Research ArticleCancer

Acute kidney injury promotes development of papillary renal cell adenoma and carcinoma from renal progenitor cells

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Science Translational Medicine  25 Mar 2020:
Vol. 12, Issue 536, eaaw6003
DOI: 10.1126/scitranslmed.aaw6003
  • Fig. 1 Recurrence-free survival in patients with pRCC according to the presence or absence of a postoperative AKI episode.

    (A and B) Recurrence-free survival of patients with pRCC based on the presence or absence of a postoperative AKI episode in (A) a single-center study (n = 65 no postoperative AKI and n = 18 postoperative AKI) and (B) a multicentric study (n = 43 no postoperative AKI and n = 46 postoperative AKI). Censored patients and patients at risk are shown. Kaplan-Meier curves are compared by log-rank test (7.143 and 5.36, respectively) and Cox regression [HR(A) = 3.6 (1.3 to 10.2); HR(B) = 3.4 (1.1 to 10.3)]. Statistical significance was set for a P value <0.05. PO AKI, postoperative AKI.

  • Fig. 2 Long-term follow-up reveals that AKI induces papillary tumors in mice.

    (A) Ultrasound image with power Doppler mode (red) of a wild-type mouse kidney (displayed in transverse plane) at 36 weeks after IRI (representative of n = 3 mice). The region of interest (dashed line) indicates tumoral tissue. Scale bar. 1 mm. (B) Length (micrometers) of the lesions in wild-type mouse kidneys at 36 weeks after IRI (n = 5 mice). Bar indicates median value. (C to F) Representative histological images with H&E staining of papillary tumors from wild-type mouse kidneys after IRI. (C) Type 1 papillary tumor. (D) Type 2 papillary tumor. (E) Nuclear atypias. (F) Foamy macrophages within a papillary lesion. Scale bars, 25 μm. (G) Percentages of mice at 4 weeks (n = 8), 12 weeks (n = 9), and 36 weeks (n = 9) after IRI presenting with papillary adenomas (pAs) and pRCCs. Numbers above graph represent P values. (H) Percentage of type 1 versus type 2 papillary tumors in wild-type mice at 36 weeks after IRI (n = 4 mice). Bars indicate mean values. No significant difference between the groups. (I) Representative confocal image of a type 1 papillary tumor from Pax8/Confetti mouse kidneys at 36 weeks after IRI. Scale bar, 25 μm. (J) Split confocal images of CK7 staining (orange) of a type 1 papillary tumor from Pax8/Confetti mouse kidneys at 36 weeks after IRI. Scale bar, 25 μm. (K) Split confocal images of CK7 staining (orange) of a type 2 papillary tumor from Pax8/Confetti mouse kidneys at 36 weeks after IRI. Scale bar, 25 μm. (L) Detail of nuclear atypias in a type 2 papillary tumor from Pax8/Confetti mouse kidneys at 36 weeks after IRI. Scale bar, 25 μm. (M) Clonal analysis in type 1 and 2 papillary tumors from Pax8/Confetti mouse kidneys at 36 weeks after IRI (n = 7 mice). Bars indicate mean value. (N and O) 3D reconstruction of representative pRCC in Pax8/Confetti mouse kidneys at 36 weeks after IRI. (N) Collagen IV (COL4) staining (cyan). Arrows indicate invasion. (O) Von Willebrand Factor (VWF) staining (purple). Arrows indicate tumor vascularization. Scale bars, 25 μm. DAPI (4′,6-diamidino-2-phenylindole) (white) counterstains nuclei. Signals for fluorescent Confetti proteins are as follows: green fluorescent protein (GFP), green; red fluorescent protein (RFP), red; yellow fluorescent protein (YFP), yellow; and cyan fluorescent protein (CFP), blue. Statistical power was assessed using Fisher's exact test (G) or Mann-Whitney test (H). N.S Not significant (P > 0.05).

  • Fig. 3 NOTCH1 overexpression is specific for human type 2 pRCC and predicts poor prognosis.

    (A) Log2 RPPA signal for proteins differently expressed in tumor stage T1-T2 (black boxes, n = 29 patients) versus T3-T4 (pink boxes, n = 66) pRCC. (B to D) Overall survival of patients with pRCC based on high (≥50th percentile, n = 62) or low (˂50th percentile, n = 61) expression of different proteins: NOTCH1 (B), PTEN (C), or SMAD4 (D). (E) Log2 RPPA signal for proteins different in tumor type 1 (black box, n = 58) versus type 2 (red box, n = 49) pRCC. (F) NOTCH1 log2 RPPA signal in patients with pRCC of clusters C1, C2a, C2b, and C2c/CIMP (n = 71, 27, 18, and 9, respectively). (G) NOTCH1 log2 RPPA signal in patients with nonmutated (black boxes) or mutated (green boxes) MET (nonmutated n = 110, mutated n = 15), FH (nonmutated n = 4, mutated n = 5), CDKN2A (nonmutated n = 88, mutated n = 37), SETD2/BAP1/PBRM1 (nonmutated n = 111, mutated n = 11), or NRF2-ARE pathway (nonmutated n = 108, mutated n = 17). (H) Differences in overall survival of patients with type 1 or 2 pRCC based on high (≥50th percentile, type 1 n = 14 and type 2 n = 40) or low (˂50th percentile, type 1 n = 44 and type 2 n = 9) NOTCH1 protein expression. β-cat, β-catenin. RPPA, reverse-phase protein array. Box-and-whisker plots: line, median; box, 25 to 75%; whiskers, minimum or maximum. Statistical significance was calculated by Mann-Whitney test. Kaplan-Meier curves are compared by log-rank test and Cox regression [HR(B) = 4.0 (1.1 to 14.3); HR(C) = 3.3 (1.0 to 10.8); HR(D) = 3.2 (1.0 to 10.1); HR(F) = 4.6 (1.0 to 20.5)]. Numbers above graphs represent P values.

  • Fig. 4 NICD1 overexpression in mouse tubular cells induces clonal type 2 papillary tumors.

    (A) Percentages of Pax8/NICD1 mice at 4 weeks (n = 8), 12 weeks (n = 10), and 36 weeks (n = 16) presenting with papillary adenomas (pAs) and pRCCs. Statistical power was assessed using Fisher's exact test: a versus c, P = 0.004; a versus e, P = 0.0001; c versus e, P = 0.2642; and b versus d, P = 0.0128; b versus f, P = 0.002; d versus f, P = 0.6924. (B) Length (micrometers) of the lesions in Pax8/NICD1 mouse kidneys at 36 weeks (n = 5 mice). Bar (red) indicates median value. (C) Ultrasound image with power Doppler mode (red) of a wild-type mouse kidney (displayed in transverse plane) at 36 weeks (representative of n = 4 mice). Inset: High magnification of the ultrasound appearance of a tumor area (dashed line). Scale bars, 1 mm. (D) Representative image of H&E staining of papillary adenoma from Pax8/NICD1 mouse kidney at 36 weeks. Scale bar, 25 μm. (E) Type 2 papillary carcinoma from Pax8/NICD1 mouse kidney at 36 weeks. Scale bar, 100 μm. (F) H&E staining of foamy macrophages within a papillary lesion from Pax8/NICD1 mouse kidney at 36 weeks. Scale bar, 25 μm. (G) Representative image of a carbonic anhydrase IX (CAIX) negative papillary tumor (brown) from Pax8/NICD1 mouse kidney at 36 weeks. Scale bar, 25 μm. (H) BCL-2–positive papillary tumor (brown) from Pax8/NICD1 mouse kidney at 36 weeks. Scale bar, 25 μm. (I) Representative type 2 papillary tumor from Pax8/NICD1/Confetti mouse kidney at 36 weeks. Scale bar, 25 μm. (J) Representative type 2 papillary tumor from Pax8/NICD1/Confetti mouse kidney at 36 weeks. Scale bar, 25 μm. (K) Clonal analysis of papillary tumors in Pax8/NICD1/Confetti mice at 36 weeks (n = 6 mice). Bars indicate mean values. (L and M) 3D reconstruction of representative lesions in Pax8/NICD1/Confetti kidney. (L) COL4 staining (cyan). Arrows indicate invasion. (M) VWF staining (purple). Arrows indicate vascularization of the tumor. Scale bars, 100 μm. (N and O) Low-power images of a papillary tumor with rhabdoid features. (N) H&E staining. (O) PAX2 staining (brown). Scale bars, 100 μm. (P to T) Histologic images of a subcutaneous metastasis. (P) H&E staining. In the upper part of the image, subcutaneous adipose tissue; in the lower part, metastatic tissue with rhabdoid features. (Q) PAX2 staining (brown). (R) Vimentin staining (brown). (S) MUC1 staining (brown). (T) PanCK staining (brown). Scale bars, 25 μm. DAPI (white) counterstains nuclei. Signals for fluorescent Confetti proteins are as follows: GFP, green; RFP, red; YFP, yellow; and CFP, blue.

  • Fig. 5 AKI accelerates clonal type 2 papillary tumor development in mice.

    (A) Representative image of activated NOTCH1 staining (brown) and lotus tetraglonolobus lectin (LTA; blue) in healthy uninduced Pax8/NICD1 mouse kidney at 1 day. Scale bar, 25 μm. (B and C) Representative images of activated NOTCH1 staining (brown) and LTA (blue) in uninduced Pax8/NICD1 mouse kidney 1 day (B) and 7 days (C) after IRI. Scale bars, 25 μm. (D) Representative image of activated NOTCH1 staining (brown) and LTA (blue) in healthy induced Pax8/NICD1 mouse kidney at 1 day. Scale bar, 25 μm. (E) Representative image of activated NOTCH1 staining (brown) and LTA (blue) in induced Pax8/NICD1 mouse kidney 1 day after IRI. Scale bar, 25 μm. (F) Graph of the reciprocal intensity of activated NOTCH1 per nucleus in uninduced and induced Pax8/NICD1 mice 1 day after IRI (n = 4 mice for each group). a. u., arbitrary units. Bars indicate mean values. (G) Papillary tumor with foamy macrophages in the lumen, present in Pax8/NICD1 mice 4 weeks after IRI. Scale bar, 25 μm. (H) PAX2-positive papillary tumor (brown) from Pax8/NICD1 mouse kidneys 4 weeks after IRI. Scale bar, 25 μm. (I) Percentages of induced Pax8/NICD1 mice with or without IRI presenting with pAdenomas and pRCCs (pAs/pRCCs, n = 5 uninduced and n = 11 induced mice) and CKD (n = 7 uninduced and n = 5 induced mice) after 4 weeks. (J) Percentages of type 1 versus type 2 papillary tumors in Pax8/NICD1 mice 4 weeks after IRI (n = 5 mice). Bars indicate mean values. (K) Representative papillary tumor from Pax8/NICD1/Confetti mouse kidney 4 weeks after IRI. Scale bar, 25 μm. DAPI (white) counterstains nuclei. (L) Clonal analysis of papillary tumors from Pax8/NICD1/Confetti mice 4 weeks after IRI (n = 5 mice). Bars indicate mean values. (M and N) 3D reconstruction of representative lesions in Pax8/NICD1/Confetti kidneys after IRI. (M) COL4 staining (cyan). Arrow indicates invasion. (N) VWF staining (purple). Arrows indicate vascularization. DAPI (white) counterstains nuclei. Signals for fluorescent Confetti proteins are as follows: GFP, green; RFP, red; YFP, yellow; and CFP, blue. Scale bars, 25 μm. Statistical power was assessed using Fisher's exact test (I) or Mann-Whitney test (F and J); numbers above graphs represent P values.

  • Fig. 6 Human renal progenitors are transformed by NOTCH1 overexpression.

    (A and B) Representative confocal images of healthy human kidney labeled with CD133 (green) and VCAM-1 (red) (n = 5 patients). (A) Glomerulus. (B) Tubules (arrows indicate CD133+VCAM-1+ cells). Scale bars, 25 μm. (C) Graph representing the evolution over time of the percentages of CD133+ and CD133 populations (n = 3 biological replicates). (D) Graph representing the number of CD133 and CD133+ cells over time (n = 3 biological replicates). Bars indicate median value. Statistical power was assessed using t test; number above graph represents P value. (E) Representative cell division analysis of CFDA-SE–labeled CD133+ cells at day 10, out of three biological replicates. In the graph, the far right peak indicates undivided cells, and each peak toward the left-hand side represents one cell division or generation. The percentages of cells in each division are shown in the graphs. (F) Representative fluorescence-activated cell sorting analysis for the contemporaneous expression of CD133 and VCAM-1 in CD133+ fraction (right) and corresponding isotype controls (left) (n = 3 biological replicates). IgG2a, immunoglobulin G2a. (G and H) Representative H&E (G) and confocal (H) images of human renal progenitors infected with ZsGreen1-mock (n = 3). In red, phalloidin staining. Scale bars, 25 μm. (I and J) Representative H&E (I) and confocal (J) images of human renal progenitors infected with ZsGreen1-NICD1 (n = 3) (black arrow indicates nuclear groove). In red, phalloidin staining. Scale bars, 25 μm. (K to M) 3D reconstruction of a confocal z-stack of human renal progenitors infected with either ZsGreen1-mock (K) or ZsGreen1-NICD1 (L and M) and showing tubular morphology with a lumen (K) or growth toward inside the tubule lumen (L), generating a tumor-like mass (M) (one representative image of n = 3 biological replicates each). In red, phalloidin staining. Scale bars, 50 μm. DAPI counterstains nuclei. (N) Representative confocal images of biopsies from patients with AKI (n = 4). In red, CD133; in green, activated NOTCH1. Scale bar, 25 μm. (O) Higher magnification of a representative confocal image of a biopsy from a patient with AKI (n = 4). In red, CD133; in green, activated NOTCH1. Scale bar, 25 μm. (P) UMAP representation of 3327 cells, with 1479 pRCC (blue) and 1848 human renal progenitor (orange) cells. (Q) Expression of canonical PT1 signature and renal progenitor (PROM1) genes. (R) Violin plots of scRNAseq data from pRCC (blue) (32) and from human renal progenitor cell cultures (orange), showing the expression of selected marker genes. In red, genes of the PT1 signature. VCAM1, vascular cell adhesion molecule 1; EPCAM, epithelial cell adhesion molecule; SLC17A3, solute carrier family 17 member 3; and SLC7A13, solute carrier family 7 member 13; CFDA-SE, carboxyfluorescein diacetate succinimidyl ester.

  • Fig. 7 AKI induces papillary tumors by promoting clonal expansion of renal progenitors in mice.

    (A) Representative histological image with H&E staining of a type 2 papillary carcinoma present in Pax2/NICD1 mice 4 weeks after IRI. Scale bar, 25 μm. (B) Representative confocal image of type 2 papillary tumor of Pax2/NICD1/Confetti mice 4 weeks after IRI. Scale bar, 25 μm. (C and D) 3D reconstruction of representative lesions in Pax2/NICD1/Confetti kidneys 4 weeks after IRI. (C) COL4 staining (cyan). Arrows indicate invasion. (D) VWF staining (purple). Arrows indicate vascularization of the tumor. Scale bars, 25 μm. (E) Percentages of Pax2/NICD1 and Pax8/NICD1 mice presenting with pAdenomas and pRCCs (pAs/pRCCs) (n = 11 for Pax2/NICD1 and n = 11 for Pax8/NICD1) and CKD (n = 6 for Pax2/NICD1 and n = 5 for Pax8/NICD1) 4 weeks after IRI. (F) Percentages of type 1 versus type 2 papillary tumors in Pax2/NICD1 mice 4 weeks after IRI (n = 6 mice). Bars indicate mean values. (G) Clonal analysis of papillary tumors in each Pax2/NICD1/Confetti mouse 4 weeks after IRI (n = 7 mice). Bars indicate mean value. (H) Graph of the number of papillary tumors per kidney section in Pax2/NICD1 (n = 6) and Pax8/NICD1 (n = 6) mice 4 weeks after IRI. Bars indicate mean values. No significant difference between the groups. (I) Graph of the number of papillary tumors per kidney section in Pax2/NICD1/Confetti mice treated with vehicle (n = 5 mice) or DAPT (n = 5 mice) and euthanized 4 weeks after IRI. Bars indicate mean values. (J) Representative confocal image of activated NOTCH1 (cyan) in Pax2/Confetti mice 2 days after IRI (n = 4). Arrows indicate Pax2+NOTCH1+ cells. Scale bar, 25 μm. (K) Percentages of Pax2/Confetti mice at 4 weeks (n = 8), 12 weeks (n = 3), and 36 weeks (n = 9) after IRI presenting with papillary adenomas (pAs) and pRCCs. (L) Clone frequency analysis in papillary tumors of Pax2/Confetti mouse kidneys at 36 weeks (n = 7 mice). Bars indicate mean values. (M) Percentages of type 1 versus type 2 papillary tumors in Pax2/Confetti mice at 36 weeks (n = 5 mice). Bars indicate mean values. (N) Graph of the numbers of papillary tumors per kidney section in Pax2/Confetti mice treated with vehicle (n = 7) or DAPT (n = 5) and euthanized 36 weeks after IRI. Bars indicate mean values. (O and P) Representative confocal images of type 1 (O) and type 2 (P) papillary tumors of Pax2/Confetti mice 36 weeks after IRI. Scale bars, 25 μm. (Q and R) 3D reconstruction of representative lesions in Pax2/Confetti kidney 4 weeks after IRI. (Q) COL4 staining (cyan). Arrows indicate invasion. (R) VWF staining (purple). Arrows indicate vascularization of the tumor. Scale bars, 25 μm. DAPI (white) counterstains nuclei. Signals for fluorescent Confetti proteins are as follows: GFP, green; RFP, red; YFP, yellow; and CFP, blue. Data are means ± SEM. Statistical power was assessed using Fisher's exact test (E and K) or Mann-Whitney test (F, H, I, M, and N); numbers above graphs represent P values.

  • Table 1 AKI episodes are a risk factor for the development of pRCC but not for ccRCC in humans.

    Clinical characteristics of pRCC, ccRCC, and controls are described. Between-group comparisons (a) were performed using the Mann-Whitney U test for continuous variables or the chi-squared test for categorical variables. Binary logistic regression analysis (b) was used for the association between previous acute kidney injury (AKI) episodes (yes/no) and pRCC development adjusted for age, gender, preoperative chronic kidney disease (CKD), and diabetes mellitus (DM). eGFR, estimated glomerular filtration rate; OR, odds ratio; RCC, renal cell carcinoma; pRCC, papillary RCC; ccRCC, clear cell RCC; CI, confidence interval; y, yes; n, no; F, female; M, male. Continuous variables are presented as mean ± SD. P values ≤0.05 are indicated in bold.

    pRCC
    (n = 56)
    Controls
    (n = 101)
    P (a)
    Previous AKI
    episodes
    12/56 (21.4%)7/101 (6.9%)0.008
    Age at surgery
    (years)
    65.3 ± 11.763.7 ± 9.90.184
    Gender (male)47/56 (83.9%)65/101 (64.4%)0.009
    Preoperative CKD
    (eGFR <60)
    10/56 (17.9%)11/101 (10.9%)0.219
    DM8/56 (14.3%)14/101 (13.9%)0.942
    Length of
    follow-up
    (years)
    4.6 ± 2.84.3 ± 2.90.571
    VariableOR(95% CI)P (b)
    AKI (y/n)3.479(1.135–10.667)0.029
    Age (years)0.979(0.947–1.012)0.213
    Gender (M/F)2.907(1.250–6.761)0.013
    CKD (y/n)1.093(0.377–3.163)0.870
    DM (y/n)0.715(0.258–1.978)0.518
    ccRCC
    (n = 75)
    Controls
    (n = 101)
    P (a)
    Previous AKI
    episodes
    9/75 (12.0%)7/101 (6.9%)0.247
    Age at surgery
    (years)
    66.6 ± 9.363.7 ± 9.90.047
    Gender (male)48/75 (64.0%)65/101 (64.4%)0.961
    Preoperative CKD
    (eGFR <60)
    12/75 (16.0%)11/101 (10.9%)0.320
    DM11/75 (14.7%)14/101 (13.9%)0.880
    Length of
    follow-up
    (years)
    4.2 ± 2.24.3 ± 2.90.916
    VariableOR(95% CI)P (b)
    AKI (y/n)1.549(0.514–4.664)0.437
    Age (years)0.971(0.939–1.004)0.085
    Gender (M/F)0.945(0.516–1.852)0.945
    CKD (y/n)1.202(0.463–3.115)0.706
    DM (y/n)0.888(0.366–2.155)0.793

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/536/eaaw6003/DC1

    Supplementary Materials and Methods

    Fig. S1. The flowchart illustrates the selection criteria for the single-center study population.

    Fig. S2. The flowchart illustrates the selection criteria for the study population from the Danish National Registry of Patients.

    Fig. S3. The flowchart illustrates the selection criteria for the multicentric study population.

    Fig. S4. AKI-driven papillary tumors originate from clonal proliferation of single TECs in mice.

    Fig. S5. NICD1 overexpression in mouse tubular cells induces papillary tumors and CKD.

    Fig. S6. AKI accelerates clonal type 2 papillary tumor development in mice.

    Fig. S7. NOTCH1 overexpression enhances cell division and promotes aberrant mitosis.

    Fig. S8. AKI induces papillary tumors by promoting clonal expansion of renal progenitors in mice.

    Fig. S9. AKI induces papillary tumors from renal progenitors in a NOTCH1-dependent manner.

    Table S1. Postoperative AKI is a risk factor for the development of pRCC recurrence in humans in a single-center study.

    Table S2. The preoperative characteristics of 594 patients treated with partial nephrectomy for kidney tumors and clinical details of 89 patients with pRCC (the RECORd1 project).

    Table S3. Postoperative AKI is a risk factor for the development of pRCC recurrence in humans in a multicentric study (RECORd1 project).

    Table S4. AKI-activated pathways are overexpressed in human pRCC in a subtype-specific manner.

    Table S5. Multivariate analysis indicates NOTCH1 as a risk factor for pRCC prognosis.

    Movie S1. 3D reconstruction of a papillary tumor in Pax8/NICD1/Confetti kidney at 36 weeks.

    References (5379)

  • The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. The flowchart illustrates the selection criteria for the single-center study population.
    • Fig. S2. The flowchart illustrates the selection criteria for the study population from the Danish National Registry of Patients.
    • Fig. S3. The flowchart illustrates the selection criteria for the multicentric study population.
    • Fig. S4. AKI-driven papillary tumors originate from clonal proliferation of single TECs in mice.
    • Fig. S5. NICD1 overexpression in mouse tubular cells induces papillary tumors and CKD.
    • Fig. S6. AKI accelerates clonal type 2 papillary tumor development in mice.
    • Fig. S7. NOTCH1 overexpression enhances cell division and promotes aberrant mitosis.
    • Fig. S8. AKI induces papillary tumors by promoting clonal expansion of renal progenitors in mice.
    • Fig. S9. AKI induces papillary tumors from renal progenitors in a NOTCH1-dependent manner.
    • Table S1. Postoperative AKI is a risk factor for the development of pRCC recurrence in humans in a single-center study.
    • Table S2. The preoperative characteristics of 594 patients treated with partial nephrectomy for kidney tumors and clinical details of 89 patients with pRCC (the RECORd1 project).
    • Table S3. Postoperative AKI is a risk factor for the development of pRCC recurrence in humans in a multicentric study (RECORd1 project).
    • Table S4. AKI-activated pathways are overexpressed in human pRCC in a subtype-specific manner.
    • Table S5. Multivariate analysis indicates NOTCH1 as a risk factor for pRCC prognosis.
    • References (5379)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). 3D reconstruction of a papillary tumor in Pax8/NICD1/Confetti kidney at 36 weeks.

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