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Establishment of renal proximal tubule cell lines derived from the kidney of p53 knockout mice.

ABSTRACT: The human cell line HK-2 is most commonly used as a model of renal proximal tubular epithelial cells (PTECs) for various studies despite the absence or low expression of transporters characteristic of parental PTECs. In an effort to develop reliable PTEC models, several human cell lines have been newly established over the last decade. In contrast, reliable mouse PTEC models are still unavailable. In this study, we established immortalized renal cortex tubule cell lines derived from p53 knockout mice and evaluated various PTEC characteristics toward the development of reliable mouse PTEC models. Here, we focus on MuRTE61, one of 13 newly established clonal cell lines. Albumin uptake in MuRTE61 cells was verified by incubation with fluorescent dye-labeled albumin. RT-PCR confirmed the expression of efflux transporter genes characteristic of PTECs in the MuRTE61 cells. MuRTE61 cells exhibited high sensitivity to treatment with cisplatin, a nephrotoxic agent, accompanied by upregulated expression of the uptake transporter Slc22a2 gene. Furthermore, MuRTE61 cells consistently formed spheroids with a lumen and apicobasal polarity in three-dimensional Matrigel cultures. Apical brush border microvilli were also observed in the spheroids by transmission electron microscopy. These data validate that MuRTE61 can be characterized as a reliable mouse PTEC line. In future, detailed analysis of reliable mouse and human PTEC lines will provide an accurate extrapolation of results of experiments using mice and humans, and may help resolve apparent inconsistencies between mouse and human nephrotoxicity.

SUBMITTER: Sasaki H

PROVIDER: S-EPMC6368522 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Establishment of renal proximal tubule cell lines derived from the kidney of p53 knockout mice.

Sasaki Hayato H Sugiyama Makoto M Sasaki Nobuya N

Cytotechnology 20190102 1

The human cell line HK-2 is most commonly used as a model of renal proximal tubular epithelial cells (PTECs) for various studies despite the absence or low expression of transporters characteristic of parental PTECs. In an effort to develop reliable PTEC models, several human cell lines have been newly established over the last decade. In contrast, reliable mouse PTEC models are still unavailable. In this study, we established immortalized renal cortex tubule cell lines derived from p53 knockout ...[more]

PMID: 30603921

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Project description:The multiligand receptors megalin (Lrp2) and cubilin (Cubn) and their endocytic adaptor protein Dab2 (Dab2) play essential roles in maintaining the integrity of the apical endocytic pathway of proximal tubule (PT) cells and have complex and poorly understood roles in the development of chronic kidney disease. Here, we used RNA-sequencing and CRISPR/Cas9 knockout (KO) technology in a well-differentiated cell culture model to identify PT-specific transcriptional changes that are directly consequent to the loss of megalin, cubilin, or Dab2 expression. KO of Lrp2 had the greatest transcriptional effect, and nearly all genes whose expression was affected in Cubn KO and Dab2 KO cells were also changed in Lrp2 KO cells. Pathway analysis and more granular inspection of the altered gene profiles suggested changes in pathways with immunomodulatory functions that might trigger the pathological changes observed in KO mice and patients with Donnai-Barrow syndrome. In addition, differences in transcription patterns between Lrp2 and Dab2 KO cells suggested the possibility that altered spatial signaling by aberrantly localized receptors contributes to transcriptional changes upon the disruption of PT endocytic function. A reduction in transcripts encoding sodium-glucose cotransporter isoform 2 was confirmed in Lrp2 KO mouse kidney lysates by quantitative PCR analysis. Our results highlight the role of megalin as a master regulator and coordinator of ion transport, metabolism, and endocytosis in the PT. Compared with the studies in animal models, this approach provides a means to identify PT-specific transcriptional changes that are directly consequent to the loss of these target genes.NEW & NOTEWORTHY Megalin and cubilin receptors together with their adaptor protein Dab2 represent major components of the endocytic machinery responsible for efficient uptake of filtered proteins by the proximal tubule (PT). Dab2 and megalin expression have been implicated as both positive and negative modulators of kidney disease. We used RNA sequencing to knock out CRISPR/Cas9 cubilin, megalin, and Dab2 in highly differentiated PT cells to identify PT-specific changes that are directly consequent to knockout of each component.

Project description:Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis.

Project description:BackgroundSustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown.MethodsWe studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell-specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell-specific deletion of amphiregulin's releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice.ResultsYes-associated protein 1 (YAP1)-dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell-specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell-specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis.ConclusionsOur results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.

Dataset Information

Establishment of renal proximal tubule cell lines derived from the kidney of p53 knockout mice.

Publications

Establishment of renal proximal tubule cell lines derived from the kidney of p53 knockout mice.

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