Separation of avian kidney tubules and glomeruli for in vitro culture

Summary A combination of mechanical and enzyme treatments allows the separation of viable avian kidney tubule fragments and glomeruli.     

Changes in Glomerular Cross-Sectional Area Induced by Platelet Activating Factor

Isolated glomeruli from normal rats were incubated with platelet-activatingfactor 10^–6 M at variable incubation times (8, 15, 30and 45 min) and with different concentrations (0 to 10^–6M) for 20 min. In addition, the platelet-activating factor effect(10^–6 or 10^–8 M, 20 min) was tested in the presenceof BN-52021 (10 or 50 µg/ml), verapamil (10^–5 M),acetylsalicylate of lysine (10^–3 M), and in a free-calciummedia with EGTA 2 mM. Glomerular microphotographs were takenbefore and 20 min after adding the substances, and glomerularcross-sectional area was measured using a computerised technique. Platelet-activating factor induced a significant time-dependentreduction in glomerular cross-sectional area, from a concentrationof 10^–8 M. BN-52021, verapamil, and the free-calcium mediainhibited platelet-activating factor-induced reduction of glomerularcross-sectional area, but acetylsalicylate of lysine did not.Platelet-activating factor-induced reduction in glomerular cross-sectionalarea seems to be dependent on the interaction of platelet-activatingfactor with a specific glomerular receptor, with a subsequentmodification of the intracellular concentration of calcium.Arachidonic acid metabolites from the cyclo-oxygenase pathwaydo not seem to be involved in these phenomena. Results suggest that platelet-activating factor could modulateglomerular filtration rate not only by inducing changes in systemicor intrarenal haemodynamics, but also by modifying the filtrationsurface, thus reducing the Kf.     

利用图像直方图特征峰分割肾小球图像

本文根据肾小球医学图像的特点 ,提出了一种新的基于直方图特征峰的图像分割方法。通过定位图像的特征峰 ,从而有效地对肾小球医学图像进行阈值化。经实验证明 ,本文提出的算法能快速、准确地分割肾小球图像。     

PGE2 binding sites and PG-stimulated cyclic AMP accumulation in rat isolated glomeruli and glomerular cultured cells

[3H]PGE2 specifically bound to isolated glomeruli. The KD value and the number of sites were 80 nM and 528 fmoles/mg respectively. PGE1 and PGE2 resulted in equipotent inhibition of binding whereas PGI2 was markedly less active. It was not possible to demonstrate specific receptors for PGE2 in glomerular mesangial and epithelial cultured cells. PGE1, PGE2 and PGI2 (0.1-100 microM) stimulated cyclic AMP concentration both in isolated glomeruli and glomerular cultured cells. Basal cyclic AMP in epithelial cells was greater than in mesangial cells or glomeruli. The cyclic AMP accumulation in the presence of PGs was greatest in mesangial cells. Maximum stimulation was in the range 300-1400%. For the three preparations, PGE2 and PGE1 produced a greater effect than PGI2. ED50 values were identical for PGE1 and PGE2 (5 microM for epithelial cells and glomeruli, 20 microM for mesangial cells). ED50 value for PGI2 were lower than those for PGE1 or PGE2 (0.2, 2 and 5 microM for glomeruli, epithelial cells and mesangial cells, respectively). The effects of the three PGs were not additive when tested at maximally effective concentrations. These results demonstrate that PGE1, PGE2 and PGI2 stimulate glomerular and cellular cyclic AMP. A relationship between [3H]PGE2 binding sites and this biological effect has not been established. The physiological events secondary to the increase in glomerular cyclic AMP are also yet to be determined.     

A review of the role of enhanced apoptosis in the pathophysiology of cystinosis

The role of lysosomal cystine in development of the phenotype in cystinosis is problematic, in that the cystine is effectively isolated from the rest of cellular metabolism. Several models have been proposed, but most do not provide a mechanism for such an interaction. During early apoptosis the lysosomes are permeablized, providing such access. We have shown that lysosomal cystine enhances apoptosis in cultured normal and cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, that the process occurs via mixed disulfide (cysteinylation) formation, and that PKC delta is involved. Further, the "swan neck" deformity of proximal renal tubules, long a hallmark of cystinosis, is explicable via this model, as is the renal failure that results from progression of tubule cell loss to atubular glomeruli. Modification of this process by other genes may explain the milder forms of the disease.     

Super-Enhancer-Associated Transcription Factors Maintain Transcriptional Regulation in Mature Podocytes

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肾小球分离与纯化在肾脏病学中的应用

目的:发明一种快速易行的肾小球分离技术,为肾脏病学研究服务.方法:小鼠麻醉后,经体循环灌注四氧化三铁溶液,使肾小球内的毛细血管中充满四氧化二铁溶液.然后取出肾脏切碎,用collagenase A消化,细胞器滤过,磁铁吸附并收集含铁的肾小球.光镜、透射和扫描电镜观察肾小球的正常形态和结构.常规方法提取RNA和蛋白质,并进行qRT-PCR,micro RNA(miRNA)基因芯片和Western blot等定量检测.结果:该方法可从新生小鼠和成体小鼠的肾脏中高质量快速提纯不同发育阶段(发育早期、中期或晚期)及成熟的肾小球,分离后的肾小球光镜形态结构以及超微结构均保存完好.常规方法提取的RNA和蛋白质可用于qRT-PCR,Western blot检测以及miRNA基因芯片等分子生物学的定性和定量分析研究.结论:该研究所述的肾小球分离技术费用低廉,简便易行,为基础与临床探索肾小球对各种肾脏疾病的基因调控作用和发育分子生物学研究提供了重要的方法和技术手段.     

Wnt/Frizzled family members mediate olfactory sensory neuron axon extension

A comprehensive model has yet to emerge, but it seems likely that numerous mechanisms contribute to the specificity of olfactory sensory neuron (OSN) axon innervation of the olfactory bulb. Elsewhere in the nervous system the Wnt/Fz family has been implicated in patterning of anterior-posterior axes, cell type specification, cell proliferation, and axon guidance. Because of our work describing cadherin-catenin family member expression in the primary olfactory pathway, and because mechanisms of Wnt-Fz interactions can depend in part on catenins, we were encouraged to explore Wnt-Fz expression and function in OSN axon extension. Here, we show that OSNs express Fz-1, Fz-3, and Wnt-5a, whereas olfactory ensheathing cells (OECs) express Wnt-4. Fz-7 is also expressed in the olfactory nerve by cells that delineate large axon fascicles, but are negative for OEC markers. Fz-1 showed a developmental downregulation. However, in adults it is expressed at different levels across the olfactory epithelium and in restricted glomeruli across the olfactory bulb, suggesting an important role in the formation and maintenance of OSN connections to the olfactory bulb. Reporter TOPGAL mice demonstrated that some OECs located in the inner olfactory nerve layer can respond to Wnt ligands. Of further interest, we show here with in vitro assays that Wnt-5a increases OSN axon outgrowth and alters growth cone morphology. Our data point to a key role for Wnt/Fz molecules in the development of the mouse olfactory system, providing complementary mechanisms required for OSN axon extension and coalescence.     

Evidence for parathyroid hormone sensitive adenylate cyclase in rat glomeruli

Isolated rat glomeruli contain an adenylate cyclase system. The amount of cyclic AMP formed increased progressively with incubation time. The rate of cyclic AMP formation increased linearly with glomerular protein concentration. This adenylate cyclase system was temperature and pH dependent. There was no evidence for saturation of the enzyme with substrate up to 10^?2 M ATP. Adenylate cyclase was strikingly activated by fluoride (10^?2 M). Purified bovine parathyroid hormone (PTH) and synthetic 1–34 bovine PTH fragment both stimulated adenylate cyclase activity: maximum activity was 3.9 to 5.4 basal activity and k_m close to 10^?7 M. Epinephrine and isoproterenol produced a slight stimulation whereas salmon calcitonin, glucagon, norepinephrine and antidiuretic hormone were inactive. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role for this hormone in regulation of glomerular activity.