Iron-Hepcidin Dysmetabolism,Anemia and Renal Hypoxia,Inflammation and Fibrosis in the Remnant Kidney Rat Model

Anemia is a common complication of chronic kidney disease (CKD) that develops early and its severity increases as renal function declines. It is mainly due to a reduced production of erythropoietin (EPO) by the kidneys; however, there are evidences that iron metabolism disturbances increase as CKD progresses. Our aim was to study the mechanisms underlying the development of anemia of CKD, as well as renal damage, in the remnant kidney rat model of CKD induced by 5/6 nephrectomy. This model of CKD presented a sustained degree of renal dysfunction, with mild and advanced glomerular and tubulointerstitial lesions. Anemia developed 3 weeks after nephrectomy and persisted throughout the protocol. The remnant kidney was still able to produce EPO and the liver showed an increased EPO gene expression. In spite of the increased EPO blood levels, anemia persisted and was linked to low serum iron and transferrin levels, while serum interleukin (IL)-6 and high sensitivity C-reactive protein (hs-CRP) levels showed the absence of systemic inflammation. The increased expression of duodenal ferroportin favours iron absorption; however, serum iron is reduced which might be due to iron leakage through advanced kidney lesions, as showed by tubular iron accumulation. Our data suggest that the persistence of anemia may result from disturbances in iron metabolism and by an altered activity/function of EPO as a result of kidney cell damage and a local inflammatory milieu, as showed by the increased gene expression of different inflammatory proteins in the remnant kidney. In addition, this anemia and the associated kidney hypoxia favour the development of fibrosis, angiogenesis and inflammation that may underlie a resistance to EPO stimuli and reduced iron availability. These findings might contribute to open new windows to identify putative therapeutic targets for this condition, as well as for recombinant human EPO (rHuEPO) resistance, which occurs in a considerable percentage of CKD patients.     

Hepcidin-25 in Diabetic Chronic Kidney Disease Is Predictive for Mortality and Progression to End Stage Renal Disease

Background

Anemia is common and is associated with impaired clinical outcomes in diabetic chronic kidney disease (CKD). It may be explained by reduced erythropoietin (EPO) synthesis, but recent data suggest that EPO-resistance and diminished iron availability due to inflammation contribute significantly. In this cohort study, we evaluated the impact of hepcidin-25—the key hormone of iron-metabolism—on clinical outcomes in diabetic patients with CKD along with endogenous EPO levels.

Methods

249 diabetic patients with CKD of any stage, excluding end-stage renal disease (ESRD), were enrolled (2003–2005), if they were not on EPO-stimulating agent and iron therapy. Hepcidin-25 levels were measured by radioimmunoassay. The association of hepcidin-25 at baseline with clinical variables was investigated using linear regression models. All-cause mortality and a composite endpoint of CKD progression (ESRD or doubling of serum creatinine) were analyzed by Cox proportional hazards models.

Results

Patients (age 67 yrs, 53% male, GFR 51 ml/min, hemoglobin 131 g/L, EPO 13.5 U/L, hepcidin-25 62.0 ng/ml) were followed for a median time of 4.2 yrs. Forty-nine patients died (19.7%) and forty (16.1%) patients reached the composite endpoint. Elevated hepcidin levels were independently associated with higher ferritin-levels, lower EPO-levels and impaired kidney function (all p<0.05). Hepcidin was related to mortality, along with its interaction with EPO, older age, greater proteinuria and elevated CRP (all p<0.05). Hepcidin was also predictive for progression of CKD, aside from baseline GFR, proteinuria, low albumin- and hemoglobin-levels and a history of CVD (all p<0.05).

Conclusions

We found hepcidin-25 to be associated with EPO and impaired kidney function in diabetic CKD. Elevated hepcidin-25 and EPO-levels were independent predictors of mortality, while hepcidin-25 was also predictive for progression of CKD. Both hepcidin-25 and EPO may represent important prognostic factors of clinical outcome and have the potential to further define “high risk” populations in CKD.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92.5 ±3.0) g/L and 0.29±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92. 5±3.0) g/L and 0.29 ±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

人红细胞生成素单克隆抗体的制备、鉴定及应用研究

用rhEPo作为抗原,免疫BALB／c小鼠,取其脾细胞与x63Ag8．653小鼠骨髓瘤细胞融合,再碱性PAGE方法进一步分离并纯化的rhEpo,包被Pvc板,对杂交瘤用ELlSA方法进行筛选,获得两株稳定分泌抗hEPO单抗的杂交瘤细胞株。经鉴定分别属于IgG1、IgG2b,轻链均为k链,Kd分别为5．53×10^-10mol／L和1．34×1O^-10mol／L．用western blot方法证明两者对hEPO具有高度韵专一性．能特异地识别rhEPO和尿源hEPO。所制备单抗可作为亲和层析的配体,用于再生障碍性贫血病人尿中EPO及哺乳类工程细胞所表达的hEPO的分离、纯化,并可用于hEPO的定量检测．     

Preischemic targeting of HIF prolyl hydroxylation inhibits fibrosis associated with acute kidney injury

Acute kidney injury (AKI) due to ischemia is an important contributor to the progression of chronic kidney disease (CKD). Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia-inducible factors (HIF), which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. While activation of HIF protects from ischemic cell death, HIF has been shown to promote fibrosis in experimental models of CKD. The impact of HIF activation on AKI-induced fibrosis has not been defined. Here, we investigated the role of pharmacologic HIF activation in AKI-associated fibrosis and inflammation. We found that pharmacologic inhibition of HIF prolyl hydroxylation before AKI ameliorated fibrosis and prevented anemia, while inhibition of HIF prolyl hydroxylation in the early recovery phase of AKI did not affect short- or long-term clinical outcome. Therefore, preischemic targeting of the PHD/HIF pathway represents an effective therapeutic strategy for the prevention of CKD resulting from AKI, and it warrants further investigation in clinical trials.     

The HIF signaling pathway in osteoblasts directly modulates erythropoiesis through the production of EPO

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.     

Stabilization of hypoxia-inducible factor 1α by cobalt chloride impairs podocyte morphology and slit-diaphragm function

Glomerular podocytes are the major components of the renal filtration barrier, and altered podocyte permselectivity is a key event in the pathogenesis of proteinuric conditions. Clinical conditions such as ischemia and sleep apnea and extreme physiological conditions such as high-altitude sickness are presented with renal hypoxia and are associated with significant proteinuria. Hypoxia is considered as an etiological factor in the progression of acute renal injury. A sustained increase in hypoxia-inducible factor 1α (HIF1α) is a major adaptive stimulus to the hypoxic conditions. Although the temporal association between hypoxia and proteinuria is known, the mechanism by which hypoxia elicits proteinuria remains to be investigated. Furthermore, stabilization of HIF1α is being considered as a therapeutic option to treat anemia in patients with chronic kidney disease. Therefore, in this study, we induced stabilization of HIF1α in glomerular regions in vivo and in podocytes in vitro upon exposure to cobalt chloride. The elevated HIF1α expression is concurrence with diminished expression of nephrin and podocin, podocyte foot-processes effacement, and significant proteinuria. Podocytes exposed to cobalt chloride lost their arborized morphology and cell-cell connections and also displayed cytoskeletal derangements. Elevation in expression of HIF1α is in concomitance with loss of nephrin and podocin in patients with diabetic nephropathy and chronic kidney disease. In summary, the current study suggests that HIF1α stabilization impairs podocyte function vis-à-vis glomerular permselectivity.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

Renal protection in chronic kidney disease: hypoxia-inducible factor activation vs. angiotensin II blockade

The 5/6(th) nephrectomy or ablation/infarction (A/I) preparation has been used as a classic model of chronic kidney disease (CKD). We observed increased kidney oxygen consumption (Q(O2)) and altered renal hemodynamics in the A/I kidney that were normalized after combined angiotensin II (ANG II) blockade. Studies suggest hypoxia inducible factor as a protective influence in A/I. We induced hypoxia-inducible factor (HIF) and HIF target proteins by two different methods, cobalt chloride (CoCl(2)) and dimethyloxalyglycine (DMOG), for the first week after creation of A/I and compared the metabolic and renal hemodynamic outcomes to combined ANG II blockade. We also examined the HIF target proteins expressed by using Western blots and real-time PCR. Treatment with DMOG, CoCl(2), and ANG II blockade normalized kidney oxygen consumption factored by Na reabsorption and increased both renal blood flow and glomerular filtration rate. At 1 wk, CoCl(2) and DMOG increased kidney expression of HIF by Western blot. In the untreated A/I kidney, VEGF, heme oxygenase-1, and GLUT1 were all modestly increased. Both ANG II blockade and CoCl(2) therapy increased VEGF and GLUT1 but the cobalt markedly so. ANG II blockade decreased heme oxygenase-1 expression while CoCl(2) increased it. By real-time PCR, erythropoietin and GLUT1 were only increased by CoCl(2) therapy. Cell proliferation was modestly increased by ANG II blockade but markedly after cobalt therapy. Metabolic and hemodynamic abnormalities were corrected equally by ANG II blockade and HIF therapies. However, the molecular patterns differed significantly between ANG II blockade and cobalt therapy. HIF induction may prove to be protective in this model of CKD.     

慢性心力衰竭患者内源性促红细胞生成素的改变及临床意义

目的：探讨内源性促红细胞生成素（EPO）在慢性心力衰竭（CHF）及CHF贫血发病中的作用及临床价值。方法;采用放射性免疫分析法测定117例CHF患者和40例非CHF患者血浆EPO水平,分析EPO水平与心功能分级、贫血以及CHF患者预后的关系。结果：心功能II-IV患者EPO水平显著上升,与对照组比较均有显著性差异（P〈0．05）;EPO水平随着心功能分级增高而逐渐上升,在各级间比较有显著性差异（P〈0．05）;CHF伴贫血患者EPO水平随着心功能分级增高和贫血程度的加重而逐渐上升,在各级间比较有显著性差异（P〈0．05）;死亡组EPO水平显著高于存活组,而Hb水平显著低于存活组,相比较有显著性差畀（P〈0．05）。结论：CHF患者和CHF伴贫血患者内源性EPO水平升高,EPO水平的上升与CHF患者病情严重程度有关,并且是CHF患者预后不良的预测指标。     

Prevalence of Anemia in Chronic Kidney Disease in the United States

Anemia is one of the many complications of chronic kidney disease (CKD). However, the current prevalence of anemia in CKD patients in the United States is not known. Data from the National Health and Nutrition Examination Survey (NHANES) in 2007–2008 and 2009–2010 were used to determine the prevalence of anemia in subjects with CKD. The analysis was limited to adults aged >18 who participated in both the interview and exam components of the survey. Three outcomes were assessed: the prevalence of CKD, the prevalence of anemia in subjects with CKD, and the self-reported treatment of anemia. CKD was classified into 5 stages based on the glomerular filtration rate and evidence of kidney damage, in accordance with the guidelines of the National Kidney Foundation. Anemia was defined as serum hemoglobin levels ≤12 g/dL in women and ≤13 g/dL in men. We found that an estimated 14.0% of the US adult population had CKD in 2007–2010. Anemia was twice as prevalent in people with CKD (15.4%) as in the general population (7.6%). The prevalence of anemia increased with stage of CKD, from 8.4% at stage 1 to 53.4% at stage 5. A total of 22.8% of CKD patients with anemia reported being treated for anemia within the previous 3 months–14.6% of patients at CKD stages 1–2 and 26.4% of patients at stages 3–4. These results update our knowledge of the prevalence and treatment of anemia in CKD in the United States.     

The role of erythropoietin and its receptor in growth,survival and therapeutic response of human tumor cells: From clinic to bench — a critical review

Recombinant human erythropoietin (rhEPO) has been used clinically to alleviate cancer- and chemotherapy-related anemia. However, recent clinical trials have reported that rhEPO also may adversely impact disease progression and survival. The expression of functional EPO receptors (EPOR) has been demonstrated in many human cancer cells where, at least in vitro, rhEPO can stimulate cell growth and survival and may induce resistance to selected therapies.     

Erythropoietin is a hypoxia inducible factor-induced protective molecule in experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3⁺/CD4⁺ regulatory T cells and decrease of IFN-γ⁺/CD4⁺ Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies.     

High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant

The highly glycosylated peptide hormone erythropoietin (EPO) plays a key role in the regulation of erythrocyte maturation. Currently, marketed EPO is produced by recombinant technology in mammalian cell cultures. The complementary DNA (cDNA) for human EPO (hEPO) was transiently and stably expressed in the moss Physcomitrella patens wild-type and Δ-fuc-t Δ-xyl-t mutant, the latter containing N -glycans lacking the plant-specific, core-bound α1,3-fucose and β1,2-xylose. New expression vectors were designed based on a Physcomitrella ubiquitin gene-derived promoter for the expression of hEPO cDNA. Transient expression in protoplasts was much stronger at 10 than at 20 °C. In Western blot analysis, the molecular size of moss-produced recombinant human EPO (rhEPO) was identified to be 30 kDa, and it accumulated in the medium of transiently transformed protoplasts to high levels around 0.5 µg/mL. Transgenic Physcomitrella Δ-fuc-t Δ-xyl-t mutant lines expressing EPO cDNA showed secretion of rhEPO through the cell wall to the culture medium. In 5- and 10-L photobioreactor cultures, secreted rhEPO accumulated to high levels above 250 µg/g dry weight of moss material after 6 days. Silver staining of rhEPO on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) taken from the bioreactor culture demonstrated a high purity of the over-expressed secreted rhEPO, with a very low background of endogenous moss proteins. Peptide mapping of rhEPO produced by the Physcomitrella Δ-fuc-t Δ-xyl-t mutant indicated correct processing of the plant-derived signal peptide. All three N -glycosylation sites of rhEPO were occupied by complex-type N -glycans completely devoid of the plant-specific core sugar residues fucose and xylose.     

EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα⁺) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.