Song K, Zhuo J, Chai S Y, Mendelsohn F A
Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Victoria, Australia.
Kidney Int. 1992 Sep;42(3):639-46. doi: 10.1038/ki.1992.329.
A method was developed to localize active renin in dog kidney sections using autoradiography to detect in vitro binding of the radiolabeled renin inhibitor, 125I-H77. Light fixation by prior perfusion of the kidney with paraformaldehyde was used to immobilize renin without denaturing its binding activity. Snap frozen sections were cut on a cryostat and incubated with 125I-H77. Dry film autoradiography revealed discrete binding of 125I-H77 to the vascular pole of glomerulus as well as diffuse binding to the outer medulla and to the cortex. Binding of 125I-H77 to other aspartyl proteases in the latter two regions was then suppressed by addition of the aspartyl protease inhibitor, N-acetyl-pepstatin (1 microM). This revealed only the juxtaglomerular binding and successfully suppressed binding of the radioligand to other sites. Light microscopic emulsion autoradiography revealed highly selective discrete labelling of the juxtaglomerular apparatus. Competition for this 125I-H77 binding by a series of structurally different renin inhibitors showed a close correspondence between their reported inhibitory potency for renin and potency in the binding system. This strongly suggests that the radioligand binds to the active site of renin immobilized in the kidney. These results demonstrate a new method to localize active renin in tissues using in vitro autoradiography and radioinhibitor binding. The method shows promise for localization and quantitation of tissue renin in extra renal tissues.
已开发出一种方法，用于在犬肾切片中定位活性肾素，该方法利用放射自显影术检测放射性标记的肾素抑制剂125I-H77的体外结合情况。通过事先用多聚甲醛灌注肾脏进行轻度固定，以固定肾素而不使其结合活性变性。在低温恒温器上切割速冻切片，并与125I-H77一起孵育。干板放射自显影显示125I-H77与肾小球血管极有离散结合，以及与外髓质和皮质有弥散性结合。然后通过添加天冬氨酸蛋白酶抑制剂N-乙酰胃蛋白酶抑制剂（1 microM）来抑制125I-H77与后两个区域中其他天冬氨酸蛋白酶的结合。这仅显示了近球小体结合，并成功抑制了放射性配体与其他位点的结合。光学显微镜乳胶放射自显影显示近球小体高度选择性的离散标记。一系列结构不同的肾素抑制剂对这种125I-H77结合的竞争显示，它们报道的对肾素的抑制效力与结合系统中的效力密切对应。这强烈表明放射性配体与固定在肾脏中的肾素活性位点结合。这些结果证明了一种利用体外放射自显影和放射性抑制剂结合在组织中定位活性肾素的新方法。该方法在肾外组织中定位和定量组织肾素方面显示出前景。
