Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses
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
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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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 3

ATRRPTC–/– mice have enhanced DNA damage and apoptosis after cisplatin injection.

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ATRRPTC–/– mice have enhanced DNA damage and apoptosis after cisplatin i...
(A) Representative images of γH2AX-stained kidney sections from ATRCtrl and ATRRPTC–/– mice 96 hours after saline or cisplatin injection. Scale bars: 50 μm. Dot plots show quantification of γH2AX+ cells, percentage of cells with nucleus-wide γH2AX+ staining or γH2AX+ foci, and γH2AX+ cells in cortex versus outer medulla. Saline: ATRCtrl (n = 3), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 9), ATRRPTC–/– (n = 11). (B) Representative images of cleaved caspase 3–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 cleaved caspase 3+ cells. Saline: ATRCtrl (n = 3), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 9), ATRRPTC–/– (n = 11). (C) Immunostaining for cleaved caspase 3 and DAPI in kidneys from ATRCtrl and ATRRPTC–/– mice 96 hours after saline or cisplatin injection. Upper panels: Stitched images represent approximately 25% of the kidney cross-sectional area (original magnification, ×200). Lower panels: Higher-magnification images from the boxed regions in the stitched (upper) images (scale bar: 500 μm). Dot plot shows quantification of cleaved caspase 3 staining from the whole kidney section images in C relative to the LTL+ area of uninjured kidney tissue. Saline: ATRCtrl (n = 4), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 6), ATRRPTC–/– (n = 8). (D) Representative images of Ki67-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 Ki67+ cells. Saline: ATRCtrl (n = 3), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 7), ATRRPTC–/– (n = 9). (E) Representative images of F4/80- and Ki67-stained and α-SMA– and Ki67-stained sections of injured kidneys from ATRRPTC–/– mice 96 hours after cisplatin injection. Scale bar: 50 μm. (F) Representative Western blot. Each lane represents 1 sample from an individual mouse. Dot plots show quantification of Western blot band intensity for p53 and p21, 96 hours after cisplatin injection. ATRCtrl (n = 6), ATRRPTC–/– (n = 6). Also shown is a dot plot of quantitative RT-PCR analysis of p21 mRNA levels in ATRCtrl and ATRRPTC–/– kidneys. Saline: ATRCtrl (n = 3), ATRRPTC–/– (n = 4); cisplatin: ATRCtrl (n = 6), ATRRPTC–/– (n = 7). Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t test (A, B, D, and F, for cisplatin ATRCtrl vs. cisplatin ATRRPTC–/–) and 1-way ANOVA followed by Tukey’s post-hoc test (C). *P < 0.05, **P < 0.01, ***P <0.001, and ****P < 0.0001. See the complete unedited blots in the supplemental material.