), Toshio Miyata (University of Tokai, Isehara, Japan), and Stuart J. Shankland (University of Washington, Seattle, WA, USA) for their continuous support. I also thank Mrs. Asako Kashihara for her secretarial assistance. I dedicate this review to my mentor, Dr. William G. Couser (University of Washington, Seattle, WA, USA), for whom I have the highest admiration and respect. Correspondence to:M. Nangaku     

Immunodysregulation,polyendocrinopathy, enteropathy,X-linked (IPEX) syndrome: an unusual cause of proteinuria in infancy

We report on a 6-month-old child presenting with chronic diarrhea, failure to thrive, eczema, autoimmune hemolytic anemia (AIHA), insulin-dependent diabetes mellitus (IDDM), hypoalbuminemia, and proteinuria. Renal biopsy showed membranous glomerulonephritis. A diagnosis of Immunodysregulation, polyendocrinopathy, enteropathy, x-linked (IPEX) syndrome was subsequently confirmed by DNA analysis, which demonstrated the presence of a mutation in exon 2 of the FOXP3 gene (303–304 del TT). Proteinuria secondary to membranous glomerulonephritis is a novel feature of IPEX syndrome. Membranous glomerulonephritis went into remission after the patient had received hematopoietic stem cell transplantation (HSCT).     

Passenger lymphocyte syndrome in renal transplantation: A systematic review of published case reports

BackgroundPassenger lymphocyte syndrome (PLS) is an immune-mediated hemolysis that occurs after ABO-mismatched kidney transplantation. PLS is caused by donor lymphocytes producing antibodies to recipient red blood cells, resulting in hemolysis. The incidence of PLS has been reported to be approximately 20% in patients with ABO-mismatched groups. Nevertheless, there is no comprehensive review of PLS following renal transplantation. In this review, we systematically summarized the data of patients with PLS after renal transplantation to help clinicians diagnose and treat more effectively.MethodsA systematic review was conducted using PubMed, Embase, and Web of Science. All relevant data were collected, including age, sex, and clinical and immune parameters.ResultsA total of 91 published cases were identified. The age ranged from 9 to 70 years old and 58.2% were male. Eighty-six cases were only kidney transplantations, one was liver-kidney transplantation, three were pancreas-kidney transplantations, and one was intestinal-kidney transplantation. Of these cases, 27 received kidneys from deceased donors, whereas 40 received kidneys from living donors. Most patients showed immune hemolysis dominated by anaemia, which was significantly improved after symptomatic support treatment, such as blood transfusion and erythropoietin injection.ConclusionPLS is an immune-mediated disease that can occur in patients with ABO-mismatched renal transplantation, which commonly causes hemolysis, although death or deformities of the graft can also occur in patients with the disorder. Symptomatic supportive treatment is an effective treatment scheme at present, but more effective treatment and prevention schemes still need to be explored.     

Expression of a soluble complement inhibitor protects transgenic mice from antibody-induced acute renal failure

Crry is a potent complement regulator in rodents that inhibits C3 convertases. In rats, intrarenal arterial injection of anti-glomerular endothelial cell (GEN) antibodies leads to complement-dependent microvascular injury and acute renal failure. In this study, a mouse variant of this model and the effects of complement inhibition were examined. Transgenic mice that overexpressed soluble Crry systemically and in their kidneys were studied. Anti-GEN IgG was injected intravenously into eight Crry transgenic mice and seven transgene-negative littermates (which were used as control animals). Thirty h after injection, blood urea nitrogen (BUN) levels were 30.3 +/- 4.4 and 114.8 +/- 23.5 mg/dl for transgene-positive and -negative animals, respectively (P = 0.012). Four of five transgene-negative animals with BUN levels of > 100 mg/dl were anuric; the remaining animal exhibited minimal albuminuria and no detectable urinary C3. In animals with renal failure, glomerular capillary collapse and tubular necrosis were observed. There was significant tubular staining for C3 in transgene-negative animals, with cellular and basal distributions, both of which were statistically greater than those in transgene-positive animals. Tubular cell C3 staining was strongly correlated with BUN values (r = 0.83, P < 0.001), as was C9 staining (r = 0.56, P = 0.037), suggesting that complement activation to the C5b-9 membrane attack complex had a casual role in renal failure. Thus, systemic injection of anti-GEN antibodies into mice leads to acute renal failure, with glomerular and tubular injury. Animals that overexpress soluble Crry in renal tubules and elsewhere are protected from the acute renal failure that occurs in this model, which ultimately seems to develop because of complement activation focused on tubules.     

沉默c-myc重组腺病毒载体的构建

目的设计、构建并筛选出转染至人骨肉瘤细胞系OS-9901，对c-myc沉默效果最佳的复制缺陷型重组腺病毒质粒pAd-c-myc．短发夹RNA（short hairpinRNA，shRNA）包装出表达c-myc-shRNA的重组腺病毒，并测定其滴度。方法设计有shRNA结构的3对单链寡核苷酸（ss oligos），经变性退火为双链寡核苷酸（dsoligos），插入穿梭质粒pENTR／U6载体，测序正确后，经Lipofectamine^TM2000阳离子脂质体介导入人骨肉瘤细胞系OS-9901，采用RT-PCR筛选对c-myc沉默效果最佳的穿梭质粒pENTR／U6-shRNA，然后与腺病毒骨架质粒行同源重组，筛选出正确重组子。采用293A细胞包装出表达c-myc-shRNA的复制缺陷型重组腺病毒，观察其细胞病变效应（cytopathiceffect，CPE），采用病毒颗粒法（viral particles，vP）和50％组织培养感染剂量法（50％tissue culture infective dose，TCID50）测定病毒滴度。结果电泳验证后的dsoligos插入穿梭质粒pENTR／U6载体，测序结果提示构建的pENTR／U6-shRNA质粒正确。从3对dsoligos通过RT-PCR方法筛选出对c-myc沉默效果最佳的穿梭质粒pENTR／U6-shRNA，经PacI酶切线性化后同源重组成功构建了介导c-myc-shRNA复制缺陷型重组腺病毒，CPE和空泡现象3d开始出现，6d更加明显。采用VP法测定的第1代腺病毒滴度为5．23&#215;109VP／mL，经3～4代扩增后可达2．26&#215;10^12VP／mL。TCID50证实病毒滴度为10^-3.8／0．1mL。结论通过RNA干扰技术，体外成功构建了介导shRNA-c-myc复制缺陷型重组腺病毒。     

CCAAT/enhancer-binding protein-d is induced in mesangial area during the early stages of anti-Thy1.1 glomerulonephritis and regulates cell proliferation and inflammatory gene expression in cultured rat mesangial cells

Background Interleukin (IL)-6, cyclooxygenase (COX)-2, and monocyte chemoattractant protein (MCP)-1 contribute to renal injury. The promoter regions of these genes contain CCAAT/enhancer-binding protein (C/EBP)-binding sites. In this study, we investigated the role of C/EBP-δ in mesangial cells (MCs). Methods In an in vivo study, anti-Thy 1.1 glomerulonephritis rats were generated and C/EBP-δ, IL-6, COX-2, and MCP-1 expressions were assessed by immunohistochemistry. In an in vitro study, cultured MCs were transfected with non-silencing (NS) short interfering RNA (siRNA) or C/EBP-δ siRNA. Subsequently, after stimulation with IL-1β, C/EBP-δ, IL-6, COX-2, and MCP-1 mRNA expression levels were evaluated using real-time polymerase chain reaction (PCR). IL-6 concentration in the culture medium was determined by enzyme-linked immunosorbent assay. In addition, cell proliferative activity against IL-1β or platelet-derived growth factor-BB was assessed by bromodeoxyuridine incorporation. Results In the in vivo study, C/EBP-δ, IL-6, COX-2, and MCP-1 were expressed in the mesangial region of anti-Thy 1.1 glomerulonephritis rats on day 1. In the in vitro study, IL-1β increased C/EBP-δ mRNA levels in NS siRNA-transfected MCs (7.3-fold), but no increase was evident in C/EBP-δ siRNA-transfected MCs. IL-6, COX-2, and MCP-1 mRNA levels in C/EBP-δ siRNA-transfected MCs were all lower than those in NS siRNA-transfected MCs (decreases of 57.7%, 85.7%, and 69.3%, respectively). The IL-6 concentration in the culture medium from C/EBP-δ siRNA transfected MCs (7.37 ± 4.3 pg/ml) was also lower than that in the culture medium from NS siRNA-transfected MCs (25.2 ± 3.4 pg/ml). Cell proliferative activity in C/EBP-δ siRNA-transfected MCs was lower than that in NS siRNA transfected MCs. Conclusions C/EBP-δ was induced in the mesangial region during the early stages of anti-Thy1.1 glomerulonephritis. C/EBP-δ contributes to inflammatory gene expression and MC proliferation.     

Synchronous expression of contractile proteins in mesangial cells and interstitial cells in IgA nephropathy

Background. Phenotypic changes in glomerular mesangial cells and interstitial cells are considered to be closely associated with the progression of glomerulosclerosis and renal fibrosis in IgA nephropathy. To further evaluate the relevance of phenotypic changes, we examined the expression of four different contractile proteins as markers of the phenotypic changes. Methods. The expression of nonmuscle myosin heavy chain isoform (SMemb), α-smooth muscle actin (α-SMA), caldesmon (CaD), and tropomyosin (TM) was evaluated by indirect immunofluorescent studies in renal biopsy specimens from 21 patients with IgA nephropathy. The relationships with the histological parameters and clinical parameters were analyzed. Results. Pronounced expression (score of 2 or more) of contractile proteins was observed in the majority of the patients (57%–90%) compared with controls. There was no significant relationship between the glomerular expression score of any contractile protein and the proliferation index or sclerotic index. Daily urinary protein excretion in the group expressing low levels of caldesmon in the glomerulus was significantly lower than that in the group expressing high levels (P < 0.05). There were significant correlations among the glomerular expressions of these contractile proteins, except for TM (P < 0.05). All contractile proteins were expressed in tubulointerstitial lesions, and only SMemb expression was detected in tubular epithelial cells. The interstitial scores of all contractile proteins increased in parallel with the degree of tubulointerstitial lesion (P < 0.05). Conclusions. Contractile proteins appeared to be coordinately expressed in the glomerulus and the interstitium in IgA nephropathy, which may reflect the mechanical stress involved with glomerulonephritis. Received: December 21, 1998 / Accepted: March 1, 1999