Title: Clinical Lectureship in Renal Medicine
Funder: Medical Research Council

Abstract: Aims Develop a novel technique for isolating proximal tubule cell messenger RNA from whole kidney organ tissue Charting the translational profile of the proximal tubule cell in models of chronic kidney disease (CKD) Identification of new cell signalling pathways and networks relevant to the natural history of renal injury and fibrosis Identification of new potential therapeutic targets within the proximal tubule cell for improving outcomes in kidney fibrosis Importance - CKD Chronic kidney disease (CKD) poses an increasing global disease burden and contributes to the leading cause of death worldwide - cardiovascular (CV) disease. Regardless of the type and even treatment of initiating kidney injury, CKD often manifests histologically with consistent features of tubular atrophy, progressive fibrosis and loss of normal architecture. The severity of such histological change remains one of the most reliable predictors of progression to end-stage renal disease and proximal tubular cell (PTC) dysfunction is recognised to play a key role in the fibrotic final common pathway of CKD. An understanding of PT cell gene expression in the diseased kidney is crucial for advancing methods of diagnosis, predicting progression and development of new therapeutic targets to improve outcomes for patients with renal transplants and CKD. The relevance of the proximal tubule in CKD Targeted injury to the proximal tubule alone can manifest all of the hallmark features of progressive CKD - diptheria toxin injury through Six2-driven Cre expression caused significant renal cortex capillary rarefaction, interstitial fibrosis and glomerulosclerosis. A recent genome wide association study of almost 24,000 patients reported 3 top-ranking gene polymorphisms that associated with worsened kidney function; NAT8, SLC7A9, SLC22A2 and TBX2 – all of which predominate in the renal proximal tubule. Translational profiles in complex organs. Historically, translation profiling has been performed across whole organ tissue samples but this is limited by the presence multiple specialised cell types that each have unique roles and patterns of gene expression. Previous techniques to isolate specific cell populations are imprecise and include manual or laser capture microdissection or enzyme disassociation and FACS. These approaches have produced some insights but remain vulnerable to issues surrounding contamination of isolates with other cell-types and artifactual gene expression changes elicited by the extraction processes making interpretation of transcriptional or translational profiling unreliable. Ribosomal capture The “RiboTag” mouse has a targeted mutation of the ribosomal protein L22 (Rpl22) locus harbouring a loxP-flanked wild-type (WT) C-terminal exon 4 followed by an identical C-terminal exon 4 that is tagged with three copies of the haemagglutinin (HA) epitope before a STOP codon. When the RiboTag mice are bred to Cre-expressing mice, offspring have the floxed WT exon 4 deleted, allowing the downstream HA-tagged exon 4 to be expressed. HA-tagged ribosomes bound to mRNA can then be immunoprecipitated and the translational profiles within a defined cell-type can be analysed using RT-qPCR, RNA sequencing or microarray analysis. Study design The KAPiCre mouse strain has been successfully characterized and utilised by the applicant in previous studies to target DDAH1 gene deletion in PTCs. Mice expressing either the Ribotag or KAPiCre transgene individually are phenotypically normal and produce offspring in expected normal Mendelian ratios. Litters suitable for characterisation of the strain are expected in mid 2015. The KAPiCre/Ribotag strain will be subjected to two mouse models of renal disease; unilateral ureteric obstruction (UUO) and folate nephropathy. Ribosomal mRNA from renal proximal tubules will be isolated at three time points spanning the development of renal fibrosis and expression analysis will be performed using RNA sequen


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RENAL AND UROGENITAL