Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.
Type 2 diabetic (T2DN) and hypertensive nephrosclerosis (HN) are the main causes of chronic kidney disease (CKD). To obtain novel insights into pathogenetic mechanisms, potentially suggesting common therapeutic procedures, we comparatively nalysed transcriptomes of kidney biopsies obtained from patients with T2DN or HN and control archived formalin-fixed and paraffin-embedded (FFPE) kidney biopsies.FFPE kidney biopsies from adult patients were selected from the Norwegian Kidney Biopsy Registry. Specimens from patients with HN, T2DN and normal-appearing control renal biopsies (Ctrl) (n = 6 for each group) were assessed. Total RNA was extracted from 10 μm whole kidney sections and processed for RNA sequencing, while immunohistochemistry (IHC) was performed on 3-μm tissue sections. Enzyme-Linked ImmunoSorbent Assay (ELISA) targeting the AXL receptor tyrosine kinase was performed on serum from separate cohorts with HN and T2DN/T1DN.Gene expression analysis showed 769 differentially expressed genes (DEG) for T2DN and 284 HN genes (log fold change > 1, adjusted P < 0.05). Only 39 of the HN genes were uniquely DEG compared to T2DN. Cluster analysis on the RNA seq data separated diseased and normal tissues, also with a distinct overlap between HN and T2DN profiles. Detailed analysis of gene expression profiles revealed upregulation of pathways related to partial epithelial to mesenchymal transition, including extracellular matrix organisation in both diseases. AXL and vimentin expression was also enhanced at the protein level in both HN and T2DN biopsies, as detected by immunohistochemistry (IHC). AXL abundance was also increased in serum from T2DN/T1DN and HN patients. Pathways related to inflammation was also overexpressed, particularly related to TH1 and TH2 helper cell pathways. The presence of CD4 and CD8 positive T- cells were confirmed by IHC.Similarities of T2DN and HN proteomes suggest common pathogenetic mechanisms and the possibility to envisage targeted treatments addressing both main causes of CKD.
The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfβ), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.
Objective: We aimed at investigating to which degree hypertensive and diabetic nephropathy show evidence of concurrent inflammation and partial epithelial-to-mesenchymal transition (EMT), including elevated expression of EMT promoting AXL receptor tyrosine kinase. Design and method: Formalin-fixed and paraffin-embedded (FFPE) kidney biopsies from adult patients with hypertensive nephropathy (HN, n = 6), type 2 diabetic nephropathy (DN, n = 6), and normal appearing tissue (n = 6) were recruited from the Norwegian Kidney Biopsy Registry. Total RNA was extracted from whole tissue sections using High Pure FFPE-tissue RNA Isolation kit (Roche). Total RNA inputs of 37.8–1324 ng per sample were used for library preparation and sequenced as 75 bp paired end on Illumina NextSeq500. Differentially expressed genes were analysed with Limma-Voom (Bioconductor) and formed the basis for calculation of a generic EMT score, principal component analysis (PCA) and ingenuity pathway analysis (IPA). Soluble AXL (sAXL) in serum samples (n = 14/group) from patients with HN and healthy controls was quantified by ELISA (Human Axl Duoset, R&D). Results: PCA separated diseased from normal tissue with a considerable overlap in expression profiles of HN and DN. The generic EMT score was higher in both HN and DN as compared to normal tissue. Expression of the EMT promoting receptor tyrosine kinase AXL was increased in both diseases. Immunohistochemistry confirmed increased AXL and vimentin, while E-cadherin was decreased in diseased tissues. Accordingly, sAXL levels were elevated in serum samples from HN patients compared to controls. IPA showed all top five canonical pathways to be involved in inflammation. Accordingly, expression deconvolution analyses provided evidence of eosinophils, natural-killer-T-cells, granulocytes and CD8+ effector-memory T-cells and myeloid-dendritic cells in HN. E.g. the CCL5-CCR5 axis represents a therapeutic target for seven existing drugs. Top scoring pathways common for both DN and HN included the iCOS-iCOSL signalling in T helper cells, Th1 & Th2 activation pathway and the Th1/Th2 pathway. Higher presence of the phagosome formation pathway in DN and the T cell receptor signalling pathway in HN differentiated the two pathologies. Conclusions: HN and DN show an increased mesenchymal phenotype and inflammation. AXL receptor tyrosine kinase represents a novel therapeutic target, especially in HN.
