Urine mRNA Profiling for Renal Allograft Surveillance

该项目的总体目标是开发一种能够预测、诊断和预测人肾移植排斥反应的非侵入性诊断试验。在一项探索性研究中，我们发现肾移植受者尿液细胞中编码细胞毒蛋白穿孔素和颗粒酶B的基因表达水平可作为肾移植急性排斥反应的诊断指标。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。本研究的具体目的是：具体目的：1.验证尿液细胞信使核糖核酸图谱诊断亚临床排斥反应的假设。我们将检测穿孔素、颗粒酶B、Fas配体、CD3-epsilon Chain(CD3)、干扰素-γ(干扰素-γ)和白介素15(IL-15)在诊断亚临床排斥反应中的敏感性和特异性。具体目的2.检验以下假设：a)连续尿样中mRNA水平的变化预测急性排斥反应的发生，b)mRNA谱预测移植肾功能。我们将确定是否可以通过连续尿样中测量的mRNA水平的变化来预测急性排斥反应的发生。除了特定目的1中列出的mRNAs外，我们还将检测尿细胞中Bcl2、A20、血红素加氧酶-1(HO1)、Bax和转化生长因子-β1(TGF-β1)的mRNA表达水平，并验证这些mRNA水平预测移植肾功能的假设。具体目的3.研究尿液细胞的信使核糖核酸图谱是否可用于临床急性排斥反应的诊断，并确定信使核糖核酸图谱是否可预测皮质类固醇抗排斥治疗的反应性，以及在鉴定肾功能进行性下降的风险方面是否有预测作用。我们将确定颗粒酶B、穿孔素和CD3、干扰素-γ和IL-15的mRNA表达水平是否可以诊断临床急性排斥反应，并且与皮质激素敏感的急性排斥反应相比，激素抵抗型急性排斥反应的表达水平更高。我们还将确定细胞毒蛋白、Bax和转化生长因子-β1的高表达是否与移植肾功能下降有关，而细胞毒蛋白、Bax和转化生长因子-β1的高表达缺乏以及细胞毒蛋白、Bax和转化生长因子-β1的高表达以及Bcl-2、A20和HO1的高表达与稳定的肾功能有关。

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.