Renal Epo Research Articles

Effects of pro- and antioxidative compounds on renal production of erythropoietin.

The most important stimulus for the enhanced synthesis of erythropoietin (Epo) is a lowered O2 tension in the tissue. However, the mechanism by which an impaired O2 supply is transduced into appropriate Epo production is still not fully understood. Recently, studies in human hepatoma cells (line HepG2) indicate that reactive O2 species are involved in the signal transduction from the cellular O2 sensor to the Epo gene. To clarify the role of reactive O2 species in the regulation of Epo synthesis in the kidney, the principal Epo-producing organ in vivo, we investigated the influence of potent pro- and antioxidants on Epo production in isolated perfused rat kidneys. Under normoxic conditions, the iron chelator desferrioxamine and the antioxidant vitamin A increased renal Epo production, mimicking hypoxic induction. In contrast, supplementation of the perfusion medium of hypoxically perfused kidneys with the prooxidant compounds H2O2 or pyrogallol caused a significant reduction of Epo synthesis. The inhibition of Epo formation by reactive O2 species could be completely antagonized by desferrioxamine and the hydroxyl radical-(OH*)-scavenger tetramethylthiourea. Vitamin A also antagonized the H2O2-dependent inhibition of hypoxically induced Epo synthesis. Interestingly, the addition of the antioxidant vitamin A to hypoxically perfused kidneys also induced Epo production significantly. Our data strongly support the idea that reactive O2 species, especially H2O2, are part of the signaling chain of the cellular O2-sensing mechanism regulating the renal synthesis of Epo.

THE EFFECT OF DIFFERENT IMMUNOSUPPRESSION THERAPY ON ERYTHROPOIETIN PRODUCTION IN RENAL TRANSPLANTATION

51 Anemia in chronic renal failure is due to erythropoietin (Epo) deficiency that is expected to normalize after successful kidney transplantation. We previously studied 40 children with excellent graft function for 12 months post kidney transplantation and found a mean hematocrit (Hct) -2 SD from the mean for age and sex. Human hepatoma (Hep3B) cells were chosen as a model to study the effect of immunosuppression therapy on Epo production. Cyclosporine(CSA), Azathioprine (AZA), FK506, and Mycophenolate Mofetil (MPM), under 21% O2 and 1% O2 (hypoxic) conditions were tested. Epo levels were measured in mU/mg protein ± SEM and compared to baseline conditions. Both CSA(10µg/mL) and AZA (40µg/mL) inhibited Epo production under 21% O2, (p<.03, p<.002) and hypoxic conditions (p<.02, p<.05) respectively. Increased concentration of CSA and AZA (100µg/mL) resulted in greater inhibition. FK506 (.0001 -1 µM) and MPM(.01-10µM) did not inhibit Epo production. CSA added to high dose AZA or MPM depressed Epo production further than either agents alone in 21% O2(p<.002 and p<.007) and 1% O2 (p<.004 and p<.0001). We then measured Hct %, plasma Epo, and ferritin, in 4 pediatric renal transplant recipients, ages 4-8 years, 3 were on CSA and one on FK506 at 1, 2, 3, 6, 9 months post transplantation. Simultaneous blood trough levels for CSA were done (whole blood TDx), and for FK506 (whole blood MEIA). No acute rejection episodes or blood transfusions occurred in any patient. Mean Hct% for all patients pre transplant, and then at 1, 3, 6, 9 months post transplantation were 36%, 33%, 38%, 37%, and 38%. All patients had adequate ferritin levels (range of 30-167 ng/ml) in the first 6 months post transplantation. Mean serum creatinine at 6 months was 0.65±2 mg/dl. Epo levels were compared to each Hct value according to a Reference Regression Graph. An inverse linear relationship between log Epo levels and Hct (R2 = 0.341, p=.0002) was found that was less steep than for healthy adults, and similar to patients with renal failure. No correlation was found between CSA or FK506 levels and Hct. Conclusion: CSA and AZA suppress in-vitro Epo production while FK506 and MPM appear to have no effect. The Epo levels produced after transplantation is inappropriately low compared to Hct. We speculate that the mild anemia in children post renal transplantation is due to inhibition of renal interstitial Epo production related to immunosuppression therapy, specifically CSA. The effect of FK506 and MFM on Epo production in-vivo needs further study.

THE EFFECT OF DIFFERENT IMMUNOSUPPRESSION THERAPY ON ERYTHROPOIETIN PRODUCTION IN RENAL TRANSPLANTATION

51 Anemia in chronic renal failure is due to erythropoietin (Epo) deficiency that is expected to normalize after successful kidney transplantation. We previously studied 40 children with excellent graft function for 12 months post kidney transplantation and found a mean hematocrit (Hct) -2 SD from the mean for age and sex. Human hepatoma (Hep3B) cells were chosen as a model to study the effect of immunosuppression therapy on Epo production. Cyclosporine(CSA), Azathioprine (AZA), FK506, and Mycophenolate Mofetil (MPM), under 21% O2 and 1% O2 (hypoxic) conditions were tested. Epo levels were measured in mU/mg protein ± SEM and compared to baseline conditions. Both CSA(10μg/mL) and AZA (40μg/mL) inhibited Epo production under 21% O2,(p<.03, p<.002) and hypoxic conditions (p<.02, p<.05) respectively. Increased concentration of CSA and AZA (100μg/mL) resulted in greater inhibition. FK506 (.0001 -1μM) and MPM (.01-10μM) did not inhibit Epo production. CSA added to high dose AZA or MPM depressed Epo production further than either agents alone in 21% O2 (p<.002 and p<.007) and 1% O2 (p<.004 and p<.0001). We then measured Hct%, plasma Epo, and ferritin, in 4 pediatric renal transplant recipients, ages 4-8 years, 3 were on CSA and one on FK506 at 1, 2, 3, 6, 9 months post transplantation. Simultaneous blood trough levels for CSA were done (whole blood TDx), and for FK506 (whole blood MEIA). No acute rejection episodes or blood transfusions occurred in any patient. Mean Hct% for all patients pre transplant, and then at 1, 3, 6, 9 months post transplantation were 36%, 33%, 38%, 37%, and 38%. All patients had adequate ferritin levels (range of 30-167 ng/ml) in the first 6 months post transplantation. Mean serum creatinine at 6 months was 0.65±.2 mg/dl. Epo levels were compared to each Hct value according to a Reference Regression Graph. An inverse linear relationship between log Epo levels and Hct(R2 = 0.341, p=.0002) was found that was less steep than for healthy adults, and similar to patients with renal failure. No correlation was found between CSA or FK506 levels and Hct. Conclusion: CSA and AZA suppress in-vitro Epo production while FK506 and MPM appear to have no effect. The Epo levels produced after transplantation is inappropriately low compared to Hct. We speculate that the mild anemia in children post renal transplantation is due to inhibition of renal interstitial Epo production related to immunosuppression therapy, specifically CSA. The effect of FK506 and MFM on Epo production in-vivo needs further study.

Effects of antioxidant vitamins on renal and hepatic erythropoietin production

An important role in O2 sensing has been assigned to microsomal and membrane-bound b-type cytochromes which generate regulatory reactive O2 species (ROS). Recently, ROS have been shown to suppress the in vitro synthesis of erythropoietin (Epo). We investigated the potential of the antioxidant vitamins A, E and C to enhance renal and hepatic Epo production. Renal effects were studied in isolated serum-free perfused rat kidneys. In control experiments without antioxidant vitamins, Epo secretion amounted to 441 +/- 23 mU/g kidney (mean +/- SEM, N = 5) during the three hour period of hypoxic perfusion (arterial pO2 35 mm Hg). Epo secretion significantly increased to 674 +/- 92 mU/g kidney (N = 7) when vitamins A (0.5 microgram/ml), E (0.5 microgram/ml) and C (10 micrograms/ml) in combination were added to the perfusion medium. The effects of the single vitamins were studied in Epo-producing hepatoma cell cultures (lines HepG2 and Hep3B). Vitamin A induced a dose-dependent increase (half-maximal stimulation at 0.2 microgram/ml) in the production of immunoreactive Epo during 24 hours of incubation (such as 680 +/- 51 U Epo/g cell protein in HepG2 cultures with 3 micrograms/ml retinol acetate compared to 261 +/- 15 U/g in untreated controls; N = 4). In contrast, vitamin E (tested from 0.05 to 500 micrograms/ml) and vitamin C (tested from 2 to 200 micrograms/ml) did not increase Epo production in hepatoma cell cultures. Thus, while vitamins E and C may have the potential to protect cells from oxidative damage, vitamin A exerts a specific stimulation of Epo production. Preliminary evidence suggests that this effect of vitamin A involves increased mRNA levels of hypoxia-inducible factor 1 alpha (HIF-1 alpha).

Immunohistochemical colocalization of the α-subunit of neutrophil NADPH oxidase and ecto-5′-nucleotidase in kidney and liver

In kidney and liver, fibroblasts and fibroblast-like cells, respectively, are sources of erythropoietin (Epo) formation, and these cells also bear a number of other similarities. Renal Epo expression is localized in peritubular type 1 fibroblasts of the cortical labyrinth, and in the liver, apart from parenchymal cells, transcription is found in Ito cells. Both the renal peritubular cells and Ito cells contain ecto-5'-nucleotidase (5'NT). It had been suggested that 5'NT is involved in the oxygen sensing mechanism via a hydrolysis of AMP to adenosine, which in turn may stimulate EPO synthesis. However, the molecular mechanism of the cellular response to hypoxia is currently not well understood. Based on the notion that a heme protein probably acts as the oxygen sensor, it has recently been proposed that a b-type cytochrome as part of the neutrophil NADPH oxidase may influence intracellular superoxide levels depending on local oxygen tension. Superoxide levels were otherwise shown to determine the EPO production in hepatoma cell lines. By double immunofluorescence labeling the alpha-subunit of cytochrome b558 (alpha-SU) and 5'NT were simultaneously localized in rat kidney and liver, and in the kidney Epo mRNA and alpha-SU were double-labeled. Positive signal for alpha-SU was found in the majority of renal peritubular fibroblasts in the cortex and outer medulla, and in Ito cells. In both organs, the cells that coexpress 5'NT and Epo mRNA also contain an immunoreactivity for alpha-SU. In these cells, cytochrome b558 as part of an NADPH oxidase may be involved in a presumptive oxygen sensing mechanism using H2O2 as a possible second messenger for EPO gene regulation.

Erythropoietin production in rats with post-ischemic acute renal failure

To study the role of erythropoietin (Epo) in the pathogenesis of anemia in acute renal failure (ARF), organ Epo mRNA was measured by RNase protection assay in rats with ARF induced by a one hour-occlusion of the left renal artery. Hematocrit was significantly decreased two hours, 24 hours and one week after renal artery occlusion. A significant reduction in serum haptoglobin at two hours and an increase in serum LDH at 24 hours indicated that hemolysis was the likely cause of the initial fall in hematocrit. However, despite the reduced hematocrit, serum Epo concentrations were not significantly different from controls, suggesting that the anemia is maintained because of lack of an appropriate Epo response. Right renal Epo mRNA levels were not significantly different in all groups, but Epo mRNA levels in post-ischemic kidneys were 50 to 67% lower than in contralateral kidneys. However, Epo mRNA in the post-ischemic kidney was increased sixfold by acute hemorrhage, a rise comparable to the ninefold increase observed in contralateral kidneys. In ARF rats exposed to 7.5% O2 for four hours, right kidney Epo mRNA increased 200-fold over normoxic levels, to a value similar to sham-operated hypoxic controls. Epo mRNA in the post-ischemic kidney also increased 200-fold, to 50% of the level in the contralateral kidney. Hepatic Epo mRNA levels were elevated to comparable levels in both groups. In this ARF model, mild anemia is associated with relative Epo deficiency. In the post-ischemic kidney, a substantial capacity for Epo production is retained but the sensitivity of the Epo response to blood oxygen availability is significantly reduced.

Regulation of erythropoietin gene expression in fetal sheep by glucocorticoids.

Erythropoietin (Epo) gene expression in ovine fetal liver and kidneys was measured by competitive RT-PCR in situations in which fetal glucocorticoid status was altered. Bilateral adrenalectomy at 120 +/- 0.3 days gestation (term is 145-150 days) caused a significant (P < 0.05) 5-fold increase in renal Epo messenger RNA (mRNA) levels at 145 +/- 1 days compared to those in age-matched controls. With cortisol replacement in adrenalectomized fetuses, renal Epo mRNA levels dropped to 17% of this values (P < 0.05). Cortisol infusion (230 micrograms/h for 48 h) at 108 +/- 1 day decreased renal Epo gene expression significantly (P < 0.01) to 23% of the control value; dexamethasone treatment of the ewe at midgestation (0.76 mg/h for 48 h) also decreased fetal, but not adult, renal Epo mRNA levels (to 12% of control value; P < 0.01). Fetal and maternal liver Epo mRNA levels were unaffected by glucocorticoid status at any stage of pregnancy. Thus, glucocorticoids can influence fetal renal, but not maternal, Epo gene expression. In the presence of high concentrations of fetal glucocorticoids, plasma Epo values were consistently 4-5 mU/ml, close to the sensitivity of the assay, whereas in seven adrenalectomized fetuses, the plasma Epo value was 9.1 +/- 1.4 mU/ml.

Characterization of the Han:SPRD rat model for hereditary polycystic kidney disease

The Han:SPRD rat model for inherited polycystic kidney disease (PKD) was characterized (clinical parameters, morphology, immunohistochemistry and in situ hybridization). Homozygous animals died of uremia after three to four weeks with severe cystic transformation of virtually all nephrons and collecting ducts (serum urea: 616 +/- 195 mg/dl; kidney-to-body weight ratio: > 20%). In heterozygotes, slow progression of the disease led to death between the 12th and 21st month (median: 17 months; serum urea levels above 200 mg/dl). Kidney enlargement was moderate, and cysts were restricted to the cortex and outer medulla. Immunohistochemical markers showed that approximately 75% of the cysts were derived from the proximal tubule. Cystic transformation started in the proximal tubule with a sharp onset of basement membrane alteration and a loss of epithelial differentiation restricted to small focal areas. In these areas, alpha 1(IV) collagen and laminin B1 mRNA were enhanced as revealed by isotopic and non-isotopic in situ hybridization. Fibroblasts underlying the affected tubular portions were involved in matrix overexpression resulting in subepithelial accumulation of immunoreactive collagen IV and laminin. In later stages of cystic transformation distal nephron segments were affected as well. A reversal in epithelial polarity as judged from Na,K-ATPase-immunoreactivity was not observed. Renal immunoreactive renin-status was significantly decreased. Hematocrit was lowered in heterozygotes (40.4 +/- 5.8 vol% compared to 46.7 +/- 1.99 vol% in controls; P < 0.05) and total renal EPO mRNA was reduced to 36 +/- 14% of the mean value of control animals, whereas serum EPO levels were not significantly altered. We conclude that the Han:SPRD rat is a useful model for the study of human ADPKD since both diseases are similar in several aspects. The model is particularly suitable for the study of epithelial-mesenchymal interactions at the beginning of tubular cystic transformation.

Stimulation of erythropoietin in renal insufficiency by hypobaric hypoxia

Patients with renal anaemia show inadequate levels of immunoreactive erythropoietin (Epo) related to the degree of anaemia. The purpose of our study is to compare the degree of stimulation of Epo by means of hypobaric hypoxia in normal controls and patients with renal anaemia. Baseline Epo concentrations were found to be 11.1 +/- 2.0 U/l in 10 healthy volunteers and 11.4 +/- 4.6 U/l in six patients with renal anaemia. After exposure to hypobaric hypoxia equivalent to 4560 m above sea level for a duration of 3.5 h, we observed a significant increase in serum Epo in healthy volunteers to 22.8 +/- 9.1 U/l (P < 0.005), while there was no increase in patients with renal anaemia: 12.3 +/- 5.2 U/l (P < 0.2). Our results show that in patients with renal anaemia serum Epo concentrations are comparable to those of normal controls, but inadequate in view of the concomitant degree of anaemia. Stimulation by acute hypobaric hypoxia was not possible in patients with renal insufficiency as opposed to normal controls. From these data it can be concluded that either Epo production is working at maximum capacity under baseline conditions, or an additional hybobaric stimulus is not able to influence a disturbed set point of the oxygen sensor regulating Epo synthesis.

Impaired Erythropoietin Production in Mice Treated With Cyclosporin A

Because recent data indicate that erythropoietin (Epo) production is defective in allogeneic bone marrow transplant (BMT) patients, we investigated the role of the immunosuppressant, nephrotoxic, agent cyclosporin A (CsA) on renal Epo production using an animal model. Mice were injected with 1.0 to 40.0 mg/kg/d CsA for 15 days. Thereafter, circulating Epo levels were evaluated in both intact animals and in mice made anemic with phenylhydrazine (PHZ). Serum Epo levels measured in CsA-treated animals were then compared with the predicted levels, which had been calculated in a reference population of normal, either intact or anemic, mice. In CsA-treated, intact animals both hematocrit and serum Epo levels were not significantly different from controls. However, serum Epo levels in CsA-treated, anemic mice were significantly lower than those expected in a control population of untreated, anemic mice with similar degrees of anemia. No significant increase in serum creatinine was recorded even at the highest doses of CsA used, nor were we able to document signs of renal toxicity by histologic examination of the kidneys. Therefore, therapeutical doses of CsA appear to affect the production of Epo under conditions in which the demand of the hormone is increased, as in response to anemia. We suggest that a subclinical kidney toxicity produced by CsA might have a role in the pathogenesis of the impaired Epo production observed in BMT patients, and may contribute to a delayed erythroid engraftment in at least some BMT patients.

Impaired erythropoietin production in mice treated with cyclosporin A

Because recent data indicate that erythropoietin (Epo) production is defective in allogeneic bone marrow transplant (BMT) patients, we investigated the role of the immunosuppressant, nephrotoxic, agent cyclosporin A (CsA) on renal Epo production using an animal model. Mice were injected with 1.0 to 40.0 mg/kg/d CsA for 15 days. Thereafter, circulating Epo levels were evaluated in both intact animals and in mice made anemic with phenylhydrazine (PHZ). Serum Epo levels measured in CsA-treated animals were then compared with the predicted levels, which had been calculated in a reference population of normal, either intact or anemic, mice. In CsA-treated, intact animals both hematocrit and serum Epo levels were not significantly different from controls. However, serum Epo levels in CsA-treated, anemic mice were significantly lower than those expected in a control population of untreated, anemic mice with similar degrees of anemia. No significant increase in serum creatinine was recorded even at the highest doses of CsA used, nor were we able to document signs of renal toxicity by histologic examination of the kidneys. Therefore, therapeutical doses of CsA appear to affect the production of Epo under conditions in which the demand of the hormone is increased, as in response to anemia. We suggest that a subclinical kidney toxicity produced by CsA might have a role in the pathogenesis of the impaired Epo production observed in BMT patients, and may contribute to a delayed erythroid engraftment in at least some BMT patients.

Organ distribution of erythropoietin messenger RNA in normal and uremic rats

We used RNAase protection assays to measure low levels of erythropoietin messenger RNA (EPO mRNA) in the organs of unstimulated rats, and to compare basal and stimulated levels of EPO mRNA in the kidneys and extrarenal organs of rats rendered uremic by subtotal nephrectomy, with pair-fed controls. Using this sensitive assay, EPO mRNA was measured in the kidneys of unstimulated control animals and was detectable, at lower levels, in the liver and lung. After exposure to hypoxia, there was a 150-fold increase in renal EPO mRNA. Hepatic EPO mRNA was also greatly increased and accounted for 39 +/- 10% of the total. The small quantity of EPO mRNA in lung did not increase, but EPO mRNA became detectable in spleen. Animals subjected to subtotal nephrectomy became uremic and anemic (hematocrit 0.32 +/- 0.04 vs. 0.43 +/- 0.04 in controls, P = 0.002), but serum EPO concentrations were not significantly increased (32 +/- 9 vs. 24 +/- 6 mU/ml, P = 0.14). However, after hypoxic exposure, uremic animals increased serum EPO concentrations greatly, although the response was less than in controls (349 +/- 82 vs. 1009 +/- 238 mU/ml, P = 0.002). After hypoxia, extrarenal EPO mRNA levels in uremic animals were similar to controls. In particular, the large hepatic potential for EPO mRNA synthesis was unchanged but accounted for a greater proportion (84 +/- 5%) of the total EPO mRNA. The renal EPO mRNA content was reduced, as expected, after subtotal nephrectomy, but increased 50-fold after hypoxia. In this model of chronic renal failure, despite anemia, a large potential for EPO synthesis exists in liver and remnant kidney.

Physiologic Regulation and Tissue Localization of Renal Erythropoietin Messenger RNA

Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

Physiologic regulation and tissue localization of renal erythropoietin messenger RNA

Although erythropoietin (Epo) is produced primarily by the kidneys in response to hypoxia, the precise cell type(s) and mechanisms by which these cells regulate production are poorly understood. In the experiments we report, the kinetics of renal Epo production in response to acute hypoxia and the intrarenal localization of cellular Epo synthesis were studied at the level of Epo mRNA. Erythropoietin mRNA expression was determined by Northern blot analysis of rat kidney RNAs using a probe derived from the mouse Epo gene. Renal Epo mRNA content increased as early as 1 hour after initiation of hypoxia and continued to accumulate during 4 hours of stimulation. Discontinuation of the hypoxic stimulus resulted in rapid decay of mRNA levels. Kidney and plasma Epo levels measured by radioimmunoassay paralleled, with respective lag times, the changes in renal Epo mRNA content, suggesting that Epo production in response to acute hypoxia represents de novo synthesis and is regulated by changes in Epo mRNA. Northern blot analysis of RNAs extracted from separated glomerular and tubular tissue fractions revealed Epo mRNA in the tubular fraction, whereas glomerular tissue did not contain Epo mRNA. Thus, the site of cellular Epo synthesis is located in the renal tubule or its interstitium and not in the glomerular tuft.

Increased Immunoreactive Erythropoietin in Serum and Kidney Extracts of Rats With Ni3S2-Induced Erythrocytossis

Radioimmunoassay of erythropoietin (epo) was performed upon serum samples and kidney extracts from rats that received intrarenal injections of nickel subsulfide (Ni/sub 3/S/sub 2/, 20 mg/rat). Serum epo concentrations were significantly increased from 1 to 4 weeks after Ni/sub 3/S/sub 2/ injection; the highest values occurred at 3 weeks, when serum epo in 5 Ni/sub 3/S/sub 2/-treated rats averaged 230 mU/ml (range = 94 - 370 mU/ml); for comparison, serum epo in 21 vehicle-treated controls averaged 19 mU/ml (range = <14 - 56 mU/ml, P <0.01). In kidney extracts of 5 rats killed 3 weeks after Ni/sub 3/S/sub 2/ injection, epo concentrations averaged 970 mU/g (range = 210 - 1880 mU/g); for comparison, epo in kidney extracts of 6 controls averaged 99 mU/g (range = 40 - 140 mU/g, P <0.005). These radioimmunoassays confirm previous bioassay findings that serum epo concentrations are significantly increased in rats with Ni/sub 3/S/sub 2/-induced erythrocytosis. Contrary to bioassay findings, this study demonstrates increased immunoreactive epo concentrations in kidney extracts of Ni/sub 3/S/sub 2/-treated rats, suggesting that renal epo in Ni/sub 3/S/sub 2/-treated rats occurs in a form that lacks biological activity in vivo. 15 references, 2 figures.