Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK1 cells

Ruben M. Sandoval, Kenneth W. Dunn, and Bruce A. Molitoris

Division of Nephrology, Department of Medicine, Indiana University School of Medicine, and the Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK1) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for ~20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration.

aminoglycosides; lysosomal-fluorescence quenching; nephrotoxicity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DESPITE RECENT ADVANCES IN the understanding of aminoglycoside nephrotoxicity, a complete picture of the complex cellular mechanisms involved remains elusive. One area of much speculation involves the relatively short time interval between aminoglycoside administration and protein-synthesis inhibition (2, 3). The time required for cellular uptake and sequestering of aminoglycosides to lysosomes, followed by subsequent rupture, release, and association with the protein-synthetic machinery, as previously suspected, exceeds the earliest time for which protein-synthesis inhibition has been observed.

We have previously demonstrated a novel pathway for the uptake and trafficking of aminoglycoside antibiotics to the Golgi complex by employing a fluorescent tracer of gentamicin (15). By using protocols involving both transmission electron microscopy (TEM) and confocal microscopy, we reported a fraction of the internalized aminoglycoside pool traffics to the Golgi complex. However, due to the low fluorophore-to-conjugate ratio of the probe used in this study, we were unable to determine whether Golgi complex accumulation of gentamicin resulted from direct trafficking to the Golgi complex after endocytosis, and if so, the kinetics of accumulation.

Therefore, the present study was conducted to determine the kinetics involved in the trafficking of aminoglycosides to the Golgi complex. To investigate this phenomenon, we employed confocal microscopy to follow a highly enriched form of the tracer Texas Red and gentamicin (TRG), used and characterized extensively in our previous study (15). We also utilized a technique that quenched fluorescence emission from the lysosomal compartment, thereby revealing only the fluorescence from nonlysosomal structures (7, 11, 14). This was crucial because the intense fluorescence originating from the lysosomes overwhelms fluorescence from less intense structures, making them undetectable.

Results from the kinetic studies revealed accumulation of gentamicin within the Golgi complex occurred within 15 min of incubation. Finally, cells grown on grided coverslips were used to visualize the different populations of TRG within the same field. These different populations were colocalized to both the trans-Golgi network (TGN)/trans-compartment and cis/medial Golgi-compartment probes. The data gathered suggest the fluorescent nephrotoxin sequesters equally among the compartments of the Golgi complex. We postulate accumulation of TRG within the Golgi complex may represent an intermediary locale before eventual retrograde transport into the endoplasmic reticulum (ER), or trafficking throughout the cell from the Golgi complex.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental model. Porcine kidney proximal tubule cells (LLC-PK1, ATCC, Rockville, MD) were grown on 18-mm-diameter coverslips (Fisher Scientific, Itasca, IL) for quantitative uptake experiments, or 35-mm-diameter cell culture dishes with a grided coverslip attached to the bottom (MaTek, Ashland, MA) for colocalization experiments. Cells were maintained in a 1:1 mixture of DMEM and Ham's nutrient mixture (F-12) (K-P media), supplemented with 10% fetal bovine serum and 1 mg/ml penicillin streptomycin (Sigma, St. Louis, MO). Cells were allowed to reach a state of ~40-70% confluence before being used in experimental protocols.

Lysosomal fluorescence quenching. Before aminoglycoside uptake, the LLC-PK1 cells in all experiments were preincubated in media containing 2 mg/ml horseradish peroxidase (HRP; Sigma) for 30-60 min under normal growth conditions. The cells were then washed twice briefly in normal media and incubated in normal media for 30 min under normal growth conditions. After termination of the TRG-uptake protocol and fixation, but before permeabelization, some cells were incubated for 2-5 min in a solution of diaminobenzidine (DAB), without the Nickel solution as described in the peroxidase substrate kit DAB (SK-4100, Vector Laboratories, Burlingame, CA) to form an electron-dense product inside the lysosomes. This is known to prevent fluorescence emissions from these structures (7, 11, 14).

Quantitative TRG uptake studies. After preincubation in HRP, cells were placed in K-P Media containing 2 mg/ml TRG (Molecular Probes, Eugene, OR) for 0, 15, 30, 60, and 120 min. Cells were then washed twice briefly in PBS and fixed.

Colocalization of TRG with Golgi complex probes. After HRP uptake, the cells were allowed to accumulate TRG for 60 min followed by fixation and image acquisition of total TRG fluorescence.

After image acquisition, the cells were washed in PBS, incubated in the DAB-H2O2 solution to quench lysosomal fluorescence, and washed in PBS. The trans-Golgi and trans-Golgi network portions of cells were labeled by using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl ceramide (C6-NBD-ceramide) coupled with defatted BSA (Sigma), a probe previously characterized by R. E. Pagano (12). The standard protocol for staining fixed cells was slightly modified to maximize the signal in our cell culture model. The fluorescent phospholipid was diluted in 1:1 in PBS for a final concentration of 2.5 µM. Staining was carried out for 20 min at 37°C, followed by a incubation in 1% defatted BSA for 20 min at 37°C to remove excess levels of the fluorescent phospholipid. Images of the Golgi complex probe and nonlysosomal associated TRG were then acquired.

Finally, the cells were permeabelized in PBS containing 0.05% Triton X-100 (Sigma) for 10 min and incubated in blocking buffer containing PBS with 2% defatted BSA for 30 min. Incubating the cells in the Triton-containing buffer removed all traces of the membrane-associated fluorescent phospholipid C6-NBD ceramide and allowed labeling with an FITC-labeled fluorescent probe. To label the medial and cis-portions of the Golgi complex, a FITC-conjugated Lens culinaris agglutinin (FITC-LCA; Vector Laboratories) was used at a concentration of 2-5 µg/ml in blocking buffer. This lectin predominantly labels the medial and cis-Golgi compartments of cells in polarized intestinal epithelia (13). The cells were washed, and images of the Golgi complex probe and nonextractable portions of remaining TRG pool were acquired. A timeline diagram of this protocol is found in Fig. 3B.

Cell fixation. LLC-PK1 cells were briefly washed with PBS, pH 7.4, at 37°C, then fixed in freshly thawed 4% paraformaldehyde in PBS, pH 7.4, for 1 h at room temperature or at 4°C overnight.

Image acquisition. Images were acquired on a Bio-Rad (Hercules, CA) MRC-1024 combination 2-photon, transillumination capability, Kr/Ar laser-scanning confocal microscope on a Nikon Diaphot inverted microscope platform by using either a ×100 oil-immersion objective with a numerical aperature (NA) of 1.4, or a ×60 water-immersion objective with an NA of 1.2. For quantitative studies, acquisition parameters on the emission detectors were kept identical between the different experimental groups; only the laser power was adjusted to detect lower intensity emissions. Any difference in laser power settings between groups was adjusted by using a ratio calculated from the fluorescence intensities of a reference object imaged at the different laser power settings. This ratio was then used to adjust the values that yield total integrated intensity (number of spots × average spot intensity × average spot size). To avoid the possibility of spectral overlap in the colocalization studies, the signals from the Texas red and FITC-NBD emissions were excited and acquired sequentially by using the Kr/Ar laser in a single photon mode. In these studies, the acquisition channel not used for collecting fluorescence data was set to acquire a transillumination brightfield image of the alphanumeric markers from the grid-etched coverslips. About four-hundred microliters of PBS were placed over the grided depression of the 35-mm dish, and an 18-mm diameter coverslip was placed over it to facilitate the acquisition of the transillumination brightfield image.

Image analysis and processing. All of the experimental protocols were repeated a total of three times to confirm initial findings. Moreover, time course studies for these experiments exhibit identical staining patterns between groups, with differences occurring only in the intensity of staining. The images from the colocalization studies were processed and overlaid by using Metamorph v 4.0 image-processing software (Universal Imaging, West Chester, PA). Acquired images for the quantitative studies were first processed using Metamorph v 4.0 for background subtraction by applying a 3 × 3 low-pass filter. A copy of the same initial images was then processed by using a 28 × 28-factor, 1-median filter. The resulting image was subtracted from the previous 3 × 3 low pass (low-pass median filter) to yield a background-subtracted image. A 128 × 128-pixel area was taken from each of the images where the cells were confluent so as not to skew the resulting data due to inclusion of nonconfluent areas lacking any fluorescence (10 random fields were acquired for each treatment). After background subtraction the images were quantified as previously described (5), to yield a total integrated intensity value for fluorescence (in arbitrary units) by multiplying the values for number of spots, average intensity, and average spot size.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Quenching of fluorescence originating from lysosomes was a crucial step in detecting TRG emission from organelles with smaller, less intense accumulations. In Fig. 1, the dramatic decrease in TRG fluorescence can be seen between unquenched (A) and quenched (B) images. Acquisition parameters changed due to the decrease in fluorescence intensity of the quenched samples; usually, detection of these samples required a threefold increase in laser emissions. Permeabelizing the cells to allow penetration of the FITC-conjugated lectin to label the Golgi complex also caused a decrease in the TRG fluorescence intensity in all cell groups. Costaining of both unquenched and quenched samples produced slight differences in the staining patterns. As expected, the vast majority of the TRG (red) in the unquenched cells (Fig. 1A) was localized to vesicular structures previously shown to be lysosomes (arrowheads) and could be seen as bright-red punctate spots that did not colocalize with the Golgi complex marker. A small portion of the TRG did colocalize with the Golgi complex (arrows), appearing yellow, around the perinuclear area. In contrast, the fluorescent TRG in the lysosomal quenched cells (Fig. 1B) colocalized with the FITC-lectin Golgi complex marker (arrows), appearing yellow. Quenching of TRG lysosomal emissions also allowed us to determine how quickly aminoglycosides accumulated in the Golgi complex via colocalization with the fluorescent lectin. In Fig. 1 (C-F), both the untreated time point 0 (C) and 15 min of TRG exposure (D) showed no detectable levels of TRG accumulation. However, for the 30 (E)- and 60-min (F) time points, there was enough accumulation of TRG in the Golgi complex to be detected. For both of these time points, the remaining TRG colocalized with the Golgi complex marker.


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Fig. 1.   Texas red and gentamicin (TRG) fluorescence retained after lysosomal quenching is localized to the Golgi complex within 30 min. LLC-PK1 cells were preloaded with horseradish peroxidase (HRP) incubated with 2 mg/ml TRG (red) for 1 h and then stained with an FITC-conjugated lectin from Lens culinaris (FITC-LCA) to localize the Golgi complex (green; A). The TRG signal (red) exhibited typical lysosomal staining pattern (A). The majority of the fluorescent signal did not colocalize with the cis/medial Golgi complex (arrowheads). However, some colocalization (yellow) did occur around the perinuclear area. When similarly treated cells were processed for lysosomal fluorescence quenching (B), a dramatic decrease in the amount of cellular TRG fluorescence was seen. The fluorescence remaining after quenching lysosomal accumulated fluorescence colocalized (yellow) primarily with the cis/medial Golgi complex marker FITC-LCA (arrows). Cells incubated in media containing 2 mg/ml TRG (red) for 0 (C), 15 (D), 30 (E), and 60 min (F) were processed for lysosomal fluorescence quenching and stained to localize the cis/medial Golgi complex (green). Accumulation of TRG in the cis/medial Golgi complex was undetectable at the untreated (C) and 15-min (D) time points. Colocalization was first detected at 30 min (E, arrows). By 60 min (F), colocalization was readily detectable (arrows). Bar = 20 µm.

Because permeabelizing cells to localize the Golgi complex with the FITC-conjugated lectin decreased the overall staining intensity of TRG, a study was undertaken to quantify TRG fluorescence at different points of uptake without permeabelizing the cells. The images from this study (Fig. 2, A-D, Total) exhibited a typical TRG staining pattern sequestered within punctate vesicular structures for cells showing total fluorescence at all time points [15 (A), 30 (B), 60 (C), and 120 min (D)] except for the 0-min time point that accumulated no TRG (data not shown). In cells processed for lysosomal fluorescence quenching (Fig. 2, A-D, quenched), the majority of the retained signal did not appear as bright punctate spots, as is typical of lysosomal accumulation. The signal was confined mainly around the perinuclear area and its staining pattern was comparable to Golgi complex morphology (Fig. 2 A-D, quenched, *). The quantitative data from this study (Fig. 3A) showed that a pool of nonlysosomal-associated TRG was detected within 15 min of accumulation in these nonpermeabelized cells, although this was detected by using laser emissions that were approximately threefold higher than those used for the 30-, 60-, and 120-min time points. This fluorescently weaker pool of TRG had an integrated intensity value of ~20% of that seen for total TRG fluorescence for the corresponding time point.


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Fig. 2.   Quantitative analysis that confirms a nonlysosomal-associated pool of TRG appears after 15 min of uptake. Images from LLC-PK1 cells incubated in media containing 2 mg/ml TRG for 15 (A), 30 (B), 60 (C), and 120 min (D) showing total fluorescence (total) and fluorescence after lysosomal-fluorescence quenching (quenched) were used to obtain quantitative fluorescence data (graph in Fig. 3A). No detectable fluorescence was seen at the zero time point (data not shown), where cells were briefly exposed to TRG. A nonlysosomal-associated pool of TRG was first detected at 15 min of exposure (A). Laser emissions needed to detect TRG accumulations for this pool were ~3 times higher that those needed to detect this same pool in lysosomal fluorescence-quenched cells with longer incubation periods. For all time points, images showing total fluorescence exhibited the typical staining pattern for TRG localized within punctate, vesicular structures. In the images showing the staining pattern in lysosomal fluorescence-quenched cells (A-D), the residual fluorescence appeared perinuclear in localization, and reminiscent of Golgi complex staining in cells (*). Bar = 20 µm.



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Fig. 3.   A pool of nonlysosomal-associated TRG was readily detectable after 15 min of exposure in unpermeabelized cells by using parameters described in MATERIALS AND METHODS. The images from Fig. 2 were quantified to yield a value for total integrated fluorescence (a product of the average number of spots, average spot size, and average spot intensity) ± SE, for both total fluorescence and cells with fluorescence resistant to quenching (A). Cells with fluorescence resistant to lysosomal quenching exhibited ~20% of the fluorescence seen in cells with total fluorescence throughout the span of uptake.

Having determined that a portion of the gentamicin internalized rapidly, trafficked to, and sequestered within the Golgi complex, we next set out to determine within which compartment of the Golgi complex accumulation of TRG occurred. The timeline diagram in Fig. 4 graphically demonstrates the protocol used to meticulously document different distributions of TRG, accumulated after 60 min of uptake (lysosomal vs. nonlysosomal associated) within the exact same cell, as well as colocalization with the two different Golgi complex probes. A group of cells showing total TRG fluorescence in Fig. 5A (inset) was processed for lysosomal fluorescence quenching and imaged (B) along with its NBD-ceramide counterpart (D). Next, the cells were imaged after membrane permeabelization (C) and stained with FITC-LCA (E). The dramatic decrease in the fluorescence within the same cells among Fig. 5, A-C demonstrated how effectively the lysosomal fluorescence quenching protocol worked. Certain portions of the Golgi complex-accumulated TRG was associated with a detergent-insoluble component (Fig. 5, B and C, cells 1 and 4, arrows), whereas others (Fig. 5, B and C, cells 2 and 3, arrowheads) lost a majority of their accumulation on detergent extraction. In general, both detergent-soluble and -insoluble accumulations of TRG showed a high degree of colocalization with both Golgi complex probes, suggesting accumulation occurred within both the TGN/trans-Golgi portions and cis/medial portions of the Golgi complex. Only cell 1 in Fig. 5B appeared to have localized all of the Golgi complex-associated TRG exclusively with the cis/medial portion as can be demonstrated by the lack of association with the NBD-ceramide marker in Fig. 5D (cell 1, arrow).


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Fig. 4.   The protocol used for the colocalization study is graphically demonstrated. Images were acquired after 60 min of TRG uptake, after quenching and staining with NBD-ceramide, and after permeabelization and staining with FITC-LCA. During each of the image-acquisition sessions, a reference brightfield image of the grid-etched marker was taken so colocalization comparisons could be made using the exact same field. DAB, diaminobenzidene.



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Fig. 5.   Colocalization experiments of total and lysosomal quenched TRG fluorescence demonstrate strong colocalization between residual TRG after lysosomal fluorescence quenching and both Golgi complex probes. LLC-PK1 cells imaged during the initial portion of the colocalization protocol exhibited typical lysosomal TRG accumulation after 60 min of exposure (A). After a group of cells showing total TRG fluorescence was processed for lysosomal fluorescence quenching (inset in A), lysosomal quenching dramatically decreased fluorescence of the same field, and residual staining was primarily localized to perinuclear structures in certain cells (B, cells 1, 2, 3, and 4, arrows and arrowheads). These residual structures localized with both the TGN/trans-compartment probe NBD-ceramide (D), and the cis/medial-compartment probe FITC-LCA (E, cells 1, 2, 3, and 4, arrows and arrowheads). On membrane extraction (C), portions of the residual TRG fluorescence seen in B were washed away (cells 2 and 3, arrowheads), yet other portions remained (cells 1 and 4, arrows). As with the membrane-bound accumulations, the detergent-insoluble pool of TRG colocalized with both Golgi complex probes, but to a greater extent with the FITC-LCA (D and E, cells 1 and 4, arrows). Bar = 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rapid uptake and accumulation of aminoglycosides within proximal tubule cells is responsible for the cascade of events leading to acute nephrotoxicity (1, 6, 21). Studies from multiple laboratories have shown cellular uptake occurs primarily through endocytosis after binding to acidic phospholipids and the multiligand receptor megalin (1, 9, 18, 23). Once internalized, the bulk of this pool is sequestered within the lysosomal compartment. In a previous study, we demonstrated accumulation of the aminoglycoside gentamicin in the Golgi complex within 8 h (15). Our present results indicate this accumulation occurs via direct trafficking of TRG to the Golgi complex from the surface membrane. This newly emerging pathway from the surface membrane to the Golgi complex is exciting because cellular effects secondary to aminoglycoside toxicity, such as a decrease in protein synthesis, occur quickly (8, 16, 17). These rapid intracellular effects are not likely attributable to sequestering of aminoglycosides within the lysosomal pool proceeded by subsequent rupture, release, and association with the protein-synthetic machinery because previous animal studies have demonstrated a lack of proximal tubular morphologic injury by light microscopy even after 2 days of gentamicin administration (2).

This study confirms and expands on data previously describing this new pathway of gentamicin trafficking (15). Here, we show trafficking occurs rapidly and directly to the Golgi complex and, through the use of lysosomal fluorescence quenching and image analysis, determines the sequestration kinetics of this pool. We hypothesized that gentamicin was directly targeted to the Golgi complex after endocytosis. To test this hypothesis, our first challenge was to delineate the earliest time point when gentamicin could be detected within the Golgi complex. Using a highly purified TRG fluorescent probe, with a 1:1 ratio of TRG, we were able to detect trafficking to the Golgi complex within as little as 30 min via direct colocalization with a specific Golgi complex marker. Even earlier detection was possible at 15 min in unpermeabelized cells by comparison of the staining pattern to established Golgi complex morphology. These data strongly suggest a pathway exists that directly shuttles aminoglycosides, after cellular uptake, to the Golgi complex where it can affect mechanisms such as protein sorting and protein synthesis. In our previous study, we were probably unable to detect a TRG-Golgi association at early time points because of the previous TRG probe's low fluorescence stoichiometry and the overwhelming intensity emanating from lysosomal accumulations of TRG. Detecting and quantifying dimly fluorescent objects in the presence of more intense structures are extremely problematic. Setting acquisition parameters to detect the dimmer objects generally causes oversaturation of detectors (20). The resulting images have no discernible details, just a sea of bright white signal. Therefore, utilization of a protocol for quenching TRG fluorescence emanating from the lysosomal pool was crucial for our studies (1, 11, 14).

We next set out to determine where within the Golgi complex gentamicin trafficked. The two markers used, NBD-ceramide and FITC-LCA, have been extensively characterized and binding has been established within the TGN/Trans compartments and cis/medial compartments of the Golgi complex, respectively (12, 13). As expected, the two markers exhibited some degree of overlap in certain cytosolic regions around the nucleus; however, they also revealed distinct staining areas. Without three dimensional rendering it was difficult to determine which of the Golgi-complex markers occupied the greatest region. Accumulations of detergent-soluble and -insoluble pools of TRG seemed to accumulate equally among the two Golgi complex probes, suggesting a steady retrograde transport of the aminoglycoside through the Golgi complex occurred after 60 min of uptake. It is possible that this steady transport of TRG continues not only through the Golgi complex but may reach the ER. However, in the present study, we were unable to detect any appreciable accumulations of TRG within the ER, as fluorescence from this pool is most likely undetectable via confocal fluorescence microscopy at these short time points.

A mechanism for retrograde transport of toxins after endocytosis from the Golgi complex to the ER has been recently reported for other toxins. Both Shiga and Ricin toxins exploit this pathway, and their toxicity is dependent on accumulation within the ER and subsequent release into the cytosol via the same mechanisms that allow small glycopeptides to translocate the ER (8). The unique structure of gentamicin, an aminoglycoside composed of three O-linked aminated carbohydrate moieties, may be best suited to utilize all of the mechanisms mentioned above. Its carbohydrate component may be responsible for the retrograde transport to components throughout the Golgi complex. Whether or not further retrograde movement to the ER occurs, the aminoglycoside is in a position to directly affect protein synthesis or may conceivably become expelled into the cytosol via the mechanisms that translocate small glycopeptides into the cytosol (8). Detectability of TRG, either ER associated or free within the cytosol, through fluorescence microscopy or biochemical techniques involving cell fractionation, would be extremely problematic because of its low concentrations.

In conclusion, through the use of a fluorescent probe with a markedly improved fluorescence, and a protocol that eliminates fluorescence from the lysosomal pool, we demonstrated and quantified rapid association of gentamicin with the Golgi complex consistent with direct trafficking from the surface membrane. Moreover, the use of compartment-specific markers to the Golgi complex indicated that accumulation of TRG occurs within all the components of the Golgi complex. Having reached this compartment via direct retrograde trafficking within such a short period of time, it is conceivable that transport to the ER follows where disruption of protein synthesis could then occur.


    ACKNOWLEDGEMENTS

These studies were supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-41126), and the Veterans Affairs Research Service.


    FOOTNOTES

Address for reprint requests and other correspondence: B. A. Molitoris, Indiana Univ. School of Medicine, 1120 South Dr., Fesler Hall Rm. 115, Indianapolis, IN, 46202 (E-mail: bmolitor{at}iupui.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 May 2000; accepted in final form 12 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 279(5):F884-F890
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