Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure

The goal of this research was to develop a strategy to couple stem cell and gene therapy for in vivo delivery of erythropoietin (Epo) for treatment of anemia of ESRD. It was shown previously that autologous bone marrow stromal cells (MSCs) can be genetically engineered to secrete pharmacologic amounts of Epo in normal mice. Therefore, whether anemia in mice with mild to moderate chronic renal failure (CRF) can be improved with Epo gene-modified MSCs (Epo+MSCs) within a subcutaneous implant was examined. A cohort of C57BL/6 mice were rendered anemic by right kidney electrocoagulation and left nephrectomy. In these CRF mice, the hematocrit (Hct) dropped from a prenephrectomy baseline of approximately 55% to 40% after induction of renal failure. MSCs from C57BL/6 donor mice were genetically engineered to secrete murine Epo at a rate of 3 to 4 units of Epo/10(6) cells per 24 h, embedded in a collagen-based matrix, and implanted subcutaneously in anemic CRF mice. It was observed that Hct increased after administration of Epo+MSCs, according to cell dose. Implants of 3 million Epo+MSCs per mouse had no effect on Hct, whereas 10 million led to a supraphysiologic effect. The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCs rose to a peak 54+/-4.0 or 63+/-5.5%, respectively, at 3 wk after implantation and remained above 48 or 54% for >19 wk. Moreover, mice that had CRF and received Epo+MSCs showed significantly greater swimming exercise capacity. In conclusion, these results demonstrate that subcutaneous implantation of Epo-secreting genetically engineered MSCs can correct anemia that occurs in a murine model of CRF.     

Inducing host acceptance to encapsulated xenogeneic myoblasts

BACKGROUND: Cell encapsulation holds promise for the chronic delivery of recombinant proteins such as erythropoietin. Encapsulated xenogeneic mouse C2C12 myoblasts display long-term survival in the central nervous system whereas they do not in the subcutaneous tissue, suggesting that encapsulation only partially prevents affector and effector mechanisms of the host immune response. Transient immunosuppression with FK506 at the time of subcutaneous implantation leads, however, to their long-term survival. The nature of this acceptance was further investigated in this report. METHODS: Fischer rats were rendered unresponsive to encapsulated murine C2C12 myoblasts secreting mouse erythropoietin by either a 1- or 4-week initial treatment of FK506. To examine the extent of xenograft acceptance, animal were challenged with a second implant 9 weeks after the initial implantation. RESULTS: Challenging animals treated only 1 week with FK506 led to rejection of both primary and secondary implants. Animals administered FK506 for 4 weeks accepted both implants over the period investigated. However, these animals rejected unencapsulated xenogeneic cells injected at a later time, highlighting the requirement of the polymer membrane for immune protection. Developed unresponsiveness to encapsulated xenogeneic myoblasts lasted over extended periods (at least 7 months), in the absence of both immunosuppression and stimulating xenoantigens. CONCLUSIONS: These findings reveal that host acceptance of encapsulated but not unencapsulated xenogeneic myoblasts can be developed in the subcutaneous tissue after transient FK506 immunosuppression. This may have direct clinical relevance as it enables capsules to be replaced without additional immunosuppression, facilitating long-term cell-based therapies.     

Effect of anemia and renal cytokine production on erythropoietin production during blood-stage malaria

BACKGROUND: Renal dysfunction and severe anemia are clinical complications of blood-stage malaria. Erythropoietin (Epo) is a hormone produced by the kidney and plays an essential role in stimulating erythrocyte production. Renal dysfunction in malaria is associated with changes in renal cytokine levels, which may affect the production of Epo and the alleviation of anemia. METHODS: Resistant C57BL/6 (B6) and susceptible A/J mice were infected with Plasmodium chabaudi AS. The levels of Epo and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and the degree of anemia was determined by hematocrit. Regression analyses were employed to estimate the influences of anemia and renal cytokines on the production of Epo during infection. RESULTS: A/J mice developed higher peak parasitemia, more severe anemia, and succumbed as compared to B6 mice, which survived the infection. B6 mice had higher levels of renal tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-10, whereas A/J mice had higher levels of IL-12p70, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and Epo. Regression analyses revealed that kidney Epo levels were influenced most strongly by changes in hematocrit levels. In addition, albeit to a much weaker degree, kidney Epo levels correlated negatively with GM-CSF levels but positively with IL-10 levels. CONCLUSION: Blood-stage malaria infection modulates the production of renal pro- and anti-inflammatory cytokines in resistant versus susceptible strains of mice differentially. However, despite the fluctuations of renal cytokines, the degree of anemia is the main determinant for Epo production during blood-stage malaria while kidney cytokines may exert secondary influences.     

Long-term host unresponsiveness to encapsulated xenogeneic myoblasts after transient immunosuppression

BACKGROUND: Encapsulating cells prevents the immune destruction of allogeneic cells in the subcutaneous site as well as allogeneic and xenogeneic cells in the central nervous system. However, when encapsulated xenogeneic cells are implanted s.c., they may be subject to rejection by the host. METHODS: Murine C2C12 myoblasts engineered to secrete mouse erythropoietin (mEpo) were used to evaluate the response of control versus FK506-treated xenogeneic recipients (Fischer rats) to encapsulated myoblasts implanted in the s.c. site. RESULTS: Encapsulated C2C12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats. Devices transplanted into nude rats induced a sustained increase in the hematocrit, associated with an extended viability of the encapsulated cells. Short-term immunosuppression with FK506, for periods lasting either 1, 2, or 4 weeks after implantation, permitted the long-term survival of encapsulated C2C12 mEpo cells in Fischer rats. Animals increased their hematocrits to more than 70% and maintained these levels for 13 weeks, independent of the duration of FK506 treatment. Unencapsulated C2C12 mEpo cells injected i.m. in immunosuppressed animals were rejected over this same period. CONCLUSIONS: Encapsulation alone cannot protect xenogeneic myoblasts from immune destruction in the s.c. site. These results highlight the importance of combining the technique of cell encapsulation with transient immunosuppression to achieve long-term survival of xenografted myoblasts in a peripheral immunoreactive site.     

Effects of erythropoietin-gene electrotransfer in rats with adenine-induced renal failure

BACKGROUND: We previously demonstrated that erythropoietin (Epo) expression increases in five-sixths nephrectomized rats, after muscle-targeted gene transfer by in vivo electroporation, using plasmid DNA expressing rat Epo (pCAGGS-Epo). Here, we apply this method to a rat model with severe anemia associated with chronic renal failure; these rats have hematocrit levels in the 30-35% range, similar to those in humans with end-stage renal disease. METHODS: Wistar rats were treated to produce adenine-induced uremia. The uremic rats were then treated with muscle-targeted gene transfer using pCAGGS-Epo. Some uremic rats died from chronic renal failure; one of these was dissected, and the kidneys were histologically examined. For the remaining rats, we measured body weight and blood pressure, and obtained blood samples regularly. RESULTS: The uremic rats showed severe anemia, with hematocrit levels at 32.6 +/- 3.3%. Epo-gene transfer increased Epo expression and serum Epo levels, and also increased the hematocrit levels to 64.5 +/- 4.8%. The dose of pCAGGS-Epo used in this study did not induce severe hypertension. CONCLUSIONS: Continuous Epo-gene expression improves the anemia associated with chronic renal failure, and without severe side effects. Our results support the potential use of gene electrotransfer for human gene therapy applications.     

Impaired erythropoietin production in liver transplant recipients: the role of calcineurin inhibitors.

Anemia is common following liver transplantation. Because cyclosporine inhibits erythropoietin (Epo) production in experimental models, we investigated whether Epo production was impaired in liver transplant recipients receiving a cyclosporine- or tacrolimus-based immunosuppressive regimen. First, serum Epo levels were measured before and 1 year after transplantation in 35 liver transplant recipients. Second, serum Epo levels were compared in a large series of liver transplant recipients with stable graft and renal functions: 27 receiving a cyclosporine-based and 31 receiving a tacrolimus-based immunosuppressive regimen. A reference group was made up of 22 blood donors and 21 nontransplanted subjects with iron-deficiency anemia. Serum Epo levels were significantly lower after than before liver transplantation, especially in cyclosporine-treated patients. Serum Epo concentrations correlated with hematocrit values in both transplant recipients and control subjects. Using multiple linear regression models, the polynomial relationship between hematocrit and serum Epo values was similar to the control group in patients under tacrolimus, whereas Epo production was significantly reduced in patients under cyclosporine-based immunosuppression. Hematocrit values and the type of calcineurin inhibitor were the only parameters independently related to Epo levels. In conclusion, cyclosporine, but not tacrolimus, inhibits Epo production at the doses used in clinical practice.     

Defective calcium signalling in uraemic platelets and its amelioration with long-term erythropoietin therapy.

BACKGROUND: Chronic renal failure (CRF) is associated with prolonged bleeding time and impaired platelet adhesion and aggregation. Erythropoietin (Epo) administration improves platelet adhesion/aggregation and ameliorates prolongation of bleeding time in CRF. However, the mechanisms of improved platelet function after Epo therapy have not been fully elucidated. The present study examined the hypothesis that the improved uraemic platelet function after Epo therapy is, in part, due to correction of the platelet calcium signalling. METHODS: Rats were randomized into four groups after 5/6 nephrectomies to produce CRF. The Epo-treated CRF group received Epo, 150 U/kg, twice weekly for 6 weeks to prevent anaemia; the felodipine and Epo-treated CRF group received Epo but was kept normotensive by felodipine treatment; the placebo-treated CRF group received placebo injections and became anaemic; and the iron-deficient CRF group received Epo but was kept anaemic by dietary iron-deficiency. A group of sham-operated rats was included as normal control. Basal and thrombin-stimulated platelet cytosolic calcium ([Ca(2+)](i)) were determined using a Ca(2+)-sensitive dye (fura-2). RESULTS: Platelets from placebo-treated CRF group exhibited a profound attenuation of thrombin-stimulated surge in [Ca(2+)](i), which is the final pathway of platelet activation. Long-term Epo administration led to a normalization of the thrombin-induced rise in platelet [Ca(2+)](i) in the CRF animals, independent of either haematocrit or blood pressure values. Further studies revealed that improved Ca(2+) signalling with Epo is associated with increased Ca(2+) uptake and expanded Ca(2+) stores in the platelets. CONCLUSIONS: The defective Ca(2+) signalling in uraemic animals and its improvement with chronic Epo therapy provides the biochemical basis of the previously reported platelet dysfunction and prolonged bleeding time in uraemic patients and animals, and their amelioration with chronic Epo therapy.     

Regulatory mechanisms of erythropoietin levels in dialysis patients

We described a radioimmunoassay system for measuring blood erythropoietin (Epo) levels using recombinant human Epo and evaluated the regulatory mechanisms of Epo in dialysis patients. A satisfactory dose-response relationship for Epo levels was observed within the wide range of 3-250 mU/ml with a sensitivity of approximately 5 mU/ml. The Epo levels were found to be 16.1 +/- 8.6 mU/ml (m +/- SD) in 422 uremic patients on maintenance dialysis and 17.1 +/- 7.2 mU/ml in 86 normal subjects. The Epo levels were not statistically different between the two groups, although the hematocrit was significantly lower in the dialysis patients. No correlation was observed between the Epo levels and hematocrit in the dialysis patients. Patients with polycystic kidneys had higher hematocrits and Epo levels than patients with chronic nephritis. No changes in Epo levels were observed with age and with the period of dialysis. Diurnal variations in the Epo levels revealed a significant increase after hemodialysis, and an acute reduction in hematocrit following massive hemorrhage raised the Epo levels up to 3,180 mU/ml. These findings indicate that the Epo levels in dialysis patients are inappropriately low for the severity of anemia and suggest that a negative feedback mechanism of the hematocrit on the Epo secretion may exist in uremic patients as well as in normal subjects and that the threshold for Epo secretion might be 'reset' at a low hematocrit level.     

Possible role of soluble erythropoietin receptors in renal anemia

Recombinant human erythropoietin(rHuEpo) is effective for the treatment of renal anemia associated with chronic renal failure(CRF). However, we have encountered some patients with CRF who have sometimes developed a resistance to rHuEpo. This resistance can be due to iron or folate deficiency, aluminum toxicity, hyperparathyroidism, or auto-antibodies for rHuEpo. In this study, we focused on the soluble erythropoietin receptor(sEpoR), which can bind to rHuEpo. To demonstrate the possibility that the sweeping of rHuEpo by sEpoR results in resistance to rHuEpo, we performed a bioassay using the rHuEpo-dependent cell line, UT7/EPO. The results showed that recombinant mouse sEpoR(rmsEpoR) can reduce the proliferation of UT7/EPO induced by rHuEpo in a dose-dependent manner. We consider that this cell line could be a useful tool in a bioassay to detect the inhibitory factor(s) against Epo. We selected sera from three groups of patients with renal anemia associated with CRF who were receiving hemodialysis three times a week: the first was a patient group that needed a high dose of rHuEpo(7,500-9,000 unit/dialysis), the second was a patient group that needed an intermediate dose of rHuEpo (4,500 unit/dialysis), the third was a patient group that needed a low dose of rHuEpo(below 1,500 unit/dialysis). Interestingly, the proliferation of UT7/EPO determined with [3H]-thymidine incorporation was reduced by the addition of sera from the first group, but not by the addition of sera from the third group. These results suggested that serum sEpoR may play an important role in signal transduction via EpoR on erythroid progenitor in CRF patients.     

活血复肾胶囊对慢性肾衰竭大鼠肾性贫血的影响

目的:探讨活血复肾胶囊对慢性肾衰竭(chronic renal failure, CRF)过程中肾性贫血的影响.方法:采用腺嘌呤制作大鼠CRF模型,分为4组:正常对照组、模型对照组、活血复肾胶囊组及川芎嗪组,治疗4周后分别观察血红蛋白(Hb)、红细胞压积(Hct)、促红细胞生成素(EPO)等的变化.结果:在模型对照组中Hb、Hct、EPO水平明显低于正常对照组,采用活血复肾胶囊治疗后血红蛋白水平明显升高.结论:活血复肾胶囊对CRF肾性贫血具有治疗作用.     

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.     

Colony-promoting activity in mice kidneys with phenylhydrazine hemolytic anemia

Aqueous anemic mice kidney extracts (MKE) were assessed colony-promoting activity (CPA) of hematopoietic progenitor cells in serum-free cultures stimulated by interleukin-3 and erythropoietin (Epo). Mice with hemolytic anemia followed by phenylhydorazine (PHZ) injection for 3 days showed a decrease in the hematocrit (25.4%) and an increase in serum Epo by 14-fold of the control on day 3 after the treatment. At 3 days, the total number of hematopoietic progenitor cells in the bone marrow of PHZ mice decreased by 67% of the control, while these cells in the spleen increased to 22-fold of the control on day 3 and 55-fold on day 6. A significant increase in CPA was observed in MKE prepared from PHZ mice kidneys. Additionally, bone marrow suppressive anemia induced by 5-fluorouracil resulted in enhanced CPA the same as for PHZ mice, but in contrast, anemia with suppression of Epo-production due to nephrotoxicity induced by cisplatin caused a decrease in CPA. These results suggest that CPA in MKE correlates with hematopoietic conditions, and may have a definite role in hematopoiesis through the function of the kidney.     

Colony-Promoting Activity in Mice Kidneys with Phenylhydrazine Hemolytic Anemia

Aqueous anemic mice kidney extracts (MKE) were assessed colony-promoting activity (CPA) of hematopoietic progenitor cells in serum-free cultures stimulated bv interleukin-3 and erythropoietin (Epo). Mice with hemolytic anemia followed by phenylhydorazine (PHZ) injection for 3 days showed a decrease in the hematocrit (25.4%) and an increase in serum Epo by 14-fold of the control on day 3 after the treatment. At 3 days, the total number of hematopoietic progenitor cells in the bone marrow of PHZ mice decreased by 67% of the control, while these cells in the spleen increased to 22-fold of the control on day 3 and 55-fold on day 6. A significant increase in CPA was observed in MKE prepared from PHZ mice kidneys. Additionally, bone marrow suppressive anemia induced by 5-fluorouracil resulted in enhanced CPA the same as for PHZ mice, but in contrast, anemia with suppression of Epo-production due to nephrotoxicity induced by cisplatin caused a decrease in CPA. These results suggest that CPA in MKE correlates with hematopoietic conditions, and may have a definite role in hematopoiesis through the function of the kidney.     

Specific unresponsiveness to skin allografts in burns

We have examined the potential to provide long-term or even permanent wound coverage in a mouse model of a 30% total body surface area burn using skin allografts. Treatment of the recipient mouse with rabbit anti-mouse thymocyte serum (ATS) followed by donor bone marrow infusion induces a state of specific unresponsiveness to the skin allograft without the need for chronic immunosuppression. Specifically, a B6AF1 mouse receives a burn on Day -2 relative to grafting, ATS on Day -1, and Day +2, a skin allograft from a C3H/He mouse on Day 0, and infusion of C3H/He donor bone marrow on Day +6. We studied three groups of burned mice: Group I, allograft control (n = 5); Group II, allograft plus ATS (n = 12); and Group III, allograft plus ATS and bone marrow infusion (n = 15). Mean graft survival was compared using a one-way analysis of variance and a Student-Newman-Keuls post hoc test. There was no statistical difference in animal mortality among any of the three groups, and there was no evidence of infectious morbidity. Mean skin allograft survival was as follows: Group I, 9 days; Group II, 29 days; and Group III, 66 days (P less than 0.05 vs Group I and II). Nine animals in Group III had intact hair bearing grafts at 90 days when the study was terminated. This study suggests the potential use of induced specific unresponsiveness to skin allografts for wound coverage in thermal injury without use of chronic immunosuppression. In our animal study this was accomplished without increased mortality or apparent infectious morbidity.     

The effects of ACE inhibitor treatment and ACE gene polymorphism on erythropoiesis in chronic hemodialysis patients

Aggravation of anemia in chronic renal failure patients by angiotensin-converting enzyme inhibitors (ACEIs) has been attributed to the inhibition of angiotensin II which facilitates erythropoietin(Epo) production. This study was aimed at evaluating whether ACEIs aggravate anemia in maintenance hemodialysis patients and to investigate the influence of ACE gene polymorphism on erythropoiesis in these patients. Ninety-one hemodialysis patients were divided into 2 groups, based on whether or not they were administered ACEIs, into the ACEI group(n = 24) and the non-ACEI group(n = 67), and comparisons were made of the doses of recombinant human Epo(rHuEpo) administered, the hematocrit(Hct) and the plasma Epo concentrations. Among the patients in the non-ACEI group, only 17 did not receive rHuEpo, while all of the patients in the ACEI group received rHuEpo. The average dose of rHuEpo was 102.7 +/- 45.4 IU/kg/week in the ACEI group and 57.8 +/- 55 IU/kg/week in the non-ACEI group and the difference between the two groups was statistically significant. A statistically significant difference in the Hct was also observed between the two groups: the mean Hct in the ACEI group was 28.7 +/- 2.9% while that in the non-ACEI group was 31.1 +/- 3.7%. The plasma Epo concentrations were significantly lower in the ACEI group than in the non-ACEI group. No significant differences in the rHuEpo dose and Hct were observed between the three ACE genotype classes in either the ACEI or the non-ACEI group, however, there was a significant difference among the three genotypes in the non-ACEI group in regard to the plasma Epo concentrations; patients with the DD genotype had higher concentrations than those with the DI or II genotypes. These data suggest that anemia in maintenance hemodialysis patients is worsened by ACEIs as a result of the suppression of Epo production. Although it has been suggested that the endogenous Epo concentrations in maintenance hemodialysis patients are associated with ACE gene polymorphism, no significant influence of the ACE genotype on the rHuEpo dose or Hct was evident. Therefore, it is possible that exacerbation of anemia by ACEIs in the patients receiving rHuEpo is a result of an inhibited bone marrow response to Epo.     

Dose response to a single intramuscular injection of recombinant adeno-associated virus-erythropoietin in monkeys

BACKGROUND: Anemia is a significant problem in many disease states. Erythropoietin (Epo) has been used in the treatment of anemia associated with numerous chronic diseases. This study investigates the dose-response profiles of a single intramuscular (im) injection of a recombinant adeno-associated virus vector (rAAV) containing the Epo gene with the goal of achieving a sustained elevation of hematocrit (Hct). METHODS: Cynomolgus (cm) monkeys were given single injections of different doses of rAAV-cm-Epo. The biological effect of Epo gene expression was monitored by determining the Hct levels and circulating hormone levels by ELISA. Antibody to the rAAV capsid protein was also measured over the 41-week period of the experiment. RESULTS: Epo expression was noted only when 2 x 10(11) or more particles were injected. Epo was noted to be increased as soon as 1 week postinjection and was maximum in 6 to 8 weeks. This level of expression remained constant for nearly 20 weeks. Animals given the highest dose of rAAV developed a higher Hct over the first 8 weeks postinjection than those given an intermediate dose. However, the maximum levels of hemoglobin were the same. There was a weak correlation between amount of rAAV injected and capsid antibody response. CONCLUSIONS: AAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single im administration. Dose responses to rAAV-Epo are achievable, although a threshold inoculum of virus is necessary to produce an effect and the therapeutic window is narrow.     

Gene expression of LDL receptor, HMG-CoA reductase, and cholesterol-7 alpha-hydroxylase in chronic renal failure

BACKGROUND: Chronic renal failure (CRF) is associated with an atherogenic lipid profile and an increased risk of ischaemic cardiovascular disease. The associated hyperlipidaemia is reportedly ameliorated by erythropoietin (Epo) therapy. According to a recent report, rats studied 3 weeks after 5/6 nephrectomy and fed a high- protein diet exhibited increased activities of hepatic HMG-CoA reductase (HMG-CoAR) and cholesterol 7 alpha-hydroxylase (Ch-7 alpha- H), despite normal corresponding mRNA values. DESIGN AND METHODS: This study was designed to examine the effects of naturally progressing CRF of longer duration as well as those of Epo therapy on gene expressions of the key factors involved in hepatic cholesterol metabolism, i.e., LDL receptor (LDLR), HMG-CoAR, and Ch-7 alpha-H. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy), Epo-treated CRF group (given Epo 150 U/kg/twice weekly) and sham-operated, placebo- treated normal controls. They were allowed free access to regular rat chow and studied 6 weeks after surgery. Liver mRNAs and protein mass or activities of the above factors were studied. RESULTS: Plasma cholesterol concentration was significantly increased in the CRF group (P < 0.001) and was modestly lowered (P < 0.05) by Epo therapy. However, microsomal cholesterol concentration and LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA as well as HMG-CoAR activity, and Ch-7 alpha-H and LDLR protein mass measurements were virtually identical in the three groups. Thus, hepatic LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA and protein measurements in rats with CRF were similar to those of the normal control group representing an inappropriate response to the associated hypercholesterolemia. Epo therapy led to partial amelioration of CRF- associated hypercholesterolaemia with no discernible effect on hepatic tissue expression of the above factors.      

Superior survival and proliferation after transplantation of myoblasts obtained from adult mice compared with neonatal mice

BACKGROUND: Myoblast transfer therapy (MTT) is a strategy designed to compensate for the defective gene in myopathies such as Duchenne muscular dystrophy (DMD). Experimental MTT in the mdx mouse (an animal model of DMD) has used donor myoblasts derived from mice of various ages; however, to date, there has been no direct quantitative comparison between the efficacy of MTT using myoblasts isolated from adult and neonate donor muscle. METHODS: Donor normal male myoblasts were injected into Tibialis Anterior muscles of dystrophic female host mice and the survival and proliferation of male myoblasts quantitated using Y-chromosome specific real-time quantitative polymerase chain reaction. The survival of late preplate (PP6) myoblasts derived from neonatal (3-5 days old) or adult (6-8 weeks old) donor mice after MTT were compared. The influence of the number of tissue culture passages, on survival post-MTT, was also evaluated for both types of myoblasts. RESULTS: Surprisingly, superior transplantation efficiency was observed for adult-derived compared with neonatal myoblasts (both early and late passage). Extended expansion (>17 passages) in tissue culture resulted in inferior survival and proliferation of both adult and neonatal myoblasts; however, proliferation of early passage myoblasts (both adult and neonate) was evident between 3 weeks and 3 months. CONCLUSIONS: Myoblasts derived from neonatal mice were inferior for transplantation, and early passage donor myoblasts from adult mice are recommended for MTT in this model.     

同种肾组织移植治疗慢性肾功能衰竭性贫血的实验研究

以Ｗｉｓｔａｒ雄性大鼠为受体，建立慢性肾功能衰竭动物模型，将鼠婴肾组织声多点植入受体双侧后肢皮下和筋膜下。结果表明，３０天后移植物的体积由１ｍｍ＾３增至４ｍｍ＾３大小，表面血管网丰富；光镜下见肾小球、肾小管结构正常。促红细胞生成素（ＥＰＯ）着色颗粒主要分布在肾小球区，移植组着色程度明显增高。血红蛋白和４促红细胞生成素随移植的时间延长而逐渐升高，实验结果提示，此方法有可能为治疗慢性肾功能衰竭性贫血提     

Kidney and anemia in familial amyloidosis type I

BACKGROUND: Familial amyloid polyneuropathy (FAP) type I is caused by a mutated transthyretin (TTR V30M) and characterized by a sensorimotor and autonomic neuropathy. Renal, cardiac, and ocular abnormalities can also occur. Anemia has been described in previous reports, but its prevalence in Portuguese FAP patients is not precisely known. The aim of this study was to estimate the prevalence of anemia in FAP type I Portuguese patients and to evaluate the contribution of erythropoietin (Epo) to its genesis. METHODS: A retrospective cross-sectional study was undertaken to determinate the prevalence and characteristics of anemia in 165 FAP patients. For comparison analysis, 3 control groups were also evaluated, 1 group of 46 apparently healthy subjects, 1 group of 17 asymptomatic carriers of FAP-trait, and a group of 14 non-FAP patients with chronic renal insufficiency. Serum Epo levels were analyzed in all groups. RESULTS: Anemia was present in 24.8% of symptomatic FAP patients. Iron stores, B12 vitamin, and serum folate levels were normal. FAP patients presented significantly lower serum Epo levels than healthy controls (P= 0.003). Epo levels were found lower than expected for the degree of anemia and in 17.5% were undetectable. Low Epo values were observed independently of the presence of renal failure or anemia, and sometimes preceded clinical disease. CONCLUSION: Anemia in FAP type I is a common manifestation. The results clearly suggest a defective endogenous Epo production in the genesis of the anemia.