Renal Effects of Erythropoietin in Humans

Overview

Erythropoietin (EPO) is a glycoprotein produced mainly in the kidney. After its release to the bloodstream EPO binds to its receptor predominantly located within the bone marrow where erythropoiesis is stimulated. Recently, we have shown that recombinant human EPO (rHuEPO) down-regulates circulating levels of renin and aldosterone. Concomitant clearance studies revealed a decrease in proximal tubular reabsorption of sodium and water and a fall in glomerular filtration rate (GFR). These results for the first time demonstrate a link between EPO and renal function: By inhibiting proximal tubular reabsorption, which in turn results in rapid declines in GFR and renin/aldosterone levels, EPO may directly reduce the major oxygen consuming factor in the kidney. The expected result will be an increase of the oxygen tension in the environment of renal EPO producing cells, in this way initiating an appropriate signal for down-regulation of endogenous EPO synthesis when circulating levels of EPO are high. The aim of this project is to test this hypothesis by investigating the renal effects of rHuEPO in humans. In a double-blinded manner healthy subjects will be tested with placebo, or low-dose rHuEPO for two weeks, or high-dose rHuEPO for three days. Accurate sodium balance studies will be conducted together with renal clearance studies for measurements of renal plasma flow (131I-Hippuran clearance with renal venous sampling), GFR (51Cr-EDTA clearance) and the segmentel tubular handling of sodium and water (lithium clearance). EPO is the sole haematopoietic growth factor that is mainly produced in the kidneys and the project will provide new information about basic physiological issues regarding the association between renal function and the regulation of EPO synthesis.

The haematopoietic effect of EPO and rHuEPO has been known for five decades but still the exact mechanisms for regulation of EPO synthesis in the kidneys remain unclear. Recently, we confirmed our previous observation that rHuEPO in normal subjects produces arterial hypertension and a reduction in plasma volume. Moreover, the study delineated the time course of these changes: rHuEPO promptly, and before any changes in hematocrit, blood volumes and blood pressure can be detected, causes a down-regulation of the renin-aldosterone system, proximal tubular reabsorption and GFR. The effect of rHuEPO on arterial blood pressure has been demonstrated to occur independent of its haematopoietic effect and subsequent effect on blood viscosity. Recently, we reported that also short-time administration of very high doses of rHuEPO (30,000 IU/day for three days) increases arterial blood pressure and the blood pressure response to exercise to a similar extent as prolonged, low-dose rHuEPO for three month. The exact mechanisms remains unclear, but may involve rHuEPO induced release of endothelin and inhibition of eNOS mediated production of NO. The early rHuEPO induced reduction of renin and aldosterone was not caused by changes in plasma and blood volumes. A fall in intravascular volume normally leads to the opposite effect due to a decreased NaCl load to the macula densa and an increased sympathetic stimulation of the juxtaglomerular apparatus. The link between administration of rHuEPO and the renin-angiotensinaldosterone system is interesting because the production of endogenous EPO is regulated by this system. Administration of angiotensin II in humans stimulates EPO synthesis and, conversely, inhibitors of angiotensin converting enzyme and angiotensin II receptors decrease the plasma concentration of endogenous EPO. In patients with type-1 diabetes, an inherent high activity of basal renin-angiotensin system (in part governed by genetic factors) was associated with higher levels of EPO compared to patients with a low activity of basal renin-angiotensin system. Our results suggest that rHuEPO may activate an opposite pathway so as to down-regulate the activity of the renin-angiotensin-aldosterone system independent of changes in red blood cell mass, blood volumes and blood pressure. Our renal clearance data suggest that the rHuEPO-induced inhibition of the renin-aldosterone system is associated with a reduction of absolute proximal tubular reabsorption of fluid and a fall in GFR. Changes in end-proximal delivery of tubular fluid to the macula densa produce inverse changes in renin release and thus the suppression of plasma renin levels may be secondary to direct effects of rHuEPO on proximal tubular reabsorption. In addition, a decrease in proximal tubular reabsorption activates the tubuloglomerular feedback mechanism causing a parallel decrease in GFR. The exact molecular mechanisms for rHuEPO's effect on the proximal tubule remain unknown but may involve inhanced release of renal endothelin-1 which in low doses attenuates sodium reabsorption in the proximal tubule. Tubular reabsorption of sodium is the main oxygen consuming process in the kidney and around 70 % of the filtered load is reabsorbed in the proximal tubule. By inhibiting proximal tubular reabsorption, which in turn results in rapid declines in GFR and renin/aldosterone levels, rHuEPO may directly reduce the major oxygen consuming factor in the kidney, reduce the filtered load, and decrease angiotensin II and aldosterone dependent reabsorption in more distal nephron segments. Thus, we suggest that the renal effects of rHuEPO may be part of a feedback system that serves to down-regulate the endogenous renal synthesis of EPO in the presence of high levels of circulating EPO. In support of such a feedback system, evidence exists to indicate that prolonged administration of rHuEPO results in a suppression of urinary excretion of endogenous EPO, and also the renal effects of rHuEPO fits well in the hypothesis advanced by Donnelly, arguing that the kidney operates as a 'critmeter' to regulate the EPO synthesis and body haematocrit through the metabolic signal of renal tissue oxygen pressure. It has been suggested that the reduction in plasma volume induced by rHuEPO may be caused by the hyporeninemic hypoaldosteronism leading to natriuresis. In our previous study we did not perform actual sodium balance studies. However, the renal sodium loss necessary to account for the observed decrease in plasma volume is small, and it is possible that the net effect of rHuEPO was to cause a negative sodium balance during the entire 28 days treatment period. Hypotheses 1. rHuEPO decreases renal proximal tubular reabsorption, concentrations of renin and aldosterone, GFR, and overall renal perfusion. 2. In subjects on a sodium-fixed diet, rHuEPO increases the sodium excretion causing a negative sodium balance. 3. rHuEPO decreases renal oxygen consumption so as to augment oxygen tension at EPOproducing, interstitiel fibroblast-like cells in the juxtamedullary region. 4. rHuEPO decreases renal synthesis and secretion of endogenous EPO. 5. Blockade with specific endothelin antagonists (Bosantan) inhibits the renal effects of rHuEPO. Research plan and methods The project includes normal subjects in which rHuEPO is administered according to previous protocols used by our group. In separate series subjects are given either 1) placebo, 2) rHuEPO (5,000 IU) every second day in two weeks, 3) rHuEPO (30,000 IU/day) for three days. Measurements are obtained at days 4, 11, 28. The trials are planned to be conducted in a double-blinded, cross-over design by which the subjects are randomised to three consecutive trial periods with either placebo, low-dose rHuEPO or high-dose rHuEPO separated by at least six weeks.