| Background and ObjectiveKidney transplant is the best treatment for terminal stage nephropathy. Although with the improvement of operation technique and the application of new types of the immunosuppressive drugs, the complications during the perioperational period and the incidence of early acute rejection decrease significantly, simultaneously the survival of one year graft increases, the half-life period of graft has not been prolonged obviously. Therefore, much more attention has been taken on chronic allograft nephropathy.Chronic allograft nephropathy is the most common cause for the failure of long-term graft. The etiology includes immunologic factors and non-immune injury. Cold ischemia, donor kidney diseases, age, the toxicity of chronic calcineurin inhibitors and infection are involved in immunologic factors. Recently, much more attention has been taken on non-immune injury. In the clinical setting, it was found that donor kidney from the living was better than from a corpse, no matter whether in long-term or short-term. It demonstrated that the quality of donor kidney and non-immune factors were the key point.Cold ischemia injury is an inevitable pathophysiologic process in kidney transplant. As one of risk factors for chronic allograft nephropathy, the longer the cold ischemia, the more possibly it causes chronic rejection after operation. The mechanism of how cold ischemia initiating chronic allograft nephropathy is still unknown. Perhaps, it is associated with hypothermy injury, cytokines, cellular metabolism and other factors.Renal interstitial fibrosis is the major feature of chronic allograft nephropathy. Transforming growth factorβ1 (TGFβ1) is the key growth factor that promotes the progress of fibrosis. In almost all of chronic allograft nephropathy models, the expression of TGFβ1 is enhanced. TGFβ1 is also the mediator for many other fibrogenic factors (eg. Ang-II). Connective tissue growth factor (CTGF) is one of the downstream factors of TGFβ1 and the key factor for renal tubular fibrosis. In the cases of renal tubular fibrosis, chronic renal interstitial impairment and graft rejection, CTGF mRNA was expressed intensively, and the inhibition of the TGFβ1 expression might lessen tissue fibrosis. However, it's possible to induce the loss of cell growth control, immune dysregulation, and severe inflammations. Because CTGF has simple biologic effect and might only mediate the progress of TGFβ1 inducing fibrosis, the blockage of CTGF expression could be the more specific and effective method that prevents fibrosis. Along with the lack of donor, more attention has been taken on broadening the criterion of donor, the usage of marginal donor and the research on the preservation of donor kidney. Therefore, the deeply investigation of the mechanism of donor kidney injury is of considerable significance.Our aim was to investigate the effects of cryopreservation on the expression of transforming growth factor-beta 1 (TGF-β1) and connective tissue growth factor (CTGF) in the proximal renal tubular epithelial cells of Wistar rats and further evaluate the mechanism of cold ischemia injury in donor kidney .MethodA total of 40 Wistar rats were used in this study. In terms of the time of cryopreservation of donor kidney, the 40 Wistar rats were randomly divided into 5 groups.①0h group (control group),②12h group,③24h group,④3 6h group,⑤48h group. A block removal of donor kidney with in situ perfusion of cooling HC-A preservation solution was adopted. The rat kidney was put in 0-4℃cooling preservation solution at Oh, 12h, 24h, 36, 48h, respectively. The morphologic and the ultra-structural changes of proximal tubular epithelial cells in different cryopreservation time were observed under light microscope and transmission electron microscope. The expression of TGF-β1, CTGF mRNA and protein in proximal tubular epithelial cells of different cryopreservation time group were detected by in situ hybridization and immunohistochemistry analysis. PU value of positive unit was calculated by image analysator. The one-factor analysis of variance was used to compare the differences between means of every groups, and bivariate correlate analysis (coefficient of correlation of Pearson) was adopted to analyze the relativity between the PU values of TGF-β1, CTGF protein expression and the PU values of mRNA expression.Results1. The morphologic and ultra-structural changes of proximal tubular epithelial cells in different cold ischemia time:The feature in CP (cryopreservation) 12h group was similar to that in control group. In CP 24h group, part of the proximal tubular epithelial cells appeared slight degeneration. In CP 36h group, the proximal tubular epithelial cells appeared significant hydropic degeneration. In CP 48h group, the proximal tubular epithelial cells demonstrated severe hydropic degeneration and parts of the cells happened necrosis and defluxion.2. The expression of TGF-β1 and CTGF protein in proximal tubular epithelial cells in different cold ischemia time: Both TGF-β1 and CTGF protein appeared brown-color buffy grains in cytoplasm as positive unit, negative unit without buffy grains and cell nucleus in blue color. Only a small amount of TGF-β1 and CTGF protein were expressed in CP 0h group and CP12h group, TGF-β1 protein PU value was 6.37±2.77, 6.11±1.82 ( n=40,P=0.0878 , ) .respectively. CTGF PU value was 5.91±2.30,5.57±2.40 (n=40,P=0.821) , respectively. As the cold ischemia time prolonged, the protein PU value of TGF-β1 was increased at CP 24h group (10.20±3.27), CP 36h group (13.84±3.91), CP 48h group(17.17±3.96), respectively. There is the significant difference among these three groups (CP 24h compared with CP 36h group, n=40, P=0.031; CP 24h compared with 48h group, n=40, P=0.000; CP 36h compared with 48h group, n=40, P=0.047). When compared with CP 0h and 12h group, the significant difference still remained. (CP 24h compared with CP 0h group, n=40, P=0.024; CP 24h compared with CP 12h group, n=40, P=0.016; CP 36h compared with CP 0h group, n=40, P=0.000; CP 36h compared with CP 12h group, n=40, P=0.000; CP 48h compared with CP 0h group, n=40,P=0.000; CP 48h compared with CP 12h group, n=40,P=0.000). The protein PU value of CTGF was also increased at CP 24h group (10.25±2.92), CP 36h group (14.31±2.83), CP 48h group (18.11±3.94), respectively. There is also the significant difference among these three groups. (CP 24h compared with CP 36h group, n=40, P=0.012; CP 24h compared with 48h group, n=40, P=0.000,; CP 36h compared with 48h group, n=40, P=0.016). When compared with CP 0h and 12h group, the significant difference still existed. (CP 24h compared with CP 0h group, n=40, P=0.006; CP 24h compared with CP 12h group, n=40, P=0.003; CP 36h compared with CP 0h group, n=40, P=0.000; CP 36h compared with CP 12h group, n=40, P=0.000; CP 48h compared with CP 0h group, n=40, P=0.000; CP 48h compared with CP 12h group, n=40,P=0.000). In different groups, the positive correlation between the protein positive unit of TGF-β1 and CTGF existed (n=40,r=0.674, P<0.05).3. The expression of TGF-β1 and CTGF mRNA in proximal tubular epithelial cells in different cold ischemia time.Both TGF-β1 and CTGF mRNA appeared brown-color buffy grains in cytoplasm as positive unit. Only a small amount of TGF-β1 and CTGF mRNA were expressed in CP 0h group and CP12h group, TGF-β1 mRNAPU value was 5.29±2.15, 5.90±2.40 (n=40, P=0.706), respectively. CTGF mRNA PU value was 6.24±2.79, 6.51±2.43 (n=40,P=0.889) , respectively. As the cold ischemia time prolonged, the mRNA PU value of TGF-β1 was increased at CP 24h group (11.32±3.34), CP 36h group (15.47±4.21), CP 48h group (19.01±3.53), respectively. There is the significant difference among these groups (CP 24h compared with CP 36h group, n=40, P=0.014; CP 24h compared with 48h group, n=40, P=0.000; CP 36h compared with 48h group, n=40, P=0.035). When compared with CP 0h and 12h group, the significant difference still existed. (CP 24h compared with CP 0h group, n=40, P=0.001; CP 24h compared with CP 12h group, n=40, P=0.002; CP 36h compared with CP 0h group, n=40, P=0.000; CP 36h compared with CP 12h group, n=40,P=0.000; CP 48h compared with CP 0h group, n=40, P=0.000; CP 48h compared with CP 12h group, n=40, P=0.000). The mRNA PU value of CTGF was also increased at CP 24h group (15.24±3.95), CP 36h group (19.20±4.73), CP 48h group (23.09±4.40), respectively. There is also the significant difference among these groups (CP 24h compared with CP 36h group, n=40, P=0.043; CP 24h compared with 48h group, n=40, P=0.000; CP 36h compared with 48h group, n=40, P=0.046). When compared with CP 0h and 12h group, the significant difference still remained. (CP 24h compared with CP 0h group, n=40, P=0.001; CP 24h compared with CP 12h group, n=40, P=0.002; CP 36h compared with CP 0h group, n=40, P=0.000; CP 36h compared with CP 12h group, n=40, P=0.000; CP 48h compared with CP 0h group, n=40, P=0.000; CP 48h compared with CP 12h group, n=40,P=0.000). In different groups, the positive correlation between the mRNA positive unit of TGF-β1 and CTGF existed (n=40, r=0.767, P<0.05).Conclusion1. The morphologic and ultra-structural changes of proximal tubular epithelial cells in CP 12h and CP 0h were similar. In CP 24h group, parts of the proximal tubular epithelial cells appeared slight degeneration. It demonstrated that the morphologic and ultra-structural changes weren't obvious in cryopreservation 24h. It's relatively safe.2. Only a small amount of TGF-β1 and CTGF protein and mRNA were expressed in CP 0h group and CP12h group. As the cold ischemia time prolonged, the expression of TGF-β1 and CTGF at mRNA and protein level was increased gradually in CP 24h , CP 36h and CP 48h group. At the same time, in CP 24h group, part of the proximal tubular epithelial cells appeared slight degeneration . In CP 36h group, the proximal tubular epithelial cells appeared significant hydropic degeneration. In CP 48h group, the proximal tubular epithelial cells demonstrated severe hydropic degeneration and parts of the cells happened necrosis and defluxion. There is the significant correlation between the expression of TGF-β1,CTGF and the degree of cold ischemia injury. It illustrates that TGF-β1 and CTGF can promote the synthesis of extracellular matrix, and might play an important role in regeneration and reparation of renal tubular epithelial cell injury. The overexpression of TGF-β1 and CTGF might be one of the mechanisms that initiate chronic allograft nephropathy. |
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