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. 2017 Sep 21;2(18):e94716.
doi: 10.1172/jci.insight.94716.

Molecular characterization of the transition from acute to chronic kidney injury following ischemia/reperfusion

Jing Liu  1 , Sanjeev Kumar  1   2 , Egor Dolzhenko  3 , Gregory F Alvarado  1 , Jinjin Guo  1 , Can Lu  1 , Yibu Chen  4 , Meng Li  4 , Mark C Dessing  1 , Riana K Parvez  1 , Pietro E Cippà  1 , A Michaela Krautzberger  1 , Gohar Saribekyan  1 , Andrew D Smith  3 , Andrew P McMahon  1
Affiliations

Molecular characterization of the transition from acute to chronic kidney injury following ischemia/reperfusion

Jing Liu et al. JCI Insight. .

Abstract

Though an acute kidney injury (AKI) episode is associated with an increased risk of chronic kidney disease (CKD), the mechanisms determining the transition from acute to irreversible chronic injury are not well understood. To extend our understanding of renal repair, and its limits, we performed a detailed molecular characterization of a murine ischemia/reperfusion injury (IRI) model for 12 months after injury. Together, the data comprising RNA-sequencing (RNA-seq) analysis at multiple time points, histological studies, and molecular and cellular characterization of targeted gene activity provide a comprehensive profile of injury, repair, and long-term maladaptive responses following IRI. Tubular atrophy, interstitial fibrosis, inflammation, and development of multiple renal cysts were major long-term outcomes of IRI. Progressive proximal tubular injury tracks with de novo activation of multiple Krt genes, including Krt20, a biomarker of renal tubule injury. RNA-seq analysis highlights a cascade of temporal-specific gene expression patterns related to tubular injury/repair, fibrosis, and innate and adaptive immunity. Intersection of these data with human kidney transplant expression profiles identified overlapping gene expression signatures correlating with different stages of the murine IRI response. The comprehensive characterization of incomplete recovery after ischemic AKI provides a valuable resource for determining the underlying pathophysiology of human CKD.

Keywords: Molecular diagnosis; Mouse models; Nephrology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Incomplete recovery after 21 minutes of bilateral renal IRI.
(A) Kaplan-Meier survival curves for mice with different periods of renal pedicle clamping (ischemia time). Mean is presented. (B) Sample collection time points. (C) Serum creatinine (mg/dl) after sham and IRI. Mean ± SEM is presented. (D–I) H&E staining on sagittal sections of the sham and IRI kidneys. Zoomed views for rectangular regions in D–I are shown in Supplemental Figure 1, G–L. (J and K) Immunostaining on sagittal sections of the kidneys 12 months after sham and IRI. α-SMA (cyan), CD45R (red), and desmin (green). Zoomed views for the top black squares in H and I are in L and M, with the left square corresponding to the top and right square to the bottom in L and M. Zoomed views for the white squares in J and K are in N and O, with the left square corresponding to the top and right square to the bottom in N and O. Scale bar: 1 mm (D–K); 50 μm (L–O).
Figure 2
Figure 2. Continuous expression of Krt20/8/18 in injured proximal tubules.
(A–C) Immunostaining on sagittal sections of kidneys 3 days and 1 week after sham and IRI. Krt20 (red) and LTL (cyan). (D–F) Zoomed views for the rectangular regions in A–C. (G–I) Confocal immunofluorescence of Krt20 (red), Krt8/18 (green), LTL (cyan), and α-SMA (magenta), showing the outer stripe of outer medullary regions on the sagittal sections of kidneys. (J–L) Split channel images for G–I. Scale bar: 1 mm (A–C); 50 μm (D–L).
Figure 3
Figure 3. Temporal-specific gene changes after IRI through RNA-seq.
(A) Sample clustering through principle component analysis (PCA) plot. (B) Heatmap of expression profiles of genes identified in modules with induction at hours (I), hours to weeks (II), days (III), days to weeks (IV), weeks to months (V), months (VI), and downregulation (VII). (C) Histogram of –log10 of P values of DAVID gene ontology for biological processes of module I~V and VII. SHAM4h, SHAM24h, and SHAM12m: 4 hours, 24 hours, and 12 months after sham surgery; NORM3m, NORM9m, and NORM15m: age-matched no surgery controls for 0, 6, and 12 months after IRI; IRI2h, IRI4h, IRI24h, IRI48h, IRI72h, IRI7d, IRI14d, IRI28d, IRI6m, and IRI12m: 2 hours, 4 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 4 weeks, 6 months, and 12 months after IRI.
Figure 4
Figure 4. Overlap of mouse IRI with published human kidney transplant profiles.
Heatmap of expression profiles in mouse IRI samples for differentially expressed genes identified in published human kidney transplant (A) 45–60 minutes (46) and (B) 1 year after reperfusion (74). Left columns indicate relative expression in human samples. Detailed sample information for each column is the same as that in Figure 3B.
Figure 5
Figure 5. Expression patterns of representative genes from module I–VI through RNA section in situ hybridization.
Expression patterns of representative genes from module I (A), module II (B), module III (C), module IV (D), module V (E), and module VI (F). Images in the second and fourth columns are high-magnification views of regions in red rectangles. Images in insets are high-magnification views of regions in black squares (the second and fourth columns in A–E and second column in F) and the region in the yellow square (the fourth column in F). Scale bar: 1 mm (whole-kidney view); 200 μm (zoomed view for red rectangular regions); 120 μm (insets).
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