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Nephrotoxic action of a synthetic plastic material
TOXICOLOGY
AND
APPLIED
Nephrotoxic
PHARMACOLOGY
Action
14,574-583
(1969)
of a Synthetic M.
Plastic
Material1
OSKOUIZ
Department of Pharmaco logy, University of Louisville, School of Medicine, LouisviNe, Kentucky 40200 Received May 23,1968
Nephrotoxic Action of a Synthetic Plastic Material. OSKOUI, M. (1969). 14,574583. The useof polypropylene in a system for perfusing the dog kidney caused a reduction in glucose reabsorption. This was a local response limited to the denervated kidney exposed to polypropylene. A similar effect was noted in the kidney, which was infused with saline extract of this plastic material. It is suspected that chromium may be partially responsible for the renal effect. Toxicol. Appl. Pharmacol.
In the course of preparing to perfuse the kidney of a dog, we noted an effect of our perfusion system on renal function which hitherto has not been described. The nature of the perfusing tubes was altered from polypropylene to polyethylene and the disturbance in renal function disappeared. This initial observation suggested that polypropylene can be a source of nephrotoxicity. The experiments reported below attempt to identify the nature of, and the mechanisms responsible for, the disturbance in renal function. In 1954, Professor Natta of the Istitut di Chimica Industriale de1 Politecnico, Milan, Italy, synthesized polypropylene by stereospecific polymerization (see monograph by Kesser, 1960). Polypropylene is a colorless and odorless material with a density of 0.904.91. It is resistant to chemical attack, and products prepared from this plastic can be autoclaved without any harmful effects. Presently, polypropylene is used for the manufacture of films, bottles, jars, syringes, tubing, and laboratory ware. This plastic has also been used to manufacture transfusion sets and tubing for intravenous infusion of solutions. Since renal shutdown is one of the serious complications following intravenous infusions, it is reasonable to suspect that the phenomenon encountered in the dog may be the experimental counterpart of the clinical situation, METHODS Generalprocedure. Sixteen female dogs weighing lo-26 kg were used. After induction of anesthesia by intravenous injection of sodium pentobarbital(35 mg/kg), the trachea 1 Based on a thesis submitted to fulfill the requirement of the degree of Doctor of Philosophy in Pharmacology at the University of Louisville. 2 Present address: Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. 574
NEPHROTOXICITY
OF POLYPROPYLENE
575
was intubated, and the dog was placed on her back with legs securely tied to the operating table. The kidneys were exposed by a midline abdominal incision. The ureters were catheterized through an incision close to their connection to the bladder. The dog was then primed by intravenous injection of saline containing glucose (Omg/kg), creatinine (40 mg/kg), and p-aminohippuric acid (5 mg/kg). Plasma levels of these substances were sustained by constant infusion (0.25 ml/kg/min) of glucose, creatinine, and paminohippuric acid (8,l and 3 ‘A per min of the priming dose respectively) by a Beckman metering pump (Model 746) via the brachial vein. These values were 0.90-1.1 mM for creatinine, 0.08-O. 11 mM forp-aminohippuric acid, and 55-84 mM for glucose. Priming and sustaining infusions were administered intravenously to estimate renal plasma flow (RPF) and glomerular filtration rate (GFR) and to produce osmotic diuresis. Urine samples were collected in a loo-ml graduated cylinder for 10 min. Blood samples were collected 4 min after the beginning of urine collection and at 20-min intervals. The value for the lo-min sample of blood was derived by extrapolation of 20-min sample of blood. The blood samples were collected in syringes containing sodium heparin and were centrifuged for 10 min at 1500 rpm. Protein-free plasma filtrate was prepared by the addition of cadmium sulfate solution. Glucose was measured by the method of Hanes (1929), which is based on a method described earlier by Hagedorn and Jensen (1923). p-Aminohippuric acid was determined by the method of Bratton and Marshall (1939); creatinine was determined by the alkaline picrate method (Peters, 1942). Kidney perfusion (dogs nos. l-3). In this initial group of three dogs, the left kidney remained intact but the right kidney was dissected free from its surrounding tissues. The right renal vessels were stripped, and nerve fibers were cut for denervation. After three lo-min periods of clearance measurements, the dog received sodium heparin (10 mg/kg), the renal artery was cannulated and attached to tubing previously inserted into one carotid or femoral artery. Arterial blood supplied the right kidney, and clearances were again measured for three IO-min periods. Finally, an extracorporeal pump system was inserted proximal to the renal artery and 3-5 sample periods were collected. During this experiment, femoral blood pressure was recorded by a Statham transducer. The perfusion apparatus consisted of glass tubing and polypropylene connections. A reciprocating pump (Harvard Model 500-1200) provided a pulsatile pressure. During a perfusion run, perfusion pressure was recorded by a second Statham transducer and renal blood flow by a Shipley-Wilson rotameter. The perfusion pressure and flow could be altered by changing the stroke output of the pump. After the onset of the perfusion via the pump, the perfusion pressure was kept constant at approximately 90-I 10 mm Hg (blood pressure of the dog) and the renal blood flow was about 70-90 ml/min. Infusion of extract ofpolypropylene tubing (dogs nos. 4-6). In these threedogs, both kidneys remained essentially intact. The left renal artery was exposed gently while avoiding disruption of nerve fibers. After preliminary priming and collection for three lo-min periods, a bent 27-gauge needle was inserted through the aorta and into the ostium of the left renal artery for a distance of 0.5 cm. Saline infusion was started and continued for three periods, and then the saline extract of polypropylene tubing was infused at the rate of 0.9 and 2.1 ml/min into the left renal artery for five to seven clearance periods. The infusion apparatus is depicted in Fig. 1. The infusion medium was prepared by
576
OSKOUI
FIG. 1. Scheme for infusion of solution into left renal artery. 1, infusion reservoir containing extract or chemical; 2, infusion reservoir containing saline only; 3, oxygen tank; 4, mercury manometer; 5, observing tube; 6, capillary resistance; and 7, needle.
adding 500 ml of saline to 18.5 g of small pieces of polypropylene connectors. After 24 hours of incubation the saline was collected and infused with 40 ml of blood plasma added to 90 ml of saline. Infusion of cupric sulfate sohtion (dogs Nos. 7-10). The dogs were prepared as in the group receiving polypropylene extract. The present group received saline infusion
NEPHROTOXICITY
OF POLYPROPYLENE
577
as control for three periods and then an infusion of cupric sulfate solution (3.6,18, and 30 pg/ml) at the rate of 1 ml/min. Infusion of sodium dichromate solution (dogs Nos. 11-16). The last six dogs received saline infusion as control and then an infusion of sodium dichromate solution: Nos. 11-15 received 13.8 or 27.6 pg/ml of saline at the rate of 2 ml/min directly into the left renal artery. The last dog (No. 16) received an intravenous infusion of 200 pg/min. Some of the dogs had the following additional measurements: dog No. 15 had measurements of Na+ and Kf excretion by the use of a flame photometer (Eppendorff). This same dog also had measurements of T,,, p-aminohippurate instead of its clearance. The priming dose of p-aminohippuric acid was 0.3 mM/kg, the rate of the infusion of p-aminohippurate was 2 % per minute, and the plasma level of p-aminohippurate was 0.8 mM to 1.1 mM/ml. Dog No. 14 had p-aminohippurate omitted from its clearance tests. Creatine was also given in a priming dose of 40 mg/kg, and 1% of this was infused per minute. Creatine plus creatinine was measured in the urine and a tungstic acid filtrate of plasma by acidifying the samples with ~/4 hydrochloric acid, autoclaving at 15 lb for 30 min, neutralizing with NaOH, and adding alkaline picrate for determination of creatinine. To permit calculation of tubular reabsorption of creatine, its proteinbinding was studied. To 4 ml of normal dog blood plasma, 15.26 PM creatine in 0.2 ml was added and ultrafiltration was performed with the apparatus described by Toribara et al. (1957). Analysis indicated that the creatine was completely free to filter. In this experiment, the plasma level of creatine was 0.54-0.46 PM/ml.
RESULTS
In Situ Perfusion of the Right Kidney The first group of experiments consisted of comparing the behavior of the intact left kidney with that of the experimental right kidney that was exposed to the plastic tubing. During three phases of experimentation, the left kidney remained intact, but the right kidney underwent the following procedures : denervation, cannulation and autoperfusion with blood from the common carotid, and perfusion by pump. Denervation. The first three dogs were subjected to denervation of the right kidney. The results of dog No. 1 are summarized in Table 1. During denervation, the measurements of urine volume, creatinine clearance, p-aminohippurate (PAH) clearance, and T, glucose were about equal for both kidneys. The results for dog No. 2 were about the same, but in the third dog, the denervated kidney had higher values than the intact kidney. The elevated values for renal function in the denervated kidney of the third dog are unexplained. It was, however, important to denervate the right kidney at the onset of perfusion so that the consequences of subsequent insertion of plastic tubing could be identified as a local phenomenon in the kidney. Insertion of polypropylene tubing. The next period of observation followed the cannulation of the right renal artery and supplying it with blood from the left common carotid. In all three dogs, there was a reduction in T,,, glucose for the right kidney. The urine volume, creatinine clearance, and PAH clearance for the right kidney also were reduced. For the left kidney, only creatinine clearance and T,,, glucose were reduced but the intensity of the fall was less than that for the right.
Mean %A
105-l 15 115-125 125-135 135-145 145-155
1.43 -62.4
Mean %A
Right perfused with pump, left intact
1.3 1.5 1.5
60-70 70-80 80-90
4.65 4.30 4.60 4.70 4.60 4.57 +23.5
1.90 1.60 1.50 1.50 1.55
1.61 -57.6
4.9 +32.4
4.80 4.95 4.95
5.24 -76.1
5.8 5.3 5.3 5.0 4.8
5.7 -74.2
5.9 5.8 5.3
21.96
3.70
3.8
Right 22.9 22.6 20.4
Right perfused without pump, left intact
1 INTACT
AND
16.76 -35.2
18.6 16.8 16.6 16.4 15.4
21.13 -18.3
22 21 20.4
25.86
27.4 25.9 24.3
Left
Creatinine clearance (ml/min)
KIDNEY
3.35 3.75 3.90
Left
LEFT
3.60 3.80 4.00
O-10 lo-20 20-30 ~Mean
left
Right
Right denervated, intact
Time (min)
WITH
(ml/min)
DOG
Urine volume
IN THE ANFSTHETIZED
procedure
FUNCTION
Experimental
RENAL
TABLE KIDNEY
26.56 -65.4
26.0 29.2 25.7 25.0 26.9
26.83 -65
29.0 26.8 24.7
76.6
80.8 75.3 73.8
Right
PAH clearance
RIGHT
75.1 -11.2
76.1 70.7 80.6 77.0 71.1
87.76 +3.8
98.1 86.0 79.2
84.6
73.3 78.5 102.0
Left
(ml/min>
PERFUSED
No.
1)
6.2
-95
2.6
4.9 +0.36 8.0 2.0 t1.6
-89
15.0 5.4 +1.8
55.4
56.52 65.70 43.74
Right
-T,,, Glucose
(DOG
24.5 -52
24.7 28.4 28.1 24.0 17.1
32.7 -36
43.7 29.7 24.7
51.18
75.42 28.26 49.86
Left
(mg/min)
NEPHROTOXICITY
OF POLYPROPYLENE
579
Perfusion of the right denervated kidney. The final phase of observation consisted of the use of a perfusion pump to maintain renal blood flow. For half an hour, the two kidneys were compared. The most important difference seen in all three dogs was a reduction in reabsorption of glucose in the perfused kidney compared to the intact kidney. The creatinine clearance, PAH clearance, and urine volume of the denervated kidney were also reduced, compared to the intact kidney. Extract of Polypropylene and Spectrographic Analysis
The next step was to examine the saline-extract prepared from 18.5 g of polypropylene tubing. A total volume of 500 ml was used for extraction, and 90 ml was added to 40 ml of blood plasma for infusion into the left renal artery. The results of one dog (No. 4) typical of the two others are summarized in Table 2. The most important effect of infusion of saline extract was a fall in glucose reabsorption, and there were no significant changes in urine volume, creatinine clearance, and PAH clearance. The ability of the saline extract to reduce glucose absorption of one kidney raised the next question as to the possible constituent responsible for the change. The tubing was analyzed by emission spectrography. The analysis revealed the following metals in decreasing order of concentration in parts per million: magnesium = 0.8-1.6; aluminum and silicon = 0.4-2.4; boron, titanium, and chromium = 0.2-2.4; copper = 0.6-1.2; cadmuim = 0.5-1.0; and iron = 0.2-0.8. Magnesium, iron, and boron are contained in the graphite which was added to permit spectrography of the plastic. Copper and chromium were selected for additional investigation. Solutions of Suspected Metals Cupric suZfate.The next four dogs (Nos. 7-10) were used to examine the effects of infusion into the left renal artery of cupric sulfate solution in the following concentrations : 3, 6,18, and 30 pg/ml saline. In three dogs there was no important effect on renal function after infusion of 3-36 pg/min for 1 hour. However, in the last dog (No. lo), 60 pg/min was infused and there was a decrease in clearances of creatinine, PAH, and glucose for both kidneys (Table 2). Since these changes appeared not only in the side infused with cupric sulfate, but also in the other side a generalized circulatory effect of cupric ion can be proposed to explain these renal effects. Sodium dichromate. The last group of six dogs were devoted to the investigation of the effect of sodium dichromate on renal function. The following solutions were infused into the renal artery: 13.8 and 27.6 pg/ml saline. The lowest dose of 13.8 pg/min did not influence urine formation, creatinine clearance, PAH clearance, or T, glucose. The infusion of 27.6 ug/min caused a fall in glucose reabsorption selectively in one dog (No. 12, Table 2). The dose of 55.2 pg/min was administered to the next three dogs, and all three showed a fall in glucose reabsorption. The additional measurement of T,,z creatine in one dog (No. 14) and of T,,, PAH in another dog (No. 15) revealed no influence of sodium dichromate on these aspects of renal function. In the last dog (No. 16) sodium dichromate was infused intravenously at a concentration of 50 and 100 pg/min saline. After the infusion of a dose of 400 pg/min, there was a fall in glucose reabsorption of both kidneys measured together but no change in urine volume. creatinine clearance, and PAH clearance.
No. 12, sodium dichromate solution
No. 10, cupric sulfate solution
No. 4, saline extract of polypropylene
Dog No., type of infusion
RENAL
IN THREE
clearances)
Of
Mean (no.
ANESTHETIZED
Both sides Mean (3) Left side infused Mean (5) %A Left side infused Mean (2) %A Mean (3) Both sides Left side infused Mean (3) %A Left side infused Mean (4) %A Both sides Mean (3) Left side infused Mean (5) %A Left side infused Mean (5) %A
Experimental Procedure
FUNCTION
RIGHT
3.37 3.6 -t6.8 3.4 +0.9
3.26 1.92 -41.1 1.44 -56
1.83 2.08 +13.7 1.92 +4.9
Right
INTACT
AND
17.74 16.94 -4.5 16.8 -5.3
Right
21.6 3.50 5.1 19.5 +45.7 -9.7 17.8 4.4 +25.7 -17.6
KIDNEY
23.6 22.2 -6 18.8 -20.3
23.8 16.6 -30.3 13 -45.4
INFUSED
Right
88.9 89.0 +O.l 73.9 -16.9
83.8 45.7 -45.5 30.5 -63.6
96.2 87.1 -9.5 70.8 -26.4
74.0 42.5 -42.6 29.1 -60.7
84.1 82.5 -2 67.5 -19.8
Left
PAH clearance (ml/min)
LEFT
19.25 77.4 18.97 71.6 -7.8 -1.5 18 64.1 -6.5 -17.4
Left
Creatinine clearance (ml/min)
KIDNEY
25.9 3.05 2.26 17.3 -33.2 -26 1.90 13 -37.3 -49.8
2.24 2.8 +25 2.7 $20.5
Left
Urine volume (ml/min)
DOGSWITH
TABLE 2
66.6 66.9 +0.5 48.2 -27.6
97.7 78.4 -19.8 78.6 -19.5
58.30 54.50 -7.0 59.4 +1.8
67.9 51.2 -24.6 39.6 -41.7
96.6 91.0 -5.8 76.8 -20.5
60.90 27.30 -55.2 41.2 -32.3
hcdmin) __Right Left
-T,,,, Glucose
58 47 46
38 109 128 98
4 (18.0) 5 (12.5) 6 (14.8)
(26.5) (19.5) (10.9) (18.72)
(16.82) (18.4) (16.8) (18.64) (14.55) (14.8)
7 8 9 10
11 12 13 14 15 16
+32 -7 -20
-7 f32 +90
-36 -54 +24.0
Period 1 %A
-28 -28 -12 -31
+34 -45 -20
-2.0 +63 +111
-52 -0.3 - 55
Period 2 %A
OF VARIOUS
81 68 73 98 45 224
84 77 57 97
61 57 61
55.4 45 57
Control (msbin)
Sodium Sodium Sodium Sodium Sodium Sodium
Cupric Cupric Cupric Cupric dichromate dichromate dichromate dichromate dichromate dichromate
sulfate sulfate sulfate sulfate
Plastic ext. Plastic ext. Plastic ext.
Perfusion Perfusion Perfusion
Period 1 procedure
PROCEDURES ON ---T,,, GLUCOSE
3
+27 -25 -40 -ml0 -53 -24
-11 +54 +24 -6
-55 f25 f13
side*
%A -89 -11 -39.0
Treated Period 2 procedure
following
the experi-
-8 -42 -65 ~32 43 -21
Sodium Sodium Sodium Sodium Sodium Sodium
dichromate dichromate dichromate dichromate dichromate dichromate
-33 i-65 -t8 20
-32 t17 +58
-95 -64 -89
O’A l-3
Cupric sulfate Cupric sulfate Cupric sulfate Cupric sulfate
Plastic ext. Plastic ext. Plastic ext.
Pump Pump Pump
IN ANESTHETIZED DOGS
a Figures for each dog represent mean values for three control lo-min measurements. * Figures for each dog represent mean value for three to seven IO-min measurement. The first period represents the time immediately mental procedure; the second period covers about 0.5-l hour later after initiation of the experimental procedure.
67 -It1 72 f5 111 P~5 72 -39 Both sides measured together
51.2 40.4 44
Control bg/min>
l(12.6) 2 (13.2) 3 (12.5)
Dog No. and weight (kg)
OF EFFECTS
Intact side”
SUMMARY
TABLE
z
3 s 2 52 s 2 2 z $ ; 8 ;s
z
582
OSKOUI
DISCUSSION
The most important feature of our results is the effect of various procedures on glucose reabsorption (T, glucose) in each of the 16 dogs. The results summarized in Table 3 include the behavior of the kidney exposed to experimental treatment, contrasted with the intact kidney. The results show a definite fall in glucose reabsorption for the perfused kidney, kidney infused with saline extract of polypropylene, and the kidney infused with concentrated sodium dichromate solution. In all instances, the intact kidney either showed an elevation in T,,, glucose or a fall which is less intense than the fall seen in the treated side. The kidneys exposed to cupric sulfate did not show a consistent fall in T, glucose. The results for the dogs receiving sodium dichromate suggest that chromium which may be present in polypropylene tubing can explain the renal effects of perfusing the kidney using this material. The effect is limited to inhibition of reabsorption of glucose by the renal tubules, whereas the mechanisms relating to electrolyte, creatinine, and PAH excretion are not altered. The phenomenon that we have noted in the dog receiving sodium dichromate is similar to the glycosuria noted by others in the following situations (a) The glycosuria produced in a variety of experimental animals following administration of chromic acid was first reported by Kossa in 1902. Subsequently, potassium chromate (Frank, 1913; Luzzato, 1914; Mosinger et al., 1954) and potassium dichromate (MacNider, 1912; Schou, 1944; Helpler and Simonds, 1946) were reported to produce nephritis and glycosuria in animals. (b) Human cases of poisoning with chromates have been reported by Lohr (1904) and Major (1922). Both reports state the appearance of glycosuria unrelated to hyperglycemia. None of these reports in man or animals has included a systematic analysis of renal function. Our experiments reported above describe for the first time a predominant interference in absorption of glucose by the renal tubules following the intravenous administration of high doses of chromium salt. However, our perfusion system contained four polypropylene connectors and tubing totaling 12.5 g. The estimated total amount by emission spectrography was 0.10 mg of sodium dichromate. The minimal rate of infusion of sodium dichromate into the renal artery which reduced glucose reabsorption was 27.6 pg/min. Thus it seems unlikely that the chromium content alone of the plastic was responsible for the observed effect. ACKNOWLEDGMENT
The author is grateful to Drs. Peter K. Knoefel and K. C. Huang for fruitful discussionsand valuable suggestions during this investigation. The author also wishes to expressindebtedness to the generous grant of the University of Louisville during the conduct of this work. REFERENCES BRATTON, A. C., and MARSHALL, E. K. (1939). A new coupling component for sulfanilamide determination. J. Biol. Chem. 128, 537-550. FRANK, E. (1913). Uber experimentelle und der klinische Glykosurien renalen Ursprungs. I. Einleitung: abnorme Zuckerdichtigkeit der Niere; Phlorizindiabetes. Arch. Exptl. Puthol. Pharmacol. 72, 387-443. HAGEDORN, H. C., and JENSEN, B. N. (1923). Zur Mikrobestimmung des Blutzuckers Mittels Ferricyanid. Biochem. Z. 135,46-58.
NEPHROTOXICITYOF POLYPROPYLENE
583
HANES,C. S. (1929). XIV. An application of the method Hagedorn and Jensen to the determination of larger quantities of reducing sugars. &&em. J. 23, 99-106. HELPLER,0. E., and SIMONDS,J. P. (1946). Experimental nephropathies. IV. Glycosuria in dogs poisoned with many1 nitrate, mercury bichloride, and potassium dichromate. A/.clr. Pathol. 41, 42-49.
KESSER, T. 0. J. (1960). Polypropylene. Reinhold, New York. KOSSA,J. ( 1902). Ueber Chromslure-Diabetes. Arch. Ges. Ph,,siol. 88, 627-637. LOHR, A. ( 1904). Ueber einen Fall acuter Chromvergiftung mit spontaner Glykosurie, geheilt durch die von R. v. Jaksch empfohlene Magenaussptilung mit salpetersaurem Silber. Berl. Klin. Wochschr. 41, 749-758.
Luzzaro, R. (1914). Die Glykosurie bei experimentellen Nephritiden. Z. Exptl. Pathol. 16, 1S-67. MACNIDER, W. D. B. (1912). A study of the renal epithelium in various types of acute experiment nephritis and of the relation which exists between the epithelial changes and the total output of urine. J. Med. Res. 26,79-126. MAJOR,R. H. (1922). Studies on a case of chromic acid nephritis. Bull. Johns Hopkins Hasp. 33, 56-67.
MOSINGER,M., FIORENTINE,H., and QUICKE,J. (1954). Diabete experimental par intoxication au chromate neutre de potassium. Compt. Rend. Acad. Sci. 148, 1453-1455. PETERS, J. H. (1942). The determination of creatinine and creatine in blood and urine with the photometric calorimeter. J. Biol. Chem. 146, 179-186. SCHOU,P. (1944). Experimental studies on kidney function during sulphate diuresis. 4. Investigations on kidney function in rabbits with chronic tubular nephritis A. M. Frandsen. Acta Physiol. Stand. 7, 200-208.
TORIBARA,T. Y., TEREPKA,A. R., and DEWEY,P. A. (1957). The ultrafiltrable calcium of human serum. I. Ultrafiltration methods and normal values. J. Clin. Invest. 36. 738-748.
AND
APPLIED
Nephrotoxic
PHARMACOLOGY
Action
14,574-583
(1969)
of a Synthetic M.
Plastic
Material1
OSKOUIZ
Department of Pharmaco logy, University of Louisville, School of Medicine, LouisviNe, Kentucky 40200 Received May 23,1968
Nephrotoxic Action of a Synthetic Plastic Material. OSKOUI, M. (1969). 14,574583. The useof polypropylene in a system for perfusing the dog kidney caused a reduction in glucose reabsorption. This was a local response limited to the denervated kidney exposed to polypropylene. A similar effect was noted in the kidney, which was infused with saline extract of this plastic material. It is suspected that chromium may be partially responsible for the renal effect. Toxicol. Appl. Pharmacol.
In the course of preparing to perfuse the kidney of a dog, we noted an effect of our perfusion system on renal function which hitherto has not been described. The nature of the perfusing tubes was altered from polypropylene to polyethylene and the disturbance in renal function disappeared. This initial observation suggested that polypropylene can be a source of nephrotoxicity. The experiments reported below attempt to identify the nature of, and the mechanisms responsible for, the disturbance in renal function. In 1954, Professor Natta of the Istitut di Chimica Industriale de1 Politecnico, Milan, Italy, synthesized polypropylene by stereospecific polymerization (see monograph by Kesser, 1960). Polypropylene is a colorless and odorless material with a density of 0.904.91. It is resistant to chemical attack, and products prepared from this plastic can be autoclaved without any harmful effects. Presently, polypropylene is used for the manufacture of films, bottles, jars, syringes, tubing, and laboratory ware. This plastic has also been used to manufacture transfusion sets and tubing for intravenous infusion of solutions. Since renal shutdown is one of the serious complications following intravenous infusions, it is reasonable to suspect that the phenomenon encountered in the dog may be the experimental counterpart of the clinical situation, METHODS Generalprocedure. Sixteen female dogs weighing lo-26 kg were used. After induction of anesthesia by intravenous injection of sodium pentobarbital(35 mg/kg), the trachea 1 Based on a thesis submitted to fulfill the requirement of the degree of Doctor of Philosophy in Pharmacology at the University of Louisville. 2 Present address: Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. 574
NEPHROTOXICITY
OF POLYPROPYLENE
575
was intubated, and the dog was placed on her back with legs securely tied to the operating table. The kidneys were exposed by a midline abdominal incision. The ureters were catheterized through an incision close to their connection to the bladder. The dog was then primed by intravenous injection of saline containing glucose (Omg/kg), creatinine (40 mg/kg), and p-aminohippuric acid (5 mg/kg). Plasma levels of these substances were sustained by constant infusion (0.25 ml/kg/min) of glucose, creatinine, and paminohippuric acid (8,l and 3 ‘A per min of the priming dose respectively) by a Beckman metering pump (Model 746) via the brachial vein. These values were 0.90-1.1 mM for creatinine, 0.08-O. 11 mM forp-aminohippuric acid, and 55-84 mM for glucose. Priming and sustaining infusions were administered intravenously to estimate renal plasma flow (RPF) and glomerular filtration rate (GFR) and to produce osmotic diuresis. Urine samples were collected in a loo-ml graduated cylinder for 10 min. Blood samples were collected 4 min after the beginning of urine collection and at 20-min intervals. The value for the lo-min sample of blood was derived by extrapolation of 20-min sample of blood. The blood samples were collected in syringes containing sodium heparin and were centrifuged for 10 min at 1500 rpm. Protein-free plasma filtrate was prepared by the addition of cadmium sulfate solution. Glucose was measured by the method of Hanes (1929), which is based on a method described earlier by Hagedorn and Jensen (1923). p-Aminohippuric acid was determined by the method of Bratton and Marshall (1939); creatinine was determined by the alkaline picrate method (Peters, 1942). Kidney perfusion (dogs nos. l-3). In this initial group of three dogs, the left kidney remained intact but the right kidney was dissected free from its surrounding tissues. The right renal vessels were stripped, and nerve fibers were cut for denervation. After three lo-min periods of clearance measurements, the dog received sodium heparin (10 mg/kg), the renal artery was cannulated and attached to tubing previously inserted into one carotid or femoral artery. Arterial blood supplied the right kidney, and clearances were again measured for three IO-min periods. Finally, an extracorporeal pump system was inserted proximal to the renal artery and 3-5 sample periods were collected. During this experiment, femoral blood pressure was recorded by a Statham transducer. The perfusion apparatus consisted of glass tubing and polypropylene connections. A reciprocating pump (Harvard Model 500-1200) provided a pulsatile pressure. During a perfusion run, perfusion pressure was recorded by a second Statham transducer and renal blood flow by a Shipley-Wilson rotameter. The perfusion pressure and flow could be altered by changing the stroke output of the pump. After the onset of the perfusion via the pump, the perfusion pressure was kept constant at approximately 90-I 10 mm Hg (blood pressure of the dog) and the renal blood flow was about 70-90 ml/min. Infusion of extract ofpolypropylene tubing (dogs nos. 4-6). In these threedogs, both kidneys remained essentially intact. The left renal artery was exposed gently while avoiding disruption of nerve fibers. After preliminary priming and collection for three lo-min periods, a bent 27-gauge needle was inserted through the aorta and into the ostium of the left renal artery for a distance of 0.5 cm. Saline infusion was started and continued for three periods, and then the saline extract of polypropylene tubing was infused at the rate of 0.9 and 2.1 ml/min into the left renal artery for five to seven clearance periods. The infusion apparatus is depicted in Fig. 1. The infusion medium was prepared by
576
OSKOUI
FIG. 1. Scheme for infusion of solution into left renal artery. 1, infusion reservoir containing extract or chemical; 2, infusion reservoir containing saline only; 3, oxygen tank; 4, mercury manometer; 5, observing tube; 6, capillary resistance; and 7, needle.
adding 500 ml of saline to 18.5 g of small pieces of polypropylene connectors. After 24 hours of incubation the saline was collected and infused with 40 ml of blood plasma added to 90 ml of saline. Infusion of cupric sulfate sohtion (dogs Nos. 7-10). The dogs were prepared as in the group receiving polypropylene extract. The present group received saline infusion
NEPHROTOXICITY
OF POLYPROPYLENE
577
as control for three periods and then an infusion of cupric sulfate solution (3.6,18, and 30 pg/ml) at the rate of 1 ml/min. Infusion of sodium dichromate solution (dogs Nos. 11-16). The last six dogs received saline infusion as control and then an infusion of sodium dichromate solution: Nos. 11-15 received 13.8 or 27.6 pg/ml of saline at the rate of 2 ml/min directly into the left renal artery. The last dog (No. 16) received an intravenous infusion of 200 pg/min. Some of the dogs had the following additional measurements: dog No. 15 had measurements of Na+ and Kf excretion by the use of a flame photometer (Eppendorff). This same dog also had measurements of T,,, p-aminohippurate instead of its clearance. The priming dose of p-aminohippuric acid was 0.3 mM/kg, the rate of the infusion of p-aminohippurate was 2 % per minute, and the plasma level of p-aminohippurate was 0.8 mM to 1.1 mM/ml. Dog No. 14 had p-aminohippurate omitted from its clearance tests. Creatine was also given in a priming dose of 40 mg/kg, and 1% of this was infused per minute. Creatine plus creatinine was measured in the urine and a tungstic acid filtrate of plasma by acidifying the samples with ~/4 hydrochloric acid, autoclaving at 15 lb for 30 min, neutralizing with NaOH, and adding alkaline picrate for determination of creatinine. To permit calculation of tubular reabsorption of creatine, its proteinbinding was studied. To 4 ml of normal dog blood plasma, 15.26 PM creatine in 0.2 ml was added and ultrafiltration was performed with the apparatus described by Toribara et al. (1957). Analysis indicated that the creatine was completely free to filter. In this experiment, the plasma level of creatine was 0.54-0.46 PM/ml.
RESULTS
In Situ Perfusion of the Right Kidney The first group of experiments consisted of comparing the behavior of the intact left kidney with that of the experimental right kidney that was exposed to the plastic tubing. During three phases of experimentation, the left kidney remained intact, but the right kidney underwent the following procedures : denervation, cannulation and autoperfusion with blood from the common carotid, and perfusion by pump. Denervation. The first three dogs were subjected to denervation of the right kidney. The results of dog No. 1 are summarized in Table 1. During denervation, the measurements of urine volume, creatinine clearance, p-aminohippurate (PAH) clearance, and T, glucose were about equal for both kidneys. The results for dog No. 2 were about the same, but in the third dog, the denervated kidney had higher values than the intact kidney. The elevated values for renal function in the denervated kidney of the third dog are unexplained. It was, however, important to denervate the right kidney at the onset of perfusion so that the consequences of subsequent insertion of plastic tubing could be identified as a local phenomenon in the kidney. Insertion of polypropylene tubing. The next period of observation followed the cannulation of the right renal artery and supplying it with blood from the left common carotid. In all three dogs, there was a reduction in T,,, glucose for the right kidney. The urine volume, creatinine clearance, and PAH clearance for the right kidney also were reduced. For the left kidney, only creatinine clearance and T,,, glucose were reduced but the intensity of the fall was less than that for the right.
Mean %A
105-l 15 115-125 125-135 135-145 145-155
1.43 -62.4
Mean %A
Right perfused with pump, left intact
1.3 1.5 1.5
60-70 70-80 80-90
4.65 4.30 4.60 4.70 4.60 4.57 +23.5
1.90 1.60 1.50 1.50 1.55
1.61 -57.6
4.9 +32.4
4.80 4.95 4.95
5.24 -76.1
5.8 5.3 5.3 5.0 4.8
5.7 -74.2
5.9 5.8 5.3
21.96
3.70
3.8
Right 22.9 22.6 20.4
Right perfused without pump, left intact
1 INTACT
AND
16.76 -35.2
18.6 16.8 16.6 16.4 15.4
21.13 -18.3
22 21 20.4
25.86
27.4 25.9 24.3
Left
Creatinine clearance (ml/min)
KIDNEY
3.35 3.75 3.90
Left
LEFT
3.60 3.80 4.00
O-10 lo-20 20-30 ~Mean
left
Right
Right denervated, intact
Time (min)
WITH
(ml/min)
DOG
Urine volume
IN THE ANFSTHETIZED
procedure
FUNCTION
Experimental
RENAL
TABLE KIDNEY
26.56 -65.4
26.0 29.2 25.7 25.0 26.9
26.83 -65
29.0 26.8 24.7
76.6
80.8 75.3 73.8
Right
PAH clearance
RIGHT
75.1 -11.2
76.1 70.7 80.6 77.0 71.1
87.76 +3.8
98.1 86.0 79.2
84.6
73.3 78.5 102.0
Left
(ml/min>
PERFUSED
No.
1)
6.2
-95
2.6
4.9 +0.36 8.0 2.0 t1.6
-89
15.0 5.4 +1.8
55.4
56.52 65.70 43.74
Right
-T,,, Glucose
(DOG
24.5 -52
24.7 28.4 28.1 24.0 17.1
32.7 -36
43.7 29.7 24.7
51.18
75.42 28.26 49.86
Left
(mg/min)
NEPHROTOXICITY
OF POLYPROPYLENE
579
Perfusion of the right denervated kidney. The final phase of observation consisted of the use of a perfusion pump to maintain renal blood flow. For half an hour, the two kidneys were compared. The most important difference seen in all three dogs was a reduction in reabsorption of glucose in the perfused kidney compared to the intact kidney. The creatinine clearance, PAH clearance, and urine volume of the denervated kidney were also reduced, compared to the intact kidney. Extract of Polypropylene and Spectrographic Analysis
The next step was to examine the saline-extract prepared from 18.5 g of polypropylene tubing. A total volume of 500 ml was used for extraction, and 90 ml was added to 40 ml of blood plasma for infusion into the left renal artery. The results of one dog (No. 4) typical of the two others are summarized in Table 2. The most important effect of infusion of saline extract was a fall in glucose reabsorption, and there were no significant changes in urine volume, creatinine clearance, and PAH clearance. The ability of the saline extract to reduce glucose absorption of one kidney raised the next question as to the possible constituent responsible for the change. The tubing was analyzed by emission spectrography. The analysis revealed the following metals in decreasing order of concentration in parts per million: magnesium = 0.8-1.6; aluminum and silicon = 0.4-2.4; boron, titanium, and chromium = 0.2-2.4; copper = 0.6-1.2; cadmuim = 0.5-1.0; and iron = 0.2-0.8. Magnesium, iron, and boron are contained in the graphite which was added to permit spectrography of the plastic. Copper and chromium were selected for additional investigation. Solutions of Suspected Metals Cupric suZfate.The next four dogs (Nos. 7-10) were used to examine the effects of infusion into the left renal artery of cupric sulfate solution in the following concentrations : 3, 6,18, and 30 pg/ml saline. In three dogs there was no important effect on renal function after infusion of 3-36 pg/min for 1 hour. However, in the last dog (No. lo), 60 pg/min was infused and there was a decrease in clearances of creatinine, PAH, and glucose for both kidneys (Table 2). Since these changes appeared not only in the side infused with cupric sulfate, but also in the other side a generalized circulatory effect of cupric ion can be proposed to explain these renal effects. Sodium dichromate. The last group of six dogs were devoted to the investigation of the effect of sodium dichromate on renal function. The following solutions were infused into the renal artery: 13.8 and 27.6 pg/ml saline. The lowest dose of 13.8 pg/min did not influence urine formation, creatinine clearance, PAH clearance, or T, glucose. The infusion of 27.6 ug/min caused a fall in glucose reabsorption selectively in one dog (No. 12, Table 2). The dose of 55.2 pg/min was administered to the next three dogs, and all three showed a fall in glucose reabsorption. The additional measurement of T,,z creatine in one dog (No. 14) and of T,,, PAH in another dog (No. 15) revealed no influence of sodium dichromate on these aspects of renal function. In the last dog (No. 16) sodium dichromate was infused intravenously at a concentration of 50 and 100 pg/min saline. After the infusion of a dose of 400 pg/min, there was a fall in glucose reabsorption of both kidneys measured together but no change in urine volume. creatinine clearance, and PAH clearance.
No. 12, sodium dichromate solution
No. 10, cupric sulfate solution
No. 4, saline extract of polypropylene
Dog No., type of infusion
RENAL
IN THREE
clearances)
Of
Mean (no.
ANESTHETIZED
Both sides Mean (3) Left side infused Mean (5) %A Left side infused Mean (2) %A Mean (3) Both sides Left side infused Mean (3) %A Left side infused Mean (4) %A Both sides Mean (3) Left side infused Mean (5) %A Left side infused Mean (5) %A
Experimental Procedure
FUNCTION
RIGHT
3.37 3.6 -t6.8 3.4 +0.9
3.26 1.92 -41.1 1.44 -56
1.83 2.08 +13.7 1.92 +4.9
Right
INTACT
AND
17.74 16.94 -4.5 16.8 -5.3
Right
21.6 3.50 5.1 19.5 +45.7 -9.7 17.8 4.4 +25.7 -17.6
KIDNEY
23.6 22.2 -6 18.8 -20.3
23.8 16.6 -30.3 13 -45.4
INFUSED
Right
88.9 89.0 +O.l 73.9 -16.9
83.8 45.7 -45.5 30.5 -63.6
96.2 87.1 -9.5 70.8 -26.4
74.0 42.5 -42.6 29.1 -60.7
84.1 82.5 -2 67.5 -19.8
Left
PAH clearance (ml/min)
LEFT
19.25 77.4 18.97 71.6 -7.8 -1.5 18 64.1 -6.5 -17.4
Left
Creatinine clearance (ml/min)
KIDNEY
25.9 3.05 2.26 17.3 -33.2 -26 1.90 13 -37.3 -49.8
2.24 2.8 +25 2.7 $20.5
Left
Urine volume (ml/min)
DOGSWITH
TABLE 2
66.6 66.9 +0.5 48.2 -27.6
97.7 78.4 -19.8 78.6 -19.5
58.30 54.50 -7.0 59.4 +1.8
67.9 51.2 -24.6 39.6 -41.7
96.6 91.0 -5.8 76.8 -20.5
60.90 27.30 -55.2 41.2 -32.3
hcdmin) __Right Left
-T,,,, Glucose
58 47 46
38 109 128 98
4 (18.0) 5 (12.5) 6 (14.8)
(26.5) (19.5) (10.9) (18.72)
(16.82) (18.4) (16.8) (18.64) (14.55) (14.8)
7 8 9 10
11 12 13 14 15 16
+32 -7 -20
-7 f32 +90
-36 -54 +24.0
Period 1 %A
-28 -28 -12 -31
+34 -45 -20
-2.0 +63 +111
-52 -0.3 - 55
Period 2 %A
OF VARIOUS
81 68 73 98 45 224
84 77 57 97
61 57 61
55.4 45 57
Control (msbin)
Sodium Sodium Sodium Sodium Sodium Sodium
Cupric Cupric Cupric Cupric dichromate dichromate dichromate dichromate dichromate dichromate
sulfate sulfate sulfate sulfate
Plastic ext. Plastic ext. Plastic ext.
Perfusion Perfusion Perfusion
Period 1 procedure
PROCEDURES ON ---T,,, GLUCOSE
3
+27 -25 -40 -ml0 -53 -24
-11 +54 +24 -6
-55 f25 f13
side*
%A -89 -11 -39.0
Treated Period 2 procedure
following
the experi-
-8 -42 -65 ~32 43 -21
Sodium Sodium Sodium Sodium Sodium Sodium
dichromate dichromate dichromate dichromate dichromate dichromate
-33 i-65 -t8 20
-32 t17 +58
-95 -64 -89
O’A l-3
Cupric sulfate Cupric sulfate Cupric sulfate Cupric sulfate
Plastic ext. Plastic ext. Plastic ext.
Pump Pump Pump
IN ANESTHETIZED DOGS
a Figures for each dog represent mean values for three control lo-min measurements. * Figures for each dog represent mean value for three to seven IO-min measurement. The first period represents the time immediately mental procedure; the second period covers about 0.5-l hour later after initiation of the experimental procedure.
67 -It1 72 f5 111 P~5 72 -39 Both sides measured together
51.2 40.4 44
Control bg/min>
l(12.6) 2 (13.2) 3 (12.5)
Dog No. and weight (kg)
OF EFFECTS
Intact side”
SUMMARY
TABLE
z
3 s 2 52 s 2 2 z $ ; 8 ;s
z
582
OSKOUI
DISCUSSION
The most important feature of our results is the effect of various procedures on glucose reabsorption (T, glucose) in each of the 16 dogs. The results summarized in Table 3 include the behavior of the kidney exposed to experimental treatment, contrasted with the intact kidney. The results show a definite fall in glucose reabsorption for the perfused kidney, kidney infused with saline extract of polypropylene, and the kidney infused with concentrated sodium dichromate solution. In all instances, the intact kidney either showed an elevation in T,,, glucose or a fall which is less intense than the fall seen in the treated side. The kidneys exposed to cupric sulfate did not show a consistent fall in T, glucose. The results for the dogs receiving sodium dichromate suggest that chromium which may be present in polypropylene tubing can explain the renal effects of perfusing the kidney using this material. The effect is limited to inhibition of reabsorption of glucose by the renal tubules, whereas the mechanisms relating to electrolyte, creatinine, and PAH excretion are not altered. The phenomenon that we have noted in the dog receiving sodium dichromate is similar to the glycosuria noted by others in the following situations (a) The glycosuria produced in a variety of experimental animals following administration of chromic acid was first reported by Kossa in 1902. Subsequently, potassium chromate (Frank, 1913; Luzzato, 1914; Mosinger et al., 1954) and potassium dichromate (MacNider, 1912; Schou, 1944; Helpler and Simonds, 1946) were reported to produce nephritis and glycosuria in animals. (b) Human cases of poisoning with chromates have been reported by Lohr (1904) and Major (1922). Both reports state the appearance of glycosuria unrelated to hyperglycemia. None of these reports in man or animals has included a systematic analysis of renal function. Our experiments reported above describe for the first time a predominant interference in absorption of glucose by the renal tubules following the intravenous administration of high doses of chromium salt. However, our perfusion system contained four polypropylene connectors and tubing totaling 12.5 g. The estimated total amount by emission spectrography was 0.10 mg of sodium dichromate. The minimal rate of infusion of sodium dichromate into the renal artery which reduced glucose reabsorption was 27.6 pg/min. Thus it seems unlikely that the chromium content alone of the plastic was responsible for the observed effect. ACKNOWLEDGMENT
The author is grateful to Drs. Peter K. Knoefel and K. C. Huang for fruitful discussionsand valuable suggestions during this investigation. The author also wishes to expressindebtedness to the generous grant of the University of Louisville during the conduct of this work. REFERENCES BRATTON, A. C., and MARSHALL, E. K. (1939). A new coupling component for sulfanilamide determination. J. Biol. Chem. 128, 537-550. FRANK, E. (1913). Uber experimentelle und der klinische Glykosurien renalen Ursprungs. I. Einleitung: abnorme Zuckerdichtigkeit der Niere; Phlorizindiabetes. Arch. Exptl. Puthol. Pharmacol. 72, 387-443. HAGEDORN, H. C., and JENSEN, B. N. (1923). Zur Mikrobestimmung des Blutzuckers Mittels Ferricyanid. Biochem. Z. 135,46-58.
NEPHROTOXICITYOF POLYPROPYLENE
583
HANES,C. S. (1929). XIV. An application of the method Hagedorn and Jensen to the determination of larger quantities of reducing sugars. &&em. J. 23, 99-106. HELPLER,0. E., and SIMONDS,J. P. (1946). Experimental nephropathies. IV. Glycosuria in dogs poisoned with many1 nitrate, mercury bichloride, and potassium dichromate. A/.clr. Pathol. 41, 42-49.
KESSER, T. 0. J. (1960). Polypropylene. Reinhold, New York. KOSSA,J. ( 1902). Ueber Chromslure-Diabetes. Arch. Ges. Ph,,siol. 88, 627-637. LOHR, A. ( 1904). Ueber einen Fall acuter Chromvergiftung mit spontaner Glykosurie, geheilt durch die von R. v. Jaksch empfohlene Magenaussptilung mit salpetersaurem Silber. Berl. Klin. Wochschr. 41, 749-758.
Luzzaro, R. (1914). Die Glykosurie bei experimentellen Nephritiden. Z. Exptl. Pathol. 16, 1S-67. MACNIDER, W. D. B. (1912). A study of the renal epithelium in various types of acute experiment nephritis and of the relation which exists between the epithelial changes and the total output of urine. J. Med. Res. 26,79-126. MAJOR,R. H. (1922). Studies on a case of chromic acid nephritis. Bull. Johns Hopkins Hasp. 33, 56-67.
MOSINGER,M., FIORENTINE,H., and QUICKE,J. (1954). Diabete experimental par intoxication au chromate neutre de potassium. Compt. Rend. Acad. Sci. 148, 1453-1455. PETERS, J. H. (1942). The determination of creatinine and creatine in blood and urine with the photometric calorimeter. J. Biol. Chem. 146, 179-186. SCHOU,P. (1944). Experimental studies on kidney function during sulphate diuresis. 4. Investigations on kidney function in rabbits with chronic tubular nephritis A. M. Frandsen. Acta Physiol. Stand. 7, 200-208.
TORIBARA,T. Y., TEREPKA,A. R., and DEWEY,P. A. (1957). The ultrafiltrable calcium of human serum. I. Ultrafiltration methods and normal values. J. Clin. Invest. 36. 738-748.