Combined interleukin 1 and tumour necrosis factor α blockade in rat crescentic anti-glomerular basement membrane glomerulonephritis

SUMMARY: Studies in experimental models have established that blockade of either interleukin 1 (IL‐1) or tumour necrosis factor α (TNF‐α) is effective in suppressing crescentic glomerulonephritis. However, it is not known whether simultaneous blockade of both cytokines will provide additional disease suppression compared with that produced by single cytokine blockade. We have addressed this question in a study of accelerated crescentic anti‐glomerular basement membrane (GBM) glomerulonephritis in the rat. Groups of six animals were treated with an IL‐1 receptor antagonist (IL‐1ra), TNF‐α‐binding protein (TNFbp), IL‐1ra + TNFbp (combined) or saline (control) from the time of anti‐GBM serum injection until being killed, 10 days later. Saline‐treated animals developed crescentic glomerulonephritis with tubulointerstitial damage, heavy proteinuria and renal impairment. Compared with saline, treatment with either IL‐1ra or TNFbp alone resulted in significant suppression of crescent formation (3.0% and 3.3%, respectively, vs. 21.0%; both P < 0.001 vs. control), tubulointerstitial leucocytic infiltration (262 ± 31 and 282 ± 32 cells/mm² vs. 481 ± 71 cells/mm²; both P < 0.001 vs. control) and proteinuria (167 ± 44 and 164 ± 23 mg/24 h vs. 279 ± 36 mg/24 h; both P < 0.001 vs. control) and prevented the loss of renal function. Combined IL‐1ra and TNFbp treatment resulted in a virtually identical degree of disease suppression as individual cytokine blockade in terms of crescent formation (2.7%), interstitial leucocytic infiltration (274 ± 45 cells/mm²), proteinuria (190 ± 18 mg/24 h) and renal function preservation. In conclusion, this study has demonstrated that blockade of either IL‐1 or TNF‐α alone substantial suppresses experimental crescentic glomerulonephritis to a similar extent to that achieved by simultaneous blockade of both cytokines. These findings provide a rationale for the use of cytokine monotherapy, rather than multiple cytokine blockade, in the treatment of human crescentic glomerulonephritis.     

Endotoxins modulate chronically tumour necrosis factor {alpha} and interleukin 6 release by uraemic monocytes

We examined in vivo the release of tumour necrosis factor alpha(TNF) and interleukin 6 (IL-6) by uraemic monocytes upon stimulationwith endotoxin-contaminated bicarbonate concentrate. Twelveuraemic patients underwent 1-month-subsequent periods of standardhaemodialysis (SHD) with cuprophane (CU), a high-complement-activatingmembrane (6 patients), or haemodiafiltration (HDF) with polyacrylonitrile(PAN), a low-complement-activating membrane (6 patients), byusing a dialysate prepared with either non-sterile bicarbonateconcentrate tanks (phase 1) or sterile bicarbonate concentratebags (phase 2). TNF and IL-6 concentrations were determinedin monocyte supernatants by ELISA; endotoxin levels in bicarbonateconcentrates were measured by a chromogenic limulus amoebocytelysate (LAL) assay. A significant increase in LAL reactivity was found in bicarbonateconcentrate tanks compared to sterile bags (P<0.001). Non-steriledialysate caused a significant (P<0.001) predialytic increasein monocyte TNF release as compared to controls and nondialyseduraemic patients. One month treatment with sterile bicarbonatesignificantly decreased TNF predialytic activity in monocytesupernatants (P<0.001) to levels closer to those of non-dialyseduraemic patients. A similar decrease was observed for IL-6 production.Dialytic treatment induced a further increase in both TNF andIL-6 production, particularly in phase 1. When uraemic patientswere examined separately according to the different dialyticprocedures (SHD-CU or HDF-PAN), the use of sterile dialysate(phase 2) caused a significant decrease of predialysis TNF releasein both SHD CU patients (24.1±8.4 pg/ml versus 55.3±5.7pg/ml, P<0.001) and HDF PAN-treated patients (16.6±5.3pg/ml versus 29.1±5.4pg/ml, P<0.005), so that thedifferences between the dialytic procedures were completelyabolished. In conclusion, TNF and IL-6 release may be induced by endotoxin-contaminateddialysate during haemodialysis. The use of sterile bicarbonatecan ameliorate the bioincompatibility of CU membranes and probablyinfluences the biocompatibility of PAN membranes. Therefore,regardless of the type of dialyser used, all attempts to obtainan ultrapure dialysate are important to optimize dialytic treatment,in order to attenuate the chronic monocyte activation whichoccurs during haemodialysis.     

Aldosterone induces CTGF in mesangial cells by activation of the glucocorticoid receptor.

BACKGROUND: Aldosterone contributes substantially to cardiac and renal injury by acting on target cells not involved in the regulation of salt and water balance. The profibrotic protein connective tissue growth factor (CTGF) has been identified as one of the target proteins of aldosterone. However, the molecular mechanisms of aldosterone-mediated CTGF induction have not been characterized. METHODS: Mesangial cells were treated with aldosterone or dexamethasone. CTGF expression was characterized at the mRNA and protein level. Translocation of the glucocorticoid receptor (GR) was detected by immunocytochemistry and by Western blotting. RESULTS: Aldosterone and dexamethasone induced CTGF at the mRNA and protein level in a time- and concentration-dependent manner. Specific antagonists of the mineralocorticoid receptor, spironolactone, canrenoate or eplerenone, did not inhibit CTGF induction. However, inhibition of the GR by RU486 prevented dexamethasone-as well as aldosterone-induced CTGF expression, indicating the importance of the GR in aldosterone-mediated regulation of CTGF. This notion was confirmed by translocation of the GR to the nucleus upon stimulation with aldosterone. CONCLUSIONS: CTGF is a functional target of aldosterone in mesangial cells, but aldosterone-induced CTGF gene expression is not directly mediated by the mineralocorticoid receptor.     

正常妊娠与自然流产小鼠模型蜕膜组织和血清中肿瘤坏死因子及其受体的表达

目的比较正常妊娠与自然流产小鼠模型蜕膜组织肿瘤坏死因子-α(TNF-α)及其受体1(TNFR1)和血清可溶性肿瘤坏死因子受体1(sTNFR1)的表达,探讨其与不明原因自然流产的关系。方法建立正常妊娠小鼠模型CBA×BALB/C和自然流产模型CBA×DBA/2。采用免疫组化SABC法测定两组模型孕13 d蜕膜组织TNF-α、TNFR1表达水平;酶联免疫吸附试验(ELISA)测定两组模型孕13 d血清sTNFR1表达水平。结果与正常妊娠模型相比,自然流产模型蜕膜组织中TNF-α、TNFR1表达显著升高(P<0.01);血清sTNFR1表达水平也高于正常妊娠模型(P<0.05)。结论TNF-α、TNFR1、sTNFR1可能与自然流产的发生发展有关。某些病理情况引发的蜕膜TNF-α、TNFR1表达增加也许是自然流产发生的原因之一,sTNFR1水平升高可能对妊娠具有自我保护和自我稳定的生理意义。     

Use of porcine tumor necrosis factor receptor 1-Ig fusion protein to prolong xenograft survival

BACKGROUND: Delayed rejection of xenografts is a major hurdle that needs to be addressed to achieve long-term engraftment in the pig-to-primate transplant setting. Both vascular and avascular xenografts are susceptible to a delayed rejection process that comprises humoral and cellular responses. Tumor necrosis factor (TNF) is believed to play a role in this process by promoting cell activation, apoptosis and the recruitment of inflammatory cells. To address this problem, we engineered the donor cell in such a way that it could block both human and porcine TNF. METHODS: We produced a recombinant fusion protein containing the extracellular domain of the porcine TNF-Receptor 1 and an IgG Fc moiety (pTNFR1Ig). We first evaluated by flow cytometry the pTNFR1Ig capacity to prevent TNF alpha-induced expression of SLAI, SLAII, VCAM-1, ICAM-1 and E-selectin on the cell surface of porcine aortic endothelial cells (PAEC). The effect on TNF alpha-mediated cell death was also assessed by propidium iodide staining after incubating PAEC with TNF alpha plus cycloheximide for 24 h. PAEC and porcine fibroblasts were subsequently engineered by retroviral infection to express and secrete pTNFR1Ig and their resistance to the TNF alpha effects was tested in vitro. Finally, we transplanted mock-control and pTNFR1Ig-expressing PAEC under the kidney capsule of BALB/c mice in the absence of immunosuppression and examined the degree of rejection at 2 and 3 weeks post-transplantation. RESULTS: Treatment with pTNFR1Ig resulted in a very potent blockade of human, porcine and murine TNF alpha activity on porcine cells. It inhibited the upregulation of all cell surface markers of activation tested as well as the TNF alpha-mediated cell death. Moreover, pTNFR1Ig-expressing PAEC showed prolonged engraftment in a pig-to-mouse xenotransplant model. CONCLUSIONS: Incorporation of strategies that block TNF may prove useful in the development of xenografts resistant to delayed rejection.     

Functional endothelin 1 receptors on human glomerular podocytes and mesangial cells.

To determine if endothelin 1 (Et1) receptors are present in human glomeruli, and which glomerular cells possess these receptors, 125I Et1 binding to isolated glomeruli and cultured glomerular mesangial and epithelial cells was studied. The latter were identified as podocytes. We demonstrated that Et1 binds specifically and reversibly to isolated human glomeruli and to cultured glomerular mesangial and epithelial cells. Scatchard analysis of competitive inhibition of 125I Et1 binding gave the following results (m +/- SEM, n = 3): isolated glomeruli, Kd = 4.2 +/- 2.1 x 10(-10) M, Bmax = 8.1 +/- 1.2 x 10(10) sites/mg protein; mesangial cells, Kd = 5.2 +/- 1.5 x 10(-10) M, Bmax = 1.87 +/- 0.49 x 10(4) sites/cell; epithelial cells, Kd = 7.2 +/- 1.5 x 10(-10) M, Bmax = 2.46 +/- 0.15 x 10(4) sites/cell. These receptors seem to be functional, since in both mesangial and epithelial cells Et1 induces a rapid and transient increase in intracellular [Ca2+]i. All these results indicate that Et1 may regulate glomerular filtration rate through an autocrine-paracrine pathway on mesangial cells and on podocytes.     

Concomitant treatment of MBT-2 bladder tumour by tumour necrosis factor alpha and interferon alpha in conjunction with delayed type hypersensitivity immunotherapy

Summary In our previous study [9], we reported the anti-tumour effect of TNF on mouse bladder tumour (MBT-2) both in vivo and in vitro. Inoculation of a single dose of TNF alone caused significant but transient tumour growth inhibition. Subsequent repeated doses of TNF did not sustain or augment the antitumour effect. The current experiments were undertaken to assess the anti-tumour activity of (i)-concomitant treatment of TNF-A and IFN-A against MBT-2 bladder tumour and (ii)-concomitant TNF+IFN-A treatment in conjunction with T-DTH (delayed-type hypersensitivity) immunotherapy. Systemic administration of multiple doses of TNF+IFN-A in vivo caused initial partial tumour regression followed by tumour growth inhibition up to 14 days following treatment. This combined treatment showed an enhanced anti-tumour effect compared to TNF-A treatment alone. Immunotherapy of MBT-2 tumour-bearing mice with T-DTH immune effector cells alone did not cause significant tumour growth inhibition. In contrast, concomitant administration of both T-DTH effector cells and TNF+IFN-A in MBT-2 tumour-bearing mice resulted in significant tumour growth inhibition for up to 16 days. The immune effector cells conferring immunotherapy were isolated from the spleens of tumour-immunized, DTH-primed animals and were characterized as Lyt 1⁺2^- helper/DTH T cells (CD4⁺ phenotype). These cells mediate both DTH response to MBT-2 tumour antigens as well as anti-MBT-2 tumour protection. In vitro treatment of the immune cells with TNF-A resulted predominantly in the proliferation of Lyt 1⁺ T cells versus Lyt 2⁺ cells. The anti-tumour effect of TNF+IFN-A can be augmented by immunotherapy possibly via the immune capacity of tumour sensitized T-DTH effector cells.     

醛固酮通过线粒体源性氧化应激促进表皮生长因子受体活化及肾小球系膜细胞增殖

目的 研究活性氧（ROS）在醛固酮（ALDO）诱导的表皮生长因子受体（EGFR）信号通路活化及系膜细胞增殖中的作用并探讨ROS的来源。 方法 体外培养人肾小球系膜细胞,应用3H-胸腺嘧啶（3H-TdR）掺入法和细胞计数测定系膜细胞增殖;荧光探针2,7-二氯二氢荧光素乙酰乙酸（DCFDA）检测细胞内ROS 的产生;Western印迹法检测EGFR活化。 结果 ALDO可显著促进肾小球系膜细胞增殖,应用100 nmol/L ALDO刺激系膜细胞24 h后,3H-TdR掺入量及细胞数分别是基础状态下的2.63倍和2.15倍。盐皮质激素受体（MR）阻断剂依普利酮（EPLE）几乎完全阻断ALDO诱导的系膜细胞增殖（P < 0.01）,而糖皮质激素受体（GR）阻断剂RU-486对ALDO诱导的细胞增殖无显著抑制作用。ALDO明显促进肾小球系膜细胞ROS产生,100 nmol/L ALDO刺激60 min,系膜细胞内ROS释放是对照组的2.14倍,EPLE显著抑制ALDO诱导的系膜细胞ROS产生（P < 0.01）。线粒体复合体 I抑制剂鱼藤酮（ROT）几乎完全阻断ALDO诱导的ROS产生（P < 0.01）,NADPH 氧化酶抑制剂夹竹桃麻素（apocynin）和二联苯碘（DPI）对ALDO诱导ROS产生的抑制率为30%~35%（P < 0.05）,而黄嘌呤氧化酶抑制剂别嘌醇、环氧化酶抑制剂吲哚美辛、脂氧化酶抑制剂去甲二氢化愈创木酸、细胞色素P450氧化酶抑制剂酮康唑以及一氧化氮合成酶抑制剂N-硝基-L-精氨酸甲酯对ALDO诱导的ROS产生均无明显影响。乙酰半胱氨酸（NAC）和ROT对ALDO诱导系膜细胞增殖的抑制率为75%~80%,apocynin和DPI的抑制作用仅为25%~30%。ALDO可显著活化系膜细胞EGFR,ALDO刺激60 min,EGFR活性是对照组的4.95倍,EPLE和NAC几乎完全阻断ALDO诱导的EGFR磷酸化（P < 0.01）,而NAC和EGFR拮抗剂AG1478则阻断ALDO诱导的系膜细胞增殖（P < 0.01）。 结论 ALDO通过氧化应激依赖的EGFR磷酸化促进肾小球系膜细胞增殖,ALDO诱导的系膜细胞氧化应激主要来源于线粒体。     

Tumour necrosis factor soluble receptors I and II and interleukin-1 receptor antagonist in acute pyelonephritis in relation to bacterial virulence-associated traits and renal function

Urinary tract infections activate both mucosal and systemicinflammatory responses reflected by elevation of cytokine concentrationsin serum and urine. We determined urine and serum concentrationsof tumour necrosis factor soluble receptors I and II (sTNFRI and sTNFR II) and interleukin-1 receptor antagonist (IL-1ra)in 41 women with acute pyelonephritis caused by Escherichiacoli, 2 weeks after the infection, during a subsequent episodeof cystitis or asymptomatic bacteriuria and also later whenthe same patients were free from bacteriuria. Concentrationsof sTNFR I, sTNFR II and IL-1ra were related to the expressionof five virulence markers of E. coli, glomerular filtrationrate (GFR) and to the concentration of C-reactive protein (CRP)in serum. Patients with acute pyelonephritis had elevated serumconcentrations of sTNFR I and sTNFR II compared to healthy women(P<0.001 for both comparisons). The concentrations of sTNFRI and sTNFR II in urine were significantly higher in patientswith acute pyelonephritis compared to controls (P<0.001 inboth cases). The concentration of sTNFR II in urine was higherin patients infected by E. coli producing haemolysin (P=0.05)and in patients infected by E. coli expressing hydrophobic properties(P=0.05) compared to patients infected by strains without thesevirulence traits. Patients who had high concentrations of sTNFRII in serum during acute pyelonephritis had lower GFR at follow-up(r=–0.48, P=0.05). Patients who responded with a markedincrease in CRP had higher sTNFR I and sTNFR II in urine (r=0.58,P<0.01 and r=0.48, P<0.01, respectively). The concentrationsof sTNFR I and sTNFR II in serum and urine decreased duringfollow-up and were lower 2 weeks after the infection when allpatients were free from bacteriuria. IL-1ra in serum was elevatedduring pyelonephritis (P<0.001) while that in urine was significantlylower compared to controls (P<0.001). It is concluded thatthe     

Dorsal root sensitivity to interleukin-1 beta, interleukin-6 and tumor necrosis factor in rats

The release of inflammatory cytokines caused by a disrupted disc may play a critical role in pain production at nerve endings, axons, and nerve cell bodies. Herniated disc tissue has been shown to release inflammatory cytokines such as interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor (TNF), and other algesic chemicals. This study was designed to characterize the effects of these proinflammatory cytokines on the somatosensory neural response at the dorsal root level in rats. It is hypothesized that their effects on nerve endings in disc and adjacent tissue contribute to low-back pain, and the effects on dorsal root axons and ganglia contribute to radiculopathy and sciatica. Surgically isolated sacral dorsal roots were investigated by electrophysiologic techniques. IL-1beta, IL-6, or TNF (100 ng, each) were applied onto the dorsal roots. Neural responses and mechanosensitivity of the receptive fields were evaluated over time. The results showed that 3 h after each cytokine application, the neural activity was statistically decreased. The mechanical sensitivity of the receptive fields increased at 90 min following IL-1beta or TNF application, and returned to normal more than 3 h after IL-1beta application. IL-1beta, IL-6, and TNF may be neurotoxic to dorsal root axons. Furthermore IL-1beta and TNF may sensitize the peripheral receptive fields. This study suggests that dorsal roots may be impaired by these proinflammatory cytokines.     

肾移植术后监测血清、尿液和肾组织液中肿瘤坏死因子的临床意义

为探讨肾移植术后肿瘤坏死因子（ＴＮＦα）在移植肾排斥反应（ＡＲ）诊断与鉴别诊断中的意义，采用液相竞争放射免疫法动态监测６０例肾移植患者术后血清、尿液及肾组织液中ＴＮＦα水平的变化。结果：ＡＲ组ＴＮＦα水平在血清、尿液及肾组织液中均显著升高，以肾组织液中升高最明显，比临床症状的出现早１～２天；ＣｓＡ肾中毒组ＴＮＦα水平轻度升高，但无统计学意义；急性肾小管环死（ＡＴＮ）组肾组织液及尿液中ＴＮＦα水平均显著升高；急性感染组仅血清中ＴＮＦα水平显著升高。结论：动态监测血清、尿液和肾组织液中ＴＮＦα水平能较好地早期诊断和鉴别诊断ＡＲ、急性感染、ＣｓＡ肾中毒和ＡＴＮ。     

Interleukin 4 enhances osteoblast macrophage colony-stimulating factor,but not interleukin 6, production

To determine if interleukin 4's (IL-4) recently discovered skeletal effects could be explained by its effects on osteoblasts, we have examined IL-4's impact on macrophage colony stimulating factor (M-CSF) and interleukin 6 (IL-6) secretion by the murine osteoblastic cell line MC3T3-E1. Interleukin-4 increased colony-forming activity in MC3T3 supernatants two-threefold with colony cytomorphology, cytohistochemistry, and blockade of the effect by anti-M-CSF antibody, indicating that the IL-4-induced activity was M-CSF. MC3T3 M-CSF supernatant activity increased in a time-dependent manner with positive IL-4 effects seen after a 24-hour exposure. The maximal IL-4 effective dose was 100 U/ml where conditioned media from IL-4-treated cells contained twofold more M-CSF than control cells (400 U/ml versus 200 U/ml M-CSF) as detected by a sandwich M-CSF ELISA. Northern blots showed that IL-4 (200 U/ml) rapidly increased steady-state M-CSF mRNA levels with maximal induction observed by 2 hours followed by a decline to near basal levels by 24 hours. IL-4 also dose dependently increased M-CSF mRNA levels with maximal induction (fourfold) seen at 100 U/ml IL-4. In contrast to its impact on MC3T3 M-CSF production, IL-4 (200 U/ml) did not stimulate MC3T3 IL-6 secretion whereas IL-1 (1 pM) stimulated a 500-fold increase in MC3T3 IL-6 release. When utilized to treat newborn calvarial osteoblast-enriched cultures, IL-4 dose dependently augmented M-CSF production, with the maximal effect seen at 200 U/ml where IL-4-treated, osteoblast-conditioned media contained almost 500 U/ml M-CSF, compared with 200 U/ml M-CSF in controlconditioned media. These observations indicate that the range of IL-4 cellular targets in skeletal tissues include osteoblastic cells, and that this cytokine increases osteoblast expression of M-CSF, a hematopoietic cytokine pivotal for monocyte/macrophage differentiation. Furthermore, IL-4's impact on osteoblast-produced M-CSF levels is selective because IL-6 levels were unaltered by IL-4 treatment.     

Administration of interleukin-1 receptor antagonist ameliorates renal ischemia-reperfusion injury.

Interleukin (IL)-1 is a major contributor to inflammation and apoptosis during ischemia/reperfusion (I/R) injury. Its deleterious effects are primarily mediated by the activation of nuclear factor-kappaB (NF-kappaB). Receptor-binding and signaling of IL-1 can be blocked by the IL-1 receptor antagonist (IL-1ra). The aim of our study was to characterize effects and mechanisms of IL-1ra administration on inflammation, apoptosis, and infiltration in renal I/R injury. Renal ischemia was induced in Lewis rats by clamping of the left renal artery for 45 min. Kidneys were removed for histological and molecular analysis 24 h or 5 days after reperfusion. IL-1ra ameliorated I/R induced renal injury and inflammation. Furthermore, the number of apoptotic tubular cells was lower in IL-1ra-treated animals 24 h after ischemia, which was paralleled by a Bax/Bcl-2 mRNA ratio towards anti-apoptotic effects. IL-1ra reduced the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA at 24 h and 5 days and that of intracellular adhesion molecule-1 (ICAM-1) expression at 24 h in the ischemic reperfused kidneys. Our results indicate that IL-1ra treatment ameliorates renal I/R injury and this protective effect might be mediated by reduced induction of NF-kappaB mediated MCP-1, ICAM-1, and a decreased ratio between Bax and Bcl-2 mRNA expression.     

Musosal tumour necrosis factor α in diverticular disease of the colon is overexpressed with disease severity

Aim Inflammation occurs in diverticular disease (DD), but there is little information on inflammatory cytokines such as tumour necrosis factor α (TNF‐α). The aim of this study was to assess TNF‐α expression in DD and to see whether it is related to the severity of the disease. Method Twenty‐four patients with symptomatic DD were divided into those with acute uncomplicated diverticulitis (AUD) (12 patients) and those with symptomatic uncomplicated diverticular disease (SUDD) (12 patients). Twelve further patients with asymptomatic diverticulosis (AD), six with segmental colitis associated with diverticulosis (SCAD), with ulcerative colitis (UC) and six healthy individuals (HC) were enrolled as controls. TNF‐α expression in the colonic mucosa was assessed by the amount of mRNA codifying for the synthesis of TNF‐α. Results TNF‐α expression was significantly higher in AUD than in HC (P = 0.0007), in AD (P = 0.0001) and in SUDD (P = 0.0179). It was significantly higher also in SUDD than in HC (P = 0.0007) and in AD (P = 0.0001). TNF‐α expression in AUD did not differ significantly from that in UC (P = 0.0678) and SCAD (P = 0.0610). It was significantly higher in UC, SCAD and AUD than in SUDD (P = 0.0007, P = 0.0001, P = 0.0179). Conclusion TNF‐α expression in DD seems to be related to the severity of the disease. In particular, it appears to be overexpressed in DD with inflammation (AUD and SUDD) compared with DD without (AD).     

Interleukin-1α and tumour necrosis factor-α modulate the production of monocyte chemoattractant protein-1 by cultured human glomerular visceral epithelial cells

Summary: Mediators released by glomerular macrophages may stimulate glomerular visceral epithelial cells (GVEC) to produce cytokines, growth factors or extracellular matrix components. This study describes that human GVEC produce monocyte chemoattractant protein-1 (MCP-1), a monocyte-specific chemotactic factor, and the effects of interleukin-lα (IL-1α) and tumour necrosis factor-α (TNF-a) on the production of MCP-1 by GVEC.
We observed that the intensity of MCP-1 staining in GVEC is stronger in membranous nephropathy and glomerulosclerosis than in normal kidneys. Various cell lines of GVEC produced significant amounts of MCP-1, as assessed by inhibition radio-immunoassay. the presence of IL-1α and TNF-α during culture of GVEC enhanced the production of MCP-1 in a dose- and time-dependent manner. Glomerular visceral epithelial cells in culture express mRNA for MCP-1 and the expression is upregulated 2.0- and 1.4-fold in the presence of optimal concentration of IL-1α and TNF-α, respectively. De novo synthesis of MCP-1 is supported by the observation that MCP-1 production is fully inhibited by cydoheximide. Monocyte chemoattractant protein-1 isolated from GVEC supernatants exhibits a molecular size of 12 and 10 kDa as determined by gel filtration chromatography. Both sizes of MCP-1 is chemotactically active for monocytes.
This study shows increased MCP-1 production by cultured human GVEC after stimulation with the inflammatory cytokines IL-1α and TNF-α. the expression of MCP-1 in GVEC was found to be upregulated in membranous nephropathy and glomerulosclerosis. These findings suggest that MCP-1 may be involved in glomerular injury in these diseases. the possible role of MCP-1 in the pathogenesis of human glomerulonephritis is discussed.     

Polymeric IgA increases the synthesis of macrophage migration inhibitory factor by human mesangial cells in IgA nephropathy.

BACKGROUND: It has been suggested that polymeric IgA (pIgA) or IgA immune complexes play a significant pathogenic role in IgA nephropathy (IgAN). Macrophage migration inhibitory factor (MIF) shares many activities with other pro-inflammatory cytokines. In human glomerulonephritis, including IgAN, glomerular expression of MIF is found to correlate with progressive renal injury. We hypothesized that deposition of pIgA within the kidney may lead to enhanced synthesis of MIF by mesangial cells. METHODS: In this study we examined the effect of pIgA and monomeric IgA (mIgA) from randomly selected patients with IgAN in clinical quiescence on the gene expression and protein synthesis of MIF in cultured human mesangial cells (HMC). RESULTS: Both pIgA and mIgA from IgAN patients or matched healthy controls increased MIF gene expression and protein synthesis in a dose-dependent fashion. The magnitude of MIF protein induction by pIgA (100 microg/ml) was similar to that of tumour necrosis factor-alpha (TNF-alpha) at 10 pg/ml. In all subjects, the induction of MIF was higher for pIgA when compared with mIgA (P < 0.01). Furthermore, the up-regulation of MIF synthesis by either pIgA or mIgA was significantly higher in IgAN patients than in healthy controls (P < 0.05). Similarly, pIgA and mIgA were able to induce TNF-alpha gene expression and protein synthesis in mesangial cells. Incubation of mesangial cells with neutralizing antibody to TNF-alpha reduced the MIF synthesis induced by pIgA. CONCLUSION: We demonstrate that pIgA is capable of inducing MIF and TNF-alpha production in HMC, which may play a major pathogenic role in IgAN. Induction of MIF can be partially blocked by neutralizing antibody to TNF-alpha, suggesting the possibility that up-regulation of MIF synthesis in HMC is mediated via an amplifying proinflammatory loop involving TNF-alpha.     

TRPC1通道参与调节肾小球系膜细胞的收缩

目的探讨离体和在体情况下TRPC1通道是否参与肾小球系膜细胞的收缩。方法通过计算细胞表面积来观察肾小球系膜细胞的收缩情况。分别利用荧光标记的菊粉和对氨基马尿酸（PAH）的血浆清除率计算大鼠肾小球滤过率（GFR）和肾血浆流量。结果在肾小球系膜细胞的培养液中加入血管紧张素Ⅱ（AngⅡ）10min后，细胞表面积减少（37．2％±4．0％），系膜细胞的收缩反应可因TRPC1基因敲除而受到显著抑制（20．1％±3．0％）。给大鼠注射AngⅡ（1．7ng·min^-1·100g^-1BW）可引起GFR下降，注射TRPC1抗体（300μg／L）显著抑制AngⅡ引起GFR下降（P〈0．05）。但与TRPC1抗体相同浓度的免疫球蛋白，不能抑制AngⅡ引起的GFR下降。另外，TRPC1抗体不影响AngⅡ引起的血压改变和肾血流量的下降。结论本实验表明TRPC1通道在调节肾小球系膜细胞收缩功能方面发挥了重要的作用。     

Ticlopidine inhibits activation of mitogen-activated protein kinase by platelet-derived growth factor in cultured rat renal mesangial cells

Background We previously found that ticlopidine inhibits the proliferation of cultured rat mesangial cells that is induced by fetal bovine serum. This study was designed to examine the effects of ticlopidine on platelet-derived growth factor (PDGF)-induced DNA synthesis and mitogen-activated protein (MAP) kinase activation in such mesangial cells to clarify the mechanism of the antiproliferative action. Methods Glomerular mesangial cells were isolated from rat kidneys, and cells were incubated with various combinations of ticlopidine, PDGF, epidermal growth factor, phorbol 12-acetate 13-myristate (PMA), cilostazol (a phosphodiesterase inhibitor), and H-89 (a cAMP-dependent protein kinase A [PKA] inhibitor). A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell count involving the trypan blue exclusion test were performed for determination of cell viability. DNA synthesis and MAP kinase activity were assessed by means of a tritiated thymidine ([³H]thymidine) incorporation assay and the Biotrak^TM MAP kinase assay system, respectively. Results Ticlopidine (1 μmol/L) significantly inhibited both PDGF-induced DNA synthesis and MAP kinase activation. Also, 1 μmol/L ticlopidine substantially blocked PMA-induced MAP kinase activation. Pretreatment with H-89 did not abolish the ability of ticlopidine to inhibit PDGF-induced MAP kinase activation, while H-89 pretreatment significantly reserved the inhibitory action of cilostazol on PDGF-induced MAP kinase activation. Conclusion These results suggest that ticlopidine might inhibit PDGF-induced DNA synthesis after MAP kinase activation by intercepting the signal transduction from c-Raf-1 to MAP kinase, independent of the cAMP-PKA pathway.