CD2AP在大鼠肾病模型中的表达及意义

目的：观察CD2-associated protein(CD2AP)在大鼠氨基核苷肾病模型。肾小球中的表达变化及其意义。方法：通过建立大鼠氨基核苷肾病模型，采用免疫组织化学技术观察CD2AP在不同时间点。肾病模型大鼠。肾小球中的表达和分布变化。结果：①。肾小球足细胞CD2AP的表达在。肾病模型建立的早期即有下调；在大鼠肾病模型蛋白尿的高峰期，CD2AP的表达明显下降；在肾病模型大鼠的疾病恢复期，CD2AP的表达逐步恢复正常；②肾小球足细胞CD2AP表达量的变化与肾病模型大鼠24h尿蛋白定量的变化存在着负相关。结论：①CD2AP是判断。肾小球足细胞损伤的一个早期重要指标；②CD2AP在足细胞中表达量的改变可能是肾小球滤过屏障功能异常的病理生理基础。     

Reduced podocyte expression of alpha3beta1 integrins and podocyte depletion in patients with primary focal segmental glomerulosclerosis and chronic PAN-treated rats

Integrins attach cells to extracellular matrix (ECM) and mediate signals from ECM to cells or from cells to ECM. They regulate cell functions, including adhesion, migration, cell cycle regulation, and differentiation. Podocytes may detach from the glomerular basement membrane (GBM) and be excreted in the urine, and proteinuria is found in patients with primary focal segmental glomerulosclerosis (FSGS); both may be associated with loss of alpha3beta1integrins. In this study, we have examined the podocyte number in patients with primary FSGS and normal controls, and the alpha3- and beta1-integrin subunits expression of podocytes in patients with primary FSGS and chronic puromycin aminonucleoside (PAN)-treated rats by the morphometric, immunoperoxidase histochemical, and immunoelectron microscopic examination. We also measured their expression serially in rats that received repeated PAN injection. The results showed that the podocyte number was significantly decreased in patients with primary FSGS than in normal control (P < 0.05). The immunostaining score showed that both alpha3- and beta1-integrin subunits on podocytes in patients with primary FSGS were significantly lower than in normal controls (both P < 0.01). The number of immuno-gold particles of alpha3- and beta1-integrins at the effaced foot process area of patients with primary FSGS were also significantly decreased than that of normal controls (both P < 0.05). The immunostaining score of both alpha3- and beta1-integrin subunits was negatively correlated with the degree of glomerular sclerosing score and the amount of daily protein loss, and they were positively correlated with the number of podocytes. Chronic 12-week PAN-treated rats showed similar findings with decreased immunostaining expression and immuno-gold particles of alpha3-integrin on podocytes than in normal control (both P < 0.05). The chronic PAN-treated rats also showed a trend toward gradually decreased immunostaining expression of alpha3-integrin subunit on podocyte during the progress from normal to FSGS state. These studies indicate that podocyte expression of alpha3- and beta1-integrin subunits is significantly reduced in humans with primary FSGS and chronic PAN-treated rats, before the morphological changes of FSGS are observed. The decreased podocyte expression of alpha3beta1 integrins is closely related with podocyte depletion, glomerular sclerosis, and daily protein loss in patients with primary FSGS.     

Involvement of puromycin‐sensitive aminopeptidase in proteolysis of tau protein in cultured cells,and attenuated proteolysis of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) mutant tau

In tauopathies, tau protein is hyperphosphorylated, ubiquitinated, and accumulated in the brain; however, the mechanisms underlying this accumulation remain unclear. To gain an understanding of the role of proteases in the metabolism of tau protein, in the present study we evaluated the effects of protease inhibitors in SH‐SY5Y human neuroblastoma cells and COS‐7 cells transfected with the tau gene. When cells were treated with 0.1–10 µmol/L of lactacystin and 1.0–20 µmol/L of MG‐132 (inhibitors of proteasome), 0.1–10 µmol/L of CA‐074Me (a cathepsin inhibitor), and 0.1–2 µmol/L of puromycin (a puromycin‐sensitive aminopeptidase (PSA) inhibitor) for up to 24 h, there were no significant changes in tau protein levels. However, pulse‐chase experiments demonstrated that the proteolysis of tau protein in SH‐SY5Y cells was attenuated following treatment of cells with 200 nmol/L puromycin. Increased tau protein levels were also observed in SH‐SY5Y cells treated with short interference (si) RNA to PSA to inhibit the expression of PSA. These data suggest that PSA is a protease that catalyses tau protein predominantly in SH‐SY5Y cells. The protein metabolism of tau‐containing mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17) was also investigated using pulse‐chase experiments. The results indicate attenuated proteolysis of tau in cells transfected with mutant tau genes after 48 h. Further immunocytochemical analysis and subcellular fractionation experiments revealed that the mutations did not alter the intracellular distribution of tau and suggested that impaired accessibility of tau to PSA is unlikely to account for the attenuated proteolysis of tau protein. Western blotting with phosphorylation‐dependent antibodies revealed that phosphorylation levels of tau at Thr²³¹, Ser³⁹⁶, and Ser⁴⁰⁹ were increased in cells transfected with V337M, R406W, and R406W mutant tau genes, respectively. Together, the data suggest that attenuated proteolysis of FTDP‐17 mutant tau may be explained by increased phosphorylation levels, resulting in resistance to proteolysis.     

Mast cells in renal inflammation and fibrosis: Lessons learnt from animal studies

Mast cells are hematopoietic cells involved in inflammation and immunity and have been recognized also as important effector cells in kidney inflammation. In humans, only a few mast cells reside in kidneys constitutively but in progressive renal diseases their numbers increase substantially representing an essential part of the interstitial infiltrate of inflammatory cells. Recent data obtained in experimental animal models have emphasized a complex role of these cells and the mediators they release as they have been shown both to promote, but also to protect from disease and fibrosis development. Sometimes conflicting results have been reported in similar models suggesting a very narrow window between these activities depending on the pathophysiological context. Interestingly in mice, mast cell or mast cell mediator specific actions became also apparent in the absence of significant mast cell kidney infiltration supporting systemic or regional actions via draining lymph nodes or kidney capsules. Many of their activities rely on the capacity of mast cells to release, in a timely controlled manner, a wide range of inflammatory mediators, which can promote anti-inflammatory actions and repair activities that contribute to healing, but in some circumstances or in case of inappropriate regulation may also promote kidney disease.     

不规则趋化蛋白在嘌呤霉素氨基核苷肾病模型中的表达

目的：检测CX3C族趋化因子不规则趋化蛋白(fractalkine,Fkn)在嘌呤霉素氨基核苷(puromycin aminonucleoside，PAN)肾病中的表达，进一步阐明肾病的发病机制，为肾病的治疗提供新的靶点。方法：从Wistar大鼠的颈静脉给予PAN，对照组给予等量生理盐水，在不同的时间点观察PAN引起的尿蛋白改变和肾脏炎性细胞浸润情况，并于第l、3、5、7、10天处死动物，制作肾组织匀浆和冰冻切片，分别用于RT-PCR和免疫组化研究，观察在：PAN注射的不同时间点肾组织中Fkn mRNA和蛋白质的表达情况；同时在体外用白介素-1β（IL-1β）刺激培养的肾小管上皮细胞观察Fkn的表达。结果：：PAN注射后第5天尿蛋白开始升高，持续增加直至第10天，同时伴有肾间质中CD4^ T细胞，CD8^ T细胞和单核／巨噬细胞的增加。RT-PCR和免疫组化均显示Fkn在第7、第10天表达升高，第3天主要分布在肾小球，以后主要在肾小管上皮细胞表达。用IL-1β刺激后1h体外培养的肾小管上皮细胞即开始表达FknmRNA，在4～6h达到高峰。结论：本文首次观察到Fkn在PAN肾病模型中表达增高，其特征为先出现于肾小球，后移行至肾小管，并早于肾组织中白细胞浸润及蛋白尿的发生。提示：Fkn可能是肾病发病和进展过程中的另一重要的相关分子。     

Salvia przewalskii extract of total phenolic acids inhibit TLR4 signaling activation in podocyte injury induced by puromycin aminonucleoside in vitro

Background: TLR4 signaling is known to be involved in podocyte injury. We have previously shown that Salvia przewalskii extract of total phenolic acids (SPE) and its active monomer salvianolic acid B (SalB) and rosmarinic acid (RA) protect podocytes from injury induced by PAN. In the present study, we test whether SPE inhibits TLR4 signaling.

Methods: The conditionally immortalized mouse podocytes were treated with SPE, SalB, RA, SalB?+?RA or tacrolimus for 30?min, followed by PAN (100?μg/mL) for 24?h. The F-actin staining with phalloidin was used to assess cytoskeletal injury in the podocytes. Western blotting and semi-quantitatives RT-PCR were used to assess the changes of the components in the TLR4 signaling pathway.

Results: (1) The F-actin stress fibers of podocytes were almost completely disrupted after PAN treatment for 24?h, and the disruption was significantly alleviated by SPE; (2) the PAN-induced elevation of mRNA levels of TLR4, MyD88 and p65 were inhibited except p65 with high-dose SalB; (3) consistently, the protein levels of TLR4, MyD88 and pp65 were significantly elevated by PAN, and SPE, SalB, RA and admixture, respectively, attenuated the elevations of TLR4 and pp65 proteins; (4) SPE and tacrolimus have a similarly strong effect on inhibition of the expression of TLR4 signaling components.

Conclusions: SPE protects podocytes from PAN-induced injury at least partly through inhibiting TLR4 signaling. SPE is as strong as tacrolimus in inhibiting TLR4 signaling in podocytes.     

嘌呤霉素肾病大鼠肾小球足细胞相关分子表达的共定位分布

目的:研究嘌呤霉素肾病大鼠肾小球足细胞相关分子在病变过程中的表达与分布的关系及变化.方法:42只SD大鼠随机分为实验组和对照组,建立嘌呤霉素肾病模型,并在24h、24h、2天、5天、10天、15天、20天七个不同的时间点,应用免疫荧光双标记的方法,通过激光共聚焦显微镜对足细胞相关分子nephrin、podocin,α-actinin-4以及CD2AP两两之间的分布关系及变化进行研究.结果:正常大鼠肾小球中CD2AP与nephrin及podocin的染色沿肾小球血管襻有部分重叠,而两者的分布模式略有不同,但这种共定位关系不随病变的发展而变化;CD2AP与α-actinin-4的分布也有部分重叠关系,且随着病变进展,其荧光重叠程度增加;α-actinin-4与nephrin及podocin在正常时有极小部分重叠,随着病程变化,两者的分布模式与分布量的变化均不同步.结论:足细胞相关分子nephrin、podocin及CD2AP之间以功能复合体的形式相互联系并参与蛋白尿的发生发展,而足细胞相关分子的分子行为可能在蛋白尿的发生中起重要作用.     

Glomerular function and morphology in puromycin aminonucleoside nephropathy in rats.

BACKGROUND: The most characteristic manifestation of minimal-change nephropathy is podocyte cell process broadening. In a previous study in children from our unit, we found an inverse correlation between foot process width, glomerular filtration rate (GFR), and filtration fraction. The aim of the present study was to determine whether this relationship also existed in the puromycin aminonucleoside (PAN) experimental model. METHODS: Sixteen Munich-Wistar-Fr?mter male rats initially weighing median 247 g (range 171-286) were used. Four rats served as controls. The other 12 rats were divided into three groups receiving daily subcutaneous injections of 1, 1.67, and 2.5 mg PAN/100 g body weight respectively, for 6 days. GFR was determined by clearance of inulin and the fractional urine albumin excretion was measured. Standard stereological methods were used to estimate the glomerular volume, the mean foot process width and the length density of slit pores. RESULTS: GFR decreased with increasing PAN doses. The glomerular volume was increased in the group receiving the lowest PAN dose, while it was decreased in the group with the highest PAN dose, compared with controls. The fractional albumin excretion and the foot process width increased and the total slit pore length decreased with increasing doses of PAN. GFR correlated directly with the glomerular volume as did the foot process width with the fractional albumin excretion. The foot process width correlated inversely with the glomerular volume as did the glomerular volume with the fractional albumin excretion, and GFR with foot process width. CONCLUSIONS: The decreased GFR found in the nephrotic rats was inversely related to foot process width and directly related to glomerular volume, confirming our previous results in children in an early stage of the nephrotic syndrome.