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The regulation of renal ammoniagenesis in the rat by extracellular factors. II. Ammoniagenesis by rat kidney slices incubating in normal acidotic sera
The Regulation of Renal Ammoniagenesis in the Rat by Extracellular Factors. II. Ammoniagenesis by Rat Kidney Slices Incubating in Normal Acidotic Sera H. G. Preuss.
K. Baird, and S. T. Eastman
Kidney slices from normal rats incubating in 100% v/v rat serum produce less ammonia than slices incubating in medium containing comparable amounts of glutamine. When lactate in concentration, expected in serum, is added to medium containing glutamine, ammoniagenesis by rat renal slices is similar in magnitude to ammoniagenesis by rat renal slices incubating in serum. In contrast, ammonia production by slices from acidotic rats is near similar whether incubation takes place in glutamine, glutamine plus lactate, or in serum (100% v/v). The source of the serum used for incubation does not matter. For the most part, ammonia production by renal slices from the same rats (control or acidotic) was indistin-
guishable whether the serum came from a control or acidotic rat. Based upon the similarity between findings with serum and with medium containing lactate, we feel that lactate and other oxidizable substrates in serum decrease glutamine ammoniagenesis. This hypothesis was strengthened when it was shown that ultrafiltrates of sera act similarly to whole sera, while dialysis of sera, to a great extent, removes many of the inhibitors. We further conclude that circulating oxidizable substrates in sera have less effect on renal ammoniagenesis by acidotic slices, and this relative insensitivity to the circulating depressors could be important in adaptive ammoniagenesis.
R
ENAL TISSUE removed from acutely and chronically acidotic rats compared to renal tissue from control rats produces more ammonia when placed adaptation is usually observed in an artificial medium. 1*2This in vitro intracellular in a balanced salt solution (pH 7.4) containing glutamine, the major precursor of renal ammonia.3.” Obviously, many other known and unknown factors present in the circulation that could influence ammoniagenesis are not included in the incubating medium. Few studies have been performed to determine how changes in the extracellular environment (circulating serum factors) might affect renal ammonia We are unaware of any studies on ammoniagenesis where slices production.5-7 were incubated in an environment composed entirely of serum (100% v/v). Recently,’ we studied the effects of renal fuels on the ammoniagenesis of incubating rat renal slices. As others,X-‘O we became aware of the complex interaction of substrates. It is known that different concentrations of substrates, as well as different combinations of substrates, can have varied effects on the metabolism of each other, It occurred to us that serum might be an ideal incubation medium as a first attempt to study renal ammoniagenesis in a natural environment. In this way, renal slices incubating in serum (100% v/v) would be exposed to a majority of the substrates and factors normally presented to the kidneys. Accordingly, we compared renal ammoniagenesis of slices incubating in sera from normal control,
From the Department of Medicine and Pathology, Georgetown University Medical School, Washington, D.C. Receivedforpublication Oclober4, 1977. Supported by NIH Grant #AM 15458. Address reprint requests 10 Harry G. Preuss. M.D.: Georgetown University Hospital. Room 2212, Washington, D.C. 20007. 0 1978 by Grune & Stratton, Inc. 0026.0495/78/2711-0006$01.00/0 Metabolism. Vol. 27. No.
11 (November).
1976
1639
1640
PREUSS. BAIRD.
acutely acidotic and chronically acidotic rats with slices incubating medium containing glutamine or glutamine plus lactate. MATERIALS We worked
with 225-275
were made acidotic weight
of 0.5 M solution).
preceding
the study;
In another
hr). A final group of rats drank Ammonium
rats. fed Purina
chloride
NH,CI
administered
them
group
on the day of study,
in an artificial
rat chow and water
a single oral dose of NH,CI
ad libitum.
dose was given 4 hr before
ml) ad libitum
by these methods
g body
a day on the day
the rats were killed
(28
and were killed on the fifth day (120 hr).
was sufficient
in the same manner,
Rats
(I mmole/lOO
of rats, the same dose was given twice
another
(I g/l00
trol rats for each group were handled
EASTMAN
AND METHODS
g Sprague-Dawley
for 2 and 4 hr by giving
AND
to create
receiving
water
metabolic
acidosis.”
Con-
by gavage or drinking
only
water. Rats were killed were placed Stadie-Riggs cubated
microtome”
Under
them
of incubation. gassing
studied,
In some studies,
with 5% CO,.
10% of the final volume.
To obtain
sera
Lactic
for incubation,
an hour after
they were drawn.
from
rats
by subtracting
slice. Medium
alone (no slice added) forms
the ammonia
formed
gassed continuously held at
and serum throughout 7.0
represented
were glutamine
and allowed
a
were in-
with the temperature
Biochemical),
(Sigma
less 99%
I .Z mM. to clot.
and control
produced
The
sera were
rats were incubated
in
by each slice in the presence of
by the same serum
very little
Tissues
to the serum
in the medium
The ammonia
was estimated
and
to give a final pH approximating
in the cold, and slices from the acidotic
serum
ammonia
during
incubating incubation
without
a
and so this
was not made in the studies using medium.
In the dialysis dialyzed
shaker
7.2 7.4 in the medium
employed
was drawn
study.
or serum (100% v/v).
metabolic
acid (Na) 40% (Nutritional
blood
were decapsulated,
under
of HCI and NaHCO,,
kidney
correction
medium
the sera were titrated
The additions
The substrates
and L (+)
by centrifugation
the sera within
medium’:’
pH approximated
quickly,
50 mg + 2 mg or 100 mg + 2 mg with
to the incubation
and 5% CO, for 90 min on a Dubnoff
- 100% pure) 0.6 mM; separated
were removed
We cut slices weighing
and added
most conditions
the duration or 7.8 after than
saline.
in 2 ml or 5 ml of bicarbonate-buffered
with 95% oxygen 37°C.
by a blow to the head. The kidneys
in cold isotonic
studies depicted
against
The dialysis
2 liters
tubing
in the first
cold medium
reportedly
retains
part of Table
(cellular
dialyzer
substances
4, one-half
tubing
of pooled sera under study was
model
with a molecular
3787-D40,
weight greater
A.H.
took place at 2” C for a period of 4 hours with at least one change in the bath occurring the dialysis.
The other
multaneously
one-half
with the dialyzed
of the pooled portion.
sera was maintained
In these studies,
Thomas
than I2.000. midway
on ice till it could
Co.).
Dialysis through
be assayed
50 mg + 2mg slices were incubated
si-
in 5 ml of
the medium. In the ultrafiltrate placed in Collodion weight greater Later,
studies depicted bags (model
in the second part of Table
3787-D40,
than 25,000. The transudate
the results
from
the ultrafiltrate
A.H.
Thomas
was collected
were compared
pooled sera, which had been refrigerated
until
4. half of another
Co.) that retain under vacuum to the results
substances
pooled
sera was
with a molecular
in a room maintained
found with the other
at 2°C.
half of the
use. As above, 50 mg i 2 mg slices were incubated
in 5
ml of medium. After
incubation,
teinized.“‘Aliquots Results nificance
slices
were
removed
of each were analyzed
are expressed
as Mmoles/g
from
the incubating
for ammonia
wet weight.
set at p < .05. Group or paired analyses
media,
and for glutamine
Statistics
and the media and lactate
are by Student’s
were
depro-
in a few studies.”
t test with
statistical
sig-
were used when appropriate.
RESULTS
Our first comparisons were made on the intracellular adaptations to acidosis (Table 1). Slices removed from rats that had received NH,CI (I mmole/lOO g body weight) 2 hr earlier produced significantly more ammonia than slices removed from normal rats when incubating in normal sera (p < 0.05) or sera 2 hr after NH&l (p < .05). At 4 hr after NH+CI. this difference between simultaneously run control and acidotic slices became more apparent and was obviously greater in slices from rats made acidotic for 28 and 120 hr. In the last
REGULATION
OF RENAL AMMONIAGENESIS
Table 1. Ammonia
It
1641
Production by Rat Renal Slices in Rat Sera Removed
at Various Intervals After Acid Loading ControlSlices COntrOl Sera 2 hr
23.8 f
1.0
26.5 f
(13) 4 hr
22.7 f
Acid Slices Acrd Sers
19.8 f
120 hr
1.3
1.0
23.7 f
30.0 f
(19) 1.5
27.5 f
13
20.3 f 0.8
(12) 19.0+ 1.5
19.8 f 2.4
27.5 f 0.9
1.0
(8) 32.1 f 1.2
(14) 37.0 f 3.8
(11) 35.0 l 1.2
18) 32.2 f
112)
1.1
(21) 1.2
(8)
18)
Acid Sera
26.5 f 0.8
(201
(8) 28 hr
COntrOl Ssra
(4)
(4)
(6)
Average and SEM depicted. Numbers in parentheses indicate number of slices from a drfferent rat that were investigated. Slices and sera were studied after 2 hr. 4 hr. 28 hr. and 120 hr receiving NH.,CI (see Materials and Methods for details). Within each group, only slices and sera from acrdotrc rats undergorng the same period of acidosis were combined. Each set of acidotic sera and slrces were run simultaneously with Sara and slices from control rats. Values are Rm/g/90
mm.
three comparisons, the trends were similar whether the sera came from control or acidotic rats. The average ammonia production by renal slices from 41 normal control rats incubating in normal control sera was 21.5 pm/g/90 min i 0.7 (SEM) (average of all values in column one of Table 1). This was decidedly lower than the production by slices incubating in 2 ml of medium containing 0.6 mM glutamine, 29.6 pm/g f 1.2 (SEM) (p < .Ol) (Table 2), but similar to slices incubating in 0.6 mM glutamine and 1.2 mM lactate, 24.3 pm/g * 2.3 (Table 2) (SEM). Kidney slices from rats, 28 hr after acid challenge, produced 34.9 pm/g it 1.2 (SEM) of ammonia in the presence of glutamine and 35.2 pm/g & 1.1 (SEM) in the presence of glutamine and lactate, which was not significantly different from the production seen in normal or acidotic sera, 32.2 pm/g f 1.0 (SEM) (Compare Tables I and 2). Comparing sera from acidotic rats with that from normal control rats, a significant stimulation by acidotic sera relative to control sera was seen only when acidotic slices were incubated in the sera from rats that had received NH,CI 2 hr Table 2. Ammonia
Production by Slices Incubating in 2 ml Medium
Substrata
Number of Observatrons
Ammonra(pm/g/90 mm)
Control Slices Glutamine 0.6 mM
4
29.6 f
Glutamine 0.6 mM + Lactate 1.2 mM
4
24.3 f 2.3 (SEM)’
1.2 (sEM)
Glutamine 0.6 mM
4
34.9 * 0.8 (SEMI
Glutamine 0.6 mM + Lactate 1.2 mM
4
35.2 f
Acid Slices 1.1 ISEM)
‘p < 0.05 compared to control slices incubating in glutamine alone. Acid slices were removed from rats made acidotic for 28 hr by receiving 3 tube feeds of 1 mmole NH.,Cl per 100 g body weight (see Materials and Methods). Ammonium excretion by rats with this form of challenge is very similar to ammonium excretion by rats drinking NH.,CI (1% w/v).”
PREUSS, BAIRD. AND
1642
Table 3. Ammonia
EASTMAN
Production by Slices incubating in 5 ml Medium or 5 ml Sera Number of
Substrate
Observations
Ammonia
(pm/g/SO
mud
Control Slices Glutamine 0.6 mM
4
Glutamine 0.6 mM + Lactate 1.2 mM
4
33.7 zk 1 0 (SEM) 20.7 i 0 8 (SEM)’
Sera
4
23.4 f 2.5 (SEM)’
Glutamine 0.6 mM
4
49 3 f 2 6 (SEM)
Glutamine 0.6 mM + Lactate 1.2 mM
4
43.5 + 2.6 ISEM)
Sera
4
48.0 zt 7.4 (SEM)
Acid Slices
‘p
< 0.01
compared to control slices incubating in glutamine alone
Sera were pooled samples from
normal control rats. Acidosis produced as described in Table 2.
earlier. The difference between 30.0 pm/g/90 min * 1.1 (SEM) and 26.5 pm/g/90 min f (SEM), was statistically significant (p < 0.02), while the difference between 2-hr sera on slices from control rats missed significance (p > .05 < .l). Sera removed from rats made acidotic for 4, 28, and 120 hr, compared to simultaneously tested control sera, had no difference in their effect on the ammoniagenesis of slices from control and acidotic rats. The amount of serum obtained from each rat was small; therefore, our initial studies were performed with 2 ml volumes. Since the amount of glutamine present in 2 ml of serum was limited and we may have experienced substrate limitations, we performed another series of studies using more serum (5 ml) and less tissue (50 mg) in each flask (Table 3). The slices were incubated in pooled sera rather than individual serum; but in every other respect, the procedures were the same as those previously described. Four observations were made for each condition. In 5 ml of medium containing both glutamine and lactate, control slices produced 20.7 pm/g * 0.8 (SEM) of ammonia, whileproducing 23.4 pm/g f I .3 (SEM) of ammonia in 5 ml of pooled control sera. The production, when glutamine was sole substrate in the medium, was 33.7 * 1.0 (SEM). Slices from 28 hr acidotic rats produced 49.3 pm/g * 3.6 (SEM) in glutamine, 43.5 pm/g * 2.6 (SEM) in glutamine + lactate, and 48.0 pm/g * 3.3 (SEM) in the presence of pooled sera. Analysis of glutamine and lactate in the serum after 90 minutes incubation with acidotic slices (n = 4) showed concentrations of 0.34 pm/ml * .07 (SEM) and 1.20 pm/ml * .I0 (SEM). When slices from normal control rats were incubated in pooled sera from the same rats, ammonia production was similar whether the initial medium pH was 7.4 or adjusted to 7.0 and 7.8. The average final pH following incubation were respectively 7.2 for that sera starting at 7.0, 7.5 for sera starting at 7.4 and 7.8 for sera starting at 7.8. In these studies, 100 mg slices were incubated in 2 ml of pooled sera. Following 4 hr dialysis, our pooled sera had an average lactate concentration of 0.13 pm/ml f 0.05 (SEM), (n = 5) compared to a lactate concentration of 1.80 pm/ml & .25 (SEM), (n = 5), found in the other half of the same pooled sera that had been stored on ice for 4 hr. The sera stored on ice were found to have a glutamine concentration of 0.8 mM prior to incubation. There was no measurable glutamine in the dialyzed portion. When rat kidney slices were incubated in
REGULATION
OF
RENAL
AMMONIAGENESIS
II
1643
dialyzed sera, the QO, averaged 3.62 pl/mg/hr (n = 2) and in regular sera was 4.54 pl/mg/hr (n = 2). This was an increase of approximately 20% and 50% over the QO, of slices incubating in Krebs medium without added substrate, 3.02 jd/mg/hr (n = 2). In slices from 10 normal control rats the average ammonia formation was 34.3 pm/g f 1.2 (SEM) in glutamine, 17.5 pm/g & 0.9 (SEM) in glutamine plus lactate (149%), 16.4 pm/g f 1.1 (SEM) (152%), in pooled sera and 23.6 pm/g * 0.7 (SEM) (131%) in dialyzed sera with added glutamine (0.8 mM). There was a significant difference in ammonia production between slices incubating in regular sera and the dialyzed sera (p < .Ol). Slices produced more ammonia in dialyzed sera. At the end of incubation, we found the final average glutamine concentrations (n = 2) to be .33 pm/ml in glutamine, .55 pm/ml in glutamine plus lactate, .45 pm/ml in regular sera and .47 pm/ml in dialyzed sera. As might be expected, the concentrations of lactate present at the end of a 90 min incubation were small when only glutamine was added to the medium or when slices ,incubated in the dialyzed medium. The concentrations averaged .08 pm/ml and .12 pm/ml respectively. Control slices incubating in serum ultrafiltrate produced less ammonia than those control slices incubating in medium containing glutamine and lactate (p < .05). This was true also when acidotic slices were used. However, the difference in ‘ammoniagenesis was less when acidotic slices were examined. The average decrease in ammoniagenesis between acidotic slices incubating in ultrafiltrate and in a medium containing only glutamine was 11.0 pm/g (125%). The average difference in the studies using normal control slices was 17.2 pm/g (162%) compared to the above (p < .05). DISCUSSION
The majority of previous studies have concentrated upon the intracellular aspects of ammonia adaptation by renal slices from acidotic rats. Less emphasis has been placed upon environmental factors, i.e., circulating extracellular regulators. Alleyne and Roobel’5 showed that renal slices preincubated in plasma (10% v/v) removed from rats 2 hr after acute acid challenge compared to slices preincubated in plasma from normal rats (10% v/v) produced more ammonia when incubated in medium containing glutamine. Studies of ammoniagenesis by slices incubating directly in serum (100% v/v) have not been reported. Also, no results have been reported using plasma or sera drawn from rats undergoing long periods ( >2 hr) of acid stress. Our most obvious finding is that slices from normal rats produce less ammonia when incubated in serum than when incubated in artificial medium containing similar amounts of glutamine (0.6 mM).16 Krebs noted this same phenomenon over 40 yr ago.” This inhibition by serum factors is not apparent in renal slices from rats that have been acidotic for 28 hr. In other words, chronically acidotic slices not only produce more ammonia than control slices in medium via intracellular adaptations, but resist the inhibition to glutamine ammoniagenesis that serum factors produce in normal slices. Major differences between the sera themselves (acidotic and control) were seen in only one of the tested conditions. Serum from rats made acutely acidotic for only 2 hr compared to serum from control rats showed modest increases in ammoniagenesis in control slices (+ 1 1%, p > 0.05 < 0.1) and acidotic slices (+ 13%,
PREUSS. BAIRD, AND
1644
Table 4.
Effects of Dialyzed Sara and Ultrafiltrates
of Sera on Ammonia
EASTMAN
Production
in Rat Kidney Slices Effects of Dialyzed Sera on Ammonlagenesls by Skes from Normal Rats Dtalvzed Sera GmOBmM
Gm08mM
Substrate
Gm 0.8 mM
SW3
LalEmM
34.3 * 1.2
17.5 f 0.9
164zt
1.1
23.6 f 0.7
Effects of Serum Ultrafiltrate on Ammontagenws by Slices from Normal and Acldotic Rats GmOBmM
Gm 0.8 mM La 1.8 mM
Normal slices
27.6 f
1.4
14.2 f 0.3
10.4 * 1.2
Acidotic slices
44.7 f 0.6
43.2 f 0.6
33.7 f 0.9
Substrate
Values are the average pm/g/90
Ultrafiltrate
min f SEM of slices from 4-8 rats.
Values of glutamine IGml and lactate (La) used in these studies are based on the values found in sera stored on ice for 4 hr Acidosis produced as described in Table 2.
p < 0.05). Although our models differ, these findings resemble those of Allyne and Roobel.‘” Therefore, our results may be due to the same unknown stimulator found in acutely acidotic plasma reported by Alleyne and Roobel.‘5 Suffice it to say, we could not see this relative stimulation by acidotic sera when the acidosis was more prolonged. In addition, no differences were apparent when control slices incubated in the same serum at pH 7.0,7.4, and 7.8. This strengthens the proposal that pH changes per se are not responsible for renal slice ammonia adaptation.’ What is in serum that inhibits renal ammoniagenesis from glutamine? Most slice studies concerned with ammoniagenesis are run in medium containing glutamine, the major ammonia precursor, as the only substrate. It has been noted in the past that oxidizable substrates such as lactate can inhibit ammoniagenesis from glutamine by rat renal slices *’ When circulating concentrations of lactate (I .2 mM)lR were added to the glutamine medium, the extent of ammoniagenesis simulated that of slices incubated in serum under comparable conditions. In a like manner, neither the presence of lactate nor serum decreased, to any great extent, ammoniagenesis by slices from acidotic rats. We chose to study lactate in detail because of its concentration in serum. However, other oxidizable substrates probably contribute to the depression. These similarities suggest to us that the oxidizable substrates in serum, such as lactate, are probably responsible for its major inhibition. Krebs arrived at a similar conclusion.” To strengthen our conclusions, we performed the studies depicted in Table 4. Initial attempts were made to dialyze oxidizable substrates from pooled sera. Although we were aware that it would be difficult to remove all substrates from the serum because of protein binding,lg some success was obtained. We reduced lactate concentrations over tenfold. However, some lactate and other oxidizable substrates probably were present, because dialyzed serum still increased oxygen consumption by rat kidney slices (t20%). With regular sera, there was a greater increase (150%) correlating with the presence of greater amounts of substrate. Importantly, slices incubated in dialyzed serum with added glutamine showed greater ammoniagenesis than slices incubated in the regular serum (p < .Ol), although less ammonia was produced compared to slices incubating in glutamine alone.
REGULATION OF RENAL AMMONIAGENESIS II
1645
Probably more cogent for our hypothesis is the similarity in trends following addition of serum ultrafiltrates and serum itself. In the second group of studies depicted in Table 4, we see that ultrafiltrates of serum depress ammoniagenesis of control slices even more than lactate. The greater depression may be due to the additional oxidizable factors present in serum (see Table I in reference 13). In contrast, the depressive effects by the ultrafiltrate are markedly lessened on acidotic slices. Although we cannot eliminate all possible causative factors, our results, taken together, suggest that lactate and other oxidizable factors play a significant role. By necessity, we initially incubated slices in small volumes of medium and serum (2 ml). A question could be raised whether the apparent resistance by acidotic slices to serum and lactate is due to substrate limitations. If during the course of incubation, medium glutamine concentrations became low when glutamine was sole substrate, production would be limited, allowing the slower ammoniagenesis by acidotic slices in the presence of lactate and serum to catch up during the latter part of incubation. In turn, a more rapid removal of oxidizable substrates such as lactate might also influence our final results. The data depicted in Fig. 1 were derived from experiments described in the first paper’” and in this one. There is no difference in ammoniagenesis by normal slices incubating in either 2 ml or 5 ml of medium containing glutamine and lactate. In I
I
I
8
I
4
2
5 MI
I
12 Medium
Fig. 1. Ammonia production by rat kidney slices in different volumes of medium. oControl slices in medium containing glutamine and lactate. lControl slices in medium containing glutamine. =Acidotic slices in medium containing glutamine. nAcidotic slices in medium containing glutamine and lactate. Values are from experiments performed in this paper and in the previous one.13
PREUSS.
1646
BAIRD.
AND
EASTMAN
all other cases, ammoniagenesis is greater in 5 ml of medium. However, ammoniagenesis by slices under all conditions is similar whether the slices incubate in 5 ml of medium or 12 ml of medium. Therefore, we would not experience substrate limitations in 5 ml of incubating medium. To rule out the possibility that substrate limitations were responsible for our findings, we repeated our studies in 5 ml volumes of media. To be doubly sure, we even added less tissue, 50 mg. With the greater volumes and lesser weights of acidotic tissue, more than one half of the glutamine in serum was still present after 90 min. Our results with acidotic slices incubating in 5 ml volumes confirmed the previous findings, i.e., that acidotic slices resist the lactate and serum inhibition of ammoniagenesis. These findings are important in at least two respects. First, although the majority of previous studies have been performed with glutamine as the sole oxidizable substrate, ammonia production in the presence of lactate is more like that seen in the natural environment, i.e., serum. Because of this, we feel that lactate should be included as a substrate in future in vitro studies concerned with ammoniagenesis. As a first approximation, lactate was studied because this is the most abundant oxidizable organic anion present in blood. However, any oxidizable substrate alone or in combination might produce the same results. Second, serum factors inhibit ammoniagenesis in control slices, but less so or not at all in acidotic slices. This resembles findings when lactate is added to a glutamine-containing medium.13 We conclude that lactate and other extracellular substrates in serum are inhibiting glutamine ammoniagenesis in renal tissue from normal control rats and that this inhibition is overcome greatly in acidotic renal tissue. Renal tissue adaptations during acidosis which would allow deinhibition of circulating factor like lactate could theoretically augment ammoniagenesis. ACKNOWLEDGMENT The authors greatly acknowledge Mend&on. and Patti Werr.
the editorial
and secretarial
assistance
of Susan
Dreux,
Betty
REFERENCES 1. Preuss HG, Weiss FR: Rate limiting factors in rat kidney slice ammoniagenesis. Am J Physiol 221:458-464, 1971 2. Relman AS, Narins RG: The control of ammonia production in the rat. Med Clin N Am 59:583-593,1975 3. VanSlyke DD, Phillips RA, Hamilton PB, et al: Glutamine as a source material of urinary ammonia. J Biol Chem 150:481-482, 1943 4. Pitts RF, Pilkington LA, deHaas JCM: N’” tracer studies on the origin of urinary ammonia in the acidotic dog. J Clin Invest 44:731-745, 1965 5. Hems DA: Biochemical aspects of renal ammonia formation in metabolic acidosis, Enzyme 20:359-380, 1975 6. Alleyne GAO: Renal metabolic response to acid-base changes. II. The early effects of metabolic acidosis on renal metabolism in the rat. J Clin Invest 49:943-95 I, 1970
7. Roxe DM, Schreiner GE, Preuss HG: Regulation of renal gluconeogenesis and ammoniagenesis by physiologic fuels. Am J Physiol 225:908891 I, 1973 8. Weidemann MJ, Krebs HA: The fuel of respiration of rat kidney cortex. Biochem J 112:149-166, 1969 9. Barac-Nieto M: Effects of lactate and glutamine on palmitate metabolism in rat kidney cortex. Am J Physiol231:14-19, 1976 10. Adler S, Preuss HG: Interrelationships between citrate metabolism, ammoniagenesis, and gluconeogenesis in renal cortex in vitro. J Lab and Clin Med 79:505-515, 1972 11. Sleeper RS, Vertuno LL, Strauss FR, et al: Effects of acid challenge on in vivo and in vitro rat renal ammoniagenesis. LifeSci 22:1561- 1572, 1978 12. Stadie WC, Riggs BC: Microtome for the
REGULATION
OF RENAL AMMONIAGENESIS
II
preparation of tissue slices for metabolic studies of surviving tissue in vitro. J Biol Chem 154:687-690, I944 13. Preuss HG, Eastman ST, Vavatsi-Manos 0, et al: The regulation of renal ammoniagenesis in the rat by extracellular factors. I. The combined effects of acidosis and physiologic fuels. Metabolism 27: 1626-1638, 1978 14. Preuss HG, Bise BW, Schreiner GE: The determination of glutamine in plasma and urine. Clin Chem 12:329-337, 1966 IS. Alleyne GAO, Roobel A: Regulation of renal cortex ammoniagenesis. I. Stimulation of renal cortex ammoniagenesis in vitro by plasma isolated from acutely acidotic rats. J Clin Invest 53:117-121, 1974
1647
16. Weiss FR, Preuss HG: Glutamine synthetase and plasma, glutamine in augmented ammoniagenesis in acidosis. Am J Physiol 218:1697-1700, 1970 17. Krebs HA: CXCVII Metabolism of amino acids. III Deamination of amino acids. Biochem J 29:1620-1644, 1935 18. Kliger AS, Eastman ST, Zachek M, et al: Effect of renal fuels on p-aminohippurate transport in rat renal cortical fragments. Metabolism 26:979-988, 1977 19. Hanson RW, Ballard FJ: Citrate, pyruvate, and lactate contaminants of commercial serum albumin. J Lipid Res 9:667-668, 1968
K. Baird, and S. T. Eastman
Kidney slices from normal rats incubating in 100% v/v rat serum produce less ammonia than slices incubating in medium containing comparable amounts of glutamine. When lactate in concentration, expected in serum, is added to medium containing glutamine, ammoniagenesis by rat renal slices is similar in magnitude to ammoniagenesis by rat renal slices incubating in serum. In contrast, ammonia production by slices from acidotic rats is near similar whether incubation takes place in glutamine, glutamine plus lactate, or in serum (100% v/v). The source of the serum used for incubation does not matter. For the most part, ammonia production by renal slices from the same rats (control or acidotic) was indistin-
guishable whether the serum came from a control or acidotic rat. Based upon the similarity between findings with serum and with medium containing lactate, we feel that lactate and other oxidizable substrates in serum decrease glutamine ammoniagenesis. This hypothesis was strengthened when it was shown that ultrafiltrates of sera act similarly to whole sera, while dialysis of sera, to a great extent, removes many of the inhibitors. We further conclude that circulating oxidizable substrates in sera have less effect on renal ammoniagenesis by acidotic slices, and this relative insensitivity to the circulating depressors could be important in adaptive ammoniagenesis.
R
ENAL TISSUE removed from acutely and chronically acidotic rats compared to renal tissue from control rats produces more ammonia when placed adaptation is usually observed in an artificial medium. 1*2This in vitro intracellular in a balanced salt solution (pH 7.4) containing glutamine, the major precursor of renal ammonia.3.” Obviously, many other known and unknown factors present in the circulation that could influence ammoniagenesis are not included in the incubating medium. Few studies have been performed to determine how changes in the extracellular environment (circulating serum factors) might affect renal ammonia We are unaware of any studies on ammoniagenesis where slices production.5-7 were incubated in an environment composed entirely of serum (100% v/v). Recently,’ we studied the effects of renal fuels on the ammoniagenesis of incubating rat renal slices. As others,X-‘O we became aware of the complex interaction of substrates. It is known that different concentrations of substrates, as well as different combinations of substrates, can have varied effects on the metabolism of each other, It occurred to us that serum might be an ideal incubation medium as a first attempt to study renal ammoniagenesis in a natural environment. In this way, renal slices incubating in serum (100% v/v) would be exposed to a majority of the substrates and factors normally presented to the kidneys. Accordingly, we compared renal ammoniagenesis of slices incubating in sera from normal control,
From the Department of Medicine and Pathology, Georgetown University Medical School, Washington, D.C. Receivedforpublication Oclober4, 1977. Supported by NIH Grant #AM 15458. Address reprint requests 10 Harry G. Preuss. M.D.: Georgetown University Hospital. Room 2212, Washington, D.C. 20007. 0 1978 by Grune & Stratton, Inc. 0026.0495/78/2711-0006$01.00/0 Metabolism. Vol. 27. No.
11 (November).
1976
1639
1640
PREUSS. BAIRD.
acutely acidotic and chronically acidotic rats with slices incubating medium containing glutamine or glutamine plus lactate. MATERIALS We worked
with 225-275
were made acidotic weight
of 0.5 M solution).
preceding
the study;
In another
hr). A final group of rats drank Ammonium
rats. fed Purina
chloride
NH,CI
administered
them
group
on the day of study,
in an artificial
rat chow and water
a single oral dose of NH,CI
ad libitum.
dose was given 4 hr before
ml) ad libitum
by these methods
g body
a day on the day
the rats were killed
(28
and were killed on the fifth day (120 hr).
was sufficient
in the same manner,
Rats
(I mmole/lOO
of rats, the same dose was given twice
another
(I g/l00
trol rats for each group were handled
EASTMAN
AND METHODS
g Sprague-Dawley
for 2 and 4 hr by giving
AND
to create
receiving
water
metabolic
acidosis.”
Con-
by gavage or drinking
only
water. Rats were killed were placed Stadie-Riggs cubated
microtome”
Under
them
of incubation. gassing
studied,
In some studies,
with 5% CO,.
10% of the final volume.
To obtain
sera
Lactic
for incubation,
an hour after
they were drawn.
from
rats
by subtracting
slice. Medium
alone (no slice added) forms
the ammonia
formed
gassed continuously held at
and serum throughout 7.0
represented
were glutamine
and allowed
a
were in-
with the temperature
Biochemical),
(Sigma
less 99%
I .Z mM. to clot.
and control
produced
The
sera were
rats were incubated
in
by each slice in the presence of
by the same serum
very little
Tissues
to the serum
in the medium
The ammonia
was estimated
and
to give a final pH approximating
in the cold, and slices from the acidotic
serum
ammonia
during
incubating incubation
without
a
and so this
was not made in the studies using medium.
In the dialysis dialyzed
shaker
7.2 7.4 in the medium
employed
was drawn
study.
or serum (100% v/v).
metabolic
acid (Na) 40% (Nutritional
blood
were decapsulated,
under
of HCI and NaHCO,,
kidney
correction
medium
the sera were titrated
The additions
The substrates
and L (+)
by centrifugation
the sera within
medium’:’
pH approximated
quickly,
50 mg + 2 mg or 100 mg + 2 mg with
to the incubation
and 5% CO, for 90 min on a Dubnoff
- 100% pure) 0.6 mM; separated
were removed
We cut slices weighing
and added
most conditions
the duration or 7.8 after than
saline.
in 2 ml or 5 ml of bicarbonate-buffered
with 95% oxygen 37°C.
by a blow to the head. The kidneys
in cold isotonic
studies depicted
against
The dialysis
2 liters
tubing
in the first
cold medium
reportedly
retains
part of Table
(cellular
dialyzer
substances
4, one-half
tubing
of pooled sera under study was
model
with a molecular
3787-D40,
weight greater
A.H.
took place at 2” C for a period of 4 hours with at least one change in the bath occurring the dialysis.
The other
multaneously
one-half
with the dialyzed
of the pooled portion.
sera was maintained
In these studies,
Thomas
than I2.000. midway
on ice till it could
Co.).
Dialysis through
be assayed
50 mg + 2mg slices were incubated
si-
in 5 ml of
the medium. In the ultrafiltrate placed in Collodion weight greater Later,
studies depicted bags (model
in the second part of Table
3787-D40,
than 25,000. The transudate
the results
from
the ultrafiltrate
A.H.
Thomas
was collected
were compared
pooled sera, which had been refrigerated
until
4. half of another
Co.) that retain under vacuum to the results
substances
pooled
sera was
with a molecular
in a room maintained
found with the other
at 2°C.
half of the
use. As above, 50 mg i 2 mg slices were incubated
in 5
ml of medium. After
incubation,
teinized.“‘Aliquots Results nificance
slices
were
removed
of each were analyzed
are expressed
as Mmoles/g
from
the incubating
for ammonia
wet weight.
set at p < .05. Group or paired analyses
media,
and for glutamine
Statistics
and the media and lactate
are by Student’s
were
depro-
in a few studies.”
t test with
statistical
sig-
were used when appropriate.
RESULTS
Our first comparisons were made on the intracellular adaptations to acidosis (Table 1). Slices removed from rats that had received NH,CI (I mmole/lOO g body weight) 2 hr earlier produced significantly more ammonia than slices removed from normal rats when incubating in normal sera (p < 0.05) or sera 2 hr after NH&l (p < .05). At 4 hr after NH+CI. this difference between simultaneously run control and acidotic slices became more apparent and was obviously greater in slices from rats made acidotic for 28 and 120 hr. In the last
REGULATION
OF RENAL AMMONIAGENESIS
Table 1. Ammonia
It
1641
Production by Rat Renal Slices in Rat Sera Removed
at Various Intervals After Acid Loading ControlSlices COntrOl Sera 2 hr
23.8 f
1.0
26.5 f
(13) 4 hr
22.7 f
Acid Slices Acrd Sers
19.8 f
120 hr
1.3
1.0
23.7 f
30.0 f
(19) 1.5
27.5 f
13
20.3 f 0.8
(12) 19.0+ 1.5
19.8 f 2.4
27.5 f 0.9
1.0
(8) 32.1 f 1.2
(14) 37.0 f 3.8
(11) 35.0 l 1.2
18) 32.2 f
112)
1.1
(21) 1.2
(8)
18)
Acid Sera
26.5 f 0.8
(201
(8) 28 hr
COntrOl Ssra
(4)
(4)
(6)
Average and SEM depicted. Numbers in parentheses indicate number of slices from a drfferent rat that were investigated. Slices and sera were studied after 2 hr. 4 hr. 28 hr. and 120 hr receiving NH.,CI (see Materials and Methods for details). Within each group, only slices and sera from acrdotrc rats undergorng the same period of acidosis were combined. Each set of acidotic sera and slrces were run simultaneously with Sara and slices from control rats. Values are Rm/g/90
mm.
three comparisons, the trends were similar whether the sera came from control or acidotic rats. The average ammonia production by renal slices from 41 normal control rats incubating in normal control sera was 21.5 pm/g/90 min i 0.7 (SEM) (average of all values in column one of Table 1). This was decidedly lower than the production by slices incubating in 2 ml of medium containing 0.6 mM glutamine, 29.6 pm/g f 1.2 (SEM) (p < .Ol) (Table 2), but similar to slices incubating in 0.6 mM glutamine and 1.2 mM lactate, 24.3 pm/g * 2.3 (Table 2) (SEM). Kidney slices from rats, 28 hr after acid challenge, produced 34.9 pm/g it 1.2 (SEM) of ammonia in the presence of glutamine and 35.2 pm/g & 1.1 (SEM) in the presence of glutamine and lactate, which was not significantly different from the production seen in normal or acidotic sera, 32.2 pm/g f 1.0 (SEM) (Compare Tables I and 2). Comparing sera from acidotic rats with that from normal control rats, a significant stimulation by acidotic sera relative to control sera was seen only when acidotic slices were incubated in the sera from rats that had received NH,CI 2 hr Table 2. Ammonia
Production by Slices Incubating in 2 ml Medium
Substrata
Number of Observatrons
Ammonra(pm/g/90 mm)
Control Slices Glutamine 0.6 mM
4
29.6 f
Glutamine 0.6 mM + Lactate 1.2 mM
4
24.3 f 2.3 (SEM)’
1.2 (sEM)
Glutamine 0.6 mM
4
34.9 * 0.8 (SEMI
Glutamine 0.6 mM + Lactate 1.2 mM
4
35.2 f
Acid Slices 1.1 ISEM)
‘p < 0.05 compared to control slices incubating in glutamine alone. Acid slices were removed from rats made acidotic for 28 hr by receiving 3 tube feeds of 1 mmole NH.,Cl per 100 g body weight (see Materials and Methods). Ammonium excretion by rats with this form of challenge is very similar to ammonium excretion by rats drinking NH.,CI (1% w/v).”
PREUSS, BAIRD. AND
1642
Table 3. Ammonia
EASTMAN
Production by Slices incubating in 5 ml Medium or 5 ml Sera Number of
Substrate
Observations
Ammonia
(pm/g/SO
mud
Control Slices Glutamine 0.6 mM
4
Glutamine 0.6 mM + Lactate 1.2 mM
4
33.7 zk 1 0 (SEM) 20.7 i 0 8 (SEM)’
Sera
4
23.4 f 2.5 (SEM)’
Glutamine 0.6 mM
4
49 3 f 2 6 (SEM)
Glutamine 0.6 mM + Lactate 1.2 mM
4
43.5 + 2.6 ISEM)
Sera
4
48.0 zt 7.4 (SEM)
Acid Slices
‘p
< 0.01
compared to control slices incubating in glutamine alone
Sera were pooled samples from
normal control rats. Acidosis produced as described in Table 2.
earlier. The difference between 30.0 pm/g/90 min * 1.1 (SEM) and 26.5 pm/g/90 min f (SEM), was statistically significant (p < 0.02), while the difference between 2-hr sera on slices from control rats missed significance (p > .05 < .l). Sera removed from rats made acidotic for 4, 28, and 120 hr, compared to simultaneously tested control sera, had no difference in their effect on the ammoniagenesis of slices from control and acidotic rats. The amount of serum obtained from each rat was small; therefore, our initial studies were performed with 2 ml volumes. Since the amount of glutamine present in 2 ml of serum was limited and we may have experienced substrate limitations, we performed another series of studies using more serum (5 ml) and less tissue (50 mg) in each flask (Table 3). The slices were incubated in pooled sera rather than individual serum; but in every other respect, the procedures were the same as those previously described. Four observations were made for each condition. In 5 ml of medium containing both glutamine and lactate, control slices produced 20.7 pm/g * 0.8 (SEM) of ammonia, whileproducing 23.4 pm/g f I .3 (SEM) of ammonia in 5 ml of pooled control sera. The production, when glutamine was sole substrate in the medium, was 33.7 * 1.0 (SEM). Slices from 28 hr acidotic rats produced 49.3 pm/g * 3.6 (SEM) in glutamine, 43.5 pm/g * 2.6 (SEM) in glutamine + lactate, and 48.0 pm/g * 3.3 (SEM) in the presence of pooled sera. Analysis of glutamine and lactate in the serum after 90 minutes incubation with acidotic slices (n = 4) showed concentrations of 0.34 pm/ml * .07 (SEM) and 1.20 pm/ml * .I0 (SEM). When slices from normal control rats were incubated in pooled sera from the same rats, ammonia production was similar whether the initial medium pH was 7.4 or adjusted to 7.0 and 7.8. The average final pH following incubation were respectively 7.2 for that sera starting at 7.0, 7.5 for sera starting at 7.4 and 7.8 for sera starting at 7.8. In these studies, 100 mg slices were incubated in 2 ml of pooled sera. Following 4 hr dialysis, our pooled sera had an average lactate concentration of 0.13 pm/ml f 0.05 (SEM), (n = 5) compared to a lactate concentration of 1.80 pm/ml & .25 (SEM), (n = 5), found in the other half of the same pooled sera that had been stored on ice for 4 hr. The sera stored on ice were found to have a glutamine concentration of 0.8 mM prior to incubation. There was no measurable glutamine in the dialyzed portion. When rat kidney slices were incubated in
REGULATION
OF
RENAL
AMMONIAGENESIS
II
1643
dialyzed sera, the QO, averaged 3.62 pl/mg/hr (n = 2) and in regular sera was 4.54 pl/mg/hr (n = 2). This was an increase of approximately 20% and 50% over the QO, of slices incubating in Krebs medium without added substrate, 3.02 jd/mg/hr (n = 2). In slices from 10 normal control rats the average ammonia formation was 34.3 pm/g f 1.2 (SEM) in glutamine, 17.5 pm/g & 0.9 (SEM) in glutamine plus lactate (149%), 16.4 pm/g f 1.1 (SEM) (152%), in pooled sera and 23.6 pm/g * 0.7 (SEM) (131%) in dialyzed sera with added glutamine (0.8 mM). There was a significant difference in ammonia production between slices incubating in regular sera and the dialyzed sera (p < .Ol). Slices produced more ammonia in dialyzed sera. At the end of incubation, we found the final average glutamine concentrations (n = 2) to be .33 pm/ml in glutamine, .55 pm/ml in glutamine plus lactate, .45 pm/ml in regular sera and .47 pm/ml in dialyzed sera. As might be expected, the concentrations of lactate present at the end of a 90 min incubation were small when only glutamine was added to the medium or when slices ,incubated in the dialyzed medium. The concentrations averaged .08 pm/ml and .12 pm/ml respectively. Control slices incubating in serum ultrafiltrate produced less ammonia than those control slices incubating in medium containing glutamine and lactate (p < .05). This was true also when acidotic slices were used. However, the difference in ‘ammoniagenesis was less when acidotic slices were examined. The average decrease in ammoniagenesis between acidotic slices incubating in ultrafiltrate and in a medium containing only glutamine was 11.0 pm/g (125%). The average difference in the studies using normal control slices was 17.2 pm/g (162%) compared to the above (p < .05). DISCUSSION
The majority of previous studies have concentrated upon the intracellular aspects of ammonia adaptation by renal slices from acidotic rats. Less emphasis has been placed upon environmental factors, i.e., circulating extracellular regulators. Alleyne and Roobel’5 showed that renal slices preincubated in plasma (10% v/v) removed from rats 2 hr after acute acid challenge compared to slices preincubated in plasma from normal rats (10% v/v) produced more ammonia when incubated in medium containing glutamine. Studies of ammoniagenesis by slices incubating directly in serum (100% v/v) have not been reported. Also, no results have been reported using plasma or sera drawn from rats undergoing long periods ( >2 hr) of acid stress. Our most obvious finding is that slices from normal rats produce less ammonia when incubated in serum than when incubated in artificial medium containing similar amounts of glutamine (0.6 mM).16 Krebs noted this same phenomenon over 40 yr ago.” This inhibition by serum factors is not apparent in renal slices from rats that have been acidotic for 28 hr. In other words, chronically acidotic slices not only produce more ammonia than control slices in medium via intracellular adaptations, but resist the inhibition to glutamine ammoniagenesis that serum factors produce in normal slices. Major differences between the sera themselves (acidotic and control) were seen in only one of the tested conditions. Serum from rats made acutely acidotic for only 2 hr compared to serum from control rats showed modest increases in ammoniagenesis in control slices (+ 1 1%, p > 0.05 < 0.1) and acidotic slices (+ 13%,
PREUSS. BAIRD, AND
1644
Table 4.
Effects of Dialyzed Sara and Ultrafiltrates
of Sera on Ammonia
EASTMAN
Production
in Rat Kidney Slices Effects of Dialyzed Sera on Ammonlagenesls by Skes from Normal Rats Dtalvzed Sera GmOBmM
Gm08mM
Substrate
Gm 0.8 mM
SW3
LalEmM
34.3 * 1.2
17.5 f 0.9
164zt
1.1
23.6 f 0.7
Effects of Serum Ultrafiltrate on Ammontagenws by Slices from Normal and Acldotic Rats GmOBmM
Gm 0.8 mM La 1.8 mM
Normal slices
27.6 f
1.4
14.2 f 0.3
10.4 * 1.2
Acidotic slices
44.7 f 0.6
43.2 f 0.6
33.7 f 0.9
Substrate
Values are the average pm/g/90
Ultrafiltrate
min f SEM of slices from 4-8 rats.
Values of glutamine IGml and lactate (La) used in these studies are based on the values found in sera stored on ice for 4 hr Acidosis produced as described in Table 2.
p < 0.05). Although our models differ, these findings resemble those of Allyne and Roobel.‘” Therefore, our results may be due to the same unknown stimulator found in acutely acidotic plasma reported by Alleyne and Roobel.‘5 Suffice it to say, we could not see this relative stimulation by acidotic sera when the acidosis was more prolonged. In addition, no differences were apparent when control slices incubated in the same serum at pH 7.0,7.4, and 7.8. This strengthens the proposal that pH changes per se are not responsible for renal slice ammonia adaptation.’ What is in serum that inhibits renal ammoniagenesis from glutamine? Most slice studies concerned with ammoniagenesis are run in medium containing glutamine, the major ammonia precursor, as the only substrate. It has been noted in the past that oxidizable substrates such as lactate can inhibit ammoniagenesis from glutamine by rat renal slices *’ When circulating concentrations of lactate (I .2 mM)lR were added to the glutamine medium, the extent of ammoniagenesis simulated that of slices incubated in serum under comparable conditions. In a like manner, neither the presence of lactate nor serum decreased, to any great extent, ammoniagenesis by slices from acidotic rats. We chose to study lactate in detail because of its concentration in serum. However, other oxidizable substrates probably contribute to the depression. These similarities suggest to us that the oxidizable substrates in serum, such as lactate, are probably responsible for its major inhibition. Krebs arrived at a similar conclusion.” To strengthen our conclusions, we performed the studies depicted in Table 4. Initial attempts were made to dialyze oxidizable substrates from pooled sera. Although we were aware that it would be difficult to remove all substrates from the serum because of protein binding,lg some success was obtained. We reduced lactate concentrations over tenfold. However, some lactate and other oxidizable substrates probably were present, because dialyzed serum still increased oxygen consumption by rat kidney slices (t20%). With regular sera, there was a greater increase (150%) correlating with the presence of greater amounts of substrate. Importantly, slices incubated in dialyzed serum with added glutamine showed greater ammoniagenesis than slices incubated in the regular serum (p < .Ol), although less ammonia was produced compared to slices incubating in glutamine alone.
REGULATION OF RENAL AMMONIAGENESIS II
1645
Probably more cogent for our hypothesis is the similarity in trends following addition of serum ultrafiltrates and serum itself. In the second group of studies depicted in Table 4, we see that ultrafiltrates of serum depress ammoniagenesis of control slices even more than lactate. The greater depression may be due to the additional oxidizable factors present in serum (see Table I in reference 13). In contrast, the depressive effects by the ultrafiltrate are markedly lessened on acidotic slices. Although we cannot eliminate all possible causative factors, our results, taken together, suggest that lactate and other oxidizable factors play a significant role. By necessity, we initially incubated slices in small volumes of medium and serum (2 ml). A question could be raised whether the apparent resistance by acidotic slices to serum and lactate is due to substrate limitations. If during the course of incubation, medium glutamine concentrations became low when glutamine was sole substrate, production would be limited, allowing the slower ammoniagenesis by acidotic slices in the presence of lactate and serum to catch up during the latter part of incubation. In turn, a more rapid removal of oxidizable substrates such as lactate might also influence our final results. The data depicted in Fig. 1 were derived from experiments described in the first paper’” and in this one. There is no difference in ammoniagenesis by normal slices incubating in either 2 ml or 5 ml of medium containing glutamine and lactate. In I
I
I
8
I
4
2
5 MI
I
12 Medium
Fig. 1. Ammonia production by rat kidney slices in different volumes of medium. oControl slices in medium containing glutamine and lactate. lControl slices in medium containing glutamine. =Acidotic slices in medium containing glutamine. nAcidotic slices in medium containing glutamine and lactate. Values are from experiments performed in this paper and in the previous one.13
PREUSS.
1646
BAIRD.
AND
EASTMAN
all other cases, ammoniagenesis is greater in 5 ml of medium. However, ammoniagenesis by slices under all conditions is similar whether the slices incubate in 5 ml of medium or 12 ml of medium. Therefore, we would not experience substrate limitations in 5 ml of incubating medium. To rule out the possibility that substrate limitations were responsible for our findings, we repeated our studies in 5 ml volumes of media. To be doubly sure, we even added less tissue, 50 mg. With the greater volumes and lesser weights of acidotic tissue, more than one half of the glutamine in serum was still present after 90 min. Our results with acidotic slices incubating in 5 ml volumes confirmed the previous findings, i.e., that acidotic slices resist the lactate and serum inhibition of ammoniagenesis. These findings are important in at least two respects. First, although the majority of previous studies have been performed with glutamine as the sole oxidizable substrate, ammonia production in the presence of lactate is more like that seen in the natural environment, i.e., serum. Because of this, we feel that lactate should be included as a substrate in future in vitro studies concerned with ammoniagenesis. As a first approximation, lactate was studied because this is the most abundant oxidizable organic anion present in blood. However, any oxidizable substrate alone or in combination might produce the same results. Second, serum factors inhibit ammoniagenesis in control slices, but less so or not at all in acidotic slices. This resembles findings when lactate is added to a glutamine-containing medium.13 We conclude that lactate and other extracellular substrates in serum are inhibiting glutamine ammoniagenesis in renal tissue from normal control rats and that this inhibition is overcome greatly in acidotic renal tissue. Renal tissue adaptations during acidosis which would allow deinhibition of circulating factor like lactate could theoretically augment ammoniagenesis. ACKNOWLEDGMENT The authors greatly acknowledge Mend&on. and Patti Werr.
the editorial
and secretarial
assistance
of Susan
Dreux,
Betty
REFERENCES 1. Preuss HG, Weiss FR: Rate limiting factors in rat kidney slice ammoniagenesis. Am J Physiol 221:458-464, 1971 2. Relman AS, Narins RG: The control of ammonia production in the rat. Med Clin N Am 59:583-593,1975 3. VanSlyke DD, Phillips RA, Hamilton PB, et al: Glutamine as a source material of urinary ammonia. J Biol Chem 150:481-482, 1943 4. Pitts RF, Pilkington LA, deHaas JCM: N’” tracer studies on the origin of urinary ammonia in the acidotic dog. J Clin Invest 44:731-745, 1965 5. Hems DA: Biochemical aspects of renal ammonia formation in metabolic acidosis, Enzyme 20:359-380, 1975 6. Alleyne GAO: Renal metabolic response to acid-base changes. II. The early effects of metabolic acidosis on renal metabolism in the rat. J Clin Invest 49:943-95 I, 1970
7. Roxe DM, Schreiner GE, Preuss HG: Regulation of renal gluconeogenesis and ammoniagenesis by physiologic fuels. Am J Physiol 225:908891 I, 1973 8. Weidemann MJ, Krebs HA: The fuel of respiration of rat kidney cortex. Biochem J 112:149-166, 1969 9. Barac-Nieto M: Effects of lactate and glutamine on palmitate metabolism in rat kidney cortex. Am J Physiol231:14-19, 1976 10. Adler S, Preuss HG: Interrelationships between citrate metabolism, ammoniagenesis, and gluconeogenesis in renal cortex in vitro. J Lab and Clin Med 79:505-515, 1972 11. Sleeper RS, Vertuno LL, Strauss FR, et al: Effects of acid challenge on in vivo and in vitro rat renal ammoniagenesis. LifeSci 22:1561- 1572, 1978 12. Stadie WC, Riggs BC: Microtome for the
REGULATION
OF RENAL AMMONIAGENESIS
II
preparation of tissue slices for metabolic studies of surviving tissue in vitro. J Biol Chem 154:687-690, I944 13. Preuss HG, Eastman ST, Vavatsi-Manos 0, et al: The regulation of renal ammoniagenesis in the rat by extracellular factors. I. The combined effects of acidosis and physiologic fuels. Metabolism 27: 1626-1638, 1978 14. Preuss HG, Bise BW, Schreiner GE: The determination of glutamine in plasma and urine. Clin Chem 12:329-337, 1966 IS. Alleyne GAO, Roobel A: Regulation of renal cortex ammoniagenesis. I. Stimulation of renal cortex ammoniagenesis in vitro by plasma isolated from acutely acidotic rats. J Clin Invest 53:117-121, 1974
1647
16. Weiss FR, Preuss HG: Glutamine synthetase and plasma, glutamine in augmented ammoniagenesis in acidosis. Am J Physiol 218:1697-1700, 1970 17. Krebs HA: CXCVII Metabolism of amino acids. III Deamination of amino acids. Biochem J 29:1620-1644, 1935 18. Kliger AS, Eastman ST, Zachek M, et al: Effect of renal fuels on p-aminohippurate transport in rat renal cortical fragments. Metabolism 26:979-988, 1977 19. Hanson RW, Ballard FJ: Citrate, pyruvate, and lactate contaminants of commercial serum albumin. J Lipid Res 9:667-668, 1968