Identification and characterization of novel glomerulus-associated genes and proteins

The kidney is responsible for sieving the circulating blood to eliminate water-soluble waste products and potentially toxic substances from the body. The filtration step occurs in specialized filtration units called glomeruli. Some renal diseases are related to specific glomerular defects, but it is highly likely that the present knowledge gained from previous studies only represents a small proportion of genes and proteins that have important roles for normal kidney function.

To identify other genes with roles for glomerular filtration function, our group developed GlomBase, which is a glomerular transcript database in which over 300 genes are highly glomerulus specific. Among those genes, several genes with highest glomerular expression were chosen for further analysis, but this thesis is mainly based on studies on three of them, dendrin, adenylate cyclase type I (Adcy1), and Crumbs homolog 2 (Crb2).

Dendrin is a cytosolic protein previously identified only in the brain. However, we localized dendrin in the kidney specifically to the glomerular podocytes. Furthermore, we generated a polyclonal antibody against this novel glomerular protein. We detected that the earliest dendrin expression during glomerular maturation is at the capillary loop stage, and that it is located in the cytoplasmic face of the podocyte slit diaphragm. Unexpectedly, inactivation of the dendrin gene in mouse did not generate any obvious phenotype. Dendrin -/- mice were born at an expected Mendelian ratio and macroscopically all organs appeared normal. By the age of 1.2 years, no signs of renal impairment have been observed in the dendrin-/- mice. Under kidney challenging conditions, dendrin -/- mice show no difference when compared with dendrin +/+ mice. Even though dendrin does not seem to be crucial for the integrity of the glomerular filtration barrier, we do find two proteins that interact with dendrin, and their biological role in podocyte is still under investigation. These results are out scope of this thesis. Adcy1 is one out of nine members of the adenylate cyclase protein family which catalyze the formation of the secondary messenger cAMP. cAMP is involved in a wide variety of cellular signaling processes, including regulation of actin cytoskeleton assembly through PKA.

Adcy1 has previously been thought to be expressed only by certain neuronal cells in the brain, but we localized Adcy1 expression to the glomerular podocytes as well. During glomerulogenesis, the Adcy1 expression was detected first at the stage when maturing podocytes develop foot processes. To study the role of Adcy1 gene in the kidney in vivo, we analyzed the kidneys of Adcy1-/- mice (mice generatedby other investigators, that without severe phenotype except mild behavioral abnormalities). We found the glomerulogenesis to proceed normally in Adcy1-/- mice, and in mature mouse, no signs of renal impairment was detected. However, challenging of the kidney with albumin overload caused severe albuminuria in Adcy1-/- mice, whereas wild type mice showed only moderate albumin leakage to the urine. Thus, Adcy1 may in fact be a susceptibility gene for proteinuria.

Crb2 is yet another novel podocyte specific protein we identified. Its Drosophila homologue Crumbs is an essential component for epithelial cells organizing apicalbasal polarity and adherent junctions. In the mouse, it is expressed only in brain, kidney and heart. In the kidney, it is specifically located in the glomerular podocyte slit diaphragm. Interestingly, inactivation of this gene led to arrest the embryonic development after E7.75 and embryonic lethality, which demonstrates the importance of this gene during early embryonic development. The Crb2-/- embryos show defects in neuroepithelium and epithelial mesenchymal transition (EMT) at the primitive streak. The function of Crb2 protein in the glomerulus will be explored later by my colleagues in studies of conditional knockout mice with podocyte specific inactivation of the Crb2 gene.

In summary, the discovery and characterization of novel glomerular genes and proteins presented in this thesis has increased our knowledge of glomerular biology as well as on the role of a glomeral gene in early embryogenesis.

List of scientific papers

I. Takemoto M, He L, Norlin J, Patrakka J, Xiao Z, Petrova T, Bondjers C, Asp J, Wallgard E, Sun Y, Samuelsson T, Mostad P, Lundin S, Miura N, Sado Y, Alitalo K, Quaggin SE, Tryggvason K, Betsholtz C (2006). "Large-scale identification of genes implicated in kidney glomerulus development and function." EMBO J. 25: 1160-1174.
https://pubmed.ncbi.nlm.nih.gov/16498405

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. 18: 689-697.
https://pubmed.ncbi.nlm.nih.gov/17251388

III. Xiao Z, Takemoto M, Chan G, Storm D, Betsholtz C, Tryggvason K, Patrakka J (2010). "Glomerular Podocytes Express Type I Adenylate Cyclase Inactivation Leads to Susceptibility to Proteinuria." Nephron Exp Nephron. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/21196775

IV. Xiao Z, Patrakka J, Nukui M, Lijun Chi, Dadi Niu, Betsholtz C, Vainio S, Tryggvason K (2010). "Crumbs homolog 2 (Crb2) is required for normal epithelialmesenchymal transition (EMT) of gastrulation process during early embryonic mouse development." [Submitted]