Intragraft Antiviral-Specific Gene Expression as a Distinctive Transcriptional Signature for Studies in Polyomavirus-Associated Nephropathy

Abstract

Background: Polyomavirus nephropathy (PVAN) is a common cause of kidney allograft dysfunction and loss. To identify PVAN-specific gene expression and underlying molecular mechanisms, we analyzed kidney biopsies with and without PVAN.

Methods: The study included 168 posttransplant renal allograft biopsies (T cell-mediated rejection [TCMR] = 26, PVAN = 10, normal functioning graft = 73, and interstitial fibrosis/tubular atrophy = 59) from 168 unique kidney allograft recipients. We performed gene expression assays and bioinformatics analysis to identify a set of PVAN-specific genes. Validity and relevance of a subset of these genes are validated by quantitative polymerase chain reaction and immunohistochemistry.

Results: Unsupervised hierarchical clustering analysis of all the biopsies revealed high similarity between PVAN and TCMR gene expression. Increased statistical stringency identified 158 and 252 unique PVAN and TCMR injury-specific gene transcripts respectively. Although TCMR-specific genes were overwhelmingly involved in immune response costimulation and TCR signaling, PVAN-specific genes were mainly related to DNA replication process, RNA polymerase assembly, and pathogen recognition receptors. A principal component analysis (PCA) using these genes further confirmed the most optimal separation between the 3 different clinical phenotypes. Validation of 4 PVAN-specific genes (RPS15, complement factor D, lactotransferrin, and nitric oxide synthase interacting protein) by quantitative polymerase chain reaction and confirmation by immunohistochemistry of 2 PVAN-specific proteins with antiviral function (lactotransferrin and IFN-inducible transmembrane 1) was done.

Conclusions: In conclusion, even though PVAN and TCMR kidney allografts share great similarities on gene perturbation, PVAN-specific genes were identified with well-known antiviral properties that provide tools for discerning PVAN and AR as well as attractive targets for rational drug design.

Figure 1

(A)- Global gene expression pattern of samples classified as PVAN were largely similar to TCMR samples when compared to normal kidney biopsies, BK viremia (BKVB), and IF/TA. (B)- Gene expression clustering demonstrates TCMR-specific gene signature. (C)- Gene expression clustering demonstrates PVAN-specific gene signature compared to normal (STA). A few genes (n=16) were also differentially expressed in the IF/TA patients compared to PVAN but this analysis is not as clean as many patients with PVAN also have associated IF/TA changes in the graft.

Figure 2

A comprehensive pathway/gene-set analysis using the software GO-Elite was performed to identified molecular pathways enriched in PVAN compared to TCMR. This resulted in enrichment of 2 distinct set of pathways. Immune system related pathways such as complement cascade, TCR signaling, innate immune system, adaptive immune system were specific to TCMR whereas PVAN associated genes were enriched with DNA replication pathways such as mRNA processing, ribosomal scanning, viral mRNA translation etc. and metabolic pathways such as urea cycle, gluconeogenesis, TCA cycle etc. were specific to PVAN demonstrating 2 distinct molecular events occurring at the time of TCMR and PVAN.

Figure 3

QPCR validations of PVAN specific genes. In order to validate PVAN specific gene expression data 4 genes (LTF, CFD, RPS15, and NOSIP) were selected for QPCR validation. Over-expression of all 4 genes in PVAN vs. STA and also in PVAN vs. TCMR was confirmed. Gene expression for; (A) lactotransferrin (LTF) in PVAN was significant when compared to AR (p=0.04) and STA (p=0.02), (B) complement factor D (CFD) in PVAN was significant when compared to AR (p=0.05) and STA (p=0.05), (C) 40 ribosomal protein S15 (RPS15) in PVAN was significant when compared to AR (p=0.002) and STA (p=0.02), (D) nitric oxide synthase interacting protein (NOSIP) in PVAN was significant when compared to AR (p=0.004) and STA (p=0.007). The first and third quartiles are at the ends of the box, the median is indicated with a horizontal line in the interior of the box, and the maximum and minimum are at the ends of the whiskers.

Figure 4

Using immunohistochemistry (IHC) validation of PVAN specific expression of LTF and IFITM1 in kidney biopsies with PVAN. We assessed the expression of 2 gene transcripts (LTF and IFITM-1) at the protein level. These transcripts were highly upregulated in kidney biopsies with PVAN as compared to TCMR and STA. A) Three representatives phenotypes from 3 representative transplant patient biopsies evaluated for the different protein stains. TCMR and STA are shown at 10× and the PVAN samples are shown at 40× magnification. Co-localization of IFITM-1 with BK viral inclusions are marked with yellow arrows in the PVAN patient. LTF also localizes in proximity to the SV40 and IFITM-1 stains in the renal tubule. (B) Semi quantitative analysis of protein expression at tubulo-epithelial cells (TEC) and mononuclear cells of patients with PVAN, TCMR and STA.