expressed differently-as means with standard deviations

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1 QUANTITATIVE HISTOCHEMISTRY OF THE NEPHRON. IV. ALKALINE PHOSPHATASE AND LACTIC DEHYDROGENASE ACTIVITIES IN RENAL TUBULAR DISEASES * By VICTOR E. POLLAK,t SJOERD L. BONTING, ROBERT C. MUEHRCKE AND ROBERT M. KARK (Frm the Departments f Bilgical Chemistry and Medicine, University f Illinis Cllege f Medicine, and Departments f Medicine, Presbyterian-St. Luke's Hspital, Ck Cunty Hspital, and Research and Educatinal Hspitals, Chicag, Ill.) (Submitted fr publicatin March 25, 196; accepted April 22, 196) The present investigatins were designed t study by a quantitative technique the distributin f enzyme activity in the varius individual anatmical and functinal units f the human nephrn. Our first bjective was t study the rle played by enzymes in the frmatin f urine, particularly in the secretin and reabsrptin f varius substances in the renal tubule. The secnd bjective was t discver enzymes in the kidney which may appear in the bld stream r in urine, and whse measurement in renal disease culd be used as an index f damage t specific structures in the nephrn. In this paper investigatins are reprted n the relatinship between islated and cmbined defects in the functin f the renal tubules and alkaline phsphatase and lactic dehydrgenase (LDH) activity in the nephrn. Assays f enzyme activity were made n the individual anatmical units f the nephrn f kidneys frm patients with familial renal glycsuria, hypphsphatasia, phsphate-lsing renal tubular disease, cystine strage disease, and the adult Fancni syndrme. The results were cmpared with thse frm kidneys f persns free f renal disease. MATERIAL AND METHODS Healthy kidneys. In five instances specimens f healthy kidneys were taken by bipsy during the curse f a lapartmy fr disease unrelated t the kidney, e.g., during elective chlecystectmy r repair f a ventral hernia. Tw specimens were taken at autpsy, 3 and 6 hurs after * Supprted in part by grants frm the Surgen General's Office, U. S. Army (Cntract n. DA-49-7-MD- 637) and the Natinal Institutes f Health, Bethesda, Md. (H-358 and H-3912). Presented in part at the Thirtyfirst Annual Meeting f the Central Sciety fr Clinical Research, Nvember, t Established Investigatr, American Heart Assciatin, supprted by the Illinis Heart Assciatin. death. All f the kidney specimens s btained were frm patients with n recent r past histry f renal disease, and in whm examinatin f the urine and bld revealed n evidence f renal disease. In nne was the primary disease ne knwn t have secndary effects n the kidney. Abnrmal kidneys. The renal tissue was remved by percutaneus renal bipsy (1) frm 5 patients (see belw). Tissue was als btained pstmrtem frm a patient with the adult Fancni syndrme (Case 1) and frm an infant with the infantile frm f hypphsphatasia. These specimens were btained and frzen within 1 and 5 hurs after death, respectively. Tw specimens f kidney tissue were analyzed frm the patient with the phsphate-lsing renal tubular disease, wh died suddenly frm a subarachnid hemrrhage. A specimen had been btained by bipsy 11 weeks befre death, the secnd was taken at autpsy 4 hurs after she died. Methds f dissectin and analysis f tissue fr enzyme activity. These have been described in detail in previus cmmunicatins frm this labratry (2, 3). Optimal cnditins fr the assay f alkaline phsphatase and LDH activities in human kidneys were used fr the assays (2, 3). The results were expressed in mles f substrate split r in mles f DPNH xidized per kilgram dry weight f tissue per hur at 37 C RESULTS Statistical handling f data (Tables I and II) In the previus tw papers the results f enzyme assays were expressed in terms f the ttal number f fragments f each structure studied (2, 3). A different frm f analysis was necessary in the present study in rder t cmpare data frm single pathlgical cases with data frm analyses f several nrmal kidneys. Therefre, in the case f healthy kidneys the results were expressed as means with standard deviatins f the means f all the healthy kidneys gruped tgether. The results fr the single pathlgical specimens were expressed differently-as means with standard deviatins f the individual renal fragments analyzed.

2 ENZYME ACTIVITIES IN RENAL TUBULAR DISEASE 1387 V4. *) Att fs.4f 4.- m e V4 )V.H ) U7* r4.4z c U).- Cd J., 6 6 4) 4 w ' ux U) w -4 U) U.v Et v M IzC 4i cds Cd 6,. N U)d 4).- U)! 4). '. : CU. N Z U) cd U u - U) = 4, Cla Ca cu ' cd. a-1 4 A 4)> I l- - -H -H -Ht - U) U) C14 % K ) 'r- i Cr-,q -H Os -Hi"L m - 6 C in en q- *d O5 5 % U) ON mt -H i- Li L 6 rz % C c'4r m -H ~ 4 ) LO) IR4 141 Li t- i i -1 c.- - r14 m - U) (N d4 % I4 (4L-i Li -4 L- C1 C d4 ('4 % - -; v- 6 % Clq 6 6 H'1'-i- C C n m- Qr-i " + -Hi -H O O ' m Or-1 m -H ON -H Li L-i i ) ' - -. (1 4 % L- ( 111- ' %r- (' 1(4 Hi r-, CN - 'C5 i l L-i C 6O % O r-i -HZ-4 C~4 L-i % Ch 4. r- 6 >L- It % % H-"14 - ) - 6 mr, r Li % U) t H- -- % Li ' ~ C; - -H~ LS-HL - C~ 11 11l 11O - O lr-, s 6 t 6 65m -2HH U) U) 6 6m 4-6s O r-i 1 tir ON -H O-H_ -H -4 C' Cd 4) * 4 ) u5 14 C.JU '-U 4U -- c 4.) CIS r. *gv* 4) C 4- U 4 ca4) CIS U), U* 4) -*-<~. A. N cn > d ) > ~* V 8 >.. = ' 4E *W 4- d -J b 'W ~ )d I

3 1388 Values remved mre than 2 standard deviatins frm the mean values btained fr healthy kidneys were regarded as significantly different statistically (p = <.5). In evaluating the functinal significance f changes in enzyme activity, hwever, it must be kept in mind that very cnsiderable changes in enzyme activity may be necessary befre functinal effects can be expected. Healthy kidneys Alkaline phsphatase. The level f alkaline phsphatase activity was fund, n a mlar basis, t be lw thrughut the nephrn, the lwest activity being in the glmeruli (Table I). The highest activity was fund in the prximal cnvlutins, and this activity was significantly greater than that in the distal cnvlutins (t = 6.65; p = <.1). Cmparatively lw levels f activity were fund in the medulla and papilla. LDH. There was a high level f activity, n a mlar basis, f this enzyme thrughut the nephrn (Table II). The enzymatic activity f the prximal and distal cnvlutins was higher than that f any ther segment f the nephrn, and as in the case f alkaline phsphatase, the lwest activity was fund in the glmeruli. In the case f bth enzymes there were n significant POLLAK, BONTING, MUEHRCKE AND KARK differences in activity between specimens taken by bipsy and at autpsy. Results in disease states Renal glycsuria. A 31 year ld bank clerk and many members f his family were knwn t have renal glycsuria. He was in excellent health and fasting bld glucse levels and ral glucse tlerance tests were nrmal. The renal functins measured were within nrmal limits, as was the histlgy f the renal bipsy. Alkaline phsphatase activity was decreased in the prximal (> 3 SD) and in the distal cnvlutins (= 3 SD), but was nt significantly altered in the glmeruli. The LDH activity was nt significantly different frm nrmal in any unit f the nephrn analyzed. Hypphsphatasia. Althugh hypphsphatasia is nt usually cnsidered t be a disease with clinically significant renal tubular dysfunctin, it was chsen fr study because f the pssible relatinship between alkaline phsphatase activity and the reabsrptin f glucse by the prximal tubule (4, 5). Kidneys frm three children with hypphsphatasia were studied. Full details f the three cases have been published elsewhere (6). The first patient, a tw and ne-half year ld ILE Lactic dehydrgenase activity in the anatmical units f nephrns frm healthy kidneys and frm the kidneys f patients with renal tubular dysfunctin (expressed in mles f DPNH xidized per kilgram dry weight f tissue per hur at 37 C) * Hypphsphatasia Phsphate- Healthy lsing adult Familial Juvenile renal Cystine Adult Fancni syndrme kidneys renal tubular strage Structure analyzed 7t glycsuria Case 1$ Case 2t Infantile diseaset disease Case 1 Case 2 Case 3t Glmeruli ± ± ± ± ±23. 5±.99 87± [46] [8] [5] [8] [7] [2] [7] Prximal cnvlutins 257± ± ± ± ± ± ± ±51. 7 [55] [7] 357± [7] [21] [7] [8] [7] Distal cnvlutins 256± ±t ± ± ± i ± [49] [5] [8] [7] [7] [7] [7] Medullary rays 231 ±25 3 [25] 249±34.4 [7] 234± ± [7] 99±23. [7] 137± ±59.4 [7] Medulla (uter and inner medullary znes) 165 ±21 3 [22] 326 ±3.8 [5] [12] [1] 2 ±26.9 [17] Papilla 175 ± ± [8] [12] Small artery ± ± ± ±t ±23. 26± ± [31] [4] [5] [2] [8] * Fr the healthy kidneys the mean value and standard deviatin are given fr the averages f each patient; fr the pathlgic kidneys the mean value and standard deviatin are given fr the fragments f tissue analyzed. The ttal number f tissue fragments analyzed is given in brackets. t Number f kidneys analyzed in each grup is shwn in italic type. Multiplied with factr 3.45 t crrect fr inhibitr present in DPNH used in these experiments (3). Autpsy specimen nly. Prximal and distal cnvlutins culd nt be dissected ut separately in this patient. II

4 white girl, presented because f lss f the deciduus teeth. There were n abnrmal clinical findings but changes cnsistent with thse seen in hypphsphatasia were fund n X-ray f the lng bnes. The serum alkaline phsphatase level was.5 Bdansky unit. The secnd patient, a three year ld white girl, had widespread bny defrmities, premature lss f deciduus teeth, and radilgical changes cnsistent with hypphsphatasia. The serum alkaline phsphatase level was.6 Bdansky unit. The third patient, a tw mnth ld white infant, had had grss skeletal defects frm birth. Seizures characteristic f hypsarrhythmia appeared at eight days. The child died f pneumnia. Physical abnrmalities included grss skeletal defrmities, with wide flat fntanelles and widely separated sutures. Radilgically, there was delayed ssificatin f the skull, demineralizatin f the spine, marked flaring f cstchndral junctins and an irregular appearance f the rib shaft. The metaphyses were demineralized and there were defrmities f the diaphyses f the lng bnes. The serum alkaline phsphatase was. and.7 Bdansky unit; calcium was 11 mg per 1 ml and inrganic phsphate was 5.2 mg per 1 ml. Overgrwth f cartilage, failure f bne resrptin, much steid, pr calcificatin and irregularity f bne trabeculae were nted pstmrtem. Calcium depsits were fund in the interstitial tissue f the kidney amng nrmal glmeruli and tubules. The tissue alkaline phsphatase activities were measured in hmgenates f liver, spleen, kidney and bne, and were fund t be frm.7 t 15 per cent f levels in tissues frm an infant f the same age wh had died in an accident (6). Serum alkaline phsphatase levels were abnrmally lw in members f the families f all three ENZYME ACTIVITIES IN RENAL TUBULAR DISEASE 1389 children. Phsph-ethanlamine was demnstrated by tw-dimensinal paper chrmatgraphy in the urine f all three patients. Nne f the three patients had glycsuria. The first patient, hwever, was fund t have aminaciduria. Extremely lw levels f alkaline phsphatase activity were fund in nephrns frm these three patients, but the levels f activity in the tw juvenile cases f hypphsphatasia were apprximately duble that fund in the infantile. All these results were significantly lwer than measurements frm tw nrmal infant kidneys, analyzed as cntrls (7). The LDH activity in the juvenile cases did nt differ significantly frm that fund in the healthy adult r infant kidneys, but there was a slight increase f LDH activity in the case f infantile hypphsphatasia. Phsphate-lsing renal tubular disease. The kidney frm a 53 year ld white advertising executive was studied n tw ccasins. She had enjyed excellent health until the age f 5, when she had a sudden pain in the chest and was fund t have several fractured ribs. Thencefrth she sustained numerus spntaneus fractures. The serum calcium was between 9.5 and 1.8 mg per 1 ml, inrganic phsphate between 1.8 and 2.5 mg per 1 ml, and alkaline phsphatase frm 3 t 4 Bdansky units. The tubular reabsrptin f phsphate was 47 per cent n a diet cntaining 1.5 g phsphate per day. It was nt affected by treatment with 5, units f vitamin D daily fr 6 days, but it increased frm 47 t 59 per cent when calcium (1.5 mg per kg) was infused. The urine specific gravity varied frm 1.2 t 1.18, and the serum nnprtein nitrgen was 12 mg per 1 ml. There was n aminaciduria r glycsuria. There was n evidence f intestinal malabsrptin. Several bne bipsies were characteristic f stemalacia, and the parathyrid glands were fund t be nrmal n explratin f the neck and mediastinum (8). The patient died frm a subarachnid hemrrhage. N mrphlgic abnrmalities were fund in the renal bipsy specimen r in the kidneys examined pstmrtem. The alkaline phsphatase activity was lw thrughut the nephrn, but least abnrmal in the glmeruli. These differences were significant in the prximal cnvlutins (> 4 SD) and in the distal cnvlutins (= 3 SD). Even lwer activities were fund in histlgically degenerated cnvluted tubules. The LDH activity was slightly but nt significantly increased thrughut the nephrn, except in the papilla. Cystine strage disease urith Fancni syndrme. Nephrns frm ne patient, aged fur and ne-half years, with cystine strage disease, were studied. Failure t thrive had been nted frm the age f 18 mnths, and plyuria and plydipsia frm the age f three years. Cystine crystals were fund in the crneas, and in the reticulendthelial cells f the bne marrw. The urine vlumes varied between 2 and 5 L daily, and the urine specific

5 139 POLLAK, BONTING, MUEHRCKE AND KARK gravity did nt exceed 1.5 during dehydratin r after injectin f Pitressin (vaspressin). There was n glycsuria but there was a prnunced aminaciduria (ttal a-amin-nitrgen excretin 1,545 mg per 24 hurs). The serum ptassium was cnsistently lw. On histlgical examinatin f the renal bipsy, slight degenerative changes were fund in the cnvluted tubules, and there was a slight increase f fuchsin-psitive and peridic acid-schiff (PAS)-psitive granules in the cytplasm. N ther abnrmality was fund. 'The father, paternal grandfather and ne sister were fund t have a heavy aminaciduria. The secnd sister was shwn t have cystine strage disease; cystine crystals were seen in crneas and bne marrw, and rickets, aminaciduria and glycsuria were nted. The alkaline phsphatase activity was significantly higher than nrmal in the glmeruli (> 2 SD) and was slightly elevated in the histlgically nrmal prximal cnvlutins (< 2 SD). Hwever, the alkaline phsphatase activity was significantly decreased in the histlgically degenerated cnvlutins. By cntrast, LDH activity was significantly decreased in glmeruli (> 3 SD), prximal cnvlutins (= 2 SD) and medullary rays (>3 SD). Adult Fancni syndrme. The kidneys frm three patients with the adult Fancni syndrme were studied. Full details f the clinical histries and metablic studies n Patients 1 and 2 will be published elsewhere (9). Althugh there were cnsiderable pathphysilgical similarities in these three patients, we reprt them belw individually because f the different underlying renal pathlgies. Patient 1, a 51 year ld man, died in cardiac and hepatic failure with pstnecrtic cirrhsis, stemalacia and atypical multiple myelma. During life the urine vlume varied frm 2 t 5 L, and the urine specific gravity frm 1.12 t The creatinine clearance was 55 ml per minute, the urea clearance was 6 per cent f average nrmal renal functin, and the excretin f phenlsulfnphthalein was 15 per cent in 2 hurs. The serum nnprtein nitrgen was 27 mg per 1 ml. The fllwing substances were excreted in excess in the urine: glucse, glycine, a-amin-nitrgen, inrganic phsphate, calcium, uric acid, and ptassium. Tubular reabsrptin f phsphate was 21 t 38 per cent. On micrscpic examinatin f the kidney, mderately severe degenerative changes were seen in the tubular cells, and there was patchy tubular atrphy. Several tubules cntained hyaline r pigmented casts, and sme f the casts cntained bilirubin. In sme tubules there was syncytial frmatin such as ccurs in multiple myelma. Cubidal crystals f unknwn cmpsitin were seen in prximal cnvluted tubules and interstitial tissue. The glmeruli were nrmal but there was diffuse fibrsis f the interstitial tissue. Patient 2, a 52 year ld husewife, was ill with stemalacia and multiple pseudfractures. Her urine vlume varied frm 1.5 t 2 L per day, the specific gravity frm 1.12 t 1.17, and the ph frm 6. t 7.5. The endgenus creatinine clearance was 21.4 ml per minute, the urea clearance was 22 per cent f average nrmal renal functin, and the excretin f phenlsulfnphthalein was 12 per cent in 2 hurs. The serum nnprtein nitrgen was 5 mg per 1 ml. She excreted the fllwing substances in excess in her urine: glucse, glycine, a-amin-nitrgen, uric acid, sdium, chlride, ptassium, and phsphate. Pyelnephritis and arterial and arterilar nephrsclersis were diagnsed by means f a renal bipsy. Several glmeruli were hyalinized and there was a mderate degree f tubular atrphy and degeneratin. Patient 3, a 38 year ld Negr husewife, was als ill with stemalacia and multiple pseudfractures, and in additin had metastases frm an stegenic sarcma. The serum calcium ranged frm 9.4 t 1.3 mg per 1 ml, and the inrganic phsphate frm 1.1 t 1.8 mg per 1 ml. The serum alkaline phsphate level was nrmal. The urine vlume varied frm 1.4 t 2. L daily, and the specific gravity frm 1.22 t The endgenus creatinine clearance was 119 ml per minute, and the serum urea nitrgen was 8 mg per 1 ml. She excreted the fllwing substances in excess in her urine: glucse, phsphate, glycine, and ther amin acids. N abnrmality was fund n histlgical examinatin f the renal bipsy specimen. Bichemical analyses f the kidneys frm these three patients revealed a marked decrease f alkaline phsphatase activity thrughut the nephrns, except fr the glmeruli, where it was nrmal. Particularly lw levels were fund in the prximal and distal cnvlutins (> 3 SD). LDH activity

6 ENZYME ACTIVITIES IN RENAL TUBULAR DISEASE was als significantly decreased in the glmeruli, prximal cnvlutins and medullary rays f Patients 1 and 2, and in the distal cnvlutins f Patient 1. Hwever, there was a slight increase f LDH activity thrughut the nephrns f the third patient, but this was significant nly in the medullary ray (> 3 SD). DISCUSSION Very little is knwn currently f the distributin and functins f enzymes in the individual anatmical and functinal units f the nephrn. The limited knwledge we pssess f the distributin f enzyme activity in the nephrn has been gleaned by histlgical staining techniques. Because these techniques yield nly qualitative infrmatin f limited value, the quantitative techniques reprted in the previus tw papers (2, 3) were applied t the study f human renal tissue. Methd f dissectin f specimens. The justificatin fr and validity f the micrdissectin technique were discussed in a previus paper (2), in which it was shwn that individual units f the healthy nephrn culd be dissected ut accurately and assayed with precisin fr enzyme activity. Several new prblems arse when nephrns frm diseased kidneys were studied. In particular, there were cnsiderable variatins in the histlgical appearance bth f glmeruli and f the individual anatmical units f the tubules. Fr example, the appearance f the prximal cnvlutins was mre hetergeneus than that in healthy kidneys. Sme appeared nrmal with brush brders, whereas thers shwed either degenerative changes r atrphy, r bth. Thus, histlgically nrmal prximal cnvlutins were mre easily identified than were histlgically abnrmal prximal cnvlutins; this might lead the dissectr, cnsciusly r uncnsciusly, t dissect ut the histlgically nrmal tubules rather than the damaged tubules. In rder t have a representative sample, every effrt was made t dissect ut fr analysis bth histlgically nrmal and histlgically abnrmal tubules whenever there were distinct variatins in the histlgical appearance f the tubules. Thse tubules which were s abnrmal that they culd nt be identified were analyzed as a separate grup -"degenerated cnvlutins." Levels f enzyme activity in renal tubular dysfunctin. The results suggest that there are sig nificant differences in enzyme activity between healthy kidneys and the nephrns in varius disease states. Alkaline phsphatase activity was fund t be decreased significantly in the cnvluted tubules in all diseases studied, with the exceptin f cystinsis. LDH activity was decreased significantly nly in tw f the three cases f adult Fancni syndrme and als in the child with cystine strage disease and Fancni syndrme. All three were excreting very large amunts f a-amin-nitrgen in the urine. The third adult with the Fancni syndrme was excreting nly a slight excess f a-amin-nitrgen; the LDH activity f the tubules was nt decreased. These findings might suggest that the lng cntinued massive amin acid depletin resulted in a decreased synthesis f enzyme prtein in these patients. Hwever, the finding f nrmal alkaline phsphatase activity in the tubules f the child with cystinsis makes this explanatin unlikely. Therefre, alternative explanatins must be sught. In lupus glmerulnephritis, fr example, where the tubular degeneratin was f a cmparatively mild degree, increased LDH activity was fund in the cnvluted tubules (1). There was n evidence f severe impairment f renal functin in this disease, and glucse, inrganic phsphate, and amin acids appeared t be reabsrbed nrmally by the tubules. Thus far, decreased LDH activity has been fund nly in nephrns frm the three kidneys with histlgically severe tubular disrganizatin, and with severe derangements f tubular functin; the lwest activity was bserved in nephrns f the mst severely damaged kidney. Thus the decreased LDH activity f these severely damaged nephrns may reflect r be a cnsequence f the severe damage sustained by the tubular cells in the basic disease prcesses f severe adult Fancni syndrme and f cystine strage disease. The cefficient f variatin within individual kidneys was invariably larger than the cefficient f variatin between individual kidneys (Table III), suggesting that the variatin frm nephrn t nephrn within a single healthy kidney was as great as, r greater than, the variatin frm healthy kidney t healthy kidney. There was little difference in the cefficient f variatin fr LDH activity in individual healthy and abnrmal kidneys, suggesting that the nephrn ppulatin f abnrmal

7 1392 POLLAK, BONTING, MUEHRCKE AND KARK TABLE III Cefficients f variatin fr alkaline phsphatase and lactic dehydrgenase activities in healthy kidneys and in kidneys frm patients with renal tubular dysfunctin Alkaline phsphatase Lactic dehydrgenase Renal tubular Renal tubular Healthy kidneys dysfunctin Healthy kidneys dysfunctin Cefficient Cefficient Cefficient Cefficient f f variatin Cefficient f Cefficient f f variatin f variatin variatin within between variatin within variatin within between within Structure analyzed individuals* individuals individuals individuals individuals individuals Glmeruli 65.3 (42.9-1) ( ) 31.9 ( ) ( ) Prximal cnvlutins 28.1 ( ) ( ) 2.4 ( ) ( ) Distal cnvlutins 48.8 ( ) ( ) 21.9 ( ) ( ) * Figures in parentheses indicate the range. kidneys was as hmgenus in this respect as was that f healthy kidneys. There was greater variatin f alkaline phsphatase activity in individual abnrmal than in individual healthy kidneys. This was due, at least in part, t the fact that the alkaline phsphatase activities were lw in the abnrmal kidneys, and the readings btained were very clse t thse f the blanks (2). Alkaline phsphatase and tubular reabsrptin f glucse. It has been suggested by sme investigatrs that alkaline phsphatase plays a significant rle in the reabsrptin f glucse frm the glmerular filtrate by the prximal cnvlutin (4, 5). If this were s, the alkaline phsphatase activity f the prximal cnvlutin might be expected t be lw in renal glycsuria. In the single patient with familial renal glycsuria studied, the alkaline phsphatase activity in the prximal cnvlutin was decreased t apprximately 5 per cent f that fund in prximal cnvlutins frm healthy kidneys. The decrease f alkaline phs- TABLE IV The effect f phlrhizin n the alkaline phsphatase and lactic dehydrgenase activity f the prximal cnvlutins in the dg * Befre After Enzyme phlrhizin phlrhizin p Alkaline phsphatase 2.6 i [9] [11] Lactic dehydrgenase i [1] [11] * Ttal number f tissue fragments analyzed is given in brackets. phatase activity was f a similar magnitude in patients with lupus nephritis (1), nne f whm had glycsuria. Mrever, in three patients with hypphsphatasia there was n glycsuria and the alkaline phsphatase activity was nly 3.5 t 9.5 per cent f that fund in healthy prximal cnvlutins. Unless an alternate pathway fr glucse reabsrptin can be invked fr patients with hypphsphatasia, these facts exclude the pssibility that alkaline phsphatase plays a key rle in the tubular reabsrptin f glucse. T btain further cnfirmatin, glycsuria was prduced in the dg by injectin f phlrhizin. Table IV shws that there was n significant difference in the activity f this enzyme befre and after injectin f phlrhizin, s that phlrhizin-induced glycsuria is nt mediated by way f inhibitin f alkaline phsphatase. SUM MARY 1. Alkaline phsphatase and lactic dehydrgenase (LDH) activities were measured quantitatively in the varius anatmical and functinal parts f the nephrns frm healthy kidneys and frm the kidneys f six patients with diseases affecting the renal tubules: familial renal glycsuria, phsphate-lsing renal tubular disease, cystine strage disease, and adult Fancni syndrme. 2. Alkaline phsphatase activity was decreased in the cnvluted tubules in all diseases studied except in cystine strage disease. LDH activity was decreased nly in tw f the three cases f

8 ENZYME ACTIVITIES IN RENAL TUBULAR DISEASE adult Fancni syndrme and in a child with cystinsis and the Fancni syndrme. A parallel decrease f bth enzymes was bserved in tw f the patients with adult Fancni syndrme. 3. The kidneys f three patients with hypphsphatasia were als studied. Nne had glycsuria. The extremely lw level f alkaline phsphatase activity fund in the prximal cnvlutins indicates that this enzyme des nt play a key rle in glucse reabsrptin. This was cnfirmed by the bservatin that prlnged phlrhizin-induced glycsuria did nt affect the level f alkaline phsphatase activity in the prximal cnvlutin f the dg. ACKNOWLEDGMENTS We wish t thank Dr. Ira Rsenthal and Dr. Thedre Schwartz fr allwing us t study patients under their care, and Dr. William R. Best fr statistical advice. We als wish t thank Miss Alta D. Tsdle, Mrs. Hendrina debruin, and Mr. Bart R. Mayrn fr technical assistance. REFERENCES 1. Kark, R. M., and Muehrcke, R. C. Bipsy f kidney in prne psitin. Lancet 1954, 1, Bnting, S. L., Pllak, V. E., Muehrcke, R. C., and Kark, R. M. Quantitative histchemistry f the 1393 nephrn. II. Alkaline phsphatase activity in man and ther species. J. clin. Invest. 196, 39, Bnting, S. L., Pllak, V. E., Muehrcke, R. C., and Kark, R. M. Quantitative histchemistry f the nephrn. III. Lactic dehydrgenase activity in man and ther species. J. clin. Invest. 196, 39, Lundsgaard, E. Die Wirkung vn Phlrrizin auf die Glucseresrptin. Bichem. Z. 1933, 264, Rche, J. Phsphatases in The Enzymes, Chemistry and Mechanism f Actin, J. B. Sumner and K. Myrback, Eds. New Yrk, Academic Press, 195, vl. I, part 1, p Rsenthal, I. M., Bnting, S. L., Hgan, W., and Pirani, C. L. Tissue alkaline phsphatase in hypphsphatasia. A. M. A. Amer. J. Dis. Child. 196, 99, Rsenthal, I. M., and Bnting, S. L. Unpublished data. 8. Reiss, E., and Schwartz, T. B. Persnal cmmunicatin. 9. Dedmn, R. E., West, J. M., and Schwartz, T. B. Pathphysilgic studies in the adult Fancni syndrme. Reprt f tw cases, ne with multiple myelma. Ann. intern. Med. Submitted fr publicatin. 1. Pllak, V. E., Bnting, S. L., Muehrcke, R. C., and Kark, R. M. Quantitative histchemistry f the nephrn. V. Alkaline phsphatase and lactic dehydrgenase activities in lupus nephritis. J. clin. Invest. 196, 39, 1394.