| Background Blood purification is the most frequently used treatment of acute renal failure(ARF) complicated by multiple organ dysfunction syndrome(MODS). However, the mortality rate of ARF with MODS remains high. The possible reason is that current renal replacement therapies restore only the glomerular filtration function and has little effect of the replacement of the tubular immunity, reabsorption, endocrine and metabolism functions of the kidney. To optimize renal substitution therapy, a bioartificial renal tubule assist device(RAD) was developed, which was composed of renal proximal tubule cells grown within a hollow fiber cartridge. The addition of the tubular cell replacement therapy in a tissue-engineered bioartificial kidney will likely improve clinical outcomes. We first constructed the RAD and finished the animal experiment with RAD in China. But in our research, we found that it was the bottleneck of RAD in basic research and clinical application to establish a large animal model of ARF and MODS in accordance with the clinical practice and the source and life of cells. So, this study was aimed to establish a composite ARF animal model with MODS and to investigate effects of the treatment with a bioartificial kidney constructed with renal cells cocultured by bone marrow mesenchymal stem cells(MSCs) in the animal model and its possible mechanism.Part I Study on establishment of pig model of multi-organ dysfunction with acute renal failure and its immune statusObjective To establish a composite pig model with ARF and MODS, and to investigate the differences of serum neutrophil gelatinase-associated lipocalin(NGAL) and proinflammatory cytokines in acute uremic pigs with or without MODS.Methods Twenty-two healthy hybridized pigs were randomly divided into three groups. Group CLP+CRA(n=12) served as the ARF and MODS model which received cecal ligation and puncture(CLP), resulting in abdominal infection plus clamping of renal artery(CRA). Group CLP(n=5) received CLP only. Group CRA(n=5) received CRA only. Vital signs and the functions of the main organs were observed. Serum NGAL, TNF-a, and IL-6 were measured at 0,4,8,24, and 48h after surgical admissions.Results The MODS model was successfully induced, which manifested as renal failure, liver dysfunction, progressive decline of cardiac function and abnormal pulmonary function. Apparent pathological changes were found in kidney, liver, lung and small intestine of group CLP+CRA. In group CLP+CRA and group CLP, serum concentrations of TNF-a reached the peak at 8h,which were higher than those of group CRA(621.50±207.31pg/ml,597.18±211.57pg/ml vs 194.82±57.06pg/ml,P<0.01).Serum IL-6 levels of group CLP+CRA and CLP were higher than those of group CRA at 24h(P<0.01). There was a significant difference in serum NGAL levels of group CLP+CRA and group CRA compared with those of group CLP at 8h(463.08±75.29ng/ml,317.42±58.51ng/mlvs 132.67±43.58ng/ml, P<0.05).Conclusions The dysfunction in organs and systems was caused by CLP-induced severe intra-abdominal infection. It was successful and steady to reproduce the large animal model which showed both MODS and ischemic ARF symptoms. ARF animals with MODS have higher serum NGAL compared with ARF animals alone. Monitoring the activities of TNF-a, NGAL and IL-6 would make great contributions in discovering MODS and evaluating the severity of MODS.Part Ⅱ Optimization of bioartificial kidney with cells and effects of tanshinone on itObjective To present an optimal co-culture system with human renal tubular cells (RTCs) and bone marrow MSCs in vitro, which would be an ideal cell source for RAD configuration, and to study its mechanism. Meanwhile, the roles of Tanshione IIA on the optimal co-culture system were also investigated.Methods The human renal proximal tubule epithelial cells(HK-2) and human bone marrow mesenchymal stem cells(MSCs) were respectively co-cultured with different proportion according to 1:1,3:1,6:1 and 10:1, respectively. The morphological and functional changes of HK-2 cell, cell survival rate, cytochrome P450 activity and cell vitality were observed in each group. Confocal laser was used to monitor the expression of megalin protein. The sodium tanshinone IIA sulfonic acid solution (including 100umol/L tanshinone IIA) was added into the optimal proportion co-culture system of HK-2 and MSCs to observe the effects of tanshinone IIA on co-culture system. Flow cytometry was used to detect and analyze the changes of cell cycle, apoptosis(Annexin V-FITC/PI method). CCK8 was used to measure the cell proliferation. The levels of IGF-1 and BMP-7 in cell culture supernatant was detected by ELISA.Results HK-2/MSCs ratio of 3:1 (group 3:1) showed the optimal configuration under microscope. The survival rate, cytochrome P450, cell vitality and the expression of megalin in group 3:1 were significantly higher than other group(all P< 0.05). Cell cycle analysis indicated that larger populations of HK-2 without MSCs treatment were accumulated in G0-G1 phase, and fewer populations of cells in G2-S phase compared with HK-2 in the co-culture group and tanshinone group(P<0.05). The cells apoptosis rate in the co-culture group and tanshinone group between 24h,48h,72h were significantly lower than that of HK-2 alone(24h:1.74±0.03%, 1.68±0.01% vs 2.75±0.02%, P<0.05). The cell vitality in the co-culture group and tanshinone group within 24h,48h were higher than HK-2 alone(P<0.05).The production of IGF-1 and BMP-7 in co-culture group and tanshinone group at 24h,48h were higher than those of HK-2 alone(P<0.05). However, no significant difference was observed between the co-culture group and tanshinone group(P>0.05).Conclusions Co-cultivation of HK-2 and MSCs at a ratio of 3:1 may preserve HK-2 morphology and function to a great degree, which could contribute to the functional RAD configuration. The optimal co-culture system with tanshinone IIA did not significantly promote cell proliferation, reduce cell apoptosis and improve its function.Part III Effect and mechanism of the treatment with modified bioartificial kidney in the pig model with multi-organ dysfunction and acute renal failureObjective To investigate the effect and mechanism of the treatment with a bioartificial kidney, which containing cocultured renal proximal tubule cells(Co-RAD), in pigs with multiple organ dysfunction and acute renal failure.Methods Pigs with MODS and ARF were treated with RAD (group A, n=5) or Co-RAD(group B, n=5) or sham RAD containing no cells (group C, n=5) or without treatment(group D, n=6). Vital signs and blood biochemical markers, including electrolytes, urea nitrogen and creatinine, serum cytokines (IL-10, TNF-a, IL-6), and arterial blood gas were measured and survival time of all the pigs was recorded.Results There was no difference of MAP between four groups before the treatment.The MAP of group A and B began to rise after 4 hours of the treatment. After 24 hours of the treatment, the MAP of group B(101.20±9.37mmHg) was higher than that of group A(93.42±7.35mmHg), group C(64.75±9.61mmHg) and group D(54.29±3.01mmHg)(P<0.05). The serum creatinine (Scr) of four groups had no difference before the treatment. After 24 hours of the treatment, the Scr of group A and B decreased obviously as compared with group C and D(P<0.05).The Scr of group A had no difference with group B. The peak level of IL-10 of group B(301.72±46.35pg/ml) was significantly higher than those of group A(261.33± 51.72pg/ml), group C(130.47±14.97pg/ml) and group D(104.11±10.89pg/ml)(P<0.05). The TNF-a concentration of four groups had no significant difference before the treatment (mean value 519.33±42.16pg/ml). After the beginning of the treatment, serum TNF-α level of group A and group B dropped gradually, which was 413.65±33.91pg/ml and 329.51±.64pg/ml(P<0.05) after 24 hours, and had significant difference between the two groups. There was no difference of the levels of serum IL-6 between pre-treatment and post-treatment in group A, group B and group C, but the levels of serum IL-6 gradually increased in group D. The average survival time in group B(124.38±17.09h) was significantly longer than that in group A (110.97±10.68h), group C(81.68±9.41h) or group D(75.29±13.71h) (P<0.05),which prolonged by 12.08%,52.28%,65.20%.Conclusions Compared with original RAD, the treatment with optimized RAD(cocultured renal tubule cells) had more advantageous in prolonging the survival time of animals with MODS and ARF. The underlying mechanism may be through inhibiting the production of pro-inflammatory cytokines and promoting the production of anti-inflammatory cytokines. |
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