Project description:The OK cell line derived from kidney of a female opossum Didelphys virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, genomic sequence for Didelphys virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, its relatedness and similarity of the transcriptome to the Didelphys virginiana species is not known. The Monodelphis domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the Didelphys virginiana OK cell line we characterized its transcriptome using de novo transcriptome assembly and alignment to the Monodelphis domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the Monodelphis domestica genome, were compared to publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT specific ion transporters and other key proteins relevant for rodent and human PT function. The sequence and expression data reported here provide a new and important resource for studies on the regulation of PT mRNA and protein expression.
Project description:Purpose:Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend on the genes expressed.We have used modern RNA-sequencing techniques to identify the gene expression profile of 14 different cell lines plus primary cultures of mouse proximal tubule and compare them to transcriptomes of native kidney proximal tubules. Methods: 14 different proximal tubule cell lines were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. Results and conclusion: Transcripts expressed in cell lines showed variable match to transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45%) with pig kidney cells (LLC-PK1) close behind (39%). Much lower percentage matches were seen for various human lines including HK-2 cells (26%) and lines from rodent kidneys (18-23%).An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created for interrogation of the data.No cell line closely matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.
Project description:We investigated the gene expression changes associated with skin pigmentation variation between Virginia opossum populations inhabiting tropical and temperate environments. We found that gene expression variation in genes with melanocytic and immune functions is associated with the degree of skin pigmentation variation. Further, we found evidence suggesting that the Wnt/ß-catenin signaling pathway might be regulating the depigmentation observed in temperate populations. We present several alternative hypotheses that may explain Gloger’s rule pattern of skin pigmentation variation in the Virginia opossum.
Project description:Loss of a kidney results in compensatory growth of the remaining kidney, a phenomenon of considerable clinical importance. However, the mechanisms involved are largely unknown. Here, we used a multi-omic approach in a mouse unilateral nephrectomy model to identify signaling processes associated with compensatory hypertrophy of the renal proximal tubule. Morphometry applied to microdissected proximal tubules showed that growth of the proximal tubule involves a marked, rapid increase in cell volume rather than cell number. Measurements of DNA accessibility (ATAC-seq), transcriptome (RNA-seq) and proteome (quantitative protein mass spectrometry) independently identified patterns of change that are indicative of activation of the lipid-regulated transcription factor, PPARα. Activation of PPARα by fenofibrate administration increased proximal tubule cell size, while genetic deletion of PPARα in mice decreased it. The results indicate that PPARα is an important determinant of proximal tubule cell size and is a likely mediator of compensatory proximal tubule hypertrophy.