Exp Toxic Pathol 1999; 51: 199-205 URBAN & FISCHER http://www.urbanfischer.de/joumals/exptoxpath

llnstitute of Molecular Biology in Medicine and Hospital Civil de Belen, CUCS. University of Guadalajara, Jalisco, Mexico, 2Department of Biology, Facultad de Qufmica UN AM, 3Department of Gastroenterology, Instituto Nacional de la Nutrici6n S.Z. Mexico, D.F.

Differential effect of CCl4 on renal function in cirrhotic and non-cirrhotic rats

ANA ROSA RINc6Ni, AMADOR COVARRUBIAS', JOSE PEDRAZA-CHAVERRf2, JORGE LUIS P003, JUAN ARMENDARIZ­BORUNDAl, and ARTURO PANDURO'

With 2 figures and 4 tables

Received: February 4, 1998; Accepted: February 24, 1998

Address for correspondence: Dr. ANA ROSA RINC6N-SANCHEZ. Institute of Molecular Biology in Medicine, University of Guadalajara, P.O. Box: 2-500, Guadalajara, Jalisco Mexico, c.P. 44281 ; Fax: 52 (3) 617- 41- 59, 614-77-43, e-mail: arrincon@mail.udg.mx

Key words: Liver regeneration; Renal function; Cirrhosis, liver; CCI4 ; Nephrotoxicity.

Abbreviations: AST: aspartate aminotransferase. ALT: alanine aminotransferase. BUN: blood urea nitrogen. GFR: glo­merular filtration rate. HRS: hepatorenal syndrome. MAP: mean arterial pressure. PP: portal pressure. PRA: plasma renin activity. PRe: plasma renin concentration.

Summary

The pathogenesis of renal function alteration associated with liver disease remains to be elucidated. Although dif­ferent experimental animal models have been utilized in order to explain such pathophysiological state, none of them have completely explained the mechanisms involved. In this study we performed differential hemodynamic, hepatic and renal function alteration studies after induction of acute liver damage via intragastric administration of a single dose of CCl4 to cirrhotic and non-cirrhotic rats . Cirrhotic rats with acute liver damage exhibited a significant decrease in mean arterial pressure followed by a decreased glomerular filtra­tion rate, urinary sodium concentration and an induction of plasma renin concentration and activity. At the same time, a significant association between oliguria and mortality was observed. The renal histopathological studies revealed glo­meruli with mesangial hypercellularity and thickening of capillary wall, but not tubular epithelial injury. All these alterations were not detected in the control group, i.e. by non-cirrhotic rats with acute liver damage. This study sug­gests that the effect of CCl4 on kidney structure and func­tion depends on the functional state of the liver. Since this experimental model of acute liver damage in cirrhotic rats presents hemodynamics and renal function alterations simi­lar to those observed in the hepatorenal syndrome in man, it could be utilized to study the pathogenesis of renal function alterations associated with liver damage.

Introduction

CC14 induced cirrhosis in rats represents an adequate experimental model of cirrhosis in man (PEREZ-T AMA YO 1983; WENSING et al. 1990a; L6PEZ-NovOA et al. 1977). In 1936, CAMERON and KARUNARATNE (1936) described the morphological changes in liver after repeated admini­stration ofCC14 to rats. Thereafter, many techniques to ad­minister CCl4 in animal species have been used (PROCTOR and CHATAMRA 1982; PEREZ-TAMAYO 1983). However, for induction of experimental cirrhosis, it is necessary to give repeated doses of CC14 regardless of the animal spe­cies and route of administration (CAMERON and KARUNA­RATNE 1936; PROCTOR and CHATAMRA 1982; PEREZ­TAMAYO 1983; WENSING et al. 1990a; L6PEZ-NovOA et al. 1977). Intraperitoneal administration of CCl4 to rats for 8 weeks, results in cirrhosis (EHRINPREIS et al. 1980; PEREZ­TAMAYO 1983; PANDURO et al. 1988) and functional altera­tions similar to "compensated cirrhosis" in man (EHRIN­PREIS et al. 1980; PEREZ-TAMA YO 1983; ELIAS et al. 1990).

Changes in renal function are associated with hepatic function alterations (WENSING et al. 1990a, b). For exam­ple, the peripheral arterial vasodilatation hypothesis em­phasizes a functional, rather than mechanical role of the liver in salt retention and ascites (SCHRIER et aI. 1988), and suggests that decreased hepatic function is associated

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with increased vascular capacitance leading to peripheral arterial vasodilatation. Subsequently, hormonal and neu­ral changes initiate renal sodium retention followed by as­cites when compensatory mechanisms become insuffi­cient (SCHRIER 1983). WENSING et al. (1990a), have shown that once liver function falls below a critical threshold, so­dium retention occurs as an acute phenomenon followed by ascites. This effect was not observed in all CC14 treated rats because of interanimal variability in development of decompensated cirrhosis (WENSING et al. 1990a, b) and regeneration.

Liver regeneration has been widely studied after intra­gastric administration of a single dose of CC14 to non-cirr­hotic rats (PEREZ-TAMAYO 1983; FAUSTO 1984; PANDURO et al.1986). After CC14 administration, rats manifest acute liver damage characterized by steatosis and cell necrosis followed by regeneration (PEREZ-TAMAYO 1983; PANDURO et al.1986). Therefore, induction of acute liver damage by intragastric administration of a single dose of CCl4 to cirr­hotic rats may provide a better definition of the associa­tion between liver and renal function alterations.

No nephrotoxic effects in rats made cirrhotic after 12 weeks of treatment with repeated small doses of CCl4,

were detected (WENSING et al. 1990a, b). However, it is not known if a single intragastric administration of CCl4

to cirrhotic rats induces tubular necrosis, which is the ty­pical nephrotoxic effect of CCl4 in non-cirrhotic rats (STRIKER et al. 1968), and associates to renal function alterations.

In a previous study, liver cirrhotic regeneration after in­duction of acute damage was observed in cirrhotic rats treated with a single intragastric dose of CCl4 (PANDURO et al. 1988). During and after the regenerative process, the rats had ascites and oliguria for a short period of time but the renal function was not analyzed. In this study, we in­vestigated whether cirrhotic rats with acute liver damage induced by a single intragastric administration of CCl4

manifest necrosis and hemodynamic and renal function alterations.

Material and methods

Induction ofliver cirrhosis: Liver cirrhosis was produced in 100 male Wistar (CUCS farm-U. de G.) rats (40-60 g ini­tial weight) by intraperitoneal injection of 0.20 ml of a mix­ture of CCl4 (Merck Company, Darmstadt, FRG) with mi­neral oil (Sigma Chemical Company, St. Louis MO, USA) 3 times per week during 8 weeks, progressively increasing the concentration of CCl4 until the fourth week of treatment (EHRINPREIS et al. 1980). The percentages of CCl4 in mine­ral oil (v/v) were as follows: week 1, 13 %;week 2, 16 %; week 3, 20 %; and weeks 4 to 8, 25 %. Age and sex matched control animals were included. The rats were maintained under day-night cycles of 12 h, fed ad libitum with rodent lab diet (PMI-500l).

Induction of acute liver damage in cirrhotic and age matched control rats: At the end of the CCl4 treatment, cirrhotic and age matched control rats were kept in indivi­dual metabolic cages. To avoid cumulative chronic effect of

200 Exp Toxic Pathol51 (1999) 3

CC14 in cirrhotic rats, the induction of acute liver damage was carried out 6 days later. Cirrhotic and age-matched con­trol rats received a single intragastric dose of a 1: 1 (v/v) mix­ture of CClicorn oil (0.5 ml per 100 g body weight) (PAN­DURO et a1.1986). Twenty four hour urine-samples at indi­cated times were collected from the different experimental groups of rats and the total urinary volume of each rat was measured and tested for electrolytes and creatinine.

Hemodynamic studies: Rats were deprived of food but not water overnight and were anesthetized with ether (BAER et at. 1993) during hemodynamic evaluation. The portal vein was exposed by median laparotomy and a polyethylene catheter was placed alongside the femoral artery for arterial pressure and heart rate measurements. Another catheter was inserted in an ileocolic vein for portal pressure measure­ment. Heart rate, arterial pressure, and portal pressure (PP) were recorded on an square wave electromagnetic mano­meter (PPG Medical Electronics, RM-300, The Netherlands). Pressure recording was performed every 5 minutes until 3 continuous stable measurements were obtained. Liver and kidney were quickly removed and a small fragment of each tissue was fixed in formalin for further histopathological analysis, as described below.

Renal and liver function tests: To perform renal and liver function tests, including renin activity, we used 35 rats that were decapitated. Serum and urine sodium were deter­mined by flame photometry (Flame photometer model 403. Instrumentation Laboratory, Lexington, MA.). Blood urea nitrogen (BUN) and creatinine were measured using an autoanalyzer (BUN analyzer 2 and creatinine analyzer 2, Beckman Instruments Inc., Fullerton CA, USA) (PEDRAZA­CHEV ARRI et al. 1993). Creatinine clearance was used to esti­mate glomerular filtration rate (GFR). Plasma renin activity (PRA) was measured by radioimmunoassay (RIA) of angio­tensin I (AI) (Du Pont New England Nuclear, Boston MA, USA) produced for endogenous renin substrate after incubation of plasma at pH 6.0 for 1 h at 37°C, as described elsewhere (IBARRA-RUBIO et al. 1990). Plasma renin concentration (PRC) was measured by RIA of Al after incubation of plasma (1 h at 37°C) with an excess of renin substrate (Plasma from 24 h nephrectomized rats) (IBARRA-RUBIO et al. 1990).

Liver damage was histologically and biochemically as­sessed by determining serum albumin, total proteins, bili­rubin, aspartate aminotransferase (AST) and alanine amino­transferase (ALT) activities.

Histological samples examination: Slices of liver and kidney were used for histological examination. Tissue blocks for light microscopy were fixed in 10 % formaldehyde in 0.1 M phosphate (pH 7.2). All samples were embedded in paraffin, cut in sections of 4 !lm thickness, and stained with hematoxylin and eosin. Optical microscopy evaluation was performed by the same researcher who was blinded to the experimental tissue to be studied. At least three animals were used at each experimental time point and fifteen glo­merulus were analyzed (40 X) in each kidney for the counting of mesangial cells.

Plasma Volume: Plasma volume was measured by the Evan's Blue dilution method (WANG 1959). A catheter was placed into the femoral artery of anesthetized rats. 0.2 ml of Evan's blue dye solution (5 % of Evan's blue dye WN in sterile isotonic saline) were injected and a blood sample was taken 15 minutes later. Plasma dye concentration was read at 610 nm on a Spectrophotometer (Beckman 650) and plasma volume determined as reported by KAUFMAN et al. (1981).

Table 1. Hepatic function studies in control, cirrhotic and cirrhotic rats with acute liver damage.

Group (n) Total protein Albumin Total bilirubin AST ALT (g/100 ml) (gllOO m!) (g/100 ml) (lUlL) (lUlL)

C (5) 6.3 ±0.9 3.5 ±0.2 1.0 ± 0.2 110± 26 35.8 ± 4.6 CR(9) 5.6 ±0.8 3.2 ±0.5 0.9 ± 0.1 182 ± 34 * 72.0 ± 8.4 * 1 (5) 5.2 ±0.2 2.7 ± 0.4 1.5 ± 0.2 * 308 ± 55 * 335 ± 16 * 2 (5) 5.5 ±0.6 2.7 ± 0.3 1.6 ± 0.4 * 524± 105 * 462 ± 32 * 3 (5) 5.4 ± 0.2 2.8 ±0.3 1.2 ± 1.5 254± 33 * 133 ± 86 7 (5) 6.6 ± 0.3 3.8 ±0.5 0.9 ± 0.1 149 ± 13 59 ± 7

Hepatic function studies were performed in age matched control rats (C), cirrhotic rats (CR) and in cirrhotic rats with acute liver damage 1,2,3 and 7 days after a single intragastric administration of CCI4 , n = number of animals, * statistically significant at p < 0.05 when compared with the control group.

Table 2. Plasma volume, water intake and renal function studies in control, cirrhotic and cirrhotic rats with acute liver damage.

Group (n) Plasma Water Urinary Urinary BUN Serum GFR volume intake volume Na+ (mg/dl) creatinine (mllmin.) (mllKg) (mllKg/h) (mllKg/h) (mEq/day) (mg/dl)

C (5) 40.15 ± 0.34 5.20 ± 1.80 2.50 ± 0.41 1.18 ± 0.4 13.3 ± 0.6 0.50 ± 0.1 1.17 ± 0.02

CR (9) 39.39 ± 0.50 5.83 ± 0.63 2.19 ± 0.24 0.86 ± 0.3 20.7 ± 2.8* 0.43 ± 0.05 0.90 ± 0.5

1 (5) 41.36 ± 1.93 3.09 ± 3.01 2.19 ± 1.32 0.37 ± 0.2* 21.6 ± 3.3* 1.10 ± 0.2* 0.41 ± 0.3 2 (5) 43.42 ± 0.21 2.96 ± 2.19 2.69 ± 2.21 0.14 ± 0.1 * 22.7 ± 3.0* 1.46 ± 0.3* 0.29 ± 0.1 * 3 (5) 46.79 ± 0.75* 3.07 ± 1.11 2.22 ± 1.35 0.56 ± 0.5 16.3 ± 2.7 0.76 ± 0.4* 0.41 ± 0.2 7 (5) 39.75 ± 2.31 5.40 ± 1.22 3.99 ± 2.02 1.34 ± 0.3* 14.0± 1.4 0.50 ± 0.1 0.67 ± 0.2

Renal function studies were performed in age matched control rats (C), cirrhotic rats (CR), and cirrhotic rats with acute liver damage 1, 2, 3 and 7 days after a single intragastric administration ofCC14 • BUN = Blood urea nitrogen, GFR = Glome-rular filtration rate, n = number of animals, * statistically significant at p < 0.05 when compared with the control group.

Statistical analysis: Results are expressed as mean ± S.D. of at least 5 determinations. Data were analyzed for statisti­cal analysis using ANOV A with Turkey test. Differences were considered to be statistically significant when p < 0.05.

Results

Hepatic and renal function studies in cirrhotic rats: Following CCl4 chronic treatment, cirrhotic rats weighed less than did age matched control rats (321 ± 60 vs. 392 ± 28 g); even though these differences were not sta­tistically significant. No significant variation in serum al­bumin, total proteins and bilirubin concentrations could be appreciated to controls. In contrast, AST and ALT in­creased 65 % and 100 % respectively (p < 0.05) (table 1).

Renal function was slightly altered in cirrhotic rats; al­though, urinary output, urinary sodium concentration and GFR were not statistically different from those corres­ponding to the control group, in spite of increased BUN (table 2). PRA and PRC values were normal in cirrhotic rats (table 3).

Table 3. Plasma renin activity and plasma renin concentra­tion in control, cirrhotic and cirrhotic rats with acute liver damage.

Group (n) PRA ng Al /ml/h PRC ng Allmllh

C (5) 2.9 ±0.5 7.2± 4.0

CR (5) 3.1 ± 0.8 8.8 ± 3.3

1 (5) 4.1 ± 1.0 25.4 ± 11.4 *

2 (5) 18.0 ± 8.0 * 106.0 ± 23.5 *

3 (5) 10.5 ± 3.8 * 54.3 ± 11.0 *

7 (5) 11.0 ± 0.4 * 16.3 ± 9.5 *

Plasma renin activity (PRA) and plasma renin concentra­tion (PRC) in control (C), cirrhotic (CR), and in cirrhotic rats with acute liver damage 1, 2, 3, and 7 days after a single intragastric administration of CCI4. Al = angioten­sin 1, n = number of animals, * statistically significant at p < 0.05 when compared with the control group.

Exp Toxic Pathol51 (1999) 3 201

Table 4. Mesangial cells in kidneys of control, cirrhotic, and cirrhotic rats with acute liver damage.

Group (n)

Control (3)

Cirrhotic (0 d) (3)

1 d (7)

2 d (3)

3 d (6)

5 d (5)

7 d (3)

Number of mesangial cells 1

50 ± 3 66±4

81 ± 9 * 90 ±4 *

104±6*

78 ±7 * 85 ± 3 *

Values are means ± S.D; 1,2,3,5 and 7 represents the days after acute liver damage. n = number of animals studied; I number of mensangial cells per 15 glomerulus; * statistically significant at p<0.05 when compared with the control group.

202 Exp Toxic Pathol 51 (1999) 3

Fig. 1. Histology of kidney of cirrhotic rats without (A) and with (B) acute liver da­mage 48 hours after CC14 administration. As shown in B, morphological changes were evident in the glomerulus displaying hyper­cellularity of messangial cells with respect to A. Hematoxylin and eosin 900 X. C shows a higher magnification (1300 X) of B in order to clearly identify the mesangiocapil­lar hypercellularity and mild thickening of the capillary loops (arrows).

Hemodynamic studies in cirrhotic rats: Portal hy­pertension in cirrhotic rats was observed, 19.0 ± 2.1 nun Hg compared to 7.7 ± 0.5 mm Hg in the age matched control group (p < 0.05), whereas MAP was lower than in controls (92.0 ± 11 mm Hg vs. 98.0 ± 2 mm Hg) (p = NS).

Histopathologic studies of cirrhotic rats with acute liver damage: Extensive liver fibrosis following chronic CC14 treatment was manifested (P ANDURO et al. 1988). At the same experimental time point, the kidneys of cirrhotic rats were histologically normal (fig. lA). On the other hand, 48 hours after cirrhotic rats received acute CC14

treatment, when liver regeneration was supposed to be completed (PANDURO et al. 1986), the kidneys of rats pre­senting acute liver damage showed glomerular mesangial hypercellularity (fig. 1B and table 4), and mild thickening of the capillary wall (fig. 1 C). Tubules appeared normal.

These histological changes were reversed 7 days after acute liver damage.

Hemodynamic studies in cirrhotic rats with acute liver damage: Portal hypertension persisted in cirrhotic rats with acute liver damage. Portal pressure in this group of animals was 13.0 ± 3 mm Hg 24 hours after CCl4 treat­ment, (control group 7.7 ± 0.5 mm Hg), showing a signi­ficant decrease at this experimental time point when com­pared to cirrhotic rats (19.0 ± 2.1 mm Hg). However, after 48 hours, there was a further increase to 16.0 ± 2 mm Hg (p < 0.05). MAP decreased significantly from 92.0 ± 11 to 69.0 ± 2 mm Hg (p < 0.05) 24 hours after administra­tion of CCl4 reaching almost normal levels after 48 hours (82 ± 10 mm Hg).

Hepatic and renal function studies in cirrhotic rats with acute liver damage: Maximum liver damage of cirrhotic rats occurred 24 and 48 hours after acute admi­nistration of CCl4 followed by replication of parenchymal cells (PANDURO et al. 1988). In this study, liver function tests revealed no significant reduction in serum albumin concentration (table 1). Neither significant changes in total serum protein were detected post CCl4 treatment. However, a statistically significant increase in total bili­rubin concentration and aminotransferases (AST and ALT) activity were detected during acute liver damage (table 1). Total bilirubin returned to control values 7 days post-CCl4 treatment, whereas AST and ALT remained elevated (table O.

Renal function alterations were also detected at the same experimental time points when cirrhotic rats mani­fested acute liver damage. Decreased urinary sodium con­centration and GFR were present 24 and 48 hours post­CCl4 treatment. BUN and serum creatinine values were, statistically, significantly higher than those of the control group (table 2). Increased activity and concentration of plasma renin were present in cirrhotic rats with acute liver damage showing a maximum peak 48 h after CCl4 treat­ment. Thereafter, activity and concentration of plasma renin decreased but still remained above control levels in surviving rats (table 3). No statistically significant chan­ges in plasma volume, water intake and urinary volume were observed in cirrhotic rats with acute liver damage after CCl4 administration (table 2). Several cirrhotic rats with acute liver damage had marked decrease of urine vo­lume, and therefore, they were analyzed separately and each parameter was correlated with the survival rate. 40 % of cirrhotic rats died 48-72 h post acute CCl4 treatment. Before dead, the rats had a marked oliguria (fig. 2).

Histopathologic, hemodynamic and renal function studies in non-cirrhotic rats with acute liver damage: Since renal function alterations occur in cirrhotic patients with hepatorenal syndrome similar to those presented in cirrhotic rats with acute liver damage, and because of the known necrotizing tubular effect of CC14 on the kidney of non-cirrhotic rats (STRIKER et al. 1968; PEDRAZA-CHA­VERRf et al. 1993), further investigations were performed to analyze hepatic and renal function , hemodynamics and pathology in non-cirrhotic rats with acute liver damage.

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2

o ~-~---~-------~ o 2 3 " 5

Days alter treatment 01 CIrrhotIC ( e) (

and non arrho\Jc ralS ( ) With CCI4

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Fig. 2. Association between oliguria and mortality in cirr­hotic rats with acute liver damage. Mortality was higher (40 %) in rats presenting marked decrease in urine excretion between 24-72 h (0) as compared with cirrhotic animals not presenting oliguria (e). Non-cirrhotic rats presenting polyuria showed a 100 % survival (0). * statistically significant at p < 0.05 when compared with the control group.

Non-cirrhotic rats with acute liver damage had higher levels of AST (753 ± 26 vs. 524 ± 105 lUlL) and ALT (762 ± 167 vs. 462 ± 32 lUlL) than did cirrhotic rats with acute live damage on the same days. The same group of animals did not show significant changes in PP and MAP 24 hours after CCl4 administration (9.0 ± 1.0 and 90.5 ± 2 mm Hg, respectively), comparing with control group values (7.7 ± 0.5 and 98.0 ± 2 mm Hg, respectively).

In contrast to cirrhotic rats with acute liver damage, non-cirrhotic rats manifested polyuria (fig. 2) and pro­teinuria (data not shown) after induction of acute liver da­mage with CCI4• Renin activity increased to a maximum at day fourfrom 2.1 ± 1.09 to 3.7 ± 2.2 ng/Allml/h; how­ever, such increase was not statistically significant. No significant changes either were detected in urinary so­dium concentration. In control rats, urinary sodium was 1.1 ± 0.2 mEq/day whereas, in non-cirrhotic CCl4 treated rats, were 1.1 ± 0.1,0.7 ± 0.4 and 0.8 ± 0.3 mEq/day at the first, second and third day, respectively.

Histological analysis of the kidney in non-cirrhotic rats with acute liver damage induced with CCl4 confirmed tu­bular injury as originally reported by STRIKER et al. and other investigators (STRIKER et al. 1968; PEDRAZA-CHA­VERRf et al. 1993).

Exp Toxic Patho151 (1999) 3 203

Discussion

In this study we observed that hemodynamic, renal and pathological alterations exhibited by cirrhotic rats with an additional CC14 -induced acute liver damage, were diffe­rent from those of non-cirrhotic rats similarly treated.

Maximal liver damage occurred 24 and 48 hours after CC14 administration to cirrhotic rats followed by replica­tion of parenchymal cells, as evidenced by the increased incorporation of 3H-thymidine around 72 hours after in­toxication (PANDURO et a1.l988). The increase of serum total bilirubin and AST and ALT activity at 48 hours, re­flected acute liver damage in CC14-treated cirrhotic rats. In agreement with previous observations no changes in total serum proteins were detected following the CC14

administration (PANDURO et al. 1986, 1988). Association between liver and renal function altera­

tions was detected in all CC14 treated cirrhotic rats. When acute liver damage occurred, hemodynamic and renal function alterations were present. Twenty-four hours post CC14-treatment, a significant decrease in MAP was re­gistered followed by a decreased GFR, and urinary so­dium concentration and induction of plasma renin con­centration and activity. This order of events is in agree­ment with other reports showing that decrease in MAP precedes onset of sodium retention (WENSING et al. 1990a). Decreased MAP in cirrhotic dogs and rats is attributed to decreased peripheral vascular resistance in the presence of normal or increased cardiac output (FERNANDEZ­MUNOz et al. 1985; BOSCH et at. 1993). Induction of plasma renin in decompensated cirrhosis is probably due to equilibrating mechanisms to avoid a fall in MAP (SCHRIER et al. 1988).

Cirrhotic rats presented portal hypertension which per­sisted after administration of CC14• However, a small de­crease in portal hypertension observed 24 h after acute intoxication may be explained by blood sequestration in liver and spleen, since an increase in wet weight of both organs was detected (data not shown). To our knowledge, no such alteration has been reported in human or animal models before, most probably because such measure­ments are not routinely performed in patients with de­compensated cirrhosis.

Repeated small doses of CC14 do not produce nephro­toxicity (L6PEZ-NovOA et al. 1980; PROCTOR and CHA­TAMRA 1982; JIMENEZ et a1.l985; WENSING et al. 1990a), as it was observed in the kidney of cirrhotic rats; however, we notice that the acute doses of CC14 used to produce tu­bular necrosis in non-cirrhotic rats did not produce tubu­lar morphological changes in cirrhotic rats. Intragastric administration of a single dose of CC14 to cirrhotic rats did not cause polyuria, proteinuria or tubular necrosis. In con­trast, glomeruli with mesangial hypercellularity and mild thickening of the capillary wall were observed with nor­mal tubuli.

CC14 is mediated through cytochrome P450-dependent metabolism (WOLF et al. 1980). The initial metabolite is the trichloromethyl free radical, which is believed to in-

204 Exp Toxic Pathol 51 (1999) 3

itiate biochemical events ultimately translated as liver cell necrosis (WOLF et al. 1980). A decrease of cytochrome p450 gene expression occurs during liver regeneration in CC14 treated rats (PANDURO et al. 1986); therefore, de­creased activity or concentration of cytochrome p450 in cirrhotic rats could lead to a reduced nephrotoxicity and explain, at least partially, why tubular morphological changes are not present in cirrhotic rats. Besides, renal excretion capacity drops after hepatic failure if a toxic substance is biotransformed in the liver before its renal excretion can take place (BARTH et al. 1996; WESTPHAL and BROGARD 1997).

No significant changes in urine volume were detected in cirrhotic rats with acute liver damage because of a large standard variation. However, when oliguric rats were ana­lyzed separately, a close correlation was observed be­tween oliguria and mortality. 40 % of the animals died du­ring the first 72 hours after CC14 administration and, be­fore dead, manifested statistically significant decrease in urine volume (p < 0.05).

Since the maximum peak of thymidine incorporation in CC14 treated cirrhotic rats occurs at 72 hours (P ANDURO et al. 1986), we infer that mortality is related to failure of the cirrhotic liver to regenerate in the presence of decreased liver function (WENSING et al. 1990a).

Such findings could also explain why decompensated cirrhosis occurs only when repeated small doses of CC14

were administered for prolonged periods of time or in the presence of cytochrome p450 system inducers (RECK­NAGEL and GLENDE 1973; WOLF et al. 1980; BRATTIN et at. 1995). But, when acute liver damage is produced in cirrhotic rats with a single dose of CC14, decompensated cirrhosis is observed with hemodynamic and renal func­tion alterations similar to those observed in the hepato­renal syndrome in man (GINES et al. 1993; ARROYO et al. 1996; BATALLER et al. 1997).

The hemodynamic and renal function alterations de­tected in CC14-treated cirrhotic rats such as decrease in MAP preceded by sodium retention and induction of plasma renin activity at a time when liver function sud­denly deteriorates, may explain the rapid formation of as­cites in all experimental animals and are consistent with the peripheral arterial vasodilatation hypothesis (SCHRIER et al. 1988).

Acknowledgments: We are grateful to Prof. IRWIN M. ARIAS, Massachusetts and M.D., Ph.D. MARCOS ROJKIND, NY for kindly revising the manuscript.

This work was supported by grants from CONACYT No. l726-PN, 05l5PM and Secretarfa de Salud (Molecu­lar Biology in Medicine Program) to A.P.

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