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Expression and Subcellular Distribution of Novel Glomerulus-Associated Proteins Dendrin, Ehd3, Sh2d4a, Plekhh2, and 2310066E14Rik

Jaakko Patrakka^*,, Zhijie Xiao^*, Masatoshi Nukui^*, Minoru Takemoto^*, Liqun He^*, Asmundur Oddsson^*, Ljubica Perisic^*, Anne Kaukinen, Cristina Al-Khalili Szigyarto, Mathias Uhlén, Hannu Jalanko, Christer Betsholtz^*,|| and Karl Tryggvason^*

^* Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, and ^|| Department of Medicine, Karolinska Institute, and Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden; and Electron Microscopy Unit, Institute of Biotechnology, and Hospital for Children and Adolescents and Biomedicum Helsinki, University of Helsinki, Helsinki, Finland

Address correspondence to: Dr. Karl Tryggvason, Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, Sweden. Phone: +46-8-5248-7720; Fax: +46-8-316-165; E-mail: karl.tryggvason{at}ki.se

Received for publication June 29, 2006. Accepted for publication December 7, 2006.

The glomerular capillary tuft is a highly specialized microcapillary that is dedicated to function as a sophisticated molecular sieve. The glomerulus filter has a unique molecular composition, and several essential glomerular proteins are expressed in the kidney exclusively by glomerular podocytes. A catalog of >300 glomerulus-upregulated transcripts that were identified using expressed sequence tag profiling and microarray analysis was published recently. This study characterized the expression profile of five glomerulus-upregulated transcripts/proteins (ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik) in detail. The expression pattern of these novel glomerular transcripts in various mouse tissues was studied using reverse transcriptase–PCR, Northern blotting, and in situ hybridization. For studying the distribution of corresponding proteins, polyclonal antibodies were raised against the gene products, and Western blotting, immunofluorescence, and immunoelectron microscopic analyses were performed. Remarkably, it was discovered that 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, however, were transcribed solely by podocytes. 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. This study provides fresh insights into glomerular biology and uncovers new possibilities to explore the role of these novel proteins in the glomerular physiology and pathology.

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