Changes in the gene expression programs of renal mesangial cells during diabetic nephropathy

Abstract

Background: Diabetic nephropathy is the leading cause of end stage renal disease. All three cell types of the glomerulus, podocytes, endothelial cells and mesangial cells, play important roles in diabetic nephropathy. In this report we used Meis1-GFP transgenic mice to purify mesangial cells from normal mice and from db/db mice, which suffer diabetic nephropathy. The purpose of the study is to better define the unique character of normal mesangial cells, and to characterize their pathogenic and protective responses during diabetic nephropathy.

Methods: Comprehensive gene expression states of the normal and diseased mesangial cells were defined with microarrays. By comparing the gene expression profiles of mesangial cells with those of multiple other renal cell types, including podocytes, endothelial cells and renal vesicles, it was possible to better define their exceptional nature, which includes smooth muscle, phagocytic and neuronal traits.

Results: The complete set of mesangial cell expressed transcription factors, growth factors and receptors were identified. In addition, the analysis of the mesangial cells from diabetic nephropathy mice characterized their changes in gene expression. Molecular functions and biological processes specific to diseased mesangial cells were characterized, identifying genes involved in extracellular matrix, cell division, vasculogenesis, and growth factor modulation. Selected gene changes considered of particular importance to the disease process were validated and localized within the glomuerulus by immunostaining. For example, thrombospondin, a key mediator of TGFβ signaling, was upregulated in the diabetic nephropathy mesangial cells, likely contributing to fibrosis. On the other hand the decorin gene was also upregulated, and expression of this gene has been strongly implicated in the reduction of TGFβ induced fibrosis.

Conclusions: The results provide an important complement to previous studies examining mesangial cells grown in culture. The remarkable qualities of the mesangial cell are more fully defined in both the normal and diabetic nephropathy diseased state. New gene expression changes and biological pathways are discovered, yielding a deeper understanding of the diabetic nephropathy pathogenic process, and identifying candidate targets for the development of novel therapies.

Figure 1

Heatmap showing genes with elevated expression in normal mesangial cells. Expression patterns of 172 genes with up-regulation in mesangial cells are compared across multiple renal compartments, including cap mesenchyme, renal vesicle, total renal cortex, podocytes, endothelial cells and renal capsule. Red indicates high, yellow represents intermediate and blue indicates low gene expression level. Most of the 172 genes are not uniquely expressed in mesangial cells, but do show significantly elevated mesangial expression. For an image that can be zoomed to visualize gene names see Additional file 2: Figure S1.

Figure 2

Immunohistochemistry showing glomerular expression patterns of some of the genes with elevated mesangial cell expression. The proteins of some genes, such as Nr2f1, show nuclear localization, as would be expected for a transcription factor, while in other cases, such as for PREX2, there is a striking extracellular localization. Data is from the human protein atlas (http://www.proteinatlas.org/).

Figure 3

Molecular function, biological processes and candidate regulatory transcription factors and microRNAs in normal mesangial cells. The central hexagons show some of the 172 genes with elevated expression in normal mesangial cells, compared to other cell types. The surrounding squares show color coded molecular functions (light green), biological processes (dark green), transcription factors (light purple) and microRNAs (dark purple), strongly associated with these genes. Lines show connections to linked genes. A. The cell adhesion biological process, with associated genes, is highlighted in yellow. B. The locomotion biological process and associated genes is highlighted in yellow. For complete lists of molecular functions, biological processes, transcription factors, microRNAs and other features see Additional file 3: Table S2.

Figure 4

Glomerular changes indb/dbdiabetic mice. Panels A, C and E are controls, and panels B, D and F are from db/db mice with diabetic nephropathy. Panels A and B show histological sections stained with hematoxylin and eosin and demonstrate glomeruli hypertrophy. Panels C and D are sections stained with Masson's trichrome stain. Note the increased staining (blue) indicating a higher level of collagen deposition in the glomeruli of diabetic mice. Panels E and F are sections stained with Periodic acid-Schiff (PAS) and show mesangial expansion in diabetic mice. All images are representative glomeruli at an original magnification of 200X.

Figure 5

Changing gene expression programs in mesangial cells ofdb/dbdiabetic nephropathy mice. The red hexagons show some of the genes up-regulated in mesangial cells of db/db diabetic mice. The dark green squares show biological processes, and light green molecular functions, and purple a microRNA, with lines showing links to related genes.

Figure 6

Immunostaining for validation of gene expression differences indb/dbglomeruli. Protein levels were examined in C (control) kidneys, panels A, C, E, G, I and K, as well as DN (diabetic nephropathy) kidneys, panels B, D, F, H, J and L. Elevated glomerular expression was observed in each case, as predicted by the microarray results. In addition, note the increased Collagen12a1 expression in the db/db tubules (panels G and H).