Renal impairment with sublethal tubular cell injury in a chronic liver disease mouse model

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet-fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms of sublethal tubular cell injury

DDC誘導性胆汁うっ滞症の進展期および回復期においてクッパー細胞は中心的な役割を果たす

京都大学0048新制・課程博士博士(医学)甲第21617号医博第4423号新制||医||1033(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 羽賀 博典, 教授 川口 義弥, 教授 妹尾 浩学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Pivotal roles of Kupffer cells in the progression and regression of DDC-induced chronic cholangiopathy

Kupffer cells (KCs) are key players in maintaining tissue homeostasis and are involved in various liver diseases. However, the roles of KCs in the pathogenesis of cholangiopathy are largely unknown. We aimed to investigate the precise roles of KCs in both the progression and regression phases of the 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC)-induced cholangiopathy model. In the early phase of DDC-induced cholangiopathy, the number of KCs significantly increased over time. Moreover, KCs were associated with abnormal phenotypic changes in other liver cells, such as hepatocytes, biliary epithelial cells, liver sinusoidal endothelial cells, and hepatic stellate cells. In contrast, KC depletion by clodronate administration suppressed the progression of the disease, and maintained the phenotypes of other cells. In the regression phase, the numbers of KCs significantly decreased, and the cells redifferentiated to their quiescent state. In contrast, KC depletion delayed the recovery of cells by maintaining other liver cells in an active state. These findings suggest that KCs play detrimental roles in the progression phase; however, they are beneficial in the regression phase by mediating interactions between other liver cells. Our data provide new insights into the roles of KCs in the pathogenesis of cholangiopathy

The relationship between renal impairment and liver dysfunction in feed-withdrawal analyses.

<p>(<b>A</b>) Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), and total bilirubin (T-Bil). (<b>B</b>) Serum levels of plasma creatinine, urine levels of urinary neutrophil gelatinase–associated lipocalin (NGAL), and urinary albumin/creatinine ratio. (<b>C</b>) Proximal tubular cells with injured brush border microvilli, and counts of autophagosomes in control (<i>white bar</i>), 3 days of DDC-fed (<i>black bar</i>), and recovery 7- or 28-day mice (3 days of DDC feeding followed by an additional 7 or 28 days of standard diet, <i>gray bar</i>). Data are presented as means ± SEM. *<i>P</i> < 0.05 compared with the control mice. ^#<i>P</i> < 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146871#pone.0146871.g001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146871#pone.0146871.g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146871#pone.0146871.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146871#pone.0146871.g008" target="_blank">8</a>, respectively.</p

Single-cell extrusion with subsequent regeneration in the kidney of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice.

<p>(<b>A</b>) A representative photograph of a single tubular cell extrusion (<i>triangles</i>) from the epithelial layer of proximal tubules at day 3. Scale bar = 10 μm. (<b>B</b>) Counts of mitotic cells per field on proximal tubules of control (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. (<b>C</b>) Counts of Ki-67-positive cells per field on proximal tubules of control (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. Data are presented as means ± SEM. *<i>P</i> < 0.05 compared with control mice. (<b>D</b>) 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.</p

Sublethal tubular cell injuries in the kidney of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice.

<p>(<b>A</b>) 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; <i>red arrowheads</i>, loss of brush border microvilli. Scale bars = 25 μm (top row), 10 μm (bottom row). (<b>B</b>) The proportion of injured cells to total tubular cells is presented as percentage; control (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. (<b>C</b>) 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; <i>asterisks</i>, mitochondria with abnormal features; <i>arrowheads</i>, intact endoplasmic reticula; <i>green</i>, immunofluorescence labeling for KIM-1; <i>red</i>, phalloidin; <i>blue</i>, 4,6-diamidino-2-phenylindole (DAPI). Scale bars = 500 nm (top row), 50 μm (bottom row). (<b>D</b>) 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 (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. Data are presented as means ± SEM. *<i>P</i> < 0.05 compared with control mice.</p

Proposed pathophysiology of renal impairment in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced chronic liver disease (CLD) model mice.

<p>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).</p

Mitochondrial damage and subsequent autophagy in the kidneys of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)–fed mice.

<p>(<b>A</b>) 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; <i>asterisks</i>, autophagosomes. Scale bar = 500 nm. (<b>B</b>) Counts of autophagosomes per 1,000× magnified field; control (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. (<b>C</b>) 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 (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. (<b>D</b>) Representative images of p62 immunostaining in kidneys of control and DDC-fed mice at days 1, 3, and 7; <i>green</i>, immunofluorescence labeling for p62; <i>red</i>, phalloidin; <i>blue</i>, diamidino-2-phenylindole (DAPI). Scale bar = 10 μm. Data are presented as means ± SEM. *<i>P</i> < 0.05 compared with control mice.</p

Sublethal injuries in the kidneys of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice are associated with oxidative stress.

<p>(<b>A</b>) 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 (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. Scale bar = 50 μm. (<b>B</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 (<i>white bar</i>) and DDC-fed mice (<i>black bar</i>) at days 1, 3, and 7. Scale bar = 50 μm. Data are presented as means ± SEM. *<i>P</i> < 0.05 compared with control mice.</p