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Recombinant Human Erythropoietin Stimulates Tubular Reabsorption of Sod ium in Anesthetized Rabbits

Noboru Nushiro, Tetsuo Sakamaki, Jin Hoshino, Tetsuya Nakamura, Hironosuke Sakamoto, Yutaka Imai * , Masahide Seino ** , Ken Omata * , Hiroshi Sekino *** , and Keishi Abe

To determine whether recombinant human erythropoietin (rHuEPO) exerts a direct vasoconstrictive effect on renal arteries or affects renal function, we measured renal hemodynamics and renal function during a 30-min intrarenal infusion of rHuEPO in anesthetized rabbits without renal failure. Intrarenal infusion of rHuEPO at a rate of 100 U/min did not alter mean arterial pressure, renal blood flow, or re- nal vascular resistance, as compared with controls treated with vehicle. There were no significant rHuEPO-associated changes in glomerular filtration rate, filtration fraction, or arterial hematocrit. However, urine volume, urinary excretion of sodium and potassium, and fractional sodium excretion were significantly reduced by intrarenal infusion of rHuEPO. These observations indicate that rHuEPO has no direct effects on mean arterial pressure or renal hemodynamics, but that it stimulates net tubular sodium reabsorption, and reduces urine volume and urinary excretion of sodium and potassium in anes- thetized rabbits without renal failure. (Hypertens Res 1995; 18: 203-207) Key Words: chronic renal failure, anemia, recombinant human erythropoietin (rHuEPO), hypertension, renal hemodynamics

Since it was molecularly cloned and a biologically sodium and potassium were examined and com- active form was expressed in mammalian cells (1, pared with the results in a vehicle-treated control 2), recombinant human erythropoietin (rJ4uEPO) group. has been produced and used clinically to treat ane- mia associated with chronic renal failure (3-5). Its Methods use is associated with a sustained, dose-dependent rise in hematocrit that effectively abolishes the Twelve female albino rabbits weighing 2.7 to 3.5 kg symptoms of anemia. The main side effect limiting were maintained on standard rabbit chow, which use of rHuEPO in dialysis and pre-dialysis patients provided 9.1 meq sodium/100 g and 63.4 meq is the development or the aggravation of hyperten- potassium/100 g, and were allowed free access to sion, which has been attributed to an increase in water. Experiments were performed after rabbits blood viscosity elicited by the rise in hematocrit (4, fasted overnight (20 h) and water was allowed ad 6) or to vasoconstriction induced by the improve- libitum. The rabbits were anesthetized with urethan ment of hypoxia (3, 7). Recently, Heidenreich et al. (450 mg/kg iv) and a-chloralose (45 mg/kg iv). (8) have reported that rHuEPO has a direct vaso- Tracheostomy was then performed, and the trachea pressor effect on renal proximal resistance vessels was intubated to allow spontaneous breathing. with internal diameters between 200 and 250 pm A catheter (PE-50) was inserted into the inferior that were obtained from Wistar-Kyoto rats without vena cava through the left femoral vein for infusion renal failure. They suggested that this direct vaso- of 5% dextrose solution. Another polyethylene pressor effect may contribute to the development of h catheter (PE-60) was inserted through the left fem- ypertension in rHuEPO-treated dialysis patients. oral artery into the abdominal aorta adjacent to the The present study was designed to determine renal arteries for monitoring of arterial pressure and whether rHuEPO has direct effects on mean arterial collection of arterial blood samples. The left renal pressure or renal hemodynamics in in vivo anesthe- artery was then exposed through a left retroperi- tized rabbits without renal failure. The effects of toneal flank incision and gently isolated. A noncan- rHuEPO on urine volume and urinary excretion of nulating electromagnetic flow probe (2.0 mm,

From the Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan, * The Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan, ** Izumigaoka Clinic, Sendai, Japan, and *** Kojinkai Hospital, Sendai, Japan. Address for Reprints: Noboru Nushiro, M. D., Ph. D., The Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi 371, Japan. Received September 1, 1994; accepted December 12, 1994. 204 Hypertens Res Vol. 18, No. 3 (1995)

Nihon Kohden, Tokyo, Japan) connected to an was infused intrarenally at a rate of 0.0656 ml/min, electromagnetic flowmeter (MF-27 Nihon Kohden) and urine samples were collected. Arterial blood was placed around the renal artery to measure renal samples were drawn 30 min after starting the blood flow. Arterial pressure was monitored with a rHuEPO infusion and at the end of the recovery pressure transducer and amplifier (Biophysiograph, period. An equivalent amount of fresh arterial 180 system, SAN-El, Tokyo, Japan). Arterial pres- blood drawn from a donor rabbit was transfused af- sure and renal blood flow measurements were re- ter collection of each 5-ml blood sample to prevent corded on a pen oscillograph. The left ureter was changes in renal blood flow. A similar procedure then cannulated with tubing for collection of urine. was followed in the time control group, but the iso- A curved needle (27G) attached to a polyethylene tonic saline vehicle, instead of rHuEPO, was in- catheter (PE-50) was then placed into the left renal fused intrarenally at a rate of 0.0656 ml/ml for 30 artery proximal to the electromagnetic flow probe min through the left renal artery catheter. for infusion of rHuEPO or vehicle. Recombinant The glomerular filtration rate was estimated from HuEPO was provided by Sankyo (Epoetin Alfa, the clearance of creatinine (Cer). Plasma and uri- Sankyo Co., Ltd., Tokyo, Japan). A priming infu- nary concentrations of creatinine were determined sion of a 5% dextrose solution was administered in- using an analyzer (VS-700S, Nihon Densi, Tokyo, travenously in a volume of up to 2% of body Japan). Plasma and urinary concentrations of urea weight. This was followed by a continuous infusion nitrogen, sodium, and potassium were determined of 5%. dextrose solution at a rate of 0.411 ml/min using an autoanalyzer (STAT/ION-IT, Nihon Tech- during and after the equilibrium period. During the nicon, Tokyo, Japan). Renal vascular resistance was equilibrium period, a priming dose of 50 mg/kg of calculated by dividing the mean arterial pressure by creatinine in 4 ml of 5% dextrose solution was renal blood flow and expressed as mmHg per millili- administered via the left femoral vein catheter. This ter per minute. The filtered sodium load (FNa) and was followed by a prolonged infusion of 0.5 net tubular reabsorption of sodium (RNa) were mg/kg/min of creatinine in 0.411 ml/min of 5% de- calculated by multiplying the glomerular filtration xtrose solution, which was continued for the dura- rate by the plasma concentration of sodium and by tion of the experiment to permit estimation of the subtracting the urinary sodium excretion from the glomerular filtration rate. Isotonic saline was in- filtered sodium load, respectively. These values are fused via a catheter into the left renal artery at a expressed as ~eq/min. rate of 0.0656 ml/min forrthe duration of the experi- ment. Experimental protocols were initiated at least Statistical Analysis 60 min after the start of the creatinine infusion to All data in figures and tables are expressed as mean permit stabilization of arterial blood pressure and ± SEM. Repeated measures analysis of variance renal blood flow. (ANOVA) for overall differences and a two-sample Rabbits (n =12) were divided into a time control t-test or Wilcoxon rank sum test was used to com- group and a rHuEPO group. Measurements were pare group differences. A p < 0.05 was considered obtained during three consecutive 10-min periods to to indicate statistical significance. serve as basal values. During each 10-min period and at the end of the third basal period, a urine Results sample and a 5-ml sample of arterial blood were obtained and the following variables were deter- Absolute values for the variables in the third basal mined: urine volume (UV); urinary excretion of period in the time control and rHuEPO groups are creatinine (UcrV), urea nitrogen (UV),unsodium summarized in Table 1. During the third basal (UNaV), and potassium (UKV); systemic arterial period, there were significant differences between hematocrit (Hct); and plasma concentrations of the two groups in PNa, UV, UNaV, and FENa, but creatinine (Pcr), sodium (PNa), and potassium (PK). not in mean arterial pressure (MAP), renal blood Thereafter, in the rHuEPO group, rHuEPO was in- flow (RBF), renal vascular resistance (RVR), heart fused intrarenally at a rate of 100 U/min in 0.0656 rate (HR), arterial hematocrit (Hct), renal plasma ml/min of isotonic saline for 30 min through the left flow (RPF), glomerular filtration rate (GFR), filtra- renal artery catheter. This infusion rate of rHuEPO tion fraction (FF), UKV, FNa, RNa, or UUNV. was chosen in based on the results of a preliminary Intrarenal infusion of rHuEPO at a rate of 100 study. In that study, the arterial concentration of U/min produced no significant changes in MAP, rHuEPO was 0.19 ± 0.014 U/ml (n = 6) and did RBF or RVR (Fig. 1). There were no significant not change significantly during the experiment in differences in these variables between the two the time control group, while the arterial concentra- groups in the recovery period. Heart rate did not tion of rHuEPO after a 30-min intrarenal infusion change significantly in the time control or rHuEPO of rHuEPO at a rate of 100 U/ml increased to 27.3 groups during the experiment (data not shown). In- ± 3.62 U/ml (n = 6), which was higher than the trarenal infusion of rHuEPO produced no signi- threshold concentration required for a pressor re- ficant changes in GFR, FF, or Hct (Fig. 2). There sponse to rHuEPO in vitro. The infusion of were no significant differences in these valiables be- rHuEPO was stopped after measurements were tween the two groups in the recovery period. obtained for three consecutive 10-min experimental The plasma concentration of sodium was higher periods. During the three consecutive 10-min recov- in the rHuEPO group than in the time control ery periods, insotonic saline, instead of rHuEPO, group in the third basal period but did not differ be- Nushiro et al: Effect of rHuEPO on Renal Function 205

Table 1. Absolute Values for Various Parameters in the Third Basal Period

Fig. 1. Mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) in the time control (0-----0, n = 6) and rHuEPO (S• , n = 6) Fig. 2. Glomerular filtration rate (GFR), filtration frac- groups. Values are mean ± SE. Basal, basal period; Ex- tion (FF), and arterial hematocrit (Hct) in the time control perimental, experimental period; Recovery, recovery I, n = 6) and rHuEPO (®, n = 6) groups. period. Values are mean ± SE. Basal, basal period; Experimental, experimental period; Recovery, recovery period. tween the two groups in the experimental or recov- decrease in UV was observed. ery periods. The plasma potassium concentration Intrarenal infusion of rHuEPO produced signi- was not significantly different between the two ficant decreases in UNaV from the basal values at groups during the experiment (data not shown). 10, 20, and 30 min, as compared with the time con- Although there was no signifiant difference between trol group (Fig. 3). Changes in UNaV during the the two groups in MAP or renal hemodynamics in recovery period were significant. the basal period, significant differences were Infusion of rHuEPO produced significant de- observed between the two groups in UV, UNaV, creases in UKV from the basal values at 10, 20, and and FENa in the third basal period. Intrarenal infu- 30 min, as compared with the time control group. sion of rHuEPO produced a gradual and significant The decrease in UKV was still significant in the decrease in UV from the basal values at 10, 20 and rHuEPO-treated group during the first recovery 30 min, as compared with the time control group period. Intrarenal infusion of rHuEPO caused a sig- (Fig. 3). Even in the recovery period, a significant nificant decrease in FENa at 30 min, as compared 206 Hypertens Res Vol. 18, No. 3 (1995)

Fig. 3. Changes in urine volume (UV), urinary sodium i excretion (UNaV), urinary potassium excretion (UKV), and 0 fractional sodium excretion (FENa) in the time control Fig. 4. Filtered sodium load (FNa), net tubular reabsorp- (I I , n=6) and rHuEPO , n=6) groups. Values are expressed as the delta change from the third tion of sodium (RNa), and urinary excretion of urea ni- basal period. Experimental, experimental period; Recov- trogen (UUNV) in the time control ([ I, n=6) and ery, recovery period. * p < 0.05, ** p < 0.01 vs. the time rHuEPO (_ , n=6) groups. Values are mean ± SE. control group. Basal, basal period; Experimental, experimental period; Recovery, recovery period. with the time control group. Even in the recovery associated with the development or worsening of period, a significant decrease in FENa was observed. hypertension in a considerable number of these pa- No significant difference was observed between tients (3, 5, 6, 12-14). The hypertension induced by the time control and rHuEPO groups in FNa, RNa, rHuEPO has been attributed to an increase in or UUNV in the basal, experimental, or recovery blood viscosity, strongly related to the increase in periods (Fig. 4). In the experimental and recovery hematocrit (4, 6), or to the vasoconstriction caused periods, rHuEPO tended to increase RNa, but the by improvement of hypoxia (3, 7). Heidenreich et increase was not significant. al. (8) recently reported that rHuEPO had a direct vasopressor effect on renal and mesenteric resis- Discussion tance vessels of normotensive Wistar-Kyoto rats in vitro and that the threshold concentration required We previously demonstrated that this in vivo model to increase the pressor response to rHuEPO was 10 was useful for study of the effects of vasoactive sub- Ulml. They suggested that the vasopressor activity stances on renal circulation and function (9-11). of rHuEPO may contribute to the development of Using this model, the present study was designed to hypertension in rHuEPO-treated dialysis patients determine whether rHuEPO has direct effects on and may amplify or aggravate other mechanisms re- renal hemodynamics or renal function in anesthe- sposible for the development of hypertension. tized rabbits without renal failure. The present re- In the present study, there were significant differ- sults demonstrated that rHuEPO had no direct ences between the time control and rHuEPO effects on MAP or renal hemodynamics in anesthe- groups in PNa, UV, UNaV, and FENa in the basal tized rabbits without renal failure, but that it stimu- period. The reason for this is not clear. Body lated net tubular sodium reabsorption, possibly by a weight, surgical maneuver, priming volume of the direct tubular effect, and reduced urine volume and 5% dextrose solution, and the time schedule for the urinary excretion of sodium and potassium. experiment were not different between the two Recombinant HuEPO has been found to reverse groups. Other conditions, such as sodium status and anemia in hemodialysis patients, but it has been water intake, might have differed. There were no Nushiro et al: Effect of rHuEPO on Renal Function 207 significant differences between the time control and Culture, Japan. rHuEPO groups in MAP, RBF, RVR, GFR, FF, or Hct in the experimental period. The renal plasma concentration of rHuEPO, estimated by renal plas- References ma flow, was higher than 27 U/ ml, which exceeds 1. Jacobs K, Shoemaker C, Rudersdorf R, et al: Isola- the threshold concentration identified by Heiden- tion and characterization of genomic and cDNA reich et al. for a pressor response to rHuEPO in clones of human erythropoietin. Nature 1985; 313: vitro. Radioimmunoassay measurements of plasma 806-809. concentrations of human erythropoietin in normal 2. Lin FK, Suggs S, Lin CH, et al: Cloning and expres- sion of the human erythropoietin gene. Proc Nail individuals have ranged from 0.003 to 0.02 U/ml. Acad Sci USA 1985; 82: 7580-7584. When 3000 U of rHuEPO was administered three 3. Eschbach JW, Egrie JC, Downing MR, Browne JK, times weekly following dialysis, plasma concentra- Adamson JW: Correction of anemia of end-stage re- tions of human erythropoietin ranged from 0.02 to nal disease with recombinant human erythropoietin. 0.04 U/ml, which is much lower than the threshold N Engl J Med 1987; 316: 73-78. concentration required for a pressor response to 4. Mayer G, Thum J, Cada EM, Stummvol HK, Graf rHuEPO in vitro. Recombinant HuEPO has been H: Working capacity is increased following recom- found to increase the intracellular free calcium con- binant human erythropoietin treatment. Kidney Int centration in single early human erythroid precur- 1988; 34: 525-528. 5. Winearls CG, Oliver DO, Pippard MJ, Reid C, sors (15) and to induce differentiation and prolifera- Downing MR, Cotes PM: Effect of human erythro- tion of erythroid cells. The threshold concentration poietin derived from recombinant DNA on the ane- of rHuEPO needed to induce an increase in in- mia of patients maintained by chronic haemodialysis. tracellular free calcium was 0.2 U/ml, and the pres- Lancet 1986; ii: 1175-1178. ence of extracellular calcium was not necessary to 6. Raine AEG: Hypertension, blood viscocity, and car- induce the observed increase in intracellular free diovascular morbidity in renal failure: implications of calcium, suggesting that rHuEPO had no direct erythropoietin therapy. Lancet 1988; is 97-99. effects on MAP or renal hemodynamics, despite an 7. Kamata K, Marumo F, Onoyama K: Hemodynamic rHuEPO-stimulated increase in intracellular free mechanism of the elevation in blood pressure fol- calcium concentration in renal vascular smooth mus- lowing the improvement of anemia with recombinant human erythropoietin. Jpn Circ J 1991; 55: 649-656. cle cells. Mechanisms other than a direct vasopres- 8. Heidenreich S, Rahn KH, Zidek W: Direct vasopres- sor effect, such as chronic stimulation of differentia- sor effect of recombinant human erythropoietin on tion and proliferation of vascular smooth muscle renal resistance vessels. Kidney Int 1991; 39: cells, may be involved in the pathogenesis of hyper- 259-265. tension induced by chronic rHuEPO administration. 9. Seino M, Abe K, Nushiro N, et al: Role of the renal Intrarenal infusion of rHuEPO produced de- kinin-prostaglandin system in diltiazem-induced na- creases in UV, UNaV, UKV, and FENa, suggesting triuresis. Am J Physiol 1986; 250: F197-F202. that it exerted a direct tubular effect that stimulated 10. Seino M, Abe K, Nushiro N, Yoshinaga K: Nifedi- sodium reabsorption. However, possible effects of pine chances the vasodepressor and natriuretic effects of atrial natiuretic peptide. Hypertension 1988; 11: rHuEPO on humoral factors, such as angiotensin II 34-40. and aldosterone, cannot be excluded. Increased 11. Nushiro N, Abe K, Seino M, Yoshinaga K: Interac- urinary excretion of urea nitrogen (UUNV)has been tion between ANP and amiloride in renal tubular found to be an indication of inhibition of tubular sodium handling in anesthetized rabbits. Am J Phy- sodium reabsorption, related to washout in the siol 1988; 254: F521-F526. medullary nephron (16). The lack of effect of the 12. Bommer J, Alexiou C, Buhl UM, Eifert J, Ritz E: intrarenal rHuEPO infusion on UUNV in the pres- Recombinant human erythropoietin therapy in ent experiment further suggests the absence of hemodialysis patients-dose determination and clinical rHuEPO-associated hemodynamic effects. The site experience. Nephrol Dial Transplant 1987; 2: of action of rHuEPO in the renal tubules remains 238-242. 13. Jacquot Ch, Haguet MF, Lefebvre A, Berthelot JM, to be defined. Peterlongo F, Castaigne JP: Recombinant erythro- In conclusion, the present study showed that poietin and blood pressure. Lancet 1987; ii: 1083. rHuEPO had no direct effects on MAP or renal 14. Tomson CRV, Yenning MC, Ward MK: Blood hemodynamics, but that it stimulated the tubular pressure and erythropoietin. Lancet 1988; is 351. reabsorption of sodium, and reduced urine volume 15. Miller BA, Scaduto RCJr, Tillotson DL, Botti JJ, and urinary excretion of sodium and potassium in Cheung JY: Erythropoietin stimulates a rise in in- anesthetized rabbits without renal failure. tracellular free calcium concentration in single early human erythroid precursors. J Clin Invest 1988; 82: 309-315. Acknowledgements 16. Roman RJ, Cowley AWJr: Characterization of a new model for the study of pressure-natriuresis in the rat. This work was supported by a Grant-in-Aid (745- Am J Physiol 1985; 248: F190-F198. 03454246) from the Ministry of Education, Science and