METABOLIC CHANNELING IN EXPERIMENTAL NEPHROSIS

Antibodies capable of producing renal damage in rats appear in the serum of rabbits immunized by repeated injections of an extract of rat kidneys (1). Heymann and Lund (2) and Ehrich, Forman, and Seifter (3) have developed a technique for the preparation of a potent antikidney serum, and the latter have demonstrated that, with the proper dosage of such a serum, an acute disease is produced in the rat which, from the viewpoint of specific renal pathology, closely resembles lipide nephrosis in man. The course of this experimentally produced disease state is dramatic: albuminuria begins 24 to 48 hours after the injection of antikidney serum and is followed by hyperlipemia, hypoproteinemia, and massive edema. This albumin-deficit state offers an opportunity to study the metabolic adjustments which follow the loss of large quantities of a specific protein in the urine. Our metabolic studies were carried out both in intact young animals and in vitro on rat liver. Glycine-2-Cl4 was used as a labeling agent. The liver glycogen content of the nephrotic rats was found to be low, and fasting hypoglycemia was demonstrated. These results, in combination with studies of lipide metabolism described in Paper II (4), have clarified many aspects of the metabolic state in experimental nephrosis.

(Received for publication, September 3, 1957) Experimental nephrosis in rats is characterized by albuminuria, which amounts in 1 day to more than twice the total circulating albumin of control rats (2). Evidence that the urinary albumin represents newly synthesized protein was obtained in the earlier experiments (2), in which the specific radioactivity of urinary protein was found to be several times higher than that of the plasma albumin of the normal rat after the injection of glycine-2-C14.
The conclusion drawn from these observations was that the synthesis of plasma albumin in the nephrotic rat is accelerated. Since the interpretation of isotope incorporation data is complicated by the problem of the size of the available amino acid pool, a more direct approach to the question of the rate of albumin synthesis by the liver of the nephrotic rat was sought.
A net synthesis of plasma albumin by chicken liver slices has been demonstrated in work by Peters and Anfinsen (3), but mammalian livers have not been investigated in this manner until recently, in a study by Campbell and Stone (4) which came to our attention after our experiments had been completed.
In the present work the net synthesis of plasma albumin by normal rat liver slices is demonstrated, and it is furthermore shown that the liver of the nephrotic rat synthesizes albumin in vitro at almost twice the normal rate.

Methods
The general procedure employed in these experiments was similar to that of Peters and Anfinsen (3). Male albino rats of the Wistar strain, weighing between 100 and 175 gm., were used. The animals were allowed food ad l&turn.
Rat plasma albumin was obtained by the alcohol fractionation technique of Ulrich, Li, and Tarver (5). In the first series of experiments (reported in Tables II and III) the final product obtained by this technique was used for the immunization of rabbits by the method of Freund and McDermott (6), with use of a total of 5 mg. of rat albumin IN NEPHROSIS. IV for each rabbit.
In the -second series of experiments, the final albumin precipitate, obtained by the method of Ulrich et al. (5), was redissolved and all the steps of the isolation procedure were repeated. The latter preparation was electrophoretically homogeneous when studied in the classical Tiselius cell at pH 8.6 in barbiturate buffer, ionic strength of 0.1. The first preparation was examined only by paper electrophoresis at pH 8.6, and showed less than 2 per cent impurity as judged by staining with brom phenol blue. For the determination of albumin, Kunkel and Ward's quantitative immunochemical method was employed (7). In this procedure, it was found helpful to heat the washed precipitate for 1 minute at 100" in 0.1 ml. of 4 N HCl. This was followed by the addition of 0.5 ml. of 1 N NaOH before addition of the buffered ninhydrin reagent of Moore and Stein (8). A linear relationship between Klett readings in a volume of 10 ml. and the amount of albumin added was obtained over the range of 5 to 50 y of albumin. Based on the weight of the lyophilized albumin preparation used for immunization, the slope of the standard curve was 13.0 Klett units per microgram in the case of the antiserum obtained from the first albumin preparation, whereas the antiserum obtained against the second preparation gave a slope of 18.5 Klett units per microgram of albumin. Rat liver slices, each 0.5 mm. thick and about 150 mg. in wet weight, were incubated for 45 minutes at 37" in 10 volumes of medium (Table I) to reduce the initial albumin content.
The slices were then removed from the medium, blotted, and weighed.
After several slices were taken for the determination of the initial albumin content, portions of the remainder   were incubated in fresh medium (two slices per 2.0 ml.) for 2 hours at 37". For the determination of the albumin content, two different procedures were employed.
In the first of these (Tables II and III), the entire vessel contents were homogenized in a final volume of 9 ml. with 0.9 per cent NaCl and centrifuged for 10 minutes at 25,000 r.p.m. in a Spinco No. 40 rotor.
A clear supernatant fluid was obtained by puncturing the plastic tube near the bottom and allowing it to drip into a .test tube. The supernatant fluid was then analyzed for albumin by the immunochemical method.
In the second series of experiments (Table IV), the albumin contained in (a) the medium, (b) the supernatant fluid from the saline homogenate of the slices, and (c) the particulate precipitate obtained after centrifugation of the saline homogenate was estimated separately.
The medium and the slices of liver were centrifuged at 2500 r.p.m. at 5" in an International refrigerated centrifuge, and an aliquot of the medium was removed for analysis.
The tube was then allowed to drain and 9 ml. of saline were added, and the contents of the tube homogenized, and then centrifuged for 10 minutes at 25,000 r.p.m.
The supernatant fluid was transferred to another centrifuge tube and the precipitate was homogenized with 9 ml. of 0.4 per cent sodium deoxycholate (pH 7.8) and returned to the original centrifuge tube. Both the saline supernatant layer and the deoxycholate solution of the particles were then spun for 10 minutes at 25,000 r.p.m., and the supernatant fluid was collected after the tube was punctured as before.
Nephrosis was induced by the intravenous injection of 0.5 ml. per 100 gm. of body weight of a sheep antikidney serum.' In the experiments reported in Table IV, a second injection was given 24 hours after the first to produce rats with a severe degree of nephrosis.
The proteinuria in the first group of rats was greater than 200 mg. per 100 gm. of body weight per day, while that in the second group (Table IV) was generally twice this amount.

Results
The optimal in vitro conditions for the net synthesis of albumin by rat liver slices in these experiments resemble closely those described for the chicken liver slice system studied by Peters and Anfinsen (3). As shown in Table II, sodium ion appeared necessary and bicarbonate buffer was superior to phosphate. Divalent cations were also needed. Oxygen was found not to be required for the release of albumin from the slice into the medium, but was necessary for albumin synthesis.
No consistent effects were obtained by adding.glucose or amino acid mixtures to the medium, and accordingly no substrates were added in the reported experiments. Addition of 0.005 M glycine-2-C4 to the medium resulted in extremely high labeling of the albumin, the specific activity of which was more than 10 times greater than the acid-precipitated liver proteins, indicating a very active synthesis.
These experiments are not reported in detail here, since they were in complete agreement with those reported by Peters (9) in experiments with chicken liver slices.
The most recent experiments of Peters (lo), indicating that cellular particles contain a great deal of albumin (56 per cent of the total tissue albumin), raise the question of whether the net increase in saline-extractable albumin is a valid measure of the net synthesis of albumin, since merely the release of particulate albumin within the tissue cells could account for a net increase in saline-soluble albumin.
Peters reported (10) that all cellular fractions lose albumin upon incubation of liver slices, but that a net increase in albumin was still observed in chicken liver slices. The results in the normal control livers shown in Table IV indicate that in our experiments there was no significant change in the saline-soluble albumin within the slice nor in the deoxycholate extract of the particles after 2 hours of incubation at 37". The major increment in albumin appeared in the medium.
In view of these findings, the experiments in which only the saline-soluble albumin was measured (Tables II and III) appear to be a valid measure of the net synthesis of albumin.
By comparing the data on the synthesis of albumin by the liver slices from the normal rats in Tables III and IV, it is apparent that the initial saline-soluble albumin content of the washed slices was the same in both series of experiments, but that the net synthesis of albumin in the experiments in Table III was 322 y per gm. in 2 hours, while that calculated from the data in Table IV was 730 y per gm. We believe that this difference in results is referable to the fact that two different antisera were employed in the two series. In view of the fact that the rat albumin preparation used for immunization in the second series of experiments was probably more homogeneous, as well as the finding by Campbell and Stone (4) of a rate of albumin production of 720 y per gm. in 2 hours by normal rat liver, it would appear that the values obtained in our second series of experiments (Table IV) are more reasonable.
We may conclude, therefore, that the net synthesis of albumin by rat liver slices in vitro is of the order of 0.7 mg. per gm. in 2 hours at 37".
A comparison of the net synthesis of albumin by liver slices from control and nephrotic rats is shown in Tables III and IV. There was no difference in the initial albumin content of normal and nephrotic liver slices after the preliminary washing period.
In both series of experiments, the syn-thesis of albumin by the liver slices from the nephrotic rats was increased by 81 per cent over that by liver slices from the controls. The duration and severity of the nephrosis did not appear to affect the results significantly in these experiments.
The experiments reported in Table IV are  particularly revealing.
The increment in albumin during the incubation period was entirely in the medium in the control livers.
No significant changes occurred in either the saline-soluble albumin or in the particulate albumin.
In the livers of nephrotic rats, the increment in albumin was almost entirely in the medium, but there was also a statistically significant increase of 13 per cent in the particulate albumin.
In view of the recent evidence pointing to the cellular particles as the sites of albumin synthesis (lo), it would seem as if the cellular particles in the nephrotic liver are actually synthesizing albumin at a rate slightly faster than it can be released into the cell sap and finally into the surrounding medium.

DISCUSSION
The present experiments, in agreement with those of Campbell and Stone (4), demonstrate that a net synthesis of plasma albumin occurs in rat liver slices incubated at 37". The optimal ionic composition of the medium for albumin synthesis in rat liver slices was found to be almost identical with that described for the chicken liver (3), although we have employed a medium with a higher potassium ion concentration.
Significantly, sodium ion is necessary.
Such a medium favors glycogenolysis (ll), and since no increase in albumin synthesis was found after the addition of glucose or amino acid mixtures, endogenous sources of amino acids and of energy in the form of carbohydrate do not appear to be limiting factors in albumin synthesis in vitro. It is interesting that, even in the perfused rat liver, Jensen and Tarver (12) were unable to demonstrate a requirement for exogenous amino acids in the synthesis of plasma proteins.
The findings of Peters (lo), that much of the cellular albumin is present in the particles and is not extracted by saline, were confirmed for the rat liver in the present experiments. From Peters' data it can be calculated that about 56 per cent of the total liver albumin is in the particles, while in our experiments the value obtained was 64 per cent. Peters reported that in the chicken liver all cellular fractions lost albumin to the medium upon incubation of the slices. In the present experiments with rat liver slices, there was a 28 per cent loss of saline-soluble albumin during incubation of the slices, but no change in the particulate albumin. This finding lends validity to our first experiments (Table III) and those of Campbell and Stone (4), in which only saline-soluble albumin was measured as an index of net albumin synthesis, with the view of comparing control and nephrotic rats, as in our work, or control liver and liver tumor as in the work of Campbell and Stone. The extremely high rate of incorporation of labeled glycine in our experiments, as in those of Campbell and Stone (4) and Peters and Anfinsen (3), clearly demonstrates the active synthesis of plasma albumin by liver slices. The specific activity of the isolated albumin is of such magnitude as to render the idea of the transformation of a preexisting protein molecule into plasma albumin highly unlikely.
An increased synthesis of plasma albumin by liver slices from nephrotic rats is demonstrated in these experiments.
Recently, Gitlin, Janeway, and Farr (13) reported that, in several children with nephrosis, the major metabolic alteration encountered was an increase in the rate of albumin catabolism, with little alteration in the rate of synthesis.
It is probably unjustifiable to compare nephrosis in nephrotic children with the situation in the experimentally nephrotic rat. Moreover, the experiments of Gitlin and his colleagues, who use I 131-labeled albumin (14), are open to some question in view of the findings of Gordon (14) and others, which indicate that 113'-labeled albumin or even alcohol-precipitated albumin may not be catabolized in the same manner or at the same rate as normal albumin. Indeed, more than lo-fold differences in the rates of catabolism of certain IL31-labeled albumin preparations were reported by Gordon (14). The plasma albumin production in the severely nephrotic rat has been estimated to be 3-to 4-fold greater than normal (2). In Paper III (15), we have obtained an estimate of the rate of plasma albumin synthesis by the liver in intact normal and nephrotic rats.
The calculated values (15) were 1.4 mg. per gm. per hour (normal) and 2.5 mg. per gm. per hour (severely nephrotic subject), or 78 per cent higher in the nephrotic subject. It is instructive to compare these in vivo rates of synthesis with the in vitro rates in the present study.
The in vitro rates are (Table IV, Column 10) ,0.73 mg. per gm. per 2 hours (normal liver slices) and 1.32 mg. per gm. per 2 hours (nephrotic liver slices), or 81 per cent higher in the nephrotic liver.
These in vitro rates of net albumin synthesis in both the control and the nephrotic rat liver slices are 26 per cent of those deduced from the data in the intact animal. SUMMARY A net synthesis of plasma albumin has been shown to occur in rat liver slices. For optimal in vitro activity, sodium, magnesium or calcium, and bicarbonate ions were necessary in the medium.
The synthesis of albumin was increased by 81 per cent in liver slices from rats with experimental nephrosis.
The rate of albumin synthesis by liver in vitro compares very favorably with that deduced for intact rats, both normal and nephrotic.