Of Tubular Fluid in Anuria Due to Mercury Poisoning

The Role of “Leakage” of Tubular Fluid in Anuria Due to Mercury Poisoning

Norman Bank, … , Bertrand F. Mutz, Hagop S. Aynedjian

J Clin Invest. 1967;46(5):695-704. https://doi.org/10.1172/JCI105570.

Research Article

The role of “leakage” of tubular fluid in anuria produced by mercury poisoning was studied in rats by micropuncture techniques. After an initial brisk diuresis, almost all animals were completely anuric 24 hours after HgCl2 injection. Lissamine green injected intravenously in the early stage of anuria appeared in the beginning of the proximal tubule, but the color became progressively lighter as the dye traversed the proximal convolutions. The dye was barely visible in the terminal segments of the proximal tubule; it did not appear at all in the distal tubules. These observations suggest that the proximal epithelium had become abnormally permeable to Lissamine green.

Tubular fluid to plasma inulin (TF/PIn) ratios and inulin clearance were measured in individual nephrons at three sites: early proximal tubule, late proximal tubule, and distal tubule. It was found that TF/PIn ratios were abnormally low in the late proximal and distal tubules. Inulin clearance was normal at the beginning of the proximal tubule but fell by more than 60% by the late proximal convolutions. Thus, the proximal tubule had also become permeable to inulin.

We conclude from these observations that anuria in mercury poisoning can occur in the presence of a normal glomerular filtration rate. The absence of urine flow appears to be due to complete absorption of the filtrate through an excessively permeable tubular […]

Find the latest version: https://jci.me/105570/pdf Journal of Clinical Investigation Vol. 46, No. 5, 1967

The Role of "Leakage" of Tubular Fluid in Anuria Due to Mercury Poisoning * NORMAN BANK,t BERTRAND F. MUTZ, AND HAGOP S. AYNEDJIAN (From the Department of Medicine, New York University School of Medicine, New York, N. Y.)

Summary. The role of "leakage" of tubular fluid in anuria produced by mercury poisoning was studied in rats by micropuncture techniques. After an initial brisk diuresis, almost all animals were completely anuric 24 hours after HgCl2 injection. Lissamine green injected intravenously in the early stage of anuria appeared in the beginning of the proximal tubule, but the color became progressively lighter as the dye traversed the proximal convolutions. The dye was barely visible in the terminal segments of the proximal tubule; it did not appear at all in the distal tubules. These observations suggest that the proximal epithelium had become abnormally permeable to Lissamine green. Tubular fluid to plasma inulin (TF/PIn) ratios and inulin clearance were measured in individual nephrons at three sites: early proximal tubule, late proximal tubule, and distal tubule. It was found that TF/PN ratios were abnormally low in the late proximal and distal tubules. Inulin clearance was normal at the beginning of the proximal tubule but fell by more than 60% by the late proximal convolutions. Thus, the proximal tubule had also become permeable to inulin. We conclude from these observations that anuria in mercury poisoning can occur in the presence of a normal glomerular filtration rate. The absence of urine flow appears to be due to complete absorption of the filtrate through an excessively permeable tubular epithelium. The driving force af- fecting this fluid absorption is probably the colloid oncotic pressure of the peritubular capillary blood.

Introduction back diffusion of the filtrate through a damaged tu- The pathologic physiology of anuria in acute bular epithelium. To our knowledge, only three renal failure remains obscure in spite of extensive studies have been published in which these various study. The three mechanisms that have been pos- possibilities were examined by direct observation tulated are: a severe reduction in renal blood flow of the kidney in zrvo in animals with experimental and glomerular filtration rate (GFR), obstruction acute renal failure (1-3). Richards reported of the tubules by casts or edema, and excessive that, in the anuric stage of cyanide or mercury poisoning in frogs, flow of fluid from the glomeru- *Submitted for publication July 12, 1966; accepted lus into the beginning of the proximal tubule was January 4, 1967. This work was supported by grants from the National normal or even increased (1). The failure of any Heart Institute (HE-05770), the American Heart Asso- urine to appear in the ureter was attributed to ciation, and the Life Insurance Medical Research Fund. gross leakage of tubular fluid from more distal tCareer Scientist of the Health Research Council of segments of the proximal tubule. More recently, New York City. Address requests for reprints to Dr. acute renal Norman Bank, Dept. of Medicine, New York Uni- Oken and his co-workers, studying versity School of Medicine, 550 First Ave., New York, failure due to mercury poisoning (2) and hemo- N. Y. globinuria (3) in rats, found GFR per nephron 695 696 BANK, MUTZ, AND AYNEDJIAN to be significantly reduced. They concluded that anuria in these two experimental models resulted from intrarenal vasoconstriction and a subsequent mOsm/Kg H20 600d fall in filtration rate. In the present study, we have reinvestigated the role of leakage of tubular fluid in anuria too~ ~~~a caused by mercury poisoning. The techniques mEq/L ' t l used were rapid sequence photography of the surface tubules after intravenous Lissamine green 250-. injection, and quantitative measurements of inu- 350 lin clearance at various points along the nephron. mEq/L 50> The findings support the view that the permeability of the tubules is markedly increased and non- selective during the anuric stage of mercury URINE 25 poisoning. Glomerular filtration rate, on the other hand, as evaluated by inulin clearance measured in early segments of the proximal tubule, was within the normal range. We conclude, therefore, HgCL2 DAYS that the mechanism of anuria in these animals was injection excessive back diffusion of the glomerular filtrate FIG. 1. URINE VOLUME, SODIUM, POTASSIUM, AND OS- through abnormally permeable tubules. MOLALITY AFTER HgCls INJECTION. The rats were fed a sodium-free diet for 3 or 4 days before HgCl2 injection and continued on the diet for the 8 days afterward. The Methods vertical lines indicate the observed range. Male white rats weighing between 225 and 300 g were fed a sodium-free diet for 3 or 4 days before the induc- polyethylene Lubing and the bladder emptied by syringe tion of acute renal failure. The reason for eliminating at the beginning and end of each experiment in order to sodium from the diet was to determine whether a reab- determine whether there was any urine flow from either sorptive defect for sodium occurred during the course kidney. Mean arterial blood pressure was measured of the illness. After 3 or 4 days on the diet, a control in all experiments by a U-tube mercury manometer con- urine specimen was collected in a metabolic cage. Tap nected through its sidearm to a catheter secured in the water was allowed ad libitum during the collection of carotid artery. Only those experiments were accepted urine, but food was withheld. Water intake as well as in which mean blood pressure remained between 100 and urine output was measured in each animal. Acute renal 130 mm Hg. Approximately 4 to 5 ml of isotonic NaCl failure was then induced by subcutaneous injection of was given intravenously during surgical preparation of HgCls solution in a dosage of 3.0 to 3.5 mg per kg body the rat to replace body fluid losses and to preclude the weight of the mercuric ion. At this dosage, all animals possibility of salt depletion. A constant infusion of iso- survived at least 48 hours, and 60% underwent a diure- tonic NaCl was administered at 0.025 ml per minute sis and recovered after a period of anuria. During the throughout the experiment. first 16 hours after HgCln injection, a marked diuresis Lissamine green experiments. In 15 rats, 24 to 48 occurred, but beginning at about 20 hours after injection hours after HgCl2 administration, the surface of the kid- and lasting for an additional 24 to 30 hours, the rats ney was photographed sequentially after rapid intravenous were either totally anuric or severely oliguric. All Lis- injection of 0.04 to 0.07 ml of 10%o Lissamine green, as samine green and micropuncture studies during the anuric described by Steinhausen (5). In these animals, the stage of the disease were carried out between 24 and 48 renal capsule was stripped away to improve the clarity hours after Hg++ injection. In 22 animals that survived of the photographs, and the kidney was bathed in con- the anuric period, daily urine collections were continued tinuously flowing saline warmed to 370 C. The photo- for up to 2 weeks, and in 8 rats of this group, observa- graphic equipment consisted of a Leitz Ultropak ob- tions on the function of surface tubules were made with jective and illuminating system, a Zeiss Ukatron elec- the aid of Lissamine green at various intervals during tronic flash unit (operated at 60 watt-seconds), and a this time. The measurements made on all urine speci- Leica camera back with a battery-operated motor for mens were volume, sodium and potassium concentrations automatic film advance. High speed Ektachrome film (internal standard flame photometery), and osmolality (ASA 160) was used throughout. Pictures were taken (Fiske osmometer). at various intervals after Lissamine green injection, de- The surgical preparation of the rats was as described pending upon the appearance of the dye in the struc- previously (4). The left ureter was catheterized with tures seen on the surface of the kidney. TUBULAR LEAKAGE IN MERCURY POISONING 697

NORMAL MERCURY FIG. 2. 698 BANK, MUTZ, AND AYNEDJIAN Measurement of TF/PJn ratios and inulin clearance curred, the average urine volume being 18 ml. per nephron. In eight rats, 24 to 30 hours after Hg++ Potassium constituted the major urinary cation injection, TF/Pln ratios and inulin clearance were measured at various points along the proximal and distal during the diuresis, as it did in the control period. tubules. A priming dose of 25 Ac "4C-labeled inulin Water ingestion paralleled or exceeded urine out- carboxylic acid' was given intravenously, and a sus- put during the 16-hour period, the average intake taining dose of 0.4,c per minute in isotonic saline was being 25 ml. Starting at about 20 to 24 hours, started 30 minutes before tubular fluid collections were urine flow fell to zero in almost all animals and begun. Early proximal segments were identified by in- was greatly reduced in the others. Between 24 jecting Lissamine green intravenously and observing the first intratubular appearance of the dye. Late proximal and 48 hours after Hg++ injection, the animals segments were identified as those clustered about promi- were virtually anuric, even though water ingestion nent branching capillaries (6), and distal tubules by the remained normal in many of them. Urine output thin, glistening appearance of their epithelium. Tubular resumed in 22 of the 35 animals after this period fluid collections were made from each of these three loci and exceeded control volumes on the third and in each animal. A column of heavy castor oil stained with Sudan black was maintained just distal to the col- fourth days after Hg++. Sodium excretion was lection site to assure that all fluid coming from the glo- markedly increased during the early recovery pe- merulus was aspirated into the pipette. The period of riod in spite of the absence of sodium in the diet. collection was carefully timed with a stopwatch and the Urinary potassium excretion, on the other hand, volume of the collected fluid measured as described by was lower than in the control period, as was total Windhager and Giebisch (7). The inulin concentration in tubular fluid and plasma was measured by liquid scin- urinary osmolality. Between the fourth and tillation counting as previously described (8). Plasma seventh days, urinary sodium, osmolality, potas- inulin concentration at the midpoint of each tubular fluid sium, and volume all returned to control levels. collection was determined from the plasma inulin curve, Although the entire anuric and recovery periods blood samples being collected from the cut tail at 30- were telescoped as compared with the usual course minute intervals during the experiment. The plasma inulin concentrations were corrected for a plasma water of acute renal failure in man (9), the pattern of re- content of 94%. Inulin clearance per nephron was cal- ciprocal changes in urinary sodium and potassium culated from the expression TF flow X TF/PIn. At concentrations and the reduction of urinary osmo- the end of each tubular fluid collection, the nephron was lality are typical of the findings in man (10). injected with colored latex and the site of the collection with determined more precisely by microdissection. Appearance of the kidney and observations In five normal rats also prepared on a sodium-free diet Lissamine green (Figure 2). At 24 to 28 hours for 3 or 4 days, the potent diuretic furosemide 2 was ad- after 3.0 to 3.5 mg per kg of Hg++ was injected ministered intramuscularly in a dose of 40 mg per kg subcutaneously, the epithelial lining of the proxi- body weight. Urine output and water intake were mea- mal tubules appeared poorly defined, pale, and sured for the next 24 hours after which time TF/Pln granular in those segments closest to prominent ratios and inulin clearance along the nephron were measured as described above. branching surface blood vessels. These blood vessels, one of which can be seen clearly in the upper left photograph in Figure 2, have been identified Results as efferent arterioles and the adjacent tubular Urine volume, sodium, potassium, and osmo- segments as the most distal portions of the proxi- lality (Figure 1). The data obtained from a total mal convolutions present on the surface of the of 35 rats followed for various lengths of time kidney (6). Other tubular segments, presum- up to 8 days after HgCl2 injection are shown in ably proximal to the abnormal ones, appeared Figure 1. All animals were maintained on a so- more normal. When they were examined between dium-free diet for 3 or 4 days before Hg++ ad- 30 and 48 hours after Hg++ injection, a more dif- tubules was ministration and were continued on the diet dur- fuse involvement of the proximal seen, with almost all segments showing a loss of the ing the 8 days afterward. During the first 16 usual sharp definition characteristic of the normal hours after Hg++ injection, a marked diuresis oc- proximal epithelium. Proximal tubule lumina 1 New England Nuclear Corp., Boston, Mass. could be seen throughout the anuric stage of the 2 The furosemide was kindly supplied by Hoechst Phar- disease, but their diameters often could not be maceuticals, Inc., Cincinnati, Ohio. judged accurately because of the granularity and TUBULAR LEAKAGE IN MERCURY POISONING 699 opacity of the overlying epithelium. In contrast termined. In five of the eight, no urine appeared to the normal kidney, only a few patent distal tu- in either the ureteral catheter draining the experi- bules could be recognized in kidneys examined mental kidney or in the bladder during the 3 to 4 between 24 and 48 hours, but their thinner more hours of the experiment. In the other three ani- glistening epithelium appeared normal. mals, a volume of urine too small to fill the ure- The appearance of Lissamine green after rapid teral catheter was produced by the experimental intravenous injection is illustrated in Figure 2. kidney. The urinary to plasma inulin (U/P11) A sequence of pictures of a normal kidney is ratios in these three animals were 9.56, 3.95, and shown at the left from top to bottom, and one of a 1.91, respectively. Hg++-poisoned kidney is shown on the right. The The control group consisted of five normal rats latter experiment was carried out 24 hours after fed a sodium-free diet for 3 or 4 days and given Hg++ injection. At the time of study, the animal furosemide intramuscularly 24 hours before mi- had been totally anuric for 8 hours. Moreover, no cropuncture study. Urine output on the day pre- urine appeared in either the left ureteral catheter ceding the experiment averaged 26.4 ml and water or bladder during the experiment. In both ani- ingestion, 26.0 ml. In contrast to the Hg++-in- mals, Lissamine green appeared in the surface jected animals, urinary sodium concentration rose peritubular capillaries 4 seconds after its intrave- markedly during the diuretic period to 43 mEq nous injection. The dye next appeared in the first per L. During micropuncture on the following segment of the proximal tubule 7 seconds after day, each of the five control rats excreted urine at injection. After this point, however, the color in- nondiuretic rates, the average flow from the ex- tensity decreased progressively in the Hg+- perimental kidney being 8.1 ul per minute per kg. poisoned animal as the dye traversed the proximal The U/P1,, ratio in this group averaged 484. convolutions. Toward the end of the proximal The TF/P10 ratios are shown in Figure 3 tubule, in those segments nearest to the prominent plotted on the ordinate against sites of collection branching arterioles, only a diffuse light green on the abscissa. The ratios were strikingly low in color could be seen. In contrast, in the normal both the proximal and distal tubules of the anuric rat, the dye intensity remained essentially un- animals, as compared with the control animals and changed from the beginning to the end of the prox- with normal values reported by numerous investi- imal tubule. The marked difference between the gators (7, 8, 11-13). The distal TF/Pin ratios normal and diseased kidneys can be best appreci- found in the furosemide-treated rats are somewhat ated by comparing adjacent photographs in Fig- lower than those previously reported for non- were after ure 2, which taken at comparable times diuretic normal rats, perhaps due in part to the Lissamine green injection. In addition to these observations, we noted that Lissamine green never reappeared in the distal tubules of the anuric animals, as it does in normal rats (5). This was the 500 S Hgq- POWNED X4.RC RATS 0 0 NON -DI~ElC CONTROL RATS case even with the few patent distal tubules that 0 were recognized by their characteristic thin epi- 4.00- 0 00 observations were made in 14 thelium. Similar TF/P,, 0 0 other anuric rats. 3.00~~~~~~~~~~ ratios and inulin clearance per nephron TF/PI, 0 00 (Figure 3, Tables I and II). In eight rats studied at 24 to 30 hours after Hgr+ injection, TF/Pi. 2DO 0 * ratios were measured at various points along the 00 * 0 nephron, and inulin clearance per nephron was de- 1.00 W,,^l, ,* ., 10 aD 30 40 s0 670 0 20 40 60 80 l00 PER CENT PROXMAL TUBLE PER CENT DISTAL TUW..E FIG. 2. APPEARANCE OF KIDNEY SURFACE IN VIVO DUR- ING PASSAGE OF LISSAMINE GREEN. In both the normal FIG. 3. TUBULAR FLUID TO PLASMA INULIN (TF/Pi.) and anuric animals, the pictures were taken at 4, 7, 15, RATIOS IN ANURIC Hg++-POISONED RATS, AND NONDIURETIC and 25 seconds, respectively, after intravenous injection CONTROLS. A striking inability to concentrate inulin is of 10% Lissamine green. Magnification 110 X. evident in the anuric animals. 700 BANK, MUTZ, AND AYNEDJIAN

TABLE I TABLE II Inulin clearance measured at various points along the nephron Inulin clearance measured at various points in a mercury-poisoned anuric rat* along the nephron* Inulin Early Late TF TF flow clearance proximal proximal Distal Time sample TF/Pin rate per nephron Location nt/min/kg nt/min/kg nl/min/kg minutes nl/ nl/ % length min/kg min/kg Hg++ 110 426t 40.5 426.2 28.6 +20.6 0 Infusion: NaCl, 150 mEq per L at 0.125 ml per minute rats p <0.001$ 40 Inulin-14C, 25 uc intravenously Control 41 Infusion: NaCl, 150 mEq per L; inulin-14C, 17 puc per rats 103±421 88.9±26.0 91.2420.8 ml at 0.025 ml per minute 0.1

* Abbreviations: TF/Pi. = tubular fluid to plasma inulin ratio; ious collection sites. A typical experiment on an nl = nanoliters; % length = per cent of total length of proximal or distal tubule. anuric rat is shown in detail in Table I, and the data from all eight anuric and five control animals large sodium diuresis on the preceding day and a are summarized in Table II. Inulin clearance subsequent decrease in medullary hypertonicity. measured in early proximal segments was not In addition to TF/PIn ratios, tubular fluid flow significantly different from that measured in the rates were measured in these experiments, and control animals or that previously reported from inulin clearance per nephron was calculated for var- this laboratory in which random collections from

FIG. 4. APPEARANCE OF KIDNEY SURFACE IN VIVO AFTER CLINICAL RECOVERY FROM HgC1S-INDUCED ACUTE RENAL FAILURE. Intraluminal cellular debris obstructs many tubule segments. The internal diameter of the functioning tubules is 25%o larger than normal. Magnification 65 X. TUBULAR LEAKAGE IN MERCURY POISONING 701 the proximal tubule had been made (11). When urine flow was due to dehydration secondary to measured in the late proximal segments, however, the large diuresis on the preceding day, since water inulin clearance averaged only 37% of the early intake during the diuretic period exceeded urine proximal values and in the distal tubule fell to output. To exclude this possibility more rigor- 26%. In the control rats, the site of tubular fluid ously, however, we injected normal sodium-de- collection had no apparent influence upon calcu- prived control rats with furosemide, which induced lated inulin clearance per nephron. a significantly greater diuresis than did the HgCl2 Recovery stage (Figure 4). In eight rats that and, moreover, resulted in considerable loss of had recovered from acute renal failure, Lissamine sodium in the urine.3 In contrast to the Hg++- green photographic studies were carried out at poisoned animals, all furosemide-treated rats ex- periods ranging from 5 to 17 days after Hg++ in- creted quantities of urine during the acute ex- jection. Urinary sodium, potassium, and osmo- periments that were easily measurable. Thus, the lality had all returned to control levels by this absence of urine flow in the Hg++-injected rats time. In spite of the apparently normal urine, cannot be attributed to dehydration. Finally, in however, examination of the surface of the kidney three of the Hg++-poisoned animals in which a revealed numerous tubules that contained what small amount of urine did appear in the ureteral appeared to be cellular debris. Lissamine green catheter, U/P1n ratios averaged only 5.14. In did not pass through these segments, and we as- the control rats with larger urine flows, U/PI, sumed that such nephrons were partially or com- ratios averaged 484. These observations are completely blocked. Adjacent tubules through which patible with a diagnosis of acute renal failure in Lissamine green did flow were significantly di- the HgCl2-injected rats. lated. A typical example of the appearance of the In the experiments with Lissamine green in the surface of the kidney 17 days after Hg++ injection anuric animals, the dye appeared in the capillaries is shown in Figure 4. The heterogeneity of the on the surface of the kidney and in the beginning nephron population is clearly evident. The aver- of the proximal tubule in what was judged to be a age luminal diameter of the functioning tubules in normal amount and time after its intravenous in- this field was 29.5 pu, as compared with an average jection. However, as the dye progressed along of 23.5 in the normal rats. These observations the proximal convolutions, its concentration ap- suggest that tubular obstruction, which can occur peared to decrease markedly so that in most cases during the anuric stage of mercury poisoning (2), only a pale diffuse coloration was visible in the may persist for some time after the diuresis and terminal segments on the surface of the kidney. could possibly account for certain functional ab- Moreover, no dye was ever seen to reappear in normalities found long after clinical recovery from the distal tubules as it does in normal rats, where, an episode of acute renal failure (14, 15). in the nondiuretic state, it is seen in its most con- centrated form (5). It seems likely that these Discussion observations were due to an increase in the perme- Several observations indicate that the Hg++-inability of the tubules to Lissamine green rather jected rats were in a true state of acute renal fail- than to some alteration in the hydrodynamics of ure when Lissamine green and micropuncture tubular fluid flow. Although it is theoretically studies were carried out. First, after an initial possible that an abnormally slow moving column of diuresis induced by the HgCl2 injection, no urine tubular fluid might allow longitudinal diffusion of was excreted for periods ranging from 4 to 24 Lissamine green and therefore dilution of the dye, hours before study, in spite of the fact that water 3 The fact that the HgCl-induced diuresis was accom- intake remained normal in most animals. Fur- panied by a large increase in K+ excretion but not in Na+ thermore, during the acute studies where one excretion could be explained by a proximal site of action ureter was catheterized and the bladder emptied by of the compound. Inhibition of proximal Nat reabsorp- little or no urine appeared from either tion could result in a large distal Na-K exchange, es- syringe, pecially since the rats were salt-deprived. Furosemide, kidney, even though 4 to 5 ml of saline had been on the other hand, has been shown to act principally on administered during the surgical preparation of the ascending limb of the loop of Henle (16), and thus the animal. It seems unlikely that the absence of increased Nat excretion in spite of salt deprivation. 702 BANK, MUTZ, AND AYNEDJIAN this has not been observed during acute reductions those measured in the proximal and distal tubules in GFR produced by controlled hemorrhage (17, of the nondiuretic control animals. That these 18), constriction of the abdominal aorta (13), or low TF/P1n ratios were not due to retention of increased ureteral pressure (13). With very se- water in the lumen but rather to leakage of inulin vere reductions in filtration rate and slowing of out of the lumen is indicated by the inulin clear- tubular fluid flow produced by hematin injection, ance per nephron measurements summarized in color intensity is increased in the proximal tubule, Table II. In the control animals, inulin clearance and the dye is almost black when it reaches the was essentially uniform when measured at vari- distal tubule (19). Finally, the rate of tubular ous sites along the nephron. In the experimental fluid flow in early segments of the proximal tu- animals, however, an average fall in inulin clear- bule was within normal limits in the Hg++-poisoned ance of 63%o was found when samples were col- rats, as indicated by the inulin clearance measure- lected from the late proximal tubule, and a fur- ments in Table II. Thus, the loss of Lissamine ther fall was observed in the distal tubule. The green color observed in these experiments cannot average value for distal tubular inulin clearance be accounted for by a reduction in filtration rate may be misleadingly high, however, as it repre- or in tubular fluid flow. Although retention of sents collections from what appeared to be a re- water in the tubular lumen as occurs, for ex- duced number of patent distal tubules. An ap- ample, in partially or completely obstructed neph- parent reduction in the number of recognizable rons might lead to dilution of the dye, this expla- distal tubules could be the result of complete ab- nation seems unlikely, since the proximal tubules sorption of the glomerular filtrate more proximally were not obviously dilated and it has been demon- in many of the nephrons, with subsequent collapse strated that intraluminal pressure is not elevated of the distal lumina. In any case, the data indi- in Hg++-produced anuria (2). The observations cate that much of the filtered inulin had left the lu- are most compatible, therefore, with the view that men along the length of the proximal tubule, and the tubules had become excessively permeable to that even more was lost before fluid reached the Lissamine green. Since there was a concentra- distal convolution. These observations thus pro- tion gradient of the dye between tubular lumen and vide a physiological counterpart for histological peritubular capillary blood (the vascular phase of and histochemical studies that have shown the ma- dye passage is over before the tubular phase be- jor site of injury in mercury poisoning to be the gins, as seen in Figure 2), dye might have been more distal segments of the proximal convoluted lost by diffusion down a concentration gradient. tubule and the pars recta (20-22). The site of increased permeability appeared to be From the data in Table II, it should be noted the more distal segments of the proximal convo- that inulin clearance measured in the early proxi- luted tubule in the earliest stage of the disease mal tubule was essentially the same in the anuric studied, but deeper segments such as the pars animals as in the control rats; both values agree recta and loops of Henle may also have been in- closely with those previously reported for normal volved. Our observations are in agreement with rats (11). Thus, glomerular filtration rate was those of Richards (1), who described leakage of apparently within the normal range in the totally phenolsulphonphthalein from the proximal tubules anuric animals. This observation also agrees of both excised and in situ frog kidneys that had with that of Richards, who noted a brisk flow of been poisoned with HgCl2. fluid from the glomerulus into the beginning of Further evidence of loss of selective permeability the proximal tubule in anuric mercury-poisoned of the tubules was found in the micropuncture ex- frogs (1). Since our data demonstrated a major periments. Measurement of TF/P1. ratios along disruption of tubular permeability, we assumed the proximal and distal convoluted tubules showed that anuria in these animals was due to complete a striking inability to concentrate inulin. The absorption of a normally formed glomerular fil- highest ratio found in the proximal tubule was trate along the length of the nephron. The site only 1.40 and that in the distal tubule, 2.08, and of final absorption of the filtrate and collapse of many measurements were not significantly higher the lumen was presumably distal to the surface than 1.0. These values differ markedly from proximal convolutions, since in the early stages of TUBULAR LEAKAGE IN MERCURY POISONING 703 total anuria fluid could still be aspirated from the flow fell to oliguric levels soon after drug adminis- most distal portions of the proximal tubule on the tration. Furthermore, since they injected the Hg surface of the kidney. intramuscularly, it seems reasonable that renal The mechanism of complete absorption of the vasoconstriction might have persisted for longer glomerular filtrate through an abnormally perme- periods than after intravenous injection. The re- able epithelium is not entirely clear. Since mer- duction in single nephron GFR that they found at cury poisoning probably inhibits active sodium 6 hours might have been due, therefore, to a pro- transport (23), some other driving force must have longed effect on the renal blood vessels. The toxic been responsible for passive movement of water effect on the tubules that we observed at 24 to 48 across the epithelium. The only other force of hours after Hgj injection had apparently not yet sufficient magnitude to accomplish this is the col- become manifest at 6 hours.4 Because our animals loidal oncotic pressure of the peritubular capillary responded to a smaller dose of Hgw with a large blood, which is equivalent to approximately 35 cm diuresis, we assumed that renal blood flow and of water (2). Normally, colloidal oncotic pressure GFR were not severely reduced during the first plays little or no role in influencing water ab- hours after the injection. From this interpreta- sorption (24), presumably because of the perme- tion of the two studies, we conclude that, in high ability characteristics of the interposed cell mem- dosages, HgCl2 may produce oliguria or even branes. In mercury nephrotoxicity, however, anuria by causing a reduction in blood flow and membrane permeability might increase to the GFR. This effect should be transient (25, 26), point where this considerable force can be exerted however, and should not in itself produce the ab- more directly on the fluid in the tubular lumen. normalities in urinary electrolyte composition seen The observations made in this study differ in in patients with acute renal failure (10) and in several respects from those reported by Flanigan our rats. The more prolonged anuria produced and Oken (2), who also studied mercury poisoning by Hg++ poisoning, which can occur in the pres- in rats by micropuncture techniques. These au- ence of a normal filtration rate, appears to be due thors found a reduction in individual nephron fil- to a toxic effect on the tubules leading to increased tration rates averaging 55% when measured at 6 permeability and complete absorption of the glo- hours after a dose of HgCl2 approximately three merular filtrate. times larger than we used. TF/P1, ratios in their animals rose progressively along the length of the References proximal tubule and were indistinguishable from 1. Richards, A. N. Direct observations of change in those measured in normal rats. The reason for function of the renal tubule caused by certain these differences between their study and ours is poisons. Trans. Ass. Amer. Phycns 1929, 44, 64. clear, but they could be explained by 2. Flanigan, W. J., and D. E. Oken. Renal micro- not entirely puncture study of the development of anuria in the a dual effect of Hgr on the kidney at different dose rat with mercury-induced acute renal failure. J. levels and by the time after Hg++ injection when clin. Invest. 1965, 44, 449. micropuncture experiments were carried out. It 3. Oken, D. E., M. L. Arce, and D. R. Wilson. Gly- has been found that a variety of mercury com- cerol-induced hemoglobinuric acute renal failure flow in the rat. I. Micropuncture study of the devel- pounds cause a sharp reduction in renal blood opment of oliguria. J. clin. Invest. 1966, 45, 724. and glomerular filtration rate shortly after their 4. Bank, N. Relationship between electrical and hy- intravenous injection (25, 26). This effect, which drogen ion gradients across rat proximal tubule. is thought to be due to renal vasoconstriction, is Amer. J. Physiol. 1962, 203, 577. transitory unless the dose is high enough to cause 5. Steinhausen, M. Eine Methode zur Differenzierung the reduc- proximaler und distaler Tubuli der Nierenrinde a fall in blood pressure. In this case, Ratten in vivo und ihre Anwendung zur tion in renal blood flow and GFR lasts for several von hours and may prevent the expected diuresis (25). 'The possibility that a reduction in renal blood flow have occurred shortly produced by large doses of Hg++ may actually afford some That such a response may protection to the tubules is suggested by the fact that only after Hg++ injection in the rats studied by Flanigan 33% of Flanigan and Oken's rats became totally anuric and Oken is suggested by the fact that their animals in 24 hours. With smaller doses of Hg++, almost all of had little or no initial diuresis. Instead, urine the rats in this study were anuric at 24 hours. 704 BANK, MUTZ, AND AYNEDJIAN Bestimmung tubularer Str6mungsgeschwindig- 16. Suki, W., F. C. Rector, Jr., and D. W. Seldin. The keiten. Pflugers Arch. ges. Physiol. 1963, 277, 23. site of action of furosemide and other sulfona- 6. Gertz, K. H., J. A. Mangos, C. Braun, and H. D. mide diuretics in the dog. J. clin. Invest. 1965, Pagel. On the glomerular tubular balance in the 44, 1458. rat kidney. Pfluigers Arch. ges. Physiol. 1965, 17. Steinhausen, M., A. Loreth, and S. Olson. Mes- 285, 360. sungen des tubularen Harnstromes, seine Bezie- 7. Windhager, E. E., and G. Giebisch. Micropuncture hungen zum Blutdruck und zur Inulin-Clearance. study of renal tubular transfer of sodium chloride Pfluigers Arch. ges. Physiol. 1965, 286, 118. in the rat. Amer. J. Physiol. 1961, 200, 581. 18. Steinhausen, M. Personal communication. 8. Bank, N., and H. S. Aynedjian. A micropuncture 19. Gessler, U., and K. Schroder. Experimental studies study of renal bicarbonate and chloride reabsorp- on the GFR during acute anuria. Presented at tion in hypokalaemic alkalosis. Clin. Sci. 1965, 29, the Third International Congress of Nephrology, 159. Washington, 1966. 9. Swann, R. C., and J. P. Merrill. The clinical course 20. Rodin, A. E., and C. N. Crowson. Mercury nephro- of acute renal failure. Medicine (Baltimore) toxicity in the rat. I. Factors influencing the lo- 1953, 32, 215. calization of the tubular lesions. Amer. J. Path. 10. Meroney, W. H., and M. E. Rubini. Kidney function 1962, 41, 297. during acute tubular necrosis: clinical studies and 21. Rodin, A. E., and C. N. Crowson. Mercury nephro- a theory. Metabolism 1959, 8, 1. toxicity in the rat. II. Investigation of the in- 11. Glabman, S., H. S. Aynedjian, and N. Bank. Mi- tracellular site of mercury nephrotoxicity by cor- cropuncture study of the effect of acute reductions related serial time histologic and histoenzymatic in glomerular filtration rate on sodium and water studies. Amer. J. Path. 1962, 41, 485. reabsorption by the proximal tubules of the rat. 22. Taylor, N. S. Histochemical studies of nephrotox- J. clin. Invest. 1965, 44, 1410. icity with sublethal doses of mercury in rats. 12. Gottschalk, C. W. Renal tubular function: lessons Amer. J. Path. 1965, 46, 1. from micropuncture. Harvey Lect. 1963, series 23. Kleinzeller, A., and J. H. Cort. The mechanism of 58, 99. action of mercurial preparations on transport 13. Rector, F. C., Jr., F. P. Brunner, and D. W. Seldin. processes and the role of thiol groups in the cell Mechanism of glomerulotubular balance. I. Ef- membrane of renal tubuler cells. Biochem. J. 1957, fect of aortic constriction and elevated uretero- 67, 15. pelvic pressure on glomerular filtration rate, frac- 24. Giebisch, G., R. M. Klose, G. Malnic, W. J. Sullivan, tional reabsorption, transit time, and tubular size and E. E. Windhager. Sodium movement across in the proximal tubule of the rat. J. clin. Invest. single perfused proximal tubules of rat kidneys. 1966, 45, 590. J. gen. Physiol. 1964, 47, 1175. 14. Finkenstaedt, J. T., and J. P. Merrill. Renal func- 25. Farah, A. Renal vascular changes produced by the tion after recovery from acute renal failure. mercurial diuretic salyrgan. Naunyn-Schmiede- New Engl. J. 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