p38 MAPK phosphorylation and NF-kappa B activation in human crescentic glomerulonephritis.

BACKGROUND: p38 mitogen-activated protein kinase (p38 MAPK) followed by the activation of NF-kappa B participates in the intracellular signal transduction and production of cytokines and chemokines. The pathophysiological roles of p38 MAPK and NF-kappa B in human glomerulonephritis, however, remain to be investigated. METHODS: We investigated the phosphorylated p38 MAPK (p-p38 MAPK) and activated NF-kappa B immunohistochemically in the kidneys of 34 patients with crescentic glomerulonephritis and 26 control patients with thin basement membrane disease and minimal change nephrotic syndrome. We also explored the co-localization of p-p38 MAPK with CCR5, the signal of which leads to p38 MAPK activation. Furthermore, urinary levels of MIP-1 alpha, the cognate ligand for CCR5, were determined by enzyme-linked immunosorbent assay. RESULTS: p-p38 MAPK-positive cells and activated NF-kappa B-positive cells were mainly detected in crescentic lesions, tubular epithelial cells, and interstitial mononuclear infiltrates. The number of p-p38 MAPK-positive cells in patients with crescentic glomerulonephritis was higher than that in control patients. The number of p-p38 MAPK-positive cells in glomeruli was well correlated with the percentage of cellular crescents, the number of CD68-positive cells, and urinary MIP-1 alpha levels. In addition, the number of activated NF-kappa B-positive cells was well correlated with the number of p-p38 MAPK-positive cells in glomeruli. Dual staining revealed that most of CCR5-positive cells were positive for p-p38 MAPK. Finally, p-p38 MAPK-positive cells and activated NF-kappa B-positive cells decreased during glucocorticoid therapy-induced convalescence. CONCLUSIONS: We conclude that the phosphorylation of p38 MAPK associated with the activation of NF-kappa B may be involved in the upregulation of intrarenal MIP-1 alpha and the utilization of CCR5 signalling, which may result in human crescentic glomerulonephritis.     

Blockade of p38alpha MAPK ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis

The p38 mitogen-activated protein kinase (MAPK) pathway is a pro-inflammatory signal transduction pathway. The aim of this study was to examine the role of this pathway in acute renal inflammation. Immunostaining localized components of the p38 MAPK pathway (p38alpha, p-p38, p-ATF-2) in normal glomeruli, to podocytes, and occasional endothelial cells. This study identified an eightfold increase in glomerular activation of p38 MAPK (phosphorylated p38, p-p38) within 3 h of the induction of rat anti-glomerular basement membrane (GBM) glomerulonephritis and localized p-p38 and p-ATF-2 to infiltrating neutrophils, with increased staining of podocytes and endothelial cells. The relevance of these findings to human acute inflammatory renal disease was determined by examination of biopsy specimens. In patients with post-infectious glomerulonephritis, there was an increased number of positive p-p38 glomerular cells, including p-p38 staining of infiltrating neutrophils, compared with normal human kidney. In rats, administration of a specific p38 MAPK inhibitor, NPC 31145, before induction of anti-GBM disease prevented a loss of renal function and substantially reduced proteinuria. The reduction in renal injury was attributed to a 55% reduction in glomerular neutrophil infiltration and a 68% reduction in platelet accumulation. This was associated with an abrogation of glomerular P-selectin immunostaining and inhibition of glomerular P-selectin gene expression. In summary, this study has localized the components of the p38 MAPK pathway to cells in normal and diseased rat and human kidney and identified a number of important mechanisms by which signaling through the p38 MAPK pathway induces inflammatory renal disease. Blockade of the p38 pathway may be a novel therapeutic strategy for the treatment of acute renal inflammation.     

大鼠新月体肾炎中的细胞增殖与凋亡

目的探讨大鼠新月体肾炎时肾脏细胞凋亡与细胞增殖。方法分别用免疫组化（ＰＣＮＡ）和原位末端标记法检测增殖和凋亡的细胞。结果浸润肾脏的巨噬细胞及肾脏固有细胞，包括肾小管上皮细胞均发生凋亡，在细胞性、细胞－纤维素性新月体中及肾小管间质损害严重处凋亡明显，凋亡的肾小管上皮细胞数与肾小管病理损害程度高度相关。结论大鼠新月体肾炎时肾脏巨噬细胞和固有细胞均发生凋亡，凋亡可能在进行性肾损害中起着重要作用     

Activation of p38MAPK signaling cascade in a VSMC injury model: role of p38MAPK inhibitors in limiting VSMC proliferation.

INTRODUCTION: P38 mitogen-activated protein kinase (MAPK) has a crucial role in regulating signaling pathways implicated in the cellular events leading to restenosis. We examine p38MAPK activation in response to vascular cell injury, its biological effects and determine whether selective p38MAPK inhibitors, SB220025/SB203580, decrease vascular smooth muscle cell (VSMC) proliferation. METHODS: Human aortic VSMCs were cultured and wounds made on the monolayers to elicit mitogenic responses and induce p38MAPK activation. P38MAPK inhibitor pretreatment, at varying doses (1-100 microM) and treatment duration was used to block p38MAPK phosphorylation. Cytotoxicity, viability, proliferation and apoptosis were determined and expression of p38MAPK/phospho-p38MAPK was obtained by chemiluminiscent immunoblot analysis. RESULTS: Phosphorylation of p38MAPK depended on injury severity and was inhibited by both p38MAPK inhibitors, but not by SB202474, a specific antagonist of p38MAPK inhibitors. VSMCs treated with p38MAPK inhibitors showed a dose-dependent decrease in viable cell number, apoptosis and proliferation, reversing the deleterious effects of p38MAPK activation comparable to controls (p < 0.05). CONCLUSIONS: This wound injury model activates the p38MAPK-signaling cascade in VSMC and causes cell proliferation that can be abrogated by pre-incubation with p38MAPK selective synthetic inhibitors in a time and dose-dependent manner. SB220025 used here for the first time in VSMC reveals itself to be a stronger p38MAPK inhibitor than SB203580 and being a second generation inhibitor may be the preferred drug for novel therapeutic maneuvers.     

血管内皮细胞中NF-κB和p38MAPK的激活

高糖在血管内皮细胞中引起氧化应激将激活细胞内一系列应激激活的信号通路，包括NF-κB、p38MAPK等，主要结果是以不同基因表达产物引起内皮细胞功能紊乱，在糖尿病慢性血管并发症的病因学中起重要作用。近年来有许多研究报道了NF-κB和p38MAPK信息通路在高糖引起的内皮细胞凋亡中的作用，为防治糖尿病血管并发症提出了新的靶向治疗策略。     

JNK、p38 MAPK在移植静脉血管重塑过程中的表达研究

目的　研究c Jun氨基末端激酶 (JNK)和p38蛋白激酶 (MAPK)在移植静脉血管重塑过程中的表达。方法　Wistar大鼠 80只 ,建立自体移植静脉模型 ,术后随机分为 6h ,1、3、7,1 4、2 8、4 2、5 6d等 8组 ,于相应时点取材 ,逆转录聚合酶链反应 (RT PCR)检测JNK和p38MAPK的mRNA表达 ,Western蛋白印迹检测JNK和p38的蛋白产物及磷酸化蛋白产物表达 ,原位杂交和免疫组化方法定位mRNA及蛋白产物表达 ,脱氧核苷酸转移酶末端标记法 (TUNEL)检测血管平滑肌细胞 (VSMC)凋亡的变化。结果　移植静脉术后 6h ,JNK和p38的mRNA表达增强 ,在术后 1 4d达到高峰 ,表达值分别为(2 6± 1 0 ) %和 (5 9± 2 6 ) %,与各时点比较差异有统计学意义 (P <0. 0 1 )。JNK、p38的蛋白产物表达在1 4～ 2 8d达高峰 ,在 5 6d时仍维持一定表达量 (1 .4～ 1 . 2 )。原位杂交及免疫组化提示阳性表达多位于移植血管中层或增生内膜中的血管平滑肌细胞 (VSMC) ,p38与凋亡呈正相关 (r =0 . 892 2 ,P <0. 0 1 )。结论　JNK和p38MAPK通路的激活是移植静脉内膜增生以及血管重塑的关键环节 ,可能成为新的治疗靶点。     

The mechanism of TDP‐43 gene expression on inflammatory factors and the JNK and p38 MAPK signalling pathways in ischaemic hypoxic stress dependence

In this study, the mechanism of TDP‐43 gene expression on inflammatory factors and Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways in ischaemic hypoxic stress dependence was investigated. Sixty SD rats were selected and divided into the control group, the osteoarthritis (OA) model group, and the TDP‐43‐mMSCs+OA group. In the OA model group and the TDP‐43‐mMSCs+OA group, OA was established by collagenase injection. Western blotting assays were used to detect the expression of TDP‐43 in cartilage tissues of each rat. The secretion of tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in the serum of rats was determined by enzyme‐linked immunosorbent assay (ELISA). The formation of cytoplasmic stress granules (SGs) and the expression of receptor for activated c‐kinase 1 (RACK1) were detected by Western blotting assays in each group of rats. The expression of MTK1 and MAPKKK phosphorylation and changes in the JNK and p38 MAPK signalling pathways were detected by Western blotting assays. Compared with the control group, the expression of TDP‐43 in the cartilage tissue of rats in the OA model group was significantly decreased. The expression of TDP‐43 in the cartilage tissue of rats in the TDP‐43‐mMSCs+OA group was significantly higher than that of the control group and the OA model group, which indicates that TDP‐43‐mMSC transplantation was successful. Enzyme‐linked immunosorbent assay results showed that the plasma TNF‐α and IL‐1β levels in the OA model group were significantly increased (P < 0.01) when compared with the control group. However, the secretion of TNF‐α and IL‐1β in the serum of the TDP‐43‐mMSCs+OA group was significantly lower than that of the model group (P < 0.01) but still higher than the control group. This indicates that overexpression of TDP‐43 reduces the inflammatory response induced by OA. Western blotting assays showed that the amount of cytoplasmic SGs in the cartilage tissue of rats in the OA model group was significantly decreased when compared with the control group. The amount of SGs in the cartilage of rats in the TDP‐43‐mMSCs+OA group was significantly higher than that of the model group. The expression of RACK1 in the cartilage tissue of rats in the OA model group was significantly higher than that of the control group. Overexpression of the TDP‐43 gene can interfere with the secretion of inflammatory factors and inhibit the activation of the JNK and p38 MAPK signalling pathways by ischaemic hypoxia stress. Thus, the molecular mechanism of chondrocytopathic lesions was reversed, which provided a new theoretical basis for the treatment of OA.     

Activation of fibroblastic reticular cells in kidney lymph node during crescentic glomerulonephritis

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p38丝裂原活化蛋白激酶在大鼠慢性非细菌性前列腺炎模型中的表达

目的探讨p38丝裂原活化蛋白激酶(p38 MAPK)在慢性非细菌性前列腺炎(CNP)大鼠模型中的表达,与细胞因子肿瘤坏死因子-α(TNF-α)、环氧合酶-2(COX-2)、基质金属蛋白酶-9 (MMP-9)的关系及它们在发病机制中可能的作用。方法将成年雄性SD大鼠去势后皮下注射苯甲酸雌二醇连续4周建模,对照组不做任何处理。HE染色,光镜下观察两组前列腺组织的病理学表现,免疫组化检测两组TNF-α、MMP-9、COX-2、磷酸化p38(p-p38)蛋白的表达,逆转录-聚合酶链反应(RT-PCR)检测p38 mRNA的水平变化。结果光镜下对照组前列腺组织无炎症反应,模型组表现为明显的炎症反应。对照组前列腺组织TNF-α、MMP-9、COX-2、p-p38蛋白表达量极少,而模型组明显增加,两组差异有统计学意义(P<0.05)。对照组p38 mRNA的表达为模型组的37.3%。结论在大鼠CNP中p38 MAPK信号转导途径被激活。细胞因子TNF-α、MMP-9、COX-2的表达可能是通过p38 MAPK调节。这为CNP的研究和治疗提供了一条新的途径。     

Intrarenal localization of interleukin-1{beta} mRNA in crescentic glomerulonephritis

II-1ß is a potent proinflammatory peptide, and inducesexpression of other cytokines which are involved in the immuneresponse. Kidney biopsies from nine patients with crescenticGN were studied by in-situ hybridization to determine the siteof expression of II-1ß mRNA. Biopsies from nine patientswith non-proliferative renal disease were studied as negativecontrols, and tonsillar tissue was studied as a positive control.An II-1ß cDNA probe was ³²P-labelled by random primersand hybridized to paraffin-embedded tissue sections after de-waxing.II-1ß mRNA was expressed in tonsil, but not in negativecontrols. Positive mRNA expression was seen in four of the ninecrescentic biopsies. This was observed in interstitial cellswith morphological characteristics of macrophages adjacent totubular cells, in cells within the glomerular tuft, and in tubularepithelial cells. II-1ß mRNA is expressed in renaltissue in crescentic GN. Tubular and interstitial expressionof II-1ß mRNA appears of equal prominence to glomerularexpression. Intrarenal cytokine synthesis may be involved inthe pathogenesis of crescentic glomerulonephritis.     

脊髓背角p38MAPK活化在大鼠神经病理性痛形成中的作用

目的观察大鼠慢性压迫性损伤（CCI）术后脊髓背角p38丝裂原激活蛋白激酶（p38 MAPK）活化水平的变化,探讨p38MAPK在神经病理性痛形成中的作用。方法SD大鼠70只,随机分为3组：对照组（n=14）、假手术组（n=14）和CCI组（n=42）。结扎大鼠左侧坐骨神经建立CCI模型,通过von Frey纤维测定大鼠神经结扎侧后足缩足反射阈值（MWT）。对照组和假手术组于术后3 d,CCI组于术后3、7、14 d各随机处死8只大鼠,取脊髓背角,采用免疫组化法计数总p38MAPK（t-p38MAPK）和磷酸化p38MAPK（p-p38MAPK）阳性细胞和总细胞,计算阳性细胞率。对照组和假手术组于术后3 d,CCI组于术后3、7、14 d各处死6只大鼠,取脊髓背角,采用Western blot法测定t-p38MAPK和p- p38MAPK蛋白表达水平。结果与对照组和假手术组比较,CCI组各时点MWT降低（P〈0.05或0.01）,t-p38MAPK阳性细胞率和蛋白表达水平差异无统计学意义（P〉0.05）,p-p38MAPK阳性细胞率和蛋白表达水平升高（P〈0.05）。结论外周神经损伤后,脊髓背角p38MAPK活化水平增加,p38MAPK通路激活,可能参与了大鼠神经病理性痛的形成。     

紫云英苷对膝关节骨性关节炎大鼠JNK/p38MAPK信号通路的影响

目的 探究紫云英苷对膝关节骨性关节炎（KOA）大鼠的保护作用以及对JNK/p38MAPK信号通路的影响。方法 90只SD大鼠随机分为假手术组、模型组、塞来昔布组（18 mg/kg）、紫云英苷低（25 mg/kg）、中（50 mg/kg）、高（100 mg/kg）剂量组,每组15只。改良Huith法建立KOA模型,术后灌胃6周。游标卡尺测量大鼠膝关节直径,足趾容积仪测量大鼠足趾容积;ELISA法检测血清IL-1β、IL-6、TNF-α水平;HE染色观察膝关节软骨组织病理变化;ELISA法检测软骨组织TGF-β1、MMP-3水平;Western Blot法检测p-JNK、JNK、p-p38MAPK、p38MAPK蛋白表达。结果 与假手术组比较,模型组大鼠软骨组织表面被破坏,组织层变薄,细胞数量减少且排列紊乱,潮线被大量破坏;膝关节直径与足趾容积明显变大（P＜0.05）;血清中IL-1β、IL-6、TNF-α水平以及软骨组织中TGF-β1、MMP-3水平,p-JNK/JNK、p-38MAPK/p38MAPK比值显著升高（P＜0.05）。与模型组比较,塞来昔布组与紫云英苷各剂量组软骨组织病变明显减轻;膝关节直径与足趾容积明显缩小（P＜0.05）;血清中IL-1β、IL-6、TNF-α水平以及软骨组织中TGF-β1、MMP-3水平,p-JNK/JNK、p-38MAPK/p38MAPK比值显著降低（P＜0.05）,且紫云英苷各剂量组间具有剂量效应（P＜0.05）;紫云英苷高剂量组与塞来昔布组比较差异无统计学意义（P＞0.05）。结论 紫云英苷可能通过抑制JNK/p38MAPK信号通路激活,减轻炎症反应,改善KOA大鼠膝关节软骨组织损伤。     

Myofibroblasts and the progression of crescentic glomerulonephritis

Background: The cellular and humoral factors involved in the pathogenesis of glomerulosclerosis and renal fibrosis following a crescentic glomerulonephritis have not been fully elucidated. Myofibroblasts and transforming growth factor-{beta} (TGF-{beta}) have been implicated in the development of experimental and clinical renal fibrosis. We have attempted to identify these mediators in crescentic glomerulonephritis and determine their role in the progression of the disease. Patients and methods. We have studied retrospectively 21 patients with crescentic and necrotizing glomerulonephritis (CNG) with emphasis on the renal expression (detected by immunohistochemistry) of myofibroblasts (&agr;-smooth muscle actin⁺ cells), TGF-{beta} and collagen (III and IV) as well as their relationship with the clinical outcome of these patients. In situ hybridization histochemistry was applied to determine the site of synthesis of TGF-{beta}1 and collagen III. All the patients were treated by immunosuppression and followed up for a median period of 14 months. Results: Myofibroblasts and TGF-{beta} were detected in the crescents as well as in the periglomerular and tubulointerstitial areas in CNG biopsies. TGF-{beta}1 was also detected within renal tubular cells. The percentage of glomeruli with fibrotic and fibrocellular crescents was positively correlated with the severity of Bowman's capsule disruption (r-0.631, P<0.01) and with the intensity of myofibroblast expression in the interstitium (r-0.504, P<0.05). Strong interstitial immunostain for myofibroblasts and TGF-{beta} was also noted in association with interstitial fibrosis. In situ hybridization revealed the site of synthesis of TGF-{beta}1 to be the renal tubular cells of patients with CNG. By contrast, the site of synthesis of collagen III appeared to be confined to interstitial cells surrounding vessels, tubules and the glomeruli in a distribution identical to that of myofibroblasts. There was a significant positive correlation between the number of interstitial &agr;-SMA⁺ cells and both interstitial TGF-{beta} (r=0.591, P<0.01) and interstitial collagen IV (r=0.588, P<0.01). In addition, the number of interstitial &agr;-SMA⁺ cells and the extent of immunostain for collagen IV were positively correlated with the final serum creatinine (r=0.517, P<0.05 and r=0.612, P<0.01 respectively) and partially predicted functional outcome (R²=26.7% and 37.5% respectively) as well as the response to treatment. An association was observed between periglomerular myofibroblasts and the generation of fibrotin and fibrocellular crescents. Conclusion: These observations suggest a causal link between myofibroblasts and fibrotic crescent formation. We also believe that interstitial myofibroblasts are actively involved in the pathogenesis of interstitial fibrosis in CNG.     

Preexisting crescentic glomerulonephritis in the renal allograft

Two patients who received cadaveric renal transplants from the same donor were found to have similar diffuse proliferative glomerular lesions on renal biopsies performed for delayed graft function. The donor had no known disease, but had subtle abnormalities on preprocurement urinalysis. In retrospect, lupus nephritis is suspected in the donor. The authors detail the course of these 2 patients, review the literature on preexisting glomerular lesions, and address issues with regard to donor selection.