The proteinuria of industrial lead intoxication

ENVIRONMENTAL RESEARCH 41, 440-446 (1986)

The Proteinuria of Industrial Lead Intoxication C. V. VACCA, J. D. HINES, AND P. W. HALL III Department of Medicine, Case Western Reserve University, Cleveland Metropolitan General Hospital, Cleveland, Ohio 44109 Received May 1985 Studies of protein excretion were undertaken in seven males, aged 35-42 years, who had more than 5 years exposure to industrial lead and had clinically established Pb intoxication. Heavy metal intoxication with Cd and Hg causes proximal tubular abnormalities, i.e., aminoaciduria, glycosuria, phosphaturia. Similar abnormalities occur in Pb intoxication except that the nature of the proteinuria remains controversial. Studies of urinary proteins included 24-hr urine protein excretion, dextran gel separations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 132microglobulin (B2M) measurements. Creatinine clearances, and serum BzM concentrations were normal. Urine total protein distribution by SDS-PAGE and the B2M excretion rate were also normal. These data imply that the nephrotoxicity of Cd and Hg are different than that of Pb. We speculate on what might account for this difference. This study suggests that when examining a population exposed to Pb, the finding of tubular proteinuria should alert investigators to search for the presence of other toxic agents. © 1986AcademicPress, Inc.

INTRODUCTION Industrial workers employed in the manufacture of batteries and paints, and as lead pouters and smelters, are at risk to develop toxicity from heavy metals such as Cd, Hg, and Pb. Intoxication with these metals is known to cause renal tubular abnormalities such as aminoaciduria, phosphaturia, and glycosuria (Clarkson and Kench, 1956; Chisolm, 1962; Sun et al., 1966). In addition, Cd and Hg intoxication are associated with a decreased ability of the proximal tubule to take up filtered low-molecular-weight proteins (LMW) such as lysozyme, IgG light chains, and 132-microglobulin (B2M). This tubular proteinuria is an early indication of Cd toxicity (Piscator, 1966). All of these proteins are of a molecular size <30,000 D and are presumed to be freely filtered at the glomerulus. While the industrial Pb problem has been well recognized, it should be noted that those industries utilizing lead usually employ some combination of other heavy metal, making exposure to only one of them rare. Failure to take this combined exposure into account may lead to erroneous conclusions concerning the toxic effects of a specific agent. The renal effects of excessive exposure to cadmium and mercury, either by virtue of occupational exposure or ingestion of foodstuffs produced in areas contaminated by industrial effluent, have been the subject of extensive epidemiologic investigations in Sweden and Japan (Friberg et al., 1974; Gompertz et al., 1983; Iesato et al., 1977). Both of these heavy metals are associated with tubular proteinuria. Brief reports on the nature of the proteinuria associated with Pb intoxication show conflicting results. Using SDS-PAGE, Bernard et al. (1980) studied 440 0013-9351/86 $3.00 Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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19 individuals having an average Pb exposure of 12.5 years. They did not show any increased excretion of urinary proteins of any size. In a preliminary report, Manuel and Greenland (1973) demonstrated that 20 of 30 Pb workers had a minimal increase in light chain proteinuria as determined by immunoelectrophoresis; 5 were normal and the remaining 5 had minimally increased amounts of low- and high-molecular-weight proteins in their urine. The methods used were paper and agarose gel electrophoresis which do not specifically separate proteins by molecular size. Colle et al. (1980) studied 10 monkeys given 15 mg of lead acetate 6 days a week for 9 months. No consistent urinary protein excretion pattern was found. Clinical studies indicate that azotemia and diminished glomerular filtration rate occur prior to detectable proteinuria (Wedeen et al., 1975, 1979). The following study of urinary proteins was done to delineate the molecular size by dextran gel filtration, SDS-PAGE, and to quantitate the urinary excretion of B2M in Pb-intoxicated industrial workers. The results demonstrate that longterm exposure (>5 years) to increased environmental Pb did not result in any detectable alteration in protein excretion in spite of clear laboratory evidence of Pb toxicity. MATERIALS AND METHODS Studies were performed on seven employees of a lead burning company in Cleveland who were diagnosed as having lead intoxication. The exposure was to "white" lead, a highly purified form of lead, being processed for use in electroplating. The criteria for establishing the diagnosis of Pb intoxication were defined as elevated blood Pb levels (normal <45 txg/dl), elevated free erythrocyte protoporphyrin (FEP; normal 40-60 ixg/100 ml rbc), diminished erythrocyte ~-aminolevulinic acid dehydratase activity, sideroblastic anemia. All subjects were male, aged 35-42 years, and met these criteria. One of the patients, No. 3, presented with evidence of peripheral neuropathy which was determined to be secondary to Pb intoxication. The other six men were asymptomatic and were investigated because of the common exposure of more than 5 years duration. The subjects were admitted to the hospital metabolic ward for EDTA chelation therapy. Twenty-four-hour urine samples were collected and adjusted to pH 7 using sodium hydroxide. Blood samples were obtained at the completion of the urine collection period. The tests performed on these specimens included: 1. Urinary total protein by the Biuret technique (Goa, 1953). 2. Molecular weight distribution of urinary proteins by two methods: A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Shapiro et al. (1967). Standards were applied at concentration of 2 mg/ml, and samples were concentrated to 5 mg/ml total protein concentration using dialysis and lyophylization then reconstituted in electrophoresis buffer. The sample volume applied was the same for all standards and urine samples. A 10% gel concentration was used to enhance the separation of low-molecular-weight proteins. B. Sephadex G 50, 2.5 x 100-cm column gel chromatography, by the method previously reported from this laboratory (Hall et al. 1982).

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TABLE 1 INDIVIDUAL DATA BY SUBJECT FOR AGE, YEARS OF EXPOSURE, BLOOD Pb, URINE PROTEIN, SERUM B2M , URINE B2M , CREATININE CLEARANCE, AND FREE ERYTHROCYTE PROTOPORPHYRIN

Subject

Age

Exposure (years)

1 2 3 4 5 6 7

41 40 34 37 33 42 42

>5 16 >5 >10 >5 >5 >5

B Pb U Pro (~xg/dl) (rag/24 hr)

S BzM (rag/l)

U BaM (txg/mg Cr)

C Cr (ml/min)

FEP (~xg/rbc)a

210.0 390.0 300.0 240.0 124.0 83,5 185.6

60.0 44.0 47.0 42.0 133.0 80.0 73.0

1.78 2.40 2.20 1.50 2.20 1.10 1.72

0.068 0.260 0.060 0.050 0.038 0.092 0.056

99.9 95.4 116.0 115.0 110.0 112.0 114.0

801 1061 790 1895 619 540 1174

Mean SD

219.0 103.9

59.8 41.3

1.84 0.46

0.089 0.077

108.9 8.0

1088 580

Normal values

<45.0

50.0 to 150.0

1.00 to 2.40

<0.350

90.0 to 140.0

40 to 60

Note. B Pb, blood lead; U Pro, urine protein; S BzM, serum BzM; U BzM, urine B2M; C Cr, creatinine clearance; FEE free erythrocyte protoporpbyrin. a Actual value ixg/100 ml rbc.

3. B2M concentration was determined in serum and urine by the radioimmunoassay method previously reported from this laboratory (Hall et al., 1982). 4. Creatinine concentration in serum and urine using the standard autoanlayzer technique. 5. Blood lead concentrations determined by atomic absorption spectroscopy (Fernandez, 1975). 6. Free erythrocyte protoporphyrin levels by the extraction methods of Sassa et al. (1973) and Granick et al. (1972). RESULTS

Table 1 lists the subject ld No., age, years of Pb exposure, blood Pb concentration (B Pb), 24-hr urine protein excretion (U Pro), serum B2M (S B2M), 24-hr urine B2M excretion (U B2M), creatinine clearance (C Cr), and free erythrocyte protoporphyrin levels. All values, with the exception of the blood lead concentrations and FEP levels were within the normal ranges. The results of these studies showed that each individual excreted normal amounts of protein and that the distribution by molecular size was also normal. We chose subject 2 to illustrate the urinary protein separation by S D S - P A G E and dextran gel column chromatography, because he had the longest industrial exposure, his B Pb was the highest, and his FEP level was one of the most elevated. His U Pro excretion was within normal limits. In Fig. 1, samples 1-6 are standards ranging from 60,000 to approximately 14,000 Da (left to right: bovine serum albumin, ovalbumin, pepsin, trypsinogen, [3-1actoglobulin, egg white lysozyme, and 7 is empty). Sample 8 is a combination

443

PROTEINURIA OF LEAD INTOXICATION

1 I

2

3

4

5

6

7

Standards

8

9 ,l

i0 Pb

Ii

12 ,

N

FIG. 1. Protein distribution utilizing S D S - P A G E f o r standards (1-8). Urine proteins of subject 2 (9-11). Urinary protein distribution froma normal subject (12). For a detailed description see text.

of the six standards. Because of the closeness of the molecular weights of some standards, only five bands are distinguishable in this combined sample. Four of the five visible bands represent proteins that are of a molecular size of 45,000 Da or less, therefore this pattern simulates a "tubular proteinuria" pattern. The 9th sample was the urine of subject 2 when he had his highest B Pb. The protein distribution is diffuse and exhibits no distinct bands which would represent proteins of a molecular size <50,000 Da. The 10th sample demonstrates urine protein distribution of subject 2 approximately 2 weeks after EDTA chelation therapy (B Pb = 100 ~xg/dl) and the 1lth sample 1 month later (B Pb = 100 txg/dl). No bands are obvious in samples 9 and 10. A band at the application point is seen in sample 11 which represents proteins >50,000 Da. The patterns seen in samples 9, 10, and 11 are typical of the patterns seen in normal urines using this technique. It is apparent that even the most severely Pb-intoxicated subject did not have tubular proteinuria by SDS electrophoresis. Figures 2a and b are provided to allow comparison of the dextran gel separations of an individual with "tubular proteinuria" resulting from industrial cadmium exposure (Fig. 2a) to subject 2 at the time his B Pb was 390 ~zg/dl (Fig. 2b). Figure 2a shows a protein peak corresponding to >30,000 Da and also two LMW peaks. The second peak is composed of proteins of approximately 22,000 Da and the third peak of proteins approximately 12,000 Da. The third peak contained BzM by radioimmunoassay. The urinary proteins from subject 2 (Fig. 2b) shows a primary peak corresponding to proteins >30,000 Da, and is void of LMW peaks. DISCUSSION Previous studies reviewed in the introduction (Clarkson and Kench, 1956; Chisolm, 1962; Sun et al., 1966) have shown that Pb, Hg, and Cd induce glycosuria and aminoaciduria, indicators of proximal tubular dysfunction. Cd and Hg intoxication are also associated with tubular proteinuria (Iesato et al., 1977; Pesce et

444

VACCA, HINES, AND HALL

I"80~ a

TUBULAR PROTE[NURIA

0'90iA

150 1.20

200

Millilifers

250

300

b EXPOSED PROTEINURIA

~ 0.90 c oa

~0.60 0

0.30

J 150

I>3o,ooodi 200

Milliliters

I | 11,8oo~ j 250 300

FIG. 2. (a) Dextran gel separation of urinary proteins from a patient with tubular proteinuria resulting from industrial Cd exposure. For a detailed description see text. (b) Dextran gel separation of urinary proteins from subject 2. For a detailed description see text.

al., 1977), another manifestation of proximal tubular abnormality. This study of urinary protein excretion in seven industrial lead-intoxicated workers did not show any abnormalities in the renal handling of low-molecular-weight proteins. The normal urinary protein excretion pattern shown here is evidence of this fact and indicates that the proximal-tubular protein-reabsorptive mechanisms are intact. Glomerular permeability appeared to be unaffected as evidenced by normal 24-hr urinary protein excretion. It is interesting to speculate as to why tubular proteinuria is not seen in chronic lead intoxication. One of the postulated etiologies of tubular proteinuria relates to an overproduction of LMW, as in light chain nephropathy, where overproduction of light chains increases the filtered load resulting in interference of the uptake of L M W (Fang, 1985). Cd and Hg induce liver synthesis of metallothionein (MT) but Pb does not (Lee et al., 1983). This significantly increases the circulating MT. This protein, having a molecular weight of 6000 Da, appears to be the major transport protein for these two heavy metals. Cd complexed with MT is freely filtered by the glomerulus. Once filtered, the C d - M T is picked up by receptor sites on the brush border and internalized to be digested by lysosomal enzymes (Foulkes, 1982) where C d - M T interferes with the uptake of small proteins such as B2M. The mechanism of this interference would not appear to be a simple competitive inhibition as the molar quantities of B2M in the filtrate are several

PROTEINURIA OF LEAD INTOXICATION

445

orders of magnitude greater than C d - M T even in the pretreated animals with increased MT production. Mego and Cain (1982) showed that Cd also interferes with primary lysosome formation. Foulkes (1974) has demonstrated that a single bolus injection of CdC12 into the renal artery of a rabbit is 100% recoverable from the renal vein. He speculated that Cd transiently binds to plasma proteins and/or endothelial sites, thus preventing the Cd from being filtered. The studies of Nordberg and co-workers (1975) showed that comparable amounts of CdC12 and C d - M T did not have the same toxic effects on the mouse kidney. The latter demonstrated a more pronounced effect on the renal tubule. These data and those of Squibb et al. (1979) suggest that Cd must be filtered to be nephrotoxic. The route by which Pb gets into the proximal tubule is unknown. It is transported inside the red blood cell, bound to a small protein (Raghavan and Gonick, 1970). Plasma concentrations are, therefore, low. The electron microscopic studies of Sun et al. (1966) have shown that the toxic effect of Pb results in mitochondrial changes that coincide with aminoaciduria. We conclude from our studies that tubular proteinuria is not an early manifestation of toxicity to industrial Pb exposure. The finding of tubular proteinuria in a population exposed to Pb should make investigators look for the presence of other toxins in the environment. ACKNOWLEDGMENT The authors acknowledge Miss Kathy A. Savanick for her expert secretarial assistance in the preparation of this manuscript.

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amino-levulinic acid dehydratase, and porphyrinogen synthetase in microliter samples of whole blood: Applications to metabolic defects involving the heme pathway. Proc. Natl. Acad. Sci. USA 69, 2381-2385. Hall, R W., Chung-Park, M., Vacca, C., London, M., and Crowley, A. (1982). The renal handling of beta 2-microglobulin in the dog. Kidney Int. 22, 156-161. Iesato, K., Wakashin M., Wakashin, Y., and Tojo, S. (1977). Renal tubular dysfunction in Minimata Disease: Detection of renal tubular antigen and beta 2-microglobulin in the urine. Ann. Intern. Med. 86, 731-737. Lee, Y. H., Shaikh, Z. A., and Tohyama, C. (1983). Urinary metallothionein and tissue metal levels of rats injected with cadmium, mercury, lead, copper or zinc. Toxicology 27, 337-345. Manuel, T., and Greenland, T. B. (1973). Immunoelectrophoresis: A convenient method of studying "minimal changes" in proteinuria. In "Protides of the Biological Fluids--21st Colloquium" (H. Peeters, Ed.), pp. 393-400. Pergamon, Brugge/Oxford/New York. Mego, J. L., and Cain, J. A. (1982). An effect of cadmium on heterolysosome formation and function in mice. Biochern. Pharrnacol. 24, 1227-1232. Nordberg, G. E, Goyer, R., and Nordberg, M. N. (1975). Comparative toxicity of cadmium-metallothionein and cadmium chloride on mouse kidney. Arch. Pathol. 99, 192-197. Pesce, A. J., Hanenson, I., and Sethi, K. (1977). Beta 2-microglobulinuria in a patient with nephrotoxicity secondary to mercuric chloride ingestion. Clin. Toxicol. 11(3), 309-315. Piscator, M. (1966). Proteinuria in chronic cadmium poisoning. II1. Electrophoretic and immunoelectrophoretic studies on urinary proteins from cadmium workers, with special reference to the excretion of low molecular weight protein. Arch. Environ. Health 12, 335-344. Raghavan, S. R. V., and Gonick, H. C., (1970). Isolation of low-molecular-weight lead-binding protein from human erythrocytes. Proc. Soc. Exp. Biol. Med. 155, 164-167. Sassa, S., Granick, J. L., Granick, S., Kappas, A., and Levere, R. D. (1973). Studies in lead poisoning. I. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorometry in detection of chronic lead intoxication in subclinical range. Biochern. Med. 8, 135-148. Shapiro, A. L., Vinuela, E., and Maizel, J. V., Jr. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Cornmun. 28, 815-820. Squibb, K. S., Ridlington, J. W., Carmichael, N. G., and Fowler, B. A. (1979). Early cellular effects of circulating cadmium-thionein on kidney proximal tubules. Environ. Health Perspect. 28, 287-296. Sun, C. N., Goyer, R. A., Mellies, M., and Yin, M. W. (1966). The renal tubule in experimental lead intoxication. Arch. Pathol. 82, 156-163. Wedeen, R. R, Maesaka, J. K., Weiner, B., Lipat, G. A., Lyons, M. M., Vitale, L. E, and Joselow, M. M. (1975). Occupational lead nephropathy. Amer. J. Med. 59, 630-641. Wedeen, R. R, Mallik, D. K., and Batuman, V. (1979). Detection and treatment of occupational lead nephropathy. Arch. Intern. Med. 139, 53-57.