James W. Fisher

Relationship Between Renal Blood Flow and Erythropoietin Production In Dogs.

Summary Reduction in renal artery blood flow in dogs with the use of a modified Goldblatt clamp to a range of 22.4-35.4% of preconstriction values for 96 hours resulted in a significant increase in plasma levels of erythropoietin at 12 hours. When renal blood flow was reduced to a level 43.5-50% of initial flow erythropoietin titers were also elevated at 12 hours, whereas, reduction to a range of 57.5-71% of initial values only resulted in a slight rise in erythropoietin levels at 24 hours. The mechanism of enhanced erythropoietin elaboration following reduction of renal blood flow is postulated to be the result of renal hypoxia.

Inhibition of Erythropoietic Effects of Hormones by Erythropoietin Antisera in Mildly Plethoric Mice.

Summary Antisera to erythropoietin (Anti-ESF) was found to block completely the erythropoietic effects of 3,5,3′-triiodothyronine (T3), ACTH, and testosterone in mildly plethoric mice. The effects of T3, ACTH, and testosterone were also markedly dampened by increased plethora. The hormones studied increased radioactive iron in red cells of mildly plethoric mice without producing a significant alteration in plasma iron. These findings indicate that ACTH, testosterone and T3 stimulate erythropoiesis indirectly by increasing the elaboration of endogenous erythropoietin, or else they require a certain level of erythropoietin in the recipient animal for their erythropoietic effects.

Effects of cobalt on activity of sheep erythropoietin in rat kidney homogenates.

Summary Rat kidney homogenates were found to inactivate sheep erythropoietin. This inactivation was partially prevented by addition of rather high concentrations of cobalt to the kidney suspensions before homogenization. Several dosages of cobalt alone or in combination with a rat kidney homogenate were tested in polycythemic mice and an unusually high dose was required to produce a significant increase in Fe50) incorporation in red cells. The recovery of erythropoietin from normal kidney homogenates was markedly increased by addition of an amount of cobalt which had in itself only a slight erythropoi-etic effect. The mechanism for this effect of cobalt as it relates to a possible antagonism of a renal erythropoietic inhibitory factor is discussed.

RENAL TRANSPLANTATION AND EXTRACTS AND ERYTHROPOIESIS

The kidney as a major source of erythropoietin was first proposed by Jacobson et al.1 in experiments on the rat. Naets2 confirmed this proposal for the dog. This view has been supported by other investigator^?-^ While a major role of the kidney in erythropoiesis appears established, the site of erythropoietin production within the kidney remains unsettled. Osteen,s Hirashima and Takaku? as well as others, suggested the juxtaglomerular apparatus as the site of erythropoietin production, thus placing this function within the cortex. Fisher et aLIO using the fluorescent antibody technique, also supported a cortical origin for the hormone by detecting fluorescence in the area of the glomerulus. Pennington,ll on the other hand, derived erythropoietin activity from both cortex and medulla of the hog kidney through the application of extraction procedures. In a preliminary presentation, using a transplantation procedure, Muirhead et al.12 suggested that both renwortical and renomedullary tissue appeared to support erythropoiesis. Since the latter findings could not be confirmed, a different scheme was developed for the transplantation approach. It is the purpose of this presentation to relate the results of the modified transplantation approach as well as to present preliminary results on the separation of erythropoietin from various regions of the kidney.

PRELIMINARY PURIFICATION OF SHEEP AND MOUSE ERYTHROPOIETIN BY VERTICAL FLAT BED DISCONTINUOUS ELECTROPHORESIS IN ACRYLAMIDE GEL

Introduction Some investigators have been able to accomplish partial purification of plasma and urinary erythropoietin by combining ion exchange, precipitation, and absorption methods.lD2 However, the erythropoietin available for experimental studies still contains contaminating proteins. In order to study the direct effects of erythropoietin on erythroid cells, it is of the utmost importance to purify erythropoietin completely. It should be possible to have this substance isotopically labeled for the study of its metabolic distribution and sites of localization and to have it available as a pure protein for immunologic and structural studies. The work described in this report is the result of a two step purification of sheep and mouse plasma erythropoietin with the use of a discontinuous vertical flat bed acrylamide gel electrophoresis technique.Y