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Phlda3, a urine-detectable protein, causes p53 accumulation in renal tubular cells injured by cisplatin

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Abstract

Measurable indicators of renal injury are required for the assessment of kidney function after toxicant challenge. In our previous study, pleckstrin homology-like domain, family A, member 3 (Phlda3) was a most greatly up-regulated molecule downstream from p53, culminating with kidney tubular injury. This study investigated the positive feedforward effect of Phlda3 on p53 in an effort to explain the largest increase of Phlda3 in injured tubules and the potential of its urine excretion. qRT-PCR assays confirmed a rapid and substantial increase in Phlda3 messenger RNA (mRNA) in the kidney cortex of mice treated with a single dose of cisplatin. Cisplatin overexpression of Phlda3 was verified by gene set analyses of three different microarray databases. In the immunohistochemistry, Phlda3 staining intensities were augmented in the tubules as kidney injury worsened. Moreover, the urinary content of Phlda3 was increased after cisplatin treatment, as were those of other kidney injury markers (Kim-1 and Timp-1). By contrast, cisplatin failed to increase Phlda3 mRNA in the liver despite hepatocyte necrosis and ensuing increases in serum transaminase activities. In NRK52E tubular cells, siRNA knockdown of Phlda3 enhanced the ability of cisplatin to increase p-Mdm2 presumably via Akt, enforcing the interaction between Mdm2 and p53. Consistently, a deficiency in Phlda3 abrogated p53 increase by cisplatin, indicating that Phlda3 promotes p53 accumulation. Phlda3 overexpression had the opposite effect. In addition, treatment with cyclosporine A or CdCl2, other nephrotoxicants, increased Phlda3 mRNA and protein levels in NRK52E cells, as did cisplatin treatment. Overall, Phlda3 may cause p53 accumulation through a feedforward pathway, facilitating tubular injury and its urine excretion.

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Abbreviations

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

BUN:

Blood urea nitrogen

Kim-1:

Kidney injury molecule 1

NAG:

N-acetyl-beta-d-glucosaminidase

Phlda3:

Pleckstrin homology-like domain

family A:

Member 3

qRT-PCR:

Quantitative real-time polymerase chain reaction

SCr:

Serum creatinine

Timp-1:

TIMP metallopeptidase inhibitor 1

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Acknowledgments

This work was supported by a grant [10182KFDA992] from Korea Food & Drug Administration in 2010–2012. The authors wish to thank Dr. Se Jin Hwang in the College of Medicine, Hanyang University, for histological evaluation.

Conflict of interest

The authors declare that they have no conflict of interest.

Author contributions

The overall study was conceived and designed by CGL and SGK; CGL analyzed data; AM and HSK contributed reagents or analysis tools; CGL and SGK wrote the paper.

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Authors and Affiliations

  1. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-742, South Korea

    Chan Gyu Lee & Sang Geon Kim

  2. School of Pharmacy, Sungkyunkwan University, Suwon, South Korea

    Yoon Jong Kang & Hyung Sik Kim

  3. College of Pharmacy, Duksung Women’s University, Seoul, South Korea

    Aree Moon

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  1. Chan Gyu Lee
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  2. Yoon Jong Kang
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  3. Hyung Sik Kim
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  4. Aree Moon
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  5. Sang Geon Kim
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Correspondence to Sang Geon Kim.

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Supplementary Figure 1

Immunoprecipitation and immunoblotting analysis for Mdm2 and p53. NRK52E cells were treated with 30 μM cisplatin for the indicated times. (JPEG 55 kb)

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Lee, C.G., Kang, Y.J., Kim, H.S. et al. Phlda3, a urine-detectable protein, causes p53 accumulation in renal tubular cells injured by cisplatin. Cell Biol Toxicol 31, 121–130 (2015). https://doi.org/10.1007/s10565-015-9299-4

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  • DOI: https://doi.org/10.1007/s10565-015-9299-4

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