The renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat angiotensinogen. This substrate was also examined in rats after bilateral nephrectomy, which is known to increase plasma angiotensinogen, and in rats treated with colchicine, which inhibits serum protein secretion. In normal rat liver, light microscopy showed the presence of immunoreactive material in a very few cells. The number of stained hepatocytes rose in rats treated with colchicine or after bilateral nephrectomy. Immuno-staining increased further when rats were both nephrectomized and colchicine treated. In the kidney, angiotensinogen was specifically located as granular formations in nephrocytes of the proximal tubule but never in the granular cells of the juxtaglomerular apparatus. The localization of these granular formations under the brush border suggests that angiotensinogen is reabsorbed from the glomerular ultrafiltrate rather than synthesized in the kidney.

The distribution of type IV collagen and laminin was studied by immunocytochemistry during rat gonadal morphogenesis and postnatal development of the testis and epididymis. Immunostaining appeared as early as the 12th day of gestation along the basement membranes of the mesonephric-gonadal complex. The connection between some mesonephric tubules and coelomic epithelium was seen between the 12th and 13th day of gestation. Discontinuous immunostained basement membranes delineated the differentiating sexual cords in 13-day-old fetuses; this process probably began in the inner part of the gonadal ridge. The seminiferous cords surrounded by a continuous immunoreactive basement membrane are separated from the coelomic epithelium by the differentiating tunica albuginea in 14-day-old fetuses. During the postnatal maturation of epididymis and testis, the differentiation of peritubular cells is accompanied by a progressive organisation of the extracellular matrix into a continuous basement membrane. This change is associated with a gradual condensation of peritubular cells inducing an increase of immunostaining. In adult animals, the tubular wall of epididymis is thicker than the lamina propria of seminiferous tubules. Both type IV collagen and laminin immunostaining paralleled during ontogenesis at the light-microscope level.

The differentiation of renin containing cells was studied by immunocytochemistry in normal rat fetuses by the use of highly specific renin, angiotensin I and II antisera. Renin synthesizing cells were detectable as early as the 15th day of gestation outside the nephrogen territories within the walls of mesonephrotic-gonadic and renal arteries. Intrarenal differentiation began at the 17th day and progressed along the intrarenal arterial tree. AII immunostaining appeared concomitantly in the renin containing cells and developed considerably during ontogenesis, suggesting intracellular biosynthesis. It can be suggested that in the fetus newly synthesized AII may contribute to the early systemic and renal blood pressure regulation.

Angiotensin II and angiotensin III, the active peptides of the renin-angiotensin system, are produced by a cascade of enzymatic reactions, whose initial step is the reaction between renin and its substrate, angiotensinogen. In plasma, the concentration of angiotensinogen is a limiting factor: the Km of the enzymatic reaction is between 1 and 2 microM depending on the species. It is therefore of interest to measure its level in plasma and tissues and to examine the main factors which may influence its synthesis and release. The complete purification of angiotensinogen has made possible the preparation of specific antibodies which cross-react with both angiotensinogen and its residue, des-angio I-angiotensinogen, and are currently used in radioimmunoassays and immunohistochemical studies. A small amount of angiotensinogen is stored in hepatic cells, where it can be detected by immunofluorescence and measured by radioimmunoassay. It is also present in proximal tubular cells of the kidney, probably reabsorbed from glomerular filtrate, but it is absent from juxtaglomerular cells. Several hormones are able to increase liver synthesis of angiotensinogen and its release. Thyroxine, angiotensin II, dexamethasone, ethinyl-estradiol and binephrectomy increase both synthesis and release. Adrenalectomy and converting-enzyme inhibition are accompanied by an increased peripheral consumption of plasma angiotensinogen, and by accumulation of des-angio I-angiotensinogen whose metabolism and role are unknown. The major role of angiotensinogen in renal hemodynamics is demonstrated by its effects on the isolated perfused kidney, an experimental observation which parallels the clinical observation of women on estroprogestative therapy, whose renal blood flow is reduced, even in the absence of a detectable increase in their blood pressure. A better knowledge of renin substrate structure in various species is a necessary requirement for the design of inhibitory analogs of angiotensinogen which will have application for the treatment of hypertension and oedema.