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Kaliopenic nephrosis: protective action of l -ornithine-l -aspartate
CLINICA CHIMICA ACTA
CCA
4856
KALIOPENIC
NEPHROSIS
: PROTECTIVE
ACTION
OF L-ORNITHINE-
L-ASPARTATE
W. P. RAAB
Vienna University
Medical
School,
Department
of Medical
Chemistry,
Vienna
(Austria)
(Received October 26, 1971)
SUMMARY
In rats, renal changes in potassium ed by determinations alkaline phosphatase,
depletion
(kaliopenic
nephrosis)
were record-
of renal enzyme excretion (urinary lactate dehydrogenase, and “leucine aminopeptidase” activities). In animals which
were treated with intraperitoneal injections of L-ornithine-L-aspartate (zoo mg/kg), the increase in urinary lactate dehydrogenase and alkaline phosphatase activities was significantly lower than in untreated potassium-depleted controls. The mechanisms by which L-ornithine-L-aspartate could exert its protective action in kaliopenic nephrosis
are briefly
discussed.
INTRODUCTION
In animal experiments, L-ornithine-L-aspartate gave protection against toxic effects of various types, mainly against those which affect the liver metabolism1p3. Liver damage in potassium depletion, too, was less in rats treated with L-ornithineL-aspartate4. Potassium depletion is not only accompanied by disturbances of liver metabolism, but provokes also severe nephrosis (kaliopenic nephrosis). The question was raised whether the administration of L-ornithine-L-aspartate would lower renal damage in potassium depletion. It has been shown in previous investigations 5*6 that determinations of renal enzyme excretion (determinations of urinary enzymatic activities) are the most sensitive method to collect information on the intensity of renal damage; especially in kaliopenic nephrosis, marked elevation of renal enzyme excretion was found at a stage where morphologic alterations were still absent. In this investigation, experiments are described comparing the intensity of kaliopenic nephrosis in normal rats and in rats treated with L-ornithine-L-aspartate, Iusing the renal enzvme excretion technique.
Clin. Chim.
Acta,
'Jo (1972)
zjg-242
240
RAAB
MATERIAL AND METHODS Animals The experiments were performed on male rats of the strain FW 49, Biberach. The animals weighed 250 + 25 g. 24-h urine specimens were collected in metabolic cages. At the beginning of each 24-h urine collection period, 10.0 ml of water were given by stomach tube in order to ascertain sufficient diuresis. Altogether, 35 animals I.
were used. Normal urinary enzyme activities were determined as the mean value of two consecutive days at the beginning of the experimental series. 2. Potassium depletion Potassium depletion was elicited in 30 rats by feeding standard laboratory diet with 4% sodium polystyrene sulfonate. This resin exchanges potassium against sodium and-as has been shown previously-causes marked hypokaliemia5v6. Fifteen rats out of this group received daily injections of saline (1.0 ml 0.15 M saline), the remaining 15 animals were given L-ornithine-L-aspartate (cf. point 3). 3. L-Omithilte-L-asjbartate For the three weeks of potassium depletion, 15 rats received dailyintraperitoneal injections of L-ornithine-L-aspartate-generously supplied by hlerz & Co., Frankfurt -in a dosage of 200 mg/kg body weight. The substance was administered in saline. For controlling the influence of L-ornithine-L-aspartate on renal enzyme excretion, 5 normal rats were given daily injections 4. Controls As controls potassium
served the untreated
of this substance over three weeks.
(saline-treated)
rats under the conditions
depletion and the 5 rats under L-ornithine-L-aspartate
Determinations
of enzymatic activities
of
treatment.
in urine
In all of the rats, renal enzyme excretion was determined at the beginning of the experiments and after three weeks. From the 24-h urine portion, 5 ml were centrifuged (5 min at 4000 rev./min). A sample was taken for the determination of “leucine aminopeptidase” (LAP, E.C. 3.4.1.2,) the rest was dialyzed against running tap water for3 h. In this portion, lactate dehydrogenase (LDH, E.C. 1.1.1.21) and alkaline phosphatase (AP, E.C. 3.1.3.1) activities were determined. Standard methods of clinical chemistry were applied for determinations of enzymatic activities (details cf 8). RESULTS Controls As compiled in Table I, normal values of urinary enzymatic activities in these series amounted to 161 :i_31 mU LDH, 177 & 24 mU AP, and 47 5 12 mU LAP. All values were calculated on the total 24-h urine portion. It has been shown previously6 that saline remains without significant influence on urinary enzymatic activities.
I.
2. L-Omithine-L-aspartate As with saline, no significant alterations of renal enzyme excretion were recorded in rats under treatment with L-ornithine-L-aspartate.
TREATMENT TABLE RENAL
FOR KALIOPENIC
241
NEPHROSIS
I ENZYME
EXCRETION
I?+ RATS
WITH
XALIOPESIC
IUEPHROSIS, WITH AND WITHOUT
PRETREAT-
MENT WITH L-OXNITHINE-L-ASPARTATE zoo mg/kg bodyweight. Lactate dehydrogenase = LDH, alkaline phosphatase aminopeptidase” == LAP. All values given in mU per 24-h urine specimens. __ ._._
__-.-
h’o. of rats
LDH
AP
LAP
35 5 15
161 * 31 176 * 40 578 rt 104 (-260%)
177 i 24 154 -rt 32 481 + 63 (+17o%)
47 It 12 51 & 18 85 i: 19 (‘,So%)
15
452 i 88 (i-180%)
292 * 47 (-#65%)
75 J- 16 (+6o%)
Normal controls (mean values of two consecutive days) Omithine-aspartate control Kaliopenic nephrosis Kaliopenic nephrosis and pretreatment ornithine-aspartate
= AP, “leucine
with
3. Potassium depletion
In confirmation of previous data 576,potassium depletion provokes statistically significant changes in urinary enzymatic activities; urinary LDH activity rose to 3.6 fold, urinary AP activity rose to 2.7 fold, and urinary LAP activity to 1.8 fold of normal values (cf. Table I). 4. Potassium
depletion in L-ornithine-L-aspartate-treated
rats
In rats treated with r.-ornithine-L-aspartate, potassium depletion provoked comparatively lower increases in urinary enzymatic activities: LDH rose to 2.8 fold, AP rose to 1.65 fold, and LAP to 1.6 fold. In the case of urinary LDH and AP activities, the difference between treated animals and plainly potassium-depleted controls proved statistically significant (9 ~0.02). Details in Table I and Fig. I.
Controls kaliopenic sis
wfth nephm-
Retreatment with crnithine - aspartate kaliopenic nephfuses
Fig. 1. Inhibition of increase in renal enzyme excretion caused by kaliopenic nephrosis by pretreatment with r_-ornithine-1-aspartate (dose: zoo mg/kg body weight). Left columns: renal enzyme excretion in kaliopenic controls; right columns: renal enzyme excretion in kaliopenic rats pretreated with I--ornithine_L-aspartate. For comparative purposes, in the left columns normal enzyme excretion is marked with an N. Lactate dehydrogenase = LDH, alkaline phosphatase = AP, “leucine aminopeptidase” = LAP. All values were calculated on the total 24-h urine portion. &fin. Chim. Acta, 39
(1972)
2yj--242
RAAX
242 DISCUSSIOK
Renal enzyme excretion, the determination of which has proved to be a valuable tool for handling various problems in clinical chemistry7, permits-among others-the registration of changes in renal tubular cells. Potassium depletion which provokes the well-known kaliopenic nephrosis is regularly accompanied by a marked rise in urinary LDH, AP, and LAP activities5y6. It should be recalled that there are considerable variations in urinary enzyme output among rats of different strains, of different age groups and of different sexes. Therefore, in every e~perinlental series, “normal” values (mean of two consecutive days prior to administration of test substances) have to be determined separately. Normal values in the series reported here correspond largely to similar series run in this laboratory. The results presented
here demonstrate
that the administration
of L-ornithine-
L-aspartate, a substance which is generally known as liver-protecting agent@, lowers the severity of renal tubular changes in l~~pokaliemia. The mechanism by which L-ornithine-L-aspartate exerts such effect remains still to be elucidated. Determinations of liver potassium content ruled out the possibility that L-ornithine-L-aspartate might decrease potassium
’
Ornithlne Fig.
I. Chemical
loss of the organism4.
form&
Aspartic
acid
of ornithiile-nspnrtate.
In the organisms, ornithine-aspartate, the formula of which is given in Fig. 2, is split into ornithine and aspartic acid. These products stimulate urea cycle and activate citrate cycle. It is assumed that these effects account for the general detoxicating action of L-ornithine-L-aspartate which was shown in endogenous hyperammonieand in general exhaustion liberating numerous toxic mia”, in drug intoxication3 metabolitesl .In respect to kaliopenic nephrosis, L-ornithine-r.-aspartate might increase resistance of tubular epithelia by accelerating ,4TP synthesis or it might interfere with the toxic consequences of potassium depletion by its influence on NH,+t-, NH,+IIlbalance. REFERENCES 1 L. CUTISELLI, L. SORREKTIKO,C.TRAMONTI, fovsch., zo (1970) 1064.
C!in.
Chim.
4cta,
39 (1972)
zj9--242
F. SALVATOREASD
F. CEURA~GOLO,AI'Z?ZL~~~~~~~!~-
CCA
4856
KALIOPENIC
NEPHROSIS
: PROTECTIVE
ACTION
OF L-ORNITHINE-
L-ASPARTATE
W. P. RAAB
Vienna University
Medical
School,
Department
of Medical
Chemistry,
Vienna
(Austria)
(Received October 26, 1971)
SUMMARY
In rats, renal changes in potassium ed by determinations alkaline phosphatase,
depletion
(kaliopenic
nephrosis)
were record-
of renal enzyme excretion (urinary lactate dehydrogenase, and “leucine aminopeptidase” activities). In animals which
were treated with intraperitoneal injections of L-ornithine-L-aspartate (zoo mg/kg), the increase in urinary lactate dehydrogenase and alkaline phosphatase activities was significantly lower than in untreated potassium-depleted controls. The mechanisms by which L-ornithine-L-aspartate could exert its protective action in kaliopenic nephrosis
are briefly
discussed.
INTRODUCTION
In animal experiments, L-ornithine-L-aspartate gave protection against toxic effects of various types, mainly against those which affect the liver metabolism1p3. Liver damage in potassium depletion, too, was less in rats treated with L-ornithineL-aspartate4. Potassium depletion is not only accompanied by disturbances of liver metabolism, but provokes also severe nephrosis (kaliopenic nephrosis). The question was raised whether the administration of L-ornithine-L-aspartate would lower renal damage in potassium depletion. It has been shown in previous investigations 5*6 that determinations of renal enzyme excretion (determinations of urinary enzymatic activities) are the most sensitive method to collect information on the intensity of renal damage; especially in kaliopenic nephrosis, marked elevation of renal enzyme excretion was found at a stage where morphologic alterations were still absent. In this investigation, experiments are described comparing the intensity of kaliopenic nephrosis in normal rats and in rats treated with L-ornithine-L-aspartate, Iusing the renal enzvme excretion technique.
Clin. Chim.
Acta,
'Jo (1972)
zjg-242
240
RAAB
MATERIAL AND METHODS Animals The experiments were performed on male rats of the strain FW 49, Biberach. The animals weighed 250 + 25 g. 24-h urine specimens were collected in metabolic cages. At the beginning of each 24-h urine collection period, 10.0 ml of water were given by stomach tube in order to ascertain sufficient diuresis. Altogether, 35 animals I.
were used. Normal urinary enzyme activities were determined as the mean value of two consecutive days at the beginning of the experimental series. 2. Potassium depletion Potassium depletion was elicited in 30 rats by feeding standard laboratory diet with 4% sodium polystyrene sulfonate. This resin exchanges potassium against sodium and-as has been shown previously-causes marked hypokaliemia5v6. Fifteen rats out of this group received daily injections of saline (1.0 ml 0.15 M saline), the remaining 15 animals were given L-ornithine-L-aspartate (cf. point 3). 3. L-Omithilte-L-asjbartate For the three weeks of potassium depletion, 15 rats received dailyintraperitoneal injections of L-ornithine-L-aspartate-generously supplied by hlerz & Co., Frankfurt -in a dosage of 200 mg/kg body weight. The substance was administered in saline. For controlling the influence of L-ornithine-L-aspartate on renal enzyme excretion, 5 normal rats were given daily injections 4. Controls As controls potassium
served the untreated
of this substance over three weeks.
(saline-treated)
rats under the conditions
depletion and the 5 rats under L-ornithine-L-aspartate
Determinations
of enzymatic activities
of
treatment.
in urine
In all of the rats, renal enzyme excretion was determined at the beginning of the experiments and after three weeks. From the 24-h urine portion, 5 ml were centrifuged (5 min at 4000 rev./min). A sample was taken for the determination of “leucine aminopeptidase” (LAP, E.C. 3.4.1.2,) the rest was dialyzed against running tap water for3 h. In this portion, lactate dehydrogenase (LDH, E.C. 1.1.1.21) and alkaline phosphatase (AP, E.C. 3.1.3.1) activities were determined. Standard methods of clinical chemistry were applied for determinations of enzymatic activities (details cf 8). RESULTS Controls As compiled in Table I, normal values of urinary enzymatic activities in these series amounted to 161 :i_31 mU LDH, 177 & 24 mU AP, and 47 5 12 mU LAP. All values were calculated on the total 24-h urine portion. It has been shown previously6 that saline remains without significant influence on urinary enzymatic activities.
I.
2. L-Omithine-L-aspartate As with saline, no significant alterations of renal enzyme excretion were recorded in rats under treatment with L-ornithine-L-aspartate.
TREATMENT TABLE RENAL
FOR KALIOPENIC
241
NEPHROSIS
I ENZYME
EXCRETION
I?+ RATS
WITH
XALIOPESIC
IUEPHROSIS, WITH AND WITHOUT
PRETREAT-
MENT WITH L-OXNITHINE-L-ASPARTATE zoo mg/kg bodyweight. Lactate dehydrogenase = LDH, alkaline phosphatase aminopeptidase” == LAP. All values given in mU per 24-h urine specimens. __ ._._
__-.-
h’o. of rats
LDH
AP
LAP
35 5 15
161 * 31 176 * 40 578 rt 104 (-260%)
177 i 24 154 -rt 32 481 + 63 (+17o%)
47 It 12 51 & 18 85 i: 19 (‘,So%)
15
452 i 88 (i-180%)
292 * 47 (-#65%)
75 J- 16 (+6o%)
Normal controls (mean values of two consecutive days) Omithine-aspartate control Kaliopenic nephrosis Kaliopenic nephrosis and pretreatment ornithine-aspartate
= AP, “leucine
with
3. Potassium depletion
In confirmation of previous data 576,potassium depletion provokes statistically significant changes in urinary enzymatic activities; urinary LDH activity rose to 3.6 fold, urinary AP activity rose to 2.7 fold, and urinary LAP activity to 1.8 fold of normal values (cf. Table I). 4. Potassium
depletion in L-ornithine-L-aspartate-treated
rats
In rats treated with r.-ornithine-L-aspartate, potassium depletion provoked comparatively lower increases in urinary enzymatic activities: LDH rose to 2.8 fold, AP rose to 1.65 fold, and LAP to 1.6 fold. In the case of urinary LDH and AP activities, the difference between treated animals and plainly potassium-depleted controls proved statistically significant (9 ~0.02). Details in Table I and Fig. I.
Controls kaliopenic sis
wfth nephm-
Retreatment with crnithine - aspartate kaliopenic nephfuses
Fig. 1. Inhibition of increase in renal enzyme excretion caused by kaliopenic nephrosis by pretreatment with r_-ornithine-1-aspartate (dose: zoo mg/kg body weight). Left columns: renal enzyme excretion in kaliopenic controls; right columns: renal enzyme excretion in kaliopenic rats pretreated with I--ornithine_L-aspartate. For comparative purposes, in the left columns normal enzyme excretion is marked with an N. Lactate dehydrogenase = LDH, alkaline phosphatase = AP, “leucine aminopeptidase” = LAP. All values were calculated on the total 24-h urine portion. &fin. Chim. Acta, 39
(1972)
2yj--242
RAAX
242 DISCUSSIOK
Renal enzyme excretion, the determination of which has proved to be a valuable tool for handling various problems in clinical chemistry7, permits-among others-the registration of changes in renal tubular cells. Potassium depletion which provokes the well-known kaliopenic nephrosis is regularly accompanied by a marked rise in urinary LDH, AP, and LAP activities5y6. It should be recalled that there are considerable variations in urinary enzyme output among rats of different strains, of different age groups and of different sexes. Therefore, in every e~perinlental series, “normal” values (mean of two consecutive days prior to administration of test substances) have to be determined separately. Normal values in the series reported here correspond largely to similar series run in this laboratory. The results presented
here demonstrate
that the administration
of L-ornithine-
L-aspartate, a substance which is generally known as liver-protecting agent@, lowers the severity of renal tubular changes in l~~pokaliemia. The mechanism by which L-ornithine-L-aspartate exerts such effect remains still to be elucidated. Determinations of liver potassium content ruled out the possibility that L-ornithine-L-aspartate might decrease potassium
’
Ornithlne Fig.
I. Chemical
loss of the organism4.
form&
Aspartic
acid
of ornithiile-nspnrtate.
In the organisms, ornithine-aspartate, the formula of which is given in Fig. 2, is split into ornithine and aspartic acid. These products stimulate urea cycle and activate citrate cycle. It is assumed that these effects account for the general detoxicating action of L-ornithine-L-aspartate which was shown in endogenous hyperammonieand in general exhaustion liberating numerous toxic mia”, in drug intoxication3 metabolitesl .In respect to kaliopenic nephrosis, L-ornithine-r.-aspartate might increase resistance of tubular epithelia by accelerating ,4TP synthesis or it might interfere with the toxic consequences of potassium depletion by its influence on NH,+t-, NH,+IIlbalance. REFERENCES 1 L. CUTISELLI, L. SORREKTIKO,C.TRAMONTI, fovsch., zo (1970) 1064.
C!in.
Chim.
4cta,
39 (1972)
zj9--242
F. SALVATOREASD
F. CEURA~GOLO,AI'Z?ZL~~~~~~~!~-