Fig 1.
Renal dysfunction with tubular injuries in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice.
Plasma creatinine levels, plasma blood urea nitrogen (BUN) levels, urinary neutrophil gelatinase-associated lipocalin (urinary NGAL) levels, urinary albumin/creatinine ratio, arterial blood bicarbonate, and arterial blood pH in control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. Data are presented as means ± SEM. *P < 0.05 compared with control mice. NS, not significant.
Fig 2.
Kidneys of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice do not show definitive morphological changes on routine histological examination.
(A) Representative macroscopic photographs of the kidneys. Scale bar = 5 mm. (B) Kidney weights in control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. (C) Histopathological images of kidneys of control and DDC-fed mice at days 1, 3, and 7 (top row, hematoxylin and eosin [H&E] staining; bottom row, periodic acid-Schiff [PAS] staining). PT, proximal tubules; DT, distal tubules; G, glomeruli. Scale bars = 100 μm. (D) Counts of Ly-6G/-6C-positive neutrophils and leukocyte common antigen (LCA)–positive lymphocytes in control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. Data are presented as means ± SEM. NS, not significant; ND, not detected. (E) Quantitative reverse transcription polymerase chain reaction analyses for tumor necrosis factor-α (Tnf-α), interleukin-1β (Il-1β), interleukin-6 (Il-6), and tissue inhibitor of metalloproteinase 1 (Timp1) mRNA in control and DDC-fed mice at days 3 and 7. Values are normalized to Gapdh expression. The mRNA values were analyzed by the two-tailed Student’s t test. Data are presented as means ± SD. *P < 0.05 compared with control mice.
Table 1.
Morphological changes of the kidney and liver in DDC-fed mice.
Fig 3.
Sublethal tubular cell injuries in the kidney of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice.
(A) Semi-thin sections with toluidine blue staining (top row) and transmission electron microscopic (TEM) (bottom row) images of proximal tubules in control and DDC-fed mice at days 1, 3, and 7; red arrowheads, loss of brush border microvilli. Scale bars = 25 μm (top row), 10 μm (bottom row). (B) The proportion of injured cells to total tubular cells is presented as percentage; control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. (C) TEM images of intracellular organelles (top row) and immunofluorescent analysis for KIM-1 (bottom row) in proximal tubules of control and DDC-fed mice at days 1, 3, and 7; asterisks, mitochondria with abnormal features; arrowheads, intact endoplasmic reticula; green, immunofluorescence labeling for KIM-1; red, phalloidin; blue, 4,6-diamidino-2-phenylindole (DAPI). Scale bars = 500 nm (top row), 50 μm (bottom row). (D) KIM-1 staining on renal tubules of the control and DDC-fed mice was digitally analyzed and presented as percentage positive staining area per field; control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. Data are presented as means ± SEM. *P < 0.05 compared with control mice.
Fig 4.
Single-cell extrusion with subsequent regeneration in the kidney of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice.
(A) A representative photograph of a single tubular cell extrusion (triangles) from the epithelial layer of proximal tubules at day 3. Scale bar = 10 μm. (B) Counts of mitotic cells per field on proximal tubules of control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. (C) Counts of Ki-67-positive cells per field on proximal tubules of control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. Data are presented as means ± SEM. *P < 0.05 compared with control mice. (D) Immunohistochemical staining for Ki-67 in the proximal tubules of control and DDC-fed mice at days 1, 3, and 7. Scale bar = 100 μm.
Fig 5.
Mitochondrial damage and subsequent autophagy in the kidneys of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)–fed mice.
(A) Representative transmission electron microscopic images of autophagosomes in the proximal tubules of kidneys from control and DDC-fed mice at days 1, 3, and 7; asterisks, autophagosomes. Scale bar = 500 nm. (B) Counts of autophagosomes per 1,000× magnified field; control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. (C) LC3-I and LC3-II protein expressions in the kidneys of control and DDC-fed mice at days 1, 3, and 7 by immunoblot analysis; β-actin served as a loading control; control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. (D) Representative images of p62 immunostaining in kidneys of control and DDC-fed mice at days 1, 3, and 7; green, immunofluorescence labeling for p62; red, phalloidin; blue, diamidino-2-phenylindole (DAPI). Scale bar = 10 μm. Data are presented as means ± SEM. *P < 0.05 compared with control mice.
Fig 6.
Sublethal injuries in the kidneys of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice are associated with oxidative stress.
(A) Representative images of 4-hydroxy-2-nonenal (4-HNE) immunostaining in the kidneys of control and DDC-fed mice at days 1, 3, and 7. 4-HNE staining on kidney tissue was digitally analyzed and presented as percentage of positive staining area per field; control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. Scale bar = 50 μm. (B) Representative images of 3-nitrotyrosine (3-NT) immunostaining in the kidneys of control and DDC-fed mice at days 1, 3, and 7. The ratio of 3-NT-positive cells to total tubular cells are presented as a percentage; control (white bar) and DDC-fed mice (black bar) at days 1, 3, and 7. Scale bar = 50 μm. Data are presented as means ± SEM. *P < 0.05 compared with control mice.
Fig 7.
Sublethal injury leads to interstitial fibrosis and fibroblast activation after long-term 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding.
(A) Plasma creatinine levels and urinary albumin/creatinine ratio in control (white bar) and DDC-fed mice (black bar) at days 7, 14, and 56. Data are presented as means ± SEM. *P < 0.05 compared with the control mice. Control and 7 days of DDC-fed mice samples were reproduced from Fig 1; data were acquired using n ≥ 3 mice/group for the albumin/creatinine ratio. (B) Light and electron microscopic images of kidneys of control and DDC-fed mice at days 7, 14, and 56; top row, reticulin staining; middle row, Azan staining; bottom row, transmission electron microscopy (TEM); red arrowheads, accumulation of immature collagen fibers; black asterisks, accumulation of mature collagen fibers (blue staining); arrows, peritubular collagen fibers; white asterisk, myofibroblast with released collagen fibers. Scale bars = 25 μm (top row), 100 μm (middle row), and 2 μm (bottom row).
Fig 8.
The relationship between renal impairment and liver dysfunction in dose-response analyses.
(A) Serum levels of total bilirubin (T-Bil) and plasma creatinine in control (white bar), 0.1% DDC-fed (black bar), and 0.5% DDC-fed mice (gray bar) at days 1, 3, and 7. Data are presented as means ± SEM. #P < 0.05 compared with the 0.1% DDC-fed mice. NS, not significant. Control and 0.1% DDC-fed mice samples were reproduced from Fig 1. (B) Representative photographs of livers and kidneys of 0.1% (left row) and 0.5% (right row) DDC-fed mice at day 7 (hematoxylin and eosin [H&E] staining). PT, proximal tubules; DT, distal tubules; G, glomeruli; arrows, porphyrin plugs; arrowheads, ductular reaction. Scale bars = 100 μm.
Fig 9.
The relationship between renal impairment and liver dysfunction in feed-withdrawal analyses.
(A) Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), and total bilirubin (T-Bil). (B) Serum levels of plasma creatinine, urine levels of urinary neutrophil gelatinase–associated lipocalin (NGAL), and urinary albumin/creatinine ratio. (C) Proximal tubular cells with injured brush border microvilli, and counts of autophagosomes in control (white bar), 3 days of DDC-fed (black bar), and recovery 7- or 28-day mice (3 days of DDC feeding followed by an additional 7 or 28 days of standard diet, gray bar). Data are presented as means ± SEM. *P < 0.05 compared with the control mice. #P < 0.05 compared with three days of DDC-fed mice; NS, not significant. Control mice samples in creatinine, NGAL, and brush border injury, and control and 3 days of DDC-fed mice samples in T-Bil, urinary albumin/creatinine ratio, and autophagosomes were reproduced from Figs 1, 3, 5 and 8, respectively.
Fig 10.
Proposed pathophysiology of renal impairment in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced chronic liver disease (CLD) model mice.
DDC feeding leads to renal impairment with sublethal tubular cell injury. The tubules show: 1) the mitochondrial injuries and consequent oxidative stress with the generation of reactive oxygen species (ROS). 2) The injury is prevented from progressing to cell death through two defense mechanisms: autophagy and single-cell extrusion with regeneration. 3) The kidney remains in a state of sublethal injury with loss of brush border and mitochondrial injury (early phase). However, 4) activation and proliferation of myofibroblasts appear after long-term feeding (late phase), leading to an increased production of collagen fibers and progression to chronic kidney disease (CKD).