- Email: [email protected]
Metabolic studies of glycerol-induced acute renal failure in the rat
EXPERIMENTAL
AND
Metabolic ANNA Department
MOLECULAR
Studies L. TRIFILLIS,
of Pathology,
PATHOLOGY
35, l- 13 (1981)
of Glycerol-Induced in the Rat’ MYONG
University
W. KAHNG,
of Maryland Received
School
September
Acute
Renal
AND BENJAMIN of Medicine,
Baltimore,
Failure
F. TRUMP Maqland
21201
19. 1980
Glycerol was used to induce acute renal failure (ARF) in rats. It caused a decrease of kidney ATP levels to 48% of control at 6 hr which remained low up to 48 hr. ATP+ADP+AMP(AXP) was 55% of control at 6 and 24 hr and 64% at 48 hr. Lactate levels were fourfold control levels at 0.25, 1, and 6 hr, and twofold at 24 and 48 hr. Hemoglobinemia and hemoglobinuria occurred within 0.25 hr. By light microscopy, casts at 6 hr, necrosis of renal cortex at 24 hr, and necrosis with some regeneration at 48 hr were seen. Fructose at the same dose also lowered ATP and AXP levels to a similar degree as glycerol within the same time intervals, but hemoglobinemia, hemoglobinuria, necrosis, or ARF was not evident. Dihydroxyacetone at the same dose maintained ATP and AXP at control values with no hemoglobinemia, hemoglobinuria, necrosis, or ARF. Both fructose and dihydroxyacetone resulted in high lactate levels (sevenfold) within 0.25 hr. Saline loading prior to glycerol injection ameliorated ARF but did not improve the AXP levels or the morphologic lesion. Results indicate that glycerol-induced ARF is not solely the result of a hyperosmolar effect, adenosine release, casts, or pigments. The changes in AXP (particularly ATP) levels reflect an early metabolic disturbance which is part of the process and may predispose toward ARF in the presence of additional factors.
INTRODUCTION Acute renal failure (ARF) has been categorized into two general groups based upon the initiating event, ischemic and nephrotoxic (Heptinstall, 1974). The ischemit lesion is associated with crush injury, hemorrhage, and hypotension, while the nephrotoxic lesion is associated with agents such as aminoglycosides, HgC12, CCll, and ethylene glycol. Experimental animal models have been developed to simulate these clinical situations. While no animal model of ARF is strictly comparable to the circulatory type of human ARF, the intramuscular (im) injection of glycerol has been proposed as a model of ARF closely resembling the syndrome of ARF in man (Preuss et al., 1975; Torres et al., 1975). Although many investigations have been concerned with functional (Oken et al., 1966; Ayer Pt al., 1971) and morphological (Finckh, 1957; Carroll et al., 1965) alterations in glycerol-induced ARF, little attention has been paid to renal metabolic perturbations which may be operative in the initiation or maintenance of ARF. In the present study, we have examined the changes in renal concentrations of adenine nucleotides and lactate for up to 48 hr following glycerol-induced ARF. In order to gain further insight into the underlying mechanism of glycerolinduced ARF, the metabolic effects of saline loading prior to glycerol treatment, fructose, and dihydroxyacetone were studied. Saline loading prior to glycerol treatment has been shown to ameliorate glycerol-induced ARF (Thiel et al., 1970). Fructose is known to cause a rapid decrease in adenine nucleotide levels similar to is similar to that caused by glycerol (Burch et al., 1970); and dihydroxyacetone 1 This is contribution No. 886 from the Cellular Pathobiology Laboratory and was supported in part by NIH Grant AM 15440-08A2. Part of this work was presented at the 1980 FASEB Meetings in Anaheim. Calif. 1 0014-4800/81/040001-13$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
2
TRIFILLIS,
KAHNG,
AND
TRUMP
glycerol in size and structure. In light of the metabolic, functional, and morphological results obtained, a possible pathogenetic mechanism of glycerol-induced ARF is discussed. MATERIALS AND METHODS Animal and tissue treatment. Male Sprague-Dawley rats weighing 220-320 g were fed standard Purina laboratory chow and were water deprived for 16 hr prior to treatment. Rats were divided into six experimental groups: (1) Control (1% NaCl, 10 ml/kg body wt, im, hindlimb); (2) saline-loaded control (1% NaCl instead of drinking water for 4 weeks prior to sacrifice); (3) glycerol (50 g/100 ml saline or 5.4 M glycerol, 10 ml/kg body wt, im); (4) saline-loaded glycerol (1% NaCl instead of drinking water 4 weeks prior to 5.4 M glycerol, 10 ml/kg body wt, im); (5) fructose (5.4 M fructose, 10 ml/kg body wt, im); (6) dihydroxyacetone (5.4 M dihydroxyacetone, 10 ml/kg body wt, im). Fifteen minutes prior to treatment, all animals were anesthetized with sodium pentobarbital (35 mg/kg body wt, intraperitoneally). Kidneys and blood were sampled at 0, 0.25, 1, 6, 24, and 48 hr after treatment. The left kidneys were gently decapsulated and freeze-clamped in situ with aluminum tongs precooled in liquid N,. They were then immersed and trimmed under liquid Nz for metabolite assays. The right kidneys were bisected longitudinally and immersion-fixed in 4% formaldehyde and 1% glutaraldehyde (4F- 1G) buffered with sodium phosphate, pH 7.2 (McDowell and Trump, 1976), processed for light microscopy and stained with H&E and PAS. Blood samples for creatinine determination were taken from the aorta. Serum creatinine levels were used as an index of renal function. Tissue extract preparation. The freeze-clamped tissue was ground with a mortar and pestle under liquid NZ, weighed, and homogenized in 4 vol of 0.6 N HClO,. The homogenate was centrifuged at 1200,g for 15 min and the supernatant was neutralized with 2 M KHC03 to pH 6.0. The clear supernatant was used for the metabolite assays. Metabolite assays. ATP, ADP, AMP, and lactate were measured by specific enzymatic methods coupled with NADH or NADPH and monitored by the decrease or increase in absorbance at 340 nm (Bergmeyer, 1974) on a Gilford 240 spectrophotometer. Serum creatinine levels were measured on a Beckman Creatinine Analyzer II. Values are expressed as mean 2 1 SD, and statistical comparisons were obtained using the unpaired Student t test. RESULTS Functional Changes Glycerol treatment resulted in hemoglobinemia and hemoglobinuria within 0.25 hr of administration. Creatinine levels were slightly increased at 6 hr and significantly elevated (P < 0.001) at 24 and 48 hr to levels consistent with ARF (Table I). Saline loading prior to glycerol treatment resulted in a similar pattern of hemoglobinemia and hemoglobinuria, but lower creatinine levels at 24 and 48 hr when compared to glycerol treatment alone (Table II). Fructose and dihydroxyacetone treatment did not result in hemoglobinemia or hemoglobinuria. Creatinine levels were within normal limits at all time periods studied (Tables III and IV).
2 k k + k
0.25 0.19' 0.16' 0.09/ 0.12/
1.51 2 0.29’
2.48 1.94 1.46 1.18 1.29
ATPb
0.58 0.63 0.59 0.41 0.37 0.42
0.12 0.06* 0.05* 0.05d
k 0.01’ r 0.10”
k 2 2 2
ADP 2 + 5 f
0.05 0.03" 0.07* 0.07*
0.15 2 0.04’ 0.19 2 0.05”
0.25 0.32 0.28 0.25
AMP 2 2 k +
0.28 0.24d 0.19' 0.13/
1.81 2 0.11’ 2.12 2 0.25f
3.31 2.88 2.33 1.82
AXP 2 + + k -r-
0.13 0.02f 0.49f 0.32' 0.30"
0.81 ? 0.27”
0.39 1.59 1.19 1.48 0.82
Lac k ? f 5 k
0.02 0.02f 0.03f O.O4f 0.03*
0.81 ” 0.05*
0.84 0.78 0.76 0.76 0.82
EC
TABLE I Lactate, and Serum Creatinine following Glycerol Injection”
3.0 r 0.70f 3.5 k 2.20'
1.0 + 0.301
0.33 * 0.15 NM NM
Serum treat (mg/dl)
UGlycerol (50% or 5.4 M, 10 ml/kg body wt) was injected intramuscularly. h The results of adenine nucleotides and lactate are expressed as @mole/g wet tissue and the mean + 1 SD. Abbreviations used: ATP, adenosine-5’-triphosphate; ADP, adenosine-5’-diphosphate; AMP, adenosine-5’-monophosphate; AXP = ATP + ADP + AMP; Lac, lactate; Great, creatinine; NM, not measured, EC, energy charge = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]). (’ Number of experiments. d Significantly different from control value, P < 0.05. r Significantly different from control value, P < 0.01. r Significantly different from control value, P < 0.001. * Not significantly different from control value.
0.25 (4) 1 (4) 6 (3) 24 (4) 48 (3
Control (9)’
Time after injection (W
Content of Adenine Nucleotides,
w
2.33 1.78 1.08 1.04 0.94 1.23
t 0.20 F 0.05” zt 0.19’ t 0.13’ t 0.23’ + 0.08'
ATP”
0.63 0.62 0.60 0.31 0.35 0.47
2 i k ? 2 2
ADP 0.04 0.02* 0.03’ 0.07’ 0.10” 0.10"
0.21 0.34 0.41 0.13 0.22 0.31
2 0.03 i 0.04” k 0.06” + 0.03” +- 0.09* ir 0.06*
AMP 3.17 2.75 2.09 1.49 1.52 2.00
k 0.19 k 0.05” zk 0.16’ 2 0.20,’ + 0.08’ 2 0.18'
AXP 0.53 1.51 1.22 0.61 0.58 0.71
t + t zt t t
Lac 0.05 0.50” 0.59” 0.27” 0.18” 0.24"
-c + 2 -c k +
EC 0.84 0.77 0.66 0.81 0.74 0.73
TABLE II Lactate, and Serum Creatinine following Saline Loading Prior to Glycerol Injection”
0.01 0.02’ 0.05” 0.02” 0.02” 0.03'
0.30 -c 0.10 NM NM NM 1.37 * 0.2oc 1.50 + 0.44"
Serum treat (mg/dl)
” Glycerol (50% or 5.4 M. 10 ml/kg body wt) was injected intramuscularly after loading with 1% NaCl for 4 weeks. ” The results of adenine nucleotides and lactate are expressed as pmole/g wet tissue and the mean 2 1 SD. For abbreviations, see Table I. V Number of experiments. rl Significantly different from control value, P < 0.05. ” Significantly different from control value, P < 0.01. ’ Significantly different from control value, P < 0.001. * Not significantly different from control value.
Control (3)’ 0.25 (3) 1 (3) 6 (3) 24 (3) 48 (3)
Time after injection (hr)
Content of Adenine Nucleotides,
;;i
$ z 9 9 5
F ”G;
z ;
ADP
0.58 2 0.12 0.58 k 0.15* 0.46 k 0.13* 0.50 0.67 0.82
ATP”
2.48 + 0.25 1.41 2 0.06’ 1.26 ?z 0.20’ 1.45 1.66 2.08
0.25 k 0.05 0.27 2 0.03’ 0.25 2 0.04’ 0.21 0.33 0.44
AMP 3.31 k 0.28 2.49 + 0.29’ 1.98 2 0.23’ 2.15 2.66 3.34
AXP 0.39 k 0.13 3.41 2 0.52’ 5.22 2 0.13’ 1.62 0.86 0.93
Lac
0.33 k 0.15 0.37 r 0.15* 0.63 + 0.38” 0.65 0.25 0.10
Serum treat bu.ddl)
see Table I.
0.84 k 0.02 0.78 k 0.04+’ 0.76 ? 0.02’ 0.79 0.76 0.74
EC
” Fructose (100% or 5.4 M, 10 ml/kg body wt) was injected intramuscularly. b The results of adenine nucleotides and lactate are expressed as pmole/g wet tissue and the mean ? 1 SD. For abbreviations, c Number of experiments. d Significantly different from control value, P < 0.05. ” Significantly different from control value, P < 0.01. ’ Significantly different from control value, P < 0.001. * Not significantly different from control value.
48 (2)
6 (2) 24(2)
Control (9)’ 0.25 (3) 1 (3)
Time after injection (hr)
Content of Adenine Nucleotides,
TABLE III Lactate, and Serum Creatinine following Fructose Injection”
F ;: z s i z 2 u
8
2 g s 0 2 s i
5
2.48 1.99 1.84 1.83 1.92 2.02
2 k k k k 2
ATP”
0.24 0.12’ 0.20’ 0.16’ 0.21’ 0.18’
0.58 0.61 0.60 0.62 0.51 0.67
2 2 ‘-’ k k k
ADP 0.12 0.14* 0.23* 0.24* 0.19* 0.32*
0.25 0.30 0.25 0.27 0.19 0.27
5 2 2 k + +
AMP 0.15 0.10* 0.06* O.lO* 0.04* 0.09*
3.31 2.90 2.69 2.72 2.62 2.96
+ 0.28 2 0.25” k 0.35’ % 0.45“ k 0.41” 2 0.45”
AXP 0.39 2.70 2.23 0.59 0.46 0.53
2 2 k 2 + 2
Lac 0.13 0.14’ 0.94’ 0.20” 0.23* 0.13*
TABLE IV Lactate, and Serum Creatinine following Dihydroxyacetone
0.84 0.80 0.80 0.80 0.84 0.80
? 2 -t + 2 +
EC 0.02 0.04* 0.05* 0.05* 0.03* 0.06*
Injection”
0 Dihydroxyacetone (50% or 5.4 M, 10 ml/kg body wt) was injected intramuscularly. * The results of adenine nucleotides and lactate are expressed as wmole/g wet tissue and the mean ? 1 SD. For abbreviations, c Number of experiments. ’ Significantly different from control value, P < 0.05. (i Significantly different from control value, P < 0.01. ’ Significantly different from control value, P < 0.001. * Not significantly different from control value.
Control (9)’ 0.25 (3) 1 (3) 6 (3) 24 (3) 48 (3)
Time after injection (W
Content of Adenine Nucleotides,
see Table I.
0.33 0.40 0.63 0.47 0.17 0.27
k + i 2 2 k
0.15 0.3* 0.15” 0.11* 0.06* 0.12*
Serum treat (mg/dl)
METABOLIC
Morphologic
STUDIES
OF
GLYCEROL-INDUCED
ARF
7
Changes
Control tissues fixed immediately after sampling showed the typical light microscopic appearance of immersion-fixed kidney with collapsed lumens (Figs. 1 and 2). At 0.25 hr after glycerol injection, the kidneys showed mild focal edema and congestion. At 1 hr, homogeneous, eosinophilic, PAS-negative material tilled the lumens of tubules, collecting ducts, and vessels of the cortex and outer medulla. Hyaline droplets were present in the cytoplasm of tubule cells. At 6 hr, numerous casts (presumably heme pigments) filled the lumens of the cortex and the outer medulla. At 24 hr, most of the tubules and collecting ducts were filled with casts (PAS negative). Cortical necrosis occurred particularly in the proximal tubule. Intratubular deposits were also evident. At 48 hr, the morphologic picture resembled that of 24 hr with the addition of casts to the inner medulla (Fig. 3). At this time, numerous mitoses of tubule cells were evident in the cortical region. These morphologic features after glycerol treatment have been previously described (Finckh, 1957). Saline loading prior to glycerol treatment exhibited light-microscopic features similar to those of glycerol treatment alone (Fig. 4). Fructose and dihydroxyacetone treatment resulted in morphologic features similar to those of controls, i.e., no tubular necrosis or intratubular debris was evident at any time period examined. Metabolic
Changes
Changes in adenine nucleotide, lactate, and creatinine levels after glycerol treatment are shown in Table I. ATP levels decreased significantly (78% of control, P < 0.01) 0.25 hr after glycerol treatment, continued to decrease significantly (P < 0.001) at 1 hr, and reached a minimum at 6 hr (48% of control) with no recovery at 48 hr. ADP levels decreased only slightly at 6 hr (71% of control, P < 0.05), while AMP levels increased transiently at 0.25 hr and decreased slightly at 24 and 48 hr (P < 0.05). Changes in total adenine nucleotide (AXP) levels were mainly reflective of changes in ATP levels. The energy charge (Atkinson, 1968) dropped slightly at 0.25 hr and fully recovered at 24 hr. Lactate levels were highest at 0.25 hr (fourfold control levels, P < 0.001) and significantly elevated at all time intervals studied. Saline loading for 4 weeks prior to glycerol treatment resulted in changes in adenine nucleotides, lactate, and creatinine levels as shown in Table II. Control levels of saline-loaded animals did not differ significantly from those of waterdrinking animals. ATP levels decreased significantly (76% of control, P < 0.01) within 0.25 hr of glycerol treatment, reached a minimum at 24 hr, and remained low at 48 hr. ADP levels were significantly decreased (P < 0.01) at 6 hr and remained low at 48 hr. AMP levels were significantly increased (P < 0.05) after 0.25 hr, remained increased at 1 hr, and finally decreased to a minimum at 6 hr. AMP levels returned to control levels at 24 hr. Changes in AXP levels were mainly reflective of changes in ATP levels. The energy charge decreased slightly at 0.25 hr, reached a minimum at 1 hr and showed an upward but variable trend (this paralleled the AMP variation) throughout the remaining time periods. Lactate levels reached threefold control levels within 0.25 hr and returned to control levels at 6 hr.
8
TRIFILLIS,
FIG. FIG. FIG. FIG.
1. 2. 3. 4.
Control rat Control rat Rat kidney Kidney of
KAHNG,
AND
TRUMP
kidney cortex showing pars convoluta and kidney outer medulla showing pars recta. 48 hr after Gly treatment showing cortical saline-loaded rats 48 hr after Gly treatment
glomeruli. x 190. x 190. necrosis. x 190. showing cortical necrosis.
x 190.
Fructose treatment resulted in changes in adenine nucleotides, lactate, and creatinine levels as depicted in Table III. ATP levels dropped to 57% of control in 0.25 hr and reached a minimum at 1 hr. Some tendency toward recovery was evident at 48 hr. AXP levels reached a minimum at 1 hr but had recovered by 48
METABOLIC
STUDIES
OF
GLYCEROL-INDUCED
ARF
9
hr. ADP and AMP levels did not change significantly until 48 hr when they increased slightly above control values. The energy charge was slightly decreased at all time intervals. Lactate levels increased dramatically to IO-fold control levels within 0.25 hr, decreased to Qfold control levels at 1 hr, and remained significantly elevated (P < 0.001) at all other time periods studied. Dihydroxyacetone injection resulted in a slight decrease in the ATP level at 0.25 hr which was maintained throughout the time periods studied. ADP and AMP levels did not change significantly at any time period. Changes in AXP levels closely paralleled those in ATP levels. The energy charge was maintained throughout. Lactate levels increased to sevenfold control values at 0.25 hr and returned to control values at 24 hr. Table IV depicts these results. DISCUSSION Glycerol-Induced
ARF
The mechanism of glycerol-induced ARF has not been elucidated, but it is generally agreed that this model has an ischemic component which is important in its pathogenesis. Data showing decreased renal blood flow (Preuss et al., 1975) with preferential cortical hypoperfusion (Ayer et al., 1971), and vasoconstriction of large renal vessels (Solez et al., 1976) along with micropuncture data (Oken et al., 1966) which suggest decreased glomerular filtration, strongly implicate alterations in renal hemodynamics as an important mechanism in the pathogenesis of glycerol-induced ARF. Furthermore, aggravation of glycerol-induced ARF with indomethacin (Torres et al., 1975), a known augmentor of renal vasoconstriction and inhibitor of prostaglandin synthesis, also suggests a vasoconstrictive component. It has been shown, for example, that PGE, infusion into the renal artery reverses the intrarenal hemodynamic changes produced by angiotensin II, and it has been postulated that renal PGE, may play a protective role against excessive influence of vasoconstrictor agents (McGiff et al., 1974). In addition to the hemodynamic component, it has been proposed that circulating heme proteins may be nephrotoxic to ischemic renal tissue (Carroll et al., 1965; Preuss et al., 1975). The following discussion of experimental results will clarify many of the current concepts and provide further information regarding the mechanism of glycerol-induced ARF. Functional and Morphologic Changes The functional and morphologic results of these experiments indicated that glycerol injection had produced ARF. Serum creatinine levels of 3.0 and 3.5 mg/dl were reached 24 and 48 hr, respectively, after treatment. Severe hemoglobinemia and hemoglobinuria indicated a fulminant red blood cell hemolysis which probably resulted from a Gibbs-Donnan equilibrium effect. Glycerol freely penetrates the red cell membrane but is not readily metabolized by red cells (Smith, 1950). The morphologic findings were also consistent with the glycerol-induced ARF lesion previously described by several investigators (Finckh, 1957; Carroll et al., 1965; Cuppage and Tate, 1968). Saline loading prior to glycerol administration resulted in serum creatinine levels of 1.37 and 1.50 at 24 and 48 hr, respectively. These experiments indicate an incomplete amelioration of the functional lesion of glycerol-induced ARF. The morphologic lesions of intratubular casts and severe tubular necrosis, however, were not modified by saline loading. This fact argues against tubular obstruction
10
TRIFILLIS,
KAHNG,
AND
TRUMP
as the sole operative pathogenetic mechanism but does not preclude a role for tubular obstruction in the pathogenesis of ARF. The protective effect of saline loading could be the result of several factors including an increase in urinary excretion and/or renin depletion as postulated by proponents of the renin-angiotensin hypothesis (Oken et al., 1966; Thiel et al., 1970; Chedru et al., 1972). Passive backleak of glomerular filtrate through necrotic tubular epithelia or as a result of tubular obstruction may explain the slight elevation in serum creatinine levels observed after saline loading. The functional and morphological changes after fructose and dihydroxyacetone treatment were similar to untreated controls and indicated that ARF did not result within the time periods studied. Metabolic Changes The rapid decrease in kidney AXP and ATP levels indicated an early metabolic disturbance in energy-generating systems as a result of glycerol treatment. This metabolic disturbance has been previously documented by several investigators (Burch et al., 1970; Woods and Krebs, 1973), although the specific problem of glycerol-induced ARF was not addressed. It is known that glycerol is readily phosphorylated to glycerol-3-phosphate in liver and kidney (Burch et al., 1970). The relatively rapid formation of a phosphorylated intermediate(s) which is not readily metabolized further results in excess trapping of inorganic orthophosphate (Pi) and degradation of ATP (Burch et al., 1970). The shift in the ATPADP-AMP equilibrium toward AMP results in a lowered energy charge. This activates AMP deaminase which causes a decrease in the total adenine nucleotide pool and restores the energy charge through irreversible degradation of AMP to inosine-5’-monophosphate (IMP; Chapman et ctf. (1976)). Also, low ATP and Pi activates 5’-nucleotidase which degrades AMP to adenosine and provides another pathway for removal of AMP. Figure 5 (Woods et al., 1970) demonstrates these degradative pathways of AMP. The minimal decrease in the energy charge after glycerol treatment with its complete recovery by 24 hr indicated an ability to reach a new equilibrium (Atkinson, 1968) via AMP degradative pathways, thereby preserving the energy charge despite low ATP levels. The rapid fourfold increase in lactate levels indicated that anaerobic glycolysis was stimulated in response to an oxygen deficit, perhaps a result of decreased renal blood flow due to vasoconstrictive or other events, and subsequent activation of phosphofiuctokinase. An increased amount of substrate in the form of a glycolytic intermediate such as glyceraldehyde-3-phosphate could channel some of the pyruvate to lactate as an effect of the law of mass action. Adenine nucleotide changes in the saline-loaded animals reflected a similar metabolic disturbance as a direct effect of glycerol treatment. The fact that lactate levels were not quite as elevated and returned to normal long before those of glycerol-treated renin-intact animals indicated relatively less stimulation of anaerobic glycolysis. This would be expected as a result of the improved perfusion and oxygenation in renin-depleted animals over that of renin-intact animals. Thus, the lactate data may reflect the status of the renin-angiotensin system as manifested through renal hemodynamic alterations. The effect of fructose treatment on ATP levels was initially even more deleterious than those of glycerol treatment and presumably was the result of the Pitrapping mechanism previously discussed (Maenpaa et al., 1968; Burch et al., 1970; Woods et al., 1970). Despite a decreased steady-state level, it is possible
METABOLIC
STUDIES
OF GLYCEROL-INDUCED
Hypoxanthine’+
ARF
11
Ribose-1-P
I
02
Xanthine
02
FIG.
5. Pathway of AMP degradation (modified from Woods et al., 1970).
that the turnover rate of ATP was high. Fructose may have been readily metabolized via the glycolytic pathway as evidenced by a rapid increase in lactate to lO-fold control levels within 0.25 hr and 13-fold at 1 hr with a significant elevation at all time periods examined. The tremendous anaerobic glycolytic activity quite possibly resulted in a high turnover rate of ATP and aided in the attempt at ATP recovery to control levels at 48 hr and served to offset the initial disturbances in adenylate metabolism. The failure of fructose to produce ARF within 48 hr, despite the early metabolic disturbances, could be related to the fact that this lesion did not persist and showed early signs of reversal with recovery of AXP levels at 48 hr. Furthermore, the failure of fructose to produce ARF may be related to the lack of additional insults such as hemoglobinemia and cast formation concomitant with decreased ATP levels. In any case, the failure of fructose to produce ARF indicated that early adenosine release was not the sole factor in the pathogenesis of ARF. The effect of dihydroxyacetone treatment was as expected. This compound is very similar to glycerol in size (MW = 90) and structure but is readily metabolized by entering directly into the glycolytic pathway at the triose-phosphate level (Burch et al., 1970). Consequently, the decrease in ATP levels was insignificant and the energy charge was maintained throughout the 48-hr period studied. The sevenfold increase in lactate levels within 0.25 hr gave evidence for the ability of dihydroxyacetone to be readily metabolized. In summary, the metabolic, functional, and morphologic data obtained in this study clarify many aspects of the pathogenesis of glycerol-induced ARF. For example, the effect of glycerol is not merely a hyperosmolar one since the same
12
TRIFILLIS,
KAHNG,
AND TRUMP
concentration of dihydroxyacetone, a structurally similar molecule with an identical molecular weight, did not produce ARF or a significant reduction in AXP. Adenosine release with its ability to elicit renal vasoconstriction (Osswald et al., 1978; Miller et nl., 1978) does not seem to be the sole operative mechanism in glycerol-induced ARF, since fructose did not cause ARF in spite of an initial decrease in ATP and AXP. Hemoglobin and/or hemoglobin degradation products released as a result of glycerol administration are probably not solely responsible for the renal lesion, since saline loading prior to glycerol treatment resulted in significant functional amelioration despite the persistence of hemoglobinemia and hemoglobinuria. Furthermore, tubular obstruction by pigment casts and/or tubular necrosis seem to be ruled out as having a primary role in the pathogenesis of ARF, since functional amelioration occurred after saline loading despite the persistence of the morphologic lesion. While it appears that none of the above factors can be implicated as the sole pathogenetic mechanism of glycerol-induced ARF, it should be emphasized that hyperosmolarity, adenosine release, hemoglobin degradation products, tubular obstruction, and tubular necrosis may augment renal insufficiency, and any one factor may predominate during the initiation or maintenance phase of glycerolinduced ARF. Moreover, a persistent early metabolic lesion seems to be an integral part of this process. While this metabolic derangement may not be sufficient in and of itself to cause ARF, it may play a significant role in the predisposition toward ARF in the presence of additional insults. Hypothesis for the Mechanism of Glycerol-Induced
ARF
The results of these experiments, therefore, are consistent with the following modification of Flamenbaum’s (1973) hypothesis of ARF: Glycerol injection results in an altered metabolic state as reflected by significantly decreased ATP and AXP levels. Since ATP plays a key role in cell economy, a significant drop in ATP results in further metabolic consequences and results in tubular dysfunction such as altered tubular handling of sodium and H20. This in turn may result in a tubular feedback mechanism which activates the renin-angiotensin system and causes increased arteriolar resistance. The vasoconstrictive component may be further augmented by increased adenosine release as a consequence of AMP degradation. The resultant decrease in effective filtration through redistribution and reduced renal blood flow produces oliguria which may be augmented and perpetuated by intratubular obstruction with casts and necrotic debris. A diversity of operative pathogenetic mechanisms seems to be responsible for the initiation and maintenance of glycerol-induced ARF. In this study, a persistent metabolic derangement precedes the functional and morphologic alterations as part of the process and may predispose toward ARF in the presence of additional factors. These findings further reiterate the need for a multidisciplinary approach to the study of the pathogenesis of ARF with particular emphasis on its early metabolic alterations. REFERENCES ATKINSON, D. E. (1968). The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochrmisrp 7, 4030-4034. AYER, G., GRANDCHAMP. A., WYLER, T., and TRUNIGER, B. (1971). Intrarenal hemodynamics in glycerol-induced myohemoglobinuric acute renal failure in the rat. Circ. Res. 29, 128- 135. BERGMEYER, H. U. (1974). “Methods of Enzymatic Analysis,” Vols. 2 and 4. Academic Press, New York.
METABOLIC
STUDIES
OF
GLYCEROL-INDUCED
ARF
13
BURCH, H. B., LOWRY, 0. H., MEINHARDT, L., MAX, P., and CHYU. K. (1970). Effect of fructose, dihydroxyacetone, glycerol, and glucose on metabolites and related compounds in liver and kidney. J. Bid. Chem. 245, 2092-2102. CARROLL, R., KOVACS, K., and TAPP, E. (1965). The pathogenesis of glycerol-induced renal tubular necrosis. J. Pathol. Bacterial. 89, 573-580. CHAPMAN, A. G., MILLER, A. L., and ATKINSON, D. E. (1976). Role of the adenylate deaminase reaction in regulation of adenine nucleotide metabolism in Ehrlich ascites tumor cells. Cancer Res. 36, 1144- 1150. CHEDRU, M. F., BAETHKE, R., and OKEN, D. E. (1972). Renal cortical blood flow and glomerular filtration in myohemoglobinuric acute renal failure. Kidney Int. 1, 232-239. CUPPACE, F. E., and TATE, A. (1968). Repair of the nephron in acute renal failure: Comparative regeneration following various forms of acute tubular injury. Pathol. Microbial. 32, 327-344. FINCKH, E. S. (1957). The indirect action of subcutaneous injections of glycerol on the renal tubules in the rat. J. Pathol. Bacterial. 78. 197-202. FLAMENBAUM, W. (1973). Pathophysiology of acute renal failure. Arch. Intern. Med. 131, 911-928. HEPTINSTALL, R. H. (1974). Acute renal failure. In “Pathology of the Kidney,” Vol. 2, pp. 781-820. Little, Brown, Boston. MXENPA,&, P. H., RAIVIO, K. O., and KEKOMAKI, M. P. (1968). Liver adenine nucleotides: Fructose-induced depletion and its effect on protein synthesis. Science 161, 1253- 1254. MCDOWELL, E. M., and TRUMP, B. F. (1976). Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med. 100, 405-414. MCGIFF, J. C., CROWSHAW, K., and ITSKOVITZ, H. D. (1974). Prostaglandins and renal function. Fed. Proc.
33, 39-47.
MILLER, W. L., THOMAS, R. A., BERNE, R. M., and RUBIO, R. (1978). Adenosine production in the ischemic kidney. Circ. Res. 43, 390-397. OKEN, D. E., ARCE, M. L., and WILSON, D. R. (1966). Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of oliguria. J. C/in. Invest. 45, 724-735. OSSWALD, H., SCHMITZ, H. J., and KEMPER, R. (1978). Renal action of adenosine: Effect of renin secretion in the rat. Arch. Pharmacol. 303, 95-99. PREUSS,H. G., TOURKANTONIS, H., Hsu, S. H., SHIM, P. S., BARZYK, P.,TIo, F., and SCHRIERNER, G. E. (1975). Early events in various forms of experimental acute tubular necrosis in rats. Lab. Invest.
32, 286-294.
SMITH, A. U. (1950). Prevention of hemolysis during freezing and thawing of red blood cells. Lancer 2, 910. SOLEZ, K., ALTMAN, J., RIENHOFF, H. Y., ANTHONY, R. R., FINER, P. M., and HEPTINSTALL, R. H. (1976). Early angiographic and renal blood flow changes after HgCI, or glycerol administration. Kidney Int. 10, S-153-S-159. THIEL, G., MCDONALD, F. D., and OKEN, D. E. (1970). Micropuncture studies of the basis for protection of renin depleted rats from glycerol-induced acute renal failure. Nephron 7, 67-79. TORRES, V. E., STRONG, C. G., ROMERO, J. C., and WILSON, D. M. (1975). Indomethacin enhancement of glycerol-induced acute renal failure in rabbits. Kidney Int. 7, 170- 178. WOODS, H. F., EGGLESTON, L. V., and KREBS, H. A. (1970). The cause of accumulation of fructosel-phosphate on fructose loading. Biochem. J. 119, 501-510. WOODS, H. F., and KREBS, H. A. (1973). The effect of glycerol and dihydroxyacetone on hepatic adenine nucleotides. Biochem. J. 132, 55-60.
AND
Metabolic ANNA Department
MOLECULAR
Studies L. TRIFILLIS,
of Pathology,
PATHOLOGY
35, l- 13 (1981)
of Glycerol-Induced in the Rat’ MYONG
University
W. KAHNG,
of Maryland Received
School
September
Acute
Renal
AND BENJAMIN of Medicine,
Baltimore,
Failure
F. TRUMP Maqland
21201
19. 1980
Glycerol was used to induce acute renal failure (ARF) in rats. It caused a decrease of kidney ATP levels to 48% of control at 6 hr which remained low up to 48 hr. ATP+ADP+AMP(AXP) was 55% of control at 6 and 24 hr and 64% at 48 hr. Lactate levels were fourfold control levels at 0.25, 1, and 6 hr, and twofold at 24 and 48 hr. Hemoglobinemia and hemoglobinuria occurred within 0.25 hr. By light microscopy, casts at 6 hr, necrosis of renal cortex at 24 hr, and necrosis with some regeneration at 48 hr were seen. Fructose at the same dose also lowered ATP and AXP levels to a similar degree as glycerol within the same time intervals, but hemoglobinemia, hemoglobinuria, necrosis, or ARF was not evident. Dihydroxyacetone at the same dose maintained ATP and AXP at control values with no hemoglobinemia, hemoglobinuria, necrosis, or ARF. Both fructose and dihydroxyacetone resulted in high lactate levels (sevenfold) within 0.25 hr. Saline loading prior to glycerol injection ameliorated ARF but did not improve the AXP levels or the morphologic lesion. Results indicate that glycerol-induced ARF is not solely the result of a hyperosmolar effect, adenosine release, casts, or pigments. The changes in AXP (particularly ATP) levels reflect an early metabolic disturbance which is part of the process and may predispose toward ARF in the presence of additional factors.
INTRODUCTION Acute renal failure (ARF) has been categorized into two general groups based upon the initiating event, ischemic and nephrotoxic (Heptinstall, 1974). The ischemit lesion is associated with crush injury, hemorrhage, and hypotension, while the nephrotoxic lesion is associated with agents such as aminoglycosides, HgC12, CCll, and ethylene glycol. Experimental animal models have been developed to simulate these clinical situations. While no animal model of ARF is strictly comparable to the circulatory type of human ARF, the intramuscular (im) injection of glycerol has been proposed as a model of ARF closely resembling the syndrome of ARF in man (Preuss et al., 1975; Torres et al., 1975). Although many investigations have been concerned with functional (Oken et al., 1966; Ayer Pt al., 1971) and morphological (Finckh, 1957; Carroll et al., 1965) alterations in glycerol-induced ARF, little attention has been paid to renal metabolic perturbations which may be operative in the initiation or maintenance of ARF. In the present study, we have examined the changes in renal concentrations of adenine nucleotides and lactate for up to 48 hr following glycerol-induced ARF. In order to gain further insight into the underlying mechanism of glycerolinduced ARF, the metabolic effects of saline loading prior to glycerol treatment, fructose, and dihydroxyacetone were studied. Saline loading prior to glycerol treatment has been shown to ameliorate glycerol-induced ARF (Thiel et al., 1970). Fructose is known to cause a rapid decrease in adenine nucleotide levels similar to is similar to that caused by glycerol (Burch et al., 1970); and dihydroxyacetone 1 This is contribution No. 886 from the Cellular Pathobiology Laboratory and was supported in part by NIH Grant AM 15440-08A2. Part of this work was presented at the 1980 FASEB Meetings in Anaheim. Calif. 1 0014-4800/81/040001-13$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
2
TRIFILLIS,
KAHNG,
AND
TRUMP
glycerol in size and structure. In light of the metabolic, functional, and morphological results obtained, a possible pathogenetic mechanism of glycerol-induced ARF is discussed. MATERIALS AND METHODS Animal and tissue treatment. Male Sprague-Dawley rats weighing 220-320 g were fed standard Purina laboratory chow and were water deprived for 16 hr prior to treatment. Rats were divided into six experimental groups: (1) Control (1% NaCl, 10 ml/kg body wt, im, hindlimb); (2) saline-loaded control (1% NaCl instead of drinking water for 4 weeks prior to sacrifice); (3) glycerol (50 g/100 ml saline or 5.4 M glycerol, 10 ml/kg body wt, im); (4) saline-loaded glycerol (1% NaCl instead of drinking water 4 weeks prior to 5.4 M glycerol, 10 ml/kg body wt, im); (5) fructose (5.4 M fructose, 10 ml/kg body wt, im); (6) dihydroxyacetone (5.4 M dihydroxyacetone, 10 ml/kg body wt, im). Fifteen minutes prior to treatment, all animals were anesthetized with sodium pentobarbital (35 mg/kg body wt, intraperitoneally). Kidneys and blood were sampled at 0, 0.25, 1, 6, 24, and 48 hr after treatment. The left kidneys were gently decapsulated and freeze-clamped in situ with aluminum tongs precooled in liquid N,. They were then immersed and trimmed under liquid Nz for metabolite assays. The right kidneys were bisected longitudinally and immersion-fixed in 4% formaldehyde and 1% glutaraldehyde (4F- 1G) buffered with sodium phosphate, pH 7.2 (McDowell and Trump, 1976), processed for light microscopy and stained with H&E and PAS. Blood samples for creatinine determination were taken from the aorta. Serum creatinine levels were used as an index of renal function. Tissue extract preparation. The freeze-clamped tissue was ground with a mortar and pestle under liquid NZ, weighed, and homogenized in 4 vol of 0.6 N HClO,. The homogenate was centrifuged at 1200,g for 15 min and the supernatant was neutralized with 2 M KHC03 to pH 6.0. The clear supernatant was used for the metabolite assays. Metabolite assays. ATP, ADP, AMP, and lactate were measured by specific enzymatic methods coupled with NADH or NADPH and monitored by the decrease or increase in absorbance at 340 nm (Bergmeyer, 1974) on a Gilford 240 spectrophotometer. Serum creatinine levels were measured on a Beckman Creatinine Analyzer II. Values are expressed as mean 2 1 SD, and statistical comparisons were obtained using the unpaired Student t test. RESULTS Functional Changes Glycerol treatment resulted in hemoglobinemia and hemoglobinuria within 0.25 hr of administration. Creatinine levels were slightly increased at 6 hr and significantly elevated (P < 0.001) at 24 and 48 hr to levels consistent with ARF (Table I). Saline loading prior to glycerol treatment resulted in a similar pattern of hemoglobinemia and hemoglobinuria, but lower creatinine levels at 24 and 48 hr when compared to glycerol treatment alone (Table II). Fructose and dihydroxyacetone treatment did not result in hemoglobinemia or hemoglobinuria. Creatinine levels were within normal limits at all time periods studied (Tables III and IV).
2 k k + k
0.25 0.19' 0.16' 0.09/ 0.12/
1.51 2 0.29’
2.48 1.94 1.46 1.18 1.29
ATPb
0.58 0.63 0.59 0.41 0.37 0.42
0.12 0.06* 0.05* 0.05d
k 0.01’ r 0.10”
k 2 2 2
ADP 2 + 5 f
0.05 0.03" 0.07* 0.07*
0.15 2 0.04’ 0.19 2 0.05”
0.25 0.32 0.28 0.25
AMP 2 2 k +
0.28 0.24d 0.19' 0.13/
1.81 2 0.11’ 2.12 2 0.25f
3.31 2.88 2.33 1.82
AXP 2 + + k -r-
0.13 0.02f 0.49f 0.32' 0.30"
0.81 ? 0.27”
0.39 1.59 1.19 1.48 0.82
Lac k ? f 5 k
0.02 0.02f 0.03f O.O4f 0.03*
0.81 ” 0.05*
0.84 0.78 0.76 0.76 0.82
EC
TABLE I Lactate, and Serum Creatinine following Glycerol Injection”
3.0 r 0.70f 3.5 k 2.20'
1.0 + 0.301
0.33 * 0.15 NM NM
Serum treat (mg/dl)
UGlycerol (50% or 5.4 M, 10 ml/kg body wt) was injected intramuscularly. h The results of adenine nucleotides and lactate are expressed as @mole/g wet tissue and the mean + 1 SD. Abbreviations used: ATP, adenosine-5’-triphosphate; ADP, adenosine-5’-diphosphate; AMP, adenosine-5’-monophosphate; AXP = ATP + ADP + AMP; Lac, lactate; Great, creatinine; NM, not measured, EC, energy charge = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]). (’ Number of experiments. d Significantly different from control value, P < 0.05. r Significantly different from control value, P < 0.01. r Significantly different from control value, P < 0.001. * Not significantly different from control value.
0.25 (4) 1 (4) 6 (3) 24 (4) 48 (3
Control (9)’
Time after injection (W
Content of Adenine Nucleotides,
w
2.33 1.78 1.08 1.04 0.94 1.23
t 0.20 F 0.05” zt 0.19’ t 0.13’ t 0.23’ + 0.08'
ATP”
0.63 0.62 0.60 0.31 0.35 0.47
2 i k ? 2 2
ADP 0.04 0.02* 0.03’ 0.07’ 0.10” 0.10"
0.21 0.34 0.41 0.13 0.22 0.31
2 0.03 i 0.04” k 0.06” + 0.03” +- 0.09* ir 0.06*
AMP 3.17 2.75 2.09 1.49 1.52 2.00
k 0.19 k 0.05” zk 0.16’ 2 0.20,’ + 0.08’ 2 0.18'
AXP 0.53 1.51 1.22 0.61 0.58 0.71
t + t zt t t
Lac 0.05 0.50” 0.59” 0.27” 0.18” 0.24"
-c + 2 -c k +
EC 0.84 0.77 0.66 0.81 0.74 0.73
TABLE II Lactate, and Serum Creatinine following Saline Loading Prior to Glycerol Injection”
0.01 0.02’ 0.05” 0.02” 0.02” 0.03'
0.30 -c 0.10 NM NM NM 1.37 * 0.2oc 1.50 + 0.44"
Serum treat (mg/dl)
” Glycerol (50% or 5.4 M. 10 ml/kg body wt) was injected intramuscularly after loading with 1% NaCl for 4 weeks. ” The results of adenine nucleotides and lactate are expressed as pmole/g wet tissue and the mean 2 1 SD. For abbreviations, see Table I. V Number of experiments. rl Significantly different from control value, P < 0.05. ” Significantly different from control value, P < 0.01. ’ Significantly different from control value, P < 0.001. * Not significantly different from control value.
Control (3)’ 0.25 (3) 1 (3) 6 (3) 24 (3) 48 (3)
Time after injection (hr)
Content of Adenine Nucleotides,
;;i
$ z 9 9 5
F ”G;
z ;
ADP
0.58 2 0.12 0.58 k 0.15* 0.46 k 0.13* 0.50 0.67 0.82
ATP”
2.48 + 0.25 1.41 2 0.06’ 1.26 ?z 0.20’ 1.45 1.66 2.08
0.25 k 0.05 0.27 2 0.03’ 0.25 2 0.04’ 0.21 0.33 0.44
AMP 3.31 k 0.28 2.49 + 0.29’ 1.98 2 0.23’ 2.15 2.66 3.34
AXP 0.39 k 0.13 3.41 2 0.52’ 5.22 2 0.13’ 1.62 0.86 0.93
Lac
0.33 k 0.15 0.37 r 0.15* 0.63 + 0.38” 0.65 0.25 0.10
Serum treat bu.ddl)
see Table I.
0.84 k 0.02 0.78 k 0.04+’ 0.76 ? 0.02’ 0.79 0.76 0.74
EC
” Fructose (100% or 5.4 M, 10 ml/kg body wt) was injected intramuscularly. b The results of adenine nucleotides and lactate are expressed as pmole/g wet tissue and the mean ? 1 SD. For abbreviations, c Number of experiments. d Significantly different from control value, P < 0.05. ” Significantly different from control value, P < 0.01. ’ Significantly different from control value, P < 0.001. * Not significantly different from control value.
48 (2)
6 (2) 24(2)
Control (9)’ 0.25 (3) 1 (3)
Time after injection (hr)
Content of Adenine Nucleotides,
TABLE III Lactate, and Serum Creatinine following Fructose Injection”
F ;: z s i z 2 u
8
2 g s 0 2 s i
5
2.48 1.99 1.84 1.83 1.92 2.02
2 k k k k 2
ATP”
0.24 0.12’ 0.20’ 0.16’ 0.21’ 0.18’
0.58 0.61 0.60 0.62 0.51 0.67
2 2 ‘-’ k k k
ADP 0.12 0.14* 0.23* 0.24* 0.19* 0.32*
0.25 0.30 0.25 0.27 0.19 0.27
5 2 2 k + +
AMP 0.15 0.10* 0.06* O.lO* 0.04* 0.09*
3.31 2.90 2.69 2.72 2.62 2.96
+ 0.28 2 0.25” k 0.35’ % 0.45“ k 0.41” 2 0.45”
AXP 0.39 2.70 2.23 0.59 0.46 0.53
2 2 k 2 + 2
Lac 0.13 0.14’ 0.94’ 0.20” 0.23* 0.13*
TABLE IV Lactate, and Serum Creatinine following Dihydroxyacetone
0.84 0.80 0.80 0.80 0.84 0.80
? 2 -t + 2 +
EC 0.02 0.04* 0.05* 0.05* 0.03* 0.06*
Injection”
0 Dihydroxyacetone (50% or 5.4 M, 10 ml/kg body wt) was injected intramuscularly. * The results of adenine nucleotides and lactate are expressed as wmole/g wet tissue and the mean ? 1 SD. For abbreviations, c Number of experiments. ’ Significantly different from control value, P < 0.05. (i Significantly different from control value, P < 0.01. ’ Significantly different from control value, P < 0.001. * Not significantly different from control value.
Control (9)’ 0.25 (3) 1 (3) 6 (3) 24 (3) 48 (3)
Time after injection (W
Content of Adenine Nucleotides,
see Table I.
0.33 0.40 0.63 0.47 0.17 0.27
k + i 2 2 k
0.15 0.3* 0.15” 0.11* 0.06* 0.12*
Serum treat (mg/dl)
METABOLIC
Morphologic
STUDIES
OF
GLYCEROL-INDUCED
ARF
7
Changes
Control tissues fixed immediately after sampling showed the typical light microscopic appearance of immersion-fixed kidney with collapsed lumens (Figs. 1 and 2). At 0.25 hr after glycerol injection, the kidneys showed mild focal edema and congestion. At 1 hr, homogeneous, eosinophilic, PAS-negative material tilled the lumens of tubules, collecting ducts, and vessels of the cortex and outer medulla. Hyaline droplets were present in the cytoplasm of tubule cells. At 6 hr, numerous casts (presumably heme pigments) filled the lumens of the cortex and the outer medulla. At 24 hr, most of the tubules and collecting ducts were filled with casts (PAS negative). Cortical necrosis occurred particularly in the proximal tubule. Intratubular deposits were also evident. At 48 hr, the morphologic picture resembled that of 24 hr with the addition of casts to the inner medulla (Fig. 3). At this time, numerous mitoses of tubule cells were evident in the cortical region. These morphologic features after glycerol treatment have been previously described (Finckh, 1957). Saline loading prior to glycerol treatment exhibited light-microscopic features similar to those of glycerol treatment alone (Fig. 4). Fructose and dihydroxyacetone treatment resulted in morphologic features similar to those of controls, i.e., no tubular necrosis or intratubular debris was evident at any time period examined. Metabolic
Changes
Changes in adenine nucleotide, lactate, and creatinine levels after glycerol treatment are shown in Table I. ATP levels decreased significantly (78% of control, P < 0.01) 0.25 hr after glycerol treatment, continued to decrease significantly (P < 0.001) at 1 hr, and reached a minimum at 6 hr (48% of control) with no recovery at 48 hr. ADP levels decreased only slightly at 6 hr (71% of control, P < 0.05), while AMP levels increased transiently at 0.25 hr and decreased slightly at 24 and 48 hr (P < 0.05). Changes in total adenine nucleotide (AXP) levels were mainly reflective of changes in ATP levels. The energy charge (Atkinson, 1968) dropped slightly at 0.25 hr and fully recovered at 24 hr. Lactate levels were highest at 0.25 hr (fourfold control levels, P < 0.001) and significantly elevated at all time intervals studied. Saline loading for 4 weeks prior to glycerol treatment resulted in changes in adenine nucleotides, lactate, and creatinine levels as shown in Table II. Control levels of saline-loaded animals did not differ significantly from those of waterdrinking animals. ATP levels decreased significantly (76% of control, P < 0.01) within 0.25 hr of glycerol treatment, reached a minimum at 24 hr, and remained low at 48 hr. ADP levels were significantly decreased (P < 0.01) at 6 hr and remained low at 48 hr. AMP levels were significantly increased (P < 0.05) after 0.25 hr, remained increased at 1 hr, and finally decreased to a minimum at 6 hr. AMP levels returned to control levels at 24 hr. Changes in AXP levels were mainly reflective of changes in ATP levels. The energy charge decreased slightly at 0.25 hr, reached a minimum at 1 hr and showed an upward but variable trend (this paralleled the AMP variation) throughout the remaining time periods. Lactate levels reached threefold control levels within 0.25 hr and returned to control levels at 6 hr.
8
TRIFILLIS,
FIG. FIG. FIG. FIG.
1. 2. 3. 4.
Control rat Control rat Rat kidney Kidney of
KAHNG,
AND
TRUMP
kidney cortex showing pars convoluta and kidney outer medulla showing pars recta. 48 hr after Gly treatment showing cortical saline-loaded rats 48 hr after Gly treatment
glomeruli. x 190. x 190. necrosis. x 190. showing cortical necrosis.
x 190.
Fructose treatment resulted in changes in adenine nucleotides, lactate, and creatinine levels as depicted in Table III. ATP levels dropped to 57% of control in 0.25 hr and reached a minimum at 1 hr. Some tendency toward recovery was evident at 48 hr. AXP levels reached a minimum at 1 hr but had recovered by 48
METABOLIC
STUDIES
OF
GLYCEROL-INDUCED
ARF
9
hr. ADP and AMP levels did not change significantly until 48 hr when they increased slightly above control values. The energy charge was slightly decreased at all time intervals. Lactate levels increased dramatically to IO-fold control levels within 0.25 hr, decreased to Qfold control levels at 1 hr, and remained significantly elevated (P < 0.001) at all other time periods studied. Dihydroxyacetone injection resulted in a slight decrease in the ATP level at 0.25 hr which was maintained throughout the time periods studied. ADP and AMP levels did not change significantly at any time period. Changes in AXP levels closely paralleled those in ATP levels. The energy charge was maintained throughout. Lactate levels increased to sevenfold control values at 0.25 hr and returned to control values at 24 hr. Table IV depicts these results. DISCUSSION Glycerol-Induced
ARF
The mechanism of glycerol-induced ARF has not been elucidated, but it is generally agreed that this model has an ischemic component which is important in its pathogenesis. Data showing decreased renal blood flow (Preuss et al., 1975) with preferential cortical hypoperfusion (Ayer et al., 1971), and vasoconstriction of large renal vessels (Solez et al., 1976) along with micropuncture data (Oken et al., 1966) which suggest decreased glomerular filtration, strongly implicate alterations in renal hemodynamics as an important mechanism in the pathogenesis of glycerol-induced ARF. Furthermore, aggravation of glycerol-induced ARF with indomethacin (Torres et al., 1975), a known augmentor of renal vasoconstriction and inhibitor of prostaglandin synthesis, also suggests a vasoconstrictive component. It has been shown, for example, that PGE, infusion into the renal artery reverses the intrarenal hemodynamic changes produced by angiotensin II, and it has been postulated that renal PGE, may play a protective role against excessive influence of vasoconstrictor agents (McGiff et al., 1974). In addition to the hemodynamic component, it has been proposed that circulating heme proteins may be nephrotoxic to ischemic renal tissue (Carroll et al., 1965; Preuss et al., 1975). The following discussion of experimental results will clarify many of the current concepts and provide further information regarding the mechanism of glycerol-induced ARF. Functional and Morphologic Changes The functional and morphologic results of these experiments indicated that glycerol injection had produced ARF. Serum creatinine levels of 3.0 and 3.5 mg/dl were reached 24 and 48 hr, respectively, after treatment. Severe hemoglobinemia and hemoglobinuria indicated a fulminant red blood cell hemolysis which probably resulted from a Gibbs-Donnan equilibrium effect. Glycerol freely penetrates the red cell membrane but is not readily metabolized by red cells (Smith, 1950). The morphologic findings were also consistent with the glycerol-induced ARF lesion previously described by several investigators (Finckh, 1957; Carroll et al., 1965; Cuppage and Tate, 1968). Saline loading prior to glycerol administration resulted in serum creatinine levels of 1.37 and 1.50 at 24 and 48 hr, respectively. These experiments indicate an incomplete amelioration of the functional lesion of glycerol-induced ARF. The morphologic lesions of intratubular casts and severe tubular necrosis, however, were not modified by saline loading. This fact argues against tubular obstruction
10
TRIFILLIS,
KAHNG,
AND
TRUMP
as the sole operative pathogenetic mechanism but does not preclude a role for tubular obstruction in the pathogenesis of ARF. The protective effect of saline loading could be the result of several factors including an increase in urinary excretion and/or renin depletion as postulated by proponents of the renin-angiotensin hypothesis (Oken et al., 1966; Thiel et al., 1970; Chedru et al., 1972). Passive backleak of glomerular filtrate through necrotic tubular epithelia or as a result of tubular obstruction may explain the slight elevation in serum creatinine levels observed after saline loading. The functional and morphological changes after fructose and dihydroxyacetone treatment were similar to untreated controls and indicated that ARF did not result within the time periods studied. Metabolic Changes The rapid decrease in kidney AXP and ATP levels indicated an early metabolic disturbance in energy-generating systems as a result of glycerol treatment. This metabolic disturbance has been previously documented by several investigators (Burch et al., 1970; Woods and Krebs, 1973), although the specific problem of glycerol-induced ARF was not addressed. It is known that glycerol is readily phosphorylated to glycerol-3-phosphate in liver and kidney (Burch et al., 1970). The relatively rapid formation of a phosphorylated intermediate(s) which is not readily metabolized further results in excess trapping of inorganic orthophosphate (Pi) and degradation of ATP (Burch et al., 1970). The shift in the ATPADP-AMP equilibrium toward AMP results in a lowered energy charge. This activates AMP deaminase which causes a decrease in the total adenine nucleotide pool and restores the energy charge through irreversible degradation of AMP to inosine-5’-monophosphate (IMP; Chapman et ctf. (1976)). Also, low ATP and Pi activates 5’-nucleotidase which degrades AMP to adenosine and provides another pathway for removal of AMP. Figure 5 (Woods et al., 1970) demonstrates these degradative pathways of AMP. The minimal decrease in the energy charge after glycerol treatment with its complete recovery by 24 hr indicated an ability to reach a new equilibrium (Atkinson, 1968) via AMP degradative pathways, thereby preserving the energy charge despite low ATP levels. The rapid fourfold increase in lactate levels indicated that anaerobic glycolysis was stimulated in response to an oxygen deficit, perhaps a result of decreased renal blood flow due to vasoconstrictive or other events, and subsequent activation of phosphofiuctokinase. An increased amount of substrate in the form of a glycolytic intermediate such as glyceraldehyde-3-phosphate could channel some of the pyruvate to lactate as an effect of the law of mass action. Adenine nucleotide changes in the saline-loaded animals reflected a similar metabolic disturbance as a direct effect of glycerol treatment. The fact that lactate levels were not quite as elevated and returned to normal long before those of glycerol-treated renin-intact animals indicated relatively less stimulation of anaerobic glycolysis. This would be expected as a result of the improved perfusion and oxygenation in renin-depleted animals over that of renin-intact animals. Thus, the lactate data may reflect the status of the renin-angiotensin system as manifested through renal hemodynamic alterations. The effect of fructose treatment on ATP levels was initially even more deleterious than those of glycerol treatment and presumably was the result of the Pitrapping mechanism previously discussed (Maenpaa et al., 1968; Burch et al., 1970; Woods et al., 1970). Despite a decreased steady-state level, it is possible
METABOLIC
STUDIES
OF GLYCEROL-INDUCED
Hypoxanthine’+
ARF
11
Ribose-1-P
I
02
Xanthine
02
FIG.
5. Pathway of AMP degradation (modified from Woods et al., 1970).
that the turnover rate of ATP was high. Fructose may have been readily metabolized via the glycolytic pathway as evidenced by a rapid increase in lactate to lO-fold control levels within 0.25 hr and 13-fold at 1 hr with a significant elevation at all time periods examined. The tremendous anaerobic glycolytic activity quite possibly resulted in a high turnover rate of ATP and aided in the attempt at ATP recovery to control levels at 48 hr and served to offset the initial disturbances in adenylate metabolism. The failure of fructose to produce ARF within 48 hr, despite the early metabolic disturbances, could be related to the fact that this lesion did not persist and showed early signs of reversal with recovery of AXP levels at 48 hr. Furthermore, the failure of fructose to produce ARF may be related to the lack of additional insults such as hemoglobinemia and cast formation concomitant with decreased ATP levels. In any case, the failure of fructose to produce ARF indicated that early adenosine release was not the sole factor in the pathogenesis of ARF. The effect of dihydroxyacetone treatment was as expected. This compound is very similar to glycerol in size (MW = 90) and structure but is readily metabolized by entering directly into the glycolytic pathway at the triose-phosphate level (Burch et al., 1970). Consequently, the decrease in ATP levels was insignificant and the energy charge was maintained throughout the 48-hr period studied. The sevenfold increase in lactate levels within 0.25 hr gave evidence for the ability of dihydroxyacetone to be readily metabolized. In summary, the metabolic, functional, and morphologic data obtained in this study clarify many aspects of the pathogenesis of glycerol-induced ARF. For example, the effect of glycerol is not merely a hyperosmolar one since the same
12
TRIFILLIS,
KAHNG,
AND TRUMP
concentration of dihydroxyacetone, a structurally similar molecule with an identical molecular weight, did not produce ARF or a significant reduction in AXP. Adenosine release with its ability to elicit renal vasoconstriction (Osswald et al., 1978; Miller et nl., 1978) does not seem to be the sole operative mechanism in glycerol-induced ARF, since fructose did not cause ARF in spite of an initial decrease in ATP and AXP. Hemoglobin and/or hemoglobin degradation products released as a result of glycerol administration are probably not solely responsible for the renal lesion, since saline loading prior to glycerol treatment resulted in significant functional amelioration despite the persistence of hemoglobinemia and hemoglobinuria. Furthermore, tubular obstruction by pigment casts and/or tubular necrosis seem to be ruled out as having a primary role in the pathogenesis of ARF, since functional amelioration occurred after saline loading despite the persistence of the morphologic lesion. While it appears that none of the above factors can be implicated as the sole pathogenetic mechanism of glycerol-induced ARF, it should be emphasized that hyperosmolarity, adenosine release, hemoglobin degradation products, tubular obstruction, and tubular necrosis may augment renal insufficiency, and any one factor may predominate during the initiation or maintenance phase of glycerolinduced ARF. Moreover, a persistent early metabolic lesion seems to be an integral part of this process. While this metabolic derangement may not be sufficient in and of itself to cause ARF, it may play a significant role in the predisposition toward ARF in the presence of additional insults. Hypothesis for the Mechanism of Glycerol-Induced
ARF
The results of these experiments, therefore, are consistent with the following modification of Flamenbaum’s (1973) hypothesis of ARF: Glycerol injection results in an altered metabolic state as reflected by significantly decreased ATP and AXP levels. Since ATP plays a key role in cell economy, a significant drop in ATP results in further metabolic consequences and results in tubular dysfunction such as altered tubular handling of sodium and H20. This in turn may result in a tubular feedback mechanism which activates the renin-angiotensin system and causes increased arteriolar resistance. The vasoconstrictive component may be further augmented by increased adenosine release as a consequence of AMP degradation. The resultant decrease in effective filtration through redistribution and reduced renal blood flow produces oliguria which may be augmented and perpetuated by intratubular obstruction with casts and necrotic debris. A diversity of operative pathogenetic mechanisms seems to be responsible for the initiation and maintenance of glycerol-induced ARF. In this study, a persistent metabolic derangement precedes the functional and morphologic alterations as part of the process and may predispose toward ARF in the presence of additional factors. These findings further reiterate the need for a multidisciplinary approach to the study of the pathogenesis of ARF with particular emphasis on its early metabolic alterations. REFERENCES ATKINSON, D. E. (1968). The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochrmisrp 7, 4030-4034. AYER, G., GRANDCHAMP. A., WYLER, T., and TRUNIGER, B. (1971). Intrarenal hemodynamics in glycerol-induced myohemoglobinuric acute renal failure in the rat. Circ. Res. 29, 128- 135. BERGMEYER, H. U. (1974). “Methods of Enzymatic Analysis,” Vols. 2 and 4. Academic Press, New York.
METABOLIC
STUDIES
OF
GLYCEROL-INDUCED
ARF
13
BURCH, H. B., LOWRY, 0. H., MEINHARDT, L., MAX, P., and CHYU. K. (1970). Effect of fructose, dihydroxyacetone, glycerol, and glucose on metabolites and related compounds in liver and kidney. J. Bid. Chem. 245, 2092-2102. CARROLL, R., KOVACS, K., and TAPP, E. (1965). The pathogenesis of glycerol-induced renal tubular necrosis. J. Pathol. Bacterial. 89, 573-580. CHAPMAN, A. G., MILLER, A. L., and ATKINSON, D. E. (1976). Role of the adenylate deaminase reaction in regulation of adenine nucleotide metabolism in Ehrlich ascites tumor cells. Cancer Res. 36, 1144- 1150. CHEDRU, M. F., BAETHKE, R., and OKEN, D. E. (1972). Renal cortical blood flow and glomerular filtration in myohemoglobinuric acute renal failure. Kidney Int. 1, 232-239. CUPPACE, F. E., and TATE, A. (1968). Repair of the nephron in acute renal failure: Comparative regeneration following various forms of acute tubular injury. Pathol. Microbial. 32, 327-344. FINCKH, E. S. (1957). The indirect action of subcutaneous injections of glycerol on the renal tubules in the rat. J. Pathol. Bacterial. 78. 197-202. FLAMENBAUM, W. (1973). Pathophysiology of acute renal failure. Arch. Intern. Med. 131, 911-928. HEPTINSTALL, R. H. (1974). Acute renal failure. In “Pathology of the Kidney,” Vol. 2, pp. 781-820. Little, Brown, Boston. MXENPA,&, P. H., RAIVIO, K. O., and KEKOMAKI, M. P. (1968). Liver adenine nucleotides: Fructose-induced depletion and its effect on protein synthesis. Science 161, 1253- 1254. MCDOWELL, E. M., and TRUMP, B. F. (1976). Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pathol. Lab. Med. 100, 405-414. MCGIFF, J. C., CROWSHAW, K., and ITSKOVITZ, H. D. (1974). Prostaglandins and renal function. Fed. Proc.
33, 39-47.
MILLER, W. L., THOMAS, R. A., BERNE, R. M., and RUBIO, R. (1978). Adenosine production in the ischemic kidney. Circ. Res. 43, 390-397. OKEN, D. E., ARCE, M. L., and WILSON, D. R. (1966). Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of oliguria. J. C/in. Invest. 45, 724-735. OSSWALD, H., SCHMITZ, H. J., and KEMPER, R. (1978). Renal action of adenosine: Effect of renin secretion in the rat. Arch. Pharmacol. 303, 95-99. PREUSS,H. G., TOURKANTONIS, H., Hsu, S. H., SHIM, P. S., BARZYK, P.,TIo, F., and SCHRIERNER, G. E. (1975). Early events in various forms of experimental acute tubular necrosis in rats. Lab. Invest.
32, 286-294.
SMITH, A. U. (1950). Prevention of hemolysis during freezing and thawing of red blood cells. Lancer 2, 910. SOLEZ, K., ALTMAN, J., RIENHOFF, H. Y., ANTHONY, R. R., FINER, P. M., and HEPTINSTALL, R. H. (1976). Early angiographic and renal blood flow changes after HgCI, or glycerol administration. Kidney Int. 10, S-153-S-159. THIEL, G., MCDONALD, F. D., and OKEN, D. E. (1970). Micropuncture studies of the basis for protection of renin depleted rats from glycerol-induced acute renal failure. Nephron 7, 67-79. TORRES, V. E., STRONG, C. G., ROMERO, J. C., and WILSON, D. M. (1975). Indomethacin enhancement of glycerol-induced acute renal failure in rabbits. Kidney Int. 7, 170- 178. WOODS, H. F., EGGLESTON, L. V., and KREBS, H. A. (1970). The cause of accumulation of fructosel-phosphate on fructose loading. Biochem. J. 119, 501-510. WOODS, H. F., and KREBS, H. A. (1973). The effect of glycerol and dihydroxyacetone on hepatic adenine nucleotides. Biochem. J. 132, 55-60.