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Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry
Published on: April 19, 2017
Establishing the molecular pathways involved in chronic allograft nephropathy for testing new noninvasive diagnostic
Valeria Mas1, Daniel Maluf, Kellie Archer
1Division of Transplant, Department of Surgery, Virginia Commonwealth University, Richmond, VA, USA. vrmas@hsc.vcu.edu
Background:
Chronic allograft nephropathy (CAN) is a cause of graft loss. The multistage processes that result in CAN are poorly understood. Noninvasive assays for detecting allograft dysfunction and predicting long-term outcomes are a priority in transplantation (Tx).
Methods:
Renal tissue from kidney transplant patients (KTP) with CAN (n=11) and normal kidneys (NK; n=7) were studied using microarrays. Markers resulting from the microarray analysis (transforming growth factor [TGF]-beta, epidermal growth factor receptor [EGFR], angiotensinogen [AGT]) were tested in urine (Ur) and peripheral blood (PB) samples from the CAN patients (collected at the biopsy time) using reverse-transcriptase real-time polymerase chain reaction. Ur and PB samples from long-term KTP with stable renal function (SRF; n=20) were used as control.
Results:
Assuming unequal variances between CAN and NK, using a false discovery rate of 0.005, and running 1,000 of all possible permutations, 728 probe sets were differentially expressed. Genes related to fibrosis and extracellular matrix deposition (i.e., TGF-beta, laminin, gamma 2, metalloproteinases-9, and collagen type IX alpha 3) were up-regulated. Genes related to immunoglobulins, B cells, T-cell receptor, nuclear factor of activated T cells, and cytokine and chemokines receptors were also upregulated. EGFR and growth factor receptor activity (FGFR)2 were downregulated in CAN samples. AGT, EGFR, and TGF-beta levels were statistical different in urine but not in blood samples of CAN patients when compared to KTP with SRF (P<0.001, P=0.04, and P<0.001, respectively).
Conclusions:
Genes related to fibrosis, extracellular matrix deposition, and immune response were found up-regulated in CAN. Markers resulting from the microarray analysis were differentially expressed in Ur samples of the CAN patients and in concordance with the microarray profiles.
Insights
Chronic allograft nephropathy (CAN) involves fibrosis and immune response gene upregulation. Urinary biomarkers like transforming growth factor-beta (TGF-beta) and epidermal growth factor receptor (EGFR) show promise for detecting kidney transplant dysfunction.
Area of Science:
- Nephrology
- Immunology
- Genomics
Background:
- Chronic allograft nephropathy (CAN) is a significant cause of kidney transplant loss.
- Understanding the molecular mechanisms of CAN is crucial for improving long-term graft survival.
- Noninvasive diagnostic tools for allograft dysfunction are a priority in transplantation.
Purpose of the Study:
- To identify differentially expressed genes in kidney allografts with CAN using microarray analysis.
- To validate potential urinary biomarkers for CAN detection in kidney transplant patients (KTP).
- To correlate gene expression profiles with clinical outcomes in kidney transplantation.
Main Methods:
- Microarray analysis of renal tissue from KTP with CAN and normal kidneys.
- Validation of candidate markers (TGF-beta, EGFR, AGT) in urine and blood using RT-qPCR.
- Comparison of marker levels in CAN patients versus KTP with stable renal function (SRF).
Main Results:
- 728 probe sets were differentially expressed between CAN and normal kidney samples.
- Genes associated with fibrosis, extracellular matrix deposition, and immune response were upregulated in CAN.
- Urinary levels of AGT, EGFR, and TGF-beta were significantly different in CAN patients compared to SRF controls.
Conclusions:
- CAN is characterized by upregulated genes involved in fibrosis and immune responses.
- Urinary biomarkers derived from microarray analysis show potential for noninvasive detection of CAN.
- These findings support the development of novel diagnostic strategies for kidney allograft dysfunction.
