Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses
Seiji Kishi, … , Ryuji Morizane, Joseph V. Bonventre
Seiji Kishi, … , Ryuji Morizane, Joseph V. Bonventre
Published November 1, 2019; First published October 7, 2019
Citation Information: J Clin Invest. 2019;129(11):4797-4816. https://doi.org/10.1172/JCI122313.
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Research Article Nephrology

Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses

Abstract

Maladaptive proximal tubule (PT) repair has been implicated in kidney fibrosis through induction of cell-cycle arrest at G2/M. We explored the relative importance of the PT DNA damage response (DDR) in kidney fibrosis by genetically inactivating ataxia telangiectasia and Rad3-related (ATR), which is a sensor and upstream initiator of the DDR. In human chronic kidney disease, ATR expression inversely correlates with DNA damage. ATR was upregulated in approximately 70% of Lotus tetragonolobus lectin–positive (LTL+) PT cells in cisplatin-exposed human kidney organoids. Inhibition of ATR resulted in greater PT cell injury in organoids and cultured PT cells. PT-specific Atr-knockout (ATRRPTC–/–) mice exhibited greater kidney function impairment, DNA damage, and fibrosis than did WT mice in response to kidney injury induced by either cisplatin, bilateral ischemia-reperfusion, or unilateral ureteral obstruction. ATRRPTC–/– mice had more cells in the G2/M phase after injury than did WT mice after similar treatments. In conclusion, PT ATR activation is a key component of the DDR, which confers a protective effect mitigating the maladaptive repair and consequent fibrosis that follow kidney injury.

Authors

Seiji Kishi, Craig R. Brooks, Kensei Taguchi, Takaharu Ichimura, Yutaro Mori, Akinwande Akinfolarin, Navin Gupta, Pierre Galichon, Bertha C. Elias, Tomohisa Suzuki, Qian Wang, Leslie Gewin, Ryuji Morizane, Joseph V. Bonventre

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Figure 4

Atr gene deletion in RPTCs leads to increased cleaved caspase 3 and G2/M-phase cells after cisplatin injection.

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Atr gene deletion in RPTCs leads to increased cleaved caspase 3 and G2/...
(A) Representative images of pH3-stained kidney sections from ATRCtrl and ATRRPTC–/– mice 96 hours after saline or cisplatin injection and the corresponding quantification of pH3+ nuclei. Scale bar: 50 μm. Saline: ATRCtrl (n = 3), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 6), ATRRPTC–/– (n = 6). (B) Representative images of F4/80-stained kidney sections from ATRCtrl and ATRRPTC–/– mice 96 hours after saline or cisplatin injection. Scale bar: 50 μm. Dot plot shows corresponding quantification of F4/80+ area. Saline: ATRCtrl (n = 3), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 5), ATRRPTC–/– (n = 5). Representative Western blots of cleaved caspase 3 expression in mouse RPTCs (C) and LLC-PK1 cells (D) treated with cisplatin, VE-821, or a combination of both. n = 3 independent experiments. (E) Representative images of H9 cell–derived day-64 organoids treated with either cisplatin (5 μM) or vehicle (DMSO) for 24 hours, with or without 10 μM VE-821 pretreatment. Sections of the organoids were stained for LTL, pH3, Ki67, and DAPI. Scale bar: 50 μm; inset shows a high-power magnification of a triple-positive tubule. (F) Quantitation of LTL+ tubules and (G) percentage of pH3+, Ki67+, and LTL+ cells to LTL+ cells (n = 2 ×10 high-power fields [HPF] in each treatment group). Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t test (A and B, cisplatin ATRCtrl vs. cisplatin ATRRPTC–/–) and 1-way ANOVA followed by Tukey’s post-hoc test (C, D, F, and G) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.