Gene therapy for renal anemia in mice with polycystic kidney using an adenovirus vector encoding the human erythropoietin gene

BACKGROUND: Recombinant human erythropoietin (rHuEPO) is primarily used for patients with anemia associated with end-stage renal disease. We evaluated the efficacy of EPO gene therapy using adenovirus vector for chronic renal failure mice expressing severe renal anemia. METHODS: Recombinant HuEPO gene transfer to mesothelial cells was performed in vitro and in vivo. Recombinant replication-deficient adenoviruses containing rHuEPO cDNA (AdCMVEPO), E. coli lacZ gene (AdCMVlacZ), or an nonexogenous gene (AdNull as control vector) driven by the cytomegalovirus promotor/enhancer were constructed. The oligosaccharides associated with the rHuEPO from AdCMVEPO-treated mesothelial cells were analyzed. For in vivo study, the DBA/2FG-pcy mouse, a model for human autosomal recessive polycystic kidney disease resulting in chronic renal failure with progressive anemia, was used. RESULTS: The sialylated oligosaccharides associated with the rHuEPO produced in AdCMVEPO-treated mesothelial cells occupied 78 +/- 0.7% of the total oligosaccharide pool. A single intraperitoneal administration of AdCMVEPO induced rHuEPO synthesis in the peritoneal cells and a marked increase in erythrocyte production. The maximal increase in hematocrit (43 +/- 4%) was observed on day 28, and it remained elevated for 40 days. CONCLUSION: These results indicate that intraperitoneal administration of AdCMVEPO improves renal anemia in mice with chronic renal failure and that the mesothelial cell is an appropriate target cell for gene transfer.     

Effects of dietary protein restriction and oil type on the early progression of murine polycystic kidney disease.

A paucity of research data exists on the potential for early dietary modification to directly retard cystic growth and proliferation in polycystic kidney disease (PKD). We have therefore examined the relative effects of dietary protein levels and oil type on the progression of disease in a murine model of PKD. In the first study, weanling DBA/2FG-pcy (pcy) mice were fed either a normal (NP), 25%, or low (LP), 6%, casein diet with 10% of either sunflower seed oil (SO) (containing n-6 fatty acids), or fish oil (FO) (containing n-3 fatty acids), in a 2 x 2 design. At the end of the dietary treatment, kidney weight relative to body weight was higher in mice on the NP diets. In addition, kidney phospholipid to kidney weight (mumol/g) was lower in pcy mice on NP diets, indicating that the increased kidney size was largely due to increased cyst development. Replacement of dietary SO with FO resulted in alterations in renal phospholipid fatty acid compositions: 18:2 n-6, 20:4 n-6, and 22:5 n-6 were lower, and 20:5 n-3, 22:5 n-3, and 22:6 n-3 were higher in FO-fed animals. No effect of dietary lipid type on disease progression was noted, however. In a second study, morphometric analysis revealed an 11% lower percentage cyst area and a 46% lower total cyst area (mm2) in kidney sections derived from mice on LP diets compared to NP diets. These results indicate that early dietary protein restriction in PKD prior to clinical manifestation of symptoms of the disease may have a significant impact on the pathogenesis of PKD.     

Effect of dietary soy protein and genistein on disease progression in mice with polycystic kidney disease

The effects of feeding a soy protein isolate or genistein, an isoflavonoid present in soy protein, on cyst development were examined in the DBA/2FG-pcy (pcy) mouse, an accepted animal model of polycystic kidney disease, before the appearance of clinical symptoms. In study 1, 60-day-old male pcy mice were evenly divided into two groups and fed semipurified diets, based on casein or a soy protein isolate (15 g protein/100 g diet) for 90 days. In study 2, the animals were fed a casein-based diet (25 g casein/100 g diet) with or without genistein (0.05 g/100 g diet) for 60 days. In study 1, total kidney weight and kidney weight relative to body weight were significantly reduced (by 24% to 25%) in the animals fed the soy protein-based diet, relative to the casein-fed group, as was kidney water content (by 38%). In addition, mean cyst volume, as measured by morphometry, were lower (by 25%) in kidneys from the soy protein-fed group. No differences were found between these two groups with respect to final body weight, plasma creatinine, and protein content; however, plasma urea values were significantly lower in the soy protein-fed animals. Genistein supplementation of a casein-based diet in study 2 did not reduce the renal enlargement and cyst development associated with progression of polycystic kidney disease. These results suggest that soy protein is effective in retarding cyst development in the pcy mouse and that this beneficial effect may be unrelated to its genistein content.     

The HIF-PHI BAY 85-3934 (Molidustat) Improves Anemia and Is Associated With Reduced Levels of Circulating FGF23 in a CKD Mouse Model

Fibroblast growth factor-23 (FGF23) is a critical factor in chronic kidney disease (CKD), with elevated levels causing alterations in mineral metabolism and increased odds for mortality. Patients with CKD develop anemia as the kidneys progressively lose the ability to produce erythropoietin (EPO). Anemia is a potent driver of FGF23 secretion; therefore, a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) currently in clinical trials to elevate endogenous EPO to resolve anemia was tested for effects on iron utilization and FGF23-related parameters in a CKD mouse model. Mice were fed either a casein control diet or an adenine-containing diet to induce CKD. The CKD mice had markedly elevated iFGF23 and blood urea nitrogen (BUN), hyperphosphatemia, and anemia. Cohorts of mice were then treated with a patient-equivalent dose of BAY 85-3934 (BAY; Molidustat), which elevated EPO and completely resolved aberrant complete blood counts (CBCs) in the CKD mice. iFGF23 was elevated in vehicle-treated CKD mice (120-fold), whereas circulating iFGF23 was significantly attenuated (>60%) in the BAY-treated CKD mice. The BAY-treated mice with CKD also had reduced BUN, but there was no effect on renal vitamin D metabolic enzyme expression. Consistent with increased EPO, bone marrow Erfe, Transferrin receptor (Tfrc), and EpoR mRNAs were increased in BAY-treated CKD mice, and in vitro hypoxic marrow cultures increased FGF23 with direct EPO treatment. Liver Bmp-6 and hepcidin expression were downregulated in all BAY-treated groups. Femur trabecular parameters and cortical porosity were not worsened with BAY administration. In vitro, differentiated osteocyte-like cells exposed to an iron chelator to simulate iron depletion/hypoxia increased FGF23; repletion with holo-transferrin completely suppressed FGF23 and normalized Tfrc1. Collectively, these results support that resolving anemia using a HIF-PHI during CKD was associated with lower BUN and reduced FGF23, potentially through direct restoration of iron utilization, thus providing modifiable outcomes beyond improving anemia for this patient population. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Effect of erythropoietin therapy on red cells filterability and left ventricular mass in predialysis patients

BACKGROUND: Cardiovascular complications are the leading cause of mortality in patients with end-stage renal disease. Left ventricular hypertrophy (LVH) is recognized as an independent risk factor for cardiovascular morbidity and mortality. At the onset of dialysis, more than 70% of the patients with chronic kidney disease have echocardiographic evidence of LVH. Anemia, increased red cells filterability time (RCFT), and blood viscosity are known to induce LVH. AIM: To evaluate, prospectively, the effects of erythropoietin (EPO) therapy for 20 weeks on RCFT and left ventricular mass (LVM). PATIENTS AND METHODS: Twenty uremic and anemic predialysis patients with creatinine clearance test below 35 mL/min were studied. RCFT test and three-dimensional echocardiography were performed at 0, 10, and 20 weeks. RESULTS: EPO therapy for 20 weeks did not adversely affect renal function and did not significantly change the mean blood pressure. It significantly increased the hemoglobin and fibrinogen levels, and decreased RCFT and LVM (p < .01). CONCLUSION: Although correction of anemia can contribute to regression of LVM, we speculate that an increasing number of cells with normalized viscoelastic properties and a direct effect of EPO on erythrocytes and myocardiocytes, through specific receptors, may also play an important role.     

Renal function during erythropoietin therapy for anemia in predialysis chronic renal failure patients

Recombinant human erythropoietin (r-HuEPO) therapy for anemia in chronic renal failure patients could have unfavorable renal effects since reversal of anemia can raise blood pressure and accelerate experimental glomerular injury. Thus, the effects of r-HuEPO on renal and systemic hemodynamics and the progression of renal disease were studied in predialysis chronic renal failure patients. The clearances of inulin and p-aminohippurate, fractional excretions of albumin and immunoglobulin G, cardiac output, plasma renin activity and aldosterone concentration were assessed at baseline, after short-term r-HuEPO (n = 4) or placebo (n = 4) therapy, and after long-term r-HuEPO for all patients (n = 8). In addition, the slope of l/serum creatinine with time was determined before and during continued r-HuEPO therapy. In contrast to placebo therapy, hematocrit increased with r-HuEPO from 32 to 37% after 7.6 +/- 2.7 weeks (mean +/- SD). Antihypertensive drug therapy was increased in 2 patients in each group. Renal function, cardiac output, plasma renin activity and aldosterone did not change significantly in either group. After 18 +/- 9 weeks of therapy for all patients, hematocrit increased from 31 to 39%. Antihypertensive drug therapy was increased in 5 patients and decreased in 1. Renal function decreased while proteinuria tended to increase. Cardiac output, plasma renin activity and aldosterone did not change. During 37 +/- 22 weeks of r-HuEPO therapy, the slope of l/serum creatinine did not worsen in any patient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythropoietin, but not the correction of anemia alone, protects from chronic kidney allograft injury

Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury.     

肾血康对肾性贫血大鼠外周血象与红细胞脆性及EPO含量的影响

目的:探讨肾血康治疗肾性贫血大鼠的药理、药效.方法:将SD大鼠随机分为空白组、模型组及中药组,以中药肾血康对腺嘌呤所致慢性肾衰竭大鼠进行治疗,并比较大鼠治疗前后的外周血象、EPO含量、红细胞脆性等各项指标.结果:肾血康能降低肾衰竭大鼠血中Scr及BUN,降低红细胞脆性,而升高其血中Hb、RBC、HCT、EPO水平.结论:肾血康能有效改善腺嘌呤所致慢性肾衰竭大鼠的贫血症状,防止肾小球硬化,延缓肾功能的进一步恶化.     

Bioavailability of iron in hemodialysis patients treated with erythropoietin: evidence for the inhibitory role of aluminum

The dose of recombinant human erythropoietin (r-HuEPO) required to correct the anemia of end-stage renal disease (ESRD) varies among patients. The response to r-HuEPO is impaired if absolute or relative iron deficiency exists. Aluminum may cause a microcytic anemia in patients with ESRD, but the mechanism remains incompletely defined. Twenty-two patients in the Canadian Multicentre EPO trial were studied for 6 months. In this randomized double-blind placebo-controlled trial, free erythrocyte protoporphyrin (FEP) was used as an indicator of iron-deficient deficient erythropoiesis. The relationship of FEP to the estimates of iron availability (serum iron, transferrin saturation, ferritin) and iron utilization (corrected reticulocyte count, hemoglobin) was evaluated by multiple linear regression analysis. The effect of aluminum on FEP was evaluated by adjusting the statistical model for this variable. All patients were iron replete as assessed by serum ferritin. FEP was not related to serum aluminum before administration of r-HuEPO, but it was significantly correlated with aluminum in the treated group. In hemodialysis patients treated with r-HuEPO, the proportion of the variability explained by the parameters of iron utilization and iron availability was 0.27. The effect of aluminum increased this to 0.59. In hemodialysis patients not receiving r-HuEPO, the proportion of variability in FEP explained by the model increased from 0.16 to 0.28 by adjusting for aluminum. The data support the hypothesis that aluminum interferes with the bioavailability of stored iron for erythropoiesis and thus may result in a microcytic anemia in patients with ESRD or may blunt their response to r-HuEPO therapy.     

Recombinant human erythropoietin independence in chronic hemodialysis patients: clinical features, iron homeostasis and erythropoiesis

AIMS: Recombinant human erythropoietin (r-HuEPO) is widely used to correct renal anemia in uremic patients. Interestingly, some chronic hemodialysis (HD) patients can maintain high hemoglobin level without the need of r-HuEPO. The aim of this study is to investigate clinical features, iron metabolism and erythropoiesis of these r-HuEPO-independent HD patients. METHODS: r-HuEPO independence was defined in dialysis patients as hemoglobin greater than 12 g/dl and no use of r-HuEPO for at least 6 months. An age- and sex-matched group was selected for comparison. Their underlying diseases, duration of hemodialysis therapy, efficacy of dialysis (Kt/V), normalized protein catabolic rate (nPCR) and body mass index (BMI) were recorded. Laboratory data including: hemoglobin, albumin, high sensitivity C-reactive protein, serum iron, total iron binding capacity, transferrin saturation, ferritin, intact parathyroid hormone, soluble transferrin receptor (sTfR), serum EPO, cortisol, testosterone, aluminum and leptin levels were measured. Renal sonography was also performed in each patient to evaluate renal cyst formation. RESULTS: About 2.3% of all HD patients (21/888; M : F = 18 : 3) were r-HuEPO-independent. These patients had significantly longer HD duration and higher serum EPO and sTfR levels, and lower transferrin saturation rate than dependent groups. Correlation analysis revealed that hemoglobin level strongly correlated with HD duration, serum sTfR and EPO levels. Levels of sTfR were positively related with serum EPO levels and BMI. Multivariate regression analysis showed that level of sTfR was the only independent factor related to r-HuEPO independence. CONCLUSION: R-HuEPO independence is rare among chronic hemodialysis patients. Factors contributing to this dependence are complex and multiple. Level of serum sTfR parallels erythropoiesis and is the most significant factor associated with r-HuEPO independence in chronic HD patients.     

Recombinant human erythropoietin treatment protects the cardio-renal axis in a model of moderate chronic renal failure

Chronic kidney disease (CKD) patients develop anemia because of the low kidney erythropoietin (EPO) production, thus promoting cardiovascular complications. The degree of renal insufficiency might determine the moment to start recombinant human erythropoietin (rhEPO) therapy, but the molecular basis for these options deserves better elucidation. This study aimed to clarify the cardio-renal effects of earlier rhEPO therapy in rats with moderate chronic renal failure (CRF). Four groups of rats were evaluated for 15 weeks (control; rhEPO ? 50 IU/kg/week; CRF ? 3/4 nephrectomy; CRF + rhEPO) to assess renal and hematology data, EPO levels, blood pressure, heart rate, peripheral catecholamines contents, serum-transforming growth factor-β1 (TGF-β1), kidney gene expression of EPO, Caspase 9 (Casp9), and vascular endothelial growth factor (Vegf). This model of moderate CRF showed moderate and corrected anemia, hypertension, tachycardia, sympathetic overactivity, and increased serum TGF-β1 content. The remnant kidney showed a proliferative profile, with hypertrophy, downregulated gene expression of EPO, and upregulated gene expression of Vegf and Casp9. rhEPO treatment promoted erythrocytosis and prevented tachycardia and catecholamines increment, with a rise of serum TGF-β1. Furthermore, the decreased kidney gene expression of EPO and the overexpression of Casp9 were prevented, demonstrating a renoprotective action on the remnant kidney. In conclusion, rhEPO therapy promotes a protective effect on the cardio-renal axis, which might be mainly attributed to its pro-proliferative and anti-apoptotic properties. These findings might recommend its use in earlier stages of CRF, acting as an erythropoiesis stimulating agent, to efficiently correct not only the anemia, one of the major complications in these patients, but also the succeeding adverse cardio-renal effects.     

Hemodynamic effects of partial correction of chronic anemia by recombinant human erythropoietin in patients on dialysis

Eighteen patients on chronic hemodialysis with renal anemia were treated with recombinant human erythropoietin (r-HuEPO). Hemodynamic parameters in the resting state were determined before and after successful treatment. Posttreatment cardiac index was decreased (3.3 v 2.8 L/min/m2), whereas diastolic blood pressure (72 v 79 mm Hg) and calculated peripheral resistance (2,230 v 2,860 dyne.cm.s-5) were increased significantly when compared with the pretreatment period. We conclude from our study that the increase of blood pressure as seen in patients on dialysis, who are effectively treated with r-HuEPO, is due to an increase in peripheral resistance. This increase overrules the decrease of cardiac index and might well be a result of peripheral vasoconstriction due to improved oxygen availability.     

肾性贫血动物模型的制作

目的:应用适量马兜铃酸(aristolochic acid,AA)探索创建肾性贫血小鼠模型。方法:6周龄雄性C57BL/6小鼠分3组,正常对照组;马兜铃酸腹腔注射组:AA/2d组(马兜铃酸3 mg·kg^-1·2 d^-1)和AA/3d组(3 mg·kg^-1·3 d^-1),共给药6周。造模第6、9、12周观察肾功能、贫血指标、肾脏病理评分、肾纤维化及肾组织EPO的蛋白表达情况。结果:给药6周时,血肌酐AA/2d组(29.9±1.5)μmol/L和AA/3d组(31.0±1.9)μmol/L均明显高于对照组(8.7±1.4)μmol/L,P<0.001;Hb水平AA/2d组(88.3±3.1)g/L、AA/3d组(85.7±7.4)g/L都明显低于对照组(148.7±4.9)g/L,P<0.001;HCT值AA/2d组、AA/3d组分别为0.30±0.01、0.29±0.02还是都明显低于对照组(0.51±0.02),P<0.001;肾脏病理呈现明显的肾间质纤维化,肾组织中EPO蛋白表达水平下降,提示肾性贫血模型成功。连续观察9、12周,上述变化持续存在,但AA/2d、AA/3d组间的临床病理指标无统计学意义。结论:3 mg·kg^-1·3 d^-1马兜铃酸连续给药6周可成功建立合格稳定的肾性贫血小鼠模型,该模型临床表现为不可逆的肾衰竭和贫血,肾组织中的促红素表达下降,肾脏病理表现为慢性肾间质纤维化。     

Congenic mice provide in vivo evidence for a genetic locus that modulates intrinsic transforming growth factor β1-mediated signaling and bone acquisition

Osteoporosis, the most common skeletal disorder, is characterized by low bone mineral density (BMD) and an increased risk of fragility fractures. BMD is the best clinical predictor of future osteoporotic fracture risk, but is a complex trait controlled by multiple environmental and genetic determinants with individually modest effects. Quantitative trait locus (QTL) mapping is a powerful method for identifying chromosomal regions encompassing genes involved in shaping complex phenotypes, such as BMD. Here we have applied QTL analysis to male and female genetically‐heterogeneous F₂ mice derived from a cross between C57BL/6 and DBA/2 strains, and have identified 11 loci contributing to femoral BMD. Further analysis of a QTL on mouse chromosome 7 following the generation of reciprocal congenic strains has allowed us to determine that the high BMD trait, which tracks with the DBA/2 chromosome and exerts equivalent effects on male and female mice, is manifested by enhanced osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and by increased growth of metatarsal bones in short‐term primary culture. An insertion/deletion DNA polymorphism in Ltbp4 exon 12 that causes the in‐frame removal of 12 codons in the DBA/2‐derived gene maps within 0.6 Mb of the marker most tightly linked to the QTL. LTBP4, one of four paralogous mouse proteins that modify the bioavailability of the transforming growth factor β (TGF‐β) family of growth factors, is expressed in differentiating MSC‐derived osteoblasts and in long bones, and reduced responsiveness to TGF‐β1 is observed in MSCs of mice homozygous for the DBA/2 chromosome 7. Taken together, our results identify a potential genetic and biochemical relationship between decreased TGF‐β1–mediated signaling and enhanced femoral BMD that may be regulated by a variant LTBP4 molecule. © 2012 American Society for Bone and Mineral Research.     

Blood rheology and hypertension in hemodialysis patients treated with erythropoietin

Fifteen hemodialysis patients suffering from stable anemia were treated with recombinant human erythropoietin (r-HuEPO). Within 16 weeks, hematocrit values increased from 23.7 +/- 1.2 to 35.7 +/- 0.2%. Simultaneously, mean predialytic blood pressure rose significantly from 131/79 to 139/85 mm Hg. Three out of 15 patients developed frank hypertension and had to be put on antihypertensive therapy. When the hematocrit was lowered again from 36.3 +/- 1.8 to 30.5 +/- 1.2% in these 3 patients, blood pressure was attenuated and the antihypertensive medication could be reduced or abolished. With rising hematocrit values, whole blood viscosity increased at both low (+42%) and high shear rates (+33%) without reaching the values seen in healthy subjects. By contrast, plasma viscosity was already elevated in hemodialysis patients prior to r-HuEPO treatment and showed only a slight, but insignificant increase during r-HuEPO treatment. Since whole blood viscosity is one factor that determines vascular resistance, it is conceivable that the development of hypertension during correction of the renal anemia is, at least partly, due to an increment of blood viscosity.     

A hereditary model of slowly progressive polycystic kidney disease in the mouse

There are two known forms of hereditary polycystic kidney disease (PKD) in humans. Although both forms initiate early in life, autosomal recessive PKD is rapidly progressive to kidney failure shortly after birth whereas autosomal dominant PKD is slowly progressive, taking many years to end stage. Research in this field has been limited by the availability of suitable animal models of PKD. Recently the C57BL/6J-cpk mouse has been used to study the pathogenesis of rapidly progressive hereditary PKD. The study presented here describes a slowly progressive PKD in the DBA/2-pcy mouse. The disease trait is transmitted in an autosomal recessive pattern and was localized to chromosome 9 through linkage to the dilute coat color and transferrin genes. Whereas some cystic changes were seen in fetal and newborn affected mice, renal enlargement did not develop until after 8 weeks of age and azotemia did not develop until after 18 weeks of age. Renal cysts were identified in all segments of the nephron and collecting duct and progressively enlarged with age. Individual cysts were found to be lined by a single layer of epithelial cells in most areas, with focal polyps and mounds of cells principally in collecting duct cysts. Early stages of cyst formation were associated with some abnormalities of tubular and glomerular basement membranes and accelerated eruption of incisors. Late stages of the disease were characterized by azotemia and chronic renal interstitial inflammatory infiltrates in all affected animals and cerebral vascular aneurysms in a few. We conclude that the DBA/2-pcy mouse has a form of renal cystic disease that appears similar in many respects to that seen in the dominant form of human PKD.     

红细胞生成素对模拟急性肾损伤微环境下培养骨髓间充质干细胞增殖的影响及机制探讨

目的 观察经红细胞生成素（EPO）干预后,体外模拟急性肾损伤（AKI）微环境下培养骨髓间充质干细胞（MSC）的分裂增殖情况,并探讨产生这种变化的可能机制。 方法 抽取C57BL/6小鼠的骨髓,经Percoll密度梯度离心联合贴壁培养法分离纯化出小鼠MSC（mMSC）,以流式细胞仪鉴定。夹闭雄性C57BL/6小鼠双侧肾蒂30 min再开放30 min的方法制作AKI鼠模型,即刻取双侧肾脏皮质制作缺血再灌注（IR）肾脏匀浆上清。取扩增第3代的mMSC分组培养：A组：含10%胎牛血清的低糖DMEM培养基;B组：含10%胎牛血清的低糖DMEM培养基＋IR肾脏匀浆上清,体外模拟AKI微环境干预mMSC;C组：含10%胎牛血清的低糖DMEM培养基＋IR肾脏匀浆上清+不同浓度EPO（1、5、10、50 U/ml）。培养1、3、5、7 d。CCK-8法检测各组培养mMSC的增殖,TUNEL法检测mMSC凋亡,Western印迹法检测mMSC表面EPO受体（EPOR）及增殖凋亡相关信号通路蛋白的表达。 结果 CCK-8法检测显示,经IR肾脏匀浆上清干预后,不同时间点mMSC的增殖效应显著减弱（P ＜ 0.01）,而TUNEL检测表明其胞核染色阳性细胞的百分比显著高于A组（P ＜ 0.01）;给予EPO干预后,mMSC的增殖能力增强,胞核染色阳性细胞百分比降低,具有浓度依赖效应。Western印迹结果显示,第3代mMSC表面存在EPOR表达,培养5 d后EPOR相对表达量为0.092±0.015。EPO以剂量和时间依赖性降低AKI微环境下凋亡相关蛋白caspase-3的表达,同时上调凋亡抑制蛋白Bcl-2的表达。用10 U/ml EPO干预5 d后,相对于B组,磷酸化蛋白酪氨酸激酶2及磷酸化信号转导和转录因子的表达均显著上调（均P < 0.01）。 结论 EPO能促进体外AKI微环境干预下培养mMSC的增殖,减轻其凋亡,该效应经EPOR介导,并与增殖凋亡相关信号通路蛋白有关。     

Alterations in sex hormones and sexual function of patients with renal failure treated with recombinant human erythropoietin.

Since it has been reported that correction of anemia in long-term hemodialysis patients by using human recombinant erythropoietin (r-HuEPO) is associated with improve sexual function, we conducted the present study to evaluate the changes in sex hormones as well as sexual function after r-HuEPO administration (1500 to 4500 IU per dialysis) for a year in patients on regular hemodialysis. Thirteen patients receiving regular hemodialysis entered this study. Their median age was 43 years. Along with correction of anemia (the hematocrit increased from 20 to 28%), testosterone (T) increased from 2.4 +/- 0.1 to 2.6 +/- 0.2 ng/ml, follicular stimulating hormone (FSH) increased (29 +/- 5 to 73 +/- 7 mIU/ml), luteinizing hormone increased (69 +/- 14 to 160 +/- 21 IU/ml) and prolactin decreased (all changes are significant at p less than 0.05). However, the improvement of sexual function was not remarkable. Only 25% of the uremic patients treated with r-HuEPO showed amelioration of this function. From the present data, it does not seem likely that therapy with r-HuEPO induces directly amelioration of sexual function through changes in sex hormones.     

Polycythemia is associated with bone loss and reduced osteoblast activity in mice

Summary

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO).

Introduction

PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO.

Methods

Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2^V617F knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO.

Results

Compared to wt, both JAK2^V617F and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P?<?0.01) suggesting a more severe bone phenotype than JAK2^V617F. Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice.

Conclusions

This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.