Plasma erythropoietin level in rats after kidney proximal tubular impairment.

The involvement of proximal kidney tubules in erythropoietin production and/or in its control has been a subject of controversy among researchers. The present study addresses the problem whether acute tubular impairment affects erythropoietin production and thus alters the basic hematological parameters. For this purpose rats with hypobaric hypoxia-stimulated (42.55 kPa) erythropoesis were studied and the rat model of gentamicin-induced (50 mg/kg daily for 15 days) tubular damage was employed. According to the study protocol 4 different groups of rats were used: control rats, rats exposed to hypobaric hypoxia, rats treated with gentamicin prior to hypoxic exposure and finally rats given a dose of 50 U human recombinant erythropoietin for 2 days following treatment with gentamicin. The evaluated hematological parameters included: hemoglobin and hematocrit levels, reticulocyte count and plasma creatinine concentration. Histological analysis of kidney sections was also used. INCSTAR (USA) immunoassay was employed to determine plasma erythropoietin level. Evidence (histological and laboratory-elevated plasma creatinine) of distinct tubular lesions was found in rats injected with gentamicin. Hemoglobin and hematocrit levels were decreased in rats treated with gentamicin but a rise in reticulocyte count was seen on day 3 after hypoxic exposure--39@1000 +/- 10 vs. 5@1000 +/- 1.3 on day 0 (p < 0.001). Reduced reticulocyte counts on day 0 were not seen only in rats injected with human recombinant erythropoietin--36.33@1000 (p < 0.01). Hypoxia-induced increase in plasma erythropoietin was inhibited in animals treated with gentamicin--15.3 mU/ml (on day 0). Both hypoxic exposure and erythropoietin administration were associated with a rise in plasma erythropoietin on day 0--40.5 +/- 4.4 mU/ml and 42.7 +/- 4.1 mU/ml respectively, which normalised on day 3. The results of the study support the hypothesis that the erythropoietin response in rats after stimulation is related to the functional condition of the proximal kidney tubules.

Modulation of erythropoiesis in rat bone marrow erythroblastic islands by cyclooxygenase inhibition.

We designed our study to explore how the inhibition of prostaglandins (PGs) could affect erythropoiesis in bone marrow erythroblastic islands (EIs). To this end, we used hypoxic-stimulated rats-hypobaric hypoxia (42.55 kPa/6 h)-pretreated or not with indomethacin (4 mg/kg/3 days). Blood sampling was done at 0 h, 24 h, and 72 h after hypoxia. The study included estimations of the plasma erythropoietin (EPO) level (by radioimmunoassay), peripheral blood, number of EI from classes I to V per femur, rate of immature cell's differentiation into erythroblasts, and rate of repeated participation of macrophages in new EI reconstruction. Plasma EPO rose significantly (p < 0.01) in all hypoxic rats: 40.5+/-10.15 mU/ml and 46.75+/-16.28 mU/ml and at 0 h versus 13.83+/-6.82 mU/ml in controls. An increased rate of cell differentiation into erythroblasts in EIs (p < 0.01), an enhanced reconstruction in involuted EIs, and a reduced number of maturing EIs (p < 0.01) were observed in all hypoxic animals. However, in indomethacin-pretreated rats, the stimulation of bone marrow erythropoiesis was better expressed. Our results favor the concept that PG inhibition does not attenuate the erythropoietic response to hypoxia and support the hypothesis about the important role of EI macrophages as a local regulator of bone marrow erythropoiesis.

Relationship between absolute and relative hematocrit changes and bone marrow response in rats.

The object of the present study was to evaluate the physiologic mechanisms regulating erythropoiesis by monitoring the changes in reticulocyte count in the circulation and the formation of erythroblastic islands in the bone marrow following hypertransfusion and dehydration. Forty-eight male Wistar rats (110-130 grams) were transfused with 70% isogenic suspension of washed and packed red cells at a dose of 2.5 ml/100 g body weight intravenously on two consecutive days (absolute erythrocytosis) or deprived of water for three days to produce relative erythrocytosis and then injected i.v. with saline at a dose of 2.5 ml/100 g body weight with water provided at libitum. Hematocrit was measured using the microhematocrit method. Reticulocytes were enumerated using the new methylene blue stain. Plasma EPO levels were measured radioimmunologically and a bone marrow suspension was prepared for morphological examination and absolute erythroblastic islands determination. We observed a marked suppression of erythropoiesis following the transfusion as evidenced by the elevation in hematocrit values up to 0.64 +/- 0.04, a decrease in reticulocyte count and plasma EPO reduction--13.5 +/- 3.4 mU/ml (P < 0.05) and a suppression of EO formation in the bone marrow (P < 0.01). The hematocrit of the dehydrated rats was elevated during the first 24 hours (P < 0.001). Reticulocytes, as well as EO, decreased on day 3 down do 53.6 x 10(9)/l +/- 9.8 x 10(9)/l (P < 0.001) and 153.6 x 10(3) +/- 21.5 x 10(3)/femur (P < 0.001), respectively. The EPO level was measured simultaneously--16.3 +/- 4.2 mU/ml versus 24.6 +/- 5.3 mU/ml at day 1 (P < 0.001). Following rehydration, a fast increase in the hematocrit value with a concomitant increase in EO and the reticulocyte counts was observed. Our results show that the absolute and relative changes in hematocrit values are paramount in the feedback mechanism for erythroid homeostasis.

Development of bone marrow erythroblastic islands in hypoxic rats with intact or damaged kidney tubules.

Keeping in mind the renal origin of erythropoietin (EPO), we designed our study in order to estimate the role of kidney damage in the development of bone marrow erythropoiesis in erythroblastic islands (EI). The experiment was performed comparing intact rats with untreated and gentamicin-pretreated rats (50 mg/kg/15 days) exposed to hypobaric pressure (42.55 kPa/6 h) to stimulate hypoxia. Blood samples were taken following a 2-week period. The study included an estimation of plasma EPO levels by RIA, the number of peripheral blood parameters and bone marrow EI (classes I to V/femur), the rate of erythroid differentiation into erythroblasts, and the rate of repeated participation of macrophages in new EI reconstruction. Plasma EPO increased to 52.88 mU/ml (p < 0.01) and 23.45 mU/ml at 0 h immediately following hypobaric exposure in untreated and gentamicin-treated rats, respectively, as compared to 14.25 mU/ml in intact animals. Bone marrow recovery patterns were markedly expressed in untreated hypoxic animals throughout the observed period. The rate of erythroid differentiation into erythroblasts in EI was increased (p < 0.01) while the number of maturing EI decreased (p < 0.01); increased reconstruction was observed in involuted EI. Less pronounced stimulation of erythropoiesis was observed in hypoxic gentamicin-treated rats. The direct impact of the hypoxic stimulus on the erythropoietic bone marrow tissue was considered significant for the erythropoietic response via activation of macrophages. These data support the hypothesis that EI central macrophages play an important role as local regulators of bone marrow erythropoiesis.