Erythropoietin response to acute hypobaric or anaemic hypoxia in gentamicin-administered rats (original) (raw)
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The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin β pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression...
Temporal pattern of erythropoietin titers in kidney tissue during hypoxic hypoxia
Pfl�gers Archiv European Journal of Physiology, 1982
Plasma titers of erythropoietin (Ep) are known to increase initially during hypoxia and to return then towards prehypoxia values. To find out if this pattern of plasma Ep might be related to changes in the production of the hormone, I have compared plasma with kidney Ep titers in hypoxic rats. Rats were exposed to hypoxia in a hypobaric chamber at 0.42 atm for various time intervals for up to 4 days. Kidney Ep titers were assayed in extracts from kidneys that had been flushed free of blood in situ. It was found that kidneys of normal rats do not store significant amounts of Ep. Kidney Ep titers increased transiently during hypoxia. They reached maximum values after 6h and then declined to almost undetectable levels at continued hypoxia. In the plasma, maximum values were found after 12-18h of hypoxia. Additional studies were done on the effects of discontinuous hypoxia. It was found that, even after 3 days of previous hypoxia exposure, plasma and kidney Ep titers increased again in rats when these were maintained intermittently in normoxia for 18 h. It is concluded that the rise and fall in plasma Ep titers during hypoxia reflect similar changes in kidney Ep production.
Demonstration of high levels of erythropoietin in rat kidneys following hypoxic hypoxia
Pfl�gers Archiv European Journal of Physiology, 1981
Controversial hypotheses exist as to whether hypoxic kidneys produce biologically active erythropoietin (Ep) or an inactive erythropoietic factor that generates Ep from plasma protein in the blood. To clarify the role of the kidney in Ep production we attempted to extract Ep from kidneys of normal and of hypoxia exposed (6 h at 0.42 atm) Sprague-Dawley rats. Ep was measured in the microsomal fraction of kidney homogenates, using the exhypoxic polycythemic mouse assay for Ep. The Ep content was also determined in kidneys that were flushed free of blood with isotonic phosphate-buffer prior to extirpation. We found 0.04 U Ep/g in blood-depleted kidneys of normal rats. Upon exposure of the animals to hypoxia the Ep level increased to 0.92 U/g kidney. Ep levels were significantly higher in the kidney cortex than in the medulla. The erythropoietic activity in renal extracts was not enhanced after incubation of samples with homologous serum. Ep extracted from hypoxic kidneys behaved identically with plasma-Ep in the following biochemical tests: heat stability, affinity chromatography, with wheat germ lectin, ion exchange chromatography, molecular sieve chromatography, and neuraminidase inactivation. These studies support the hypothesis that kidney cortex cells are capable of producing biologically active Ep.
Distribution of erythropoietin producing cells in rat kidneys during hypoxic hypoxia
Kidney …, 1993
Distribution of erythropoietin producing cells in rat kidneys during hypoxic hypoxia. We have used in situ hybridization to determine the localization and distribution of cells expressing the erythropoietin (EPO) gene in kidneys of rats exposed to reduced oxygen tensions to characterize the control of renal EPO formation during hypoxic hypoxia. Animals were subjected to severe hypoxia (7.5% 02) for4, 8 and
Lowered plasma erythropoietin in hypoxic rats with kidney tubule lesions
Blut, 1988
The role of the kidney tubules in the renal formation of erythropoietin is incompletely understood. Therefore, the capability to produce erythropoietin in response to hypoxia was studied in rats with tubular lesions. Nephron damage was induced in two different ways. First, rats were treated with the nephrotoxic aminoglycoside gentamicin (67.5 mg/kg and day) for 14 days. The animals were then subjected to simulated altitude (6,800 m) for 6 h. The resulting plasma erythropoietin concentration was significantly lower (0.5 IU/ml) than in saline treated control rats exposed to hypoxia (1.0 IU/ml). Second, unilateral hydronephrosis was induced by ureteral ligation. The contralateral kidney was removed immediately before the animals were exposed to siulated altitude for 6 h. The plasma erythropoietin concentration in the ureterligated rats did not increase above the value (0.3 IU/ml) in hypoxia exposed anephric rats. These results indicate that the production of erythropoietin is reduced following tubular injury. Tubule cells may diretly produce the hormone or interfere with the O2-sensing mechanisms controlling its synthesis. The latter hypothesis would seem to be supported by our failure to demonstrate in vitro erythropoietin production by the two established kidney tubule cell lines, LLC-PK1 and PK-15.
Protective effect of Epo on oxidative renal injury in rats with cyclosporine nephrotoxicity
Pediatric Nephrology, 2008
The aim of our study was to determine the effect of recombinant human erythropoietin (rhEPO) on cyclosporine (CsA) nephrotoxicity. Twenty-six female Wistar rats were injected with 15 mg/kg subcutaneous CsA and intraperitoneal saline/rhEPO for 28 days. Four groups were formed: Group 1 (n = 5), a control group; Group 2 (n = 7), CsA + saline; Group 3 (n = 7), CsA + low dose (20 U/kg per day) rhEPO; Group 4 (n = 7), CsA + high dose (100 U/kg per day) rhEPO. Body weights, creatinine clearance, urinary protein/creatinine, hematocrit, serum creatinine levels, histopathological parameters, apoptosis and lipid peroxidation tests were compared between the three groups. Body weights and renal functions were similar in Groups 2, 3 and 4 rats but significantly lower than the values found for the control group at the end of the study. The hematocrit was significantly different between the four groups, showing a positive association with the strength of the injected rhEPO doses. Tubular and arteriolar damage was significantly lower in Groups 3 and 4 rats than in Group 2 rats, while chronic changes were similar between the three groups. TUNEL-positive cells and thiobabarbituric acid reacting substances(TBARS) levels were significantly higher in Group 2 rats, whereas superoxide dismutase levels were significantly lower in Group 2 rats than in those of the other three groups. Low or high dose rhEPO had no significant protective effects on body weight, renal functions, chronic fibrotic changes, but both doses reduced tubular and arteriolar changes, apoptotis and oxidative stress.
Erythropoietin protects against ischaemic acute renal injury
Nephrology Dialysis Transplantation, 2004
Background. Erythropoietin (EPO) has recently been shown to exert important cytoprotective and antiapoptotic effects in experimental brain injury and cisplatin-induced nephrotoxicity. The aim of the present study was to determine whether EPO administration is also renoprotective in both in vitro and in vivo models of ischaemic acute renal failure. Methods. Primary cultures of human proximal tubule cells (PTCs) were exposed to either vehicle or EPO (6.25-400 IU/ml) in the presence of hypoxia (1% O 2), normoxia (21% O 2) or hypoxia followed by normoxia for up to 24 h. The end-points evaluated included cell apoptosis (morphology and in situ end labelling [ISEL], viability [lactate dehydrogenase (LDH release)], cell proliferation [proliferating cell nuclear antigen (PCNA)] and DNA synthesis (thymidine incorporation). The effects of EPO pre-treatment (5000 U/kg) on renal morphology and function were also studied in rat models of unilateral and bilateral ischaemia-reperfusion (IR) injury. Results. In the in vitro model, hypoxia (1% O 2) induced a significant degree of PTC apoptosis, which was substantially reduced by co-incubation with EPO at 24 h (vehicle 2.5±0.5% vs 25 IU/ml EPO 1.8±0.4% vs 200 IU/ml EPO 0.9±0.2%, n ¼ 9, P<0.05). At high concentrations (400 IU/ml), EPO also stimulated thymidine incorporation in cells exposed to hypoxia with or without subsequent normoxia. LDH release was not significantly affected. In the unilateral IR model, EPO pre-treatment significantly attenuated outer medullary thick ascending limb (TAL) apoptosis (EPO 2.2±1.0% of cells vs vehicle 6.5±2.2%, P<0.05, n ¼ 5) and potentiated mitosis (EPO 1.1±0.3% vs vehicle 0.5±0.3%, respectively, P<0.05) within 24 h. EPO-treated rats exhibited enhanced PCNA staining within the proximal straight tubule (6.9±0.7% vs vehicle 2.4±0.5% vs sham 0.3±0.2%, P<0.05), proximal convoluted tubule (2.3±0.6% vs vehicle 1.1±0.3% vs sham 1.2±0.3%, P<0.05) and TAL (4.7±0.9% vs vehicle 0.6±0.3% vs sham 0.3±0.2%, P<0.05). The frequency of tubular profiles with luminal cast material was also reduced (32.0±1.6 vs vehicle 37.0±1.3%, P ¼ 0.05). EPO-treated rats subjected to bilateral IR injury exhibited similar histological improvements to the unilateral IR injury model, as well as significantly lower peak plasma creatinine concentrations than their vehicle-treated controls (0.04±0.01 vs 0.21±0.08 mmol/l, respectively, P<0.05). EPO had no effect on renal function in sham-operated controls. Conclusions. The results suggest that, in addition to its well-known erythropoietic effects, EPO inhibits apoptotic cell death, enhances tubular epithelial regeneration and promotes renal functional recovery in hypoxic or ischaemic acute renal injury.
A study on the Effect of Erythropoietin Treatment on Healing of Renal Damage in Male Albino Rats
Bulletin of Egyptian Society for Physiological Sciences, 2009
This study investigated the healing effect of erythropoietin treatment on renal damage in male albino rats. This work was carried out on 24 male albino rats, divided into four equal groups. Group (1) Control group: injected by 0.2ml saline intraperitonealy (2) Mercuric chloride (HgCl 2) treated group: rats were injected intraperitonealy by single dose(3mg/kg) of HgCl 2 , group(3): Erythropoietin (Epo) treated group: rats were treated by intraperitoneal injection of Epo (1000u/kg) /day for 2 weeks, and group (4) HgCl 2 and Epo treated group: rats were injected by single dose of HgCl 2 and Epo for 2 weeks. At the end of experimental period, rats were sacrificed and blood samples were collected and sera were separated for estimation of serum levels of creatinine, urea, glutathione peroxidase, glutathione concentration, malondialdehyde and haematocrit value. The abdomen was dissected and kidney was excised and fixed in formalin for histopathological examination. The results showed in HgCl2 treated group, significant increase in serum creatinine, urea and malondialdehyde levels, and significant reduction in glutathione concentration, glutathione peroxidase and haematocrit value (HV) levels compared with control. Epo treated group showed significant reduction in serum levels of creatinine, and malondialdehyde, and significant increase in HV value levels, compared with the control. HgCl 2 and Epo treated group showed, signification reduction in malondialdehyde, creatinine and urea and significant increase in glutathione concentration, glutathione peroxidase and HV compared with HgCl 2 group. Histopathological examination showed necrosis of renal tubular epithelium and dilated proximal and distal tubules and wide Bowman's capsule in HgCl 2 treated group. HgCl 2 and Epo treated group showed improvement of renal tubular epithelium, mild dilatation of Bowman's capsule and bone marrow derived cells. It is concluded that, Epo treatment improved renal damage due to HgCl2 and promote healing of renal tissue, and it is recommended to be used in chronic renal disease.