Urine mRNA Profiling for Renal Allograft Surveillance

该项目的总体目标是开发一种非侵入性诊断测试，用于预测、诊断和预后人类肾移植排斥反应。 在一项探索性研究中，我们发现肾移植受者尿细胞中编码细胞毒性蛋白穿孔素和颗粒酶B的mRNA表达水平可诊断人肾移植急性排斥反应。 我们现在建议进一步发展和完善急性排斥反应的非侵入性诊断。 本次调查的具体目标是：具体目标1。检验尿细胞mRNA表达谱可诊断亚临床排斥反应的假设。 我们将测量在方案活检时获得的尿液标本中穿孔素、颗粒酶B、Fas配体、CD 3 β链（CD 3）、干扰素γ（IFN-γ）和白细胞介素-15（IL-15）的mRNA，并确定mRNA水平诊断亚临床排斥反应的灵敏度和特异性。具体目标2。检验以下假设：a）连续尿样中mRNA水平的改变预测急性排斥反应的发生，和B）mRNA谱预测肾移植功能。 我们将确定急性排斥反应是否可以通过连续尿液标本中mRNA水平的变化来预测。 除了特定目标1下列出的mRNA外，我们还将测量尿细胞中Bcl-2、A20、血红素加氧酶-1（HO 1）、Bax和转化生长因子-β 1（TGF-β 1）的mRNA表达水平，并检验mRNA水平预测肾移植功能的假设。具体目标3。研究尿细胞的mRNA谱是否可诊断临床急性排斥反应，并确定mRNA谱是否可预测皮质类固醇抗排斥治疗的反应性，以及识别肾功能进行性下降风险的同种异体移植物。 我们将确定颗粒酶B、穿孔素和CD 3、IFN-γ和IL-15的mRNA表达水平是否可诊断临床急性排斥反应，并且与皮质类固醇敏感性急性排斥反应相比，皮质类固醇耐药急性排斥反应的mRNA表达水平是否更高。 我们还将确定细胞毒性蛋白、Bax和TGF-β 1的mRNA过表达是否与移植物功能下降相关，而细胞毒性蛋白、Bax和TGF-β 1的mRNA过表达以及Bcl-2、A20和HO 1的mRNA过表达的缺乏是否与肾功能稳定相关。

The overall objective of this project is to develop a noninvasive diagnostic test that is predictive, diagnostic and prognostic of human renal allograft rejection. In an exploratory study, we found that the levels of expression of mRNA encoding cytotoxic proteins perforin and granzyme B in urinary cells of renal allograft recipients are diagnostic of acute rejection of human renal allografts. We now propose to further develop and refine the noninvasive diagnosis of acute rejection. The specific aims of this investigation are: Specific aim 1. To test the hypothesis that mRNA profiling of urinary cells is diagnostic of subclinical rejection. We will measure mRNA for perforin, granzyme B, fas ligand, CD3 epsilon chain (CD3), interferon gamma (IFN-gamma), and interleukin-15 (IL-15) in urine specimens obtained at the time of protocol biopsies and determine the sensitivity and specificity of mRNA levels in diagnosing subclinical rejection. Specific aim 2. To test the hypotheses that: a) alterations in mRNA levels in sequential urine specimens predict the development of acute rejection, and b) mRNA profiles predict renal allograft function. We will determine whether acute rejection episodes can be predicted by alterations in mRNA levels measured in sequential urine specimens. In addition to mRNAs listed under Specific Aim 1, we will measure level of expression of mRNA for Bcl-2, A20, hemeoxygenase-1 (HO1), Bax, and transforming growth factor-beta1 (TGF-beta1) in urinary cells and test the hypothesis that the mRNA levels predict renal allograft function. Specific aim 3. To investigate whether mRNA profiles of urinary cells are diagnostic of clinical acute rejection and to determine whether mRNA profiles are prognostic with respect to responsiveness to antirejection therapy with corticosteroids, and in identifying allografts at risk for progressive decline in renal function. We will determine whether the level of expression of mRNA for granzyme B, perforin, and CD3, IFN-gamma and IL-15 are diagnostic of clinical acute rejection and are higher in those with corticosteroid resistant acute rejections compared to corticosteroid sensitive acute rejections. We will also determine whether hyperexpression of mRNA for cytotoxic proteins and for Bax and TGF-beta1 is associated with graft functional decline whereas lack of hyperexpression of mRNA for cytotoxic proteins, Bax and TGF-beta1 and hyperexpression of mRNA for Bcl-2, A20 and HO1 is associated with stable renal function.