Enhanced renoprotective effect of IGF-1 modified human umbilical cord-derived mesenchymal stem cells on gentamicin-induced acute kidney injury

The therapeutic action of umbilical cord-derived mesenchymal stem cells (UC-MSCs) against acute kidney injury (AKI) has been demonstrated by several groups. However, how to further enhance the renoprotective effect of UC-MSCs and improve the therapy effect, are still unclear. In this study, we mainly investigated whether insulin-like growth factor-1 (IGF-1)-modified UC-MSCs hold an enhanced protective effect on gentamicin-induced AKI in vivo. Our results indicated that the IGF-1 overexpression could enhance the therapeutic action of human UC-MSCs, and the AKI rats treated with IGF-1-overexpressed UC-MSCs (UC-MSCs-IGF-1) showed better recovery of biochemical variables in serum or urine associated with renal function, histological injury and renal apoptosis, compared with AKI rats treated with normal UC-MSCs. RNA microarray analysis indicated that some key genes in the signal pathways associated with anti-oxidation, anti-inflammatory, and cell migratory capacity were up-regulated in UC-MSCs-IGF-1, and the results were further confirmed with qPCR. Furthermore, a series of detection in vitro and in vivo indicated that the UC-MSCs-IGF-1 hold better anti-oxidation, anti-inflammatory, and cell migratory capacity for IGF-1 overexpression. Thus, our study indicated that enhancement of UC-MSCs bioactivities with IGF-1 overexpression could increase the UC-MSCs therapeutic potential and further developed a new therapeutic strategy for the treatment of AKI.


Materials and Methods
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
The protocol was approved by the Committee on the Ethics of Animal Experiments and Human Subject Research of School of Pharmaceutical Science, Jilin University.
All operations were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. The participants have provided the written informed consent to participate in this study.

Isolation and characterization of human UC-MSCs
Human umbilical cords were obtained after caesarean section of normal pregnancies at First Hospital of Jilin University. The UC-MSCs were extracted as the reference 1 .

Establishment of IGF-1 overexpressing cell lines
To generate IGF-1 overexpressing vectors, the IGF-1-coding sequences were obtained by reverse transcription PCR and further cloned into pMXs-based retroviral plasmid After 2 days of culture, the medium of each group was collected for secretory cell function analysis. Vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-7 (BMP-7), and interleukin-10 (IL-10), which were secreted by UC-MSCs and relieved kidney injury, were measured using an ELISA Kit (Abcam, United Kingdom) following the manufacturer's instructions.

Real-time quantitative PCR
Total RNA was extracted with Trizol (Invitrogen). Then, 2 mg of RNA was reverse transcribed using RT-PCR kit (Takara) and qPCR was performed using a Thermal Cycler Dice TM Real Time System and SYBR Green Premix EX Taq TM (Takara). In our study, β-actin was used for Real-time quantitative PCR (qPCR) normalization and all items were measured in triplicate. All primer sequences (5'-3') are as follows:

Preparation and treatment of AKI model
Male node rats, weighing 250 g to 300 g, were purchased from Vital River Laboratories (Beijing, China). All rats were housed in the room with constant temperature and a 12 hours dark-12 hours light cycle. The node rats were fed a standard diet. The method used to prepare or treat the AKI model was similar to our previous research 3,4 . In brief, animals were divided into six groups (six rats per and Fas) and anti-apoptotic gene (Bcl-2) in the kidney tissues of each group were further detected using real-time qPCR, and some apoptotic genes were also confirmed with immunohistochemistry.

Immunohistochemistry
The cells or kidney tissues were fixed in 4% paraformaldehyde in PBS. The kidney tissues of each group were embedded in paraffin and sliced into 5 μm-thick sections.

Microarray analysis
Total RNA was extracted from UC-MSCs, UC-MSCs-vector and UC-MSCs-IGF-1 using Trizol (Invitrogen) and the RNeasy kit (Qiagen). Samples were amplified and labeled using a Aglient Quick Amp Labing Kit. Array hybridization was analyzed with the Aglient Hybridization System and followed by washing with the Aglient wash buffer kit. The Aglient DNA microarray scanner was used for array scanning.
Genes that have values greater than or equal to lower cut-off: 50.0 in 2 out of two samples were chosen for data analysis. Differentially expressed genes were identified through Fold Change filtering. Pathway Analysis and GO analysis were applied to determine the roles of these differentially expressed genes played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show distinguishable gene expression profiling among samples. The cells were incubated with DMEM/F12 (Hyclone) plus 10% FBS (Gibco) in the co-culture system, and exposed to 30 μM H 2 O 2 for 1 day and 2 days respectively.

Knockdown of IGF-1 in UC-
Finally, proliferation index of HK2 cells in each group was detected with CCK-8, and the HK2 cells co-cultured with noting were used as the control group.
Anti-inflammatory potential assay 11 The inflammatory components in the kidney tissues of each group were detected with ELISA. Similar to anti-oxidation capacity assay, the supernatant from kidney tissue was collected for the detection. Herein, monocyte/macrophage chemotactic protein-1 (MCP-1), Macrophage inflammatory protein 2 (MIP-2), regulated upon activation normal T cell expressed and presumably secreted (RANTES), and transforming growth factor-β (TGF-β) and were examined for evaluation of inflammatory reaction.
In addition, to further detect the anti-inflammatory potential of UC-MSCs, UC-MSCs-vector and UC-MSCs-IGF-1, a co-culture system was established using trans-well. Similar to anti-oxidation capacity assay, UC-MSCs, UC-MSCs-vector and UC-MSCs-IGF-1 were seeded on the upper side of a porous polycarbonate membrane in co-culture with murine macrophage RAW264.7 cell line (ATCC, USA) respectively.
The RAW264.7 cells were seeded at 1×10 5 cells/well in a 24-well plate. Keep the ratio of RAW264.7 cells and UC-MSCs at 5:1. The cells were incubated with DMEM/F12 (Hyclone) plus 10% FBS (Gibco) in the co-culture system, and exposed to lipopolysaccharide (100 ng/ml, Sigma) for 24 hours. Finally, the expression of genes associated with inflammatory response (TNF, IL-6 and COX-2) in RAW264.7 cells of each co-culture group was detected with qPCR, and the RAW264.7 cells co-cultured with noting were used as the control group.

Cell migration assay
To evaluate the effect of IGF-1 overexpression on migratory ability of UC-MSCs in vivo, anti-human nucleus antigen (hNA, Millipore) was used to detect the location of 12 human mesenchymal stem cells in kidney tissue of each group with immunohistochemistry. The number of hNA-positive cells was further counted in ten fields for each group, and the average value was used to evaluate the migratory ability of UC-MSCs, UC-MSCs-vector and UC-MSCs-IGF-1.
To investigate the influence of cell proliferation on the migration, mitomycin C was used to block proliferation as the reference 3 . The migration of UC-MSCs was still measured using Tran-swell plates as the previous study 3

Statistical analysis
The results were expressed as means ± SEM, and statistical analysis was performed using SPSS 17.0. The differences among groups were analyzed by one-way ANOVA followed by t-test. P＜0.05 was considered statistically significant.