uPAR isoform 2 forms a dimer and induces severe kidney disease in mice
Changli Wei, … , M. Amin Arnaout, Jochen Reiser
Changli Wei, … , M. Amin Arnaout, Jochen Reiser
Published February 7, 2019
Citation Information: J Clin Invest. 2019;129(5):1946-1959. https://doi.org/10.1172/JCI124793.
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Research Article Nephrology

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

Abstract

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding β3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin β3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for β3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

Authors

Changli Wei, Jing Li, Brian D. Adair, Ke Zhu, Jian Cai, Michael Merchant, Beata Samelko, Zhongji Liao, Kwi Hye Koh, Nicholas J. Tardi, Ranadheer R. Dande, Shuangxin Liu, Jianchao Ma, Salvatore Dibartolo, Stefan Hägele, Vasil Peev, Salim S. Hayek, David J. Cimbaluk, Melissa Tracy, Jon Klein, Sanja Sever, Sanford J. Shattil, M. Amin Arnaout, Jochen Reiser

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Figure 2

Detection of msuPAR2 in adipocytes, serum, and urine.

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Detection of msuPAR2 in adipocytes, serum, and urine. (A) Schematic of m...
(A) Schematic of msuPAR2-Tg construction and msuPAR2-Tg mouse treatment. (B) qPCR analysis of muPAR2 in fat tissues. The value was calculated as a ratio of muPAR2 differential expression between littermate controls (WT) and msuPAR2-Tg mice over that of housekeeping gene GAPDH. muPAR2 mRNA was increased significantly compared with littermate controls. Mann-Whitney U test. **P < 0.01. (C) Localization of msuPAR2 in adipocytes. As msuPAR2 carries the c-Myc tag, immunohistochemistry was performed with a rabbit anti-Myc antibody. msuPAR2 was seen in adipocytes of msuPAR2-Tg mice, but not in littermate control mice. Scale bar: 50 μm. (D) Detection of msuPAR2 in circulating blood in msuPAR2-Tg mice. Albumin-depleted sera were separated by NuPAGE gel and processed for Western blot with rabbit anti-msuPAR2 antibody. P, recombinant msuPAR2 protein; B, blank without protein samples. Lane 1, uPAR KO sera; lane 2, msuPAR1-Tg sera; lanes 3 to 7, sera from different msuPAR2-Tg mice. Images shown are representatives of 3 different experiments. Red arrow indicates msuPAR2. (E) Detection of msuPAR2 in urine. Processed urine samples were separated by SDS-PAGE and blotted with a customized rabbit anti-msuPAR2 antibody. A band at 10–15 kDa (highlighted in red rectangle) was identified in msuPAR2-Tg but not in msuPAR1-Tg nor in uPAR-KO mice. Preincubation of the antibody with msuPAR2 peptide nullified the band. (F) Verification of msuPAR2 fragment by LC-MS analysis. The msuPAR2 fragment identified by Western blot was processed for MS analysis. Multiple peptides in the N-terminal region were detected. Shown is one of these peptides (bottom panel), which matches very well with the spectrum of the peptide from recombinant msuPAR2 protein (top panel).