Ionizing radiation causes a state of oxidative stress and destroys essential cellular targets, such as DNA and membranes, leading to failure of tissue function beside cell death. Radiation-induced apoptosis has been detected in many cells and tissues that may relate to the radiation-induced expression of proteins that trigger an apoptotic response. In non-hematopoietic cells, radiation can lead to up-regulation of pro-apoptotic genes (caspase-3) and/or downregulation of anti-apoptotic genes, (such as Bcl-2) that leads to nephropathy and hematological changes even with dose fractionation strategy. The curative effect of BM-MSCs transplantation on rats has been studied before in different models; BM-MSCs can express growth factors that facilitate survival, and improve the structure and function of damaged kidney. EPO is a glycoprotein hormone used in the treatment of anemia associated with kidney diseases. It is used in cancer patients undergoing chemotherapy as an alternative way to minimize blood transfusions. EPORs mediate the proliferation and apoptosis of a number of non-hematopoietic cells by the downstream PI3K/Akt pathway. Therefore, the present study was undertaken to: (i) Explore the therapeutic efficacy of BM-MSCs and/ or EPO in kidney injury induced by γ-irradiation. (ii) Confirm whether BM-MSCs and/ or EPO exhibit effects involving PI3K/Akt anti-apoptotic signaling pathway. (iii)Investigate the possible mechanisms by which EPO could help to regenerate kidney tissues by BM-MSCs. Summary and conclusion 6 - 2 These purposes were fulfilled by determination of some biochemical markers: 1- Whole blood: Complete Blood Count (CBC), differential leucocyte count. 2- Serum: Kidney functions (urea, creatinine, BUN). 3- Renal tissue: ➢ Lipid peroxides as an oxidative stress marker expressed as malondialdehyde (MDA). ➢ Anti-oxidant content: reduced glutathione (GSH) content and total antioxidant capacity (TAC). ➢ Apoptotic marker: Caspase-3. ➢ Anti-apoptotic marker: Bcl2. ➢ Erythropoietin signaling markers: erythropoietin (EPO), the gene expression of erythropoietin receptor (EPOR), phosphorylated Janus kinase 2 (p-JAK2), phosphorylated phospho-inositol-3 kinase (p-PI3K) and phosphorylated protein kinase B (p-Akt). ➢ Histopathological examinations for renal tissue: Hematoxylin and Eosin (H & E stain). Experimental design Preliminary study was conducted in two doses of fractionated γirradiation on the normal rats (6Gy group): total dose of 6Gy as 2Gy*3 days and (8Gy group): total dose of 8Gy as 2Gy* 4 days. Depending on the results obtained about the kidney damage, we selected the dose of total 8Gy to be used with the rest of the experiment. Normal rats and fractionated whole body γ-irradiated rats were used in the current study. Normal rats were divided into four subgroups (n=6 each) as follow: Summary and conclusion 6 - 3 Normal control group: normal rats received no treatment. BM-MSCs group: normal rats received BM-MSCs (1x106 cells/rat) as single intravenous injection. EPO group: normal rats received EPO (100 IU/ kg, i.p) every other day for 30 days. BM-MSCs + EPO group: normal rats received BM-MSCs and EPO as defined above. Moreover, the fractionated whole body γ-irradiated rats were divided into 4 subgroups (n=6 each) as follow: γ- Irradiated control group: rats exposed to fractionated whole body γirradiation (2 Gyx4 days) of total 8Gy and received no treatment. γ- Irradiated + BM-MSCs group: γ-irradiated rats received BM-MSCs (1x 106 cells per rat, i.v. once) one hour afterward last radiation dose. γ- Irradiated + EPO group: γ-irradiated rats received EPO (100 IU/ kg, i.p) every other day for 30 days starting 1 hour after last radiation dose. γ- Irradiated + BM-MSCs + EPO group: γ-irradiated rats received BMMSCs and EPO as described above. The experiment was ended after 30 days of treatments. Blood was collected for complete blood count and measuring kidney function. Renal tissue samples were used for measuring oxidative stress, apoptotic markers, EPO signaling markers and histopathological studies. Results 1-Effect of treatment with BM-MSCs and/ or EPO on normal rats Treatment of normal rats with BM-MSCs (1x106 cells/rat) as single intravenous injection and/ or EPO (100 IU/ kg, i.p) every other day for 30 days leads to decline in serum urea, BUN and increased renal GSH. Summary and conclusion 6 - 4 BM-MSCs monotherapy increased renal TAC, p-PI3K contents. EPO monotherapy decreased renal caspase-3 and EPO contents while the combined treatment increased renal TAC, Bcl2, p-PI3K and p-Akt contents in comparison to normal control rats. Histopathological study showed normal histological structure characterized by circumscribe glomeruli with normal structure of capillary tufts and Bowman's capsule. The renal tubules of both proximal and distal convoluted tubules showed intact epithelial lining and regular arrangement (score 0) in comparison with normal control group. 2-Effect of fractionated γ- radiation on normal rats Exposure of normal rats to whole body γ- radiation as fractionated dose (2Gy*4 days) revealed pancytopenia (low RBCs, WBCs and platelets), with low lymphocytes and monocytes percentage. MCHC was decreased with a noticeable elevation in MCV, serum urea, BUN, creatinine, renal MDA and caspase 3 activity. They also revealed significant drop in renal GSH, TAC and Bcl2 protein contents. Conspicuously, they revealed down-regulation of renal EPO signaling (EPO, EPOR, p-JAK2, p-PI3K and p-Akt). Histopathological study revealed shrinkage of capillary tufts with widening of Bowman's space of some glomeruli and renal tubules showed epithelial cell degeneration with marked swelling of tubular epithelial lining. Congestion and perivascular edema were also seen. This in turn caused a significant increase in histopathological scoring in comparison with normal rats. 3-Effect of treatment with BM-MSCs and/ or EPO on fractionated irradiated groups Treatment with BM-MSCs or EPO after exposure to fractionated dose (2Gy*4 days) γ- radiation improved the disrupted parameters induced by γirradiation. Therefore, whole blood RBCS, Hb, MCHC, WBCs, ALC, platelets, lymphocytes and monocytes percentage, renal GSH, TAC and Bcl2 Summary and conclusion 6 - 5 protein contents were significantly increased with up-regulation of renal EPO signaling (EPO, EPOR, p-JAK2, p-PI3K and p-Akt) compared to untreated γirradiated group. Meanwhile serum urea, BUN, creatinine, MDA, caspase3 levels were significantly reduced as compared to untreated γ-irradiated group. In addition, histopathological examination of renal tissue showed a mild protection against kidney damage with a single treatment group while in the combined treated group showed moderate or marked renal tissue protection and appeared as normal kidney structure. In addition to regenerated epithelial cells in some renal tubules and a significant decline in histopathological scoring was reported. 4-Correlations Using combined results from all irradiated groups in this study revealed that urea was positively correlated with both creatinine and MDA. However, it was negatively correlated with RBCs, WBCs, platelets, GSH and TAC content. All blood cells: (RBCs, WBCs and platelets) were correlated positively with GSH, TAC and correlated negatively with MDA. Moreover, Bcl2 and EPO signaling markers (EPO, EPOR, p-JAK2, p-PI3K and p-Akt) were correlated positively with GSH and TAC, while negatively correlated with MDA. Caspase 3 was correlated negatively with GSH and TAC, while positively correlated with MDA in renal tissues. Conclusion 1- The present study revealed that exposure to 8Gy fractionated dose γradiation (2Gy*4 days) intensify the damaging effects on the kidneys than exposure to 6Gy fractionated dose γ- radiation (2Gy*3 days). 2- BM-MSCs transplantation has a promising therapeutic/repair potential in renal regeneration in γ-irradiated rats. 3-EPO can prevent anemia by stimulating erythropoiesis in γ-irradiated rats. It also stimulated antioxidant and antiapoptotic effects mediated by the non- Summary and conclusion 6 - 6 hematopoietic PI3K/Akt anti-apoptotic signaling downstream to EPOR activation. 4- EPO enhanced BM-MSCs homing and regeneration potential and potentiated the therapeutic efficacy of BM-MSCs to restore injured hematopoietic tissue components, deteriorated kidney function parameters, antioxidants, renal tissue histopathology as well as its associated anemia. 5- Treatment with EPO and BM-MSCs together revealed a significant modulation in most tested parameters and the benefit of this therapeutic approach is the use of a smaller dose of BM-MSCs for transplantation. 6- Based on statistical evaluations using correlation analysis, the obtained data showed that; there was a correlation between oxidative stress markers with all other tested parameters in the current study. Recommendation This study recommended the combination of both (BM-MSCs and EPO) as a promising intervention for the treatment of nephropathy, hematological parameters, and other adverse effects of γ- radiation. Obviously, this combination is suggested for large scale experimental and clinical trials in the future to overcome the side effects of γ-radiation.