Document Np9Ro35jX4YkaoN0zLRo50wR

f 5 (PtpJlKj A Renal Damage After Prolonged Exposure to Cadmium An Experimental Study BENGT AXELSSON, MD, AND MAGNUS PISCATOR, STOCKHOLM Th.HAT POISONING from long ex The problems given consideration were posure to cadmium can give rise to renal the following: damage and proteinuria has been demon 1. In which part or parts of the nephron strated in man and laboratory animals.1"7 is the damage located? Exposure to cadmium results in functional 2. Is the proteinuria a symptom of tubu disorders in the renal tubules; whether it is lar damage? the proximal or distal segment that is the 3. Where in the kidney is the cadmium main site of impairment has not been estab deposited? lished. In electrophoretic examinations of 4. Is the amount deposited a measure of urinary proteins the electrophoretic pattern the degree of poisoning? was characterized by a fairly low albumin 5. Does the cadmium excretion provide a content and high 03-, /?-, and y-fractions * measure of the toxic effect ? The proteinuria is a tubular type resembling that in a number of metabolic disorders in Material and Methods volving impairment of tubular function. Experimental Conditions.--The experiments were Further evidence of tubular proteinuria was presented by Kazantzis et al. No consistent connection between the ex amined renal functions and presence of proteinuria has been found,15 but tubular performed on S3 Belgian Giant rabbits (weight 3.3-4.9 kg). These were divided into two series, A and B, and a pilot group of 15 animals, with a con trol group of ten. Series A, which constituted the chief material for the study of renal function, was divided into four groups, of seven rabbits each and' changes seem to provide the most plausible explanation of the proteinuria. It is con ceivable that there is either defective tubular reabsorption of proteins normally one control group of ten. Series B, on which most urinary and serum studies were perfonned, was divided into one group exposed to cadmium and a control group, each of which contained ten rabbits. Mean weights of the various groups at the begin occurring in the glomerulus filtrate, or a leakage or secretion of proteins from dam aged tubule cells; a combination of these mechanisms is conceivable. With the 'object of examining problems arising from clinical study, the following animal experiments were designed. A serum study was also carried out, a report of which is given elsewhere.8 Submitted for publication Dec 30, 1965; accepted Jan 3, 1066. From the Institute of Hygiene, Karolinska Tnstitutet, and the departments of General Hygiene and Industrial Hygiene, National Institute of Public Health, Stockholm. Reprint requests to Institute of Hygiene, Karolinska In* stitutet, Stockholm 60, Sweden (Dr. Piscator). ning of the experiments were about the same. The grouping is shown in Table 1 with the exposure time for each group. Cadmium was administered as a subcutaneous in jection of 0.5 ml/kg f body weight (corresponding to 0.25 mg of cadmium per kilogram) of an isotonic solution with the following composition: 82 mg of cadmium chloride and 720 mg of sodium chloride in 100 ml of sterile water. Injections were given five days a week for the period specified in Fig 1. To minimize the risk of untoward tissue reactions the site of injection was varied as much as possible- The study was started simultaneously for the various groups. Group 4 in series A, ten rabbits in series B, and the 15 in the pilot group were given cadmium chloride and the others physiological Arch Environ Health -Vo! 12, March 1966 N36921 RENAL DAMAGE--AXELSSON & P1SCAT0R 361 Ta b l e 1.--Number of Rabbits and Exposure Time in Different Groups Pilot group ConlrolS A. Group 1 2 "3 "4 Conirols B. Controls Exposed group Number of Exposure Time Rabbits (Weeks) 15 24 10 0 7 11 7 17 7 23 7 * 29 10 0 10 0 10 24 saline by subcutaneous injection. Cadmium chloride was administered to groups 3, 2, and 1 of series A in turn, at intervals of five weeks. The pilot group was used to determine the time at which the animals exposed longest (group 4) could be expected to display observable functional and morphological renal damage, apart from the proteinuria. Once a month two exposed rabbits were taken from the pilot group and one from its control group for examination of renal function. When these tests disclosed functional and morphologic tubular damage and an appreciably increased excre tion of protein in the urine {>150 mg of protein daily), the exposure of groups 1-4 was discontinued in turn, at intervals of one week. The renal func tion tests on exposed groups (1-4) were performed simultaneously with those on two or three rabbits from the control group. For series B the exposure was discontinued after 24 weeks, when the protein excretion was consid erably increased. Serum and urine specimens were taken weekly during the first three weeks of the experiment, and then every two to three weeks. Examinations were continued for 25 weeks after exposure had ceased. Seven days after the renal function had been ex amined the rabbits in series A were sacrificed by bleeding, a heart puncture being made to obtain a blood sample. The puncture was performed under pentobarbital (Nembutal) anesthesia. Renal Function Tests.--The glomerulus filtra tion rate was examined by means of a creatinine clearance test, performed largely as described by Josephson and Kallas.8 Under shallow pentobarbital anesthesia 50 ml of tepid water per kilogram of body weight was given through a stomach tube to obtain good diuresis, and a Foley catheter No. 16 or 18 was introduced into the bladder. The rabbit was then placed in a wooden box de signed so that the rabbit was seated in a natural pos ture with the head passing through a hole in the lid large enough not to prevent head movements. Tire bladder catheter left through another hole under the hindquarters of the animal. The rabbit was given 50 ml of freshly prepared 5% creatinine so lution by- subcutaneous injection. It usually re covered from the anesthetic after about half an hour, and after one hour the effect of the anesthesia had worn off. The animal usually did not display nervousness. Blood specimens were obtained by puncturing an ear vein. The first specimen was taken about two Renal Function tests. CdCl2 NaCI i|---------------------------------------------------------------------------------------------------------------------------------------------i1 0 5 10 15 20 25 Fig 1.--Plan of the exposure experiment Arch Environ Health--Vol 12, March 1966 30 week! DUP050312799 362 RENAL DAMAGE--.AXELSSON & PISCATOR hours after the creatinine injection. The urine flowed down through the catheter into a dish and was collected over three periods, of 20-40 minutes each. At the end of each period the bladder was flushed twice with 20 ml of water and twice with air. The creatinine was determined by the method de scribed by Heyrowsky, and used as a routine method at St. Erik's Hospital, Stockholm. The clearance value reported is the mean for the three periods. The function of the proximal tubules was ex amined by the glucose reabsorption test. The ex amination was performed at the same time as the creatinine clearance, and the three periods in the two tests were coincident. Thirty percent glucose solution was given by con tinuous infusion (0.375 ml/min) through a poly ethylene catheter inserted in an ear vein. Just before the infusion was begun 20 ml of a 50% so lution of glucose was given through the catheter. Blood and urine specimens were taken at the same time as those for the creatinine clearance determi nation, and the former specimens were invariably taken by puncturing a vein of the ear not used for the glucose infusion. This infusion technique gave blood sugar values that usually exceeded 300 mg/ 100 ml. The blood and urinary sugar analyses were per formed by the routine method used at St. Erik's Hospital, Stockholm." The results showed that with the sugar levels used, the upper limit for tubular reabsorption of the glucose could not be determined and the glucose during concurrent glucosuria in creased with the amount of glucose filtered. This is consistent with results reported by Decsi for a study on the rabbit.11 The reason why the rabbit differs in this respect from other species and man is ob scure. Since the reabsorption capacity is a function of the amount of glucose filtered the results of the study are reported as the relationship between the reabsorbed and filtered amounts. Urine Examinations.--The 24-hour volumes of urine were collected in metabolic cages with separa tion of feces. Urine was centrifuged and filtered be fore the various tests were performed. Glucose was assayed with diagnostic tests for glucose in urine (Clinistix and Clinitest}.* Total protein was de termined by the biuret method after precipitation of the urinary proteins with Tsuchiya reagent." The excretion of ur-amino acid-N was determined in series B by a Ninhydrin method.1 For the con trol group it was performed only after 41 and 49 weeks. For preparation of urine colloids a method de scribed earlier was used, involving concentration by ultrafiltration through a dialysis tube,+ dialysis, and freeze drying. The urine colloids were then examined by paper electrophoresis at pH 8.6, and in some cases also with starch gel electrophoresis with various buffer systems.11'15 * Ames Co., luc., SIS McXaughton Ave, Elkhart, Ind f Visking dialysis tubing, 24/32 inch For fractionation of urine colloids the pooh.; urine colloids from series B were gel filtered c. Scphadex G-200t with 0.1 M NaCl as the chicm An account of the theory and range of application of gel filtration has been published by Flodiu,5'- ,\ fraction collector was used and the fraction., ; were read at 250 and 280 mjt in a spectrophotomc , ter.)| Pooled fractions were dialyzed, freeze dried, . and examined by paper and starch gel electrophorc- , sis. Cadmium was estimated with a dithizont ; method.17 j Other Analyses.--Alkaline phosphatase activity , in the renal cortex was determined. Dry weight, used as a reference standard in reporting enzyme : activity, was determined on specimens of renal ] cortex from areas adjacent to those from j which autopsy specimens for enzyme analysis ; were taken ' (the upper pole of the right ; kidney). The enzyme activity was measured by a ; method reported by'Holmgard1 as the amount of j p-nitrophenol formed by P-nitrophe-nylphosphate j through the action of alkaline phosphatase present in the homogenate. F-nitrophenylphosphate is color less, and in the hydrolysis of the phosphate group p-nitropheno! it is liberated as a yellow salt. Color was read in a spectrophotometer? at 410 mji. The substrate was 0.42% sodium p-nitrophenylphos phate # and the standard 1 mM p-nitrophenol.*** * Results are expressed in moles of substrate split per kilogram of dry weight per hour. The cadmium in. the kidney and liver was esti mated. Specimens were taken at autopsy of about 100 mg of cortex. 100 ml of medulla, usually from the upper right pole, and about 100 ml of liver tissue. On these specimens activation analyses of cadmium were performed by a method reported by Westermark and Sj6strand.1" f'1 Because of the fairly high cost of the activation method, assays on renal medulla and liver were performed only on some animals. , . ...... Cadmium in urine and feces was determined by: the polarographic method of Cholak and Hubbard,5" which, with minor modifications, is routine at this Institute. Mineralization was effected with nitric acid and hydrogen i peroxide. The determination was made on total urine. For series B it was per formed only afterf 18. and 49 weeks; on the latter occasion it was also performed on filtrate obtained after ultrafiltration through a dialysis tube. A dry weight determination of renal cortex tis sue was performed for 'presence and extent of renal edema and to obtain, a; reference standard for re porting the activity of; alkaline phosphatase. Histological, examinatiom of renal and liver tis sue was performed omSpecimens stained with he matoxylin and eosiiT arid! by the method of Timm.21 t Pharmacia, Uppsala/ Sweden. Radi.Rac, LKB Frodufcfer,' Stockholm, Sweden I) Beckman DB 11 Beckman B # Sigma 104, Sigma Chemical Co. *' Sigma Chemical Cor.:; ft The determinations, were performed by T. Westermark and B. Sjostrand, Royal Institute of Technology, Stockholm, Sweden. . Arch Environ Health--Vol 12, March 1966 DUP050312800 1 aa^te^. I RENAL DAMAGE--AXELSSON & PISCATOR 363 liie latter to locate deposited metal. To precipitate a regular record of weight was made. It has been all inetals as sulfides specimens were fixed in alco shown that rabbits exposed to cadmium for a long hol saturated with hydrogen sulfide, after which period develop anemia,1'2*'* so hemoglobin levels tiiev were treated with silver nitrate solution. In the were determined and red cell counts performed. vicinity of metal sulfide particles silver is then pre- Results are reported elsewhere.8 vipilated in the presence of hydroquinone; the sites j at which metals were originally deposited can he Results I identified histologically from the presence of the Series A.--In series A two animals . Idack particles. Since marked hypochromic. anemia with a he died--one after eight weeks and one after molytic component developed in the course of the 14 weeks of exposure. Both are excluded experiment, and iron was conceivably stored in the from the following results. . i kidney and liver, specimens were stained for iron. Creatinin Clearance (Fig 2): Reduction in A more detailed description of methods used in the histological examination is given elsewhere/3 Examination of renal proteins: From two rabbits in series B that died during the study the kidneys mean clearance was not significant, but might be responsible for some effect Reabsorption of Glucose (Fig 2): The and liver were removed. Cadmium was assayed by decrease in the ratio of reabsorbed to fil the dithizone method. The organs were frozen and later homogenized in 0.1 M phosphate buffer (pH 7). After centrifugation, the soluble fraction was fil tered through Sephadex G-2S in distilled water; the protein fraction was then freeze dried. The pro tered amounts of glucose from 23 weeks ex posure was statistically significant (F= 8.75+). The Activity of Alkaline Phosphatase in teins were examined by paper and starch gel elec the Renal Cortex Homogenate (Fig 2): trophoresis. In addition to protein staining, the Prussian blue reaction was used for ferritin. Muramidase activity was determined qualitatively I iy eluting 5 mm oE segments from electrophoresis strips with phosphate-chloride buffer at pH 6.2 and Reduction in mean alkaline phosphatase activity in the renal cortex homogenate from 17 weeks of exposure was highly significant (F=8.52+++). changes in the transmission of a suspension of Cadmium in the Kidney and Liver (Fig muramidase substance were measured. Egg white muramidase was used as a standard. As a general check of the condition of the animals Dico Laboratories Sigma Chemical Co., St. Louis 3); Between 11 and 17 weeks of exposure deposition of cadmium in the renal cortex and liver reached a "saturation value." As to distribution in the kidney, the renal cor- , OX 100 090- ;9 070- cv r.060- c z 0 50- y lOrt- i 0 30- 020- 0'0- 0 *posur& time in weeks. Fig 2.--Filtration rate, ratio of reabsorbed to filtered glucose and activity of alkaline phos phatases in the renal cortex homogenate (series A). Arch Environ Health--Vol 12, March 1966 DUP050312801 364 RENAL DAMAGE--AXELSSON & P1SCAT0R 0 Renal corit* liver Exposure time tex contained two. to nine times more cad mium by weight than the medulla (Table 2). There was no definite change in distri bution with exposure time. Cadmium in the Urine (Fig 4): For the animals exposed for 23 and 29 weeks there was a marked increase in urinary cadmium excretion. At 23 weeks the rabbits were given about 1 mg of cadmium daily but they excreted an average of 12 mg in the urine. Excretion of Protein in the Urine (Fig 4): For 23 and 29 weeks, for the animals ex posed, there was a marked increase in amounts of protein excreted in the urine. were strikingly pale and swollen compared with controls. Histological Picture: At least two kidney sections from each animal were examined. The pathologist was not informed which series of rabbits had been exposed nor fur how long the exposed series had received cadmium injections. The results will be reported in detail; in the present connection only a brief survey of results will be pre sented. There was some overlapping be tween the various cadmium exposure groups regarding degree of morphological Excretion of Glucose in the Urine: In the group exposed for 23 weeks, glucosuria was found in two rabbits, and the same signs were found in four of those exposed for 29 weeks. For shorter exposure times no glucosuria was. detected. Dry Weight Determination of Renal Cor tex: The increase in the ratio of wet to dry weight of' renal cortex with exposure time was highly significant (F=1222+++); this indicates that the administration of cad mium resulted in an increase in the fluid content of the renal cortex. This is consist ent with the gross observations at autopsy; the kidneys of rabbits receiving cadmium Tabl e 2.--Amounts of Cadmium in the Rabbit Kidneys After Various Periods of Exposure (series A) Renal Cadmium (PPM of Wet Tissue} Duration of Exposure *-- (Weeks) Cortex Medulla Control Control 11 11 11 17 17 23 23 29 29 20 <2 <1 <2 <1 m 77 249 37 261 29 344 3 677 140 237 70 231 6S 281 72 192 23 361 17$ Arch Environ Healt i--Vol 12, March 1966 DUP050312802 RENAL DAMAGE--AXELSSON & PISCATOR 365 alteration; the description applies to the material as a whole. Control Group. No pathological changes in the kidneys nor pathologic deposits of pig ment or metal particles were found. Exposure for 11 Weeks. Mild, largely degenerative changes were found in the proximal tubules. There were distinct de bits of metal in the cells of the proximal tubules. The glomeruli were well preserved .aid displayed no abnormal alterations. There was no evidence of inflammatory reaction. Exposure for 17 Weeks. In principle the picture resembled that of the 11-week group. Deposition of metal in the tubule cells was more pronounced and degenerative changes in the tubules more severe. Exposure for 23 and 29 Weeks. There .cere fairly severe pathological alterations. Tubule cells in the proximal segments were largely exfoliated; residual cells coniained vacuoles and were granulated and partly disrupted, the nuclei being destroyed r absent. In this group the changes were not restricted to the proximal tubules but. were found also in other segments; apart from epithelial fragments and cell detritus in place in their lumina, the collecting tubules were unaffected. Most animals had no pathological alter ations of glomeruli. In some rabbits, chiefly those exposed for 29 weeks, there was slight thickening of the capsule and hyaline fibrotic thickening of the basal membrane. Special staining methods disclosed abun dant deposits of metal particles in the tubule cells, especially in the proximal seg ments. In the glomeruli, collecting tubules and renal medulla there were no patholog ical accumulations of metal. In some ani mals hemosiderin staining showed an increase in iron deposits chiefly in the proximal segments. Series B.--1. Urine Examinations: There was no difference in 24-hour volumes of urine from the exposed and control rabbits. Protein excretion had increased signifi cantly after 23 weeks of exposure (Fig 4). The same results for series B are seen in O Cadmium O Prolern - 250 11 17 23 29 Fig 4.--Urinary excretion of cadmium and protein (series A). Arch Environ Health--Vol 12, March 1966 Exposure lime in weeks. DUP050312803 36G RENAL DAMAGE--AXELSSON 6- P1SCAT0R Fig 5.--Urinary excretion of protein (series B). Exposure was discontinued at 24 weeks. Fig 5, which shows the means for protcii; excretion at various times. 'The line repre senting exposed animals is interrupted at 24 weeks when exposure was discontinued. Compared with controls there was a def initely greater increase in protein excretion after 18 weeks of exposure (Fig 5). There was a significant difference after only 12 weeks but this must be interpreted with reserve in view of the. reduction in protein excretion by the controls compared with the initial values observed after ten weeks. The fine showing the situation after exposure was discontinued constitutes the mean for five rabbits remaining after three had died . Fig 6.--Electrophoretic ! pattern for urinary pro C tein from controls (series - " ! B). (A) Time, 3 days, 64 mg protein per day; (B) time, 10 days, 124 mg protein per day; (C) time, 8 weeks, 236 mg protein per day; (D) time, 11 weeks, 23 mg protein per day. AU>.. ft t *1 Fig 7.--Eiectrophoretic pattern for urinary pro tein from a rabbit ex posed to cadmium (series B). (.A) Before expo sure, 49 rag protein per day; (B) exposed 10 days, 39 mg protein per day; (C) exposed 5 weeks, 54 mg protein per day; (D) 6 weeks after exposure: 327 mg protein per day; () 17 weeks after exposure, 59 mg protein per day; (E) 25 I weeks after exposure, 225 mg protein per day. Arch Environ Health--Vol 12, March 1966 DUP050312804 RENAL DAMAGE--AXELSSON & PISCATOR 367 Fig- 8.--Electrophoretic pattern for urinary proK-in from a rabbit ex posed to cadmium (series B). (A) Before expo-rc, 114 mg protein per ,1,-iv; (i?) exposed 10 dnrs, 115, mg protein per Jav; (C) exposed 5 wicks, (D) exposed 13 weeks, ISO mg protein p,r day; () exposed 18 weeks, 64 nig protein per day; (F) exposed 21 .weeks, 42 mg protein per day; (G) exposed 24 weeks, 77 mg protein per day; (N) 6 weeks after exposure, 363 mg protein per day; (/) 17 weeks after exposure, 372 mg protein per day; (/) 25 weeks after exposure, S>9 mg protein per day. Ml i i F fSgfSK i between 24 and 28 weeks 8 and when ex cretion values had been deducted for the animal that later developed nephrosis with an extremely high protein excretion (more than 500 mg/day). Inclusion of this ani mal in the account of protein excretion up to 24 weeks does not affect the mean excretion. 3''' ` / ', : 'HSff'Fir v*.>- Protein excretion increased during the first six weeks after exposure had been dis continued, and ten weeks later had fallen to the level of the controls. The increase after 49 weeks is not significant. The electrophoretic pattern for urinary proteins excreted by the controls is charac terized by a fairly small albumin fraction ( Fig 6). The mean for 98 determinations was 22%. In a few controls with low pro tein excretion (< 20 mg/day) extremely weak staining was obtained, and in some rabbits the albumin was not dearly sep arated. During the first weeks there was a tendency for an increase in excretion of -proteins (Fig 6ri). Most rabbits in the control and exposed groups sometime during the first months of the experiment displayed a high albumin level and a relatively high daily excretion "f protein (100-300 mg/day) (Fig 6 C); proteinuria was then assessed as orthostatic, f hiring preparation of the urine colloids a tuicoid substance was precipitated which tained positive for protein but did not 'uigrate in electrophoresis. No correction ;* J'-fc ?*>* * 1 "#r > Alb. - ' - V 1 Fig 9.--Electrophoretic pattern for pooled uri nary protein after 24 weeks of exposure (series B). (A) Original pattern. (B) Fraction found after gel filtration through Sephadex G-200. ' Cadmium exposed group Fig 10.--Excretion of a-amino acid-N for ex posed rabbits (series B). Values for controls at 41 and 49 weeks. Arch Environ Health- Vol 12, March 1966 .;; --Kill; ' , 4, ' '' '' v ii' 'i ' 1 :7' i ' - :.i.. i /i.'iVy.' V ..Ail:::', DU P050312805 368 RENAL DAMAGE--AXELSSON & P1SCAT0R the typical fraction in the anterior y-regi. , was found toward the end of the exposv!r, period or after discontinuance it was m -, found at this stage in any control. With starch gel electrophoresis a post-y. protein was found in one rabbit six week, after exposure had ceased, and in two oi series A after 29 weeks of exposure. With gel filtration the distinct fraction in the y-region was enriched. It was the hut protein to appear on the Sephadex G 20o column (Fig 9). Cadmium was present in all protein fractions. The enriched y-pn,. tein contained about 5% of totally protein- Fig 11.--Electrophoretic pattern for muramidase bound cadmium. ; and kidney protein from one exposed rabbit that The mean daily excretion of cadmium in ; died after 32 weeks (series B). (A) Muramidase; (B) kidney protein; (C) kidney protein with added series B was 53jig after 18 weeks of ex | muramidase. posure (range 16-117^) and 160 (20- Vi 400/ng) after 49 weeks--25 weeks after ex for this mucoid was made in the urinary posure had been discontinued. On this last i protein determinations, occasion less than 20/^g was obtained in the i The electrophoretic pattern for urinary ultrafiltrate in all six rabbits. ; proteins from exposed rabbits showed an After an initial transient increase in ex i initial increase in the a-fraction (Fig 7 B); cretion of a-amino-acid-N the level was t i a protein band then appeared also in fairly constant to the end of the exposure i the anterior y-region. This was found also period when there was a further increase. i for the controls at the same time (Fig At 41 and 49 weeks, the only times when j 6 A, B; Fig 7 B). The pattern was then determinations were made for the .control ; ' normalized (Fig 7 C); after 15 weeks of group, excretion by the cadmium group 1 | exposure the -/-fraction reappeared to re- was significantly higher (Fig 10). j main for the rest of the experiment (Fig Six weeks after exposure had been dis- j ; 7 D-F). The albumin fraction was consist- continued three exposed rabbits exhibited j 1 ently smalt and the maximum of proteins glucosuria but this was temporary and 17 | was in the /I-region. weeks after exposure ceased glucosuria was ) Protein excretion varied considerably af- no longer detectable. | -ter exposure was discontinued, being high Examination of Renal and Liver Proteins: j ] after 6 weeks, normal after 17, and high These tests were performed on two rabbits - 1 again after 25 weeks while the electropho- in series B that died after 25 and 28 weeks, ] retie pattern was unchanged throughout. after exposure had terminated. In the sol The other rabbits displayed largely the uble protein fraction a protein was found ^ same changes in electrophoretic pattern, with that in paper electrophoresis migrated f a single exception when it was not found on toward the cathode at pH 8.6. A murarni- 1 any occasion, and there was no increase in dase activity was recorded in this area and i protein excretion. Mean albumin content the basic protein migrated as muramidase . ranged from 10% to 20%. The lowest (Fig 11). Paper electrophoresis did not : ! values were obtained during the first weeks disclose the protein in the kidneys of normal i of the .experiment. rabbits nor in the liver of exposed ones. ! In one rabbit the electrophoretic pattern Out of the 10 mg constituting the total > | was as for the other exposed rabbits up to cadmium in one liver, 8.7 mg was found in i 41 weeks, but thereafter a picture of ne- the soluble protein fraction; in two kidneys phrosis developed (Fig 8). with totals of 2.3 and 2 mg this fraction While in the majority of exposed rabbits contained 1.3 and 1.5 mg, respectively. ! Arch Environ Health--Vol 12, March 1966 DU P050312806 RENAL DAMAGE--AXELSSON & PISCATOR 369 With paper and starch gel electrophoresis ferritin was found in both liver and kidney extracts from exposed rabbits but in no control. Correlations Between the Various Tests (Series A): No relation was found between urinary protein excretion and creatinine clearance (r= --0.26 for the whole ma terial). There was a significant association be tween glucose reabsorption and protein excretion (r=--0.65 for the whole mate rial) ; this indicates that a low ratio be tween reabsorbed and filtered amounts of glucose was usually associated with a high excretion of protein. Here, too, there was a significant correla tion between the activity of alkaline phos phatase and the excretion of protein (r=--0.56 for the whole material). This indicates that a low enzyme activity was usually associated with a high excretion of protein. N6 association was found between the amount of cadmium deposited in the renal cortex and results of renal function tests nor between cadmium content and alkaline phosphatase activity. The amount of cadmium deposited was not associated with urinary excretion of the metal. Thus, early in the exposure period, Ihere were high levels of cadmium in the renal cortex with little if any excretion of metal. Protein excretion was positively corre lated with cadmium excretion but apparently not linearly, for extremely high cadmium excretion values were not accompanied by a proportionate increase in protein excre tion. Excretion of cadmium in the urine was significantly correlated with glucose reabsorption (r=-- 0.50, n = 18) and activity of alkaline phosphatase (r=--0.41; n = 24). There was no significant correlation be tween cadmium excretion and creatinine clearance. Comment Erom the reduction in glucose reabsorpand activity of alkaline phosphatase it is evident that in the rabbit exposure to cadmium gives rise to functional disorders of the tubules. Examination of creatinine clearance dis closed no definite effect on glomerular func tion. In some i-abbits exposed for the longest period mild morphologic changes were noted in some glomeruli, but whether these and the evidence of reduction in creatinine clearance with exposure time reflect glo merular impairment is not certain. There was probably no serious functional disorder of the glomeruli. Nevertheless, if the ex posure had been continued, significant glo merular damage would probably have been found with the method used. These results are consistent with earlier observations that the rabbit suffers rehal damage in cadmium poisoning.2 No direct comparison can be made, however, since there is no earlier study in which renal function and proteinuria were examined simultaneously. If the various function tests were equiv alent from all aspects the results would indicate that the functional diso.rders were located at first, and chiefly, in the proximal tubules. If exposure had been continued it is probable that definite interference with glomerular function would also have de veloped. Histological examination indicates that this is the probable chronological order of functional disorders. According to earlier studies on protein uria in cadmium poisoning in the rabbit the chief component is not albumin but a axglobulin1 and that a- and jS-globulins are predominant.25 It has recently been found that rabbits exposed to cadmium excrete a protein of low molecular weight.28 In none of the studies mentioned was the nor mal excretion of urinary protein given special attention. In the present study, nor mal excretion of protein and the electro phoretic pattern varied considerably. There were orthostatic reactions with "albumi nuria," and even in the controls there were transient increases in a-fraction at the be ginning of the experiment--apparently ini tial reactions to the injection to which the animal later became adapted. Minden et al25 reported in rabbits exposed to cadmium that Arch Environ Health- -Vol 12, March 1966 npjrrtrreBrrt*'y DUP050312807 370 RENAL DAMAGE--AXELSSON & PISCATOR after seven weeks the albumin constituted protein has also been found in the hum;,;, 30% of the total protein and the globulins serum suggests that in the rabbit scrim; 70%. In the present study a mean of 22% low molecular weight protein may be pres of albumin was obtained for a large num ent in extremely low concentration ai>.; ber of determinations on the controls where owing to defective reabsorption will appear as the exposed animals gave a mean of in the urine. i0%-20%. The lowest values were recorded No explanation can be offered of the during the first weeks of exposure, that is appearance of a similar component in both during the acute phase of the poisoning. In controls and exposed animals during tin ' an examination of urinary proteins from .first weeks of the experiment; it is no; workers exposed to cadmium the mean known whether they are identical. albumin content was 15% against 24% for A further similarity between the tubular the controls.4 proteinuria in exposed man and the pro . When, toward the end of the exposure teinuria found in the rabbit is the presence , time, the excretion of protein began to in of a post y-protein which in man has always crease, the pattern in general became been associated with impairment of tubular j slightly more distinct than in the controls, function.4''28 j with the appearance of several peaks in the Of the other metals that cause renal | a- and jS-region. The most marked differ damage uranium1-28 and mercury salts30 ence was the distinct fraction in the an- have been found in animal experiments to ! ter.ior y-region. produce a picture reminiscent of nephritis, i By and large, however, the differences and exposure to cadmium thus gives a dif 5 between the electrophoretic patterns of the ferent picture. According to results of the : controls and the cadmium group were qual- present renal function studies and earlier i itative, being more distinct for the exposed electrophoretic studies of urinary proteins j animals. It is notable that in the case of the demonstrated proteinuria appears to be i workers exposed to cadmium no quantita- due to tubular dysfunction. j tive difference in the electrophoretic pattern On discontinuing exposure the maximum 1 was found between those with low and high protein excretion was recorded, and this protein excretion. There was, however, a was followed by a reduction. In a few rab qualitative difference, the latter groups hav bits the typical electrophoretic pattern per ing the more distinct peaks, especially in sisted, and in one of them features of the /3-region.4 nephrosis developed, including massive pertain similarities were thus apparent proteinuria and histologic evidence of glo between proteinuria in the cadmium-ex merulus alterations. This may be consistent posed rabbit and man. In the latter there is with experience from cadmium workers, a so-called tubular proteinuria, which is some of whom exhibited no changes in ascribed to defective reabsorption of pro renal function or protein excretion even teins in the glomerular filtrate.4' Many of after quite long exposure while others the proteins excreted are of low molecular showed obvious signs of renal damage after i weight and occur normally in the serum.27 only a short time. In the present study there | To judge from the results of the gel filtra- was one rabbit in which no pathological | tion experiment the distinct fraction in the signs were found in the urine at any time. * anterior y-region was probably of low | molecular weight, and presumably identical j with the one recognized by Kench et al26 I . by ultracentrifugation. The typical /3-peak | in the urine from the exposed worker was 1 found to be largely due to a low molecular } weight protein,17 about 13,500; and it is ' possible that it corresponds to the typical j fraction in the rabbit. The fact that a simitar Further evidence of tubular damage was found in an increase in excretion of a-amino acid-N, which occurred later than the pro teinuria and was less pronounced. This is consistent'with the fact that the excretion of amino-acids by cadmium workers is usually normal or moderately increased.27 Glucosuria, too, was found on discontinuing exposure or just afterwards. In cadmium j Arch Environ Health --Vol 12, March 1966 i-Xj mi; vTe rcc iu> !!!>' the |IU l I Kit ; to'i cor UVi me ica to j col tail ! g17 \ Oil j ;ve ' ice ; ' iot j till ha an ate Uil au cv \ nil V iIm mi tin i ha hi: ra; Ti \ i'l; DUP050312808 ./ . ^ - die human it serum a :iy be presralion and will appear ed o the ent in both during the it is not d. the tubular 1 the proie presence lias always of tubular uise renal ry salts 30 riments to nephritis, ives a difilts of the nd earlier ,r proteins ears to be maximum and this i few rabttern peratures of massive e of gloconsistent workers, langes in ion even le others mge after udy there thological my time, nage was " tr-aniino the proThis is ,'xcretion rkers is teased,1'7 intinuing ..idiniuin RENAL DAMAGE--AXELSSON & PISCATOR 371 workers tliere was no apparent association amounts were small, however, compared between excretion of glucose and protein.27 with the deposit of metal and had no evident The most striking finding of the prelimi relationship to them. In view of this, the nary studies of renal proteins from animals great difference in amounts of pigment in exposed to cadmium was a protein that exposed and control rabbits, and the presence migrated toward the cathode in paper elec of cadmium in the renal cortex demon trophoresis. Muramidase activity was dem strated by activation analysis, it is reason onstrated in this fraction and the protein able to suppose that the pigment is a fairly migrated as egg-white muramidase. An in reliable indication of cadmium deposition. crease in muramidase in the kidneys has This is discussed in greater detail else recently been found in experimentally in where.22 The action of deposited cadmium duced tumors.31,32 This would be due to an is not fully understood. The evidence sug increase in production of muramidase by gests that the dement exerts an inhibitory ' the reticuloendothelial system and subse- effect, as measured on the living organism, | quent increase in its tubular reabsorption.33 organ functions, and enzyme systems due to s Where Is the Cadmium Deposited in the a reversible formation of mercaptan with i Kidney? Is the Amount Deposited a Meas- SH groups in the proton component of cell [ urc of the Degree of Poisoning.--The renal enzymes. I cortex of exposed rabbits contained from The extent to which tubular function was two to nine times as much cadmium as the impaired apparently had no relationship to \ medulla. The deposits were found histolog size of cadmium deposits. One possible ex ically chiefly in the proximal segment and, planation of this is that once the critical to a lesser extent, in the distal, while the toxic amount of cadmium has been ex collecting tubules and medulla as a rule con ceeded further increase has little effect tained hardly any metal. Using an autoradio- The significance of the deposition of iron : graphic, method on the rabbit, Friberg and in the kidneys so far as damage is con 1 Odeblad34 also found that the deposits cerned is unclear. The advanced structural | were located chiefly in the cortex. On in- alterations in the absence of iron deposits * iecting 1I5Cd in the rat, Gunn and Gold35 suggest that this metal is probably -of little i found a selective accumulation in the cortex, importance compared with cadmium.8 ; die amount being related to the number of Does the Cadmium Excretion Provide a | nephrons. They considered that cadmium Measure of ike Severity of the Toxic ' has a physiological role in renal function Effect.--The deposit of cadmium in the and proposed that it is in some way associ kidneys and liver was- practically the same ated with reabsorption of water in the after 11 weeks of exposure as after 29 * tubules. Berlin and Ullberg,38 found by an weeks, although there was no appreciable I autoradiographic technique that injected increase in metal excretion until 23 weeks ! '.idmium was deposited in the proximal of exposure. At this time the kidneys ex 'ubules. Using a clearance method and stop- creted more cadmium than was being sup d"w analyses, Vander37 found that cad plied. Parallel with this the cadmium level mium increased reabsorption of sodium in in the kidneys and liver was lower in these 'be proximal tubules. rabbits than in those exposed for 17 weeks. \ .. The location of pigment just below the At 29 weeks the excretion roughly balanced , ' ial membrane of the proximal tubules in the supply and there was again an increase * b:-1illogical sections fixed in alcohol satu- in cadmium content of the liver while renal 'V<1 in hydrogen sulfide is in agreement with deposits remained largely constant. The ex ' ttntn and Neth's 38 findings for heavy metals cretion in the urine appears to play a con earring normally in kidney. Part of the siderably more important role than in the ' -:ment certainly indicates the location of feces. 'Vr metals than cadmium. A special Urinary excretion of cadmium corre | ' -ming method showed' that in some ani- lated significantly with the capacity for 1* b iron could occur at the same sites. The glucose reabsorption and alkaline phospha- i Arch Environ Health -Vol 12, March 1966 A DUP050312809 372 RENAL DAMAGE--AXELSSON 6- PJSCATOR tase activity, that is, with the function of the kg of body weight, given as subcutancnm proximal segment of the tubules, cadmium injections of cadmium chloride. One group excretion increasing as function deterio of animals was exposed for 24 weeks and rated. There was no correlation between then followed for a further 25 weeks. cadmium excretion and creatinine clearance. The results suggest that during exposure fairly large amounts of cadmium are not excreted until renal damage has been caused, and that the amount then increases with the degree of damage. To ascertain the effect of exposure on the kidneys and to examine the mechanism underlying the proteinuria, experiments were performed on renal function and urinary and renal proteins. Determinations were also made of cadmium in the kidneys The fact that cadmium excretion also and liver, excretion of cadmium in urine correlated significantly with protein excre and feces, and the cadmium level in the tion is consistent with results reported by various urinary protein fractions. Histolog hriberg.39 Ultrafiltration experiments per ical examination was performed by the formed 25 weeks after exposure had been usual methods, hemosiderin staining and, to discontinued, following a 24-week period of study the deposition of metal in the kidneys, exposure, showed that at least 95% of the also by special staining methods. excreted metal was colloid- or cellbound. Previous dialysis experiments89 yielded varying percentages for dialysable cadmium. This may be due to differences in pH and the use of different types of dialysis mem brane. It appears probable that excreted cad mium is largely bound to urinary proteins. It is logical to suppose that part of the excretion is due to desquamated tubule cells containing cadmium, evidence for which is found in results of histological examination. In a fairly advanced stage of poisoning the urinary excretion of cadmium during exposure can provide at least an approxi mate measure of tubular damage in the rabbit. This study has not provided a definitive answer to the problem of the mechanism underlying renal damage. In fact, so far as the rabbit is concerned, the problem is further complicated in that account must Results.---1. Damage to the renal tubules was found localized to the proximal seg- ment. In a number of rabbits exposed for long periods small histological alterations were seen in the glomeruli. There was no definite impairment of filtration rate. 2. Proteinuria developed characterized by a low albumin level and fairly large - and ^-fractions, and a distinct fraction in the anterior y-region. In some rabbits there was also a post y-protein. 3. The results suggest that protein uria in chronic cadmium poisoning is a sequel of impairment of the tubules. 4. Cadmium is deposited chiefly in the cortex where, to judge from histochemical examination, it is localized mainly in the proximal segment of the tubules. Metal de posits were found also in the distal tubules but not in collecting tubules, glomeruli, or stroma. also be taken of hemolysis and the possible 5. The amount of cadmium deposited was effect of hemosiderosis on the kidney, as not correlated with the severity of func well as a direct effect of cadmium on the tubules. The experiments have shown, how ever, that quite a large amount of cadmium can accumulate in the rabbit kidney without producing more than relatively mild alter ations. Summary tional impairment. 6. The excretion of cadmium in the urine, as the proteinuria, increased greatly after renal damage had been produced and was significantly correlated with the func tion of the proximal tubules. 7. A basic protein in the renal cortex of . Rabbits have been exposed for 11, 17, 23, the exposed rabbits was found by electro and 29 weeks to 0.25 mg of cadmium per phoresis. It migrated as muramidase; mura- Arch Environ Health-Vol 12, March 1966 DUP050312810 *::: j | 1 I | r I ei t RENAL DAMAGE--AXELSSON & PISCATOR 373 midase activity was demonstrated in this fraction. 8. Ultrafiltration experiments showed that after exposure had been discontinued the greater part of excreted cadmium was bound to colloids or cells. This study was supported in part by a grant from the Swedish Medical Research Council (R 198). REFERENCES 1. Friberg, I,.: Health Hazards in die Manufacture of Alkaline Accumulators With Special Reference to Chronic Cadmium Poisoning', Acta Med Scand 138 (suppl 240): 1960. 2. Friberg, L.: Chronic Cadmium Poisoning, Arch fndustr Health 20;401-407, 1969. 3. Ahlmark, A., et alt Further Investigations into Kidney Function and Proteinuria in Chronic Cadmium Poisoning. Proceedings 13th International Congress Occupational Health 291-208, 1961. 4. Piscator, M.: Proteinuria in Chronic Cadmium Poison ing: 1. An Electrophoretic and Chemical Study of Urinary and Serum Proteins From Workers With Chronic Cadmium Poisoning, Arch Environ Health 4:697-621, 1962. 6. Axelsson, B.: Kidney Damage in Workers From Long Exposure to Cadmium, to be published. 6. Kazantizis, G., et at: Renal Tubular Malfunction and Pulmonary Emphysema in Cadmium Pigment Workers, Quart J Med 32:165-192, 1963. 7. Bonnell, J.A.; Ross, J.H.; and King, E.: Renal Lesions in Experimental Cadmium Poisoning, Brit J Indust Med 17:69-80, 1960. 8. Axelsson, B., and Piscator, M.: Serum Proteins in Cadmium Poisoned Rabbits: With Special Reference to Hemolytic Anemia, Arch Environ Health, 12:874-381, 1966. 9. Josephsscm, B., and Kallas, J.: Inulin and Creatinine Clearance of Unanaesthetized Rabbits, Acta Physiol Scand 39:1-19, 1953. 10. Hultman, E.: Rapid Specific Method for Determination of Aldosaccarides in Body Fluid, Nature 183:108, 1959. 11. Dec&t, L.: Uber die Eigcatumlichkciten der Renaleu Zuckerresorption beim Kaninchen, Acta Physiol Acad Sci Hung 3:255-263, 1958. 12. Piscator, M.: Proteinuria in Chronic Cadmium Poi soning: II. The Applicability of Quantitative and Qualita tive Methods of Protein Determination for the Demonstra tion of Cadmium Proteinuria, Arch Environ Health 5: 325-332, 1962. 13. Szentirmai, A., et at: A Rapid Screening Test for Determination of Total G-Amino Acids in Urine and Serum, Clin C/ifro Acta 7:459-462, 1952. 14. Smithies, O.: Zone Electrophoresis in Starch Gels: Group Variations in the Serum Proteins of Normal Human Adults, Biochem J 61:629-641, 1955. 15. Ferguson, 1CA., and Wallace, A.L.C.: Starch-gel Electrophoresis of Anterior Pituitary Hormones, Nature 190:629-680,* 1961. 16. Flodln, P.: Dex.tran Gels and Their Applications in Gel Filtration, Dissertation University of Uppsala, Sweden, 1962. 17. Smith, J.L.; Kench, J.E.; and Lane, R.E.: Deter mination of Cadmium in Urine and Observations on Urinary Cadmium and Protein Excretion in Men Exposed to Cad mium Oxide Dost, Biochem J 61:698-701, 1955. 18. Holmgard, A.: Quantitative Analysis of Enzymes in Normal and Diseased Kidney Tissue, Scand J Clin Lab In vest 14 (suppl) :l-79 1962. 19. Westermark, T., and S; ostrand, B.: Activation Analy sis of Cadmium In Small Biopsy Samples, Int 3 Appl Radial 9:78-83, I960. 20. Cholak, J., and Hubbard, D.j Determination of Cad mium in Biological Material, Ind Eng Chem Anal Edit 16: 833-336, 1944. 21. Timm, F.: Zur Histochemie der Scbwermetalle. Das Sulfidstlberverfahren, Deutsch Z Ges Gerichtl Med 46:706, 1958. 22. Axclsson, B.; Dahlgren, S.; and Piscator, M.: Mor phological Alterations and Cadmium Deposits in the Rabbit Kidney After Long-Term Exposure to Cadmium, to be pub lished. 23. Berlin, M., and Piscator, M.; Blodd Volume in Nor mal and Cadmium-Poisoned Rabbits, Arch Environ Health 2:576*583, 1961. 24. Berlin, M., and Friberg, L.: Bone-Marrow Activity and Erythrocyte Destruction in Chronic Cadmium Poisoning, Arch Environ Health 12:478-486, 1930. 25. Minden, H.; Bruckner, Ch.; and Simon, J.: Beitrag zvar CadmSumvergiftung. Symptomatik ud ExpertmenteUe Untersuchungen. Arch Geiverbepath 17:531-546, 1859. 26. Kench, J.E.; Wells, A.R.; and Smith, J.C.: Some Observations on the Proteinuria of Rabbits Poisoned With Cadmium. 5* Ajr Med J 36:800-894, 1962. 27. Piscator, M.: Proteinuria in Chronic Cadmium Poi soning: III. Arch Environ Health 12:835-844, 1966. 28. Butler, E.A., and Flynn, F.V.: The Occurrence of Past-Gamma Protein in Urine: A New Protein Abnormal ity, 3 Clin Path 14:174478, 1961. 29. Magee, M., and Foreman, H.: Nature of Proteinuria Induced by Uranium in Rats, Anter 3 Physiol 195:354-356, 1958. 30. Coye, R.D.* et al: Experimental Proteinuria: An Elec trophoretic Study of Three Different Types in Dogs, Arch Path 60:54S-555, 1955. 31. Ferri, G.C., and Anigstein, R.: Ebriich-reacting Pro tein in Kidneys of Tumor-bearing Rats, Cancer Res 20: 16544058, 1960. 32. Perri, G.C., et al: Crystallization of a Basic Protein (Lysozyme) From Kidneys of Tumor-bearing Rate (Jensen Sarcoma), Nature 193:649-651, 1962. __ 33. Perri, G.C., et al: Role of the Kidney in Accumula tion of Egg White Muramidase in Experimental Animals,' Proc Soo Exp Biol Med 115:189-192, 1964. 34. Friberg, L., and Odehlad, E.: Localization of Cd**s in Different Organs: An Autoradiographic Study, Acta Path Microbiol Scaftd 41:96-98, 1957. 35. Gunn, S.A., and Goutd, T.C.: Selective Accumula tion of CdP by Cortex of Rat Kidney, Proc Soc Exp Biol Med 96:820-823, 1957. 36. Berlin, M., and Ullberg, S.: The Fate of Cd1* in the Mouse, Arch Environ Health 7:686-693, 1963. 37. Vander, A.J.: Cadmium Enhancement of Proximal Tubular Sodium Reabsorption, Arner 3 Physiol 203:1005, 1962. 38. Timm, F., and Neth, R.: Die Norraalca Scbwermetalle der Niere, Histochemie 1:403-419, 1959. 39. Friberg, L.: Further Investigations on Chronic Cad mium Poisoning, Arch Indastr Hyg Occnp Med 5:30-36, 1952. A ii i Arch Environ Health--Vol 12, March 1966 WSVs'W>?<^,!V1s! DUP050312811