Glomerular expression profiling and novel proteins in normal mouse kidney and adriamycin-induced nephrosis

Kidney glomeruli function as high-capacity molecular sieves through which plasma is filtered into the Bowman s space as primary urine. The glomerular filtration barrier is composed of glomerular endothelial cells, the glomerulus basement membrane and the podocyte cell layer. Dysfunction of the glomerulus is a central component of renal complications leading to end-stage renal disease. However, the molecular composition of the glomerulus and how it changes during disease are still mostly unknown. To elucidate the picture of molecules involved in the biology and pathology of the glomerulus, large-scale approaches including two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry analysis and microarray profiling were applied to normal and diseased glomeruli.

A proteome analysis of healthy glomeruli in mouse was performed using 2-DE with two different staining methods and subsequent mass spectrometric identifications. Altogether, 414 protein spots were identified, revealing 232 different proteins representing a wide spectrum of activities. Only 53 of the proteins identified here were detected in another proteome study, showing the value of analysis utilizing different methodologies. 80 of the proteins were not identified in a separate transcriptome analysis, while ten of the present proteins were identified as genes implicated in glomerulus development and function, allowing direct correlation with expression data.

Characterization of five glomerulus-upregulated transcripts/proteins, ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik was performed. The expression pattern of these novel glomerular transcripts in various mouse tissues was studied, and the distribution of corresponding proteins was examined. All five transcripts/proteins were expressed in the kidney exclusively by glomerular cells. Ehd3 was expressed only by glomerular endothelial cells. Importantly, ehd3 is the first gene ever shown to be expressed exclusively by glomerular endothelial cells and not by other endothelial cells in the kidney. Dendrin, sh2d4a, plekhh2, and 2310066E14Rik were transcribed specifically in podocytes within the glomerulus. With the use of polyclonal antibodies, dendrin, sh2d4a, and plekhh2 proteins were localized to the slit diaphragm and the foot process, whereas 2310066E14Rik protein was localized to the podocyte major processes and cell body.

Comparison of the normal glomerular transcriptome with its changes during progression of glomerular disease can yield information about molecular pathomechanisms. The adriamycin (ADR)-induced proteinuric mouse model allows the precise timing of the onset of proteinuria and of morphological changes in glomerulus. Overt proteinuria was observed from four days after ADR injection, and reached maximum at seven days. Blood urea nitrogen (BUN) gradually elevated indicating the failure of renal function. TUNEL staining of kidney section revealed an increase in apoptotic positive cells in glomeruli. Transcriptional profiling of kidney glomeruli revealed that nine p53 target genes were up-regulated probably due to DNA damage caused by ADR at four days and several glomerular enriched genes were differentially expressed indicating glomerular injury at seven days.

These studies provide fresh insights into glomerular biology and reveal new possibilities to explore the role of glomerular specific proteins in renal physiology and pathophysiology. Furthermore, these studies shed light on the pathomechanisms of proteinuria, which eventually results in end-stage kidney disease as a result of progressive glomerular damage.

List of scientific papers

I. Tryggvason S, Nukui M, Oddsson A, Tryggvason K, Jörnvall H. (2007). "Glomerulus proteome analysis with two-dimensional gel electrophoresis and mass spectrometry" Cell Mol Life Sci. 64: 3317-3335.
https://pubmed.ncbi.nlm.nih.gov/18030416

II. Patrakka J, Xiao Z, Nukui M, Takemoto M, He L, Oddsson A, Perisic L, Kaukinen A, Szigyarto CA, Uhlén M, Jalanko H, Betsholtz C, Tryggvason K. (2007). "Expression and subcellular distribution of novel glomerulus-associated proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik." J Am Soc Nephrol. 6896-6897.
https://pubmed.ncbi.nlm.nih.gov/17251388

III. Nukui M, He L, Patrakka J, Takemoto M, Betsholtz C, Tegnér J, Tryggvason K (2010). "Stage specific glomerular transcriptome profiling in ADR-induced nephropathy mice." [Manuscript]