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 1

Podocyte-specific Ercc1 deletion causes glomerulosclerosis.

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Podocyte-specific Ercc1 deletion causes glomerulosclerosis. (A) Gene set...
(A) Gene set enrichment analysis (GSEA) of gene classes according to transcript length. Shown are the shortest 5% (top left), shortest 1% (top right), longest 5% (bottom left), and longest 1% (bottom right) of genes. Bottom color-coded panel shows log2 fold-changes of microarray data in ranked order. Top panels show running enrichment score as orange line. Smallest 1% (normalized enrichment score [NES] of 2.01) and 5% (NES of 1.37) of genes are significantly enriched in upregulated genes, while longest 1% (NES of –1.69) and 5% (NES of –1.42) of genes are significantly enriched in downregulated genes in the comparison of Ercc1–/Δ 14 weeks versus WT 14 weeks (n = 4). (B) Representative electron microscopy image of 14-week-old WT and Ercc1–/Δ glomerular filtration barrier, scale bar indicating 2 μm. B, blood side; U, urinary side; asterisk, foot process (n = 4). (C) Quantitative PCR (qPCR) analysis for Ercc1 in FACS-sorted podocytes of Ercc1 ctrl, WT/pko (het), or pko mice (1-way ANOVA with Tukey’s multiple comparisons test, n = 3–6). (D) Survival of Ercc1 ctrl, WT/pko (het), and pko mice (Mantel-Cox test, n = 4–14). (E) Urinary albumin/creatinine analysis of Ercc1 ctrl and pko mice (2-way ANOVA with Šídák’s multiple comparisons test, n = 4–9). (F) Representative periodic acid–Schiff (PAS) staining of 13-week-old Ercc1 ctrl and pko mice, scale bars: 100 μm in overview, 30 μm in zoom (n = 6). (G) Kaplan-Meier curve depicting survival of Ercc1 ctrl and ipko mice (Mantel-Cox test, n = 5–6). (H) Representative Coomassie staining of Ercc1 ctrl and ipko urine 18 weeks after tamoxifen induction; bovine serum albumin was loaded as reference (n = 6). Values are in kilodaltons. (I) Representative PAS staining of Ercc1 ctrl and ipko mice 25 weeks after induction with tamoxifen (n = 6). Scale bar as in F. Scatterplots indicate mean plus 95% confidence interval. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.