Immunocytochemistry for amoxicillin and its use for studying uptake of the drug in the intestine, liver, and kidney of rats

Abstract

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 cross-reacted 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.

FIG. 1.

ELISA measurements of the binding of serially diluted anti-AMPC serum to AMPC-GA-BSA (closed circles) or BSA (open squares).

FIG. 2.

ELISA measurements showing competition between free and conjugated AMPC and AMPC-GA-BSA coated to solid phase for binding to anti-AMPC serum. The curves show the amount (percentage) of bound enzyme activity (B) for various doses of AMPC-GA-BSA (closed circles), AMPC-GA (closed squares), amoxicillin (closed diamonds), or ampicillin (closed triangles) as a ratio of that bound using the HRP-labeled second antibody alone (B₀). The concentrations of AMPC-GA-BSA and AMPC-GA were calculated by assuming that one molecule of AMPC was incorporated into a BSA molecule and that AMPC, which was used for conjugation with GA, completely reacted, respectively (18).

FIG. 3.

Reactivity of anti-AMPC-serum determined from its immunoreactivity in the binding ELISA. Activated wells prepared for the binding ELISA were incubated with various concentrations of amoxicillin (closed circles), ampicillin (open squares), cephalexin (closed triangles), or kanamycin (closed squares). The wells were reacted with NaBH₄ and then with anti-AMPC-serum (1:4,000), followed by HRP-labeled goat anti-mouse IgG (whole; 1:2,000). Note that anti-AMPC-serum is almost equally immunoreactive with amoxicillin and ampicillin.

FIG. 4.

Immunostaining for AMPC in small intestine of rats administered AMPC. Rats were orally administered AMPC at 60 mg/kg and then killed 3 h later. The AMPC ICC method was carried out following digestion of sections with protease at 0.001% at 30°C for 1 h. (a) Jejunum (lower magnification). Strong staining occurred exclusively in the absorptive epithelial cells and not in the goblet cells (arrowheads), in the crypt cells, or in other cell types of the intestine. (b) Jejunum (higher magnification). Strong staining occurred in the microvilli (arrows), in the cytoplasm, and in the nuclei (open arrowheads) of the absorptive epithelial cells but not in the goblet cells (closed arrowheads). Very weak staining occurred in unidentified cells in the lamina propria mucosae. (c) Duodenum (lower magnification). Staining was pronounced in the microvilli (arrows) of the absorptive cells, in which intracellular staining, however, was very weak, with the exception that the top villus cells (arrowheads) were strongly stained. (d) Duodenum (lower magnification). The staining was completely abolished by absorption of the anti-AMPC serum with AMPC-GA-BSA (30 μg/ml). Bars = 100 μm (a and d) and 20 μm (b and c).

FIG. 5.

Immunostaining for AMPC in liver of rats administered AMPC. (a) Lower magnification. The ICC method without the protease digestion step in the protocol showed that staining occurred in the cytoplasm of hepatocytes and in the luminal surface of the bile capillaries (arrows), intercalated portion (open arrowheads), and interlobular bile ducts (closed arrowheads). (b) Higher magnification. Sections were digested with 0.006% protease for 2 h prior to immunoreaction. Note the strong staining in the small spots (arrows) lining the bile capillaries. (c) Staining was completely abolished by absorption of the anti-AMPC serum with AMPC-GA-BSA (30 μg/ml). Bars = 20 μm (a and b) and 50 μm (c).

FIG. 6.

Immunostaining for AMPC in the kidneys of rats administered AMPC. Rats were orally administered AMPC at 15 mg/kg (g) or 60 mg/kg (a to f and h) and then killed 3 h later (a to h). The AMPC ICC method was carried out without protease digestion (a, b, and g) or following digestion of sections with protease at 0.006% at 30°C for 1 h (c, f, and h) or 2 h (d and e). (a) Wide ranges of immunostaining of AMPC occur in the nuclei, in the cytoplasm, and in the microvilli of the S3 segment cells of the proximal tubules (S3). Also, strong staining was observed in some, but not all, cells of the collecting duct cells (C). The S3 segment was identifiable by the presence of periodic acid-Schiff-positive brush borders. (b) Staining occurred moderately in some, but not all, cells of the distal convolution tubules (D) and strongly in some cells of the collecting duct cells (C). Note that some of heavily stained cells are swollen (arrows) and that adjacent cells are virtually unstained (arrowheads). G, glomeruli. (c) Many small bodies (open arrowheads) heavily stained with antibody occurred in the tubular lumens of the S3 segment. (d) The cytoplasm and the microvilli of the S1 and S2 segment cells (S1,2) were weakly stained, although the microvilli were somewhat disrupted and peeled off. Note the staining in small granules (arrowheads), which were located at the bottom of the microvilli of the S1 and S2 segment cells and which formed in a line encircling the tubule. (e) Moderately stained granules of various sizes (arrowheads) and very weakly stained microvilli (arrows). (f) A swollen cell strongly reacted with the antibody in the collecting duct cells in the medulla of the kidney. (g) Some of the convolution distal tubule cells were weakly stained. (h) The staining was completely abolished by absorption of the anti-AMPC serum with AMPC-GA-BSA (30 μg/ml). Bars = 20 μm (a, c, and d to h) and 50 μm (b).

FIG. 7.

Immunostaining for AMPC in the kidneys of rats administered AMPC. Rats were orally administered AMPC at 60 mg/kg (a and c) or 15 mg/kg (b) and then killed 3 h (a and b) or 24 h (c) later. The AMPC ICC method was carried out following digestion of sections with protease at 0.006% at 30°C for 1 h (b) or 2 h (a and c). (a to c) Renal cortex (lower magnification). (a and b) The staining pattern was characteristic of the fact that strong staining occurred extensively in the S3 segment cells of the proximal tubules. In addition, strong staining occurred intermittently in the cells of the collecting ducts. The staining intensity in the collecting duct cells was much weaker in panel b than in panel a. (c) Except for weak staining in the S3 segment cells, almost no staining of the proximal tubules occurred. G, glomeruli; C, collecting ducts. Bars = 100 μm.