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

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.

Immunocytochemistry for Vancomycin Using a Monoclonal Antibody That Reveals Accumulation of the Drug in Rat Kidney and Liver

“…The present IEM study first demonstrated that uptake of VM occurred exclusively in the cytoplasm of the principal cells but scarcely or only very slightly in the intercalated cells, the two cell types which are known to organize both collecting duct cells and convoluted distal tubule cells (30). These phenomena in drug uptake seem to generally occur, as a similar observation has been made with GM or peplomycin in our previous ICCs (18,20). However, differences, such as that most of the cells seemingly affected by VM disappeared by 24 h after administration, most likely returning back to the normal size of the cells with decreased immunostaining, while those affected by GM or by peplomycin remained morphologically affected during a few experimental days, were also seen (18,20).…”

“…Nakamura et al (33) have suggested the organic cation transporter (OCT) and P glycoprotein for VM at the basolateral and brush border membranes, respectively, of the renal tubular epithelium, both of which mediate transport of the drug crossing the epithelium in the direction of the lumen side from the blood side. Furthermore, we made observations similar to those in our previous ICCs for AMPC and peplomycin (19)(20)(21); small bodies which were extruded into the tubular lumen side from the epithelial tubular cells and which contained VM occurred. Both immunoelectron microscopy and conventional electron microscopy revealed that most of the small bodies were the fragments of the tubular cell cytoplasm or sometimes those including the nuclei but not protein droplets (data not shown).…”

“…These phenomena in drug uptake seem to generally occur, as a similar observation has been made with GM or peplomycin in our previous ICCs (18,20). However, differences, such as that most of the cells seemingly affected by VM disappeared by 24 h after administration, most likely returning back to the normal size of the cells with decreased immunostaining, while those affected by GM or by peplomycin remained morphologically affected during a few experimental days, were also seen (18,20). This may mean that VM might affect the cells less strongly than GM or peplomycin at the therapeutic dose of the drug administered in the present study.…”

“…The ICC method was carried out essentially according to our previous methods (19)(20)(21). The postfixed specimens of the liver and kidneys were subsequently embedded in paraffin in a routine way.…”

“…Controls for the immunocytochemical method included conventional staining (second-level) controls as well as a peplomycin MAb (20). Absorption controls used conjugates of VM-GMBS-BSA at a concentration of 30 g/ml and the free compounds VM, amoxicillin (AMPC), and GM at concentrations ranging between 30 g and 100 g/ml.…”