Cyclodextrin Nephrosis in the Rat D. W. Frank, DVM, PhD, J. E. Gray, DVM, and R. N. Weaver
The renal toxicity of the Schardinger dextrins, a and O-cvclodextrin, is manifested as a series of alterations in the vacuolar organelles of the proximal convoluted tubule. These changes begin as an increase of apical vacuoles and the appearance of giant lysosomes. The giant Iysosomes characteristic of cyclodextrin nephrosis are notable because of the prominent acicular microcrvstals embedded in the Ivsosomal matrix. Giant vacuoles devoid of acid phosphatase reaction product are found in advanced lesions. The vacuolar apparatus shows advanced changes prior to manifestation of lesions in mitochondria and other organelles. These obsertations indicate a role of the v-acuologenic apparatus in the nephrotic process. Intracellular concentration of toxin via the lysosomal pathway represents a perversion of the physiologic function of the proximal tubule which ultimately leads to cell death. (Am J Pathol 83:367-382, 1976)
THE RENAL PROXIMAL TUBULE functions to recover many- substances from the glomerular filtrate. A structural adaptation for this purpose is the vacuologenic apparatus which recovers and retains proteins and other substances requiring degradation before recycling. In addition to substances normally regained from the glomerular filtrate, these organelles may intercept and sequester toxic materials. Indeed, the toxid tubular nephrosis associdted w-ith several substances 1.2 is manifested because of the recycling function of the proximal tubule. This studx' describes the nephrosis in the rat kidney caused by parenteral administration of a- and O-cvclodextrin. These carbohy-drates result from fermentation of potato starch by Bacillus macerans.3 Because their natural configuration is a closed circle of 6 (a) or 7 (B) D-glucose molecules, the c-clodextrins are unusually resistant to hydrolysis bv the common amvlases. Their appeal to pharmaceutical formulation follows the theoretical clathrate model which suggests that after enclosure of a less hydrophilic compound within the lumen of the cyclodextrin, solubility increases.
The nephrosis found after cyclodextrin administration appears to result from disturbances in the vacuologenic apparatus. Ultrastructural lesions are characterized by intracellular acicular crystals and giant lvsosomes which form prior to appearance of alterations in other organellies. From the Patholog- and
Toxicology
Research Unit. The U pjohn Compan%. Kalamazoo. \Mich-
igan.
Accepted for publication January 27. 1976 Address reprint requests to Dr. Doyle W. Frank. Upjohn Company. Kalamazoo. \I 49001
Pathology and Toxicolog'
Research L-nit. The 367
368
FRANK ET AL
American Journal of Pathology
Materials and Methods Formulations of the Cycodextrins 3-C0-clodextrin ICN. K and K Laboratories. Plainv iew. N.Y. and Pierce Chemical Companv. Rockford. Ill.l was dissolxed in sx-arm phvsiologic saline at the maximum soluble concentration L1 S g 100 ml at 2.5 C A.A saturated solution of a-cv cl(dextrin Sigma Chemical Companv. St. Louis. \lo. resulted "vhen 14.5 g %"as dissolved in 100 ml "arm 50 to 70 C physiologic saline. Both solutions svere used at room temperature. Acute Intravenous Administration of the LD50 of Cycodextrins Groups of 5 male and 3 female Sprague-Da".-ley rats 'eighing 1.0 to 160g were given
the cxclodextrins intravenously. The acute LD5a s-as determined bx- the Spearman-Karber method 7 dav s later. Cyclodextrin Administration Subcutaneously Single Administration
Groups of 4 100 to 125-g rats wsere gix-en 0.225. 0.45. or 0.90 g kg 3-cs-clodextrin bx- single subcutaneous injection and killed 12. 24. 48. or 96 hours later. Similar groups "ere injected with 0.1 or 1.0 g kg a-c!-clodextrin and killed 24 or 96 hours later Salineinjected control rats "ere killed at 12 or 96 hours after injection. Repeated Administration
Groups of four 100 to 125-g Sprague-Das-ley rats received 1. 2. :3. 4. or 7 dail\ subcutaneous injections of B-cvclodextrin at 0.225. 0.4.. 0.67.5. or 0.90 g kg. Other groups of rats were given a similar regimen of a-cvclodextrin at 0.1 or 1.0 g kg. Animals "vere killed 24 hours follomsing the last injection. Rats given saline in drug-equivialent volumes were killed after the seventh daily injection. Collcto of Tissues Rats "vere perfused via the abdominal aorta by a modification of the procedure 4 described bv Griffith. The aorta "-as clamped mvith mosquito forceps at the anterior surface of the diaphragm and anterior to the iliac bifurcation. The aorta was cannulated with ani 1S-gauge needle anterior to the terminal clamp. and the posterior vena cava wsas nicked at the moment perfusion began. The abdominal viscera wzas perfused with cold 1.5 or 2% glutaraldehvde in 0.1 \I Msodium cacodvlate buffer containing 1Cc sucrose. pH 7.2. for approximately :3 minutes at 130 to 10 mm Hg pressure. The kidneys were removed and immersed in fixative. and cortical slices "vere diced into 1-mm cubes. Tissues wxere fixed for 43 minutes to 1 hour. rinsed in 0.1 \I sodium cacodylate buffer and postfixed in 1%c osmium tetroxide for 43 minutes. Areas selected after Epon embedding were examined after staining with uranvl acetate and lead citrate. Nonperfused kidney cortex "as diced and fixed in 2% glutaraldehvde in .1 \l phosphate buffer. pH 7.4. for 1 hour. Glutaraldehvde-fixed tissue "as placed in Mlillonigs fixative for 11: hours at 4 C. Tissue "as embedded in Epon s12. One-micron-thick sections were stained mvith toluidine blue. Thin sections "vere stained "ith uranvl acetate and lead citrate. Kidneys collected for light microscopy were fixed in 10%c buffered formalin and routinelv processed. Acid phosphatase actixity was demonstrated hb the osmium bridging technique 5 of Hanker. \lounted sections were examined unstainied or stained "ith uran\l acetate.
Results The intraxenous LD50 for rats given 3-c\-clodextrin is 0. ,88 g kg and 1.00 g kg for a-cyclodextrin (Table 1). A close relationship \vas found betvveen the acute toxicitv and the nephrotoxic dose.
CYCLODEXTRIN NEPHROSIS
Vol. 83, No. 2 May 1976
369
Light MicdoscopiFndgs
The light microscopic findings associated 'with either a- or O-cvclodextrin were qualitatively similar folloxving single or repeated injections of nephrotoxic doses. Lesions were confined to the kidney and first appeared in the convoluted segments of the proximal tubule. Changes observed by- light microscopy resulted from a minimal single dose of 0.67 g, kg g-cvclodextrin or 1.0 g, kg a-cyclodextrin. Because of a selective tubule response, a crude dose relationship was apparent, particularly follow-ing f-cyclodextrin administration. Repeated administration of nephrotoxic doses resulted in extensive nephrosis. In paraffin-embedded sections, the earliest microscopic lesions s-ere mideellular cvtoplasmic vacuoles found at 24 hours after injection. Vacuolative change was most evident in Epon-embedded, semi-thin sections m-hich showed, at 24 hours after injection, numerous apical vacuoles and dark staining lysosomes in the midcellular and basal level of the proximal tubule epithelium (Figure 1). Large cytoplasmic v-acuoles were found in tubules examined 48 hours after injection of the cvclodextrins (Figure 2). The giant vacuoles wvere usually empty in paraffin sections. m-hereas amorphous material w-as found in the vacuoles in Epon-embedded tissue. Occasionally at 2 days and usually by 3 days follow-ing a single injection, cells that showed marked vacuolation had disintegrated. Epithelial sloughing follomved and mineralization of exfoliated epithelium occurred (Figure 3). Many of the tubules distal to the convoluted segments of the proximal tubules were occluded with exfoliated cells and cellular debris at 3 days after injection. In semi-thin sections, long cylindrical or acicular cytoplasmic crystals wvere found in rats given 0.98 g kg 0-cyclodextrin (Figure 4). Crystals were found occasionally in intact convoluted proximal Table 1-Acute Intravenous LD5 of Sprague-Dawley Rats for a-Cyclodextrin (1.8 g/100 ml saline) and for 3-Cyclodextrin (14.5 g/100 ml)
Dose
LD5 a-Cyclodextrin 788.4
mg/kg
ml/kg
Time of death
576 900
32 50
0/10
1400
80
10/10
8 in 48 hrs 24 hrs
1820 1160
12.5 8
10/0 8/2
24 hrs 7in24hrs, 8 in 48 hrs
730
5
3-Cyclodextrin 1008
Deaths/survivors
8/2
0/10
5in24hrs,
370
FRANK ET AL
American Journal
of Pathology
tubule segments and were concentrated in sloughed epithelial cells occluding the lower nephron. Rarely were they found in distal tubule epithelium. Extra tubular lesions of the renal cortex were uncommon. A mild interstitial fibrosis, sometimes containing a mild mononuclear cell infiltrate, was occasionallv observed. Nephrosis was not apparent in rats given 1, 2, 4, or 7 daily injections of 0.1 g/kg a-cvclodextrin; light microscopic lesions wvere found in one rat given 0.225 g/kg ,-c clodextrin daily for 4 days. Daily injections of 0.45 g/kg ,B-cyclodextrin resulted in severe nephrosis and produced no deaths. All rats given 0.9 g/kg f-cvclodextrin died within 4 days and 1 died after 2 days of treatment with 1.0 g/kg a-cyclodextrin. The survivors in this group showed severe nephrosis. Cvtoplasmic crystals were easily demonstrated in the tubules of rats given repeated large doses of the cyclodextrins, and their increase was the characteristic finding of repeated large doses of the cvclodextrins. Electro M
IJltrastructural alterations found 24 hours after administration of 0.45 g/kg f-cvclodextrin were limited to the vacuologenic apparatus of the proximal tubule. The numbers of large and small apical vacuoles increased. Lvsosomes were perinuclear and basal in location and normal in size except when containing electron-lucent streaks. These lvsosomes were often deformed because of needle-like microcr-vstals which projected into the lsosomal membranes (Figures 5 and 6). Since the number of microcrvstals appeared related to drug dosage, we regarded them as representing nondegradable cyclodextrins. Ultrastructural changes in other organelles were rare; occasionally the rough endoplasmic reticulum was dilated. Proximal tubules examined 2 and 3 days after injection with 0.45 g/'kg #-cvclodextrin showed extensive structural alterations in the vacuologenic apparatus. Apical vesicles and apical vacuoles were prominent at the luminal surface. Deeper in the cells, large apical vacuoles were present; occasionally these showed interrupted membranes at points of contact wvith adjacent ly-sosomes (Figure 7). The Golgi apparatus w,as usually prominent. Lv-sosomes were distorted by long matrical microcrvstals which projected into the membrane. Some of these crystal-containing giant ly-sosomes measured 5 to 8 p in diameter (Figure 8). The identity of lvsosomes was confirmed by acid phosphatase reaction product (Figure 9). Ultrastructural alterations wvere not evident in other organelles. Evidence of irreversible injurv was found in proximal tubule epithelium at 72 hours after injection (Figure 10). Large membrane-bounded vacu-
Vol. 83, No. 2 May 1976
CYCLODEXTRIN NEPHROSIS
371
oles, presumably Ivsosomal in origin, displaced the normal intracellular organelle relationships. The contents of these vacuoles was variable, many contained faint remnants of matrix, others contained a few irregular electron-dense particles. Microcrystals were not found in giant vacuoles. Their manifestation was associated with lvsosomes containing a relatively intact matrix. The fate of the microcrystals was not determined. Rarely, microcrvstals extended into the cytoplasm from disrupted lysosomes (Figure 11). Acid phosphotase reaction product was not demonstrated in Iysosomes with an indistinct matrix or in giant vacuoles. Irreversible cellular injury was indicated by focal ballooning of mitochondria and loss of matrix. The smooth endoplasmic reticulum was aggregated in some cells. Alterations were seldom found in the rough endoplasmic reticulum, brush border, or basal membrane. Advanced nephrosis was characterized by ultrastructural changes in many organelles. The mitochondria were swollen with loss of cristae and dissolution of the matrix. Many showed focal disintegration. The rough endoplasmic reticulum was fragmented into cytoplasmic tubular remnants. Numerous lipid droplets appeared in some cells. Partiallv membrane-bounded large vacuoles were characteristic of the cells in advanced injury. Such marked change was not observed at other levels of the nephron. In the glomerular tufts, the foot processes of the epithelial cells were preserved. An occasional vacuole with granular content and indistinct membrane was observed in the epithelial cells. Distal tubules showed debris in the lumen and prominent autophagic vacuoles. Discussion Cyclodextrin nephrosis is characterized bv a series of alterations in the vacuolar apparatus of the proximal tubule. Light microscopic examination showed cvtoplasmic vacuolation, cell disintegration, and amorphous mineralization. The sequence of morphologic events occurring in the nephrotic process was best illustrated in Epon-embedded tissue. Examination of these tissues by light and electron microscopy revealed that the alterations began with an increase in apical vacuoles. As the lysosomes gained prominence, microcrvstals became evident. The characteristic finding of cyclodextrin nephrosis occurs as a giant Iysosome, with the peripheral membrane stretched and distorted to confine the large microcrystals embedded in the Iysosomal matrix. Following appearance of giant Iysosomes, large vacuoles containing irregular flocculent densities were found. Degeneration in other cell organelles, notably the mitochondria, occurred with the manifestation of giant vacuoles.
372
FRANK ET AL
American Journal
of Pathology
The apical vacuole increase found early in the renal response to the Schardinger dextrins is not uniquely associated with the cyclodextrins. Similar vacuoles are found following glomerular filtration of many substances,'1ll and are considered a nonspecific response of the proximal tubule epithelium. The acicular microcrvstals may be peculiar to the cvclodextrin. Repeated administration of large doses of the cyclodextrins resulted in numerous giant Ivsosomes distorted by enclosed microcr-stals. The manifestation of crystals were related to dose, which strongly suggests that the crystals represented drug regained from the glomerular filtrate. In cells showing degenerative mitochondrial changes, giant empty vacuoles presumablv represented defunct lvsosomes. Because previous alterations were manifested only in the vacuolar apparatus, it is unlikely that the giant vacuoles arose from other organelles. The absence of identifiable elements in these vacuoles is unusual. The fate of microcrystals that were so prominent in Ivsosomes with an intact matrix was also curious. Rarely were microcrrstals seen spilling out into the cytoplasm from ruptured vacuoles. Structural alterations of lysosomes are often accompanied by functional change which may play a significant role in the nephrotic process. Even the mild changes noted 12 in mice with Chediak-Higashi type lysosomes show delayed degradation of horseradish peroxidase in the proximal tubule. Our results show severe vacuolative change occurs before alteration of other organelles. Acid phosphatase reaction product was confined to lysosomes with a homogeneous matrix and was not demonstrated in giant vacuoles thought to represent defunct lysosomes. Janigan and Santamaria 7 found little acid phosphatase reactivity in lvsosomes of heavily vacuolated rat proximal tubule epithelium in advanced sucrose nephrosis. Mitochondrial oxidative function, as indicated by histochemical demonstration, was reduced only after decrease in lvsosomal acid phosphatase reaction product. Less acid phosphatase activity was found 9 in multivesicular bodies than in lysosomes when Trump and janigan examined the kidnevs of rats given sucrose. These observations and the results reported here indicate that severe morphologic change in the vacuolative apparatus are likely accompanied by altered function. Whether such events precipitate irreversible cell injurv remains to be established. Cvclodextrin nephrosis represents an exaggerated form of osmotic nephrosis. Established models of osmotic nephrosis show severe alteration in the vacuolar apparatus, but these changes are usually transient, and little response is observed in other organelles. Mild changes occur in the vacuolar apparatus following glucose administration. NMaunsbach 8 observed numerous apical vacuoles in the proximal tubules of rats given
Vol. 83, No. 2 May 1976
CYCLODEXTRIN NEPHROSIS
373
glucose. He also found more profound vacuolative change and Issosomal increase following injection of sucrose, dextran, and mannitol. Trump and Janigan 9 confirmed Maunsbach's results and noted that multivesicular bodies arise via coalescence between members of lysosomes. Though a spectrum of changes may occur in the vacuolar apparatus following administration of glucose, sucrose and dextran, the lesions produced are apparentlv confirmed to this system of organelles and result in no sustained injury. On the other hand, cyclodextrin nephrosis apparently represents irreversible injurv, at least when giant Ivsosomes containing crystals have formed. An explanation for the contrasting reversibility of these lesions rests with the ease of the carbohvdrate hydrolysis. Cvclodextrin is not degradable by the common amvlases, and concentration within the Iysosomes results in the toxic injury to the cell. Whether this occurs because of physical injury to the lysosomal membrane or as a result of direct or indirect metabolic dysfunction, is unknown. The nephrotoxicitv of a diverse group of compounds is associated with severe alterations in the vacuolar apparatus of the proximal tubules. Ethylenediaminetetraacetic acid (EDTA) produces giant vacuoles'2 shortly after administration and is presumably bound to lysosomal membranes. A pronounced increase in proximal tubule lysosomes occurs 2 after administration of organic mercury. The aminoglycoside antibiotic, gentamicin," produces severe nephrosis. Structural manifestation of renal toxicity occurs as the number of autophagic vacuoles, cytosegresome, and mveloid figures increases in the proximal tubule. These diverse observations indicate the integral role of the vacuolar apparatus in the nephrotic process and show how normal proximal tubule reabsorption and degradation are perverted to result in cellular injurv. References 1. Emanuelli G, Satta G, Perpignano G: Lysosomal damage and its possible relationship with mitochondrial lesion in rat kidney under obstructive jaundice. Clin Chim Acta 25: 167-172, 1969 2. Fowler BA, Brown HW, Lucier GWX, Krigman NIR: The effects of chronic oral methvl mercurv exposure on the lysosome system of rat kidney. Lab Invest 32:313-322, 1975 3. French D: The Schardinger dextrins. Adv Carbohydrate Chem 12:189-260, 1957 4. Griffith LD, Bulger RE, Trump BF: The ultrastructure of the functioning kidnev. Lab Invest 16:220-246, 1967 a. Pearse AGE: Histochemistrv, Vol 2, Third edition. London, Churchill Livingstone, 1972, p 1444 6. Diomi P, Ericsson JLE, Matheson NA, Shearer JR: Studies on renal tubular morphology and toxicity after large doses of dextran 40 in the rabbit. Lab Invest 22:355-3, 1970
374
.
8. 9.
10. 11.
12. 13.
FRANK ET AL
American Journal of Pathology
Janigan DT, Santamaria A: A histochemical study of sw-elling and vacuolation of proximal tubular cells in sucrose nephrosis in the rat. Am j Pathol :39:1753-193. 1961 Maunsbach AB, NMadden SC, Latta H: Light and electron microscopic changes in proximal tubules of rats after administration of glucose. mannitol. sucrose or dextran. Lab Invest 11:421-432, 1962 Trump BF, Janigan DT: The pathogenesis of cytologic vacuolization in sucrose nephrosis: An electron microscopic and histochemical study. Lab Invest 11:395-41 1. 1962 Doolan PD, Schwartz SL, Haves JR, NMullen JC. Cummings NB: An evaluation of the nephrotoxicity of ethylenediaminetetraacetate and diethvlenetriaminepentaacetate in the rat. Toxicol Appl Pharmacol 10:481-50, 1967 Kosek JC, Mazze RI, Cousins MtJ: Nephrotoxicity of gentamicin. Lab Invest :30:4S57, 1974 Prieur DJ, Davis WC, Padgett GA: Defective function of renal lysosomes in mice with the Chediak-Higashi syndrome. Am J Pathol 67:227-240, 1972 Schwartz SL, Johnson CB, Doolan PD: Study of the mechanism of renal vacuologenesis induced in the rat by ethylenediaminetetraacetate: Comparison of the cellular activities of calcium and chromium chelates. Mol Pharmacol 6:54-60, 1970
2
Figures 1 and 2-Renal cortex from a rat given 0.90 g/kg 0-cyclodextrin (Epon, semi-thin sections, 1-Tissue collected 24 hours after injection. Prominent apical vacuoles toluidine blue, x 450). are evident under the brush border of the proximal tubules. Mid-cellular and basal cytoplasmic 2-Tissue collected 48 hours after injection. Large cytoplasmic vacuoles vacuoles are present. are a prominent feature of the proximal tubular epithelium. Frank cellular disintegration is present (arrow).
Figure 3-Renal cortex from rat given 0.90 g/kg d cyclodextrin 3 days earlier. Obvious nephrosis characterized by cytoplasmic vacuolation, cell disintegration, and amorphous mineralization is present. (Paraffin-embedded hematoxylin, phloxine, and eosin, x 180)
Fgu 4-Renal cortex from
rat given four consecutive daily injections of 0.45 g/kg d-cyclodextrin. Tubules show severe cytoplasmic vacuolation and prominent cylindrical and acicular crystals. (Epon-embedded, semi-thin section, toluidine blue, x450)
6
1.
dik. 1%,
.
-4
I AlbJMk -.".
Mr. .a,-
I.-&*
--1
:11 11
r.''
he
4
(v A. I
'.
Y
..
Figure 5-Proximal tubular epithelium from rat given 0.45 g/kg cyclodextrin; the membrane of the lysosomal structure is distorted by the enclosed acicular microcrystal (Uranyl acetate and
lead citrate, x 16,500).
4 I&'b
p
Om *A 4
I
Id
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-)
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.
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Figure 6-Portion of renal proximal tubule cell from rat given single injection of 0.90 g/kg d3-cyclodextrin showing marked distortion of lysosomal membrane (arrow) by acicular microcrystals embedded in the matrix (Uranyl acetate and lead citrate, x 35,500).
Figure 7-Portion of renal proximal tubule cell from a rat given 0.90 g/kg 3-cyclodextrin: apical vacuoles have joined the lysosome which shows microcrystals in the matrix (Uranyl acetate and Fiu 8-Portion of renal proximal tubule cell from a rat given 0.90 lead citrate, x 23,500. g/kg i-cyclodextrin 48 hours earlier showing several giant lysosomes all distorted by enclosed microcrystals (Uranyl acetate and lead citrate, x 5700).
Figure 9-Portion of renal proximal tubule cell from a rat given 0.90 g/kg g3-cyclodextrin 48 hours earlier; lysosome contains acid phosphotase reaction product and microcrystals (65,200).
Fwe 10-Portion of proximal tubular epithelium from a rat given g/kg 3-cyclodestrin 3 days earlier. A large cytoplasmic vacuole is present. Cytoplasmic microcrystals appear to have spilled from a partially membrane-bound vacuole (arrow). Mitochondrial swelling and loss of intracristalline matrix are present. (Uranyl acetate, and lead citrate, x 12,900
The renal toxicity of the Schardinger dextrins, a and O-cvclodextrin, is manifested as a series of alterations in the vacuolar organelles of the proximal convoluted tubule. These changes begin as an increase of apical vacuoles and the appearance of giant lysosomes. The giant Iysosomes characteristic of cyclodextrin nephrosis are notable because of the prominent acicular microcrvstals embedded in the Ivsosomal matrix. Giant vacuoles devoid of acid phosphatase reaction product are found in advanced lesions. The vacuolar apparatus shows advanced changes prior to manifestation of lesions in mitochondria and other organelles. These obsertations indicate a role of the v-acuologenic apparatus in the nephrotic process. Intracellular concentration of toxin via the lysosomal pathway represents a perversion of the physiologic function of the proximal tubule which ultimately leads to cell death. (Am J Pathol 83:367-382, 1976)
THE RENAL PROXIMAL TUBULE functions to recover many- substances from the glomerular filtrate. A structural adaptation for this purpose is the vacuologenic apparatus which recovers and retains proteins and other substances requiring degradation before recycling. In addition to substances normally regained from the glomerular filtrate, these organelles may intercept and sequester toxic materials. Indeed, the toxid tubular nephrosis associdted w-ith several substances 1.2 is manifested because of the recycling function of the proximal tubule. This studx' describes the nephrosis in the rat kidney caused by parenteral administration of a- and O-cvclodextrin. These carbohy-drates result from fermentation of potato starch by Bacillus macerans.3 Because their natural configuration is a closed circle of 6 (a) or 7 (B) D-glucose molecules, the c-clodextrins are unusually resistant to hydrolysis bv the common amvlases. Their appeal to pharmaceutical formulation follows the theoretical clathrate model which suggests that after enclosure of a less hydrophilic compound within the lumen of the cyclodextrin, solubility increases.
The nephrosis found after cyclodextrin administration appears to result from disturbances in the vacuologenic apparatus. Ultrastructural lesions are characterized by intracellular acicular crystals and giant lvsosomes which form prior to appearance of alterations in other organellies. From the Patholog- and
Toxicology
Research Unit. The U pjohn Compan%. Kalamazoo. \Mich-
igan.
Accepted for publication January 27. 1976 Address reprint requests to Dr. Doyle W. Frank. Upjohn Company. Kalamazoo. \I 49001
Pathology and Toxicolog'
Research L-nit. The 367
368
FRANK ET AL
American Journal of Pathology
Materials and Methods Formulations of the Cycodextrins 3-C0-clodextrin ICN. K and K Laboratories. Plainv iew. N.Y. and Pierce Chemical Companv. Rockford. Ill.l was dissolxed in sx-arm phvsiologic saline at the maximum soluble concentration L1 S g 100 ml at 2.5 C A.A saturated solution of a-cv cl(dextrin Sigma Chemical Companv. St. Louis. \lo. resulted "vhen 14.5 g %"as dissolved in 100 ml "arm 50 to 70 C physiologic saline. Both solutions svere used at room temperature. Acute Intravenous Administration of the LD50 of Cycodextrins Groups of 5 male and 3 female Sprague-Da".-ley rats 'eighing 1.0 to 160g were given
the cxclodextrins intravenously. The acute LD5a s-as determined bx- the Spearman-Karber method 7 dav s later. Cyclodextrin Administration Subcutaneously Single Administration
Groups of 4 100 to 125-g rats wsere gix-en 0.225. 0.45. or 0.90 g kg 3-cs-clodextrin bx- single subcutaneous injection and killed 12. 24. 48. or 96 hours later. Similar groups "ere injected with 0.1 or 1.0 g kg a-c!-clodextrin and killed 24 or 96 hours later Salineinjected control rats "ere killed at 12 or 96 hours after injection. Repeated Administration
Groups of four 100 to 125-g Sprague-Das-ley rats received 1. 2. :3. 4. or 7 dail\ subcutaneous injections of B-cvclodextrin at 0.225. 0.4.. 0.67.5. or 0.90 g kg. Other groups of rats were given a similar regimen of a-cvclodextrin at 0.1 or 1.0 g kg. Animals "vere killed 24 hours follomsing the last injection. Rats given saline in drug-equivialent volumes were killed after the seventh daily injection. Collcto of Tissues Rats "vere perfused via the abdominal aorta by a modification of the procedure 4 described bv Griffith. The aorta "-as clamped mvith mosquito forceps at the anterior surface of the diaphragm and anterior to the iliac bifurcation. The aorta was cannulated with ani 1S-gauge needle anterior to the terminal clamp. and the posterior vena cava wsas nicked at the moment perfusion began. The abdominal viscera wzas perfused with cold 1.5 or 2% glutaraldehvde in 0.1 \I Msodium cacodvlate buffer containing 1Cc sucrose. pH 7.2. for approximately :3 minutes at 130 to 10 mm Hg pressure. The kidneys were removed and immersed in fixative. and cortical slices "vere diced into 1-mm cubes. Tissues wxere fixed for 43 minutes to 1 hour. rinsed in 0.1 \I sodium cacodylate buffer and postfixed in 1%c osmium tetroxide for 43 minutes. Areas selected after Epon embedding were examined after staining with uranvl acetate and lead citrate. Nonperfused kidney cortex "as diced and fixed in 2% glutaraldehvde in .1 \l phosphate buffer. pH 7.4. for 1 hour. Glutaraldehvde-fixed tissue "as placed in Mlillonigs fixative for 11: hours at 4 C. Tissue "as embedded in Epon s12. One-micron-thick sections were stained mvith toluidine blue. Thin sections "vere stained "ith uranvl acetate and lead citrate. Kidneys collected for light microscopy were fixed in 10%c buffered formalin and routinelv processed. Acid phosphatase actixity was demonstrated hb the osmium bridging technique 5 of Hanker. \lounted sections were examined unstainied or stained "ith uran\l acetate.
Results The intraxenous LD50 for rats given 3-c\-clodextrin is 0. ,88 g kg and 1.00 g kg for a-cyclodextrin (Table 1). A close relationship \vas found betvveen the acute toxicitv and the nephrotoxic dose.
CYCLODEXTRIN NEPHROSIS
Vol. 83, No. 2 May 1976
369
Light MicdoscopiFndgs
The light microscopic findings associated 'with either a- or O-cvclodextrin were qualitatively similar folloxving single or repeated injections of nephrotoxic doses. Lesions were confined to the kidney and first appeared in the convoluted segments of the proximal tubule. Changes observed by- light microscopy resulted from a minimal single dose of 0.67 g, kg g-cvclodextrin or 1.0 g, kg a-cyclodextrin. Because of a selective tubule response, a crude dose relationship was apparent, particularly follow-ing f-cyclodextrin administration. Repeated administration of nephrotoxic doses resulted in extensive nephrosis. In paraffin-embedded sections, the earliest microscopic lesions s-ere mideellular cvtoplasmic vacuoles found at 24 hours after injection. Vacuolative change was most evident in Epon-embedded, semi-thin sections m-hich showed, at 24 hours after injection, numerous apical vacuoles and dark staining lysosomes in the midcellular and basal level of the proximal tubule epithelium (Figure 1). Large cytoplasmic v-acuoles were found in tubules examined 48 hours after injection of the cvclodextrins (Figure 2). The giant vacuoles wvere usually empty in paraffin sections. m-hereas amorphous material w-as found in the vacuoles in Epon-embedded tissue. Occasionally at 2 days and usually by 3 days follow-ing a single injection, cells that showed marked vacuolation had disintegrated. Epithelial sloughing follomved and mineralization of exfoliated epithelium occurred (Figure 3). Many of the tubules distal to the convoluted segments of the proximal tubules were occluded with exfoliated cells and cellular debris at 3 days after injection. In semi-thin sections, long cylindrical or acicular cytoplasmic crystals wvere found in rats given 0.98 g kg 0-cyclodextrin (Figure 4). Crystals were found occasionally in intact convoluted proximal Table 1-Acute Intravenous LD5 of Sprague-Dawley Rats for a-Cyclodextrin (1.8 g/100 ml saline) and for 3-Cyclodextrin (14.5 g/100 ml)
Dose
LD5 a-Cyclodextrin 788.4
mg/kg
ml/kg
Time of death
576 900
32 50
0/10
1400
80
10/10
8 in 48 hrs 24 hrs
1820 1160
12.5 8
10/0 8/2
24 hrs 7in24hrs, 8 in 48 hrs
730
5
3-Cyclodextrin 1008
Deaths/survivors
8/2
0/10
5in24hrs,
370
FRANK ET AL
American Journal
of Pathology
tubule segments and were concentrated in sloughed epithelial cells occluding the lower nephron. Rarely were they found in distal tubule epithelium. Extra tubular lesions of the renal cortex were uncommon. A mild interstitial fibrosis, sometimes containing a mild mononuclear cell infiltrate, was occasionallv observed. Nephrosis was not apparent in rats given 1, 2, 4, or 7 daily injections of 0.1 g/kg a-cvclodextrin; light microscopic lesions wvere found in one rat given 0.225 g/kg ,-c clodextrin daily for 4 days. Daily injections of 0.45 g/kg ,B-cyclodextrin resulted in severe nephrosis and produced no deaths. All rats given 0.9 g/kg f-cvclodextrin died within 4 days and 1 died after 2 days of treatment with 1.0 g/kg a-cyclodextrin. The survivors in this group showed severe nephrosis. Cvtoplasmic crystals were easily demonstrated in the tubules of rats given repeated large doses of the cyclodextrins, and their increase was the characteristic finding of repeated large doses of the cvclodextrins. Electro M
IJltrastructural alterations found 24 hours after administration of 0.45 g/kg f-cvclodextrin were limited to the vacuologenic apparatus of the proximal tubule. The numbers of large and small apical vacuoles increased. Lvsosomes were perinuclear and basal in location and normal in size except when containing electron-lucent streaks. These lvsosomes were often deformed because of needle-like microcr-vstals which projected into the lsosomal membranes (Figures 5 and 6). Since the number of microcrvstals appeared related to drug dosage, we regarded them as representing nondegradable cyclodextrins. Ultrastructural changes in other organelles were rare; occasionally the rough endoplasmic reticulum was dilated. Proximal tubules examined 2 and 3 days after injection with 0.45 g/'kg #-cvclodextrin showed extensive structural alterations in the vacuologenic apparatus. Apical vesicles and apical vacuoles were prominent at the luminal surface. Deeper in the cells, large apical vacuoles were present; occasionally these showed interrupted membranes at points of contact wvith adjacent ly-sosomes (Figure 7). The Golgi apparatus w,as usually prominent. Lv-sosomes were distorted by long matrical microcrvstals which projected into the membrane. Some of these crystal-containing giant ly-sosomes measured 5 to 8 p in diameter (Figure 8). The identity of lvsosomes was confirmed by acid phosphatase reaction product (Figure 9). Ultrastructural alterations wvere not evident in other organelles. Evidence of irreversible injurv was found in proximal tubule epithelium at 72 hours after injection (Figure 10). Large membrane-bounded vacu-
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oles, presumably Ivsosomal in origin, displaced the normal intracellular organelle relationships. The contents of these vacuoles was variable, many contained faint remnants of matrix, others contained a few irregular electron-dense particles. Microcrystals were not found in giant vacuoles. Their manifestation was associated with lvsosomes containing a relatively intact matrix. The fate of the microcrystals was not determined. Rarely, microcrvstals extended into the cytoplasm from disrupted lysosomes (Figure 11). Acid phosphotase reaction product was not demonstrated in Iysosomes with an indistinct matrix or in giant vacuoles. Irreversible cellular injury was indicated by focal ballooning of mitochondria and loss of matrix. The smooth endoplasmic reticulum was aggregated in some cells. Alterations were seldom found in the rough endoplasmic reticulum, brush border, or basal membrane. Advanced nephrosis was characterized by ultrastructural changes in many organelles. The mitochondria were swollen with loss of cristae and dissolution of the matrix. Many showed focal disintegration. The rough endoplasmic reticulum was fragmented into cytoplasmic tubular remnants. Numerous lipid droplets appeared in some cells. Partiallv membrane-bounded large vacuoles were characteristic of the cells in advanced injury. Such marked change was not observed at other levels of the nephron. In the glomerular tufts, the foot processes of the epithelial cells were preserved. An occasional vacuole with granular content and indistinct membrane was observed in the epithelial cells. Distal tubules showed debris in the lumen and prominent autophagic vacuoles. Discussion Cyclodextrin nephrosis is characterized bv a series of alterations in the vacuolar apparatus of the proximal tubule. Light microscopic examination showed cvtoplasmic vacuolation, cell disintegration, and amorphous mineralization. The sequence of morphologic events occurring in the nephrotic process was best illustrated in Epon-embedded tissue. Examination of these tissues by light and electron microscopy revealed that the alterations began with an increase in apical vacuoles. As the lysosomes gained prominence, microcrvstals became evident. The characteristic finding of cyclodextrin nephrosis occurs as a giant Iysosome, with the peripheral membrane stretched and distorted to confine the large microcrystals embedded in the Iysosomal matrix. Following appearance of giant Iysosomes, large vacuoles containing irregular flocculent densities were found. Degeneration in other cell organelles, notably the mitochondria, occurred with the manifestation of giant vacuoles.
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The apical vacuole increase found early in the renal response to the Schardinger dextrins is not uniquely associated with the cyclodextrins. Similar vacuoles are found following glomerular filtration of many substances,'1ll and are considered a nonspecific response of the proximal tubule epithelium. The acicular microcrvstals may be peculiar to the cvclodextrin. Repeated administration of large doses of the cyclodextrins resulted in numerous giant Ivsosomes distorted by enclosed microcr-stals. The manifestation of crystals were related to dose, which strongly suggests that the crystals represented drug regained from the glomerular filtrate. In cells showing degenerative mitochondrial changes, giant empty vacuoles presumablv represented defunct lvsosomes. Because previous alterations were manifested only in the vacuolar apparatus, it is unlikely that the giant vacuoles arose from other organelles. The absence of identifiable elements in these vacuoles is unusual. The fate of microcrystals that were so prominent in Ivsosomes with an intact matrix was also curious. Rarely were microcrrstals seen spilling out into the cytoplasm from ruptured vacuoles. Structural alterations of lysosomes are often accompanied by functional change which may play a significant role in the nephrotic process. Even the mild changes noted 12 in mice with Chediak-Higashi type lysosomes show delayed degradation of horseradish peroxidase in the proximal tubule. Our results show severe vacuolative change occurs before alteration of other organelles. Acid phosphatase reaction product was confined to lysosomes with a homogeneous matrix and was not demonstrated in giant vacuoles thought to represent defunct lysosomes. Janigan and Santamaria 7 found little acid phosphatase reactivity in lvsosomes of heavily vacuolated rat proximal tubule epithelium in advanced sucrose nephrosis. Mitochondrial oxidative function, as indicated by histochemical demonstration, was reduced only after decrease in lvsosomal acid phosphatase reaction product. Less acid phosphatase activity was found 9 in multivesicular bodies than in lysosomes when Trump and janigan examined the kidnevs of rats given sucrose. These observations and the results reported here indicate that severe morphologic change in the vacuolative apparatus are likely accompanied by altered function. Whether such events precipitate irreversible cell injurv remains to be established. Cvclodextrin nephrosis represents an exaggerated form of osmotic nephrosis. Established models of osmotic nephrosis show severe alteration in the vacuolar apparatus, but these changes are usually transient, and little response is observed in other organelles. Mild changes occur in the vacuolar apparatus following glucose administration. NMaunsbach 8 observed numerous apical vacuoles in the proximal tubules of rats given
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glucose. He also found more profound vacuolative change and Issosomal increase following injection of sucrose, dextran, and mannitol. Trump and Janigan 9 confirmed Maunsbach's results and noted that multivesicular bodies arise via coalescence between members of lysosomes. Though a spectrum of changes may occur in the vacuolar apparatus following administration of glucose, sucrose and dextran, the lesions produced are apparentlv confirmed to this system of organelles and result in no sustained injury. On the other hand, cyclodextrin nephrosis apparently represents irreversible injurv, at least when giant Ivsosomes containing crystals have formed. An explanation for the contrasting reversibility of these lesions rests with the ease of the carbohvdrate hydrolysis. Cvclodextrin is not degradable by the common amvlases, and concentration within the Iysosomes results in the toxic injury to the cell. Whether this occurs because of physical injury to the lysosomal membrane or as a result of direct or indirect metabolic dysfunction, is unknown. The nephrotoxicitv of a diverse group of compounds is associated with severe alterations in the vacuolar apparatus of the proximal tubules. Ethylenediaminetetraacetic acid (EDTA) produces giant vacuoles'2 shortly after administration and is presumably bound to lysosomal membranes. A pronounced increase in proximal tubule lysosomes occurs 2 after administration of organic mercury. The aminoglycoside antibiotic, gentamicin," produces severe nephrosis. Structural manifestation of renal toxicity occurs as the number of autophagic vacuoles, cytosegresome, and mveloid figures increases in the proximal tubule. These diverse observations indicate the integral role of the vacuolar apparatus in the nephrotic process and show how normal proximal tubule reabsorption and degradation are perverted to result in cellular injurv. References 1. Emanuelli G, Satta G, Perpignano G: Lysosomal damage and its possible relationship with mitochondrial lesion in rat kidney under obstructive jaundice. Clin Chim Acta 25: 167-172, 1969 2. Fowler BA, Brown HW, Lucier GWX, Krigman NIR: The effects of chronic oral methvl mercurv exposure on the lysosome system of rat kidney. Lab Invest 32:313-322, 1975 3. French D: The Schardinger dextrins. Adv Carbohydrate Chem 12:189-260, 1957 4. Griffith LD, Bulger RE, Trump BF: The ultrastructure of the functioning kidnev. Lab Invest 16:220-246, 1967 a. Pearse AGE: Histochemistrv, Vol 2, Third edition. London, Churchill Livingstone, 1972, p 1444 6. Diomi P, Ericsson JLE, Matheson NA, Shearer JR: Studies on renal tubular morphology and toxicity after large doses of dextran 40 in the rabbit. Lab Invest 22:355-3, 1970
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10. 11.
12. 13.
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Janigan DT, Santamaria A: A histochemical study of sw-elling and vacuolation of proximal tubular cells in sucrose nephrosis in the rat. Am j Pathol :39:1753-193. 1961 Maunsbach AB, NMadden SC, Latta H: Light and electron microscopic changes in proximal tubules of rats after administration of glucose. mannitol. sucrose or dextran. Lab Invest 11:421-432, 1962 Trump BF, Janigan DT: The pathogenesis of cytologic vacuolization in sucrose nephrosis: An electron microscopic and histochemical study. Lab Invest 11:395-41 1. 1962 Doolan PD, Schwartz SL, Haves JR, NMullen JC. Cummings NB: An evaluation of the nephrotoxicity of ethylenediaminetetraacetate and diethvlenetriaminepentaacetate in the rat. Toxicol Appl Pharmacol 10:481-50, 1967 Kosek JC, Mazze RI, Cousins MtJ: Nephrotoxicity of gentamicin. Lab Invest :30:4S57, 1974 Prieur DJ, Davis WC, Padgett GA: Defective function of renal lysosomes in mice with the Chediak-Higashi syndrome. Am J Pathol 67:227-240, 1972 Schwartz SL, Johnson CB, Doolan PD: Study of the mechanism of renal vacuologenesis induced in the rat by ethylenediaminetetraacetate: Comparison of the cellular activities of calcium and chromium chelates. Mol Pharmacol 6:54-60, 1970
2
Figures 1 and 2-Renal cortex from a rat given 0.90 g/kg 0-cyclodextrin (Epon, semi-thin sections, 1-Tissue collected 24 hours after injection. Prominent apical vacuoles toluidine blue, x 450). are evident under the brush border of the proximal tubules. Mid-cellular and basal cytoplasmic 2-Tissue collected 48 hours after injection. Large cytoplasmic vacuoles vacuoles are present. are a prominent feature of the proximal tubular epithelium. Frank cellular disintegration is present (arrow).
Figure 3-Renal cortex from rat given 0.90 g/kg d cyclodextrin 3 days earlier. Obvious nephrosis characterized by cytoplasmic vacuolation, cell disintegration, and amorphous mineralization is present. (Paraffin-embedded hematoxylin, phloxine, and eosin, x 180)
Fgu 4-Renal cortex from
rat given four consecutive daily injections of 0.45 g/kg d-cyclodextrin. Tubules show severe cytoplasmic vacuolation and prominent cylindrical and acicular crystals. (Epon-embedded, semi-thin section, toluidine blue, x450)
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Figure 6-Portion of renal proximal tubule cell from rat given single injection of 0.90 g/kg d3-cyclodextrin showing marked distortion of lysosomal membrane (arrow) by acicular microcrystals embedded in the matrix (Uranyl acetate and lead citrate, x 35,500).
Figure 7-Portion of renal proximal tubule cell from a rat given 0.90 g/kg 3-cyclodextrin: apical vacuoles have joined the lysosome which shows microcrystals in the matrix (Uranyl acetate and Fiu 8-Portion of renal proximal tubule cell from a rat given 0.90 lead citrate, x 23,500. g/kg i-cyclodextrin 48 hours earlier showing several giant lysosomes all distorted by enclosed microcrystals (Uranyl acetate and lead citrate, x 5700).
Figure 9-Portion of renal proximal tubule cell from a rat given 0.90 g/kg g3-cyclodextrin 48 hours earlier; lysosome contains acid phosphotase reaction product and microcrystals (65,200).
Fwe 10-Portion of proximal tubular epithelium from a rat given g/kg 3-cyclodestrin 3 days earlier. A large cytoplasmic vacuole is present. Cytoplasmic microcrystals appear to have spilled from a partially membrane-bound vacuole (arrow). Mitochondrial swelling and loss of intracristalline matrix are present. (Uranyl acetate, and lead citrate, x 12,900
