Loss of genome maintenance is linked to mTOR complex 1 signaling and accelerates podocyte damage
Fabian Braun, Amrei M. Mandel, Linda Blomberg, Milagros N. Wong, Georgia Chatzinikolaou, David H. Meyer, Anna Reinelt, Viji Nair, Roman Akbar-Haase, Phillip J. McCown, Fabian Haas, He Chen, Mahdieh Rahmatollahi, Damian Fermin, Robin Ebbestad, Gisela G. Slaats, Tillmann Bork, Christoph Schell, Sybille Koehler, Paul T. Brinkkoetter, Maja T. Lindenmeyer, Clemens D. Cohen, Martin Kann, David Unnersjö-Jess, Wilhelm Bloch, Matthew G. Sampson, Martijn E.T. Dollé, Victor G. Puelles, Matthias Kretzler, George A. Garinis, Tobias B. Huber, Bernhard Schermer, Thomas Benzing, Björn Schumacher, Christine E. Kurschat
Fabian Braun, Amrei M. Mandel, Linda Blomberg, Milagros N. Wong, Georgia Chatzinikolaou, David H. Meyer, Anna Reinelt, Viji Nair, Roman Akbar-Haase, Phillip J. McCown, Fabian Haas, He Chen, Mahdieh Rahmatollahi, Damian Fermin, Robin Ebbestad, Gisela G. Slaats, Tillmann Bork, Christoph Schell, Sybille Koehler, Paul T. Brinkkoetter, Maja T. Lindenmeyer, Clemens D. Cohen, Martin Kann, David Unnersjö-Jess, Wilhelm Bloch, Matthew G. Sampson, Martijn E.T. Dollé, Victor G. Puelles, Matthias Kretzler, George A. Garinis, Tobias B. Huber, Bernhard Schermer, Thomas Benzing, Björn Schumacher, Christine E. Kurschat
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Research Article Aging Cell biology Nephrology

Loss of genome maintenance is linked to mTOR complex 1 signaling and accelerates podocyte damage

Abstract

DNA repair is essential for preserving genome integrity. Podocytes, postmitotic epithelial cells of the kidney filtration unit, bear limited regenerative capacity, yet their survival is indispensable for kidney health. Podocyte loss is a hallmark of the aging process and of many diseases, but the underlying factors remain unclear. We investigated the consequences of DNA damage in a podocyte-specific knockout mouse model for DNA excision repair protein Ercc1 and in cultured podocytes under genomic stress. Furthermore, we characterized DNA damage-related alterations in mouse and human renal tissue of different ages and patients with minimal change disease and focal segmental glomerulosclerosis. Ercc1 knockout resulted in accumulation of DNA damage and ensuing albuminuria and kidney disease. Podocytes reacted to genomic stress by activating mTOR complex 1 (mTORC1) signaling in vitro and in vivo. This was abrogated by inhibiting DNA damage signaling through DNA-dependent protein kinase (DNA-PK) and ataxia teleangiectasia mutated (ATM) kinases, and inhibition of mTORC1 modulated the development of glomerulosclerosis. Perturbed DNA repair gene expression and genomic stress in podocytes were also detected in focal segmental glomerulosclerosis. Beyond that, DNA damage signaling occurred in podocytes of healthy aging mice and humans. We provide evidence that genome maintenance in podocytes is linked to the mTORC1 pathway and is involved in the aging process as well as the development of glomerulosclerosis.

Authors

Fabian Braun, Amrei M. Mandel, Linda Blomberg, Milagros N. Wong, Georgia Chatzinikolaou, David H. Meyer, Anna Reinelt, Viji Nair, Roman Akbar-Haase, Phillip J. McCown, Fabian Haas, He Chen, Mahdieh Rahmatollahi, Damian Fermin, Robin Ebbestad, Gisela G. Slaats, Tillmann Bork, Christoph Schell, Sybille Koehler, Paul T. Brinkkoetter, Maja T. Lindenmeyer, Clemens D. Cohen, Martin Kann, David Unnersjö-Jess, Wilhelm Bloch, Matthew G. Sampson, Martijn E.T. Dollé, Victor G. Puelles, Matthias Kretzler, George A. Garinis, Tobias B. Huber, Bernhard Schermer, Thomas Benzing, Björn Schumacher, Christine E. Kurschat

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Figure 2

The podocyte-specific constitutive knockout of Ercc1 leads to foot process effacement and podocyte loss accompanied by accumulation of DNA damage.

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The podocyte-specific constitutive knockout of Ercc1 leads to foot proce...
(A) Representative electron microscopy image of 7- and 9-week-old Ercc1 ctrl and pko glomerular filtration barrier, scale bar: 2 μm. B, blood side; U, urinary side; asterisk: foot process (n = 3). (B) Representative immunofluorescence staining of synaptopodin (SNP, gray), dachshund family transcription factor 1 (Dach1, green) (75), and nuclei (Draq5, red) in sections of 9-week-old Ercc1 ctrl and pko kidneys, with quantification of podocyte number and density, scale bar indicating 10 μm (unpaired t test, n = 5). (C) Corresponding staining of SNP (gray), Dach1 (green) (75), and nuclei (red) in sections of 11-week-old Ercc1 ctrl and pko kidneys, scale bar indicating 10 μm (n = 5). (D) Representative immunofluorescence staining of SNP (gray), DNA damage marker gH2A.X (green), and Draq5 (red) in sections of 9-week-old Ercc1 ctrl and pko kidneys, with quantification of gH2A.X foci per podocyte nucleus and nuclear area, scale bar indicating 2 μm, yellow dotted line indicating nuclear border (unpaired t test, n = 5, 10 glomeruli per sample, 5 podocytes per glomerulus). (E) Representative immunofluorescence staining of SNP (gray), gH2A.X (green), and Draq5 (red) in sections of 11-week-old Ercc1 ctrl and pko kidneys, with quantification of gH2A.X foci per podocyte nucleus and nuclear area, scale bar indicating 2 μm (unpaired t test, n = 5, 10 glomeruli per sample, 5 podocytes per glomerulus). (F) Representative immunofluorescence staining of nephrin (yellow) with DAPI (gray) of Ercc1 ctrl at 13 weeks of age and pko kidneys at 7, 9, 11, and 13 weeks of age, scale bar indicating 10 μm (n = 5). All violin plots indicate median (black) and upper and lower quartile (gray). *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.