Transcriptional profiling of the kidney glomerulus

The kidney glomerulus plays a crucial role in ultrafiltration of the blood through the glomerular capillary wall. Glomerulus injury may occur in many systemic and primary kidney diseases or as secondary effects to drugs and infections. It has been shown that many known glomerular specific/enriched genes play crucial roles in the maintenance of glomerular filtration function and in the pathogenesis of the known glomerular diseases of known etiology. With functional genomics and bioinformatics approaches, the identification of additional glomerular specific/enriched gene transcripts will provide a better understanding of the glomerular physiology and function, as well as the mechanisms of glomerular diseases where causes and pathogenic processes are still poorly understood. To achieve the goal of identifying glomerular genes, we, firstly, performed comparative analysis of mouse glomerular EST libraries and whole kidney EST libraries. In this study, we found 497 glomerular enriched genes. These glomerular enriched genes were validated by different approaches. In situ hybridization confirmed the cellular location of six new glomerular markers. Apart from the mouse kidney EST library comparisons, we integrated the results of glomerular enriched genes from four additional expression-profiling platforms, and made a comprehensive catalogue of glomerular genes. Based on this, a protein-protein interaction network in glomerulus (GlomNet) was constructed. To further understanding the gene expression profile of human glomerulus, we performed transcriptional profiling of human kidney glomeruli which were obtained from renal needle biopsies taken from live renal transplant donors. By comparison with our mouse transcriptional profiling data, we observed a surprisingly large difference between the most glomerulus-specfic genes in humans and mice, respectively. Furthermore, the common glomerular enriched genes between human and mouse were classified into six different clusters according to their expression specificity in human and mouse glomeruli. To gain insight into glomerular transcriptional changes that accompany pathological processes leading to proteinuria, we analyzed gene expression profiles following glomerular injury in the mouse lipopolysaccharide (LPS)-induced proteinuria model. LPS triggered the specific gene expression pattern changes in the glomerulus and regulated several different pathways. We observed that podocyte number decreased significantly in the model. Moreover, the genes encoded collagen α1, α2 and laminin β2 chains which are expressed in the embryonic kidney glomerulus increased their expression in LPS-treated mice, suggesting remodeling of the glomerular basement membrane, caused by activation or dedifferentiation of glomerular cells. In summary, the new glomerular gene transcripts identified in this thesis provide the foundation for targeted analysis of individual genes and pathways, and the GlomNet provides a preliminary systematic view of the relationships and interactions between different components in the glomerulus. The methodology of human glomerular transcription profiling can be applied for the identification of diagnostic and prognostic markers for renal diseases. The transcriptional profile changes accompanying LPS-induced proteinuria may provide clues about mechanisms involved in proteinuria. The information from this study contributes new insights for the glomerular function, physiology and the disease mechanisms. LIST OF PUBLICATIONS I. He L, Sun Y, Patrakka J, Mostad P, Norlin J, Xiao Z, Andrae J, Tryggvason K, Samuelsson T, Betsholtz C, Takemoto M. Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis. Kidney Int. 2007 May;71(9):889-900. II. He L, Sun Y, Takemoto M, Norlin J, Tryggvason K, Samuelsson T, Betsholtz C. The glomerular transcriptome and a predicted protein-protein interaction network. J Am Soc Nephrol. 2008 Feb;19(2):260-8. III. Sun Y, Nyström J, Ebefors K, Katayama M, Lal M, Patrakka J, Pikkarainen T, He L, Tryggvason K, Haraldsson B, Betsholtz C. Comparison of human and mouse glomerular transcription profiling data. Manuscript. IV. Sun Y, He L, Takemoto M, Patrakka J, Pikkarainen T, Genové G, Norlin J, Truvé K, Tryggvason K, Betsholtz C. Glomerular transcriptome changes associated with lipopolysaccharide-induced proteinuria. Am J Nephrol. 2009; 29(6):558-70. LIST OF ABBREVIATIONS GBM Glomerular Basement Membrane CNF Congenital Nephrotic Syndrome of the Finnish Type PM Perfect match probe MM Mismatch probe SAGE Serial Analysis of Gene Expression QC Quality Control SAM Significance Analysis of Microarray PAM Partitioning Around Medoids SVM Support Vector Machine GO Gene Ontology PPI Protein-Protein Interaction LPS Lipopolysaccharide EST Expressed Sequence Tag ISH in situ hybridization IHC Immunohistochemistry HPA Human Protein Atalas database FDR False Discovery Rate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis qPCR Quantitative Polymerase Chain Reaction EM Electron Microscopy RMA Robust Multi-Array Average (RMA) ANOVA Analysis of Variance