Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury

Abstract

Fibrosis is responsible for chronic progressive kidney failure, which is present in a large number of adults in the developed world. It is increasingly appreciated that acute kidney injury (AKI), resulting in aberrant incomplete repair, is a major contributor to chronic fibrotic kidney disease. The mechanism that triggers the fibrogenic response after injury is not well understood. In ischemic, toxic and obstructive models of AKI, we demonstrate a causal association between epithelial cell cycle G2/M arrest and a fibrotic outcome. G2/M-arrested proximal tubular cells activate c-jun NH(2)-terminal kinase (JNK) signaling, which acts to upregulate profibrotic cytokine production. Treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor or the removal of the contralateral kidney, rescues fibrosis in the unilateral ischemic injured kidney. Hence, epithelial cell cycle arrest at G2/M and its subsequent downstream signaling are hitherto unrecognized therapeutic targets for the prevention of fibrosis and interruption of the accelerated progression of kidney disease.

Figure 1

Clinical-pathological features of AKI models. (a) Changes of serum creatinine over time in AKI mouse models, including moderate and severe IRI and AAN (left) and unilateral IRI (UIRI) and UUO (right) models (n = 8 for each time point). *P < 0.001, ^#P < 0.01 versus day 0 before procedure. (b) Histology of the fibrotic outcomes of the AKI models (n = 3 mice in each group, Masson's trichrome staining showing fibrosis with blue color). (c) Sircol assay of kidney collagen content in the five AKI models (n = 3 mice in each group). *P < 0.001 versus control, ^#P < 0.001 versus moderate IRI. (d) Immunostaining of collagen IV (left, green (anti–collagen IV)) and α-SMA (right, red (anti–α-SMA)) in AKI models (n = 3 mice in each group). (e) The percentage of total tissue area that stained positively for collagen IV, collagen I and α-SMA in the kidneys at 42 d (moderate IRI, severe IRI, UIRI and AAN) or 14 d (UUO). *P < 0.001 versus control, ^#P < 0.001 versus moderate IRI. Scale bars, 50 μm. Error bars represent s.d.

Figure 2

Repair of renal tubular cells in models of AKI. (a) Number (per 400× field) of Ki-67–positive (left), BrdU-positive (middle) or p-H3–positive (right) tubular cells in moderate IRI, UIRI and AAN mice (n = 3 mice at each time point in each group). *P < 0.01 versus sham, ^#P < 0.01 versus control. (b) Cell cycle distribution (G1, S and G2/M) of tubular cells in moderate IRI (left), UIRI (middle) and AAN (right) models as a function of time after the insult (n = 3 mice of each time point in each group). (c) Percentage of the proliferating (Ki-67–positive) tubular cells that are in the G2/M phase of the cell cycle in the AKI models in moderate IRI, UIRI and AAN (top) and UIRI and UUO models (bottom), showing both the injured kidney and the contralateral kidney (contr). *P < 0.001, ^#P < 0.01 versus control or sham (n = 3 mice of each time point in each group). (d) Coimmunostaining with antibodies to Ki-67 (anti–Ki-67) and p-H3 (anti–p-H3) on day-7 kidneys from the moderate and UIRI groups. (e) Coimmunostaining with antibodies to p-H3 and Kim-1 (anti–Kim-1) in UIRI mice. The tubular basement membrane is outlined. (f) Western blot analysis of cyclin D1 and cyclin B1 in isolated tubules from AKI kidneys (top) and ratio of cyclin B1 to cyclin D1 densities standardized to β-actin (bottom). *P < 0.001. (g) Percentage of BrdU-positive tubular cells that are in G2/M at various times after BrdU administration in the moderate IRI, AAN and UUO groups. *P < 0.001. (h) Staining with antibodies to BrdU and p-H3, as well as of nuclei with DAPI, in moderate IRI, AAN and UUO mice that were injected with BrdU at 12 h before killing. Scale bars, 50 μm. Error bars represent s.d.

Figure 3

Profibrogenic factor production in G2/M-arrested proximal tubular cells in vitro and in AKI models in vivo. (a) Cell cycle analysis by propidium iodide staining and flow cytometry in HK-2, LLC-PK1 or IRPTC cells at baseline (top three graphs) and after treatment with aristolochic acid at 5 μg ml⁻¹ for 48 h (bottom three graphs). (n = 10, n = 3 and n = 3 experiments for the three cell types, respectively.) (b) Quantification of mRNA levels of profibrogenic genes in HK-2, LLC-PK1 and IRPTC cells treated with aristolochic acid (AA) at 5 μg ml⁻¹ for 48 h, expressed as fold increases over controls (n = 6, n = 3 and n = 3 experiments). *P < 0.001, **P < 0.01 versus control. (c) TGF-β1 concentration (left) and fold increase in CTGF protein concentrations (right) in the supernatant of HK-2 cells treated with aristolochic acid for 48 h. *P < 0.001, ^#P < 0.05. (d) Changes in mRNA levels of profibrogenic factors in cells in various cell cycle phases with or without previous aristolochic acid treatment (n = 6). *P < 0.01, **P < 0.05 versus control G0/G1; ^#P < 0.01, ^##P < 0.05 versus control G2/M. (e) Effect of conditioned medium from G2/M-arrested HK-2 cells on cell proliferation of serum-starved fibroblasts. *P < 0.05. (n = 3.) (f) Collagen secretion (left) and collagen IV production (right) in fibroblasts treated with conditioned medium from control or G2/M-arrested HK-2 cells, presented as percentage increase (collogen) or fold increase (collagen IV) in response to CM from AA-treated HK-2 cells versus CM from untreated HK-2 cells. (n = 3.) *P < 0.01. (g) mRNA levels of profibrogenic factors as a function of time in AKI models, including moderate IRI, severe IRI, UIRI, AAN and UUO (n = 3 mice per time point in each group). *P < 0.001, **P < 0.01, ^#P < 0.05 versus control or sham (set arbitrarily to 1). (h) Western blot analysis of TGF-β1 and CTGF in isolated tubules from moderate IRI and aristolochic acid–treated mice. (i) Co-staining of CTGF or TGF-β1 with p-H3. Nuclei (N) are outlined in the top image. Scale bar, 10 μm. Error bars represent s.d.

Figure 4

Reversal of G2/M arrest rescues the fibrogenic effect in aristolochic acid-treated HK-2 cells and in the UIRI mouse model. (a) Number of proximal tubule cells per 600× fieldthat were positive for p-ATM (Ser1981) for all ofthe models on day 3 (left) and a temporal curve of p-ATM–positive cells in a 400× field for the moderate IRI and AAN insults (right, n = 3 mice per time point in each group). *P < 0.05, **P < 0.01, ^#P < 0.001 versus control or sham. (b) Western blot analysis of p-ATM, p-Chk2 (Ser68) and p-p53 (Ser15) in HK-2 cells treated with aristolochic acid (AA). (c) Effects of KU 55933 (an ATM inhibitor) on cell cycle changes (top) and profibrogenic gene expression (bottom) in HK-2cells (left), LLC-PK1 cells (middle) and IRPTC cells (right) treated with AA for 48 h (n = 3). *P < 0.01,**P < 0.05 versus control; ^#P < 0.05 versus AA. (d) Cell cycle distribution (top) and profibrogenic factor production (bottom) in HK-2 cells and LLC-PK1 cells with or without previous ATM shRNA treatment. (n = 3.) *P < 0.01, ^#P < 0.05. Symbols in the cell cycle data panels refer to the comparison of G2/M phases. (e) Percentage of proliferating tubular cells in G2/M in kidneys from UIRI mice and UIRI mice that had undergone contralateral nephrectomy (N) or PIF-α treatment 10 d after the UIRI. *P < 0.01. (f) The mRNA levels of whole-kidney Tgfb1, Ctgf, Col4a1 and Col1a1 in UIRI mice with contralateral Nx or PIF-α treatment. *P < 0.01, versus sham; ^#P < 0.05 versus UIRI. (g) The effect of Nx or PIF-α treatment on the degree of interstitial fibrosis in the UIRI kidneys (examined 42 d after UIRI injury), as indicated by staining with Masson's trichrome (left) and collagen content determined with the Sircol assay (right). Scale bar, 50 μm. *P < 0.01. Error bars represent s.d. For the mRNA levels in c and d, the control values were orbitrarily set to 1.

Figure 5

Prolonged G2/M arrest, induced by alternative strategies, causes a profibrotic phenotype both in vitro and in vivo. (a) Cell cycle changes in HK-2 and LLC-PK1 cells treated with RO3306 (RO) for 48 h. (b) Cell cycle changes (top) and profibrotic gene expression (bottom) in HK-2 and LLC-PK1 cells treated with RO3306 for 24 h (RO24 h) or 48 h (RO 48 h). (n = 3.) In one group, the RO3306 drug was washed out after 24 h and the cells examined 24 h later (RO 24 h/WO 24 h). **P < 0.001, *P < 0.01, ^#P < 0.05. (c) Cell cycle distribution of normal cycling HK-2 cells (control), cells treated with RO3306 for 24 h (RO 24 h), cells treated with RO3306 for 48 h (RO 48 h) and cells treated with RO3306 for 48 h and then collected 6 h after washout of the RO3306 (RO 48 h WO) (top) and mRNA levels of profibrogenic genes: TGFB1, CTGF, COL4A1 and ACTA2 in cells in G0/G1 or G2/M phase (bottom) (n = 3 experiments). *P < 0.001, **P < 0.05 versus control G0/G1; ^#P < 0.001, ^##P < 0.05 versus control G2/M; &P < 0.01 versus RO 48 h G2/M. (d) Cell cycle changes in HK-2 cells treated with paclitaxel at various doses for 24 h. (n = 3.) *P < 0.001. (e) The CTGF protein (top western blot) and mRNA levels of TGFB1, CTGF, COL4A1 and COL1A1 (bottom) in HK-2 cells treated with paclitaxel for 24 h. n = 5. *P < 0.001. For mRNA levels in b and e, control values were arbitrarily set to 1. (f) The cell cycle changes in proliferating tubular cells in moderate IRI models without (day 1) or with (>day 1) paclitaxel treatment. *P < 0.01, ^#P < 0.05. Symbols in the cell cycle data graphs refer to the comparison of G2/M phases. (g) Masson's trichrome staining of moderate IRI kidneys with or without paclitaxel treatment (21 d after injury, n = 3 mice in each group). Scale bar, 50 μm. (h) The serum creatinine changes in moderate IRI models with or without paclitaxel treatment. Error bars represent s.d.

Figure 6

JNK signaling activation mediates G2/M arrest–induced profibrogenic cytokines upregulation. (a) Western blot analysis of MAPK pathway activation (top) and the corresponding cell cycle distribution (bottom) of HK-2 cells treated with aristolochic acid, RO3306 or paclitaxel. (n = 3–5.) (b) Effects of the JNK inhibitor (SP600125, SP) on cell cycle distribution (top) and profibrogenic gene expression (bottom) in HK-2 cells treated with aristolochic acid, RO3306 (RO) or paclitaxel. (n = 3.) mRNA data are presented as fold induction over the untreated cells. *P < 0.05, **P < 0.001. Symbols in the cell cycle graphs refer to the comparison of G2/M phases. (c) The protein amounts of CTGF in the supernatant from HK-2 cells treated with aristolochic acid or aristolochic acid together with SP600125 (AA + SP) versus untreated cells (CON). Data are presented as fold induction over the control cells (n = 3). *P < 0.001. (d) The proliferation of fibroblasts incubated with conditioned medium (CM) from HK-2 cells treated with aristolochic acid or from HK-2 cells treated with aristolochic acid together with SP600125. (n = 3.) *P < 0.01. (e) The collagen content, expressed as percentage change over control, in the supernatant from fibroblasts incubated with CM from aristolochic acid–treated HK-2 cells or from HK-2 cells treated with aristolochic acid together with SP600125 (SP). (n = 3.) *P < 0.01. (f) Co-localization of p-JNK or p–c-jun with p–histone H3 in AAN kidney on day 7. Scale bar, 10 μm. (g) Masson's trichrome staining of UIRI kidneys with or without SP600125 treatment on day 28 after UIRI. Scale bar, 50 μm. (h) Quantitative kidney interstitial fibrosis score (left) and collagen content in the kidneys on day 28, as determined by the Sircol assay (right). *P < 0.001, ^#P < 0.05. (i) Effect of JNK inhibition on profibrogenic gene expression in UIRI-treated kidneys. Data are presented as fold induction over the untreated cells. *P < 0.001, ^#P < 0.05. Error bars represent s.d.