The Therapeutic Effects Of Combinatorial Atorvastatin Administration And Mesenchymal Stem Cells Implantation In Rat Model Of Renal Ischemia/Reperfusion Injury And Its Potential Mechanisms

Part 1:Maximum efficacy of mesenchymal stem cells in rat model of renal ischemia/reperfusion injury:renal artery administration with optimal numbersBackgrounds:Despite the potential therapeutic benefits, cell therapy in renal ischemia/reperfusion (I/R) injury is currently limited by low rates of cell engraftment after systemic delivery. Previous studies have revealed that the route of cell delivery has a crucial role in determining the efficiency of the therapy. The most commonly used routes for MSC administration are tail vein and carotid artery. In this study, we develop an alternative route-renal artery and compare the migration and therapeutic potential of mesenchymal stem cells (MSCs) in ischemic kidney after the graded doses MSC implanted via different routes.Methods:The model of renal I/R injury was induced by 45 min occlusion of the left renal pedicle and right nephrectomy in rat. Followed by reperfusion, these rats were randomly divided into the intervene group (n=60) and the control group (n=6). According to the delivery routes and different doses of CM-Dil labeled MSCs, the intervene group was divided into 10 subgroups (n=6/subgroup):tail vein (TV) group, 1×106; carotid artery (CA) group, 1×106，5×105; 1×105; 5×104; and renal artery (RA) group, 1×106,5×105;1×105; 5×104. Renal blood flow was evaluated by color and spectral Doppler ultrasound at 1h and 24h post-I/R. All the samples were collected for analysis at 24h post-I/R. The serum creatinine (Scr) level was examined; the kidney tissue stained by HE was analysed for evaluating the renal injury score; and the homing of implanted MSC in the kidney and other organs were observed under fluorescent microscopy.Results:(1) At 24h after reperfusion, animals in the control group displayed severe kidney damage:the renal injury score was 3.30±0.65 and the Scr level significantly increased compared to normal rats (5.22±0.43mg/dl vs.0.92±0.14mg/dl, P<0.05). After injection of 1×106 MSCs, the CA group showed the better therapeutic efficiency in attenuating the I/R-induced renal dysfunction than the TV group and the RA group; compared with the control group, the Scr level decreased to 4.48±0.95mg/dl (P<0.05) and renal injury score 2.85±0.76 (P<0.05). In the TV group, there was a trend towards lower Scr level (4.78±0.88mg/dl) and renal injury score (3.15±0.68); however, the differences between the TV group and the control group were not significant. A few cells were observed in the renal tissue of the CA group and fewer in the TV group. The RA group showed obviously increased renal retention of grafted MSCs compared with TV and CA group; however, the renal function was even further deteriorated as indicated by significant higher Scr level (6.51±0.58mg/dl, P<0.05)and injury score (3.35±0.51mg/dl, P<0.05) compared to the control group. (2) Among the subgroups in CA group, there was positive correlation between the injected cell dosage and the improvement of renal function, and the best therapeutic effect was observed in the CA group (1×106). The RA group displayed a U-type change of Scr level, with the lowest Scr level occurring when 1×10 MSCs were applied. Compared with CA group (1×106), RA group (1×105) performed significant better in preserving the renal functionthe (P<0.05). (3) In addition, further fluorescent microscopic found that lager number of implanted cells accumulated in glomeruli, especially in the RA group (1×106), and with the reduction of infused cell dosages, the MSCs located in the kidney decreased. The ultrasonic examination further confirmed that the relationship between the amount of MSCs homing to the kidney and renal blood flow was positive and the aggravated renal dysfunction in RA group was due to renal hypoperfusion caused by cell occlusion.Conclusion:Administration route and dosage are two critical factors determining the efficiency of cell therapy. Administration of MSCs through renal artery is the best delivery route to improve the renal retention of implanted MSCs and 1×105 MSCs injected through renal artery produces the most dramatic improvement in renal function and morphology in rat model of renal I/R injury. Too many MSCs localized to glomeruli may cause occlusion, reduce renal blood flow and then aggravated the renal damage.Part 2:The therapeutic effects of combinatorial Atorvastatin administration and mesenchymal stem cells implantation in rat model of renal ischemia/reperfusion injury and its potential mechanismsBackgrounds:The pathological environment produced by renal ischemia-reperfusion (I/R) injury, characterized by the elevation of inflammatory cytokines, is thought to be at least partially responsible for the poor survival and engraftment of injected cells and limit the benefit of MSCs. Statin has been shown to improve renal microcirculation and preserve renal function after renal I/R injury. This study aims to investigate the impacts of combinatorial Atorvastatin (Ator) perioperative administration and mesenchymal stem cells (MSCs) implantation on therapeutic effects in the rat experimental acute kidney injury.Methods:The model of renal ischemia/reperfusion injury (I/R) was induced by the release of bilateral renal pedicles clamps following 45 min of occlusion. Immediately after reperfusion, CM-Dil labeled MSCs (1×106 cells) or vehicles only were administered through the carotid artery of the animals pretreated with Ator (5mg/kg/d, n=24) or without (PBS, n=24). Treatment with Ator was started 3 days prior to MSCs transplantation and continued until time of sacrifice. All animals were divided into four groups, and each of the following groups contained twelve rats: no-Ator+no-MSCs (control), Ator+no-MSCs (Ator), no-Ator+MSCs (MSCs) and Ator+MSCs. Six rats per group were sacrificed per time point, at 24h and 72h post-I/R, respectively and collect all the samples for analysis. The serum creatinine (Scr) level and blood urea nitrogen (BUN) level were examined; the kidney tissue stained by HE was analysed for evaluating the renal injury score; the homing of implanted MSC in the kidney was observed under fluorescent microscopy; apoptosis was confirmed by terminal deoxynueleotidyl transferase(TdT)-mediated dUTP-biotin Hick end labeling (TUNEL) assay; expression and distribution of Ki-67 in the kidney were determined by immunohistochemical analysis; the expression of inflammatory-associated cytokines, and several oxidative stress-related products and enzymes in the kidney were detected by ELISA; the relative abundance of Bax, Bcl-2, Toll-liker receptor 4 (TLR4) and high-mobility group box 1(HMGB1) was analyzed by western blot.Results:(1) Post-I/R 24h, there was a marked reduction in the levels of Scr and BUN in Ator or MSCs group, compared to control group, as well as the kidney injury score (P<0.05). Furthermore, Ator+MSCs group further decreased Scr and BUN levels compared with either control group or MSCs groups (P<0.05). At 24h after renal IR injury, apoptosis of renal tubular cells in the Ator or MSCs group was obviously lower than that in control group (P<0.05) and further analysis indicated that Ator+MSCs group had a significantly reduced apoptotic index compared with MSCs group (P<0.05). The quantitative analysis of apoptosis-regulating proteins (Bax and Bcl-2) was consistent with the TUNEL. After I/R injury 72h, the frequency of Ki-67 (renal proliferation response) was higher in Ator group and/or MSC group than the control group, especially in Ator+MSC group (P<0.05). (2) Immunofluorescent analysis of renal sections revealed that post-ischemia 24h, the numbers of CM-Dil labeled cells in Ator+MSCs group were obviously higher compared with MSCs group (32.33±3.51 vs.22.67±3.79, P<0.05).The expression levels of IGF-1 in Ator+MSCs group were significantly higher than that in MSCs group. Moreover, there were trends towards up-regulation of VEGF and HGF in Ator+MSCs group compared with MSCs group. (3) Compared with the non-Ator treated groups (control group and MSCs group), the Ator treated groups (Ator group and Ator+MSCs group) revealed lower inflammatory factors (TNF-α:3020.53±907.78 vs.4020.95±1207.28, P<0.05; IL-10: 985.74±207.19 vs.1210.29±307.79, P<0.05) and increased anti-inflammatory factors (IL-10:542.23±91.25 vs.402.28±72.07, P<0.05). In regarding to the oxidative stress-related products and enzymes, which included MPO, MDA, SOD and GSH, we found that the MPO activity and the MDA concentration were lower in Ator-treated groups than non-Ator treated groups (P>0.05). With regard to the further mechanism, we found that the expression of Toll-like receptor 4 (TLR4) and high-mobility group box 1, potential mediators of innate immunity, was significantly decreased in the Ator-treated groups (P<0.05).Conclusion:Ator treatment may protect the kidney undergoing I/R injury through suppression of TLR4 signaling, creating a better environment for the survival of grafted MSCs. The extra benefit of the Ator+MSCs combined therapy may result from the Ator-mediated inhibition of oxidative stress and inflammation in the ischemic kidney.