Br. J. exp. Path (1977) 58, 220
URINARY LDH ISOENZYME 5 EXCRETION IN EXPERIMENTAL PYELONEPHRITIS R. J. CUNNINGHAM, III, H. F. CARVAJAL AND R .B. PASSEY From the Department of Pediatrics, Divisions of Nephrology and Pathology, The University of Texas Medical Branch, Galveston, Texas, 77550 Received for publication November 26, 1976
Summary.-Studies to determine the effect of kidney parenchymal infection upon urinary lactic dehydrogenase (ULDH) isoenzyme composition were performed in 12 female Sprague-Dawley rats on Day 0 and on Days 2, 5, and 10, after experimental inducement of Escherichia coli pyelonephritis. An additional group of 12 animals was subjected to similar experimental manipulations and served as sham controls. Repeated 12-h urine collections revealed lower urine osmolalities and significantly higher levels of ULDH 5 excretion in the experimental than in the sham operated animals (P < 0-05). These differences persisted for the length of the experiment (10 days). Leucocyte excretion rates were also higher in the experimental than in the sham group, and a high correlation with ULDH 5 activity was demonstrated (r = 0.815). No other evidence that the two variables may be causally related was found.
MEASUREMENTS of urinary lactic dehydrogenase have been of interest to clinical investigators since the late 1950s when it was found that total urinary LDH (ULDH) was increased in a wide spectrum of renal diseases (Walker and Dorfman, 1962; Rosalki and Wilkinson, 1959). Markert first described LDH isoenzymes (Markert and Moller, 1959) and demonstrated that although these compounds had the same substrate specificity, they differed in their physical, chemical, and immunological behaviour. In the absence of disease, normal human urine contains a predominance of ULDH 1 and ULDH 2 (fast zone enzymes) and very little slow zone activity (Carvajal, Passey and Berger, 1975; Dubach, 1966). In patients with clinical pyelonephritis the isoenzyme composition of the urine changes from a predominance of the fast zone isoenzymes to a " slow zone pattern ". The present study was undertaken to test whether or not experimental pyelonephritis in the rat resulted in a change in ULDH isoenzyme composition analogous
to the clinical situation, and to determine the feasibility of using such a model for further investigations. MATERIALS AND METHODS
Twenty-four female Sprague-Dawley rats weighing 250-300 g constituted the subjects of the study. These animals were maintained in cages on Purina rat chow for 7 days prior to inducement of bilateral kidney infections. The animals were placed in metabolic cages connected to a small refrigerator so that urine flowed from the collection chamber to containers inside the refrigerator by gravity. Two consecutive 12 h urines wrere collected on Day 0 and on Days 2, 5, and 10, following inducement of the infection. Experimental pyelonephritis was induced in 12 of the animals according to a modification of the method described by Miller and Robinson (1973). Under light anaesthesia with Penthrane, the backs of the animals were shaved. The kidneys were exposed through two flank incisions and injected in 3 separate areas with a total of 0-06 ml per kidney of an Esch. coli (075) suspension in phosphate buffer containing approximately 109 organisms/ml. Injections were accomplished by means of 27gauge needles connected to Hamilton ® syringes. Sham controls (n= 12) were injected with an equivalent amount of phosphate buffer. Following injection the incisions were closed by
221
LDH 5 EXCRETION IN EXPERIMENTAL PYELONEPHRITIS
layers writh stainless steel wire. In order to avoid contamination of the urine, food and water were withheld during the collection period. Urinary LDH isoenzyme activity of undialysed urine was assayed within 4 h of urine collection by a previously described method (Carvajal et al., 1975). In addition, a complete urinalysis, leucocyte excretion rates and osmolality (Osmete A, Precision Instruments) were obtained on all urine samples. Except for the osmolality, which was only determined on the second 12-h collection, all other measurements were performed on 24 h urines. On the 11th postoperative day, all animals were killed with ether and both kidneys were removed using sterile techniques. The intact kidneys were immediately dipped in alcohol and flamed for 5-10 seconds. Thereafter, the organs were sectioned longitudinally and swab cultures obtained. The kidneys were then homogenized in sterile saline and cultured in meat broth, blood agar, and McConkey's agar. Cultures were considered positive if an overgrowth of Esch. coli was present on all 3 media. If an organism other than Esch. coli grew out, the subject was eliminated from the study. If only 1 or 2 colonies grew out on 1 of the 3 media, the cultures were considered negative and subjects included in the study. Statistical analysis was accomplished according to the analysis of variance technique for a two-factor experiment with repeated Values of measurements of one factor. P -< 005 were considered statistically significant.
3.2/
2.8
2.4 +
/
2.0
1.2-
n
.81
Sham Experimental
1 Mean ± SEM p >0.05
.40-
2
0
10
5
Days
FIG. 1. Total LDH activity {log + 1 (mU/24 h)} in the uirine of experimental (0) and sham (*) operated animals (rats) on Day 0 and on Days 2, 5, and 10 following inducement of a kiclney infection.
3.0*
n - 20 e~~~oSham 0
/ 2.0-
1.
Experimental
iMean ± SEM ~~~ X*P(0.05
~
ss
s
/
0-
0
2
RESULTS
Twenty-three of the 24 animals survived for the 11-day period. Esch. coli was grown from kidney homogenates in all experimental animals. Three rats were excluded from the study; one experimental animal who, in addition to Esch. coli, grew enterobacter from both kidney homogenates and 2 control animals whose kidney cultures grew Esch. coli. Data from 20 animals (10 in each group) were available for analysis. There were no significant differences (on any of the 4 variables measured) between the experimental and shamoperated groups on Day 0. After inducement of pyelonephritis, all animals disclosed significant increments in total ULDH activity (Fig. 1), ULDH 5 (Fig. 2), and leucocyte excretion rates (Fig. 3). The latter 2 variables were higher in the
-20
* o
5
10
Days
FIG. 2. LDH isoenzyme 5 activity {log + 1 (mU/24 h)} in the urine of the same animals described in Fig. 1.
8.0*
n 20 Sham
0
Experimental
-
7.5-
*
Mean
±
SEM
*
7.0-
-~--
-
//
6.5-
-----
--
-
/
6.0 5.5-
IA
5.04. u %-
-
§
0
5
2
Days
Leucocyte excretion rate {log x 105 (cells/24 h)} in the urine of the same animals described in Fig. 1.
FIG. 3.
10
222
R. J. CUNNINGHAM, H. F. CARVAJAL AND R. B. PASSEY 3.3
,2
*
3.1
2! E ^
3.
=O7
2.
_-?
-..
----------- 5
n -20 Sham Experimental i Mean i SEM
2.
*
o
2.7
*P
URINARY LDH ISOENZYME 5 EXCRETION IN EXPERIMENTAL PYELONEPHRITIS R. J. CUNNINGHAM, III, H. F. CARVAJAL AND R .B. PASSEY From the Department of Pediatrics, Divisions of Nephrology and Pathology, The University of Texas Medical Branch, Galveston, Texas, 77550 Received for publication November 26, 1976
Summary.-Studies to determine the effect of kidney parenchymal infection upon urinary lactic dehydrogenase (ULDH) isoenzyme composition were performed in 12 female Sprague-Dawley rats on Day 0 and on Days 2, 5, and 10, after experimental inducement of Escherichia coli pyelonephritis. An additional group of 12 animals was subjected to similar experimental manipulations and served as sham controls. Repeated 12-h urine collections revealed lower urine osmolalities and significantly higher levels of ULDH 5 excretion in the experimental than in the sham operated animals (P < 0-05). These differences persisted for the length of the experiment (10 days). Leucocyte excretion rates were also higher in the experimental than in the sham group, and a high correlation with ULDH 5 activity was demonstrated (r = 0.815). No other evidence that the two variables may be causally related was found.
MEASUREMENTS of urinary lactic dehydrogenase have been of interest to clinical investigators since the late 1950s when it was found that total urinary LDH (ULDH) was increased in a wide spectrum of renal diseases (Walker and Dorfman, 1962; Rosalki and Wilkinson, 1959). Markert first described LDH isoenzymes (Markert and Moller, 1959) and demonstrated that although these compounds had the same substrate specificity, they differed in their physical, chemical, and immunological behaviour. In the absence of disease, normal human urine contains a predominance of ULDH 1 and ULDH 2 (fast zone enzymes) and very little slow zone activity (Carvajal, Passey and Berger, 1975; Dubach, 1966). In patients with clinical pyelonephritis the isoenzyme composition of the urine changes from a predominance of the fast zone isoenzymes to a " slow zone pattern ". The present study was undertaken to test whether or not experimental pyelonephritis in the rat resulted in a change in ULDH isoenzyme composition analogous
to the clinical situation, and to determine the feasibility of using such a model for further investigations. MATERIALS AND METHODS
Twenty-four female Sprague-Dawley rats weighing 250-300 g constituted the subjects of the study. These animals were maintained in cages on Purina rat chow for 7 days prior to inducement of bilateral kidney infections. The animals were placed in metabolic cages connected to a small refrigerator so that urine flowed from the collection chamber to containers inside the refrigerator by gravity. Two consecutive 12 h urines wrere collected on Day 0 and on Days 2, 5, and 10, following inducement of the infection. Experimental pyelonephritis was induced in 12 of the animals according to a modification of the method described by Miller and Robinson (1973). Under light anaesthesia with Penthrane, the backs of the animals were shaved. The kidneys were exposed through two flank incisions and injected in 3 separate areas with a total of 0-06 ml per kidney of an Esch. coli (075) suspension in phosphate buffer containing approximately 109 organisms/ml. Injections were accomplished by means of 27gauge needles connected to Hamilton ® syringes. Sham controls (n= 12) were injected with an equivalent amount of phosphate buffer. Following injection the incisions were closed by
221
LDH 5 EXCRETION IN EXPERIMENTAL PYELONEPHRITIS
layers writh stainless steel wire. In order to avoid contamination of the urine, food and water were withheld during the collection period. Urinary LDH isoenzyme activity of undialysed urine was assayed within 4 h of urine collection by a previously described method (Carvajal et al., 1975). In addition, a complete urinalysis, leucocyte excretion rates and osmolality (Osmete A, Precision Instruments) were obtained on all urine samples. Except for the osmolality, which was only determined on the second 12-h collection, all other measurements were performed on 24 h urines. On the 11th postoperative day, all animals were killed with ether and both kidneys were removed using sterile techniques. The intact kidneys were immediately dipped in alcohol and flamed for 5-10 seconds. Thereafter, the organs were sectioned longitudinally and swab cultures obtained. The kidneys were then homogenized in sterile saline and cultured in meat broth, blood agar, and McConkey's agar. Cultures were considered positive if an overgrowth of Esch. coli was present on all 3 media. If an organism other than Esch. coli grew out, the subject was eliminated from the study. If only 1 or 2 colonies grew out on 1 of the 3 media, the cultures were considered negative and subjects included in the study. Statistical analysis was accomplished according to the analysis of variance technique for a two-factor experiment with repeated Values of measurements of one factor. P -< 005 were considered statistically significant.
3.2/
2.8
2.4 +
/
2.0
1.2-
n
.81
Sham Experimental
1 Mean ± SEM p >0.05
.40-
2
0
10
5
Days
FIG. 1. Total LDH activity {log + 1 (mU/24 h)} in the uirine of experimental (0) and sham (*) operated animals (rats) on Day 0 and on Days 2, 5, and 10 following inducement of a kiclney infection.
3.0*
n - 20 e~~~oSham 0
/ 2.0-
1.
Experimental
iMean ± SEM ~~~ X*P(0.05
~
ss
s
/
0-
0
2
RESULTS
Twenty-three of the 24 animals survived for the 11-day period. Esch. coli was grown from kidney homogenates in all experimental animals. Three rats were excluded from the study; one experimental animal who, in addition to Esch. coli, grew enterobacter from both kidney homogenates and 2 control animals whose kidney cultures grew Esch. coli. Data from 20 animals (10 in each group) were available for analysis. There were no significant differences (on any of the 4 variables measured) between the experimental and shamoperated groups on Day 0. After inducement of pyelonephritis, all animals disclosed significant increments in total ULDH activity (Fig. 1), ULDH 5 (Fig. 2), and leucocyte excretion rates (Fig. 3). The latter 2 variables were higher in the
-20
* o
5
10
Days
FIG. 2. LDH isoenzyme 5 activity {log + 1 (mU/24 h)} in the urine of the same animals described in Fig. 1.
8.0*
n 20 Sham
0
Experimental
-
7.5-
*
Mean
±
SEM
*
7.0-
-~--
-
//
6.5-
-----
--
-
/
6.0 5.5-
IA
5.04. u %-
-
§
0
5
2
Days
Leucocyte excretion rate {log x 105 (cells/24 h)} in the urine of the same animals described in Fig. 1.
FIG. 3.
10
222
R. J. CUNNINGHAM, H. F. CARVAJAL AND R. B. PASSEY 3.3
,2
*
3.1
2! E ^
3.
=O7
2.
_-?
-..
----------- 5
n -20 Sham Experimental i Mean i SEM
2.
*
o
2.7
*P
