Prostaglandin and thromboxane synthesis by rat glomerular epithelial cells

Isolated rat glomeruli have been shown to synthesize prostaglandin (PG) and thromboxane (Tx). In this study, we evaluated, by radioimmunoassay and radiochromatographic methods, PG and Tx synthesis by glomerular cells in culture. Transmission and scanning electron microscopy showed polygonal cells, attached by desmosomes, with surface microvilli. These features are typical of glomerular epithelial cells. Incubation of these glomerular epithelial cells with arachidonic acid (C20:4) resulted in an array of endproducts with concentrations of PGE2 greater than TxB2 greater than PGF2 alpha greater than 6-keto-PGF1 alpha . Addition of angiotensin II (AII) to the cultured glomerular cell produced almost exclusive stimulation of PGE2 with PGE2 much much greater than PGF2 alpha greater than TxB2 = 6-keto-PGF1 alpha . AII and AIII (100 micrometer to 1 micrometer ) stimulated PGE2 in glomerular epithelial cells, and the increments of PGE2, as a function of the concentration of AII or AIII, were similar. The sar1-thr8-AII analog inhibited both AII- and AIII-stimulated PGE2 synthesis. The divalent cation ionophore A23187 in concentrations of 0.2 to 2.0 micrometer increased primarily PGE2 and TxB2 synthesis with smaller increases of PGF2 alpha and 6-keto-PGF1 alpha . The relative concentrations of PG and Tx produced by rat glomerular epithelial cells, incubated with C20:4 or A23187, were similar. Our results demonstrate that: (1) the predominant cell grown in culture from the rat glomerulus, after 9 days, is the epithelial cell; (2) this cell is capable of PG and Tx synthesis; (3) stimulation of PG by AII and AIII may be mediated by the same cellular receptor, AII and AIII increase primarily the synthesis of a vasodilatory PG, PGE2; (4) exogenous substrate C20:4 or release of endogenous C20:4 by the divalent cation ionophore A23187 not only stimulates PGE2 but also the vasoconstrictor TxA2; and (5) the PG and Tx endproducts synthesized by epithelial cells may be determined by an intracellular coupling of the specific synthetic enzymes with different pools of C20:4.     

Tissue factor production by cultured rat glomerular epithelial cells

It is well known that fibrin deposition in Bowman's space inassociation with crescent formation may play an important rolein progressive glomerular injury in crescentic glomerulonephritis.Recent reports describe the presence of a procoagulant activity(PCA) in the glomeruli and its increased expression in humanand experimental nephritis. The cells that synthesize PCA havenot yet been identified. We attempted to determine if glomerularepithelial cells (GEC), one of the prominent cell populationsin the crescent, can produce PCA. The PCA of cultured rat GECwas measured by clotting and amidolytic assays. The culturedGEC yielded PCA with the characteristics of a tissue factor,and this PCA was stimulated by interleukin 1, tumour necrosisfactor-alpha, and lipopolysaccharide. We concluded that GEC produce tissue-factor-like PCA and therebymay contribute to fibrin deposition, which, along with macrophageor monocyte infiltration, leads to crescent formation in crescenticglomerulonephritis.     

FTY720诱导大鼠系膜细胞的凋亡

目的 观察新型免疫抑制剂FTY720对体外培养的正常大鼠肾小球系膜细胞（GMC）的促凋亡作用及其对细胞周期调节蛋白基因表达谱的影响,以探讨其作用机制。 方法 体外培养大鼠GMC,加入20 μmol/L FTY720,分别作用6 h、12 h、24 h、48 h后,以MTT法检测GMC增殖情况;流式细胞术检测GMC凋亡;Hoechst33258和PI染色观察凋亡细胞形态变化;琼脂糖DNA凝胶电泳法观察凋亡细胞核小体DNA的断裂现象。并通过SuperArray 实时定量PCR细胞周期基因芯片测定FTY720对细胞周期调节蛋白基因表达谱的影响。 结果加入FTY720培养6 h 后,流式细胞术检测发现,GMC出现典型细胞凋亡的亚二倍体峰;12 h后Hoechst33258和PI荧光染色观察发现亮蓝色的凋亡小体,且开始出现典型细胞凋亡形态改变;24 h后DNA琼脂糖凝胶电泳可见凋亡梯度的出现。随FTY720作用时间延长,细胞凋亡明显增加,不同时间组间细胞凋亡率差异有统计学意义（P < 0.01）。SuperArray 实时定量PCR细胞周期基因芯片发现FTY720分别显著上调系膜细胞Dnajc2、LOC688900、RGD1562436_predicted基因表达,分别达41.6、38和16倍。 结论 FTY720 在体外可呈时间依赖诱导正常大鼠GMC凋亡,其机制与影响细胞周期调节蛋白及凋亡相关基因的表达有关。     

高效的毛乳头细胞分离培养方法

目的 寻求一种洁净且无须显微镜下操作的毛乳头分离方法 ,培养高纯度的毛乳头细胞.方法 中性蛋白酶消化头皮组织,将含有毛囊的皮下脂肪层剪下切碎,并用Ⅰ型胶原酶消化,之后通过多次离心的方法 获得毛乳头;对培养出的毛乳头细胞进行形态学观察,并检测毛乳头细胞的标志物alfa-SMA和碱性磷酸酶的表达情况.结果 通过新型的两步酶消化法可获得洁净的毛乳头,培养出的毛乳头细胞标志物检测阳性.结论 新型的两步酶消化法是一种高效低劳动强度的毛乳头分离方法.     

Monoclonal antibodies to human glomerular cells: a marker for glomerular epithelial cells

A monoclonal antibody, PHM 5, directed against human glomerular epithelial cells, was prepared after immunisation of a mouse with isolated human glomeruli and fusion of spleen cells with a mouse myeloma. Binding of antibody to isolated glomeruli was detected by radioimmune indirect binding assay, and specificity for glomerular epithelial cells was shown using a four-layer peroxidase-antiperoxidase technique applied to epoxy resin sections of the human kidney cortex. PHM 5 appears to detect a human-specific cell surface carbohydrate antigen not previously described, and can be used to identify epithelial cells in renal biopsy sections and in culture outgrowths of isolated glomeruli.     

Isolation and characterization of rat glomerular epithelial cells in vitro

Rat glomeruli were isolated by a graded sieving technique, and the nature and purity of the preparation were examined by scanning electron microscopy (SEM). A great majority of the glomeruli (86.0 +/- 6.0%) were not encapsulated, and there was very little contamination of the preparations with tubular fragments. By supplementing tissue culture medium with conditioned medium (CM) and insulin, we were able to grow single cells from dissociated rat glomeruli. From these single cells, we were able to clone and maintain in culture three distinct cell types. Also, with this specialized medium, we were able to clone these three cell types from outgrowths of whole golmeruli. One of these cell types was characterized as glomerular epithelial cells (GEC). GEC, as opposed to the other two cloned cell types obtained in this study, had cilia on their surfaces and also possessed receptors for complement (C3) in vitro. Low concentrations of the aminonucleoside of puromycin (AMNS), which have been shown to be specifically cytotoxic to GEC in vivo, were found to be cytotoxic to GEC but not to the other two glomerular cells in vitro. In addition, GEC did not contain antihemophilic factor (factor VIII), a marker for endothelium, nor were they able to phagocytose polystyrene spherules in vitro, as can mesangial cells in short-term culture.     

Culture methods of glomerular podocytes

Podocytes (glomerular visceral epithelial cells) cover the exterior surface of the glomerular capillaries and contribute to the glomerular filtration membrane. Failure of podocyte function is involved in the progression of chronic glomerular disease; accordingly, research interest into podocyte biology is driven by the need for better protection and perhaps recovery of these cells in renal diseases. This review aims at summarizing available techniques for podocyte cell cultures from both the past and present, with special attention to the currently used methods. The establishment of classical primary cultures is based on isolation of glomeruli by differential sieving. Plating of glomeruli onto a collagen surface is followed by an outgrowth of cobblestone-like cells that, after replating, differentiate into arborized, mature podocytes. Currently, the majority of research studies use immortalized podocytic cell lines most often derived from transgenic mice bearing a conditional immortalizing gene. The podocytes can also be collected and cultured from healthy or diseased animal or patient urine. The urinary podocytes obtained from subjects with active glomerulopathies display higher proliferation potential and viability in vitro, perhaps due to disease-induced transdifferentiation. Finally, a list of phenotypic markers useful for identification and characterization of the cultured podocytic elements is provided.     

Leukotriene C4 and D4 contract rat glomerular mesangial cells

The sulfidopeptide leukotrienes, LTC4 and LTD4, have vasoconstrictor effects in the kidney, reducing both renal blood flow and the glomerular filtration rate. As one mechanism regulating the glomerular filtration rate, mesangial cell contraction may reduce the capillary surface area, thereby lowering the ultrafiltration coefficient. Using image analysis microscopy to quantify changes in cell morphology, we found that LTC4 and LTD4 (1 X 10(-12)M to 1 X 10(-6)M) reduced the cross-sectional area of cultured mesangial cells from rat glomeruli. The response to LTC4 and LTD4 (10(-6)M), as measured by the percentage of responding cells (30 to 35%), the maximum decrease in cross-sectional area (25 to 32%), and the time course was identical to that for angiotensin II (10(-6)M). The contraction induced by LTD4 was attenuated by an LTD4 receptor antagonist (4R,5S,6Z-nor-LTD1). Also, preincubation with colchicine prevented LTC4-induced contraction. Leukotriene B4, a non-sulfidopeptide leukotriene that stimulates chemotaxis and chemokinesis, had negligible agonist activity. Mesangial cells cultured on less adhesive teflon membranes were more responsive to LTD4 (62% of cells responded) than cells cultured on glass or polystyrene (35% of cells responded). Mesangial cell contraction was not merely a shape-change as a result of cell damage, since cellular injury was not documented by lactate dehydrogenase release and proliferation of mesangial cells was not retarded by LTC4. Furthermore, the contraction was independent of cell size. Because leukotrienes stimulate cyclooxygenase products in other cells, we examined the ability of the sulfidopeptide leukotrienes to stimulate prostaglandin and thromboxane synthesis. LTC4 and LTD4 did not stimulate PGE2 formation, the major cyclooxygenase product of rat mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometric analysis of normal glomerular epithelial cells in rat and human

It has been postulated that morphological changes of podocytes might be related to glomerular sclerotic lesions in experimental models and patients with glomerular diseases. To estimate the absolute number of podocytes in mammalian normal glomerulus, we analyzed normal glomeruli in four rats and six humans. In PAS stained light microscopic sections, at least 25 midsections of open glomeruli were photographed. Stereologic estimation was performed to obtain the following values: absolute values of glomerular volume (V), glomerular surface area (S), podocyte and intraglomerular cell number per glomerulus (P and IGC), glomerular surface area covered by one podocyte (S/P) and glomerular volume occupied by one intraglomerular cell (V/IGC). The glomerular volume, glomerular surface area and podocyte and intraglomerular cell numbers per glomerulus of human were significantly increased compared with those of the rat (V: 2.70 +/- 0.86 > 0.89 +/- 0.19, S: 4.84 +/- 1.26 > 1.88 +/- 0.26, P: 407.7 +/- 88.2 > 153.8 +/- 84.0, p < 0.01 vs rat). On the other hand, there were no significant differences in glomerular surface area covered by one podocyte and glomerular volume occupied by one intraglomerular cell between the humans and rats (S/P: 1.25 +/- 0.20, 1.29 +/- 0.05, V/IGC: 2,471 +/- 487, 2,227 +/- 201, p < 0.01 vs rat). These data were almost the same as previously reported values. It appears that these values can be considered as standards for rats and humans in morphometric analysis of the glomerulus.     

两种不同方法培养大鼠肾上腺细胞的比较

目的 观察胶原凝胶三维培养对肾上腺细胞形态和功能的影响.方法 分离SD大鼠乳鼠肾上腺细胞,于自制鼠尾型Ⅰ胶原凝胶中培养14 d,与传统平面培养的肾上腺细胞相对比,比较两者在细胞形态、细胞增殖率、蛋白表达和分泌功能方面的差异.结果 两种培养方式下,肾上腺细胞均能表达其特异性细胞色素氧化酶CYP11B1和CYP11B2.原代培养8 d后,三维胶原凝胶培养大鼠肾上腺细胞的增殖率显著高于平面培养,至第14天,胶原凝胶中培养的肾上腺细胞增殖率为16.6%,而传统平面培养的仅为2.9%.胶原凝胶中培养的肾上腺细胞分泌功能维持时间更长,第14天其培养液中皮质酮和醛固酮浓度分别为(278.33±38.20)μg/L和(133.85±38.20)ng/L,而相应的平面培养肾上腺细胞分泌皮质酮和醛固酮浓度分别为(160.35 ±28.12)μ/L和(74.03±12.12)ng/L,差异有统计学意义(P＜0.05).结论 胶原凝胶中三维培养肾上腺细胞更有利于细胞功能的维持.     

人胚嗅鞘细胞不同取材及培养方法对纯度的影响

目的通过对多种方法的比较获得较为理想的取材和纯化嗅鞘细胞的措施。方法应用3种不同的取材方法各获取20份样本,对取材引起的细胞损伤以及同一条件下培养10d后细胞纯度进行对照;在将细胞接种密度统一调整到106/ml后应用3种纯化方法各对20份样本处理并作纯度比较;同样密度和接种数量的原代细胞各取10份种植于3种不同培养皿,对细胞贴壁速度和最终纯度进行检验;对3种常用的细胞接种密度104/ml、105/ml、106/ml统一其他培养条件后作比较。结果我们从中筛选出最佳取材方法为直接挤压法,其获得的细胞纯度为82.56%,明显高于传统分离法(66.24%)和机械过滤法(61.35%);最好的纯化嗅鞘细胞方法为免疫吸附法,其细胞纯度达到94.66%,而差速贴壁法(81.59%)和化学试剂法(76.45%)均由于损失细胞太多不可取;同时发现包被培养皿对纯化细胞意义重大,P75包被组不仅培养细胞的纯度最高(87.69%),而且还有利于OECS的快速贴壁;高密度接种细胞对提高细胞的最终纯度也是非常有利,3组(接种密度分别为104/ml、105/ml、106/ml)培养10d后的p75阳性细胞率差异巨大,分别为34.61%,49.39%,71.55%。结论应用最佳取材及培养方法收获大量嗅鞘细胞将对其进一步的基础研究以及将来应用于临床奠定基础。     

Stimulation of prostaglandin production in rat glomerular epithelial cells by antidiuretic hormone

Prostacyclin (PGI2) and prostaglandin E2 (PGE2) production by rat glomerular epithelial cells in culture were stimulated by arginine vasopressin (AVP) over a dose range of 10(-9) to 10(-6) M, but only if the cells were allowed to recover from trypsin treatment. The effect of AVP was related to its pressor activity since the antidiuretic analogue of AVP, 1-deamino-8-D-Arg-vasopressin (dDAVP) had no effect. Angiotensin II and kallidin (lysyl-bradykinin) did not affect prostaglandin production by these cells. The stimulatory effect of AVP on arachidonate metabolism was inhibited by the calcium channel antagonist, nifedipine, in a dose-dependent fashion suggesting that cellular uptake of calcium was required.     

Demonstration and characterization of C3 receptors on rat glomerular epithelial cells

Receptors for C3 have been demonstrated on the glomerular podocyte in humans. There is conflicting evidence regarding the presence of C3 receptors on rat glomerular cells. Even when shown to be present, the ligand specificity of the receptor has not been determined. Decapsulated rat glomeruli obtained from male Sprague-Dawley rats weighing 50 to 100 g were placed in enriched culture media. On days four to eight, cells of epithelial morphology were observed growing out of glomeruli. Receptors for C3 were detected by rosette formation of sheep erythrocytes (E) coated with antibody (A) and complement (EAC) around the glomerular epithelial cells in culture. The EACs were prepared by incubating antibody-coated sheep erythrocytes with C5-deficient mouse serum or with individual components of complement. Results indicate the presence of two types of C3 receptors on glomerular epithelial cells--CR1 for C3b and CR2 for C3d. The functional roles of these receptors remain to be elucidated.     

Cadmium nephrotoxicity assessed in isolated rat glomeruli and cultured mesangial cells: evidence for contraction of glomerular cells.

Cadmium (Cd), an important pollutant, causes severe damage at the renal tubular level. Numerous previous studies have focused upon Cd tubular nephrotoxicity. The present study of Cd-induced glomerular damage examined the vasoactive effect of Cd in freshly isolated glomeruli and mesangial cells. Glomeruli were isolated by passing rat renal cortex pulp through calibrated sieves followed by culture for outgrowth of cells. Quantitative evaluation of glomerular and cellular contractions was performed by morphometric measurement of the area with an automatized image analyzer following different incubation times with Hanks' balanced salt solution or Cd2+. Each glomerulus or mesangial cell served as its own control. Cd lethality was measured with microassay methods (neutral red, MTT uptake, and lactate dehydrogenase release), allowing the determination of an IC50. This ranged from 35 to 60 microM. CdCl2 induced a time-dependent contractile effect on isolated glomeruli; planar surface area decreases were 6.9% (1 microM), 7.5% (0.1 microM), and 7% (0.01 microM). The decrease started as soon as Cd was in contact with glomeruli and ended 40 min later: T5 (2%), T10 (3.5%), T20 (4. 2%), T30 (6.3%), T40 (7%). Cell size reduction was 19% (1 microM), 14% (0.1 microM), and 18% (0.01 microM) and was also time-dependent. To confirm that contractile events occurred during the cell shape changes, examination of the mesangial alpha-actin network was performed concurrently. These results indicate that Cd contracts glomerular structures. This may, in part, explain the reduction in glomerular filtration seen in Cd nephrotoxicity.