Adipose Derived Mesenchymal Stem Cells Treat The Acute Kidney Injury

Cisplatin has been widely and effectively used for chemo-therapy against a number of tumor types; however, the drug induces a series of side effects in various organs, of which the major organ affected is the kidney. Cisplatin has been hypothesized to induce neph-rotoxicity especially acute kidney Injury (AKI), through triggering the apoptosis of tubular cells. Recently, for treatment of AKI, the stem cells therapy was improved.since many restrictive factors related to ethics and safe, the mesenchymal stem cells (MSC) was used in clinical therapy and research, and it has been proved that MSC has capacity to repair the kidney when cisplatin induced AKI happened.Furthermore, there was lots of datas showed MSC (bone marrow) protect kidney against AKI, with the mechanism like cells infusion or paracrine. Usually, MSC was obtained from bone marrow with making patients feel pain, besides that the quality of bone marrow depend on the patients’ states.As the results, we choose the MSC cells from human adipose tissue (AD-MSC) in this research, this potential cells have series of advantage, including extensive sources, easily to obtain, and the similar feature as MSC from bone marrow.In our research, patients’adipose tissue was obtained aroud 5-10ml, and MSC was purified and cultured using serum free kits.45 Sprague-Dawley rats were divided into three groups, which included the healthy group, controls group, those subjected to cisplatin-induced acute kidney injury (AKI) for 24h with injecting saline, and treatment group, whose rats was subjected to cisplatin-induced AKI for 24 h, followed by AD-MSC engraftment with 1-2*106 cells/1 ml/rat for one rat. The rats were sacrificed at day 5 and the effects were analyzed using various methods, including biochemical analysis(BUN、 Ccr> mALB、β2 mG), structural examina-tion (HE and PAS staining), the change of apoptosis and proliferation capacity for renal cells (TUNEL and PCNA staining),and cell tracking experiments. In addition, an in vitro experiment with NRK-52E cells was performed. The cells were divided into three groups, including the healthy control, cisplatin induction and cisplatin induction with co-culture of AD-MSCs, and were subsequently assessed with a Transwell assay. After culture for four days, the cells were lysed and the total protein extract was subjected to western blot analysis.At last, we establish the embryonic cells (ES cells) as model, a universal gene targeting plasmid, which successfully targeting any functional gene which can repair AKI in Rosa26, and Cre/LoxP system has been used to achieve the conditional targeting with model protein A in ES cells. PCR, immunofluoresent staining and western blotting was used to detect expression effects of protein A (as model).The data showed Cisplatin-induced renal dysfunction and tissue damage was recoved following AD-MSC infusion, although there were few AD-MSCs observed around the injured kidney tubules in the kidney. When the cisplatin-treated NRK-52E cells were co-cultured with AD-MSCs, the activation of p38 and BAX were inhibited, while the expression of Bcl-2 was upregulated, as compared with the cisplatin-treated NRK-52E cells that were not co-cultured. Therefore, AD-MSCs were shown to markedly improve cisplatin-induced renal failure and tubular cells necrosis through the secretion of certain factors, which subsequently inhibited the apoptosis pathway in vitro. It was hypothesized that AD-MSC secretion was trig-gered by the injured tubular cells. Thus, AD-MSCs may be important for the therapy of patients with renal injury due to the proliferation capacity increase and their antiapoptotic capacity.However, even AD-MSC can help repair renal structure, kidney function and epithilia cells to some extent, but MSC were supposed that it was still cannot recover the kidney to totally health in the experiments. So in the third part of this research, we design a "universal" gene targeting plasmid, protein A was tested to insert in the plasmid and homologous recommbinated in to ROSA26 through DNA test and western blotting, by Cre protein expression, A protein expressed in high efficiency.This plasmid and gene targeting was used to optimized the MSC treatment, it can insert some functional gene which can repair AKI kidney into ROSA26 of MSC, and make some key protein overexpression in specific time, genome and in one copy, it will be more safety and stabelly,which will help to promote the effect of therapy in the future.