POSTER NO: 480

Global gene expression profiling in a canine model of X-linked Alport syndrome: Cross-species cDNA microarray analysis provides insight into the early molecular alterations associated with the onset of this progressive renal disease

¹K. Zheng, ¹S.J. Harvey, ¹D. Chen, ²B. Jefferson, ²R.M. Jacobs, ¹P.S. Thorner
¹Division of Pathology, The Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada, ²Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada

Alport syndrome (AS) is an inherited disorder of type IV collagen characterized by progressive renal failure, ocular abnormalities and sensorineural deafness. The most common X-linked form of AS is caused by mutations in the COL4A5 gene, which encodes the alpha 5 chain of type IV collagen. Within the kidney, this primary genetic defect results in a complex cascade effect leading to the dysregulated expression of many proteins, including structural components of the glomerulus (e.g. selected collagen and laminin isoforms) and proteins which may influence progression to renal failure (e.g. TGF-beta and MMPs). A comprehensive understanding of these early molecular alterations would help to provide insight into the factors contributing to progressive nephropathy in AS. Towards this goal, gene expression profiling was carried out in a canine model of X-linked AS using cDNA microarray analysis. cDNA derived from the kidneys of normal and affected 2 month dogs (the onset of clinical disease) was used to survey a human cDNA array consisting of 19,200 ESTs. A total of ~40 genes (nearly half of which are unknown) were identified as being highly dysregulated, with the majority of these being overexpressed in affected kidney. For a number of these genes, confirmation of differential expression was obtained through Northern blot analysis, in situ hybridization and/or by immunostaining using age-matched normal and affected dog kidney at various timepoints. Known genes upregulated in affected kidney included the monocyte/macrophage chemotactic factors monocyte chemotactic protein-1 (MCP-1) and osteopontin (OPN); the cytoskeletal proteins vimentin (VIM), cardiac muscle alpha actin (ACTC), non-muscle myosin (MYH11) and ras homologue gene family member A (ARHA); and the structural proteins collagen types III (COL3A1) and XVI (COL16A1). Also overexpressed in affected kidney were clusterin (CLU), osteonectin (SPARC), immunoglobulin lambda (IgL), several metallothionine isoforms (MT1E/1F/2A) and unexpectedly, the epididymis-specific protein HE4 which displays homology to extracellular proteinase inhibitors. Some of these genes (e.g. CLU, OPN) have been previously implicated in other chronic renal diseases, whereas others (e.g. HE4), and those genes which are unknown may reflect novel pathways involved in the etiology of progressive renal disease and warrant further investigation. The identification of factors mediating the recruitment of monocytes/macrophages (e.g. MCP-1, OPN), or factors that are indicative of lymphocyte infiltration (e.g. IgL) highlight the processes of inflammatory and/or immune reactions as early contributing events in the pathogenesis of X-linked AS. In conclusion, cDNA microarray technology can be a powerful tool to identify and explore differentially expressed genes that may play a role in the pathogenesis and progression of AS as well as other chronic renal diseases. Furthermore, our results validate that human cDNA microarrays can be employed for expression profiling in experimental models of renal disease from other species for which no arrays exist.