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 1

ATR and DNA activation in human kidneys and organoids.

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ATR and DNA activation in human kidneys and organoids. (A) Representativ...
(A) Representative images of periodic acid–Schiff (PAS) and MT staining of human kidney tissue and the corresponding quantitation of MT+ areas. Scale bars: 10 μm. (B) Representative images of γH2AX- and KIM-1–stained sections of human kidneys and the corresponding quantitation of γH2AX+/KIM-1+ tubules. Scale bars: 10 μm. (C) Correlation between the number of γH2AX+/KIM-1+ tubules and eGFR. (D) Representative images of pATR- and KIM-1–stained sections of human kidney and the corresponding quantitation of pATR/KIM-1+ tubules. Scale bar: 10 μm. (E) Relationship between γH2AX and pATR expression in KIM-1+ chronically injured RPTECs. (F) Representative images of H9 cell–derived day-49 organoids treated with either cisplatin (5 μM) or vehicle (RPMI) for 24 hours. Sections of the organoids were stained for ATR, pATR, γH2AX, and LTL. Scale bar: 20 μm. Dot plots show quantitation of pATR+ nuclei (n = 6, control; n = 6, cisplatin) and γH2AX+ nuclei in the organoids (n = 6, control; n = 7, cisplatin). (G) Viability of HKC-8 cells assessed 24 hours after cisplatin treatment, with or without 10 μM VE-821 pretreatment. Viability was determined using the MTT assay. Data are expressed as a percentage of the control MTT value (n = 3). (H) Viability of HKC-8 cells was assessed by MTT assay immediately following culturing under 21% or 5% O2 for 24 hours, with or without 10 μM VE-821 pretreatment. n = 9, MCD; n = 11, CKD (A–E). Data are presented as the mean ± SEM. A 2-tailed, unpaired t test (A, B, D, and F), 1-way ANOVA with Tukey’s post hoc test (G and H), and Pearson’s correlation analysis (C and E) were used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. HM, high-magnification.