Competitive inhibition of GTTR uptake in vitro
We first verified that uptake of Texas Red-tagged gentamicin (GTTR) by MDCK cells could be competitively inhibited by an unconjugated aminoglycoside, i.e., gentamicin or kanamycin. The cellular distribution of GTTR in MDCK cells after a 30 second exposure was the same as previously described (Myrdal and Steyger, 2005
). GTTR fluorescence was diffusely distributed throughout the cytoplasm and associated with intra-nuclear structures () previously identified as RNA-containing nucleoli (Myrdal et al., 2005
). GTTR fluorescence in MDCK cells was significantly inhibited (p <0.005) by unconjugated gentamicin at all molar ratios tested (). Inhibition of GTTR fluorescence by increasing concentrations of kanamycin was less effective, with a statistical drop in GTTR fluorescence occurring only at the highest dose of kanamycin co-administration (1000:1 kanamycin:GTTR, p <0.05; ).
Unconjugated gentamicin, but not kanamycin, efficaciously decreases GTTR fluorescence in MDCK cells
Serum kinetics of GTTR
Thirty minutes following i.p. injection of GTTR alone, serum levels of the gentamicin epitope, detected by turbidimetric inhibition immunoassay (Newman et al., 1992
), reached 3.1 ± 0.48 μ
g/ml (n=12, ±s.e.m.). GTTR cannot be distinguished from native gentamicin (Wang and Steyger, 2009
). Thus, when GTTR was co-administered with unconjugated gentamicin, serum levels of the gentamicin epitope increased proportionally with increasing gentamicin dose (GT:GTTR: 10:1, 22.3±1.4 μ
g/ml; 100:1 209.3 ± 18.7 μ
g/ml; 400:1 596 ± 104 μ
g/ml; n≥3 per dose ± s.e.m). When GTTR was co-administered with increasing doses of kanamycin, GTTR levels in serum were relatively constant at 4.39 ± 0.38 μ
g/ml (n=12, ±s.e.m.), ~ 40% higher than serum GTTR levels without unconjugated aminoglycoside co-administration.
Competitive antagonism of strial GTTR uptake in vivo
To verify that GTTR uptake in vivo
is competitively antagonized by unconjugated gentamicin, we dosed mice receiving GTTR (2 mg/kg gentamicin base) with increasing doses of unlabeled gentamicin (20, 200, 600 and 800 mg/kg) for 30 minutes prior to cardiac perfusion and fixation. All animals maintained a robust heart beat until termination of the experiment. Animals treated with GTTR alone for 30 minutes displayed the same distribution of GTTR fluorescence within the stria vascularis as described previously (Wang and Steyger, 2009
). Briefly, strong GTTR fluorescence was observed within the marginal cells (), with reduced fluorescence in the intra-strial tissues (putatively the intermediate cells and intra-strial space; p < 0.005, n =8; ). Greatly reduced GTTR fluorescence was observed in the basal cells and spiral ligament (fibrocytes; data not shown) as reported previously (Wang and Steyger, 2009
Competitive inhibition of GTTR uptake by gentamicin in murine stria vasculari
Co-administration of unconjugated gentamicin with GTTR at 10:1 and 100:1 molar dilutions did not significantly (p >0.25) affect the distribution or intensity of GTTR fluorescence within the marginal cells or intra-strial tissues of the stria vascularis (). Only high molar ratios of gentamicin:GTTR (300:1, not shown, and 400:1 ) significantly decreased the intensity of GTTR fluorescence in marginal cells and intra-strial tissues compared to GTTR alone fluorescence intensities (p <0.005).
To assess whether strial uptake of GTTR is antagonized by unconjugated kanamycin, we dosed mice receiving GTTR (2 mg/kg gentamicin base) with increasing doses of unlabeled kanamycin (200, 800 and 1000 mg/kg) for 30 minutes prior to fixation. Co-administration with a 100:1 molar dilution of kanamycin:GTTR did not significantly alter the distribution or intensity of GTTR fluorescence in marginal cells or intra-strial tissues of the stria vascularis compared to GTTR-only animals (p >0.45; n=4; ). Only high molar ratios of kanamycin to GTTR (400:1 and 500:1) resulted in a statistically significant decrease in the intensity of GTTR fluorescence in marginal cells and intra-strial tissues (p <0.05; n=4 per group; ).
Competitive inhibition of GTTR uptake by kanamycin in murine stria vasculari
Competitive antagonism of renal GTTR uptake in vivo
For comparison, GTTR fluorescence in kidney tissues from the same animals were also examined. Administration of GTTR alone revealed both cytoplasmic and intense punctate fluorescence in proximal tubule cells, with weak diffuse cytoplasmic fluorescence in distal tubule cells (). Animals simultaneously dosed with GTTR and increasing molar ratios of unconjugated gentamicin displayed monotonic and statistically significant reductions in cytoplasmic GTTR fluorescence in proximal tubule cells compared to the GTTR-only treated animals (p <0.005 for all doses; ). The intensity of GTTR fluorescence at the brush border membranes of proximal tubule cells was qualitatively diminished with gentamicin administration. Punctate GTTR fluorescence in proximal tubule cells also appeared to diminish in intensity at higher molar ratios of GT (>100:1 gentamicin:GTTR), suggestive of reduced endosomal uptake of GTTR near the apical brush border membranes ().
Inhibition of renal uptake of GTTR is more efficacious with gentamicin than kanamycin
When animals were simultaneously dosed with GTTR and kanamycin, only a small, statistically insignificant reduction in cytoplasmic GTTR fluorescence was observed in proximal tubule cells at the lowest dose of kanamycin co-administration (100:1; p >0.1; ). Animals simultaneously dosed with high molar ratios of kanamycin:GTTR displayed significantly greater reductions in cytoplasmic GTTR fluorescence in proximal tubule cells (400:1 and 500:1 p <0.05; ). GTTR fluorescence at the brush border membranes of proximal tubule cells was qualitatively diminished only at very high kanamycin:GTTR molar ratios (>400:1). Punctate GTTR fluorescence in proximal tubule cells also appeared to diminish in intensity only at high molar ratios of kanamycin, suggestive of reduced uptake of GTTR in endosomes near the apical brush border.
Relative fluorescence intensity of kidney and strial tissues
Subjectively, kidney tissues displayed more intense GTTR fluorescence than strial tissues. For image acquisition of kidney proximal tubules, the confocal settings (i.e., laser setting and pinhole size) to obtain the greatest dynamic range of fluorescence intensity, limiting the number of saturated pixels ~1%, were much lower than for strial tissues. To quantify these differences, we obtained fluorescence intensity isopleths (contour lines; ) for MDCK cells exposed to two different concentrations of GTTR, 5 and 0.5 μg/mL. Each isopleth indicates the pinhole size and laser power settings required to reach a desired image intensity. MDCK cells treated with 5 μg/mL GTTR were intensely fluorescent, and required lower laser power and pinhole sizes compared to 0.5 μg/mL-treated MDCK cells that had less GTTR uptake and consequently weaker fluorescence. In , typical confocal settings for proximal tubule or strial tissues were also plotted, and demonstrate that the two tissues contain widely differing amounts of fluorescence beyond the dynamic range of the confocal system to record fluorescence using identical acquisition settings for both tissues.
Relative GTTR fluorescence in proximal tubule and marginal cells
The difference in the fluorescence emission of proximal tubules and the stria vascularis was estimated from the change in excitation laser power required to produce an equivalently bright image of a stable fluorophore, yellow fluorescent plastic. With the pinhole diameter at 3.5, a laser power of 13% (measured power 37 microwatts) produced a mean image (pixel) intensity of 76. When the pinhole was reduced 1.8, 62% (493 microwatts) laser power was required to obtain a mean image intensity of 76, a 13.2-fold difference. This pinhole factor was multiplied by the fold difference in excitation power of the laser settings used. For kidney tissues, a 12% laser setting corresponds to 33 microwatts, while the stria was excited by a laser setting of 40%, or 288 microwatts; a 8.7-fold difference. Multiplying the two factors (13.2 × 8.7) gives ~115-fold difference in fluorescence between the two tissues, indicating substantially greater in vivo uptake of GTTR by proximal tubule cells across their lumenal membrane, compared to trans-endothelial trafficking of GTTR into strial tissues.
Modeling aminoglycoside antagonism of GTTR uptake
In all experiments, the dose of GTTR was constant at 2 mg/kg, while the dose of either unconjugated aminoglycoside, the presumed competitive ligand, was varied (e.g., molar ratios of 10:1, 100:1, 400:1 of aminoglycoside:GTTR). The cytoplasmic fluorescence intensity in cells was obtained from single confocal optical planes for each dose, and normalized against GTTR-only intensity for comparison across data sets. These normalized
data were fitted to the Gaddum equation (law of mass action), the standard for analyzing reversible and surmountable dose-response curves with competitive antagonism (Kenakin, 2008
; Neubig et al., 2003
). Here, we assumed that the intensity of cytoplasmic fluorescence is proportional to level of GTTR uptake that can be antagonized by aminoglycosides. Therefore, using a simplified Gaddum equation:
are the equilibrium dissociation constants for GTTR and the antagonist, gentamicin (GT), respectively, obtained by a non-linear, least squares fit (R [statistical analysis software]; ). The constants are estimates of the concentrations at which 50% occupancy in the uptake mechanism(s) occurs, which are different for the two ligands and the two tissues. Nonlinear regression of gentamicin antagonism of normalized GTTR fluorescence in vivo
was used to obtain values for KGTTR
() and the curvefits shown in . The curvefit for marginal cells (or intra-strial tissues, not shown) is displaced to the right corresponding to the higher concentrations of gentamicin required to antagonize GTTR uptake.
Derived equilibrium dissociation constants for GTTR and gentamicin
Gentamicin antagonism of GTTR uptake is different in kidney and strial tissues
The gentamicin dose required for 50% inhibition of GTTR fluorescence in proximal tubule cells and strial tissues differs substantially. If gentamicin is a competitive antagonist of GTTR, then the inhibitory dose, ID50, can be calculated from KGT * (1 + ([GTTR]/KGTTR)), with dissociation constants obtained from the nonlinear curvefit of the data. The ID50 for the proximal tubule cells was 82 mg/kg, and that for marginal cells 832 mg/kg. In the stria vascularis, increasing gentamicin concentrations have a relatively reduced effect on GTTR fluorescence compared to proximal tubule cells where the effect is more pronounced. This corresponds to the normalized dose-fluorescence curve for proximal tubule cells approaching an asymptote at a lower molar ratio of gentamicin:GTTR compared to marginal cells ().
Kanamycin antagonism of GTTR uptake in vivo was much reduced compared to gentamicin. Nonlinear regression of kanamycin antagonism of normalized GTTR fluorescence was used to obtain values for KGTTR and KKM () and the curvefits shown in . The curvefit for marginal cells is relatively similar to that for proximal tubules, as were the ID50 for kanamycin in marginal cells, 1377 mg/kg, and proximal tubules, 1330 mg/kg.
Nonlinear regression of aminoglycoside antagonism of normalized GTTR fluorescence was also used for the in vitro data in to obtain the KGTTR, KGT and KKanamycin (KKM; ) and the curvefits shown in . The curvefit for kanamycin is very different, and flat compared to that for gentamicin. The inhibitory concentration, IC50, for gentamicin (30 μgml) was also distinctly smaller than for kanamycin (6519 μg/ml).