Document 2Q12wk3jn768X7GEOwMKgwDr

, J Reprinted from ' -rw. ' --/.,/ jf" / \ -/ }' Clinica Chimica Acta Elsevier PubBshing Company, Amsterdam - Printed in The Netherlands THE EFFECT OF EXPERIMENTAL LEAD POISONING ON SOME ENZY MATIC ACTIVITIES OF THE KIDNEY Q. C. SECCHI, L. ALESSIO a n d A. CXRLA with the technical assistance of M. SCHWEIZER Laboratory of Enzymology, Institute of Occupational Health, University of Milan (Italy)* (Received October 13, 1969} SUMMARY The alterations of lactate dehydrogenase and its isoenzyme fractions, of glucose6-phosphate dehydrogenase, and of glutamate dehydrogenase were studied in the kidneys of 12 guinea pigs poisoned with a daily dose of 120 mg/kg body weight of lead nitrate given by means of an oesophageal catheter, and in the kidneys of 12 guinea pigs made ischemic by partial constriction of the renal artery. The investiga tion was made on homogenates of kidneys in toto and on homogenates of cortex and medulla separately. In the kidneys of lead-poisoned guinea pigs and in the kidneys with ischemia as a result of stenosis of the renal artery, similar metabolic alterations were observed. In both experimental situations a marked increase in type M isoenzymes of lactate dehydrogenase (LDH, and LDHS), an increase in glucose-6-phosphate dehydrogenase up to values twice those found in normal kidneys, and a marked reduction in gluta mate dehydrogenase activity were observed. The increase of glucose-6-phosphate dehydrogenase activity was only moderately prevalent in the cortical zone, and the other enzymes examined did not undergo alteration of their percentage distribution between cortex and medulla. These results are interpreted as indicating a metabolic adaptation of the kidney to a condition of tissue hypoxia, in both experimental situations. * Requests for reprints be addressed to: CHnica del Lavoro, via S. Barnaba 8, Milano (Italy). The following abbreviations are used: G6PD: Glucose-6-phosphate dehydrogenase (D-Giucose-6-ph.osphate: NADP oxidoreductase E.C. i .x .i -49)6PGD: Phosphogluconate dehydrogenase (6-Phospho-D-gluconate:NADP oxidoreductase E.C. 1.1.1.44). LDH: Lactate dehydrogenase (L-Lactate:NAD oxidoreductase E.C.1.1.1.27). LDH,, LDH2, LDH3i LDH4, LDHa: Lactate dehydrogenase isoenzymes. G1DH: Glutamate dehydrogenase (L-Glutamate:NADP oxidoreductase E.C.i.4.1.4). Clin. Ckim. Acta, 27 {1970) 467-474 is*!* illiliji Bill iiSSi 11 limm N36933 DUP050312933 468 i INTRODUCTION s e c c h t et al. Renal involvement in human and experimental lead poisoning is clearly estab i lished by pathological and clinical findings. In the acute stage of lead poisoning, the onset of renal damage has been attri buted to acute ischemia1-6, with transitory and reversible reduction of the renal blood flow and of the glomerular filtration rate. Moreover, the presence of amino aciduria is an indication of early tubular damage, which has also been demonstrated by micro scopic and ultramicroscopic studies0-12. The prevalence of renal insufficiency in sub jects exposed for many years to lead is proof that lead-nephropathy can evolve towards an irreversible nephroangiosclerosis2-3-6-7-13-11'. In the last few years, several studies carried out on kidneys made ischemic by partial constriction of the renal artery, have drawn attention to alterations of enzy matic activities related to the metabolism of pyruvate and glucose-6-phosphate. Hess and Pearse1 showed, by histochemical methods, an increase in G6PD and 6PGD activities in the macula densa of ischemic kidneys. This finding has been confirmed by Gross and Hess1', Fisher18, Hess and Regoli18, Peart80 and Vander21*. A positive correlation between an increase in G6PD activity in the macula densa, hypertrophy of the juxta glomerular apparatus, increase in juxta-glomerular granularity, and a rise in renin synthesis have been clearly demonstrated in the ischemic kidney20-28-87. More recently, in clamped kidneys a significant increase was demonstrated in the M-type isoenzymes of LDH (LDH, and LDH6)28-29. Therefore, in the kidneys of guinea pigs poisoned with lead nitrate, we have examined the activities of those enzymes known to be modified by renal ischemia. We compared the LDH isoenzymatic pattern and G6PD activity in guinea pig kidneys made ischemic by constriction of the renal artery and in the kidneys of guinea pig poisoned with lead nitrate. At the same time we studied the activity of G1DK, an enzyme localized in the mitochondria, which are the site of the main cellular oxido- reductive processes. Since, at least as far as G6PD is concerned, the alteration in activity has been observed previously only in the macula densa, our investigation was performed not only on whole kidney, but also on cortical and medullar homogenates of the same organs. MATERIALS AND METHODS Male guinea pigs, weighing 400-500 g were divided in three groups of 12 each. The first was used as control. The second was subjected to ligature of the right renal artery as described by Schroeder et al.so. The degree of stenosis was standardized, as advised by these authors, by inserting a 0.5-mm diameter metal filament between the renal artery and the silk thread, which was gently removed after the knot had been tied. The animals were sacrificed by decapitation after 20 days, and only those animals exhibiting a weight reduction of clamped kidney below 35-40% in respect [ to the contralateral kidney were utilized for our experiments. This result was obtained in 80% of the animals treated. * Due to the low specificity of histochemical methods used the true identity of these enzymatic activities is uncertain: in fact, Capelli et al.27 propose a more generic denomination, i.e. "NADPH tetrazolium reductase". Clin, Chim. Acta, 27 (1970) 467-474 ii a* i.. DUP050312934 : 'V *,1 LEAD POISONING AND KIDNEY ENZYMES 469 The third group was poisoned, in accordance with the method of Pemis et oil.*1, with a daily dose of 120 mg/kg body weight of lead nitrate given by means of an ", ;'' oesophageal catheter. All animals were decapitated after 40-50 days of treatment. The kidneys were dissected longitudinally into equal parts, one of which was ` used for the studies on the kidney in toto. In the other half, the cortex and medulla j 1" were separated. The samples of tissue were cut into thin slices which were then twice ; , ' dipped in isotonic NH,,Cl at 40 in order to hemolyze the red cells and were afterwards : ' washed in Tris-buffered saline. The organs were finally homogenized at 40 with 9 volumes of buffered saline (v/v) in a Potter-Elvejhem tissue grinder. The homogenates v were twice frozen and dried. The protein content was determined by Lowry's method. Enzyme determinations ,, Lactate dehydrogenase (E.C.i.i.r.27--LDH) was determined by Warburg's op- . tical test according to the method of Bergmeyer et at.32. The values were expressed as'micromoles NADH/min/mg protein. The identification of LDH isoenzymes was . made by electrophoresis on Cellogel, using Veronal 0.06 M, pH 8.6 buffer. The iso enzyme fractions were identified with a method based on the reduction of tetrazolium salts according to Barnett33. The quantitative evaluation of the fractions was obtained by reading on Chromoscan photodensimeter (Joyce, Loeb). Glucose-6-phosphate dehydrogenase (E.C.i.r.1.49--G6PD) was determined by the method of Lohr and Waller84. The values were expressed as //moles NADPH /min/ mg protein. Glutamate dehydrogenase (E.C.1.4.1.4--G1DH) was determined according to Schmidt's method36. The values were expressed as micromoles NADH/min/mg protein. EXPERIMENTAL RESULTS Lactate dehydrogenase The amount of LDH activity was found to be raised in the kidneys of both TABLE I QUANTITATIVE DETERMINATION OF LACTATE DEHYDROGENASE IN KIDNEYS OF NORMAL AND LEADPOISONED GUINEA PIGS, AND IN KIDNEYS MADE ISCHEMIC BY PARTIAL CONSTRICTION OF THE RENAL ARTERY i; The values of lactate dehydrogenase of kidney homogenate in toto, expressed as micromoles NADH/min/mg protein, and of the cortical and medullar fractions, expressed as a percentage of the total activity, are given. Groups Normal SD Lead-poisoned SD Ischemic SD Kidney in toto 6.8 O.64 9-5 2.1 7-4 3-8 Cortex . (%> 41-5 1.4 39.7 z.6 43-6 1-5 Medulla (%) 58.5 1.4 60.3 2.6 56.4 r-5 Statistical evaluation: Normal/Lead-poisoned p < 0.001. Normal/Ischemic p > 0.05. Clin. Chi-m. Ada, 27 (1970) 467--474 DUP050312935 m? s e c c h i et al. ..TABLE II i CHARACTERIZATION OF LDH ISOENZYMES IN KIDNEYS OF GUINEA PIGS ! i / > /1 ' Normal and lead-poisoned guinea .pig's kidneys and kidneys made ischemic by partial constriction of the renal I; V,si Mtety were examined. Values are expressed as percentage of total activity. ferbups LDH, (HHHH) LQH^ (H H H M) LDH2 (HtlMM) LDH, (H M M M) LDHS (M M M M) ,Normal ) Kidney in toto . Cortex , Medulla ^Lead-poisoned ' : Kidney in toto ' Cortex j Medulla Ischemic Kidney in toto . Cortex V Medulla 37 (SD 4 4.2) 37 (SD 6.7) 37 (SD 4 10.5) 26 (SD 3.4) 25 (SD 1.9) 24 (SD 4 4.9) 26 (SD 3.7) . 28 (SP 4 3.7) 27 (SD 4,4) J 24 (SD 1.9) 24 (SD 2.8) 23 (SD 3.0) 22 (SD 2.6) 22 (SD i 2,6) 25 (SD 3-S) 22 (SD 4 3.7) 23 (SD 2.0) 22 (SD 4 2.3) 20 (SD i 2.0) 19 (SD 1.5) 20 (SD 4 4.4] 2% (SD 0.6) 21 (SD 1.9) 22 (SD 2.7) 22 (SD 6.9) 22 (SD . I.3) 23(SD2.6) 14 (SD 4 3.0) 14 (SD 3.5) 14 (SD 4 3.0) 19 (SD 4 2.5) 19 (SD 4 1.2) 18 (SD 2.5) 19 (SD 4 3.3) 17 (SD 4 r.7) 18 (SD 4 3.0) 5 (SD 4 2.3) 6 (SD 4 3.0) 6 (SD42.6) 12 (SD 4 3.4) r3 (SD 9- 2.6) 11 (SD 4 4.3) 11 (SD 4 3.7) 10 (SD 3.5) 10 (SD 4 2.4) ) J experimental groups. However; a greater increase was observed in the lead-treated animals than in guinea pigs subjected to partial constriction of the renal artery (Table I). The average values obtained were 6.8 /tmoles NADH/min/mg protein in the nor mal kidneys;-9.5 /zmoles NADH/min/mg protein in the kidneys of the lead-poisoned guinea !pigs; and 7.4 /tmoles NADH/min/mg protein in the ischemic kidneys. The percentage distribution of this enzymatic activity in the cortex and medulla (Table I), was not modified with respect to controls. ' In normal guinea pigs, the electrophoretic characterization of LDH isoenzyme fractions showed a prevalence of H-type isoenzymes (LDHt and LDH2) in the kidney tissue in toto\ in fact the fractions LDHX and LDH2 accounted respectively for 37.2% and 24.2% of total activity; the LDH3 fraction corresponded to 19.6% of total, while the fractions LDH,, and LDHS, were present in percentages of 13.5% and, 5.1% respectively (Table II and Fig. 1). The distribution of isoenzymes of LDH in the < l8.3/a- . LDH 4 = 14.3 <7* .$=20.1% '1 = 43 8<7* LDH- &=4.6`l* " i-i*. 4 14 2 / py&x??. 1 > 3-19''".'. 2 s 21,GVo 1 = 40 4 'L ic% i r t'' -t ! L -1 mt * kidney in toto~ * cortey Fig. 1. Electrophoretic characterization of lactate deaydrogcnasc isoenzymes in kidneys of normal guinea pigs. Clin. Chim. Acta, 27. (1970) 467-474 DUP050312936 LEAD POISONING AND KIDNEY ENZYMES 471 Fig. 2. Electrophoretic characterization of lactate dehydrogenase isoenzymes in kidneys of Icadpoisoned guinea pig. cortex and medulla was substantially the same as that observed in the krdhey tissue in toto. In the kidneys of. the lead-poisoned guinea pigs we observed a marked increase in the activity of the M-type isoenzymes (LDH4 and LDH5); the LDH4 activity was 18.5% and the LDH5 activity was 12% of total activity. The increase in the activity of M-type LDH isoenzymes was of the same order in the cortex and medulla (Table II and Fig. 2). << In the clamped kidneys, a similar alteration in the pattemof LDHisoenzymes could be recognized, with a significant rise of M-type isoenzymes. In fact, in this group of animals LDH4 activity in the kidney tissue in toto amounted to. 18.8%.'and that of LDHa to 10.7% of total activity. As in lead-treated animals,1 the inerease>df LDH, and LDHS was of the same amount'in cortex and m medulla (Table II and Fig. 3). \ Fig. 3. Electrophoretic characterization of lactate dehydrogenase isoenzymes in guinea pigs' kid neys made ischemic by constriction of the renal artery. Clin. Child. Ada, 27 {197} 467-474 DUP050312937 .{iawawlataiSjl 472 s e c c h x et al. TABLE III QUANTITATIVE DETERMINATION OF OLUCOSE-6-PHOSPHATE DEHYDROGENASE IN KIDNEYS OF NOR MAL AND LEAD-POISONED GUINEA PIGS, AND IN KIDNEYS MADE ISCHEMIC BY PARTIAL CONSTRICTION OF THE RENAL ARTERY The values of glucose-6-phosphate dehydrogenase of whole kidney homogenate expressed as micromoles 'NADPH/min/mg protein, and of the cortical and medullar fractions, expressed as a percentage of total activity, are given. Groups Normal SD Lead-poisoned SD Ischemic SD Kidney in toto 51 5-4 107 12.7 76 11.4 Cortex (%) 43 1-3 48 0.8 5 2 Medulla (%) 57 1-3 52 0.8 5 2 Statistical evaluation: Normal/Lead-poisoned Normal/Ischemic p < o.ooi. p < o.ooi. Glucose-6-phosphate dehydrogenase G6PD evaluation in kidney tissue in toto showed a marked increase of this enzymatic activity in the kidneys of lead-poisoned guinea pig and in the kidneys made ischemic by constriction of the renal artery. The values obtained were 50-io-3 //moles NADPH/min/mg protein in normal kidneys, 107.2 -io-3 //moles NADPH/ min/mg protein in kidneys of lead-poisoned guinea pigs, and 75 io~3 //moles NADPH / min/mg protein in ischemic kidneys (Table III). As can be seen from Table III, in both experimental groups the rise in activity was present in the cortex and in medulla; however the increase was clearly higher in the cortex. Glutamate dehydrogenase G1DH evaluation in the kidney tissue in toto showed a significant reduction of this enzymatic activity both in the kidneys of lead-poisoned guinea pigs and in the kidneys made ischemic by arterial stenosis. The values obtained -were 0.631/trades NAD/min/mg protein in normal kidneys, 0.484 //moles NADH/min/mg protein in TABLE IV QUANTITATIVE DETERMINATION OF GLUTAMATE DEHYDROGENASE IN NORMAL AND LEAD-POISONED GUINEA PIGS, AND IN KIDNEYS MADE ISCHEMIC BY PARTIAL CONSTRICTION OF THE RENAL ARTERY The values of glutamate dehydrogenase of whole kidney homogenate, expressed as micromoles NADH/min/mg protein, and of the cortical and medullar fractions, expressed as percentage of total activity, are given. Groups Normal SD _h Lead-poisoned SD Ischemic SD + Kidney in toto 0.63 0.19 0.48 0.15 0.48 0.14 Cortex (%) 57-2 5-5 60.5 3-5 64-2 4*5 Medulla (%) 43 5-5 40 3-5 36 4-5 Statistical evaluation: Normal/Lead-poisoned p < 0.05. Normal/Ischemic p < 0.05. 0 Clin. Ckim. Acta, 27 (1970) 467-474 DUP050312938 LEAD POISONING AND KIDNEY ENZYMES 473 kidneys of lead-treated guinea pigs and 0.482 /tmoles NADH/min/mg protein in ischemic kidneys. The percentage distribution of this enzymatic activity in the cor tical and medullar fractions of the kidneys shows a more marked reduction in activity in the medullar fraction in kidneys of both experimental groups; a distinct reduction in activity is however also observed in the ccrtical zone (Table IV). DISCUSSION The experiments here described show that in the kidneys of lead-poisoned guinea pigs and in the kidneys made ischemic by constriction of the renal artery, the same kind of alteration in enzymatic activities occurs. There is a marked increase in G6PD and LDH4, LDHS activities and an evident reduction in G1DH activity, in both experimental groups. In clamped lddneys of rats, an increase in G6PD activity was demonstrated histochemically in the macula densa by several authors18'1'-81, and has been related with an increase in renin synthesis. Recently, Ambrosi et al.33-3' demonstrated a hyperplasia of the juxta-glomerular apparatus and an increase in G6PD activity of the macula densa, also in the kidneys of lead-poisoned rabbits. In these experiments we have been able to demonstrate that th increase in G6PD activity is not exclusively found in the kidney cortex, where juxta-glomerular apparatus is localized, but can be shown also in the medulla, in both experimental groups. Since G6PD is the enzyme leading into the hexose monophosphate shunt, we interpret this finding as an expression of kidney adaptation to hypoxia. Therefore the conspicuous increase in G6PD activity might be considered not only as an index of renin synthesis but also as an indication of a more general metabolic disorder caused by hypoxia. The increase in M-type isoenzymes of LDH that we have observed in leadtreated and in ischemic kidneys is consistent with this hypothesis. It is known that the M-type LDH isoenzymes catalyze the pyruvate -* lactate reaction and that their optimum activity occurs in conditions of low 08 tension. The increased activity of these isoenzymes in the kidneys of lead-poisoned guinea pigs and in the kidneys made ischemic by constriction of the renal artery, can also be considered as an expression of metabolic adjustment of the tissue to cellular hypoxia. In such a condi tion, because of the reduced activity of the Krebs cycle, there is an accumulation of pyruvate in the tissue, which only an increased activity of M-type LDH isoenzymes * can adequately metabolize to lactate. The suggestion that in the kidney tissue of lead-poisoned guinea pigs the enzy matic alterations are related to a condition of hypoxia, is further demonstrated by the behaviour of G1DH. A marked reduction in this activity was observed in both the kidneys of lead-poisoned animals and in kidneys made ischemic by constriction of renal artery, and suggests the existence of a mitochondrial involvement in both experimental situations. In conclusion, as regards the enzymatic activities investigated, an identical behaviour was observed in the kidneys of lead-poisoned guinea pigs and in kidneys made ischemic by constriction of the renal artery. Therefore, these experiments indicate that renal hypoxia occurs in experimental lead poisoning. Clin. Chim. Acta, 27 (197a) 467-474 .! DUP050312939 474 s e c c h i et al. ACKNOWLEDGEMENTS The authors express their appreciation to Prof. E. C. Vigliani and Prof. B. Pemis for helpful discussions. REFERENCES 1 A. M. Fis h b er g , Hypertension and Nephritis, Lea & Febiger, Philadelphia, 1964. 2 M. Cr e p e t a n d F. Go b b a t o , Med. Lavoro, 45 (1954) 532. 3 M. Cr e p e t , F. Go b b a t o , P. Ma r t in o a n d F. 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Sc h mid t , in Methods of Enzymatic Analysis, Academic Press, New York, 1965, p. 752. 36 L. Amb r o s e F. Vimer c a t i, C. Di Nu n n o a n d G. De St a s io , Med. Lavoro, 60 (1969) 118. 37 C, Di Nu n n o , F. Vimer c a t i, G. De St a s io , L. Amb r o s i a n d L. St r a d a , Med. Lavoro, 60 (1969) 343- Clin. Chim. Acta, 27 (1970) 467-474 taflBK <* 1 V ieiH- BsiSf DUP050312940