The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive ...kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and super-enhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.
Chronic kidney disease (CKD) remains one of the leading causes of death in the developed world, and acute kidney injury (AKI) is now recognized as a major risk factor in its development. ...Understanding the factors leading to CKD after acute injury are limited by current animal models of AKI, which concurrently target various kidney cell types including epithelial, endothelial, and inflammatory cells. Here, we developed a mouse model of kidney injury using the Six2-Cre-LoxP technology to selectively activate expression of the simian diphtheria toxin (DT) receptor in renal epithelia derived from the metanephric mesenchyme. By adjusting the timing and dose of DT, a highly selective model of tubular injury was created to define the acute and chronic consequences of isolated epithelial injury. The DT-induced sublethal tubular epithelial injury was confined to the S1 and S2 segments of the proximal tubule rather than being widespread in the metanephric mesenchyme–derived epithelial lineage. Acute injury was promptly followed by inflammatory cell infiltration and robust tubular cell proliferation, leading to complete recovery after a single toxin insult. In striking contrast, three insults to renal epithelial cells at 1-week intervals resulted in maladaptive repair with interstitial capillary loss, fibrosis, and glomerulosclerosis, which was highly correlated with the degree of interstitial fibrosis. Thus, selective epithelial injury can drive the formation of interstitial fibrosis, capillary rarefaction, and potentially glomerulosclerosis, substantiating a direct role for damaged tubule epithelium in the pathogenesis of CKD.
Dominantly inherited missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, but its normal physiological function remains unclear. We ...previously reported that loss of LRRK2 causes impairment of protein degradation pathways as well as increases of apoptotic cell death and inflammatory responses in the kidney of aged mice.
Our analysis of LRRK2-/- kidneys at multiple ages, such as 1, 4, 7, and 20 months, revealed unique age-dependent development of a variety of molecular, cellular, and ultrastructural changes. Gross morphological abnormalities of the kidney, including altered size, weight, texture, and color, are evident in LRRK2-/- mice at 3-4 months of age, along with increased accumulation of autofluorescent granules in proximal renal tubules. The ratio of kidney/body weight in LRRK2-/- mice is increased at 1, 4, and 7 months of age (-10% at 1 month, and -20% at 4 and 7 months), whereas the ratio is drastically decreased at 20 months of age (-50%). While kidney filtration function evaluated by levels of blood urea nitrogen and serum creatinine is not significantly affected in LRRK2-/- mice at 12-14 months of age, expression of kidney injury molecule-1, a sensitive and specific biomarker for epithelial cell injury of proximal renal tubules, is up-regulated (-10-fold). Surprisingly, loss of LRRK2 causes age-dependent bi-phasic alterations of the autophagic activity in LRRK2-/- kidneys, which is unchanged at 1 month of age, enhanced at 7 months but reduced at 20 months, as evidenced by corresponding changes in the levels of LC3-I/II, a reliable autophagy marker, and p62, an autophagy substrate. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2-/- kidneys at 7 months of age but increased at 20 months. Interestingly, the age-dependent bi-phasic alterations in autophagic activity in LRRK2-/- kidneys is accompanied by increased levels of lysosomal proteins and proteases at 1, 7, and 20 months of age as well as progressive accumulation of autolysosomes and lipofuscin granules at 4, 7-10, and 20 months of age.
LRRK2 is important for the dynamic regulation of autophagy function in vivo.
Acute kidney injury predisposes patients to the development of both chronic kidney disease and end-stage renal failure, but the molecular details underlying this important clinical association remain ...obscure. We report that kidney injury molecule-1 (KIM-1), an epithelial phosphatidylserine receptor expressed transiently after acute injury and chronically in fibrotic renal disease, promotes kidney fibrosis. Conditional expression of KIM-1 in renal epithelial cells (Kim1(RECtg)) in the absence of an injury stimulus resulted in focal epithelial vacuolization at birth, but otherwise normal tubule histology and kidney function. By 4 weeks of age, Kim1(RECtg) mice developed spontaneous and progressive interstitial kidney inflammation with fibrosis, leading to renal failure with anemia, proteinuria, hyperphosphatemia, hypertension, cardiac hypertrophy, and death, analogous to progressive kidney disease in humans. Kim1(RECtg) kidneys had elevated expression of proinflammatory monocyte chemotactic protein-1 (MCP-1) at early time points. Heterologous expression of KIM-1 in an immortalized proximal tubule cell line triggered MCP-1 secretion and increased MCP-1-dependent macrophage chemotaxis. In mice expressing a mutant, truncated KIM-1 polypeptide, experimental kidney fibrosis was ameliorated with reduced levels of MCP-1, consistent with a profibrotic role for native KIM-1. Thus, sustained KIM-1 expression promotes kidney fibrosis and provides a link between acute and recurrent injury with progressive chronic kidney disease.
Human reconstructed kidney models Kishi, Seiji; Matsumoto, Takuya; Ichimura, Takaharu ...
In vitro cellular & developmental biology. Animal,
02/2021, Letnik:
57, Številka:
2
Journal Article
Recenzirano
The human kidney, which consists of up to 2 million nephrons, is critical for blood filtration, electrolyte balance, pH regulation, and fluid balance in the body. Animal experiments, particularly ...mice and rats, combined with advances in genetically modified technology have been the primary mechanism to study kidney injury in recent years. Mouse or rat kidneys, however, differ substantially from human kidneys at the anatomical, histological, and molecular levels. These differences combined with increased regulatory hurdles and shifting attitudes towards animal testing by non-specialists have led scientists to develop new and more relevant models of kidney injury. Although in vitro tissue culture studies are a valuable tool to study kidney injury and have yielded a great deal of insight, they are not a perfect model. Perhaps, the biggest limitation of tissue culture is that it cannot replicate the complex architecture, consisting of multiple cell types, of the kidney, and the interplay between these cells. Recent studies have found that pluripotent stem cells (PSCs), which are capable of differentiation into any cell type, can be used to generate kidney organoids. Organoids recapitulate the multicellular relationships and microenvironments of complex organs like kidney. Kidney organoids have been used to successfully model nephrotoxin-induced tubular and glomerular disease as well as complex diseases such as chronic kidney disease (CKD), which involves multiple cell types. In combination with genetic engineering techniques, such as CRISPR-Cas9, genetic diseases of the kidney can be reproduced in organoids. Thus, organoid models have the potential to predict drug toxicity and enhance drug discovery for human disease more accurately than animal models.
Phagocytosis of apoptotic cells by both professional and semi‐professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM‐1/TIM‐1 is a ...phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM‐1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi‐membrane organelles. KIM‐1‐mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM‐1‐mediated phagocytosis leads to pro‐tolerogenic antigen presentation, which suppresses CD4 T‐cell proliferation and increases the percentage of regulatory T cells in an autophagy gene‐dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.
Synopsis
This study reveals how KIM‐1/TIM‐1‐mediated phagocytosis down‐modulates inflammation through ATG5 and ULK1‐dependent autophagic processing of phagocytosed material to MHC I and MHC II.
Phagocytosis of apoptotic cells by KIM‐1, a non‐myeloid phagocytosis receptor, induces LC3 lipidation of resulting phagosomes.
Autophagy protein expression, ATG5, ULK1 and Beclin1, are required for LC3 targeting to phagosomes and efficient phagosome acidification and maturation.
KIM‐1 signaling via the PI3 kinase subunit p85 promotes LC3 lipidation and accumulation on phagosomes.
Phagocytosed material processed through autophagy is presented to MHC I and MHC II.
Enhanced antigen presentation resulting from autophagic processing results in a pro‐tolerogenic milieu, reducing the percentage of CD4 effector T cells while increasing the percentage of regulatory T cells.
Phosphatidylserine receptor KIM‐1/TIM‐1‐mediated phagocytosis and autophagic processing of apoptotic cells promote antigen presentation in epithelial cells to modulate inflammation.
Sensitive and specific biomarkers are needed to detect early kidney injury. The objective of the present work was to develop a sensitive quantitative urinary test to identify renal injury in the ...rodent to facilitate early assessment of pathophysiological influences and drug toxicity. Two mouse monoclonal antibodies were made against the purified ectodomain of kidney injury molecule-1 (Kim-1), and these were used to construct a sandwich Kim-1 ELISA. The assay range of this ELISA was 50 pg/ml to 5 ng/ml, with inter- and intra-assay variability of <10%. Urine samples were collected from rats treated with one of three doses of cisplatin (2.5, 5, or 7.5 mg/kg). At one day after each of the doses, there was an approximately three- to fivefold increase in the urine Kim-1 ectodomain, whereas other routinely used biomarkers measured in this study plasma creatinine, blood urea nitrogen (BUN), urinary N-acetyl-beta-glucosaminidase (NAG), glycosuria, proteinuria lacked the sensitivity to show any sign of renal damage at this time point. When rats were subjected to increasing periods (10, 20, 30, or 45 min) of bilateral ischemia, there was an increasing amount of urinary Kim-1 detected. After only 10 min of bilateral ischemia, Kim-1 levels on day 1 were 10-fold higher (5 ng/ml) than control levels, whereas plasma creatinine and BUN were not increased and there was no glycosuria, increased proteinuria, or increased urinary NAG levels. Thus urinary Kim-1 levels serve as a noninvasive, rapid, sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in pathophysiological studies and in preclinical drug development studies for risk-benefit profiling of pharmaceutical agents.
Cancer patients with malignant involvement of tumor-draining lymph nodes (TDLNs) and distant metastases have the poorest prognosis. A drug delivery platform that targets the primary tumor, TDLNs, and ...metastatic niches simultaneously, remains to be developed. Here, we generated a novel monoclonal antibody (MHA112) against peripheral node addressin (PNAd), a family of glycoproteins expressed on high endothelial venules (HEVs), which are present constitutively in the lymph nodes (LNs) and formed ectopically in the tumor stroma. MHA112 was endocytosed by PNAd-expressing cells, where it passed through the lysosomes. MHA112 conjugated antineoplastic drug Paclitaxel (Taxol) (MHA112-Taxol) delivered Taxol effectively to the HEV-containing tumors, TDLNs, and metastatic lesions. MHA112-Taxol treatment significantly reduced primary tumor size as well as metastatic lesions in a number of mouse and human tumor xenografts tested. These data indicate that human metastatic lesions contain HEVs and provide a platform that permits simultaneous targeted delivery of antineoplastic drugs to the three key sites of primary tumor, TDLNs, and metastases.
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•A novel monoclonal antibody (MHA112) against peripheral node addressin (PNAd) on high endothelial venules (HEVs).•Simultaneous targeting to primary tumor, tumor-draining lymph nodes and metastatic sites by MHA112-antineoplastic drug.•Tumor metastatic lesions contain HEVs.
Purpose
Acute kidney injury (AKI) is a common and serious adverse effect of cisplatin-based chemotherapy. However, traditional markers of kidney function, such as serum creatinine, are suboptimal, ...because they are not sensitive measures of proximal tubular injury. We aimed to determine whether the new urinary biomarkers such as kidney injury molecule-1 (KIM-1), monocyte chemotactic protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL) could detect cisplatin-induced AKI in lung cancer patients in comparison with the conventional urinary proteins such as
N
-acetyl-
β
-
d
-glucosaminidase (NAG) and β2-microglobulin.
Methods
We measured KIM-1, MCP-1, NGAL, NAG, and β2-microglobulin concentrations in urine samples from 11 lung cancer patients, which were collected the day before cisplatin administration and on days 3, 7, and 14. Subsequently, we evaluated these biomarkers by comparing their concentrations in 30 AKI positive (+) and 12 AKI negative (−) samples and performing receiver operating characteristic (ROC) curve analyses.
Results
The urinary levels normalized with urine creatinine of KIM-1 and MCP-1, but not NGAL, NAG, and β2-microglobulin in AKI (+) samples were significantly higher than those in AKI (−) samples. In addition, ROC curve analyses revealed that KIM-1 and MCP-1, but not NGAL, could detect AKI with high accuracy (area under the curve AUC = 0.858, 0.850, and 0.608, respectively). The combination of KIM-1 and MCP-1 outperformed either biomarker alone (AUC = 0.871).
Conclusions
Urinary KIM-1 and MCP-1, either alone or in combination, may represent biomarkers of cisplatin-induced AKI in lung cancer patients.
Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin ...domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1–mutant mouse (Tim-1 Δᵐᵘᶜⁱⁿ) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1 Δᵐᵘᶜⁱⁿ mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-γ and elevated serum levels of Ig and autoantibodies. However, Tim-1 Δᵐᵘᶜⁱⁿ mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1 Δᵐᵘᶜⁱⁿlpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.
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