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Department of Medicine, Division of Nephrology, Indiana University School of Medicine, and the Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana.
Correspondence to Dr. Bruce A. Molitoris, Indiana University School of Medicine, 1120 South Dr., Fesler Hall, Room 108, Indianapolis, IN 46202. Phone: 317-274-5287; Fax: 317-274-8575; E-mail: bmolitor{at}mdep.iupui.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aminoglycoside use can be complicated by well described nephrotoxic and ototoxic effects (3,4,5). Complications attributable to aminoglycoside toxicity rank as one of the most common reasons for prolonging hospital stays in the developed world (6). Nephrotoxic effects are most common and develop after relatively short periods of treatment (7,8). In many cases, these effects are reversible if administration is discontinued.
Although many hypotheses have been proposed and tested (3,9,10), the precise mechanisms of aminoglycoside-induced nephrotoxicity remain unclear. Alterations in phospholipid metabolism after aminoglycoside treatment have been described by several laboratories, including our own (11,12,13,14,15). These changes occur rapidly and persist for extended periods, even after recovery, in the rat model (11,14,15,16,17). Inhibition of lysosomal phospholipases, subsequent accumulation of phospholipids, and formation of lysosomal myeloid bodies have been implicated as direct mechanisms of nephrotoxicity (11,12,13,16,18).
Accumulating evidence has also suggested aminoglycoside-induced inhibition of protein synthesis as a mechanism of nephrotoxicity. It has been demonstrated that elevated concentrations of aminoglycosides can cause mistranslation or block incorporation of amino acids by eukaryotic ribosomes in vitro (19,20,21). In addition, concentrations of gentamicin attained in the rat renal cortex during in vivo treatment were demonstrated to inhibit subsequent in vitro microsomal protein synthesis (19,20,22). This effect was observed before a decline in renal function or significant alterations in cellular enzyme activities and morphologic features were observed.
In large part, aminoglycoside effects on protein and phospholipid synthesis and metabolism have not been determined in completely in vivo experiments. Therefore, the purpose of the studies described here was to determine the effect of aminoglycosides on these factors in completely in vivo experiments. Studies were performed at time points (1 to 3 d) when little or no alteration of cellular enzyme activities, morphologic features, or function was observed. Correlative confocal microscopic and electron microscopic (EM) studies were also performed and suggested a role for intracellular trafficking in aminoglycoside-induced nephrotoxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal Protocol
Male Sprague-Dawley rats (200 to 250 g; Harlan, Indianapolis, IN) were used
for all experiments. They were fed standard rat chow and allowed free access
to food and water during the experiments. Rats were given intraperitoneal
injections of either gentamicin (gentamicin sulfate, 40 mg/ml; Elkins-Sinn,
Cherry Hill, NJ), at a dose of 100 mg/kg [short-term gentamicin (STG)], or
vehicle (vehicle control) for 1 to 3 d. In all cases, rats were given
injections for the indicated times and euthanized the next day. The dosage
used was previously demonstrated, by our laboratory and others, to induce
reproducible and reversible nephrotoxicity
(12,15,16,24).
The times chosen ensured the occurrence of only mild toxicity
(22,25).
Serum creatinine levels were measured for the 3-d gentamicin-treated rats, as
a measure of toxicity. Additional measured parameters were enzyme activities
and membrane lipid contents (see below).
TRG Studies
In these studies, tracer amounts of Texas Red-labeled gentamicin
(i.e., 354 ng TRG/mg regular gentamicin, 0.04% TRG) were included in
the normal gentamicin solution (see above). Rats that received this solution
were treated exactly like those given gentamicin only. Only the 3-d time point
was used in these studies. Rats were anesthetized with pentobarbital, the
abdominal cavity was opened, the portal vein was catheterized, and
phosphate-buffered saline (PBS) infusion was begun. The vena cava was then
cut, and the vascular tree was flushed by continued perfusion with PBS
(approximately 200 ml). Fixation of the tissue was accomplished by perfusion
with 180 to 200 ml of freshly prepared 4% paraformaldehyde in PBS. Kidneys
were then decapsulated and cut into approximately 5-mm3 pieces, and
fixation was continued overnight at 4°C in the same fixative. The next
day, kidney pieces were washed three times with PBS and placed in 0.25%
paraformaldehyde/PBS until sectioning. Sections of 50 µm were obtained and
either photo-oxidized (see below) or mounted in Gelvatol mounting medium
(Aldrich Chemical Co., Inc., Milwaukee, WI); coverslips were sealed with clear
acrylic nail polish. TRG staining was examined by confocal microscopy (see
below).
Brush Border Membrane (BBM) Preparation and Characterization
Control and gentamicin-treated rats were lightly anesthetized with
metafane, decapitated, and exsanguinated, and their kidney cortices were
dissected and homogenized. Homogenates for all rats were prepared using
exactly the same volume of buffer and as close to the same amount of tissue as
possible. The beginning and ending weights did not vary by >5% between
control and STG-treated rats in any experiment. BBM were isolated from
cortical homogenates using Mg2+ precipitation and differential
centrifugation techniques, as described in detail elsewhere
(15,26,27,28,29).
Relative enrichments of BBM preparations were documented enzymatically using
the BBM marker leucine aminopeptidase and the basolateral membrane marker
Na+/K+-ATPase, as described previously
(15,27,28,29).
Marker enzymes used to determine contamination by other intracellular
organelles were succinate dehydrogenase (mitochondria) and KCN-resistant NADH
dehydrogenase [endoplasmic reticulum (ER)], as described previously
(15,27,28).
Biosynthetic Labeling of Proteins and Phospholipids
Control and gentamicin-treated (1 to 3 d) rats were given intraperitoneal
injections, on the day of euthanasia, of either 0.1 mCi of
[3H]leucine or 0.5 mCi of 32P (Amersham, Arlington
Heights, IL), both in 0.5 ml of normal saline solution. [3H]Leucine
was injected 1 h before euthanasia, and 32P was injected 2 h before
euthanasia. After euthanasia, cortical homogenates were made and BBM were
prepared from them as described above. In both cases, aliquots were added to
scintillation fluid (Beckman, Fullerton, CA) and counted in a Beckman LS1801
ß-counter. Incorporation of radioactivity into protein or lipid was then
determined as described below. We previously used similar procedures to
quantify phospholipid synthesis
(27).
Protein Determinations
Protein concentrations were determined using the method of Lowry et
al. (30) or the Bradford
dye binding assay (31), and
synthesis was expressed as counts incorporated per milligram of protein.
Incorporation of [3H]leucine into protein was determined by TCA
precipitation. Samples (homogenates or BBM) were incubated on ice in 10% TCA
for >1 h and were filtered using Millipore filtration techniques we
described previously
(29,32).
Each filter was rinsed, suspended in scintillation cocktail, and counted.
Alternatively, TCA-precipitated samples were centrifuged at 16,000 x
g for 10 min, and the TCA pellet was solubilized with 1 N NaOH, mixed
with scintillation cocktail, and counted.
Lipid Determinations
These determinations were performed as we described in detail previously
(15,27,28).
Briefly, lipids from 0.5 to 1.0 mg of cortical homogenate or BBM were
extracted with chloroform/methanol (1:2, vol/vol) and isolated according to
the method of Bligh and Dyer
(33). Total phospholipid
levels were determined using the method of Ames and Dubin
(34), and individual
phospholipid species were separated by two-dimensional thin layer
chromatography, using a modification
(27) of the method of Esko and
Raetz (35). Incorporation of
32P into total cellular membranes, BBM, and individual lipid
species was determined by counting aliquots after chloroform/methanol
extraction and TCA precipitation, as described for filters above (homogenates
and BBM), or counting resuspended scrapings of individual lipid species after
two-dimensional thin layer chromatography (individual phospholipid species;
see above).
Confocal Microscopy
TRG conjugate fluorescence was examined using a Bio-Rad MRC-1024 scanning
laser confocal microscope (Bio-Rad, Richmond, CA), with a Nikon x 100
1.4-NA PLAN APO objective (Nikon, Natick, MA). Samples were illuminated using
an argon-krypton laser. We successfully used this system in previous studies
(15,23).
Photo-Oxidation of TRG and EM
The protocol for and mechanism of photo-oxidation of ringed structures in
the presence of diaminobenzidine (DAB) have been described by others
(36). We previously described
our modifications of the photo-oxidation reaction
(23). An excitation filter
(filter 560DF40; Optical Omega, Brattleboro, VT) was positioned directly over
sections (see above) immersed in DAB substrate solution (Vector Laboratories,
Burlingame, CA) at 4°C. A mercury lamp suspended 30 cm above the filter
cube was used to excite the sample with 560-nm light for 2.5 min three times.
After each excitation, fresh 4°C DAB solution was added to the sample
well. Reactive species produced by photo-oxidation of the Texas Red
fluorophore react with DAB to form a dense EM-observable reaction product
wherever TRG is present. Additional processing of the photo-oxidized tissue
for EM was performed using procedures similar to those we have used before
(15,29,32).
Samples were fixed with 1% glutaraldehyde for 1 h, washed four times with PBS,
postfixed with 0.14% osmium tetroxide/0.04% tannic acid/0.50%
K3Fe(CN)6 in PBS, dehydrated with graded ethanol,
infiltrated with graded solutions of propylene oxide/Spurr's resin to 100%
Spurr's resin (incubated for 2 x 1 h at 100%), and then polymerized
overnight at 65°C. Ultrathin sections were cut using a MT6000-XL
ultramicrotome (RMC Inc., Tucson, AZ) and observed with a Philips CM-12
electron microscope (Wilmington, DE), using 80- to 100-kV accelerating
voltage.
Statistical Analyses
Values are reported as means ± SD. Data were analyzed using the
paired and unpaired t test and the two-sample t test,
assuming unequal variances. Differences were considered statistically
significant at P < 0.05 and are reported as P < 0.05,
0.03, 0.02, 0.001, or 0.0001.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similarly to previous studies (16), we observed little effect of STG treatment on several cellular homogenate enzyme activities and their enrichment in BBM preparations. The BBM marker leucine aminopeptidase exhibited no significant change in either specific activity or BBM enrichment (Table 2). Likewise, markers of two intracellular organelles, i.e., succinate dehydrogenase (mitochondria) and KCN-resistant NADH dehydrogenase (ER), did not exhibit any significant change in enrichment (Table 2). However, statistically significant alterations were observed for the basolateral membrane marker Na+/K+-ATPase. The specific activity in homogenates from STG-treated animals was reduced, whereas the BBM enrichment was increased. No alterations in enzyme activities or BBM enrichment were observed at time points earlier than 3 d (data not shown).
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The next experiments directly assessed the effects of gentamicin on in vivo protein synthesis. Rats were treated with gentamicin for 1 to 3 d and then injected with [3H]leucine 1 h before euthanasia, for determination of newly synthesized protein in homogenates and BBM preparations. As presented in Figure 1A, a significant decrease in [3H]leucine incorporation into total cellular protein was observed by the second day of treatment. Protein synthesis decreased further on the third day of treatment. When total cellular and BBM protein syntheses were compared at 3 d (Figure 1B), both were decreased by approximately 50%. Effects on protein degradation were not directly measured.
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It has been demonstrated that short-term (11,12,16,37) and long-term (15) aminoglycoside exposure produces rapid and persistent alterations in renal phospholipid contents. These changes are thought to result from inhibition of phospholipid degradation (13,37,38,39). It is also possible that synthesis and/or incorporation of phospholipids into their appropriate membranes may be affected. To explore these possibilities, experiments were performed to better characterize the effects of gentamicin on phospholipid synthesis and incorporation into membranes. Results are presented in Table 3. After treatment of rats with gentamicin for 3 d, the total phospholipid content of homogenates and BBM preparations was significantly increased (both 21%), compared with control homogenates and BBM. Although incorporation of 32P into total cellular phospholipids (i.e., synthesis) was moderately decreased (11%) and increased (7%) in gentamicin-treated homogenates and BBM, respectively, neither change was statistically significant.
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Our next experiments characterized the effects of 3 d of gentamicin treatment on individual phospholipid contents. No significant effect on the percentage composition of any phospholipid analyzed in STG cellular homogenates was observed, except for phosphatidylinositol (PI), which exhibited an increase of 34% (Figure 2A). However, when individual phospholipids of BBM preparations from STG-treated and control rats were compared, significant increases in phosphatidylcholine (PC) and PI levels (37 and 64%, respectively) were observed, whereas a significant decrease (23%) in sphingomyelin (SPH) content was observed (Figure 2B).
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To evaluate whether synthesis of individual phospholipids was being altered, rats were treated with gentamicin exactly as described above, except they were injected with 32P 2 h before euthanasia. Significant decreases in the rate of incorporation of 32P into SPH (P < 0.05) and phosphatidylserine (PS) (P < 0.05) were observed, whereas a significant increase was noted for phosphatidylethanolamine (PE) (P < 0.02) (Table 4). The same analysis performed on lipids extracted from BBM preparations from treated animals revealed only a significant decrease in 32P incorporation into SPH (P < 0.001) (Table 4). No consistent significant change in total or individual phospholipid composition or in total or individual phospholipid synthesis was observed at times earlier than 3 d (data not shown).
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Both membrane protein and phospholipid biosynthetic pathways use cytosolic enzymes. However, they are also both dependent on and intimately associated with the membranes of intracellular organelles, specifically the ER and Golgi complex. The last series of experiments was performed to morphologically characterize where gentamicin was trafficked and concentrated during STG treatment. These experiments were performed exactly as described above, except that tracer amounts of TRG were added to the native gentamicin used for injection (see Materials and Methods).
Confocal microscopic results from these experiments are presented in Figure 3; large bright fluorescent structures are clearly evident. Similar to native gentamicin, the TRG was taken up, retained, and concentrated in numerous, relatively large structures (probably lysosomes) located predominantly in the basal region of proximal tubule cells (PTC). Only minimal plasma membrane staining was observed 24 h after the final injection. No fluorescence staining was observed in proximal tubules of control animals (data not shown). Little, if any, intracellular staining was observed in glomeruli or distal tubules.
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When the fluorescent structures were examined in greater detail using photo-oxidation of TRG and EM, it became evident that TRG was associated with a variety of structures. The classic effects of gentamicin on lysosomes can be seen in Figure 4. Electron micrographs of photo-oxidized tissue sections from animals given injections of vehicle (Figure 4A), native gentamicin (Figure 4B), and TRG-spiked gentamicin (Figure 4C) are shown. It was evident that the photo-oxidation reaction itself did not induce significant reaction product in either control tissue (Figure 4A) or native gentamicin-treated tissue (Figure 4B), which contained myeloid material. However, in the tissue treated with gentamicin and tracer amounts of TRG, much of the myeloid material was darkly stained (Figure 4C). Not all lysosomes or myeloid material in the same section or cell was labeled. Much of the TRG-stained lysosomal myeloid material appeared as heavily stained, collapsed/aggregated membranes that lacked the classic concentric circular, lamellar appearance (compare Figure 4, B and C) (25,40). Whether TRG was associated with lipid, protein, or both within the membrane was not determined.
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More detailed examination revealed evidence of other intracellular
organelle staining (Figure 5).
Darkly stained structures could be observed in the basal region of cells from
adjacent proximal tubules of gentamicin/TRG-treated animals
(Figure 5B). This staining was
very well correlated with the confocal microscopic data presented in
Figure 3. Dark staining, as
seen in Figure 5B, was not
observed in vehicle-treated control samples photo-oxidized exactly like the
gentamicin/TRG-treated samples (Figure
5A). At higher magnification, it was clear that much of this
staining was lysosomal (Figure
5C). However, some of the darkly stained structures did not appear
to be lysosomal (Figure 5B). In
additional experiments, which were designed to specifically enhance this
staining with the injection of TRG into STG-treated rats 30 min before
perfusion-fixation, we observed unequivocal staining of classic Golgi stacks
(Figure 5D) and mitochondria,
according to morphologic criteria (Figure
5E). Although the results were not rigorously quantified, we
estimate that 
10% of the Golgi complexes and mitochondria observed were
stained in this manner.
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Unequivocal evidence of mitochondrial staining was observed in the TRG accumulation experiments (Figure 5, B and C) and the uptake experiments (Figure 5D). At high magnification (Figure 5E), it was clear that the outer membrane and intermembrane space were heavily stained. However, it was not clear whether the inner membrane was labeled. If labeled, it did not appear to extend deeply into the cristae of the mitochondria. Staining of the matrix was not observed in any of the heavily stained mitochondria observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a rat model with little other evidence of PTC toxicity, we demonstrated that protein synthesis was significantly inhibited after 2 d of gentamicin treatment. This inhibition increased to 50% by the third day. Furthermore, this inhibition appeared to affect BBM protein synthesis in a similar manner and to the same degree as total cellular protein synthesis. Effects on protein degradation were not directly determined. These studies agree with and expand earlier, partly in vivo studies performed somewhat differently than ours (19,20,21,22).
With the same model, we also observed disruption of individual phospholipid synthesis and accompanying inhibition of phospholipid degradation. In contrast to our studies of protein synthesis, total cellular and BBM phospholipid syntheses were not significantly affected by gentamicin treatment. However, there were significant alterations in the incorporation of 32P into individual phospholipids. Incorporation of 32P into PE was significantly increased in STG-treated homogenates, whereas incorporation into SPH and PS was reduced. Only a reduction in the incorporation of 32P into SPH in BBM preparations was observed with STG treatment. Previous in vivo studies, which were performed differently than ours, demonstrated either no change in 32P incorporation into any phospholipid in a rat model (16) or an increase in the synthesis of at least two phospholipids using a rabbit primary culture PTC model (14). In our studies, we observed no change (PC and PI in STG-treated homogenates and PC, PE, PI, and PS in STG-treated BBM), an increase (PE in STG-treated homogenates), and decreases (PS and SPH in STG-treated homogenates and SPH in STG-treated BBM) in phospholipid levels.
Similar to findings reported by others (11,12,13,16,37,38,39), our studies showed that phospholipid degradation was inhibited at early time points after gentamicin treatment (in these studies, at 3 d). We demonstrated that total phospholipid contents in homogenates and BBM preparations were increased 21%, compared with control samples. We also showed that the individual phospholipid contents of BBM were affected to a greater extent by STG treatment than were total cellular membranes, which is, to our knowledge, a new finding. The only significant effect on the percentage composition of any phospholipid in STG-treated homogenates was an increase in PI content (34%). However, in STG-treated BBM preparations, significant increases in PC and PI levels (37 and 64%, respectively) were observed, whereas a significant decrease (23%) in the SPH content was observed, compared with control samples. It was clear from these studies that any increase in phospholipid content observed as a result of gentamicin treatment was predominantly the result of the inhibition of degradation, rather than any effect on synthesis. Finally, the phospholipid degradation and synthesis effects were somewhat delayed, compared with the protein synthesis effects, and did not become statistically significant until the third day of treatment. This is relevant because the previously mentioned animal studies that examined lipid synthesis were performed at 2 d (16).
Our last, and perhaps most interesting, results were from the morphologic studies. Confocal microscopic studies revealed accumulation of fluorescently labeled gentamicin in large basal structures. It was clear that most of these structures were lysosomes. However, more sensitive EM techniques revealed transport of gentamicin not only to lysosomes but also to Golgi complexes and mitochondria, again in entirely in vivo experiments. Photo-oxidized TRG darkly labeled not only membranes of myeloid bodies within lysosomes but also membranes of Golgi cisternae and the outer membrane and intermembrane space of mitochondria.
Although disruptions of protein and phospholipid synthesis could occur at many sites, a common site for both would be at the level of the Golgi complex, ER, or cell cytoplasm, particularly in the early stages. We are aware of only one study that suggests that gentamicin may be released into the cytosol at early time points after uptake (41). In the morphologic studies we have performed, we have never observed evidence of gentamicin being released into the cytosol in living cells. Therefore, for alteration of protein and phospholipid synthesis, we think that retrograde trafficking of gentamicin to the Golgi complex and perhaps the ER must occur. Our recent cell culture studies (23) and the in vivo morphologic data presented here, as well as implications from studies performed by other investigators (12,14,24,42), support this possibility. We do not now have a good explanation for the mitochondrial staining. However, if gentamicin reaches the ER, it might be able to traffic to the mitochondria from there.
Retrograde trafficking of a number of bacterial and plant protein toxins after endocytosis is now well established (43). These protein toxins kill eukaryotic cells after translocation of an active part of the molecule from various sites into the cytoplasm, where they inactivate the 60S subunit of the ribosome or elongation factor 2 (see reference 43 and references cited therein). Gentamicin inhibits bacterial protein synthesis by binding to the 30S subunit of the bacterial ribosome and inhibiting elongation or by causing mistranslation (1,2). In general, it is thought that gentamicin is not released into the cytoplasm of kidney PTC in the early stages of toxicity. As a result, the gentamicin-induced disruption of protein and phospholipid synthesis observed in these studies seems to be produced via a different, more indirect mechanism than that of the protein toxins. We suggest that gentamicin may interact with components of the protein and/or phospholipid biosynthetic machinery from the lumen of the Golgi complex or ER to induce the alterations characterized in these studies. The possibility that aminoglycosides might directly interact with the Golgi complex or ER was suggested previously (12,24,37).
It is also possible that normal membrane traffic and fusion become increasingly perturbed during gentamicin treatment, resulting in disruption of many cellular activities and ultimately cell death. The evidence of apparent retrograde movement of gentamicin observed in this study is consistent with this possibility. Additionally, the subtle but significant alteration in Na+/K+-ATPase enrichment in BBM preparations supports the possibility of altered trafficking resulting from gentamicin treatment.
Other investigators, using similar STG treatment models, also proposed alterations in membrane traffic (12,14,16,24,42). Relevant to this concept is evidence that gentamicin treatment inhibits vesicle/lysosome fusion in vivo (42) and homotypic endosome fusion in vitro (44), as well as the suggestion that longer-term gentamicin treatment causes swelling of the ER (25,45). It has also been speculated that myeloid bodies may be formed as a result of decreased degradation of smooth ER (46). Furthermore, in a long-term model of gentamicin treatment in which rats recover from gentamicin-induced toxicity, we observed an apparent reduction of the endosomal compartment (15). This hypothesis suggests that disruption of normal trafficking and fusion could lead to aberrant trafficking and fusion. Because gentamicin is normally delivered to lysosomes and contains no known targeting sequence, altered trafficking and fusion could provide a mechanism for inducing the putative aberrant retrograde movement.
Although we do not present direct evidence to confirm this hypothesis, disruption of membrane trafficking provides a unique but consistent explanation of the results observed in this study. Significantly, it also provides an encompassing mechanism of gentamicin-induced toxicity that could explain the different effects described in detail by many investigators over several decades.
In summary, the results of our studies provide, for the first time, direct and completely in vivo evidence that gentamicin treatment inhibits protein synthesis in an animal model. They also provide new evidence that individual phospholipid synthesis and degradation are disrupted in vivo. Lastly, they provide unique evidence that gentamicin treatment causes trafficking of gentamicin to the Golgi complex and mitochondria. Together, these observations provide a mechanistic basis for gentamicin-induced nephrotoxicity. As a result of treatment, gentamicin is delivered to multiple sites, perhaps by altered trafficking and fusion. These sites then correspond with multiple sites of potential toxicity. The implication may be that the observed toxicity is the result of multiple minor toxic effects, which may act synergistically, as suggested previously (47).
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