Immunocytochemistry for Drugs Containing an Aliphatic Primary Amino Group in the Molecule, Anticancer Antibiotic Daunomycin as a Model

Matsunaga

et al. 2009

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Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin, kanamycin, or amikacin. The antiserum also detected glutaraldehyde-fixed GM, and this enabled us to develop an immunocytochemical method for detecting the uptake of GM in rat kidney. Twelve hours after a single intravenous administration of GM, immunocytochemistry revealed that GM accumulated in the S1, S2, and S3 segments of the proximal tubules, as well as in the distal tubules and collecting ducts. By 12 h after injection, the drug was detected in cytoplasmic granules of the proximal tubule cells. However, early (1 h) after injection, drug accumulation was detected in the microvilli of these cells. The distal tubules and collecting ducts contained scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and the cytoplasm reacted strongly with GM. No staining occurred in the kidneys of saline-injected control rats. These results agree with previous studies showing that GM is endocytosed in the proximal tubules and accumulates in lysosomes. Additionally, our results show that GM also accumulates in the distal tubules and collecting ducts. This was achieved by systematically varying the pretreatment conditions-an approach necessary for detecting GM in different subcellular compartments. This approach should be useful for accurately detecting the uptake and toxicity of the antibiotic in different tissues.Gentamicin (GM) is widely used as a bactericidal agent for the treatment of severe infections with gram-negative bacteria, such as Pseudomonas aeruginosa. Its antibacterial activity is due to an irreversible inhibition of bacterial protein synthesis. However, clinical use of aminoglycosides, such as GM, is limited by their ototoxicity and nephrotoxicity. The mechanisms behind the nephrotoxity have been investigated extensively, but how these drugs induce cellular malfunction and necrosis remains to be elucidated. Accurate localization of GM in cells and tissues can be expected to contribute to this effort. Previously, subcellular fractionation (27), micropuncture techniques (24), and autoradiography using radioactively labeled aminoglycosides (6, 26) have been used to study the mechanisms of nephrotoxity. Additionally, immunogold labeling methods have been used to study the subcellular distribution of aminoglycosides in proximal tubules of the nephron (1,2, 3,4,20). However, these studies have been limited to ultrastructural analysis of proximal tubule cells. Recently, we have successfully developed immunocytochemical (ICC) procedures for detecting the cellular uptake of other water-diffusible small-molecule drugs, such as daunomycin (DM) (14,15,22,23). These procedures make use of glutaraldehyde (GA)-fixed tissues, which undergo a series of pretreatments to unmask sites of accumulation of the drug...

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confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney

Matsunaga

et al. 2009

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“…ICCs for drugs necessitated a series of processes prior to the immunocytochemical reaction (16,35). Thus, in ICC for VM, we established the optimal conditions for immunostaining for VM in rat kidneys.…”

Section: Discussionmentioning

confidence: 99%

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

Yoshizaki

et al. 2012

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bWe prepared monoclonal antibodies against N-(␥-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

“…Also, it should be valuable for developing drug therapy for infections caused by intracellular parasites, including Staphylococcus aureus, since it is known that they invade host cells, grow, and evade the bactericidal potential of antimicrobial agents (33,35,41). Recently, we have successfully developed immunocytochemical (ICC) procedures for detecting cellular uptake of other water-diffusible small drugs, such as daunomycin (DM) (16,17,36,37,46,47) and gentamicin (GM) (18). These procedures make use of glutaraldehyde (GA)-fixed tissues which undergo a series of pretreatments to unmask the sites of accumulation of the drug.…”

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Immunocytochemistry for Amoxicillin and Its Use for Studying Uptake of the Drug in the Intestine, Liver, and Kidney of Rats

Miyazaki

et al. 2011

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Specific transport systems for penicillins have been recognized, but their in vivo role in the context of other transporters remains unclear. We produced a serum against amoxicillin (anti-AMPC) conjugated to albumin with glutaraldehyde. The antiserum was specific for AMPC and ampicillin (ABPC) but crossreacted weakly with cephalexin. This enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of AMPC in the rat intestine, liver, and kidney. Three hours after a single oral administration of AMPC, the ICC method revealed that AMPC distributed to a high degree in the microvilli, nuclei, and cytoplasm of the absorptive epithelial cells of the intestine. AMPC distributed in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries, and in addition, AMPC adsorption was observed on the luminal surface of the capillaries, intercalated portions, and interlobular bile ducts on the bile flow. Almost no AMPC could be detected 6 h postadministration in either the intestine or the liver. Meanwhile, in the kidney, AMPC persisted until 12 h postadministration to a high degree in the proximal tubules, especially in the S3 segment cells in the tubular lumen, in which numerous small bodies that strongly reacted with the antibody were observed. All these sites of AMPC accumulation correspond well to specific sites where certain transporter systems for penicillins occur, suggesting that AMPC is actually and actively absorbed, eliminated, or excreted at these sites, possibly through such certain penicillin transporters.Amoxicillin (AMPC) is a moderate-spectrum, bacteriolytic, ␤-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms, acting by inhibiting the synthesis of the bacterial cell wall. In chemotherapy, the ability of a drug to reach its site of action for a desired duration is dependent on absorption, distribution, metabolism, and excretion, all of which are intimately related to the transport mechanisms in the barrier epithelia (56). Actually, pharmacotherapeutic efficacy and toxicity are governed in vivo by a multitude of pharmacodynamic and pharmacokinetic factors. Recently, a variety of transporters for penicillins have been demonstrated at the molecular level, especially in the kidney and liver, in which numerous potentially toxic xenobiotics and drugs are eliminated (23). It has been postulated that the following may be involved in the transport of penicillins: in the small intestine, the proton-coupled oligopeptide transporter PEPT1 (1); in the liver, the organic anion transporter (OAT) (5, 22, 44, 54), multidrug resistance-associated protein (Mrp2) (6,26,39,42), and sodium-dependent phosphate transport protein (NPT1) (8, 21, 57); and in the kidney, the rat multispecific organic anion transporter 1 (rOAT1), rOAT2, and rOAT3 (2, 22, 23, 51, 55), Mrp2 (43), and H ϩ /peptide cotransporters (PEPT1 and PEPT2) (23-25, 38, 45, 49, 53), etc. The interaction of such transporters with ␤-lactam antibiotics has been stu...