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Distribution study of peplomycin in rat kidney revealed by immunocytochemistry using monoclonal antibodies

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Abstract

Peplomycin (PEP), an anti-tumor antibiotic related structurally to bleomycin, is widely used, especially for squamous cell carcinoma but shows renal toxicity. We prepared monoclonal antibodies (mAbs) against N-(γ-maleimidobutyryloxy)succinimide-conjugated PEP. The mAbs were monospecific for PEP, but did not react with bleomycin and other anticancer antibiotics. The mAbs enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of PEP in the rat kidney. Two hours after a single i.v. administration of PEP, ICC revealed immunostaining for PEP in irregularly shaped cytoplasmic granules of the proximal tubules in which the microvilli were also stained. Also, staining occurred in the distal tubules and collecting ducts, in both of which we observed scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and cytoplasm reacted strongly with the antibody. Twenty-four hours after injection, PEP in the proximal tubules completely vanished, but yet significant amounts of PEP remained in both the distal tubules and collecting ducts. Distribution patterns of PEP in cells of the kidneys resembled, in some ways, those of our recent ICC studies for an organic cation aminoglycoside antibiotic gentamicin. This ICC suggests that PEP taken up in the proximal tubule cells is localized in the lysosomes, and organic cation transporters and bleomycin hydrolase might be involved in entrance and/or disappearance of PEP in this cell type. Furthermore, the distal tubules and collecting ducts may be the sites readily affected by some chemotherapeutic agents.

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Abbreviations

PEP:

Peplomycin

BLM:

Bleomycin

DM:

Daunomycin

GM:

Gentamicin

ICC:

Immunocytochemistry

BSA:

Bovine serum albumin

APEP mAbs:

Anti-peplomycin monoclonal antibodies

HRP:

Horseradish peroxidase

PBS:

Phosphate-buffered saline

RT:

Room temperature

GA:

Glutaraldehyde

ELISA:

Enzyme-linked immunosorbent assay

GMBS:

N-(γ-Maleimidobutyryloxy)succinimide

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Acknowledgments

We are grateful to Y Teruya, N Kiriyama, M Takauye, Y Yamashita, S Ishii, and T Kawazoye for technical assistance throughout this study.

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Authors and Affiliations

  1. Department of Applied Life Science, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto, 860-0082, Japan

    Kunio Fujiwara & Masashi Shin

  2. Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark

    David M. Hougaard

  3. Department of Pharmacy, Taku City Hospital, 1771-4 Taku-cho, Taku-Shi, Saga, 846-0031, Japan

    Tetsuya Saita

Authors
  1. Kunio Fujiwara
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  2. Masashi Shin
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  3. David M. Hougaard
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  4. Tetsuya Saita
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Corresponding author

Correspondence to Kunio Fujiwara.

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418_2010_768_MOESM1_ESM.jpg

Fig. 1a-d. Immunostaining for PEP in the kidneys of rats 2 h (a, b, d) or 24 h (c) after PEP injection in a single intravenous dose (5 mg/kg of body weight). In ICC, enzyme digestion of section specimens was carried out with 0.004% protease at 30°C for 2 h (a, b, d) or 15 min (c). (a) Lower magnification of the renal cortex. Note that immunostaining pattern was essentially the same as that of Fig. 5a. (b) Higher magnification of the S1 and S2 segment of the proximal tubule cells. Note that immunostaining pattern was essentially the same as that of Fig. 4c. (c) Note staining occurs in the distal tubule cells in which some swollen, necrotic-like cells (arrows) are heavily stained, but not at all in the proximal tubule cells. This pattern was essentially the same as that of Fig. 4e. (d) The staining (b) was completely abolished by absorption of APEP 51-35 with 10 µg/ml of PEP. G, glomerulus; P, proximal tubule; D, distal tubule. Bars = 100 µm (a); 20 µm (b, c, d) (JPEG 1607 kb)

418_2010_768_MOESM2_ESM.jpg

Fig. 2a-c. Immunostaining for GM in the kidneys of rats 24 h (a, b) or 12 h (c) after GM injection in a single intravenous dose (16 mg/kg of body weight). In ICC, enzyme digestion of section specimens was carried out with 0.004% protease at 30°C for 2 h (a, b) or 15 min (c). (a) Lower magnification of the renal cortex. Immunostaining is strong in the S1 and S2 segments of the proximal tubules and weak in the S3 segment (medullary portions of the straight proximal tubule). (b) Higher magnification of the renal cortex. Note strong immunostaining of cytoplasmic granules in proximal tubule cells and weak staining in the nuclei of the distal tubule cells. (c) The nuclei as well as the cytoplasm of cells of the distal convoluted tubules are immunostained for GM. Note no staining cells (arrowheads) next to the heavily immunostained cells (arrows). The proximal tubule cells are GM-negative under this ICC conditions. G, glomerulus; P, proximal tubule; D, distal tubule. Bars = 100 µm (a); 20 µm (b, c) (JPEG 1349 kb)

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Fujiwara, K., Shin, M., Hougaard, D.M. et al. Distribution study of peplomycin in rat kidney revealed by immunocytochemistry using monoclonal antibodies. Histochem Cell Biol 135, 93–101 (2011). https://doi.org/10.1007/s00418-010-0768-9

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