Further Characterization of the Natriuretic Factor Derived from Kidney Tissue of Volume‐Expanded Rats: Effects on Short‐Circuit Current and Sodium‐Potassium‐Adenosine Triphosphatase Activity

Boiled homogenates of kidneys from volume-expanded and hydropenic rats were subjected Co column chroniatogra-phy. The fraction eluting within the range of partition coefficients (Kav) 0.76-0.89 (fraction III) was lyophilized and the effects of this semipurified preparation were assessed on short-circuit current (SCC) across isolated frog skin, on rat kidney cortex Na-K-ATPase activity, and on sodium excretion by the rat in vivo. At a dose of 500 ftg/ml, fraction III from expanded rat kidney inhibited SCC by 21 ± 5% (P < 0.01), whereas the same fraction from hydropenic rat kidney produced an insignificant change in SCC of 2 ± 8%. In a dose-response study, 50, 150, 500, and 1,500 pg/ml of fraction III from expanded rat kidney inhibited SCC by 4, 8, 19, and 28%, respectively; 500, 1,000, and 1,500 μg/ml inhibited Na-K-ATPase activity by 11, 22, arid 49%, respectively. An identical study with fraction III from hydropenic animals showed no significant effect in either assay. Also, fractions from expanded and hydropenic rats, eluted after fraction III (fractions IV and V), had no effect on SCC or Na-K-ATPase activity. Fraction HI also produced significant natriuresis in vivo at a dose of 500 μg/ml, confirming our observations that a natriuretic principle may be recovered from the kidneys of volume-expanded rats. We suggest that this natriuretic principle may act by reducing active sodium transport via inhibition of Na-K-ATPase.


Further Characterization of the Natriuretic Factor Derived from Kidney Tissue of Volume-Expanded Rats
Effects on Short-Circuit Current and Sodium-Potassium-Adenosine Triphosphatase Activity STANLEY D. HILLYARD, P H . D . , ESTHER LU, AND HARVEY C. GONICK, M.D.
SUMMARY Boiled homogenates of kidneys from volume-expanded and hydropenic rats were subjected Co column chroniatography. The fraction eluting within the range of partition coefficients (K av ) 0.76-0.89 (fraction III) was lyophilized and the effects of this semipurified preparation were assessed on short-circuit current (SCC) across isolated frog skin, on rat kidney cortex Na-K-ATPase activity, and on sodium excretion by the rat in vivo. At a dose of 500 ftg/ml, fraction III from expanded rat kidney inhibited SCC by 21 ± 5% (P < 0.01), whereas the same fraction from hydropenic rat kidney produced an insignificant change in SCC of 2 ± 8%. In a dose-response study, 50, 150, 500, and 1,500 pg/ml of fraction III from expanded rat kidney inhibited SCC by 4, 8, 19, and 28%, respectively; 500, i.OOOVand 1,500 M g/ml inhibited Na-K-ATPase activity by 11, 22, arid 49%, respectively. An identical study with fraction III from hydropenic animals showed no significant effect in either assay. Also, fractions from expanded and hydropenic rats, eluted after fraction III (fractions IV and V), had no effect on SCC or Na-K-ATPase activity. Fraction HI also produced significant natriuresis in vivo at a dose of 500 ^g/ml, confirming our observations that a natriuretic principle may be recovered from the kidneys of volumeexpanded rats. We suggest that this natriuretic principle may act by reducing active sodium transport via inhibition of Na-K-ATPase.
SEVERAL STUDIES now have shown that natriuretic factors can be found in both the serum and the urine of volume-expanded or uremic animals and humans. 1 " 9 These factors not only produce a natriuresis when injected into test animals but also inhibit transepithelial sodium transport in anuran membrane preparations such as the isolated frog skin or toad bladder. 1 ' 10 ' 13 The term "antinatriferic" has been used to describe this latter effect. 11 In a recent study, kaplan et a!. 13 have shown that the natriuretic factor obtained from uremic urine increases intracellular sodium content of and decreases pyruvate oxidation by isolated toad bladder cells. These findings suggest that this natriuretic factor inhibits active sodium transport either through interference with ATP generation via the tricarboxylic acid cycle or with some step in the active transport sequence, possibly the transport enzyme, Na-K-ATPase. Previous work from this laboratory 14 has demonstrated that a natriuretic factor also can be isolated from kidneys of volume-expanded rats but is not found in kidneys of hydropenic rats. In the present study we have shown in addition that this factor inhibits transepithelial sodium transport by isolated frog skin in a dose-dependent manner. Furthermore, the factor inhibits Na-K-ATPase activity of pooled rat kidney cortex homogenates with a similar dose-response relationship.

PREPARATION OF MATERIAL
Female Sprague-Dawley rats (250-300 g) were infused with 0.9% saline through a jugular venous cannula. An amount equivalent to 10% of the body weight was infused over a 1-hour period and a sustaining infusion of 0.2 ml/min was maintained for a 2nd hour. Urine flow was monitored during volume expansion through an indwelling urethral catheter; a diuresis of greater than 0.25 ml/min was taken to indicate an adequate response to volume expansion. A second group of rats was made hydropenic by water deprivation on the night before they were killed.
The kidneys from three expanded or three hydropenic rats were removed under ether anesthesia, immediately bisected and placed for 20 minutes in 30 ml of deionized boiling water acidified to pH 3.4 with acetic acid. The kidney tissue was then cooled to 0°C, blotted gently to remove excess water, decapsulated, cut into small pieces, and then weighed and replaced in the original solution, also cooled to 0°C. Sufficient deionized water was added to the mixture to give a 1:10 (wt/vol) dilution of the kidney material, and the mixture was homogenized with a Tri-R tissue homogenizer. The homogenate was centrifuged at 600 g for 15 minutes at 4°C to remove cell debris, and 20 ml of the supernatant fluid were subjected to Sephadex G-25 column chromatography (5 x 45 cm, void volume of gel bed, approximately 360 ml) with 0.1 M acetic acid as eluate. The ultraviolet absorbance of the eluting fractions was monitored at 280 nm (A 2ao ). Fractions having elution volumes of 760-830 ml, 830-900 ml, and 900-970 ml, termed fractions HI, IV, and V, respectively, were collected and lyophilized (Fig. 1). Lyophilized material was weighed to the nearest 0.1 mg and assayed immediately or stored desiccated at -20°C.

FROG SKIN ASSAY
Active sodium transport was measured as the short-circuit current (SCC) across the isolated ventral skin of Rana pipiens (Los Angeles Biologicals). The frogs were maintained in tap water at room temperature. The Ussing-type cell 15 which we employed had three separate chambers bored into the Lucite chamber halves. Each chamber had a surface area of 1 cm 2 and a volume of 4 ml. This allowed concurrent measurement of potential difference (PD) and SCC across three sections from the same piece of skin. PD was monitored continuously (open circuit) and SCC applied every 10 minutes by the method described by Sullivan et al.' 6 PD was measured via agar-Ringer bridges and paired calomel reference electrodes connected to a digital voltmeter (Orion Research, model 801). SCC was passed through Ag/AgCl electrodes connected to the chambers of agar-Ringer bridges. The Ringer's solution used in all experiments contained 111 mM NaCl, 2.0 mM KC1, 2.4 mM NaCHOj, 1.0 mM CaCl 2 , and 5.0 mM glucose. The pH was 7.6 and the osmotic pressure 220 mOsM. The frogs were doubly pithed and the ventral skin was mounted between the chamber halves. After a 60-minute equilibration period, measurements of PD and SCC were started. When the SCC values for each chamber varied by < 10% for 30 minutes, the experimental treatment was begun. If the control SCC of any of the three chambers was <20 /zA/cm 2 , or if the interchamber variation in SCC was greater than 20%, the preparation was discarded.
To determine initially which fraction contained antinatriferic activity the lyophilized material was dissolved in frog Ringer's solution to give a concentration of 500 n%/m\. Samples (4 ml) of two of the fractions were added to the inner (serosal) chamber halves bathing two of the three sections of the skin and fresh Ringer's solution was added to the serosal half of the control chamber. The pH and osmolality of the Ringer's solution were checked before and after addition of each of the lyophilized fractions and were found to be unchanged. The outer half of all three chambers was refilled with fresh Ringer's solution. PD and SCC were recorded for the following 60 minutes. At this time the Ringer's solution in inner and outer chambers bathing all three sections of the skin was replaced with fresh solution. After 30 minutes, PD and SCC again were monitored continuously. When SCC varied by < 10% for an additional 30 minutes, the remaining fraction was assayed in the manner described above. The order in which the three fractions were assayed was random.
After we found that the antinatriferic substance was located in fraction III, a dose-response relationship was established for the inhibition of SCC by this fraction. Doses of 50, 150, 500, and 1,500 fig of fraction III per ml of frog Ringer's solution were prepared and assayed as above except that two doses of fraction III were assayed relative to the control chamber during each experimental period. Changes in SCC and PD from control were evaluated after incubation for 60 minutes with each fraction.
Na-K-ATPase ASSAY Rat kidneys were dissected free of the adipose and connective tissue which constituted the capsule. We removed slices of cortex, leaving a wide corticomedullary junction to ensure that only cortical enzyme would be assayed. Homogenates of the cortex were prepared in a 1:10 (wt/vol) dilution with Tri-R tissue homogenizer at 0°C with 0.015 mM ethylenediaminetetraacetic acid (EDTA) and 2.4 mM sodium deoxycholate (DOC). Samples (1 ml) of each homogenate preparation were stored at -20°C prior to use. This constituted the enzyme preparation. Enzyme activity was assayed no earlier than 3 days and no later than 3 weeks after preparation. During this time interval we found total ATPase activity and the ratio of Na-K-ATPase activity to Mg-ATPase activity to be constant.
The frozen homogenate was thawed at 4°C and diluted to 1:10 with cold distilled water prior to the assay of ATPase, which was carried out by a procedure described previously from this laboratory" and modified as follows: For determination of total ATPase activity, incubation tubes contained 0.5 ml of substrate solution and provided final concentrations of 1 mM ATP, 1 mM Mg 2+ , 10 mM imidazole-HCl buffer, pH 7.2, 100 mM Na + , and 20 mM K + ;0.1 mlof5mM ethylene glycol bis(/3-aminoethyl ether)-yV,W-tetraacetic acid (EGTA); 0.3 ml of material from each fraction dissolved in water was added to each assay tube, and the tube was placed in the water bath at 37°C. A control was prepared in the same manner, but 0.3 ml of distilled water was substituted for the kidney fraction. To start the reaction we added 0.1 ml of the enzyme preparation. We stopped the incubation after 10 minutes by adding 1 ml of ice-cold 10% trichloroacetic acid (TCA). After centrifugation at 1,700 g for 5 minutes, we assayed 1 ml of the supernatant fluid for inorganic phosphate by the method of Fiske and Subbarow (18). Mg 2+ ATPase activity was assayed with I HIM ouabin in the incubation medium, and Na-K-ATPase was calculated as the difference between total ATPase and Mg 2+ ATPase activities. Protein determinations were obtained for each enzyme preparation by the method of Lowry et al. 19 The protein content of 0.1 ml of enzyme preparation varied from 0.160 to 0.180 mg. ATPase activity was then expressed as micromoles of inorganic phosphate per milligram of protein per hour.

RAT ASSAY IN VIVO
In order to verify the fact that fraction III possessed natriuretic activity, the natriuretic response in vivo was assayed according to the procedure described by Sealey and Laragh (type IV assay) 20 as modified in this laboratory. 14 After a 90-minute equilibration period and after three stable 15-minute control periods had been obtained, 500 ng of fraction III from kidneys of volume-expanded animals were dissolved in 0.3 ml of 0.9% saline and injected through the femoral cannula.
Glomerular filtration rate (GFR), urine flow (V), absolute sodium excretion (U Na V), and fractional excretion of sodium (FE Na ) were calculated from the average of the three 15-minute control periods and from the average of the three 15-minute periods following injection of the test fraction. Fraction III from hydropenic rats was not tested because our earlier experiments had demonstrated clearly that the fraction from expanded, but not from hydropenic, rat kidneys contained natriuretic activity. 14

STATISTICAL ANALYSIS OF RESULTS
Statistical comparisons were accomplished by Student's i-test and, where applicable, by two-way analysis of variance.

COLUMN CHROMATOGRAPHY
The pattern of ultraviolet (UV) absorption at 280 nm by the eluting fractions resembles that obtained in our earlier study in which we used a Sephadex G-25 column, 2.5 x 40 cm, but there was a greater separation between the third and fourth UV peaks. For consistency with the earlier work, the material eluting with the fourth UV peak is termed fraction IV. The fractions eluting just before and just after fraction IV are termed fractions III and V (Fig. 1). These fractions were calculated to have partition coefficients (*"") of 0.76-0.89, 0.89-1.03, and 1.03-1.17, respectively. For these calculations we used the formula K au = (V e -Vo)/K -^o), where V t is eluted volume of the gel bed, V a is void volume, and V, is total volume.
Thus fraction III of our present study is found within the same range of K au as fraction IV from our earlier work (0.72-0.94); this latter fraction had been shown to contain the natriuretic factor. 14 Sodium, potassium, and calcium ions were spread throughout fractions III, IV, and V. The distribution of Ca 2+ is shown in Figure 1. In no case, however, were the concentrations of these ions sufficiently high to affect any of the assays. The final calcium concentration in the Na-K-ATPase assay never exceeded 0.1 mM at the highest concentration of fraction III (1,500 /ig/ml). EGTA (5 mM) prevented inhibition of Na-K-ATPase when calcium was added up to a concentration of 0.2 mM.

FROG SKIN
The changes in PD and SCC produced by the experimental treatments were evaluated in relation to the values of PD and SCC for the control section of each skin by the equation 16 F Ĉ L _ b . x ioo E. C e where E and C are the experimental and control sections of the skin, respectively, and c and e refer to the control and experimental periods, respectively. The percent changes (mean ± 1 SEM) in SCC for six experiments are shown in Figure 2.
When fractions III, IV, and V, at a concentration of 500 Mg/ml, were tested immediately after lyophilization, antinatriferic activity was found only in fraction III (Fig. 2b) from expanded rats. The mean changes in SCC and PD produced by fraction III were -22.7 ± 2.2% and -22.2 ± 5.4%, respectively (P < 0.001). The mean change in resistance, calculated as the ratio of PD/SCC, 16 was -6 . 4 ± 9 . 1 % (P > 0.5). Data from a typical experiment are shown in Figure  3. Fractions IV and V from expanded rat kidneys and fraction III from hydropenic rat kidneys produced no

FIGURE 3 Results of a typical experiment demonstrating the onset of inhibition of short-circuit current (SCC) and potential difference (PD) across a section of frog skin treated with fraction III from expanded rats at a concentration of 500 fig/ml. The inhibition usually was reversed by washing the inner surface of the skin with fresh Ringer's solution.
significant inhibition of SCC (Figs. 2a, d, and e). Fraction III from expanded rat kidneys was shown to lose inhibitory activity after storage in the lyophilized form for 1 week at -20°C (Fig. 2c).
In the dose-response study with expanded fraction III (mean of five experiments) inhibition of SCC increased linearly with the log of the dose. On the other hand, hydropenic fraction III failed to produce a significant inhibition of SCC even at the highest concentration tested (Fig. 4).

Na-K-ATPase
The changes in total, Mg-activated, and Na-K-activated ATPase activity produced by hydropenic and expanded fractions III and IV (1,500 jig/ml) and fraction V (500 /ig/ml) are shown in Table 1  and V) or triplicate (fraction III). Only fraction III from expanded rats produced significant inhibition of Na-K-ATPase. Mg ATPase activity was unaffected by any of the fractions. In Figure 5 is a dose-response curve for fraction III from expanded and hydropenic rats. As with the frog skin SCC assay, Na-K-ATPase inhibition increased progressively with increases in the log of the dose of expanded fraction III but not with hydropenic fraction III. Table 2 shows that 500 fig of fraction III from expanded rat kidneys exert significant natriuretic and diuretic activity. Urine flow increased by a mean of 90% and fractional excretion of sodium by a mean of 123% after injection of the test fraction. The mean increase in fractional excretion of sodium is almost identical to that demonstrated in our previous study, in which we utilized a comparable quantity of natriuretic substance. 14

Discussion
The results of our present study indicate the kidneys of volume-expanded rats yield a fraction (fraction III) which in vitro is both antinatriferic and an inhibitor of Na-K-  ATPase, and when injected in vivo produces a diuresis and natriuresis. This fraction elutes within the same range of values for partition coefficients (0.76-0.89) and has essentially the same natriuretic potency as the natriuretic fraction obtained in our earlier study, 14 but the chromatographic locus is prior to rather than within the fourth UV peak at 2 8 0 -Two major changes were introduced in the preparation of the material for the present study: (1) A column of larger dimensions was used in order to provide sufficient material for the three bioassays. This change in column dimension may account for the alteration in the UV absorption pattern and the greater spread in salt distribution. (2) Mercaptoacetic acid and albumin, which were added to the natriuretic fraction prior to lyophilization in the first study in order to achieve stability (as originally suggested by  were omitted from the present studies because they interfere with the frog skin and Na-K-ATPase assays. It is likely that these substances did enhance stability, because natriuretic activity was demonstrable for at least 2 weeks after lyophilization of the active fraction in the first study whereas the antinatriferic activity of the fraction was lost within 1 week after lyophilization in the present study.
The effects of the natriuretic fraction in many respects resemble those of ouabain. Ouabain, which is a specific and potent inhibitor of the transport enzyme, Na-K-ATPase, decreases sodium transport by the intact kidney in parallel with a reduction in enzyme activity. 21 There is a similar close parallelism between the inhibition of SCC in anuran epithelia produced by ouabain and the reduction of Na-K-ATPase activity in that tissue." In our present study, we also have shown that the natriuretic fraction from rat kidney inhibits both SCC and kidney cortex Na-K-ATPase activity in a dose-dependent manner. These data are consistent with our previously advanced hypothesis that the natriuretic factor is an inhibitor of Na-K-ATPase." In this preliminary study" we observed the appearance of a Na-K-ATPase inhibitor in the plasma of volume-expanded dogs and rats but not in the plasma of hydropenic dogs and rats. This inhibitor eluted in the same locus from a Sephadex G-25 column as the present kidney material. Furthermore, volume expansion was shown to decrease Na-K-ATPase activity and increase ATP content of the renal cortex of intact rats. 17 Clarkson and deWardener, 24 working with a fraction recovered from the urine of salt-loaded humans, demonstrated that this factor increased the sodium content and decreased the potassium content of isolated rabbit kidney tubule fragments, further suggesting a ouabain-like effect. More recently, Kaplan et al. 1 * found that the antinatriferic fraction obtained from uremic urine increased the sodium content of, and inhibited pyruvate oxidation by, isolated toad bladder cells but failed to alter water and potassium content. They suggest that the effect of the transportinhibiting substance may be either on metabolism or on some step of the transport sequence across the serosal border of the epithelial cells. Their data would also be consistent with a direct inhibition of Na-K-ATPase in that the subsequent accumulation of ATP would be expected to act as a feedback inhibitor of glycolysis and the citric acid cycle via inhibition of phosphofructokinase and isocitrate dehydrogenase. 26 In summary, we have demonstrated that the natriuretic fraction derived from kidneys of volume-expanded rats is also an inhibitor of epithelial sodium transport and Na-K- ATPase in vitro. Further purification of the fraction will be necessary to establish whether these properties arise from a single molecular species.