Gene expression programs of mouse endothelial cells in kidney development and disease

Abstract

Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.

Figure 1. Profile plot of gene expression differences of renal endothelial cells isolated from different developmental times, compartments, and disease states.

786 probesets, each represented by a single line, color coded according to expression in embryonic cells, with red indicating high and blue representing low levels of expression. E15.5, total E15.5 kidney endothelial cell population, Cortical, endothelial cells from adult kidney cortex, with glomeruli removed, Medullary, adult medulla compartment endothelial cells, wt Glom, control adult glomerulus compartment endothelial cells, db Glom, adult glomerulus compartment endothelial cells from db/db mouse with diabetic nephropathy.

Figure 2. Heat Map displaying gene expression differences (531 probe sets) between different kidney development compartments.

Shown are End (E15.5 endothelial cells), UB (E11.5 ureteric bud), CM (P0 capping mesenchyme), and RV (P4 renal vesicle).

Figure 3. Molecular pathways analysis of genes with elevated expression in the kidney developing endothelial compartment.

Generated with GeneSpring 11.0 using the 207 genes with elevated expression in the embryonic endothelial cells. Relations between encoded proteins are shape and color coded, with dark purple square  =  member, light blue triangle  =  transport, red square  =  expression, purple square  =  regulation, blue square  =  binding, orange circle  =  promoter binding, light blue diamond  =  metabolism, pink  =  protein modification.

Figure 4. Enlarged glomeruli in seven month old db/db mice.

Panels A and C are controls, and panels B and D are from db/db mice. Panels A and B are lower magnification views showing multiple glomeruli, while panels C and D are higher magnification, more clearly showing the size difference. Sections were hematoxylin and eosin stained.

Figure 5. Heat Map of twenty two genes showing the greatest change in expression in glomerulus endothelial cells of diabetic nephropathy db/db mice.

Red indicates high level of expression, and blue low expression, with yellow intermediate. C, control samples. DN, diabetic nephropathy db/db mice. Nineteen of the genes were up regulated in db/db mice, and three genes were down regulated.

Figure 6. Immunostain validation of gene expression differences in db/db glomerulus.

Protein levels were examined in C (control) kidneys, panels A, C, E, G and K, as well as DN (diabetic nephropathy) kidneys, panels B, D, F, H, and K. Elevated expression was observed in each case. Arrows in panels D, H and K show striking signal in the vascular pole region for AGTRL1, ADAMTS9 and GPIHBP1. Arrowhead in panel D marks elevated AGTRL1 expression also observed in tubules.