Research ArticleKidney Fibrosis

Elastin imaging enables noninvasive staging and treatment monitoring of kidney fibrosis

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Science Translational Medicine  03 Apr 2019:
Vol. 11, Issue 486, eaat4865
DOI: 10.1126/scitranslmed.aat4865
  • Fig. 1 Elastin expression is increased in renal fibrosis.

    (A) Representative elastin immunofluorescence staining in healthy and fibrotic kidneys (rat UUO model) (n = 4). Quantification of the elastin+ area in the cortex is shown in the right panel. (B) Transmission electron microscopy of elastin in kidney blood vessel walls (arrowheads). The region enclosed by the dashed box is shown at higher magnification in the middle panels. Discontinued and rigid elastin fibers (open arrow) can be seen in the interstitium between collagen bundles (asterisks) in renal fibrosis (right panel). (C) Time-course study of elastin expression (protein and mRNA) using Western blot and qRT-PCR in the murine model of adenine nephropathy (n = 4 for each time point). Scale bars, 50 μm. Elastin area: *P < 0.05, t test. Elastin protein and mRNA: *P < 0.05, #P < 0.0001, one-way analysis of variance (ANOVA) test with d1.

  • Fig. 2 Elastin expression in fibrotic human kidneys is increased independent of the disease etiology.

    (A) Immunohistochemistry and (B) Western blot analysis of elastin expression and (C) qRT-PCR for elastin in human biopsies from healthy (n = 6) and fibrotic (n = 6) kidneys. (D) Renal elastin expression in major renal pathologies (n = 89). T0, protocol time zero biopsy of transplanted kidneys; Nephrectomy, nonaffected tissue from tumor resection; Membranous GN, membranous glomerulonephritis; Crescentic GN, crescentic glomerulonephritis including pauci-immune glomerulonephritis and lupus nephritis; FSGS, focal segmental glomerulosclerosis; Rejection, acute and chronic antibody and T cell–mediated renal rejection; ACKD, acquired cystic kidney disease; ADPKD, autosomal dominant polycystic kidney disease. Scale bars, 50 μm. *P < 0.05, P < 0.01, P < 0.001, #P < 0.0001, elastin expression healthy versus fibrosis, t test. Elastin expression score in human samples: one-way ANOVA test with T0 biopsy.

  • Fig. 3 Elastin imaging reflects renal fibrosis in adenine nephropathy and corresponds to elastin content in human kidney samples.

    (A to G) Murine adenine nephropathy. (A) Coronal T1-weighted MR images and (B) pseudocolor-coded images before and 24 hours after intravenous injection of ESMA and Gd-DTPA in healthy (n = 4) and adenine-induced fibrotic (n = 4) kidneys. (C and D) Quantification of normalized MRI signal intensities in cortex and medulla of ESMA- and Gd-DTPA–injected mice with healthy and fibrotic kidneys. (E and F) Gd quantification in cortex and medulla of healthy and fibrotic kidneys. (G) Gd distribution by LA-ICP-MS in healthy and fibrotic kidneys of mice with ESMA or Gd-DTPA. (H to L) Human kidney fibrosis. (H) Representative T1WI images of gelatin-embedded human kidney biopsies after incubation with ESMA or Gd-DTPA. (I and J) Quantification of normalized MR signal intensities in ESMA- and Gd-DTPA–incubated healthy (n = 4) and fibrotic (n = 4) kidneys. (K and L) Gd quantification is reflective of ex vivo binding of ESMA in healthy and fibrotic human kidneys. *P < 0.05, P < 0.01, P < 0.001, #P < 0.0001, t test.

  • Fig. 4 ESMA specifically binds to elastin in renal fibrosis.

    (A and B) Representative T1-weighted MR images and quantification of normalized MRI signal intensities of kidneys in the I/R mouse model. Mice received either standard 153Gd-ESMA (control, n = 4) or a preinjection of a 25-fold excess of 69Ga-ESMA followed by 153Gd-ESMA (competition, n = 4). (C and D) Immunofluorescence and Western blot analyses of elastin expression in mice injected with 153Gd-ESMA alone (control) or preinjected with a 25-fold excess of 69Ga-ESMA (competition). (E) Quantification of collagen I and α-SMA expression in control and competition group. (F and G) Representative images and quantification of binding of radiolabeled 99mTc-ESMA to murine kidney sections (n = 18) ex vivo. Arrowheads denote elastin expression in arteries in healthy kidneys and interstitium in fibrotic kidneys. Scale bars, 50 μm. *P < 0.05, P < 0.01, t test. ns, not significant.

  • Fig. 5 Elastin imaging enables longitudinal monitoring of fibrosis progression in mice with adenine diet–induced nephropathy.

    (A) Representative T1-weighted MR images in renal fibrosis before disease induction (week 0) as well as at the second and third week of adenine diet (n = 4). Scans were acquired before (Pre) and 24 hours after injection of the contrast agents. (B and C) MR signal quantification in the cortex and medulla 24, 48, and 72 hours after ESMA injection. (D) Western blot analysis of elastin in healthy kidneys and kidneys with adenine nephropathy–related fibrosis. (E) Immunofluorescence images of elastin expression in healthy and fibrotic kidneys (adenine day 23). *P < 0.05 compared to week 0, t test. P < 0.001, #P < 0.0001, t test.

  • Fig. 6 Elastin imaging enables monitoring of anti-fibrotic therapy response.

    (A) Representative immunohistochemical and immunofluorescence staining of collagen I, α-SMA, and elastin in kidneys from vehicle (n = 4) and CRID3-treated (n = 3) mice with adenine-induced nephropathy. (B) Quantification of collagen I expression in the cortex of vehicle- and CRID3-treated kidneys. (C) MR images of kidneys obtained 24 hours after the intravenous injection of ESMA in vehicle- and CRID3-treated mice. (D to G) Quantification of the MRI signal intensities and protein expression in the cortex (D and E) and medulla (F and G) of vehicle- and CRID3-treated mice. (H) Immunohistochemical and immunofluorescence staining of collagen I, α-SMA, and elastin in water-treated (n = 8) or imatinib-treated mice (n = 8) after I/R-induced fibrosis. (I) Quantification of collagen I expression in the cortex of water- and imatinib-treated mice. (J) MR images obtained 24 hours after ESMA injection in water- and imatinib-treated mice. (K to N) Quantification of the MRI signal intensities and protein expression in cortex (K and L) and medulla (M and N) of imatinib-treated versus vehicle-treated mice. Scale bars, 50 μm. *P < 0.05, P < 0.01, P < 0.001, t test. Mann-Whitney test in elastin protein in CRID3 and ΔCNR in imatinib-treated mice.

  • Fig. 7 Elastin imaging identifies residual renal fibrosis that is not detectable using routine kidney function measurement.

    (A) Scheme of the adenine reversal experiment: After 14 days of adenine diet, the animals received normal chow for 14 days in a “regeneration” phase. Red triangles represent injection of contrast agents, and blue and black arrows show MRI scanning before and 24 hours after injection, respectively (n = 4 ESMA, n = 4 Gd-DTPA). (B to E) Serum creatinine (B), serum urea (C), creatinine clearance (D), and systolic blood pressure (E) analyses before, during, and 14 days after terminating the adenine diet. (F and G) Representative immunohistochemical staining (F) and quantification of collagen type III expression (G) in the cortex before, during, and 14 days after terminating the adenine diet. (H and I) Immunofluorescence staining (H) of elastin fibers and their quantification (I) in kidneys before, during, and 14 days after terminating the adenine diet. (J) Analyses of elastin expression via Western blot analysis of kidney tissue before, during, and 14 days after terminating the adenine diet. (K) ESMA-based MRI and (L and M) quantification of the normalized MR signal intensities in cortex (L) and medulla (M) before, during, and 14 days after terminating the adenine diet. Scale bars, 50 μm. *P < 0.05, P < 0.01, P < 0.001, #P < 0.0001, t test. Mann-Whitney test in elastin/GAPDH protein.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/486/eaat4865/DC1

    Materials and Methods

    Fig. S1. Scheme of in vivo molecular MRI of renal fibrosis in three independent mouse models of renal fibrosis.

    Fig. S2. Confirmation of increased elastin expression in renal fibrosis.

    Fig. S3. Elastin expression in murine models of renal fibrosis.

    Fig. S4. Identification of interstitial (myo-)fibroblasts as elastin-producing cells in fibrotic kidneys.

    Fig. S5. Resident fibroblasts express elastin.

    Fig. S6. Elastin expression in CKD patients.

    Fig. S7. Staining validation by means of nonspecific secondary antibody and buffer control.

    Fig. S8. Feasibility assessment of different MRI sequences and measurement times.

    Fig. S9. Elastin imaging detects fibrosis in adenine nephropathy.

    Fig. S10. Elastin imaging detects renal fibrosis in I/R.

    Fig. S11. Elastin imaging detects renal fibrosis in UUO.

    Fig. S12. Molecular MRI captures therapy effects in renal fibrosis.

    Fig. S13. Molecular MRI analysis of renal fibrosis versus routine measurement of kidney function.

    Fig. S14. Overall study and experimental design.

    Table S1. Collection of patient fibrotic kidney samples reflecting different kidney diseases.

    Table S2. IgA nephropathy patient cohort.

    Table S3. List of primers used for qRT-PCR.

    Data file S1. Individual subject-level data.

    References (3840)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Scheme of in vivo molecular MRI of renal fibrosis in three independent mouse models of renal fibrosis.
    • Fig. S2. Confirmation of increased elastin expression in renal fibrosis.
    • Fig. S3. Elastin expression in murine models of renal fibrosis.
    • Fig. S4. Identification of interstitial (myo-)fibroblasts as elastin-producing cells in fibrotic kidneys.
    • Fig. S5. Resident fibroblasts express elastin.
    • Fig. S6. Elastin expression in CKD patients.
    • Fig. S7. Staining validation by means of nonspecific secondary antibody and buffer control.
    • Fig. S8. Feasibility assessment of different MRI sequences and measurement times.
    • Fig. S9. Elastin imaging detects fibrosis in adenine nephropathy.
    • Fig. S10. Elastin imaging detects renal fibrosis in I/R.
    • Fig. S11. Elastin imaging detects renal fibrosis in UUO.
    • Fig. S12. Molecular MRI captures therapy effects in renal fibrosis.
    • Fig. S13. Molecular MRI analysis of renal fibrosis versus routine measurement of kidney function.
    • Fig. S14. Overall study and experimental design.
    • Table S1. Collection of patient fibrotic kidney samples reflecting different kidney diseases.
    • Table S2. IgA nephropathy patient cohort.
    • Table S3. List of primers used for qRT-PCR.
    • References (3840)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data.

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