慢性移植物抗宿主病狼疮样小鼠肾组织RANTES表达及泼尼松的调控作用

目的 观察趋化因子RANTES（“regulated upon activation normal T cell expressed and secreted”）在慢性移植抗宿主病（VGHD）狼疮样小鼠肾组织中的表达及泼尼松的调控作用。方法 GVHD狼疮样小鼠模型参照有关文献建立。按随机设计原则将模型动物分两组：即模型组（A组）和泼尼松治疗组（B组）,另设正常对照组（C组）,每组均有8只动物。RANTES的蛋白表达采用免疫组织化学方法,mRNA表达采用原位杂技术检测。结果 ①模型组（A组）肾组织中RANKTES蛋白表达显著高于正常对照组（C组）（P＜0．05）,肾小球和肾小管间质中均有表达,尤以肾小管－间质表达更为丰富;而泼尼松治疗组（C组）RANTES在肾小球和肾小管－间质表达均显著减少（P＜0．05）。②A肾组织中RANTESmRNA表达显著高于C组（P＜0．05）,经泼尼松治疗后RANTESmRNA以到明显抑制。③A组中RANTES蛋白和mRNA表达呈正相关（r＝0．612,PO＜0．05）。结论 慢性GVHD狼疮样小鼠肾组织中RNNTES蛋白和mRNA表达均显著增高,泼尼松在改善肾组织病理改变的同时,对RANTES表达亦起抑制作用,提示RANTES可能参与了狼疮肾炎的发病机制,泼尼松抑制RANTES过度表达可能是其在狼疮肾炎中发挥治疗作用的机制之一。     

慢性移植物抗宿主病狼疮样小鼠肾组织CTGF表达及氟伐他汀的干预作用

目的观察结缔组织生长凶子(CTGF)在慢性移植物抗宿主病(cGVHD)狼疮样小鼠。肾组织中的表达情况及氟伐他汀的调节作用。方法建立cGVHD狼疮样小鼠模型。按随机设计原则将模型小鼠分为3组,即模型对照组(A组)、氟伐他汀干预组(B组)及正常对照组(C组),每组均有6只小鼠。CTGF蛋白检测采用免疫组织化学方法及免疫荧光法．mRNA表达采用反转录-聚合酶链反应(RT-PCR)检测：留取24 h尿,测定尿蛋白排泄量。结果模型对照组小鼠24 h尿蛋门总量明显高于氟伐他汀干预组(P＜0．05)及正常对照组小鼠(P＜0．01)；RT-PCR检测到模型对照组小鼠肾组织CTGF mRNA表达较氟伐他汀干预组小鼠明显升高(P＜0．05),而正常对照组小鼠表达量少；免疫组织化学及免疫荧光表明正常对照组小鼠仅在少数肾小管有微量CTGF表达,而模型对照组及氟伐他汀干预组小鼠肾小管及问质均有CTGF蛋白表达,且模型对照组小鼠表达量显著高于氟伐他汀干预组小鼠(P＜0．01)。结论CTGF可能参与介导狼疮肾炎的发生、发展；cGVHD狼疮样小鼠模型肾组织中CTGF蛋白及mRNA表达明显上调,提示CTGF可能在狼疮肾炎(LN)肾小球硬化及肾间质纤维化的进程中起重要作用；氟伐他汀可能通过降低CTGF表达及减少尿蛋白排泄从而缓解LN,延缓肾纤维化进程。     

狼疮样肾炎鼠白细胞介素-17的表达及其作用研究初探

目的　了解狼疮样肾炎鼠白细胞介素 17(IL 17)的表达及其作用。方法　建立一种狼疮样肾炎动物模型即鼠慢性移植物抗宿主病 (cGVHD)模型 ,用放射免疫法测定尿白蛋白水平 ;用酶联免疫吸附试验 (ELISA)法测定血、尿IL 17水平 ;用免疫组织化学方法检测IL 17蛋白在肾组织的表达 ;用逆转录 聚合酶链反应 (RT PCR)分析肾组织IL 17mRNA的表达。结果　模型鼠组较对照鼠组血IL 17水平不高 ,尿IL 17水平明显升高 [(2 2± 6 7) pg/ml比 (3 1± 2 1)pg/ml];肾小球毛细血管襻、部分系膜细胞、肾小管和间质浸润的单个核细胞均有IL 17蛋白表达 ;肾组织IL 17mRNA水平明显升高 (0 34± 0 15比 0 ) ,且肾组织IL 17+ 细胞数明显增多 [Ng :(2 5± 8)cell/gcs比 0 ;Nt:(2 3± 8)cell/mm2 比 0 ];模型鼠肾组织中IL 17+ 细胞数与尿白蛋白和尿IL 17水平呈明显正相关。结论　IL 17在狼疮样肾炎鼠肾组织表达明显增加 ,并在其发病中可能起重要作用。     

狼疮清颗粒对AHN模型大鼠肾功能和MRL/lpr小鼠肾组织NF-κBp65蛋白表达的影响

目的观察狼疮清颗粒对主动型Heymann肾炎(AHN)模型大鼠肾功能的作用及其对MRL/lpr小鼠肾组织NF-κBp65蛋白表达的影响。方法大鼠肾皮质加弗氏完全佐剂腹腔注射建立AHN大鼠模型,应用狼疮清颗粒、泼尼松、狼疮清颗粒联合泼尼松,对AHN模型大鼠进行干预治疗4 w,检测其24 h尿蛋白、尿素氮(BUN)、肌酐(Cr)及肾脏病理学变化,同时对MRL/lpr小鼠干预治疗4 w,Western印迹检测肾组织NF-κBp65蛋白表达。结果与模型组相比,各治疗组均降低AHN模型大鼠尿蛋白、BUN、Cr的水平,不同程度的改善AHN模型大鼠肾组织病理变化;显著降低MRL/lpr小鼠NF-κBp65蛋白的表达,其中以狼疮清颗粒联合泼尼松组(中西医结合治疗组)最为明显。结论狼疮清颗粒对狼疮性肾炎具有一定的治疗作用,其机制可能与抑制肾组织NF-κBp65蛋白表达有关。     

Lck在狼疮肾炎肾小管上皮细胞中表达的研究

目的 观察狼疮肾炎病理过程中淋巴细胞特异性蛋白酪氨酸激酶（Lck）在肾小管上皮细胞内的表达及白细胞介素-2（IL-2）对其表达的影响。方法 体外培养6周龄BXSB狼疮小鼠的近端肾小管上皮细胞，给予IL-2刺激，采用反转录聚合酶链反应（RT—PCR）方法及免疫印迹法检测肾小管上皮细胞中Lck mRNA和蛋白的表达，观察IL-2对Lck表达水平的影响。同时用免疫组织化学方法观察Lck蛋白在狼疮肾炎小鼠和人类肾脏组织标本肾小管上皮细胞内的表达情况。结果 体外培养的6周龄BXSB狼疮小鼠近端肾小管上皮细胞中仅有微量LckmRNA及蛋白的表达，IL-2刺激后表达水平显著增加（P〈0．05）。免疫组织化学方法显示：正常BALB／C小鼠、未发病的狼疮小鼠和人类微小病变性肾病肾脏组织的肾小管上皮细胞内均未发现明显的Lck蛋白表达，而已发病的16周龄狼疮肾炎小鼠和人类Ⅳ型狼疮肾炎肾脏组织标本的肾小管上皮细胞内则有明显的Lck蛋白表达。结论 IL-2可活化肾小管上皮细胞并诱导Lck表达，Lck可能作为细胞因子或炎症因子的重要信号分子，在小管-间质性炎症及狼疮肾炎病理过程中发挥着重要作用。     

狼疮肾炎患者肾组织白细胞介素-18表达的研究

目的　初步探讨白细胞介素 18(IL 18)在人类狼疮肾炎 (LN)肾组织炎症损伤中的作用。方法　应用免疫组织化学和原位杂交技术观察 6例正常肾组织和 18例LN患者肾组织IL 18的蛋白和基因表达量 ,并用酶联免疫吸附测定 (ELISA)法对该组患者尿IL 18水平进行检测。结果　正常肾组织有弱的IL 18mRNA及蛋白表达 ;LN患者肾组织IL 18mRNA及蛋白表达量均较正常肾组织显著增高 (IL 18mRNA :8 9± 3 4比 2 2± 0 6 ;IL 18蛋白 :8 8± 3 7比 1 4± 0 4 ) ,而且WHOⅣ型LN患者肾组织IL 18表达量显著高于非WHOⅣ型LN患者 (IL 18mRNA :10 8± 3 2比6 6± 1 7;IL 18蛋白 :11 2± 2 7比 5 6± 2 0 ) ;Spearman相关分析提示肾组织IL 18蛋白表达量与狼疮肾炎肾组织活动指数 (AI)、尿IL 18水平及肾组织损伤程度均呈正相关 (P均 <0 0 5 )。结论　肾内IL 18表达上调参与LN肾组织炎症损伤过程 ,尿IL 18水平可能作为衡量LN肾组织炎症活动程度的评估指标。     

血管性血友病因子及P-选择素在狼疮肾炎患者肾组织的表达及意义

目的 探讨血管性血友病因子(vWF)及P-选择素与狼疮肾炎的病理临床关系.方法 应用免疫组织化学方法检测71例狼疮肾炎患者肾活检组织及10份正常对照肾组织中vWF及P-选择素的表达,结果与临床病理指标进行综合分析.采用单因素方差分析,Nemenyi秩和检验,Pearson相关分析进行统计学处理.结果 vWF在正常对照组几乎无表达;狼疮肾炎组肾小球系膜基质,肾小管及间质血管内膜均可见vWF的阳性表达,以Ⅳ型表达最明显(0.30±0.10),与Ⅱ、Ⅲ型(0.12±0.05,0.22±0.14)相比,差异有统计学意义(x2=9.273.P＜0.05).正常对照组仅有极少量P-选择素表达;狼疮肾炎组肾小球系膜区、肾小管和肾间质中均有P-选择素的广泛表达,以Ⅳ、Ⅴ型显著(0.29±0.05.0.36±0.10),与Ⅱ、Ⅲ型(0.27±0.09,0.29±0.05)相比差异有统计学意义(P＜0.05).肾组织中vWF的表达水平与肾组织活动指数、尿蛋白定量(24 h)呈正相关(r分别为0,403,0.332,P均＜0.05);肾组织中P-选择素的表达水平与肾组织活动指数、尿蛋白定量(24h)、系统性红斑狼疮疾病活动度(SLEDAI)呈正相关(r分别为0283,0.453,0.297,P均＜0.05);肾组织中vWF与P-选择素的表达呈明显正相关(r=0.371,P=0.001).结论 vWF及P-选择素参与狼疮肾炎病理发展过程,联合检测vWF、P-选择素有助于对狼疮肾炎严重程度的评估,进而为临床治疗提供依据.     

单核细胞趋化蛋白-1在BXSB狼疮小鼠肾组织中表达及药物干预效应

目的研究单核细胞趋化蛋白-1(MCP-1)在BXSB狼疮小鼠肾组织中的表达情况并探讨甲泼尼龙(MPS)是否可抑制MCP-1表达而缓解狼疮肾炎(LN)。方法应用双缩脲法、反转录聚合酶链反应(RT-PCR)、免疫组织化学法及医学图像分析系统检测各组小鼠(BXSB狼疮小鼠MPS治疗组和对照组、BALB/C正常对照组小鼠)24h尿蛋白、肾组织MCP-1的表达,并分析其与24h尿蛋白之间的相关性。结果BXSB狼疮小鼠肾组织MCP-1表达较正常BALB/C小鼠增高,以肾小管更为明显,且肾小管MCP-1的表达与24h尿蛋白呈正相关(P<0.01);甲泼尼龙干预后,MCP-1表达明显减弱(P<0.01),且肾小管MCP-1表达的下降与24h尿蛋白降低呈正相关(P<0.01)。结论MCP-1可能参与介导BXSB狼疮小鼠LN的发生发展;甲泼尼龙可通过抑制肾组织MCP-1的表达,减轻蛋白尿,可能是缓解LN作用机制之一。     

慢性移植物抗宿主病狼疮样肾炎小鼠巨噬细胞移动抑制因子的表达及作用

目的：观察巨噬细胞移动抑制因子（MIF）在实验性狼疮样肾炎小鼠肾组织中的表达及作用。方法：建立移植抗宿主病（GVHD）狼疮样肾炎小鼠动物模型，用原位杂交及免疫组织化学染色法，观察MIF mRNA及蛋白质在狼疮样肾炎小鼠组织中的表达，及其与肾组织中的表达，及其与肾组织病理变化、肾功能损害的可能关系。结果：随着狼疮肾炎的发展，MIF在肾组织中的表达增多；MIF的表达与狼疮肾炎组织的活动性病变有明显的相关关系，与肾脏病理及肾功能损害有一定的相关关系。结论：MIF在介导GVHD狼疮肾炎病损中起重要作用，其表达增多可反映狼疮肾炎活动性病变和肾脏损害。     

Bcl-2、Bax在狼疮肾炎患者肾组织中的表达及意义

目的研究细胞凋亡调节蛋白(Bcl-2及Bax)在狼疮肾炎(LN)患者肾组织的表达情况及与LN肾脏病理改变的关系。方法2003-012003-04对广西医科大学第一附属医院的44例活动期LN患者用免疫组化方法检测肾组织中Bcl-2、Bax的表达,观察它们与LN肾脏病理改变的关系。结果(1)LN肾小球Bcl-2及Bax表达均增高,与肾小球细胞增殖程度及增殖细胞核抗原(PCNA)阳性细胞数呈正相关。(2)LN肾间质Bcl-2表达增高,与间质炎症细胞浸润程度正相关。(3)LN肾小管Bax表达增高,与肾小管间质病变呈正相关。结论LN肾组织存在Bcl-2、Bax蛋白异常表达。Bcl-2高表达在LN肾小球细胞增殖及肾间质炎症细胞浸润过程中可能起重要作用。Bax在肾小管过度表达与肾小管间质病变有关。     

Chronic predominant interstitial nephritis in a patient with systemic lupus erythematosus: a follow up of three years and review of the literature.

Predominant interstitial nephritis is a rare manifestation of systemic lupus erythematosus. Only seven cases have been reported in the literature. Owing to the rarity of this entity, the natural history of predominant interstitial nephritis in lupus has not been adequately recorded and an appropriate therapeutic approach has yet to be defined. In this report we present the case of a 25 year old woman with active systemic lupus erythematosus complicated by kidney failure and renal tubular acidosis due to predominant interstitial nephritis. We describe the course of her disease over a three year period. Seven additional patients with systemic lupus erythematosus and predominant interstitial nephritis are reviewed.     

Anti-macrophage-derived chemokine antibody relieves murine lupus nephritis

We observed the distribution of monocyte’s changes in damage to renal tissue and the expression of macrophage-derived chemokine (MDC) in MRL/lpr mice. The 8-week-old MRL/lpr mice were randomly assigned to receive either MDC antibodies (n = 6) or placebo (n = 6) at a once-every-other-day dose of 300 μl from week 4. We then quantified the differences in 24-h urine protein and serum creatinine concentrations, performed a histological evaluation of renal tissue and assessed the expression of MDC protein and mRNA between the two groups 4 weeks after treatment was initiated. Antibody-treated mice demonstrated significantly lower urine protein and serum creatinine concentrations and had fewer renal lesions compared with control mice. The expression of MDC protein and mRNA in renal tissues was significantly lower in the antibody-treated mice than in control mice, suggesting that the elevated expression of MDC, which led to monocyte infiltration in the kidney, may play an important role in the development of lupus nephritis. Furthermore, the anti-MDC antibodies may act to alleviate the renal lesions of murine lupus nephritis by inhibiting the infiltration of monocytes in the renal tissue of the lupus mice.     

Association of monocyte chemoattractant protein-1 with renal tubular damage in diabetic nephropathy

Monocyte chemoattractant protein-1 (MCP-1), is a chemokine that mediates renal interstitial inflammation, tubular atrophy, and interstitial fibrosis by recruiting monocytes/macrophages into renal tubulointerstitium. Recent studies have demonstrated that protein overload in renal tubular cells up-regulates MCP-1 gene and its protein expression. Therefore, we hypothesized that increased expression of MCP-1 in renal tubuli, probably triggered by an increase in the leakage of plasma protein from glomerular capillary to tubular fluid, may contribute to renal tubular damage and accelerate the progression of diabetic nephropathy. To test this hypothesis, we examined urinary excretion levels of MCP-1 and N-acetylglucosaminidase (NAG), a sensitive marker of renal tubular damage, in Japanese Type II diabetic patients with normoalbuminuria (n=29), microalbuminuria (n=25), and macroalbuminuria (n=18). The median urinary excretion level of MCP-1 in patients with macroalbuminuria (394.4 ng/g creatinine) was significantly elevated compared to the levels in patients with normoalbuminuria and microalbuminuria (159.6 and 193.9 ng/g creatinine, respectively). Furthermore, the urinary MCP-1 excretion level was positively correlated with urinary excretion levels of albumin (r=.816, P<.001) and NAG (r=.569, P<.001) in all subjects. These results suggest that MCP-1 is produced in renal tubular cells and released into urine in proportion to the degree of proteinuria (albuminuria), and that increased MCP-1 expression in renal tubuli contributes to renal tubular damage. Therefore, we conclude that heavy proteinuria itself may accelerate the progression of diabetic nephropathy by increasing the MCP-1 expression in renal tubuli.     

Serum level and urinary excretion of RANTES in patients with primary glomerulonephritis

Proteinuria plays a central role in the progression of glomerular disease, and there is growing evidence suggesting that it may determine tubular cell activation with release of proinflammatory chemokines and fibrogenic factors, leading to interstitial inflammatory reaction. Chemokines are proteins that contribute to the migration of leukocytes to sites of tissue injury. C-C chemokine receptor 5 is receptor for the C-C chemokine RANTES, which is expressed in inflammatory kidney diseases. To better understand the role of RANTES in various types of human glomerular diseases, we studied 53 patients with primary glomerular diseases (5 minimal change--MC; 4 focal glomerulosclerosis--FS; 4 membranous nephropathy--MN; 12--mesangial proliferative GN--MesPGN; 18 IgA nephropathy--IgAN; 6 membranoproliferative GN-MPGN, and 4 extracapillary GN-ExGN) and 10 healthy person. Renal biopsies were evaluated by light and immunofluorescence microscopy. RANTES concentrations in serum and urine were measured by ELISA (BIOSOURCE international kits). The treatment of patients consisted of 3 to 5 i.v. methylprednisolone pulses (1.0 g per single dose, average total 1.0 g/20 kg given alternate days) followed by oral prednisone 20 to 25 mg/day and six monthly i.v. cyclophosphamide 0.6 g/l m2/month. The study groups (except FS) showed a significantly higher concentration of RANTES in their sera compared with the control. The increase of urinary excretion for RANTES was 2-fold in patients with MN, and 8-fold in patients with ExGN but in patients with FS a significant decrease in urinary RANTES excretion was found. There was no significant differences in the urinary excretion of RANTES in other groups compared to a control group. In patient groups serum Cr showed significant correlations with interstitial volume in renal biopsy. No significant correlation was found between serum concentration of RANTES and their urinary excretion and other parameters considered (serum creatinine, urinary protein, serum protein concentration, and interstitial volume in renal biopsy). In patients with renal insufficiency (Cr > 1.3 mg%) and reduction of proteinuria > 50% after 1 year of treatment, the serum concentration and urinary secretion of RANTES was higher before treatment than in patients with protein reduction < 50%, and in patients with renal sufficiency. These results showed that patients with glomerular diseases who showed renal insufficiency and reduction of urinary protein after 1 year of immunosuppressive treatment revealed high levels of serum and urinary excretion of RANTES. It was thus suggested that the measurement of serum and urinary excretion of RANTES is useful in evaluating the degree of renal injury in patients with glomerular diseases after immunosuppressive treatment.     

Renal interstitial cells, proteinuria and progression of lupus nephritis: new frontiers for old factors

Interstitial cells, inflammatory-immune cells, tubular cells and endothelial cells of the peritubular capillaries have arisen as possible major players of the nephron damage in lupus nephritis. Increased ICAM-1, Von Willebrand factor, soluble endothelial protein C receptors and decreased ADAMS-13 point to a diffuse vascular damage. Albuminuria elicits a rapid generation of hydrogen peroxide in proximal tubular cells along with nuclear factor-kB activation, endothelin-1 and transforming growth factor (TGF-beta1) upregulation. TGF-beta1 enhances epithelial-to-mesenchymal transdifferentiation. Albuminuria also enhances the expression of macrophage chemotactic protein-1 and macrophage inflammatory protein-1alpha, thus leading to increased interstitial inflammation. TGF-beta1 and thrombospondin-1, a putative activator of TGF-beta, induce apoptosis of peritubular capillaries, as well as of glomerular endothelial cells. All these events can be counteracted by hepatocyte growth factor (HGF), which is expressed by the epithelial tubular cells and stimulates the growth of epithelial cells (mitogen), enhances the motility of epithelial cells (motogen), induces renal epithelial tubule regeneration (morphogen) and enhances angiogenesis (angiogen). The balance between TGF-beta1 and HGF could be a key to define the prognostic value of kidney histopathology at baseline and during follow-up, in lupus nephritis. Therapeutic strategies aiming at altering the biological balance in the patients are at hand to test and prove the experimental evidences.