Effect of HO-1 gene modification on proliferation and differentiation of bone marrow-derived mesenchymal stem cells under the acute kidney injury microenvironment

doi:10.3877/cma.j.issn.2095-3216.2015.04.008

Abstract

Abstract:

Objective

To investigate the effect of heme oxygenase-1 (HO-1) gene modification on the proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) under the acute kidney injury (AKI) microenvironment in vitro and the possible mechanism.

Methods

Plasmids were constructed by the gateway technology that contained HO-1 target gene (eGFP as the tracing-gene) or only eGFP as the control, and were transfected into the 293FT cells through the liposome method to get the lenti-HO-1-eGFP/puro and the lenti-eGFP/puro, which then infected BMSCs to obtain HO-1-BMSCs and eGFP-BMSCs, testing the activity and differentiation potential of transfected cells. The ischemia/reperfusion-AKI kidney homogenate supernatant (KHS) and the control N-KHS were harvested and used to cultivate with BMSCs, eGFP-BMSCs, or HO-1-BMSCs, respectively, consisting of five groups: the blank group (BMSCs group), control group (BMSCs/N-KHS group), BMSCs/AKI-KHS group, eGFP-BMSCs/AKI-KHS group, and HO-1-BMSCs/AKI-KHS group, at 37 ℃ in 5% CO₂ for 3 days. The MTT method was used to detect the growth inhibitory rate of BMSCs, flow cytometry to detect the cell apoptosis, the transmission electron microscope (TEM) to observe the cell ultrastructure changes, immunohistochemistry to detect the expression of cytokeratin 18 (CK18), and Western blot to detect the protein expressions of HO-1, phospho-AKT (pAKT), and phospho-ERK (pERK).

Results

The cell viability and differentiation potential of BMSCs were not changed by the gene modification. Compared with the BMSCs/N-KHS group, the growth inhibitory rate of the BMSCs/AKI-KHS group as well as the proportion of apoptotic cells increased significantly (t=12.581, t=16.283, P<0.05), which, after HO-1 gene modification, however, significantly decreased in the HO-1-BMSCs/AKI-KHS group (t=5.958, t=7.957, P<0.05). AKI-KHS induced ultrastructural change of the renal tubular epithelial differentiation in the cultured BMSCs with CK18 expression in the cytoplasm. The proportion of the CK18⁺ cells was the highest in the HO-1-BMSCs/AKI-KHS group (t=4.057, P<0.05). Compared with the BMSCs/AKI-KHS group, the HO-1-BMSCs/AKI-KHS had significantly increased cellular expression of HO-1 (t=4.163, P<0.05), pAKT (t_pAKT=14.305, P<0.05), and pERK (t_pERK=7.148; P<0.05).

Conclusions

In the AKI microenvironment, HO-1 gene modification could improve the proliferation of BMSCs, enhance the ability of BMSCs to differentiate into renal tubular epithelial cells, and decrease the apoptosis of BMSCs. HO-1 overexpression with the downstream AKT and ERK signaling pathway might be the possible mechanism.

Key words: Heme oxygenase-1, Bone marrow-derived mesenchymal stem cells, Acute kidney injury

Nanmei Liu, Huiling Wang, Guofeng Han, Xiuzhi Yu, Jun Tian, Weifeng Hu, Jinyuan Zhang. Effect of HO-1 gene modification on proliferation and differentiation of bone marrow-derived mesenchymal stem cells under the acute kidney injury microenvironment[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2015, 04(04): 200-207.

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References 22

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