Uptake of Polymyxin B into Renal Cells

Abstract

Polymyxin B is increasingly used as a treatment of last resort against multidrug-resistant Gram-negative infections. Using a mammalian kidney cell line, we demonstrated that polymyxin B uptake into proximal tubular epithelial cells was saturable and occurred primarily through the apical membrane, suggesting the involvement of transporters in the renal uptake of polymyxin B. Megalin might play a role in the uptake and accumulation of polymyxin B into renal cells.

TEXT

Multidrug-resistant Gram-negative infections are disseminating worldwide (1–3). This has necessitated the use of systemic polymyxin B as a treatment of last resort, despite the risk of nephrotoxicity (4–6). Polymyxin B is a polypeptide antibiotic commercially available as a mixture of several closely related cyclic amphiphilic molecules, of which polymyxin B1 (PB1) is the most abundant component (7). Our group previously studied the nephrotoxicity and renal disposition of polymyxin B in a rat model (8, 9). We showed that polymyxin B had a tendency to persist in the kidneys (8). Using a dose fractionation design, we have also demonstrated the saturation of renal uptake with a clinically relevant dosing exposure (9), which suggested that the renal uptake of polymyxin B was unlikely a passive process. In addition, histological examination showed that renal injury was predominantly confined to the proximal tubular epithelial cells of the renal cortex. Based on these results, we postulated that polymyxin B was taken up primarily by proximal tubular epithelial cells. Collectively, these observations suggest a selective uptake process into renal cells, which can play a prominent role in the nephrotoxic potential of polymyxin B. The objective of this study was to characterize the cell uptake of polymyxin B in vitro. Understanding the mechanism of renal uptake might provide useful insights into minimizing the accumulation of polymyxin B and thus reducing its nephrotoxic potential.

(This study was presented in part at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, CO, 10–13 September 2013 [10].)

Polymyxin B sulfate which met U.S. Pharmacopeia (USP) testing specifications, gentamicin sulfate (USP), and piperacillin sodium powder were purchased from Sigma-Aldrich (St. Louis, MO). A fluorescent polymyxin B derivative, polymyxin B Bodipy FL conjugate (FLPB), was purchased from Life Technologies (Grand Island, NY). Prior to each experiment, a fresh stock of each drug (100 mM, titrated to pH 5 by NaOH) was made with Dulbecco's phosphate-buffered saline (DPBS) with calcium and magnesium (Mediatech, Inc., Manassas, VA). Hanks' balanced salt solution (HBSS) was purchased from Sigma, supplemented with NaHCO₃, glucose, and HEPES, and adjusted to pH 7.4 with NaOH at room temperature. Porcine kidney proximal tubular epithelial (LLC-PK1) cells were purchased from the ATCC (Manassas, VA). The cells were grown in medium 199 (Lonza, Walkersville, MD) supplemented with 3% fetal bovine serum (Thermo Scientific, Rockford, IL), 1% minimal essential medium (MEM) nonessential amino acid solution (Cellgro, Manassas, VA), 100 U/ml penicillin, and 1,000 mg/ml streptomycin solution in a humidified atmosphere of 5% CO₂ at 37°C.

We examined the rate of drug uptake using escalating concentrations of polymyxin B. Briefly, LLC-PK1 monolayers were grown to confluence in 6-well plates. The growth medium was removed, and the cells were washed twice with HBSS. The cells were preincubated with 2 ml of HBSS for 30 min at 37°C and exposed to escalating concentrations of polymyxin B (USP) in HBSS (0.5 to 64 μg/ml) in triplicate experiments for 1 h at 37°C. The cells were washed three times with HBSS and harvested by scrubbing in 100 μl of deionized water. The cell suspensions were sonicated for 20 min. After centrifugation, the total protein content from each well was determined using a Pierce bicinchoninic acid protein assay kit (Thermo Scientific, Rockford, IL); an equal volume of 0.2% formic acid was added to the supernatants. Intracellular polymyxin B1 concentrations were measured using a validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method (11) and normalized to the total protein content of the cells. A Michaelis-Menten model was fit to the rate of polymyxin B1 uptake.

To examine vectorial transport of polymyxin B across the cell monolayer, LLC-PK1 cells were grown on membrane inserts, washed, and incubated with FLPB (1 μM) added to either the apical or the basolateral side of the cell monolayer (n = 4) for 1 h at 37°C. Control cells were incubated under the same conditions with the blank buffer without FLPB. The cells were then washed three times with DPBS and incubated with 3 ml of cell dissociation enzyme solution (TrypLE Express; Life Technologies, Grand Island, NY) until they started to detach from the inserts (10 to 15 min). Complete growth medium was added to the inserts, and the cell suspensions were centrifuged for 10 min at 4,000 × g at room temperature. The supernatants were discarded, and the cells were resuspended in 500 μl phosphate-buffered saline. The intracellular fluorescence intensity was assessed using a flow cytometer (Accuri C6; BD Biosciences, San Jose, CA) with an excitation wavelength of 488 nm and an emission range of 530 ± 15 nm. The fluorescence intensities of 100,000 events from the samples were normalized to those of the blank samples. The intracellular fluorescence intensity from cells where polymyxin B was loaded apically was set as the reference. The intracellular fluorescence intensity from cells where polymyxin B was loaded apically was compared to that from cells where the drug was loaded basolaterally. Student's t test was used, and P values of <0.05 were considered significant.

The uptake of polymyxin B was further examined in the presence of escalating concentrations of gentamicin (0.5 mM to 25 mM), a known substrate for megalin (12, 13) and piperacillin (50 mM, negative control). The cell monolayers were incubated with FLPB (2 μM) in triplicate experiments for 1 h at 37°C. Control cells were incubated under the same conditions with 2 μM or 1 μM FLPB. The intracellular fluorescence intensity was assessed as described above.

Intracellular polymyxin B1 was satisfactorily detected in all the samples. The relationship between the polymyxin B1 concentration and the rate of cellular uptake was well characterized by Michaelis-Menten kinetics (r² = 0.995), as shown in Fig. 1. A significantly higher intracellular fluorescence intensity was observed when FLPB was loaded apically, as shown in Fig. 2. The fluorescence intensity ratio of the drug loaded apically to that of the drug loaded basolaterally was 4.4 ± 1.5 (P < 0.05). The uptake of FLPB was reduced in a concentration-dependent manner in the presence of gentamicin (P < 0.05), as shown in Fig. 3. With 25 mM gentamicin, >50% of the FLPB uptake was inhibited. This finding was subsequently validated using unconjugated polymyxin B (USP) under similar experimental conditions (intracellular polymyxin B1 concentrations were assayed by UPLC-MS/MS in triplicate experiments on two different days). In contrast, no uptake inhibition was observed in the presence of piperacillin.

FIG 1.

Rate of cellular uptake of polymyxin B1 into LLC-PK1 cells. Data points represent means ± standard deviations (SD). The dotted line represents the best-fit curve through the data points. The best-fit values for K_m and V_max were 10.8 ± 1.2 μM and 45.4 ± 2.1 μM/mg/h, respectively.

FIG 2.

Uptake of FLPB through the apical and basolateral sides of LLC-PK1 cell monolayers. The bar represents the mean ± SD.

FIG 3.

Inhibition of FLPB uptake. Bars represent means ± SD. Each asterisk indicates a significant reduction in fluorescence intensity compared to that of FLPB 2 μM (P < 0.05).

Systemic use of polymyxin B is associated with a considerable risk of nephrotoxicity (4, 6, 14). Nevertheless, it will continue to be used clinically until less toxic agents against multidrug-resistant bacteria become available (15–17). We previously showed that polymyxin B has a high affinity to renal tissue, which might be correlated to its nephrotoxic potential (8, 9). Given that the nephrotoxic injury of polymyxin B was more pronounced in the renal proximal tubular epithelial cells (9), we attempted to elucidate the underlying mechanism of drug uptake into this cell type.

The cell line that we used was LLC-PK1, which is one of the most commonly used cell lines in renal physiology and pharmacology. LLC-PK1 cells have been well characterized and possess many of the properties of the human proximal tubule, including apical membrane enzymes, transports, and microvilli (18–20). Polymyxin B uptake into LLC-PK1 cells was saturable. To elucidate the vectorial absorptive transport of polymyxin B, a fluorescent polymyxin B conjugate (FLPB) was used as a surrogate to assess the intracellular uptake of the drug semiquantitatively. A pilot study showed that under similar experimental conditions, the fluorescence intensity correlated well with escalating concentrations of FLPB (data not shown). This simple experimental setup can be used as a screening method for approved drugs as drug uptake-blocking agents. The uptake of polymyxin B occurred primarily through the apical surface, which indicated that the transporter responsible for polymyxin B uptake was expressed predominantly on the apical membrane of the LLC-PK1 cells.

An earlier study demonstrated the high affinity of polymyxin B toward megalin (13), a 600-kDa endocytic receptor expressed on the clathrin-coated pits of the brush border membrane of renal proximal tubular epithelial cells (21, 22). Megalin-mediated endocytosis plays an important role in the scavenging of low-molecular-weight proteins and polybasic compounds from the glomerular ultrafiltrate. A study using megalin-deficient mice also provided strong evidence that the proximal tubular reabsorption of gentamicin is linked to megalin (23). Using a different experimental design, another recent study also provided evidence that colistin (structurally similar to polymyxin B) is a megalin ligand and that megalin plays an important role in colistin renal uptake (24). Given that polymyxin B also exhibits a strong affinity to megalin (13), we postulated that megalin may be involved in the reabsorption and internalization of polymyxin B in renal cells. The uptake of polymyxin B was reduced by gentamicin in a concentration-dependent manner, which indicates that the two drugs might share a cellular uptake pathway. However, based on the drug concentrations used, the affinity of polymyxin B to the receptor appeared to be much higher than that of gentamicin, consistent with the previous findings (13). Therefore, the concomitant administration of gentamicin and polymyxin B clinically is unlikely to significantly reduce the renal accumulation of polymyxin B. Therefore, our future work will include the screening of different megalin ligands for their relative inhibitions of polymyxin B uptake in vivo.

In summary, our evidence reveals that the uptake of polymyxin B into renal cells is carrier mediated and that the transporter megalin is possibly responsible for its uptake across the apical membrane. More in-depth investigations on the role of megalin in the internalization and accumulation of polymyxin B in renal cells are ongoing, and this research may provide valuable insights for minimizing and preventing the nephrotoxicity of polymyxin B.