A new method to localize active renin in tissues by autoradiography: application to dog kidney.
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.
Song
K
K
Department of Medicine, Austin Hospital, University of Melbourne, Heidelberg, Victoria, Australia.
Zhuo
J
J
Chai
S Y
SY
Mendelsohn
F A
FA
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D001667:Binding, Competitive; D004285:Dogs; D004789:Enzyme Activation; D007457:Iodine Radioisotopes; D007606:Juxtaglomerular Apparatus / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008297:Male; D009842:Oligopeptides / Q000378:metabolism; D010436:Pepstatins / Q000494:pharmacology; D012083:Renin / Q000097:blood / Q000378:metabolism*; D014018:Tissue Distribution; D014357:Trypsin / Q000378:metabolism; D000092502:Renin Inhibitors
D007457:Iodine Radioisotopes; D009842:Oligopeptides; D010436:Pepstatins; D012083:Renin; D014357:Trypsin; D000092502:Renin Inhibitors; C014195:Streptomyces pepsin inhibitor; C034503:H 77