Ethanol-Its Nephrotoxic Effect in the Rat David H. Van Thiel, MD, William D. Williams, Jr., MS, Judith S. Gavaler, BA, Joanna M. Little, BA, Larry W. Estes, MS, and Bruce S. Rabin, MD, PhD
The nephrotoxic effect of ethanol feeding on renal structure and function was evaluated in rats and compared to that in dextrose-fed isocaloric control animals. Alcohol-fed animals had larger kidneys than their controls. Despite this increase in renal mass, the alcohol-fed animals had a 50% reduction in creatinine clearance and a 67% reduction in osmolar clearance compared to their controls. When specific renal constituents were compared, the alcohol-fed animals were found to have twice the renal protein and a 50% increase in renal lipid. Despite these marked structural and functional differences, the light microscopic appearance of the kidneys of the two groups did not appear significantly different. In contrast, the electron microscopic differences were sub-stantial. The renal epithelial cells, particularly of the distal tubules and Henle's loops, were found to show varying degrees of cellular injury and were observed to be sloughing into the lumens. These electron microscopic observations are similar to those obtained in tubular necrosis due to a variety of nephrotoxic agents. We propose, therefore, that chronic alcohol feeding of rats produces significant renal dysfunction and abnormalities of structure such that ethanol should be considered a true nephrotoxin. (Am J Pathol 89:67-84, 1977)
ALCOHOL recently has been demonstrated to be toxic to a varietv of extrahepatic tissues. These include the heart,' the nervous system,2 the bone marrow, 3 the skeletal muscle, and the gonads.5 Unlike the wvell-studied hepatotoxic effects of alcohol, the pathogenesis of these recently recognized extrahepatic abnormalities, their rates of development, and their potential for reversibility are poorly defined. NMuch of our current understanding of the morphologic and toxic biochemical effects of ethanol on the liver wvere preceded by electron microscopic studies. The initial electron microscopic demonstration of swollen mitochondria,>'2 increased smooth endoplasmic reticulum,8-10,13.14 and focal cvtoplasmic degeneration -12 ,14,15 in animals and in men fed alcohol led directly to subsequent studies which defined the chronology of these morphologic abnormalities,8",0" 4 the failure of low-fat and high-protein diets to prevent their development,8 and the biochemical consequences of their presence. Accordingly, we have attempted to investigate the effect of ethanol on From the Division of Gastroenterology. Department of \Medicine. and the Department of Patholog\-. Universitv of Pittsburgh School of Medicine. Pittsburgh. Pennsylvania Suipported by Grant AA-01450 from the National Institutes of Health Accepted for publication May 1:3. 1977 Address reprint requests to Dr David H Van Thiel. IOOOC, Scaife Hall. Division of Gastroenterolov. Department of Medicine. L niversity of Pittsbureh. School of Medicine. Pittsburgh. PA 15261 67
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renal structure and function in pair-fed rats receiving similar diets with identical fluid, electrolyte, lipid, protein, and caloric content. The diets differed only in the number of calories presented as carbohydrates or as ethanol isocalorically substituted for each other.5 16 Materials and Methods Animals
Forty male Wistar rats age 24 days and matched for weight (mean ± SEM, 57.7 ± 0.5 g; range, 50.0 to 63.5) were obtained from Charles River Breeding Laboratories, North Wilmington, Mass. The animals were housed in individual metabolic cages and pair-fed a li(quid diet containing alcohol or an identical liquid diet in which ethanol was isocaloricallv replaced with dextrose. The diet containing alcohol consisted of 5% ethanol by volume which accounted for 36% of the total caloric content.16 On the average the animals ate 47 i 2.0 cu cm diet/day. Twenty-four hours prior to sacrifice, a stool-free 24-hour urine collection was obtained from each animal. Animals were weighed and then sacrificed by exsanguination under light ether anesthesia. The left kidney of each animal was weighed immediately after removal for determination of wet renal weight. It was then freeze-dried for 72 hours and reweighed. Prior control experiments have shown that dry renal weight does not change after 36 hours of freeze-drying. Homogenates were prepared from these freeze-dried tissues and used for subse(luent renal constituent analysis. The right kidney of each animal was removed and bisected longitudinallv in a frontal plane; half was then fixed in Bouin's solution for light microscopy. The other half was fixed in glutaraldehvde, osmiuim tetroxide, or Karnovskv's fixative for subse(quent electron microscopic analysis. Physiologic Studies
Using urine samples collected for the 24 hours prior to sacrifice and serum obtained at the time of sacrifice, renal free water, osmolar, and creatinine clearances were determined using standard methods.17 Serum and urine osmolaritv were determined by freezing point depression. Serum and urine creatinine content were determined according to the method of Chasson.18 Blood pH and alcohol content 19 as well as serum electrolyte 20 and urea nitrogen 21 were determined using standard techni(quies. Renal Constituent Analysis
Renal protein and DNA content were determined utilizing the saline homogenates prepared from the freeze-dried left kidney according to standard methods.22'23 Standard solutions were prepared from bovine serum albumin obtained from Calbiochem, Spring Valley, N.Y. and calf thvmus DNA obtained from Sigma Chemical Co., St. Louis, Mo. The renal homogenates were extracted according to the method of Folch24 and analyzed for total lipid, cholesterol, and phospholipid levels using previously described methods.24 26 Light Microscopy
After fixation in Bouin's solution for 72 hours, the tissues were dehydrated in progressive ethanol solutions, embedded in paraffin, sectioned at 5 ,, and stained with hematoxvlin and eosin for light microscopy. Slides were coded and read blindlv after all of the studies were completed. The glomeruli, proximal and distal tubules, loops of Henle, collecting ducts, renal arteries and veins, and the interstitial tissues for each animal were each individually assessed and graded (on a sale of 1 to 4) for degree of variation from the histologic appearance of kidneys obtained from 20 control animals of the same age maintained on a standard rat chow diet and fed ad libitum.
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Small pieces of the right kidney were fixed in 3.0% glutaraldehyde (Ladd Research Industries Inc., Burlington, Vt.) in a 0. 1 M cacodvlate buffer (pH 7.4), Karnovskv's fixative (half strength)"7 in cacodylate buffer of I% osmium tetroxide in 0.1 M cacodylate buffer from 2 hours at 4 C. Due to the large number of samples taken, the tissues may have remained in the fixative for as long as 3 weeks under refrigerated conditions. Only the samples that were fixed in buffered osmium tetroxide were processed immediately after fixation. Urinarv sediments from 6 animals (3 alcohol-fed rats and 3 isocaloric controls) were obtained for microscopy bv aspirating the urinarv bladder, thus avoiding any fecal contamination. The urines were centrifuged at 2,000 rev/min in an International Clinical Centrifuge for 5 minutes to remove any intact cells and then centrifuged for an additional 60 minutes at 105,000g. The final urine pellets were fixed ovemight in 3% glutaraldehvde in 0. 1 M cacodvlate buffer at 4 C. After primarv aldehvde fixation, all samples were washed for 1 hour in 0.1 M cacodylate buffer and postfixed in I% osmium tetroxide in 0.I NI cacodvlate buffer for 2 hours at room temperature, rinsed, dehydrated in a graded ethanol series, and embedded in Epon-Araldite. After polymerization, silver sections were cut with glass or diamond knives on a Sorvall MT 1 microtome, mounted on uncoated 200- or 300mesh grids, and stained with 3% uranvl acetate n and lead citrate." Grids were examined and photographed at 60 kV with a Philips EM 200 or EM 300 utilizing a double condenser system. A magnification calibration grid was photographed at various times during the studvT.
Experimental results are expressed as the mean ± standard error. The P values were obtained by use of the Student t test for the difference between the two group means. Values were considered probably significant at P < 0.05 and significant at P < 0.01.
Results Ar*alms
Both the alcohol-fed and the isocaloric control animals (Table 1) grew steadily during the study period.5 The alcohol-fed animals consistently weighed less but had larger kidneys than the isocaloric controls. In contrast to the greater wet renal weight, the drv renal weights did not differ between the two groups. When the dry renal weights were corrected for body weight, however, the alcohol-fed animals clearly had a greater renal mass per gram body weight than did the isocaloric controls. Table 1 -Body and Renal Mass Parameters
Alcohol-fed animals (N = 20)
Bodyweight(g) Wet renal weight (g) Wet renal weight (g)/body weight (100 g)
Dry renalweight(g) Dry renal weight (g)/body weight (100 g) P < 0.01.
101.2 0.87 0.86 0.188 0.15
± 3.7* ± 0.01' ± 0.03* ± 0.011
±0.001*
Isocaloric controls (N = 20) 113.0 0.51 0.45 0.175 0.10
± 3.7 ± 0.02 ± 0.01
±0.007 ± 0.01
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Physiologic Studies
The serum creatinine levels (Table 2) of the alcohol-fed animals were 50% greater than those of the isocaloric controls. Despite this difference in the levels of serum creatinine, the amount of creatinine excreted in 24 hours was similar for both groups. Therefore, the creatinine clearance was reduced in the alcohol-fed animals by 50%. In addition to the observed reduced creatinine clearance, the alcohol-fed animals had a reduced osmolar clearance compared to the isocaloric control animals. This reduction in osmolar clearance occurred despite the presence of a significant hypernatremia and hyperosmolarity in the alcohol-fed animals. Consistent with this reduction in renal function, the alcohol-fed animals had a metabolic acidosis associated with a significant anion gap which probably is due, in part, to an associated lactic acidosis, known to occur in cases of chronic alcohol ingestion.30 Despite these marked metabolic differences between the alcohol-fed and the isocaloric control animals, no significant difference was observed for serum uric acid content or blood urea nitrogen. As might be expected, however, the alcohol-fed animals had lower blood glucose levels than did the isocaloric controls. Renal Constituent Analysis
Renal tissue protein, lipid, and DNA (Table 3) were increased significantly in the alcohol-fed animals compared to their isocaloric controls. In addition to the expected increase in absolute levels of triglyceride,31 the levels of the other major classes of renal lipids were also markedly elevated. Table 2-Physiologic Differences Between Alcohol-Fed Animals And Isocaloric Controls Alcohol-fed animals (N = 10)
Creatinine (mg/dl) Urine creatinine (mg/24 hrs) Creatinine clearance (cu cm/min) Osmolar clearance (liter/24 hrs) Serum sodium (mEq/liter) Serum osmolarity (mOsmol/liter) pH Anion gap (mEq/liter) Blood alcohol (mg/dl) Uric acid (mg/dl) Blood urea nitrogen (mg/dl) Blood sugar (mg/dl) *
P < 0.01.
t P < 0.05.
0.91 7.6 0.6 0.042 54 329 7.23 36 112 3.7 15 92.7
± 0.06* ± 1.3 ± 0.1 * ± 0.010* ± 2* ± 3* ± 0.04* ± 1* ± 24* ± 0.3 ±1 ± 6.2t
Isocaloric controls (N = 10) 0.62 ± 0.04 10.5 ± 1.1 1.2
0.120 146 301 7.39 17 0 3.9 13 120.5
± 0.2 ± 0.025 ±1 ±4 ± 0.03
±2 ± 0.3 ±1 ± 9.3
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Sbta Lght No difference was observed between the appearance of the renal arteries and veins of the alcohol-fed animals and the isocaloric control animals. The distal tubules in the alcohol-fed animals appeared dilated, with markedly flattened epithelial cells, while the distal tubules in the isocaloric control animals appeared normal. Proximal convoluted tubules in the alcohol-fed animals appeared normal, with the exception of some eosinophilic staining material which was occasionally present in the lumen. This material was initially interpreted as staining artifact at the light microscopic level. Subsequent electron microscopic studies clearlv show this material to be sloughed epithelial cell cytoplasm and organelles. In all cases, the proximal convoluted tubules of the isocaloric control animals were normal. A marked interstitial edema was present in the medulla of the alcohol-fed animals, with wide separation of the individual renal tubules and a loose endematous appearance of the intervening connective tissue compared to that of the isocaloric control animals. El
M1'
SWis
Although the observed differences at the light microscopic level were not marked, it is apparent that the kidneys of the alcohol-fed animals have a different histologic appearance at the electron microscopic level compared to the appearance of kidneys from the isocaloric controls. These differences are especially striking in the renal cortex at the corticomedullarv junction; prominent differences involve the entire nephron, excluding the glomerulus. The glomeruli of kidneys obtained from the alcohol-fed animals, as well as those of the isocaloric controls, appeared normal. The glomerular capillaries and basement membranes appeared normal. Likewise, the slit membrane between podocytes and the fenestrated capillary endothelia appeared intact. No distinct changes were seen in the juxtaglomerular apparatus. Table 3-Renal Constituent Analysis Alcohol-fed animals (N = 10) Protein (mg/i 00 g body weight) Total lipid (mg/100g body weight) Cholesterol (mg/i00 g body weight) Phospholipid (mg/i00 g body weight) DNA (g/l00 g body weight) *
P < 0.01.
1 12.0 21.7
± 8.3* ± 3.31 0.028 ± 0.001* 1.082 ± 0.005* 1.34 ± 0.10*
Isocaloric controls (N = 10) 68.6 ± 4.4 14.86 ± 2.39 0.009 ± 0.001 0.356 ± 0.006 0.80 ± 0.05
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The epithelial cells of the proximal convoluted tubules exhibited marked cytoplasmic swelling with disruption of the brush border (Figure 1). Nuclei, mitochondria, and ribosomes were visualized pushing past the microvillus border filling the lumen as a consequence of cellular swelling. Fragmentation and vesiculation of the cisternae of the rough endoplasmic reticulum (RER) and swelling of mitochondria occurring in association with the presence of an electron-dense matrix were also frequently seen (Figure 2). There was no apparent proximal tubular luminal dilation independent of that caused by the mechanical distension produced as a result of the proximal epithelial cell swelling. The thin segment of the loop of Henle showed more discrete cytoplasmic changes. In the alcoholic kidney, the thin cytoplasm formed numerous excrescences which projected into the lumen. These excrescences were easily distinguished from stubby microvilli seen in the control kidney. Unusually thin epithelial cytoplasm was frequently seen detached from the basement membrane of the loop of Henle and sloughed into the lumen. In some cases, the nucleus of the tubular epithelial cells was flattened. Fibroblasts infiltrated the interstitium and deposited excess collagen diffusely along the basement membranes. The distal convoluted tubule showed the most remarkable ultrastructural changes of the entire nephron. The distal epithelial cells demonstrated a progressive change or necrotic metamorphosis (Figures 3-7). To simplify this description, the progression that occurs as a result of alcohol feeding has been divided arbitrarily into five stages. The first-stage distal tubular lesion was manifested as a grossly dilated tubular lumen with flattened epithelial lining cells (Figure 3). At this stage, the mitochondria and the number and appearance of the microvilli appeared normal. No unusual fibroblastic activity or peritubular fibrosis was observed. The second-stage lesion (Figure 4) showed a proliferation of the rough endoplasmic reticulum. The basement membrane appeared minimally thickened due to increased collagen deposition by adjacent fibroblasts. The third-stage lesion (Figure 5) showed the development of cytoplasmic epithelial cell protuberances which project into the lumen. These resembled exocytosing or sloughing microvilli. Particularly noticeable at this stage was the vesiculated rough and smooth endoplasmic reticulum and ballooning of the mitochondria. Lysosome-like bodies often appeared packed with membrane-bounded vacuoles. Abundant collagen fibers could be seen underlying the basement membrane (Figure 5). The fourth-stage lesion (Figure 6) exhibited further dilation of the rough and smooth endoplasmic reticulum that contributed to the forma-
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tion of numerous large cvtoplasmic excrescences. Blebs of cy7toplasm containing ribosomes were seen in the lumen. In the end-stage lesion (Figure 7), the distal tubular epithelial cells had completely necrosed. The basement membrane, however, did not appear to be irreversiblv damaged. The most proximal regions of the collecting ducts appeared to be affected bv alcohol in a manner similar to the distal tubules; progressive lesions were seen ranging from dilated ducts to end stage necrosis. Cells of the more distal regions of the collecting ducts and the ducts of Bellini appeared virtually unaffected bv the alcohol. The lumens of these distal regions, however, were often filled with cellular debris including fragtents of cytoplasm, free ribosomes, mitochondria, and nuclear material (Figure 8). Interstitial and perivascular fibrosis were seen about the collecting ducts (Figures 9 and 10). The earliest evidence for this new collagen formation was the local accumulation of fibroblasts. Subsequently, excess collagen accumulated around blood vessels and then spread into the interstitial tissue. Examination of urine sediments (Figure 11) collected from the bladder of alcohol-fed animals prior to sacrifice confirmed the presence of cellular debris, secretory vesicles, free membranes, and chromatin-like material within the urine as suggested by the electron microscopic appearance of the collecting ducts (Figure 8). Urine collected from control animals contained no cellular debris.
Discussion Known causes of tubular necrosis include severe trauma with shock, crushing injuries, bums, and a variety of poisons. Commonly recognized nephrotoxins include ethylene glycol, organic mercurials, arsenic, carbon tetrachloride, and certain antibiotics.32 In addition to these recognized nephrotoxins, other more frequentlv used substances are potentially nephrotoxic.l" u- In particular, Merrill suggests that excessive use of ethyl alcohol may be associated with renal failure.'4 He concludes, however, that the exact role of ethanol as a nephrotoxin remains unclear. The marked potentiation of carbon tetrachloride nephrotoxicitv by the concomitant use of ethanol is a well-established fact that provides some support for a potential nephrotoxic action of alcohol.-" In light of the evidence herein presented for biochemical and ultrastructural abnormalities induced by alcohol on kidney parenchyma, it seems reasonable to attempt to relate this pathology to that observed in other examples of tubular necrosis due to established nephrotoxins. Bio-
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chemically, azotemia, oliguria, and reduced creatinine clearance are indications of renal failure. Dilation of the distal tubules with flattening of the epithelial cells and subsequent necrosis observed in the alcohol-fed animals is histologically consistent with lesions reported to occur in tubular necrosis due to other causes.32 Moreover, since only the alcohol-fed animals demonstrate these changes, it is reasonable to assume that alcohol ingestion may have been responsible for tubular necrosis. The increase in plasma osmolarity observed for the alcohol-fed rats can be entirely explained on the basis of the observed blood alcohol levels, which would be expected to add 25 to 30 mOsmol to the measured osmolarity. The significant increase in serum sodium levels observed in the alcohol-fed animals may in part be artifactual and due to the smaller volume of distribution of the sodium ion in the lipemic alcohol serum. Of interest is the failure to observe an increase in the serum uric acid concentration of the alcohol-fed animals; chronic alcohol administration in man almost always increases the uric acid concentration. It must be stated, however, that it is unusual to observe either clinical renal failure or azotemia in alcoholics. When renal failure does occur in alcoholics, it almost always occurs in the presence of severe liver disease, which is known to produce alterations in renal circulatory dynamics. We have yet to establish such changes in this animal model. Based on the increased total renal weight and specific constituents, we had proposed that chronic alcohol feeding has induced renal hypertrophy.37 Based upon our present ultrastructural studies, however, this increase in renal weight is almost probably due both to synthesis of new cells and to loss of cells in the lumen of the kidney, presumably as a consequence of an alcohol-induced injury. Obviously, since considerable amounts of cellular debris remain in the tubular lumens, it must contribute significantly to the weight increase observed for the total kidneys. Effective evaluation of the renal pathology induced by ethanol requires comparison of the alcohol-induced lesion with that of some other wellaccepted nephrotoxins. Grossly, the kidneys obtained from the alcohol-fed animals and the animals with nephrotoxic tubular necrosis are enlarged and swollen. Moreover, presumably because of the presence of considerable interstitial edema, the cortex of both alcohol-fed animals and animals with nephrotoxic tubular necrosis is often pale compared to the cortex of isocaloric control animals and normal animals. The gross pathology of the kidneys obtained from the alcohol-fed animals is, therefore, similar to that reported to occur in typical nephrotoxic tubular necroSiS 14,32,38
In addition, in classic tubular necrosis and in the present model, the glomeruli are normal at both the light and electron microscopic lev-
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els. 1432'" Moreover, in classic tubular necrosis the proximal tubules show few if any changes at the light microscopic level. Hydropic degeneration and luminal dilation have been reported but are not uniformly present.'432'8 With the electron microscope, mitochondrial swelling, focal loss of the brush border, and the development of vacuoles are seen." In contrast, the distal tubules in classic tubular necrosis show extensive cell necrosis, cell drop-out, and dissolution of the basement membrane. 14'32,' As in the case of classic tubular necrosis, the kidneys obtained from the alcohol-fed animals show little change at the light microscopic level. At the electron microscopic level, however, marked alterations in renal structure are apparent. In the proximal convoluted tubules, structural integrity is sacrificed by swelling, which being limited by the basement membrane, pushes the cell inward, filling the lumen and disrupting the brush border. In addition, the mitochondria are frequently swollen and vacuolated. Despite these changes, no definite necrosis is seen. Although there are reports of necrosis in the thick limbs of the loop of Henle occurring in classic tubular necrosis, the thin loop seems to be relatively spared. In contrast to this classic picture, the cells lining the loops of Henle obtained from the alcohol-fed animals demonstrate obvious abnormalities with irregular cytoplasmic excrescences which extend into the lumen and suggest cellular necrosis. The distal tubules in classic tubular necrosis show luminal dilation, focal cell loss, and extensive necrosis. 4,323" Our light and electron microscopic studies in kidneys obtained from alcohol-fed animals demonstrate similar abnormalities. These include cell flattening, mitochondrial swelling, proliferation and dilation of the rough and smooth endoplasmic reticulum, the development of a microvillus border, and formation of multilocular vacuolated inclusions. In contrast, the cells lining the collecting ducts appear to be spared in classic tubular necrosis, although the lumens usually are filled with cellular debris. Our observations on kidneys obtained from alcohol-fed animals are similar. In both classic nephrotoxic tubular necrosis and the alcohol-fed kidney, the interstitium appears edematous and becomes progressively fibrotic. A specific interstitial reaction called tubular interstitial nephritis is seen occasionally in classic tubular necrosis and consists of massive infiltration of the interstitial tissue with inflammatory cells.3" This phenomenon is not observed in the alcohol-fed kidney at any stage. Similarly, extramedullarv hematopoiesis, although a common feature of classic tubular necrosis,40 is not observed in the alcohol-fed kidney. Nonetheless, the renal lesion produced by alcohol is quite similar in most of its features to the lesions described for classic cases of tubular necrosis due to a varietv of established nephrotoxic agents.
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References 1. Regan TJ, Korxenidis G, Moschos CB, Oldewurtel HA, Lehan PH, Hellems HK: The acute metabolic and hemodynamic responses of the left ventricle to ethanol. J Clin Invest 45:270-280, 1966 2. Symposium on neurologic and hepatic complications of alcoholism: On the etiology of the alcoholic neurological diseases with special reference to the role of nutrition. Am J Clin Nutr 9:370-406, 1961 3. Sullivan LW, Herbert V: Suppression of hematopoiesis by ethanol. J Clin Invest 43:2048-2062, 1964 4. Ekbom K, Hed R, Kirstein L, Astrom KE: Muscular affections in chronic alcoholism. Arch Neurol 10:449-458, 1964 5. Van Thiel DH, Gavaler JS, Lester R, Goodman MD: Alcohol induced testicular atrophy: An experimental model for hypogonadism occurring in chronic alcoholic men. Gastroenterology 69:326-332, 1975 6. Kiessling KH, Lindgren L, Strandberg B, Tobe U: Electron microscopic study of liver mitochondria from human alcoholics. Acta Med Scand 176:595-598, 1964 7. Klion FM, Schaffner F: Ultrastructural studies in alcoholic liver disease. Digestion 1:2-14, 1968 8. Lane BP, Lieber CS: Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Am J Pathol 49:593-604, 1966 9. Porta EA, Bergman BJ, Stein AA: Acute alcoholic hepatitis. Am J Pathol 46:657-689, 1965 10. Rubin E, Lieber CS: Experimental alcoholic hepatic injury in man: Ultrastructural changes. Fed Proc 26:1458-1467, 1967 11. Schaffner. F, Loebel A, Weiner HA, Barka T: Hepatocellular cytoplasmic changes in acute alcoholic hepatitis. JAMA 183:343-346, 1963 12. Svoboda DJ, Manning RT: Chronic alcoholism with fatty metamorphosis of the liver: Mitochondrial alterations in hepatic cells. Am J Pathol 44:645-662, 1964 13. Iseri OA, Gottlieb LS, Lieber CS: The ultrastructure of ethanol induced fatty liver. Fed Proc 23:579-581, 1964 14. Merrill JP: Acute renal failure. Diseases of the Kidney, Second edition. Edited by MB Strauss, LG Welt. Boston, Little, Brown & Co., 1971, p 637 15. Stein 0, Stein Y: Fine structure of the ethanol induced fatty liver in the rat. Isr J Med Sci 1:378-388, 1965 16. Lieber CS, DeCarli LM: Quantitative relationship between amount of dietary fat and severity of alcoholic fatty liver. Am J Clin Nutr 23:474-478, 1970 17. Pitts RF: Physiology of the Kidney and Body Fluids, Third edition. Chicago, Year Book Medical Publishers, Inc., 1974, p 61 18. Chasson AL, Grady HJ, Stanley MA: Determination of creatinine by means of automatic chemical analysis. Am J Clin Pathol 35:83-88, 1961 19. Bernt E, Gutman I: Ethanol determination with alcohol dehydrogenase and NAD. Methods of Enzymatic Analysis, Inc., Second edition, Vol 3. Edited by H-U Bergmeyer. New York, Academic Press Inc., 1974, p 1499 20. Berry JW, Chappell DG, Barnes RB: Improved method of flame photometry. Ind Eng Chem Anal Ed 18:19-23, 1946 21. Marsh WH, Fingerhut B, Miller H: Automated and manual direct methods for the determination of blood urea. Clin Chem 11:624-627, 1965 22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 23. Martin RF, Donohue DC, Finch LR: New analytical procedure for the estimation of DNA with p-nitrophenylhydrazine. Anal Biochem 47:562-574, 1972 24. Folch J, Lees M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipides from animal tissue. J Biol Chem 226:497-510, 1957
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23. Abel LL, Levv BB, Brodie BB, Kendall FE: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J Biol Chem 195:35 7-366, 1952 26. Dittmer JC, Lester RL: A simple specific spray for the detection of phospholipids on thin-laver chromatograms. J Lipid Res 5:126-127, 1964 27. Karnovskv MJ: A formaldehyde-glutaraldehvde fixative of high osmolalitv for use in electron miscroscopy. J Cell Biol 27:137A-138A, 1965 28. Watson NM L: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478, 1958 29. Reynolds ES: The use of lead citrate at high pH ac an electron opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963 ,30. Lieber CS: Effects of ethanol upon lipid metabolism. Lipids 9:103-116, 1974 31. Lieber CS, Spritz N, DeCarli LM: Accumulation of triglvcerides in heart and kidney after alcohol ingestion. J Clin Invest 43:1041, 1966 32. Heptinstall RH: Acute renal failure. Pathology of the Kidnev, Second edition. Boston, Little, Brown & Co., 1974, p 781 .33. Baldwin DS, Levine BB, McCluskev RT, Gallo GR: Renal failure and interstitial nephritis due to penicillin and methicillin. N Engl J Med 279:1245-1252, 1968 ,34. Lindeneg 0, Fischer S, Pedersen J, Nissen NI: Necrosis of the renal papillae and prolonged abuse of phenacetin. Acta Med Scand 165:321-328, 1959 ,35. Van Wy-k JJ, Hoffman CR: Periarteritis nodosa: A case of fatal exfoliative dermatitis resulting from "dilantin sodium" sensitization. Arch Intern Med 81:605-611, 1948 :36. Kennedy AC, Burton JA, Luke RG, Briggs JD, Lindsay RM, Allison MEM, Edward N, Dargie HJ: Factors affecting the prognosis in acute renal failure: A survey of 251 cases. Quart J M ed 42:73-86, 1973 37. Brun C: Acute Anuria: A Studv Based on Renal Function Tests and Aspiration Biopsy of the Kidney. Copenhagen, E. Munksgaard, 1954 38. Brun C: The renal lesion in acute tubulointerstitial nephropathv. Acute Renal Failure. Edited by CT Flynn. Lancaster, England, Medical and Technical Publishing Co., Ltd., 1973, p 46 39. Olsen TS, Skjoldborg H: The fine structure of the renal glomerulus in acute anuria. Acta Pathol NMicrobiol Scand 70:205-214, 1967 40. Baker SBdeC: Intravascular haematopoiesis in the renal medulla in shock. J Pathol Bacteriol 75:421-428, 1958
Adckowledgments The authors would like to express their particular thanks and appreciation to Drs. Emanuel Rubin and Thomas J. Gill III for their thoughtful review of the manuscript.
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Legends for Figures Figure 1-Proximal convoluted tubule with alcohol-induced changes. The brush border (Mv) has been disrupted, and the proximal cells are seen swelling into the lumen. Note the presence of osmiophilic reabsorptive vesicles adjacent to the fragmented brush border (arrow). N = nucleus. (Fixation with osmium tetroxide, x 5100) Figure 2-Proximal convoluted tubule with alcohol-induced changes. Discontinuity of the brush border (Mv) is due to the swelling of the proximal cells which have pushed into the lumen. Numerous mitochondria are surrounded by the interdigitating plasma membranes (P) which extend down to the basement membrane (B). Abundant collagen fibers (C) are seen next to an interstitial fibroblast (F). Note the lysosomes (arrows, L) packed with membranes. (Fixation with glutaraldehyde, x 11,300)
Figure 3-Distal tubule with alcohol-induced changes. First-stage lesion. Elongated mitochondria, microvilli, and basement membrane (B) are shown. (Fixation with Karnovsky fixative, x 4000) Figure 4-Distal tubule with alcohol-induced changes. Second-stage lesion. The flattened epithelium exhibits proliferation of the rough endoplasmic reticulum (arrow). B = basement membrane. (Fixation with glutaraldehyde, x 10,000)
Figure 5-Distal tubule with alcohol-induced changes. Third-stage lesion. The rough endoplasmic reticulum (arrow) has vesiculated. Collagen (C) is seen deposited along the basement membrane (B). Note the appearance of delicate cytoplasmic projections into the lumen (Lu). (Fixation with glutaraldehyde, x 11,300)
Figure 6-Distal tubule with alcohol-induced changes. Fourth-stage lesion. The rough endoplasmic reticulum (arrow) is dilated, and numerous protrusions of cytoplasm into the lumen (Lu) are seen. The structural integrity of the cell presumably is reduced. Blebs of cytoplasm are seen in the lumen. (Fixation with glutaraldehyde, x 700) Figure 7-Distal tubule with alcohol-induced changes. End-stage lesion. Part of the distal cell (DC) had sloughed into the lumen (Lu). (Fixation with glutaraldehyde, x 12,700) Figure 8-Collecting duct, obtained from a kidney of an alcohol-fed rat, filled with cell debris. A collecting duct lumen (Lu) is lined with normal appearing duct cells. The two populations of duct cells, light and dark, are easily seen. The debris in the lumen presumably originates from the sloughing of cells from tubules of the more proximal portions of the nephron. Tubular content includes membrane-bound as well as unbound cytoplasmic blebs with ribosomes, swollen mitochondria, and lysosomes. (Fixation with Karnovsky fixative, x 5,500)
Figure 9-Perivascular accumulation of fibroblasts. Collagen (c) deposition is seen around an atypical vasa recta (V) with endothelial cell hyperplasia in the early alcohol-induced renal lesion. Three fibroblasts (F) can be identified around this blood vessel. (Fixation with glutaral-
dehyde, x 5000)
Figure 10-Interstitial fibrosis. Excess collagen deposition is seen between interstitial cells of the end-stage lesion. (Fixation with glutaraldehyde, x 4800) Figure 11-Composite electron micrograph of pelleted urine obtained from the bladder of 3 alcohol-fed animals. A-Abundant vesicular structures. B-Free cytoplasmic membranes. C-Chromatin-like material (Cr). D-Mixture of vesicular structures and cytoplasmic membranes. (x 32,000)
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American Journal of Pathology
[End of Article]
The nephrotoxic effect of ethanol feeding on renal structure and function was evaluated in rats and compared to that in dextrose-fed isocaloric control animals. Alcohol-fed animals had larger kidneys than their controls. Despite this increase in renal mass, the alcohol-fed animals had a 50% reduction in creatinine clearance and a 67% reduction in osmolar clearance compared to their controls. When specific renal constituents were compared, the alcohol-fed animals were found to have twice the renal protein and a 50% increase in renal lipid. Despite these marked structural and functional differences, the light microscopic appearance of the kidneys of the two groups did not appear significantly different. In contrast, the electron microscopic differences were sub-stantial. The renal epithelial cells, particularly of the distal tubules and Henle's loops, were found to show varying degrees of cellular injury and were observed to be sloughing into the lumens. These electron microscopic observations are similar to those obtained in tubular necrosis due to a variety of nephrotoxic agents. We propose, therefore, that chronic alcohol feeding of rats produces significant renal dysfunction and abnormalities of structure such that ethanol should be considered a true nephrotoxin. (Am J Pathol 89:67-84, 1977)
ALCOHOL recently has been demonstrated to be toxic to a varietv of extrahepatic tissues. These include the heart,' the nervous system,2 the bone marrow, 3 the skeletal muscle, and the gonads.5 Unlike the wvell-studied hepatotoxic effects of alcohol, the pathogenesis of these recently recognized extrahepatic abnormalities, their rates of development, and their potential for reversibility are poorly defined. NMuch of our current understanding of the morphologic and toxic biochemical effects of ethanol on the liver wvere preceded by electron microscopic studies. The initial electron microscopic demonstration of swollen mitochondria,>'2 increased smooth endoplasmic reticulum,8-10,13.14 and focal cvtoplasmic degeneration -12 ,14,15 in animals and in men fed alcohol led directly to subsequent studies which defined the chronology of these morphologic abnormalities,8",0" 4 the failure of low-fat and high-protein diets to prevent their development,8 and the biochemical consequences of their presence. Accordingly, we have attempted to investigate the effect of ethanol on From the Division of Gastroenterology. Department of \Medicine. and the Department of Patholog\-. Universitv of Pittsburgh School of Medicine. Pittsburgh. Pennsylvania Suipported by Grant AA-01450 from the National Institutes of Health Accepted for publication May 1:3. 1977 Address reprint requests to Dr David H Van Thiel. IOOOC, Scaife Hall. Division of Gastroenterolov. Department of Medicine. L niversity of Pittsbureh. School of Medicine. Pittsburgh. PA 15261 67
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renal structure and function in pair-fed rats receiving similar diets with identical fluid, electrolyte, lipid, protein, and caloric content. The diets differed only in the number of calories presented as carbohydrates or as ethanol isocalorically substituted for each other.5 16 Materials and Methods Animals
Forty male Wistar rats age 24 days and matched for weight (mean ± SEM, 57.7 ± 0.5 g; range, 50.0 to 63.5) were obtained from Charles River Breeding Laboratories, North Wilmington, Mass. The animals were housed in individual metabolic cages and pair-fed a li(quid diet containing alcohol or an identical liquid diet in which ethanol was isocaloricallv replaced with dextrose. The diet containing alcohol consisted of 5% ethanol by volume which accounted for 36% of the total caloric content.16 On the average the animals ate 47 i 2.0 cu cm diet/day. Twenty-four hours prior to sacrifice, a stool-free 24-hour urine collection was obtained from each animal. Animals were weighed and then sacrificed by exsanguination under light ether anesthesia. The left kidney of each animal was weighed immediately after removal for determination of wet renal weight. It was then freeze-dried for 72 hours and reweighed. Prior control experiments have shown that dry renal weight does not change after 36 hours of freeze-drying. Homogenates were prepared from these freeze-dried tissues and used for subse(luent renal constituent analysis. The right kidney of each animal was removed and bisected longitudinallv in a frontal plane; half was then fixed in Bouin's solution for light microscopy. The other half was fixed in glutaraldehvde, osmiuim tetroxide, or Karnovskv's fixative for subse(quent electron microscopic analysis. Physiologic Studies
Using urine samples collected for the 24 hours prior to sacrifice and serum obtained at the time of sacrifice, renal free water, osmolar, and creatinine clearances were determined using standard methods.17 Serum and urine osmolaritv were determined by freezing point depression. Serum and urine creatinine content were determined according to the method of Chasson.18 Blood pH and alcohol content 19 as well as serum electrolyte 20 and urea nitrogen 21 were determined using standard techni(quies. Renal Constituent Analysis
Renal protein and DNA content were determined utilizing the saline homogenates prepared from the freeze-dried left kidney according to standard methods.22'23 Standard solutions were prepared from bovine serum albumin obtained from Calbiochem, Spring Valley, N.Y. and calf thvmus DNA obtained from Sigma Chemical Co., St. Louis, Mo. The renal homogenates were extracted according to the method of Folch24 and analyzed for total lipid, cholesterol, and phospholipid levels using previously described methods.24 26 Light Microscopy
After fixation in Bouin's solution for 72 hours, the tissues were dehydrated in progressive ethanol solutions, embedded in paraffin, sectioned at 5 ,, and stained with hematoxvlin and eosin for light microscopy. Slides were coded and read blindlv after all of the studies were completed. The glomeruli, proximal and distal tubules, loops of Henle, collecting ducts, renal arteries and veins, and the interstitial tissues for each animal were each individually assessed and graded (on a sale of 1 to 4) for degree of variation from the histologic appearance of kidneys obtained from 20 control animals of the same age maintained on a standard rat chow diet and fed ad libitum.
ALCOHOL NEPHROTOXICITY
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6Ektro
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AnaVd
Small pieces of the right kidney were fixed in 3.0% glutaraldehyde (Ladd Research Industries Inc., Burlington, Vt.) in a 0. 1 M cacodvlate buffer (pH 7.4), Karnovskv's fixative (half strength)"7 in cacodylate buffer of I% osmium tetroxide in 0.1 M cacodylate buffer from 2 hours at 4 C. Due to the large number of samples taken, the tissues may have remained in the fixative for as long as 3 weeks under refrigerated conditions. Only the samples that were fixed in buffered osmium tetroxide were processed immediately after fixation. Urinarv sediments from 6 animals (3 alcohol-fed rats and 3 isocaloric controls) were obtained for microscopy bv aspirating the urinarv bladder, thus avoiding any fecal contamination. The urines were centrifuged at 2,000 rev/min in an International Clinical Centrifuge for 5 minutes to remove any intact cells and then centrifuged for an additional 60 minutes at 105,000g. The final urine pellets were fixed ovemight in 3% glutaraldehvde in 0. 1 M cacodvlate buffer at 4 C. After primarv aldehvde fixation, all samples were washed for 1 hour in 0.1 M cacodylate buffer and postfixed in I% osmium tetroxide in 0.I NI cacodvlate buffer for 2 hours at room temperature, rinsed, dehydrated in a graded ethanol series, and embedded in Epon-Araldite. After polymerization, silver sections were cut with glass or diamond knives on a Sorvall MT 1 microtome, mounted on uncoated 200- or 300mesh grids, and stained with 3% uranvl acetate n and lead citrate." Grids were examined and photographed at 60 kV with a Philips EM 200 or EM 300 utilizing a double condenser system. A magnification calibration grid was photographed at various times during the studvT.
Experimental results are expressed as the mean ± standard error. The P values were obtained by use of the Student t test for the difference between the two group means. Values were considered probably significant at P < 0.05 and significant at P < 0.01.
Results Ar*alms
Both the alcohol-fed and the isocaloric control animals (Table 1) grew steadily during the study period.5 The alcohol-fed animals consistently weighed less but had larger kidneys than the isocaloric controls. In contrast to the greater wet renal weight, the drv renal weights did not differ between the two groups. When the dry renal weights were corrected for body weight, however, the alcohol-fed animals clearly had a greater renal mass per gram body weight than did the isocaloric controls. Table 1 -Body and Renal Mass Parameters
Alcohol-fed animals (N = 20)
Bodyweight(g) Wet renal weight (g) Wet renal weight (g)/body weight (100 g)
Dry renalweight(g) Dry renal weight (g)/body weight (100 g) P < 0.01.
101.2 0.87 0.86 0.188 0.15
± 3.7* ± 0.01' ± 0.03* ± 0.011
±0.001*
Isocaloric controls (N = 20) 113.0 0.51 0.45 0.175 0.10
± 3.7 ± 0.02 ± 0.01
±0.007 ± 0.01
VAN THIEL ET AL.
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Physiologic Studies
The serum creatinine levels (Table 2) of the alcohol-fed animals were 50% greater than those of the isocaloric controls. Despite this difference in the levels of serum creatinine, the amount of creatinine excreted in 24 hours was similar for both groups. Therefore, the creatinine clearance was reduced in the alcohol-fed animals by 50%. In addition to the observed reduced creatinine clearance, the alcohol-fed animals had a reduced osmolar clearance compared to the isocaloric control animals. This reduction in osmolar clearance occurred despite the presence of a significant hypernatremia and hyperosmolarity in the alcohol-fed animals. Consistent with this reduction in renal function, the alcohol-fed animals had a metabolic acidosis associated with a significant anion gap which probably is due, in part, to an associated lactic acidosis, known to occur in cases of chronic alcohol ingestion.30 Despite these marked metabolic differences between the alcohol-fed and the isocaloric control animals, no significant difference was observed for serum uric acid content or blood urea nitrogen. As might be expected, however, the alcohol-fed animals had lower blood glucose levels than did the isocaloric controls. Renal Constituent Analysis
Renal tissue protein, lipid, and DNA (Table 3) were increased significantly in the alcohol-fed animals compared to their isocaloric controls. In addition to the expected increase in absolute levels of triglyceride,31 the levels of the other major classes of renal lipids were also markedly elevated. Table 2-Physiologic Differences Between Alcohol-Fed Animals And Isocaloric Controls Alcohol-fed animals (N = 10)
Creatinine (mg/dl) Urine creatinine (mg/24 hrs) Creatinine clearance (cu cm/min) Osmolar clearance (liter/24 hrs) Serum sodium (mEq/liter) Serum osmolarity (mOsmol/liter) pH Anion gap (mEq/liter) Blood alcohol (mg/dl) Uric acid (mg/dl) Blood urea nitrogen (mg/dl) Blood sugar (mg/dl) *
P < 0.01.
t P < 0.05.
0.91 7.6 0.6 0.042 54 329 7.23 36 112 3.7 15 92.7
± 0.06* ± 1.3 ± 0.1 * ± 0.010* ± 2* ± 3* ± 0.04* ± 1* ± 24* ± 0.3 ±1 ± 6.2t
Isocaloric controls (N = 10) 0.62 ± 0.04 10.5 ± 1.1 1.2
0.120 146 301 7.39 17 0 3.9 13 120.5
± 0.2 ± 0.025 ±1 ±4 ± 0.03
±2 ± 0.3 ±1 ± 9.3
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Sbta Lght No difference was observed between the appearance of the renal arteries and veins of the alcohol-fed animals and the isocaloric control animals. The distal tubules in the alcohol-fed animals appeared dilated, with markedly flattened epithelial cells, while the distal tubules in the isocaloric control animals appeared normal. Proximal convoluted tubules in the alcohol-fed animals appeared normal, with the exception of some eosinophilic staining material which was occasionally present in the lumen. This material was initially interpreted as staining artifact at the light microscopic level. Subsequent electron microscopic studies clearlv show this material to be sloughed epithelial cell cytoplasm and organelles. In all cases, the proximal convoluted tubules of the isocaloric control animals were normal. A marked interstitial edema was present in the medulla of the alcohol-fed animals, with wide separation of the individual renal tubules and a loose endematous appearance of the intervening connective tissue compared to that of the isocaloric control animals. El
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Although the observed differences at the light microscopic level were not marked, it is apparent that the kidneys of the alcohol-fed animals have a different histologic appearance at the electron microscopic level compared to the appearance of kidneys from the isocaloric controls. These differences are especially striking in the renal cortex at the corticomedullarv junction; prominent differences involve the entire nephron, excluding the glomerulus. The glomeruli of kidneys obtained from the alcohol-fed animals, as well as those of the isocaloric controls, appeared normal. The glomerular capillaries and basement membranes appeared normal. Likewise, the slit membrane between podocytes and the fenestrated capillary endothelia appeared intact. No distinct changes were seen in the juxtaglomerular apparatus. Table 3-Renal Constituent Analysis Alcohol-fed animals (N = 10) Protein (mg/i 00 g body weight) Total lipid (mg/100g body weight) Cholesterol (mg/i00 g body weight) Phospholipid (mg/i00 g body weight) DNA (g/l00 g body weight) *
P < 0.01.
1 12.0 21.7
± 8.3* ± 3.31 0.028 ± 0.001* 1.082 ± 0.005* 1.34 ± 0.10*
Isocaloric controls (N = 10) 68.6 ± 4.4 14.86 ± 2.39 0.009 ± 0.001 0.356 ± 0.006 0.80 ± 0.05
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The epithelial cells of the proximal convoluted tubules exhibited marked cytoplasmic swelling with disruption of the brush border (Figure 1). Nuclei, mitochondria, and ribosomes were visualized pushing past the microvillus border filling the lumen as a consequence of cellular swelling. Fragmentation and vesiculation of the cisternae of the rough endoplasmic reticulum (RER) and swelling of mitochondria occurring in association with the presence of an electron-dense matrix were also frequently seen (Figure 2). There was no apparent proximal tubular luminal dilation independent of that caused by the mechanical distension produced as a result of the proximal epithelial cell swelling. The thin segment of the loop of Henle showed more discrete cytoplasmic changes. In the alcoholic kidney, the thin cytoplasm formed numerous excrescences which projected into the lumen. These excrescences were easily distinguished from stubby microvilli seen in the control kidney. Unusually thin epithelial cytoplasm was frequently seen detached from the basement membrane of the loop of Henle and sloughed into the lumen. In some cases, the nucleus of the tubular epithelial cells was flattened. Fibroblasts infiltrated the interstitium and deposited excess collagen diffusely along the basement membranes. The distal convoluted tubule showed the most remarkable ultrastructural changes of the entire nephron. The distal epithelial cells demonstrated a progressive change or necrotic metamorphosis (Figures 3-7). To simplify this description, the progression that occurs as a result of alcohol feeding has been divided arbitrarily into five stages. The first-stage distal tubular lesion was manifested as a grossly dilated tubular lumen with flattened epithelial lining cells (Figure 3). At this stage, the mitochondria and the number and appearance of the microvilli appeared normal. No unusual fibroblastic activity or peritubular fibrosis was observed. The second-stage lesion (Figure 4) showed a proliferation of the rough endoplasmic reticulum. The basement membrane appeared minimally thickened due to increased collagen deposition by adjacent fibroblasts. The third-stage lesion (Figure 5) showed the development of cytoplasmic epithelial cell protuberances which project into the lumen. These resembled exocytosing or sloughing microvilli. Particularly noticeable at this stage was the vesiculated rough and smooth endoplasmic reticulum and ballooning of the mitochondria. Lysosome-like bodies often appeared packed with membrane-bounded vacuoles. Abundant collagen fibers could be seen underlying the basement membrane (Figure 5). The fourth-stage lesion (Figure 6) exhibited further dilation of the rough and smooth endoplasmic reticulum that contributed to the forma-
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tion of numerous large cvtoplasmic excrescences. Blebs of cy7toplasm containing ribosomes were seen in the lumen. In the end-stage lesion (Figure 7), the distal tubular epithelial cells had completely necrosed. The basement membrane, however, did not appear to be irreversiblv damaged. The most proximal regions of the collecting ducts appeared to be affected bv alcohol in a manner similar to the distal tubules; progressive lesions were seen ranging from dilated ducts to end stage necrosis. Cells of the more distal regions of the collecting ducts and the ducts of Bellini appeared virtually unaffected bv the alcohol. The lumens of these distal regions, however, were often filled with cellular debris including fragtents of cytoplasm, free ribosomes, mitochondria, and nuclear material (Figure 8). Interstitial and perivascular fibrosis were seen about the collecting ducts (Figures 9 and 10). The earliest evidence for this new collagen formation was the local accumulation of fibroblasts. Subsequently, excess collagen accumulated around blood vessels and then spread into the interstitial tissue. Examination of urine sediments (Figure 11) collected from the bladder of alcohol-fed animals prior to sacrifice confirmed the presence of cellular debris, secretory vesicles, free membranes, and chromatin-like material within the urine as suggested by the electron microscopic appearance of the collecting ducts (Figure 8). Urine collected from control animals contained no cellular debris.
Discussion Known causes of tubular necrosis include severe trauma with shock, crushing injuries, bums, and a variety of poisons. Commonly recognized nephrotoxins include ethylene glycol, organic mercurials, arsenic, carbon tetrachloride, and certain antibiotics.32 In addition to these recognized nephrotoxins, other more frequentlv used substances are potentially nephrotoxic.l" u- In particular, Merrill suggests that excessive use of ethyl alcohol may be associated with renal failure.'4 He concludes, however, that the exact role of ethanol as a nephrotoxin remains unclear. The marked potentiation of carbon tetrachloride nephrotoxicitv by the concomitant use of ethanol is a well-established fact that provides some support for a potential nephrotoxic action of alcohol.-" In light of the evidence herein presented for biochemical and ultrastructural abnormalities induced by alcohol on kidney parenchyma, it seems reasonable to attempt to relate this pathology to that observed in other examples of tubular necrosis due to established nephrotoxins. Bio-
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chemically, azotemia, oliguria, and reduced creatinine clearance are indications of renal failure. Dilation of the distal tubules with flattening of the epithelial cells and subsequent necrosis observed in the alcohol-fed animals is histologically consistent with lesions reported to occur in tubular necrosis due to other causes.32 Moreover, since only the alcohol-fed animals demonstrate these changes, it is reasonable to assume that alcohol ingestion may have been responsible for tubular necrosis. The increase in plasma osmolarity observed for the alcohol-fed rats can be entirely explained on the basis of the observed blood alcohol levels, which would be expected to add 25 to 30 mOsmol to the measured osmolarity. The significant increase in serum sodium levels observed in the alcohol-fed animals may in part be artifactual and due to the smaller volume of distribution of the sodium ion in the lipemic alcohol serum. Of interest is the failure to observe an increase in the serum uric acid concentration of the alcohol-fed animals; chronic alcohol administration in man almost always increases the uric acid concentration. It must be stated, however, that it is unusual to observe either clinical renal failure or azotemia in alcoholics. When renal failure does occur in alcoholics, it almost always occurs in the presence of severe liver disease, which is known to produce alterations in renal circulatory dynamics. We have yet to establish such changes in this animal model. Based on the increased total renal weight and specific constituents, we had proposed that chronic alcohol feeding has induced renal hypertrophy.37 Based upon our present ultrastructural studies, however, this increase in renal weight is almost probably due both to synthesis of new cells and to loss of cells in the lumen of the kidney, presumably as a consequence of an alcohol-induced injury. Obviously, since considerable amounts of cellular debris remain in the tubular lumens, it must contribute significantly to the weight increase observed for the total kidneys. Effective evaluation of the renal pathology induced by ethanol requires comparison of the alcohol-induced lesion with that of some other wellaccepted nephrotoxins. Grossly, the kidneys obtained from the alcohol-fed animals and the animals with nephrotoxic tubular necrosis are enlarged and swollen. Moreover, presumably because of the presence of considerable interstitial edema, the cortex of both alcohol-fed animals and animals with nephrotoxic tubular necrosis is often pale compared to the cortex of isocaloric control animals and normal animals. The gross pathology of the kidneys obtained from the alcohol-fed animals is, therefore, similar to that reported to occur in typical nephrotoxic tubular necroSiS 14,32,38
In addition, in classic tubular necrosis and in the present model, the glomeruli are normal at both the light and electron microscopic lev-
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els. 1432'" Moreover, in classic tubular necrosis the proximal tubules show few if any changes at the light microscopic level. Hydropic degeneration and luminal dilation have been reported but are not uniformly present.'432'8 With the electron microscope, mitochondrial swelling, focal loss of the brush border, and the development of vacuoles are seen." In contrast, the distal tubules in classic tubular necrosis show extensive cell necrosis, cell drop-out, and dissolution of the basement membrane. 14'32,' As in the case of classic tubular necrosis, the kidneys obtained from the alcohol-fed animals show little change at the light microscopic level. At the electron microscopic level, however, marked alterations in renal structure are apparent. In the proximal convoluted tubules, structural integrity is sacrificed by swelling, which being limited by the basement membrane, pushes the cell inward, filling the lumen and disrupting the brush border. In addition, the mitochondria are frequently swollen and vacuolated. Despite these changes, no definite necrosis is seen. Although there are reports of necrosis in the thick limbs of the loop of Henle occurring in classic tubular necrosis, the thin loop seems to be relatively spared. In contrast to this classic picture, the cells lining the loops of Henle obtained from the alcohol-fed animals demonstrate obvious abnormalities with irregular cytoplasmic excrescences which extend into the lumen and suggest cellular necrosis. The distal tubules in classic tubular necrosis show luminal dilation, focal cell loss, and extensive necrosis. 4,323" Our light and electron microscopic studies in kidneys obtained from alcohol-fed animals demonstrate similar abnormalities. These include cell flattening, mitochondrial swelling, proliferation and dilation of the rough and smooth endoplasmic reticulum, the development of a microvillus border, and formation of multilocular vacuolated inclusions. In contrast, the cells lining the collecting ducts appear to be spared in classic tubular necrosis, although the lumens usually are filled with cellular debris. Our observations on kidneys obtained from alcohol-fed animals are similar. In both classic nephrotoxic tubular necrosis and the alcohol-fed kidney, the interstitium appears edematous and becomes progressively fibrotic. A specific interstitial reaction called tubular interstitial nephritis is seen occasionally in classic tubular necrosis and consists of massive infiltration of the interstitial tissue with inflammatory cells.3" This phenomenon is not observed in the alcohol-fed kidney at any stage. Similarly, extramedullarv hematopoiesis, although a common feature of classic tubular necrosis,40 is not observed in the alcohol-fed kidney. Nonetheless, the renal lesion produced by alcohol is quite similar in most of its features to the lesions described for classic cases of tubular necrosis due to a varietv of established nephrotoxic agents.
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References 1. Regan TJ, Korxenidis G, Moschos CB, Oldewurtel HA, Lehan PH, Hellems HK: The acute metabolic and hemodynamic responses of the left ventricle to ethanol. J Clin Invest 45:270-280, 1966 2. Symposium on neurologic and hepatic complications of alcoholism: On the etiology of the alcoholic neurological diseases with special reference to the role of nutrition. Am J Clin Nutr 9:370-406, 1961 3. Sullivan LW, Herbert V: Suppression of hematopoiesis by ethanol. J Clin Invest 43:2048-2062, 1964 4. Ekbom K, Hed R, Kirstein L, Astrom KE: Muscular affections in chronic alcoholism. Arch Neurol 10:449-458, 1964 5. Van Thiel DH, Gavaler JS, Lester R, Goodman MD: Alcohol induced testicular atrophy: An experimental model for hypogonadism occurring in chronic alcoholic men. Gastroenterology 69:326-332, 1975 6. Kiessling KH, Lindgren L, Strandberg B, Tobe U: Electron microscopic study of liver mitochondria from human alcoholics. Acta Med Scand 176:595-598, 1964 7. Klion FM, Schaffner F: Ultrastructural studies in alcoholic liver disease. Digestion 1:2-14, 1968 8. Lane BP, Lieber CS: Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Am J Pathol 49:593-604, 1966 9. Porta EA, Bergman BJ, Stein AA: Acute alcoholic hepatitis. Am J Pathol 46:657-689, 1965 10. Rubin E, Lieber CS: Experimental alcoholic hepatic injury in man: Ultrastructural changes. Fed Proc 26:1458-1467, 1967 11. Schaffner. F, Loebel A, Weiner HA, Barka T: Hepatocellular cytoplasmic changes in acute alcoholic hepatitis. JAMA 183:343-346, 1963 12. Svoboda DJ, Manning RT: Chronic alcoholism with fatty metamorphosis of the liver: Mitochondrial alterations in hepatic cells. Am J Pathol 44:645-662, 1964 13. Iseri OA, Gottlieb LS, Lieber CS: The ultrastructure of ethanol induced fatty liver. Fed Proc 23:579-581, 1964 14. Merrill JP: Acute renal failure. Diseases of the Kidney, Second edition. Edited by MB Strauss, LG Welt. Boston, Little, Brown & Co., 1971, p 637 15. Stein 0, Stein Y: Fine structure of the ethanol induced fatty liver in the rat. Isr J Med Sci 1:378-388, 1965 16. Lieber CS, DeCarli LM: Quantitative relationship between amount of dietary fat and severity of alcoholic fatty liver. Am J Clin Nutr 23:474-478, 1970 17. Pitts RF: Physiology of the Kidney and Body Fluids, Third edition. Chicago, Year Book Medical Publishers, Inc., 1974, p 61 18. Chasson AL, Grady HJ, Stanley MA: Determination of creatinine by means of automatic chemical analysis. Am J Clin Pathol 35:83-88, 1961 19. Bernt E, Gutman I: Ethanol determination with alcohol dehydrogenase and NAD. Methods of Enzymatic Analysis, Inc., Second edition, Vol 3. Edited by H-U Bergmeyer. New York, Academic Press Inc., 1974, p 1499 20. Berry JW, Chappell DG, Barnes RB: Improved method of flame photometry. Ind Eng Chem Anal Ed 18:19-23, 1946 21. Marsh WH, Fingerhut B, Miller H: Automated and manual direct methods for the determination of blood urea. Clin Chem 11:624-627, 1965 22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 23. Martin RF, Donohue DC, Finch LR: New analytical procedure for the estimation of DNA with p-nitrophenylhydrazine. Anal Biochem 47:562-574, 1972 24. Folch J, Lees M, Sloane-Stanley GH: A simple method for the isolation and purification of total lipides from animal tissue. J Biol Chem 226:497-510, 1957
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23. Abel LL, Levv BB, Brodie BB, Kendall FE: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J Biol Chem 195:35 7-366, 1952 26. Dittmer JC, Lester RL: A simple specific spray for the detection of phospholipids on thin-laver chromatograms. J Lipid Res 5:126-127, 1964 27. Karnovskv MJ: A formaldehyde-glutaraldehvde fixative of high osmolalitv for use in electron miscroscopy. J Cell Biol 27:137A-138A, 1965 28. Watson NM L: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478, 1958 29. Reynolds ES: The use of lead citrate at high pH ac an electron opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963 ,30. Lieber CS: Effects of ethanol upon lipid metabolism. Lipids 9:103-116, 1974 31. Lieber CS, Spritz N, DeCarli LM: Accumulation of triglvcerides in heart and kidney after alcohol ingestion. J Clin Invest 43:1041, 1966 32. Heptinstall RH: Acute renal failure. Pathology of the Kidnev, Second edition. Boston, Little, Brown & Co., 1974, p 781 .33. Baldwin DS, Levine BB, McCluskev RT, Gallo GR: Renal failure and interstitial nephritis due to penicillin and methicillin. N Engl J Med 279:1245-1252, 1968 ,34. Lindeneg 0, Fischer S, Pedersen J, Nissen NI: Necrosis of the renal papillae and prolonged abuse of phenacetin. Acta Med Scand 165:321-328, 1959 ,35. Van Wy-k JJ, Hoffman CR: Periarteritis nodosa: A case of fatal exfoliative dermatitis resulting from "dilantin sodium" sensitization. Arch Intern Med 81:605-611, 1948 :36. Kennedy AC, Burton JA, Luke RG, Briggs JD, Lindsay RM, Allison MEM, Edward N, Dargie HJ: Factors affecting the prognosis in acute renal failure: A survey of 251 cases. Quart J M ed 42:73-86, 1973 37. Brun C: Acute Anuria: A Studv Based on Renal Function Tests and Aspiration Biopsy of the Kidney. Copenhagen, E. Munksgaard, 1954 38. Brun C: The renal lesion in acute tubulointerstitial nephropathv. Acute Renal Failure. Edited by CT Flynn. Lancaster, England, Medical and Technical Publishing Co., Ltd., 1973, p 46 39. Olsen TS, Skjoldborg H: The fine structure of the renal glomerulus in acute anuria. Acta Pathol NMicrobiol Scand 70:205-214, 1967 40. Baker SBdeC: Intravascular haematopoiesis in the renal medulla in shock. J Pathol Bacteriol 75:421-428, 1958
Adckowledgments The authors would like to express their particular thanks and appreciation to Drs. Emanuel Rubin and Thomas J. Gill III for their thoughtful review of the manuscript.
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Legends for Figures Figure 1-Proximal convoluted tubule with alcohol-induced changes. The brush border (Mv) has been disrupted, and the proximal cells are seen swelling into the lumen. Note the presence of osmiophilic reabsorptive vesicles adjacent to the fragmented brush border (arrow). N = nucleus. (Fixation with osmium tetroxide, x 5100) Figure 2-Proximal convoluted tubule with alcohol-induced changes. Discontinuity of the brush border (Mv) is due to the swelling of the proximal cells which have pushed into the lumen. Numerous mitochondria are surrounded by the interdigitating plasma membranes (P) which extend down to the basement membrane (B). Abundant collagen fibers (C) are seen next to an interstitial fibroblast (F). Note the lysosomes (arrows, L) packed with membranes. (Fixation with glutaraldehyde, x 11,300)
Figure 3-Distal tubule with alcohol-induced changes. First-stage lesion. Elongated mitochondria, microvilli, and basement membrane (B) are shown. (Fixation with Karnovsky fixative, x 4000) Figure 4-Distal tubule with alcohol-induced changes. Second-stage lesion. The flattened epithelium exhibits proliferation of the rough endoplasmic reticulum (arrow). B = basement membrane. (Fixation with glutaraldehyde, x 10,000)
Figure 5-Distal tubule with alcohol-induced changes. Third-stage lesion. The rough endoplasmic reticulum (arrow) has vesiculated. Collagen (C) is seen deposited along the basement membrane (B). Note the appearance of delicate cytoplasmic projections into the lumen (Lu). (Fixation with glutaraldehyde, x 11,300)
Figure 6-Distal tubule with alcohol-induced changes. Fourth-stage lesion. The rough endoplasmic reticulum (arrow) is dilated, and numerous protrusions of cytoplasm into the lumen (Lu) are seen. The structural integrity of the cell presumably is reduced. Blebs of cytoplasm are seen in the lumen. (Fixation with glutaraldehyde, x 700) Figure 7-Distal tubule with alcohol-induced changes. End-stage lesion. Part of the distal cell (DC) had sloughed into the lumen (Lu). (Fixation with glutaraldehyde, x 12,700) Figure 8-Collecting duct, obtained from a kidney of an alcohol-fed rat, filled with cell debris. A collecting duct lumen (Lu) is lined with normal appearing duct cells. The two populations of duct cells, light and dark, are easily seen. The debris in the lumen presumably originates from the sloughing of cells from tubules of the more proximal portions of the nephron. Tubular content includes membrane-bound as well as unbound cytoplasmic blebs with ribosomes, swollen mitochondria, and lysosomes. (Fixation with Karnovsky fixative, x 5,500)
Figure 9-Perivascular accumulation of fibroblasts. Collagen (c) deposition is seen around an atypical vasa recta (V) with endothelial cell hyperplasia in the early alcohol-induced renal lesion. Three fibroblasts (F) can be identified around this blood vessel. (Fixation with glutaral-
dehyde, x 5000)
Figure 10-Interstitial fibrosis. Excess collagen deposition is seen between interstitial cells of the end-stage lesion. (Fixation with glutaraldehyde, x 4800) Figure 11-Composite electron micrograph of pelleted urine obtained from the bladder of 3 alcohol-fed animals. A-Abundant vesicular structures. B-Free cytoplasmic membranes. C-Chromatin-like material (Cr). D-Mixture of vesicular structures and cytoplasmic membranes. (x 32,000)
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VAN THIEL ET AL.
American Journal of Pathology
[End of Article]
