Long-Term Reversal of Renal Anemia in Rat by Erythropoietin Gene Modified Mesenchymal Stem Cell Transplantation Beneath the Renal Capsule, Which Is Effectively Regulated by Hypoxia.

INTRODUCTION: Patients with chronic renal failure usually require exogenous erythropoietin (EPO) to alleviate anaemia resulting from inadequate epo production by the kidneys. Although this treatment is effective, recombinant EPO could cause hypertension, its antibodies, and other side-effects. EPO gene therapy is more attractive since it could be more economical and more convenient for the long-term management of the disease. As targets for gene therapy, mesenchymal stem cells (MSCs) are more easily expanded, lower immunogenic, and more stable for expressing exogenous genes than other cells. Here we describe an approach of transplantation of the erythropoietin gene modified mesenchymal stem cells beneath the renal capsule to treate Wistar rats with relative erythropoietin deficiency.

METHODS: we constructed an EPO-expressed vector pGenesil-HRE-EPO directed by hypoxia response element (HRE)/SV40 promoter, and transduced it into MSCs prepared by adherently culturing rat bone marrow. The EPO-gene modified MSCs (Epo+) in vitro growed normally and stably secreted high level of functional EPO in hypoxia condition, which produced from 3.23 × 10(5) to 9.98 × 10(5) mIU of EPO per 10(6) cells per 24 hr. To determine the role of these MSCs to treate renal anemia in vivo, EPO+, EPO (vector pGenesil-1 or normal) MSCs were injected into rats with renal anemia beneath the renal capsule, the skin, or in muscles (4 × 10(7) cells/rat). Renal anemia rats had been induced by feeding with adenine, whose hematocrit levels were at 33.5 +/− 4.1%, the plasma Epo concentration was 6.5 ± 1.7 mU/ml. EPO concentration was determined by ELISA. During whole experiment, we measured rat body weight and blood pressure, and obtained blood samples regularly.

RESULTS: 2wk after transplantation, the hematocrit levels in anemia rats injected Epo+ MSCs beneath the renal capsule, the skin, or in muscles markedly increased to 67.2 ± 5.9%, 61.5 ± 5.7%, and 53.8 ± 5.2%, and 10wk after transplantation to 68.0 ± 5.6%, 52.7 ± 5.8%, and 40.6 ± 5.5%, respectively. Moreover, the plasma Epo concentration of anemic rats injected Epo+MSCs beneath the renal capsule, the skin, or in muscles was considerably increased to 158 ± 35 mU/ml, 136 ± 36 mU/ml, and 114 ± 42 mU/ml at 2 wk, whereas that of anemic rats injected EPO MSCs was not increased significantly. All rats injected Epo+MSCs did not have polycythemia or severe hypertension. Survival time of anemic rats injected Epo+MSCs beneath the renal capsule is longest among anemic rats. Histological examination by fluorescent microscopy showed that implanted MSCs beneath the renal capsule survived best.

CONCLUSIONS: EPO gene-modified mesenchymal stem cells can be transplanted in renal anemia rats, and can produce sufficiently active EPO to correct anemia. Transplantation of the cells beneath the renal capsule is best.