Sprague-Dawley Rats Treated with

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1 The Use of Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis to Detect Renal Damage in Sprague-Dawley Rats Treated with Gentamicin Sulfate* GERALD J. KOLAJA, DONALD A. VANDERMEER, WILLIAM H. PACKWOOD, AND PAUL S. SATOH The Upjohn Company, Drug Safety Research and Clinical Research Laboratories, Kalamazoo, Michigan Renal toxicity is a common manifestation to the exposure of laboratory animals and humans to a wide range of xenobiotics. Traditional methods for evaluating renal damage by clinical chemistry such as blood urea nitrogen (BUN) and serum creatinine are not sensitive to early, mild changes. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to measure the molecular weight spectrum of urinary proteins allows for an evaluation of the functional changes associated with renal damage. The ability of the kidney to filter and reabsorb proteins is related to the functional ability of glomeruli and the proximal tubules. Gentamicin sulfate produces injury to the S-1 and S-2 segments of the proximal tubule in laboratory animals and humans. While severe damage to the tubules is associated with increased BUN, serum creatinine, and N-acetyl-β-glucosiminadase (NAG), mild injury is not detected by these means. The evaluation of urinary proteins by SDS-PAGE demonstrated renal toxicity at a dose of 6 mg/kg after 2 days of sc treatment. The NAG: creatinine ratio was shown to be elevated after 2 days of treatment at 63 mg/kg. The use of SDS- PAGE as described in this paper provides a sensitive method for detecting renal injury. Keywords. Urinary proteins; proximal tubule cell necrosis; α 2-microglobulin INTRODUCTION Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been used to evaluate proteinuria associated with renal disease in humans (1, 14) and animals (3, 18). The molecular weight (Mr) distribution of urinary proteins is an important indicator as to the site and extent of renal damage (1, 15). High molecular weight proteins (>69,000 Mr) are associated with glomerular damage, whereas an increase in low molecular weight proteins (12,000-60,000 Mr) indicates damage to the tubules. The classification of renal injury related to the molecular weight pattern of urinary proteins has recently been reported in human renal disease (1, 14). The lengthy procedure for concentrating urine and the possibility of protein loss or modification during dialysis has limited the use of SDS-PAGE for routine use in toxicology. Recent advances in SDS-PAGE techniques such as the availability of commercially produced precast gels and improved * Address correspondence to: Dr. Gerald J. Kolaja, Drug Development Toxicology II, The Upjohn Company, 333 Portage Street, , Kalamazoo, Michigan staining procedures (1, 4, 13, 14) make it more accessible for use in the evaluation of renal effects in laboratory animals treated with xenobiotics. The use of nonconcentrated urine in this technique provides a rapid, accurate assessment of the molecular weight range of urinary proteins. Gentamicin and other aminoglycosides produce damage in the proximal tubules in laboratory animals (2, 5, 6) and humans (7, 10, 12, 16). Injury occurs primarily in the S-1 and S-2 segments of the proximal convoluted tubules. The severity of renal pathology is dose-dependent, ranging from mild tubular injury at low doses to severe renal disease accompanied by increases in blood urea nitrogen (BUN), serum creatinine, marked tubular necrosis, and death at higher doses. The detection of early, mild renal damage is not possible using standard clinical chemistry tests such as creatinine and BUN (3). The purpose of the present study was to evaluate the renal effects of gentamicin sulfate in Sprague- Dawley rats. The molecular weight range of proteins in the urine as assessed by SDS-PAGE was determined and correlated with clinical chemistry, rou-

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3 605 tine urinalysis evaluation, urinary N-acetyl-{1-glucosiminadase (NAG), and morphologic evaluation of the kidney. Dose levels that produced minimal, mild, or severe lesions were chosen. MATERIALS AND METHODS Four male and 4 female Sprague-Dawley rats were given 0 (saline control), 6, 16, and 63 mg/kg/day (BID) of gentamicin sulfate. Rats were treated for 4 consecutive days. Urine for routine urinalysis, NAG, and SDS-PAGE was collected prior to the first dose, on study day 2, and immediately prior to necropsy on study day 5. NAG was performed by standard laboratory methodology using a Dynatech MR 600 microplate reader. Results of the NAG analysis were evaluated using the paired t-test. Blood samples for clinical chemistry evaluation were collected by cardiac puncture prior to necropsy. Creatinine and BUN measurements were performed on the Coulter DA- COS analyzer. Metofane was administered in a closed chamber to induce anesthesia prior to bleeding and necropsy. Kidneys were collected at necropsy, fixed in 10% neutral-buffered formalin, processed, and embedded in methylmethacrylate. Kidneys were then sectioned at 1 Am and stained with hematoxylin and eosin. Rats were housed individually in stainless-steel cages, fed Purina rodent chow ad libitum, and given free access to water for the entire study period except for times when they were housed in metabolism cages for urine collec- were collected for 3 hr in vials tion. Urine specimens containing sodium azide (0.05% minimum final concentration). The collected urine was centrifuged at 3,000 x g for 10 min to remove particulate materials and stored frozen at - 20 C. The urine specimens were analyzed within 2 wk. Studies in our laboratory have shown the urine to be stable with these storage conditions. To evaluate urinary proteins, SDS-PAGE was performed using a modification of the techniques described by Weber (17), Matsudaira and Burgess (9), and Laemmli (8). Daiichi-sodium dodecyl sulfate polyacrylamide density gradient Mini GELS (4-20%), colloidal Coumassie blue (ProBlue~), and silver staining 1119 kits were obtained from Integrated Separation Systems (Natick, MA). Undiluted urine samples were treated with an equal volume of SDS-mercaptoethanol buffer (8) for 90 sec at 100 C. Ten III of the SDS-treated urine and standard protein markers ( kda: 2 ~g/ 10 ~1) were electrophoresed for 2.5 hr at 170 V constant voltage (30 ma/gel). After completion of the electrophoresis, the gel was fixed in 12% trichloracetic acid and stained first in ProBlue, followed by the silver staining II kits. The location of the mobility equivalent to that of albumin and its absorbance typically was used as the calibration point based on the electrophoretogram of the standard protein markers. The stained gel was then scanned in an Ultroscan XL@ laser densitometer (LKB-Pharmacia, Piscataway, NJ). The graphic data, collected in absolute binary signals, were converted to ASCII using Pharmacia s conversion utility GELCON.EXE, and the data were statistically evaluated using a LOTUS (Lotus Development) spreadsheet. Data were statistically evaluated by the paired t-test and p < was considered statistically significant. RESULTS Male and female rats that received gentamicin sulfate had dose-related morphologic alterations in the proximal convoluted tubules comparable to those previously described (5). At the lowest dose (6 mg/ kg), there was minimal focal necrosis of the proximal tubules. The rats receiving 16 mg/kg of gentamicin sulfate for 4 days had similar but more prominent histologic lesions. Rats receiving 63 mg/kg of gentamicin sulfate had widespread necrosis of the proximal convoluted tubules and histologic evidence of tubular regeneration. Evaluation of clinical chemistry tests performed after 4 days of dosing showed few indications of renal damage. BUN and serum creatinine levels were not elevated in any dose group. Other indices such as serum electrolytes, glucose, triglycerides, and serum chemistries were within the normal range for Sprague-Dawley rats at our laboratory. An increase in the NAG: creatinine ratio was seen in the highdose group at study day 2, and at day 5 showed a 3-fold increase (Table I). The results of the evaluation of the measurement of urinary proteins by SDS-PAGE are shown in Fig. 1. Both male and female control rats showed an increase in 16,000-Mr proteins during the course of the study. The increase in the male rats was greater than that of female rats. This protein is the sexlinked o 2-microglobulin, which is produced in larger amounts in male rats. In the low-dose group, both - FIG. 1.-Measurements of urinary proteins by SDS-PAGE. Control: The molecular weight range of urinary proteins from control rats from day 0 through day 5 is shown. Low dose: The rats receiving the low dose of gentamicin demonstrate an increase in 16,000- and 20,000-Mr proteins. Medium dose: A larger increase in the 16,000- and 20,000-Mr proteins is seen in the medium-dose rats at days 2 and 5. The increase in male rats is more pronounced than in females. High dose: Rats receiving the high dose of gentamicin show increased levels of low molecular weight proteins, although not as marked as in the medium-dose group.

4 606 TABLE L-Urinary NAG/creatinine from gentamicintreated rats. n = 8. ± = standard deviation. * Significant at p < male and female rats showed an increase in the 16,000-Mr protein at day 5 and 20,000-Mr proteins at day 2. In the medium-dose group, both male and female rats showed an increase in 16,000- and 20,000-Mr proteins at days 2 and 5. Proteins with a molecular weight of 26,000 were also increased at day 2. The high-dose group showed an increase in 16,000- and 20,000-Mr proteins in male and female rats at days 2 and 5 and an increase in the 26,000- Mr proteins at day 5. The increase in these proteins in the high-dose rats was not as great as that seen in the medium-dose group. DISCUSSION The results of this study indicate that the evaluation of urinary proteins by SDS-PAGE provides a sensitive indicator of proximal tubule damage in gentamicin-treated rats. In the absence of immunochemical identification of protein bands, the following proteins can be listed as potential urinary proteins attributed to these peaks: Tamm Horsfall glycoprotein (80 kda), albumin (68 kda), transferrin (72 kda), y-globulin chains (40-50 kda), almicroglobulin (26-33 kda), retinol binding protein (21.4 kda), and a2-microglobulin (16-22 kda). Some proteins such as retinol binding protein and.b2-microglobulin were too low in concentration or low in molecular weight to assess accurately in this study. Elevation of low molecular weight proteins between 16,000 and 26,000 have been reported previously to be an indication of tubular damage (1, 3, 11). In this study, this elevation of low molecular weight urinary proteins correlated with the histologic appearance of proximal tubule necrosis. The detection of renal injury by SDS-PAGE was more sensitive in detecting renal damage than standard clinical chemistry techniques such as BUN, serum creatinine, and NAG. BUN and serum creatinine were not elevated in this study; NAG showed a small increase in the high-dose group at day 2 and a 3-fold increase at day 5. SDS-PAGE detected changes that correlated with morphologic changes in the lowdose group after 4 days of dosing. The elevation of low molecular weight urinary proteins was less in the high-dose group rats than that seen in the medium-dose group. This finding is not explained by this study but may be due to a decrease in glomerular filtration rate in rats that had more extensive proximal tubular necrosis. The most likely source of low molecular weight proteins in the urine following gentamicin toxicity is the reduced capacity of the proximal tubules to reabsorb protein (1, 3). Other possible sources of elevated urinary proteins include proteins from necrotic tubular cells and extra renal sources such as proteins from neoplasms or extra renal necrosis (1). The amount of filtered protein in the urine is directly related to the glomerular filtration rate. When this rate is altered, either due to systemic vascular effects or severe tubular necrosis, alterations in filtered proteins may occur (3). The technique for performing SDS-PAGE to measure urinary proteins has been substantially improved. Double staining of the gel with colloidal Coumassie dye and silver stain allowed detection of as many as 35 protein bands. Interassay precision of electrophoretic mobility of proteins was <4% confidence of variance (CV; CV SD/mean = x 100), whereas that of peak heights by densitometry was < 15% CV. The use of nonconcentrated rat urine that has not been dialyzed reduces the time to prepare samples and obtain results as well as avoids loss or modification of protein by dialysis and concentration. Using the 6-gel device (ISS Corporation), 66 specimens can be assayed in 3 hr or 142 specimens assayed in 1 working day. SDS-PAGE provides a means for detecting and following early, mild renal damage in laboratory animals. Combined with standard clinical chemistry procedures, histopathology, the evaluation of renal blood flow, and glomerular filtration rate, SDS-PAGE provides a highly efficient and sensitive evaluation of renal toxicity. ACKNOWLEDGMENT We wish to acknowledge Ms. Denise Frailey for on the sam- performing the SDS-PAGE procedure ples in this study. REFERENCES 1. Bernard A and Lauwerys RR (1991). Proteinuria: Changes and mechanisms in toxic nephropathies. Crit. Rev. Toxicol. 21: Bernard A, Viau C, Ouled A, Tulkens P, and Lauwerys R (1986). Effects of gentamicin on the renal uptake of endogenous and exogenous proteins in conscious rats. Toxicol. Appl. Pharmacol. 84: Bovee KC (1986). Renal function and laboratory evaluation. Toxicol. Pathol. 14: Halman J, Miller J, Fowler JSL, and Price RG (1986). Renal toxicity of propyleneimine: Assessment by noninvasive techniques in the rat. Toxicology 41:

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