CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival
Suma Yaddanapudi, … , Sanja Sever, Jochen Reiser
Suma Yaddanapudi, … , Sanja Sever, Jochen Reiser
Published September 12, 2011
Citation Information: J Clin Invest. 2011;121(10):3965-3980. https://doi.org/10.1172/JCI58552.
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Research Article Nephrology

CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival

Abstract

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-β1–dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-β1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-β response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.

Authors

Suma Yaddanapudi, Mehmet M. Altintas, Andreas D. Kistler, Isabel Fernandez, Clemens C. Möller, Changli Wei, Vasil Peev, Jan B. Flesche, Anna-Lena Forst, Jing Li, Jaakko Patrakka, Zhijie Xiao, Florian Grahammer, Mario Schiffer, Tobias Lohmüller, Thomas Reinheckel, Changkyu Gu, Tobias B. Huber, Wenjun Ju, Markus Bitzer, Maria P. Rastaldi, Phillip Ruiz, Karl Tryggvason, Andrey S. Shaw, Christian Faul, Sanja Sever, Jochen Reiser

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Figure 6

Dendrin is a transcription factor of CatL.

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Dendrin is a transcription factor of CatL. (A and B) Nuclear dendrin ind...
(A and B) Nuclear dendrin induced transcription from the CatL promoter, but not from the CatB promoter. HEK 293 cells were cotransfected with pSEAP reporter vector containing the promoter of interest, dendrin, and Metridia luciferase to normalize for transfection efficiency. Mutant dendrin lacking its nuclear localization signal (dNLS) was ineffective. Promoter activity and response to dendrin depended on a region between bp –1,215 and bp –339, as revealed by partial deletion promoter constructs. ***P < 0.001. (C) Strategy used to identify the dendrin binding site within the CatL promoter. The promoter fragment between bp –1,215 and –339 was divided into 4 parts. EMSA with these unlabeled DNA fragments, visualized by SYBR green, revealed dendrin binding to fragment F (data not shown), which was then further divided into overlapping fragments to fine-map the dendrin binding site. (D) EMSA demonstrating specific dendrin binding to one of the biotin-labeled 60-bp oligonucleotides that were completely abolished by a 200-fold excess of unlabeled oligonucleotide. (E) The 60-bp oligonucleotide that exhibited dendrin binding was divided into 3 overlapping 24-bp oligonucleotides to further narrow the dendrin binding site. Mutation of 3 nucleotides in the 5′ region, but not in the central or 3′ region, of the 24-bp oligonucleotide 4-1 completely abolished dendrin binding, as demonstrated by the inability of a 200-fold excess of unlabeled mutated oligonucleotide to compete with the labeled WT oligonucleotide.