Analysis of segmental renal gene expression by laser capture microdissection
Analysis of segmental renal gene expression by laser capture microdissection
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DOI：
10.1046/j.1523-1755.2000.00824.x
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发表时间：
2000-01-01
影响因子：
19.6
通讯作者：
Star, RA
中科院分区：
文献类型：
--
作者：
Kohda, Y;Murakami, H;Star, RA
背景显微解剖技术极大地帮助了正常肾生理学的研究，该技术描绘了沿沿着肾单位和不同肾单位群体之间的异常功能和细胞异质性。由于组织坏死或纤维化，显微切割很困难，因此这些技术并没有广泛用于研究肾损伤。我们开发了一种程序来检测特定的基因表达在特定位置的肾脏组织切片。采用激光捕获显微切割技术（LCM）的解剖特异性和逆转录-聚合酶链反应（RT-PCR）的敏感性进行比较。LCM/RT-PCR在2%的单个肾小球中检测到podoplanin的mRNA，在6%的单个近曲小管横截面中检测到大鼠碱性氨基酸转运蛋白的mRNA，在8个近曲小管横截面中检测到肾素的mRNA。LCM/RT-PCR可以从肾组织切片中分离出纯的近曲小管、近直小管和粗升支，但不能分离出纯的集合管。LCM/RT-PCR定位缺血再灌注诱导的KC/IL-8诱导主要位于髓质粗升支，并检测1例膜性肾小球肾炎患者肾小球中转化生长因子-β（TGF-β）mRNA。当与适当的激光光斑尺寸一起使用时，LCM/RT-PCR可以测量肾小球或肾单位特定部分的基因表达，并可以研究肾脏疾病动物模型中稳态mRNA水平的变化。这种方法的应用，局限性和改进进行了讨论。
Background. The study of normal renal physiology has been greatly aided by microdissection techniques that have delineated the exceptional functional and cellular heterogeneity both along the nephron and between different nephron populations. These techniques are not widely used to study renal injury as microdissection is difficult because of tissue necrosis or fibrosis. We developed a procedure to detect specific gene expression in specific locations of the kidney in histologic sections.Methods. The anatomic specificity of laser capture microdissection (LCM) was employed with the sensitivity of reverse transcriptase-polymerase chain reaction (RT-PCR).Results. LCM/RT-PCR detected mRNA for podoplanin in 2% of a single glomerulus, rat basic amino acid transporter in 6% of a single cross-section of proximal straight tubule, and renin in eight proximal convoluted tubule cross-sections. LCM/RT-PCR could isolate pure populations of proximal convoluted tubules, proximal straight tubules, and thick ascending limbs from renal histologic sections, although pure collecting ducts could not be isolated. LCM/RT-PCR localized ischemia reperfusion-induced induction of KC/interleukin-8 primarily to the medullary thick ascending limb, and detected transforming growth factor-beta (TGF-beta) mRNA in glomeruli of a patient with membranous glomerulonephropathy.Conclusions. When used with an appropriate laser spot size, LCM/RT-PCR can measure gene expression in glomeruli or specific parts of the nephron and can study alterations in steady-state mRNA levels in animal models of renal disease. The applications, limitations, and refinements of this approach are discussed.