British Journal of Industrial Medicine 1992;49:226-232

Renal mechanisms in the cardiovascular effects ofchronic exposure to inorganic mercury in rats

Marco Carmignani, Paolo Boscolo, Luciano Artese, Goffredo Del Rosso, Giovanni Porcelli,Mario Felaco, Anna Rita Volpe, Giovanni Giuliano

AbstractMale weanling Wistar rats received 200 pg/miof mercury (Hg), as HgCl,, in drinking waterfor 180 days. At the end of the treatment,systemic arterial blood pressure was augmen-ted, cardiac inotropism was reduced, and heartrate was unchanged. Light and electronmicroscopical studies of the kidney showed amesangial proliferative glomerulonephritis inabout 80% of the glomeruli. Tubular cellsshowed reduction of the acid phosphataseactivity, which was related to functionalabnormalities of the lysosomes. In the 24 hoururine samples oftheHg exposed rats, there wasslight reduction of kallikrein activity, butevident proteinuria was not present in allsamples. Plasma renin activity was reduced,that of angiotensin I-converting enzyme wasaugmented, and plasma aldosterone concen-trations were unchanged. Mercury wasaccumulated mostly in the kidney of the Hgtreated animals; and the content of Hg in theheart was higher than in the brain. These datashow that chronic exposure to Hg acts on thekidney with complex mechanisms of toxicity;these contribute to modify systemichaemodynamics.

Department of Cell Biology and Physiology, Univer-sity of L'Aquila, 67100 Coppito, ItalyM CarmignaniInstitute of Occupational Medicine, G D'AnnunzioUniversity, 66100 Chieti, ItalyP BoscoloInstitute ofHuman Pathology and Social MedicineL ArteseInstitute of NephrologyG Del RossoInstitute of Biology and GeneticsM FelacoInstitute of Pharmacology, Catholic University,00168 Rome, ItalyM Carmignani, G Porcelli, A R VolpeInstitute of Occupational Medicine, University ofFlorence, 50100 Florence, ItalyG Giuliano

Epidemiological and clinical studies have shownincreased incidence of arterial hypertension andnephropathy with proteinuria and enzymuria inmercury (Hg) exposed workers."Z Although it wasfound that the kidney has a greater capacity thanother organs to accumulate Hg78 there is, however,no experimental evidence that renal storage of Hgactivates mechanisms of toxicity leading to arterialhypertension or cardiovascular disorders.Animal studies have found that chronic exposure

to inorganic Hg affects some mechanisms regulatingcardiovascular homeostasis. Male Sprague-Dawleyrats, treated with 50pg/ml Hg as mercuric chloride(HgCl,) in drinking water for 320 or 350 days,showed an increase in cardiac inotropism, hyporeac-tivity of baroreflex mechanisms, and changes incardiovascular reactivity after stimulation of theperipheral a- or ,B-adrenoreceptors79; arterial bloodpressure was significantly increased only in the ratsexposed to the metal for 350 days.89 These findingssuggested that Hg interferes with both influx andintracellular availability of calcium ions (Ca2+) forcontractile mechanisms in cardiac and vascularmyocells by an action on receptor operated Ca2channels in the plasma membrane and on cyclicnucleotide pathways. Light and electron micros-copical observation of the kidneys of these ratsshowed lysis of lysosomes in the tubular cells andminor morphological alterations in the glomeruli(segmental thickening of the basal membrane withcorresponding focal fusion of foot processes).9

In a recent experiment, male Wistar rats received50 or 200 ug/ml of Hg (as HgCl,) in the drinkingwater for 350 days from weaning. Blood pressure wasincreased in the rats exposed to both doses of Hg,whereas cardiac inotropism was augmented only inthe animals exposed to the lower dose.'°0 Exposureto Hg specifically reduced cardiovascular reactivityto stimulation of either baroreceptors or cardiac andvascular a,-adrenoreceptors; there was also anincrease of the effects after activation of the car-diovascular DA2-dopaminergic and f,- and fl,2-adren-ergic receptors. Light microscopical observation ofthe kidney showed glomerulonephritic lesions in30% of the glomeruli of the rats exposed to 50 ppm

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Hg and in almost all the glomeruli of the animalstreated with 200 ppm Hg; in the glomeruli thickeningof the basal membrane and hypercullularity withproliferation of the mesangial matrix were found; inthe tubuli, there was hydropic degeneration of thecells and some casts.101'Other experimental studies have confirmed that

subacute or chronic exposure to Hg, at doses able toaffect cardiovascular function, may induce renalalterations. For example, Wistar rats, treated withdoses of inorganic Hg that ranged from 50 to 200 ug/100 g body weight by subcutaneous route three timesa week for a period of two to 10 months, showed amembranous glomerulonephritis." In another study,Wistar rats that received 150 jMg of HgCl2 per 100 gbody weight by subcutaneous injection three times aweek for 27 weeks showed a mesangial glomerulone-phritis with deposition of IgG, IgM, and com-plement component C, in the mesangium." Otherstrains of rats treated with HgCl2 presented differentimmunological alterations dependent upon geneticvariables.'4 Brown Norway rats, treated withdifferent doses of HgCl2 for different periods, hadautoimmune abnormalities that included lym-phoproliferation, production of antiglomerularbasement membrane and anti-DNA antibodies,increased serum IgE concentrations, and polyclonalincrease in total IgG concentration, as well as cir-culating immune complexes. On the basis of theseresults, it was suggested that Hg stimulates autoreac-tive T-helper cells, inhibits T-suppressor cells, andinduces polyclonal activation of f-lymphocyte cells.After the initial period of exposure there was spon-taneous remission of these changes, which suggeststhat the T-suppressor cells may recover from inhibi-tion and regulate the Hg induced autoimmune res-ponses. On the other hand, Lewis rats, treated withHgCl2 in experimental protocols similar to those ofthe studies on Brown Norway rats, showed impairedregulation of immunological function and immuno-suppression.'415 Finally, rabbits and mice treatedwith HgCl2 showed several autoimmune disordersand glomerulonephritis.'4The purpose of this study was to investigate some

mechanisms of renal toxicity possibly involved in thedysfunction of cardiovascular function, in ratsexposed to doses ofHg known to induce both arterialhypertension and autoimmune lesions in the kidney.

Materials and methodsSixteen male weanling Wistar rats were randomlydivided into two equal groups, housed in stainlesssteel cages, and fed a standard laboratory diet. Onegroup received 200 pg/ml of Hg (as HgCl2) indeionised drinking water for 180 days and the othergroup was kept as a control. At the end of theexposure all the rats were placed in metabolism cagesfor the collection of 24 hour urine samples.

The animals were anaesthetised with a singleintraperitoneal injection of sodium thiopental (50mg/kg body weight) in order to perform haemodyn-amic measurements. The trachea was cannulated toallow spontaneous breathing and polyethylene cath-eters (containing sodium heparin, 100 USP units/ml)were placed in the left femoral artery to record aorticblood pressure. Systolic and diastolic blood pressurewere measured by means of a P23Db Stathampressure transducer (Statham Medical Instruments,Los Angeles, CAL) and averaged electronically. ABiotronex BL 620 derivative computer (BiotronexLaboratories, Inc., Kensington, MA) was used fordetermining the maximum rate of rise of the leftventricular isovolumetric pressure (dP/dt), an indexof cardiac inotropism. In this regard, a calibrated 3Fcatheter tip pressure transducer (Millar Instru-ments, Houston, TX), inserted in the right commoncarotid artery and advanced in the left ventricle, wasused to determine dP/dt.' "1 The computer wasadjusted to minimise the expression of preload andafterload according to Crawford et all6 and Davidsonet al.'7 Heart rate was measured by a Beckmancardiotachometer coupler, which was triggered by anR-peak of the lead II electrocardiogram. Thecardiovascular parameters were continouslymonitored on a Beckman type RM Dynographrecorder (Beckman Instruments, Inc, Schiller Park,ILL) after stabilisation for 30 minutes.

Blood samples were collected from the left com-mon carotid artery for determining plasma reninactivity (PRA),'8 plasma angiotensin I-convertingenzyme (ACE) activity,'9 and plasma aldosteroneconcentrations.20 Kallikrein activity,2' and creatinine,total protein, sodium, potassium, and calcium con-centrations were determined in the 24 hour urinesamples.

Samples of renal tissue were snap frozen in liquidnitrogen for histochemical and immunofluorescenceexamination; cryostat sections were prepared fromthese samples for demonstrating acid phosphataseactivity by the method of Gomori.22 Sections 3 gmthick were cut in a cryostat, acetone fixed, washed inphosphate buffer solution (PBS, pH 7 4), and stainedwith monospecific antisera to IgA, IgG, IgM, C3, Cld,albumin, and fibrinogen (Boehringer MannheimBiochemica, Mannheim, Germany), washed in PBS,and mounted in buffered glycerine.2' Other renalsamples were excised for light and electron micros-copical observation, as previously described.24Specimens of several tissues were also excised forhistopathological observation by light microscopy.

Samples of blood, brain, heart, and kidney wereprepared for determination of Hg content. Mercurywas analysed by flameless atomic spectrophotometryafter special digestion of tissues and blood.25 The Hgcontent was quoted as the wet weight of blood andtissues.

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Data were compared by Student's t test. Resultswere considered significant at p < 0 05.

ResultsBody weight and general appearance of the animalswere not affected by Hg exposure. In the Hg exposedrats, systolic and diastolic blood pressure were sig-nificantly increased, whereas dP/dt was reduced andheart rate was unchanged (table 1).

In the Hg treated animals plasma renin activity(PRA) was decreased, plasma ACE activity augmen-ted, and plasma aldosterone concentrations wereunmodified compared with the control rats (table 2).Moreover, urinary kallikrein activity and urinarycreatinine concentration were slightly but significan-tly reduced after exposure to Hg. Proteinuria wasevident only in some of the Hg exposed rats. Concen-trations of 24 hour urinary sodium, potassium, andcalcium were similar to those ofthe controls (table 3).

Table 1 Blood pressure, heart rate, and maximum rate ofrise of left ventricular isovolumetric pressure (dP/dt) in ratschronically exposed to Hg

Control (n = 8J Exposed (n = 8)(mean (SE)) (mean (SE))

Svstolic pressure (mm Hg) 131 (6) 151 (3)*Diastolic pressure (mm Hg) 96 (7) 123 (6)*Heart rate (beatsmin) 242 (18) 248 (25)dP dt (mm Hg& s) 4421 (332) 3066 (227)

*Significantly different from control, p < 0 05.

Table 2 Plasma renin activity (PRA), plasma angiotensinI-converting enzyme (ACE) activity, and plasmaaldosterone concentration in rats chronically exposed to Hg

Control Exposed(mean (SE)) (mean (SE))

PRA (ng/ml/h) 33-3 (3 2) 16-9 (6 0)*Plasma ACE (nmole/ml) 0-14 (0-1) 0-30 (0 1)*Plasma aldosterone (pg/ml) 491 (112) 453 (103)

*p < 0 05; significantly different from control.For both groups n = 6 for PRA and ACE, and n = 4 for plasmaaldosterone.

Table 3 Twenty four hour urinary kallikrein activity andcreatinine, urea nitrogen, protein, sodium, potassium, andcalcium concentrations in rats chronically exposed to Hg

Control (n = 8) Exposed (n = 8)(mean (SE)) (mean (SE))

Kallikrein (mamidolitic U) 6070 (550) 4500 (370)*Creatinine (jig) 17 8 (1 2) 13 0 (1l8)*Urea nitrogen (jig) 386 (45) 358 (49)Protein (mg) 8 4 (1 4) 26 4 (16 2)Sodium (mEq) 1 2 (0 05) 1 4 (0 1)Potassium (mEq) 1 3 (0 1) 1 4 (0-1)Calcium (jig) 1 4 (0 3) 1 1 (0 2)

*p < 0 05; significantly different from control.

Light microscopical observation showed altera-tions in about 80% of the glomeruli of the Hgexposed rats (fig 1). Glomeruli showed hyper-cellularity, deposition of amorphous material in themesangium, and thickening of the basal membrane.Examination with electron microscopy did not showmodifications in the glomerular capillary wall, butonly deposits of amorphous material in the mesan-gium; deposition ofamorphous material and thicken-ing ofthe vascular wall were found in the interstitiumby light microscopy. The immunofluorescence tech-nique showed deposition of IgM, but not of IgA,IgG, C3, Cld, albumin, or fibrinogen in the glomeruliof the Hg exposed rats (fig 2). Light focal hydropicdegeneration of the tubular cells and amorphousmaterial in some lumens were found (by light micros-copy) after Hg treatment (fig 3). At electron micros-copy lysosomes of all the tubular cells showed areduction of electrondensity and irregular borders,but not lysis. The acid phosphatase activity of thesecells, mostly localised in the lysosomes,26 wasreduced. Histopathological examination of thearteries and ofthe kidney showed that exposure to Hginduced thickening of the wall ofthe renal vessels andfocal thickening of the middle layer of the abdominalaorta.We found that Hg was accumulated much more in

the kidney than in the brain and in the heart ofthe Hgexposed animals. The heart appeared to store moreHg than the brain (table 4).

DiscussionIn this study, unlike Sprague-Dawley rats exposedto 50 ppm of Hg in drinking water for 320 or 250days,79 Wistar rats treated with 200 ppm of Hg for180 days showed a reduction in cardiac inotropism.The high concentrations ofHg found in the heart ofWistar rats treated with 200 ppm ofHg may explainthe reduction seen in the present experiment. In thisregard, the ratio between the Hg stored in the heartand that accumulated in brain or kidney was three orfour times higher in Wistar than in Sprague-Dawleyrats.910 On the other hand, the increase in bloodpressure seen in Wistar rats may be explained only bya vasoconstrictor effect ofHgwith a consequent rise inthe total peripheral resistance. This rise is likely to berelated to greater release of noradrenaline frompostganglionic adrenergic neurons and to baroreflexhyposensitivity, as suggested in previous studies.7'0On the basis of these experiments, the increase intotal peripheral resistance does not seem to dependon effects of Hg causing alterations of sympatheticnerve activity or vascular responsiveness after activa-tion of the a,-, 22, and f2-adrenoreceptors and DA2-dopaminergic receptors in blood vessels. On theother hand, the cardiovascular alterations found inthe Wistar rats exposed to 200 ppm of Hg cannot beexplained by increased reactivity to stimulation of

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Figure 3 Proximal tubular cells of an Hg exposed rat showing hydropic degeneration, deposition of ialine material in lumens,and inflammatory cells (mostly lymphocytes) in the interstitium (PAS x 250).

Table 4 Concentration ofHg in blood, brain, heart, andkidney of rats chronically exposed to Hg

Control(mean (SE))

Exposed(mean (SE))

Blood pg/g) 0-94 (0-27) < 0.03*Brain (,ug/g) 2-27 (0-73) <0-06*Heart (pg/g) 4-12 (1 16) <0.07*Kidney (pg/g) 160-25 (27-16) <0-20*

*p < 0-05; significantly different from control.All values are expressed on a wet weight basis (n = 4 in bothgroups for blood and brain and n = 6 in both groups for heart andkidney).

the peripheral f1 and f2-adrenoreceptors, as it wasfound in the Sprague-Dawley rats exposed to 50 ppmofHg for 320 or 350 days.7' BecauseHg was shown toreduce the availability of Ca"+ for contractilemechanisms in both cardiac and vascular myocells(by an interaction with both receptor operated Ca"+channels and cyclic nucleotide pathways), it may bethat the decrease of cardiac inotropism found in thisstudy is the result of a local toxic effect of Hg thatinvolves myocardial contractile processes.7-10Both reduction of creatinine excretion and

proteinuria found in some of the Hg exposed Wistar

rats seem to depend upon a mild form of glomerulo-nephritis induced by Hg. In this regard, rats exposedto Hg showed several forms of glomerulonephritissuch as membranous,"' a mesangial,1' or an immunecomplex type of glomerulonephritis.'5 The hyper-cellularity and deposition of amorphous material inthe mesangium, without alterations in the glomerularvessels, indicate that Hg caused a mesangialproliferative glomerulonephritis in the Wistar ratsused in this research.27 Differences in duration ofexposure, doses, and routes of administration of Hg,strains of animals, and other variables may accountfor the ability of Hg to induce various types ofglomerulonephritis. On the other hand, autoimmunemechanisms involved in these glomerular lesionsmay also be related to the morphological alterationsoflysosomes seen in the tubular cells and to reductionof the acid phosphatase activity, which is localised inthese lysosomes.27 Thus as lysis of lysosomes wasfound in tubular cells of Hg exposed Sprague-Dawley rats, it may be that circulating Hg bindingproteins, derived from the lysis of lysosomes, play apart in determining the different Hg induced immun-ological and pathological alterations found both inhumans4 and experimental animals."' '5

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Renal mechanisms in the cardiovascular effects of chronic exposure to inorganic mercury in rats

A surprising result of this experiment was theincrease of plasma ACE activity in the presence ofreduced PRA. It is possible, therefore as in leadexposed humans and laboratory animals,' that Hginitially increases PRA and, whenever the exposure isprolonged, reduces the activity of this plasmaenzyme. Moreover, considering the biochemical andfunctional relations between the plasma renin andurinary kallikrein systems,"' the possibility also existsthat reduction of urinary kallikrein activity andreduction of PRA are interdependent. On the otherhand, reduction of urinary kallikrein alone was notseen in Sprague-Dawley rats chronically exposed to50 ppm of Hg'1 and in asymptomatic Hg exposedworkers,32 but only in cadmium exposed rats" andhuman subjects.'2 Another explanation for theincrease in plasma ACE activity and reduction ofPRA may be derived from the recent revision of theclassic concept of the circulating renin angiotensinsystem (RAS).' In this respect, the most importantfindings are that (a) the major site of production ofangiotensin I (Al) and II (A2) is peripheral tissues,(b) the primary role of the circulating RAS is that ofdelivering renin and angiotensinogen to tissues, and(c) in tissues (including vessel wall and kidney) thereis synthesis of angiotensinogen, renin or renin-likeenzymes, and ACE. It is apparent that this localproduction (and degradation) ofAl and A2 is subjectto tissue specific mechanisms of regulation, whichchange local A2 concentration without reference tothe plasma concentrations of renin or A2. Thus Hgmay interfere with these local tissue specific mechan-isms such as the extent of tissue uptake of renin,angiotensinogen Al and A2, and the local tissueconcentration of ACE. In other words, the effects ofHg on plasma ACE activity and PRA may be theresult of different actions of Hg on plasma and tissueRAS. According to these concepts, the normal con-centrations of plasma aldosterone in Hg exposed ratsmay reflect the cumulative effects ofHg on the adrenalglands, RAS, and renal kallikrein system (influencingboth synthesis and release of aldosterone)."'This study confirms that the kidney is one of the

main targets of the toxic effects seen after chronicexposure to Hg. In particular, through differentmechanisms (including those of autoimmunity), Hgcauses changes in the RAS and the kallikrein-kininsystem that contribute to modify systemichaemodynamics. Experimental variables (such asdose ofexposure, route of administration, duration oftreatment, sex of the animals, and environmentalfactors) may have influenced or modulated the effectsof Hg.

We thank Mr Giuseppe Ripanti for his skilfulassistance.

Requests for reprints to: Marco Carmignani,Department of Cell Biology and Physiology, ViaVetoio, 67010 Coppito, L'Aquila, Italv.

1 Gobbato F, Chiesura P. La nefropatia da piombo. MinervaNefrologica 1968;75:12-24.

2 Buchet JP, Roels H, Bernard A, Lauwerys R. Assessment ofrenal function of workers exposed to inorganic lead, cadmiumor mercury vapor. J Occup Med 1980;22:741-50.

3 Stonard MD, Chater BV, Duffield DP, Nevitt AL, O'SullivanJJ, Steel GT. An evaluation of renal function in workersoccupationally exposed to mercury vapor. Int Arch OccupEnviron Health 1983;52:177-89.

4 Foa V, Caimi L, Amanti L. Patterns of some lysosomal enzymesin the plasma and of proteins in urine of workers exposed to

inorganic mercury. Int Arch Occup Environ Health1976;37:1 15-24.

5 Bernard A, Roels H, Buchet JP, Lauwerys R. Comparison bysodium dodecyl sulphate polyacrylamide gel electrophoresisof urinary proteins excreted by workers exposed to cadmium,mercury or lead. Toxicol Lett 1980;5:219-22.

6 Barregard L, Hultberg B, Schiitz A, Sallsten G. Enzymuria inworkers exposed to inorganic mercury. Int Arch OccupEnviron Health 1988;61:65-9.

7 Carmignani M, Finelli VN, Boscolo P. Mechanisms in car-

diovascular regulation following chronic exposure ofmale ratsto inorganic mercury. Toxicol Appl Pharmacol 1983;69:442-50.

8 Carmignani M, Boscolo P. Cardiovascular homeostasis in rats

chronically exposed to mercuric chloride. Arch Toxicol1984;(suppl 7):383-6.

9 Carmignani M, Boscolo P, Preziosi P. Renal ultrastructuralalterations and cardiovascular functional changes in rats

exposed to mercuric chloride. Arch Toxicol 1989;(suppl13):353-6.

10 Boscolo P, Carmignani M, Giuliano G, Preziosi P. Peripheralcatecholaminergic mechanisms and baroreflex pathways are

involved in vascular and cardiac effects of long-term exposureto inorganic mercury in rats. In: Strano A, Novo S, eds.Advances in vascular pathology. Amsterdam: Elsevier SciencePublishers 1989:1061-6.

11 Boscolo P, Carmignani M, Artese L, et al. New experimentalstudies on the cardiovascular effects of chronic mercuryexposure. In: Anke N, Baumann W, Braunlich H, Brucker C,Groppel B, Grin M, eds. Sixth international trace elementssymposium 1989. Jena: Wissenschaftliche Publikationen derFriedrich-Schiller-Universitat, 1989:1638-46.

12 Bariety J, Druet P, Laliberte F, Sapin C. Glomerulonephritiswith y and PI C-globulin deposits induced in rats by mercuricchloride. Am J Pathol 1971;65:293-302.

13 Makker SP, Aikawa M. Mesangial glomerulopathy with deposi-tion of IgG, IgM and C3 induced by mercuric chloride. A new

model. Lab Invest 1979;41:45-50.14 Hirsch F, Couderc J, Sapin C, Fournier G, Druet P. Polyclonal

effect of HgCl, in the rat; its possible role in an experimentalautoimmune disease. Eur J Immunol 1982;12:620-5.

15 Druet P, Pelletier R, Hirsch F, et al. Mercury-induced autoim-mune glomerulonephritis in animals. Contributions to

Nephrology 1988;61:120-30.16 Crawford W, Teamy RJ, Hawthorne EW. Increased myocardial

contractility during the initial period of hypertension in dogsfollowing renal artery constriction. Federation Proceedings.1973;32:351.

17 Davidson DM, Covell JW, Mallock CI, Ross J, Jr. Factorsinfluencing indices of left ventricle contractility in the cons-

cious dog. Cardiovasc Res 1974;8:299-312.18 Freedlender AE, Goodfriend TL. Renin and the angiotensins.

In: Jaffe BM, Behrman HR, eds. Methods of hormone radio-immunoassay. New York: Academic Press 1979:889-913.

19 Volpe AR, Porcelli G, Di Iorio M. Determination of kininase Iand II activities in human urine by HPLC. J Chromarogr1989;414:427-31.

20 Abraham GE, Garza AR, Manlimos FS. Radioimmunoassay ofsteroids. In: Abraham GE, ed. Handbook of radioimmunoassay.New York: Dekker M, Inc 1977:591-618.

21 Morita T, Kato H, Iwanaga S, Takada K, Kimura T, SakakibaraS. New fluorogenic substrates for a-thrombin, factor Xa,kallikreins and urokinase. J Biochem Tokio 1977;82:1495-8.

22 Gomori G. Microscopic histochemistry: principles and practice.

231

on June 1, 2020 by guest. Protected by copyright.

http://oem.bm

j.com/

Br J Ind M

ed: first published as 10.1136/oem.49.4.226 on 1 A

pril 1992. Dow

nloaded from

Carmignani, Boscolo, Artese, Del Rosso, Porcelli, Felaco, Volpe, Giuliano

Chicago: The University of Chicago Press, 1952.23 Paul WE. Fundamental immunology. 2nd edition. New York:

Raven Press, 1989.24 Boscolo P, Carmignani M, Sacchettoni-Logroscino G, et al.

Ultrastructure of the testis in rats with blood hypertensioninduced by long-term lead exposure. Toxicol Lett1988;41:129-37.

25 Krause LA, Henderson R, Shotwell HP, Culp DA. The analysisof mercury in urine, blood, water and air. Am Ind Hyg Assoc J197 1;32:331-7.

26 Thorlacius-Ussing 0, M0ller Graabaek P. Simultaneous ultra-structural demonstration of heavy metals (silver, mercury)and acid phosphatase. Histochem J 1986;18:639-46.

27 Glassock RJ, Cohen AH. Immunologically mediated renaldisease. In: Klahr S, Massry S, eds. Contemporary nephrology,Vol 3. New York: Plenum Medical Book Company, 1985.

28 Boscolo P, Carmignani M. Neurohumoral blood pressureregulation in lead exposure. Environ Health Perspect1988;78:101-6.

29 Victery M. Evidence for effects of chronic lead exposure on

blood pressure in experimental animals: an overview. EnvironHealth Perspect 1988;78:71-6.

30 Scicli AG, Carrettero AO. Renal kallikrein-kinin system. KidneyInt 1986;29:120-30.

31 Boscolo P, Carmignani M, Menini E, et al. Urinary vanillylman-delic acid and kallikrein and cardiovascular parameters in ratschronically exposed to different toxic metals. Acta MedicaRomana 1985;23:208-15.

32 Roels HA, Lauwerys R, Buchet JP, Bernard AM, Lijnen P, VanHoute G. Urinary kallikrein activity in workers exposed tocadmium, lead, or mercury vapour. Br J Ind Med1990;47:331-7.

33 Boscolo P, Porcelli G, Carmignani M, Finelli VN. Urinarykallikrein and hypertension in cadmium-exposed rats. ToxicolLett 1981;7:189-94.

34 Campbell DJ. Circulating and tissue angiotensin systems. J ClinInvest 1987;79:1-5.

Accepted 15 July 1991

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