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 7

Podocytes accumulate DNA damage during human FSGS.

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Podocytes accumulate DNA damage during human FSGS. (A) Uniform manifold ...
(A) Uniform manifold approximation and projection (UMAP) of a single-nucleus sequencing data set (47) depicting 24 cell types in analyzed control, MCD, and FSGS biopsies. FIB/Immune, fibroblasts/immune cells; C-TAL/MD, thick ascending limb of loop of Henle; PT-1–6, proximal tubular cells; DTL, descending thin limb; PC-CNT, connecting tubule; EC-PT, endothelial cell; FIB, fibroblast; DCT, distal convoluted tubule; IC-A, intercalated cell A; vSMC/MC-1, vascular smooth muscle cell/muscle cell 1; TAL, thick ascending limb of loop of Henle; T_Cell, T cells; MAC/MON, macrophage/monocyte; MD, medullary cell; vSMC/MC-2, vascular smooth muscle cell/muscle cell 2; PEC, parietal epithelial cell; GC-EC, glomerular endothelial cell; IC-B, intercalated cell B; POD, podocyte. (B) Bubble plot indicating the differences in DNA repair and mTORC1 target gene expression in podocytes between living donor (LD) kidney samples and MCD and FSGS biopsies obtained through single-nucleus sequencing (47). Bubble size indicates percentage of podocytes expressing the target gene, bubble color indicates expression level, and gray line indicates the split between DNA repair and mTORC1 target genes. (C) Selected scatterplots of ERCC1 and ERCC4 expression data of single podocytes obtained from LD, MCD, and FSGS biopsies. (D) Selected scatterplots of RNA polymerase 1–3 subunit POLR2K and RNA polymerase 2 subunit POLR2J of single podocytes obtained from LD, MCD, and FSGS biopsies. (E) Representative immunofluorescence staining of SNP, gH2A.X, and Draq5 in sections of human MCD and FSGS biopsies, yellow dotted line indicating nuclear border, scale bar indicating 2 μm with quantification of gH2A.X foci per podocyte nuclear area and per podocyte nucleus in human MCD and FSGS biopsies (unpaired t test, n = 4, 4 glomeruli per sample, 5 podocytes per glomerulus). All violin plots indicate median (black) and upper and lower quartile (gray). **P ≤ 0.01. ***P ≤ 0.001.