Epithelial-to-mesenchymal transition gene expression is associated with early tubulointerstitial damage in pediatric renal transplant recipients

Matthew Vitalone¹, Maarteen Naesens², Li Li¹, Sue Hsieh¹, Tara Sigdel¹, Minnie Saral^1.

¹Pediatrics, Stanford University Medical School, Stanford, CA, USA; ²Nephrology and Renal Transplantation University Hospitals, Leuven, Belgium.

Introduction: Tubulointerstitial damage (TID) is a key feature of chronic renal allograft loss. One proposed mechanism attributing to this chronic fibrotic injury is epithelial-to-mesenchymal transition (EMT), however its impact and influence on TID is still controversial as it has recently been shown to be unrelated to early TID in adult renal allograft recipients. This phenomenon has yet to be investigated in pediatric renal transplant recipients or late TID, a question we investigated in this study.

Methods: This study used RNA isolated from protocol renal transplant biopsies at 3m (n=23), 6m (n=48), 12m (n=21), 24m (n=45) from 83 pediatric renal allograft recipients. Total RNA was extracted using Trizol, analysed using the Bioanalyser, converted to cRNA and hybridised to the Affymetrix U133+2.0. 401 probes representing 154 EMT associated genes were identified from the literature and analysed for differential gene expression using SAM comparing TID versus noTID. Differentially expressed genes are FDR<10%.

Results: Comparing TID vs. noTID at 3m we identified 69 probes that represented 57 genes, which simplifies to 35 up-regulated and 22 down-regulated genes. SMAD, NOTCH, SKI, SMURF, TGFBR were all upregulated at 3m suggesting a pro-EMT profile. In addition, a number of growth factors and a few MAPKs were both up and downregulated, CDH1 up-regulated and cell cycling molecules down-regulated also at 3m, suggesting a shift in the EMT equilibrium and activation through alternate mechanisms. The EMT gene expression profile exhibited a major positive shift at 6mths post-transplant with upregulation of 33 genes (49 probes) consisting of SMAD, SMURF, NFKB, TLN, several integrins, MAPKs, growth factor receptor and cell cycle molecules. No EMT related genes were downregulated at 6mths. no EMT related gene expression was observed at either 12m or 24m post-transplant, suggesting a series of time dependent mechanisms that are initiated early post-transplant in pediatric renal allograft recipients.

Conclusions: EMT gene expression in protocol biopsies with TID appeared to be time dependent, starting early, peaking at 6m and resolving by 12m post-transplant. The mechanisms driving EMT in pediatric renal allograft recipients are un-clear, but they would appear to be quite different to those involved in the adult transplant setting.