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Acute renal failure induced by carbon tetrachloride in rats with hepatic cirrhosis Fernando Jaramillo-Juarez;1 Maria Luisa Rodriguez-Vazquez;1 Ana Rosa Rincon-Sanchez;2# Maria Consolacion Martinez;1 Genaro G. Ortiz;3 Javier Llamas;1 Francisco Anibal Posadas;1 Jose L. Reyes4 1 Universidad Autonoma de Aguascalientes, Aguascalientes, Mexico.DC animals had severe hypoglycemia, there was an extensive liver fibrosis, and glomeruli had hypercellularity and tubules showed extensive vacuolization in cirrhotic and DC rats.(Na+ -K+ )-ATPase is located at the basolateral membrane complex attached to actin cytoskeleton and is essential for the transtubular vectorial reabsorption of sodium. Studies in humans and experimental animals provided strong evidence that ADH plays a major role in the pathogenesis of water retention in cirrhosis. Longitudinal studies in rats with cirrhosis and ascites have shown a chronological relationship between ADH hypersecretion and impaired water excretion.33,34 In addition, kidneys from cirrhotic rats with ascites show increased gene expression of aquaporin-2, the ADH-regulated water channel.35 In most patients with ascites free-water clearance is reduced.31 Our data are in accordance with this report. Water retention contributes to dilutional hyponatremia observed in patients with advanced liver disease.36,37 Hypoxic injury to tubular cells represents an early event in acute renal failure. Our data showed that both, renal hemodynamics and proximal tubular function were damaged in DC with RF rats. Persistent renal hypoxia may favor occurrence of tubular damage associated to ATP depletion, as shown in this study. In regard to renal vasoconstriction during RF, sympathetic axis can be stimulated by three different mechanisms: a) activation of pressure receptors in response to hypotension, b) non-volume-dependent hepatic baroreceptors and c) secondary to metabolic changes like catecholamines secretion in response to hypoglycemia.By contrast, both groups, cirrhotic and DC rats with RF, showed renal cortical ATPases (Total, Mg++-dependent and Na+ -K+ -dependent) that were significantly unaffected. Na+ -K+ -ATPase activity changed from a control value of 23 +- 3.3 to 32.5 +- 4.6 and 32.7 +- 10, nmol Pi min-1 mg-1, in cirrhotic and DC rats with RF respectively. These findings suggest that low tissue concentration of ATP was probably related to nucleotide altered synthesis. Increased glucose and phosphate urinary excretions in DC with RF rats However, glucose urine/plasma ratio and phosphate urine/plasma ratio were markedly higher in both groups, cirrhotic and DC rats with RF, as compared to control values, indicating marked urinary losses of glucose and phosphate. Free-water clearance decreased significantly in DC rats with RF when compared to control and cirrhotic group Figure 1. Histopathologic evaluation of rat liver and kidney. When additional acute liver damage was induced in cirrhotic rats, intense hemodynamic and renal functional alterations ensued. In contrast, renal hemodynamics did not change in cirrhotic rats, similarly to what occurs in compensated cirrhosis in humans. Renal response to decreased blood pressure is altered in severe liver disease with activation of sympathetic system and increased synthesis of several renal vasoconstrictor compounds (endothelin, thromboxane A2 and F2 -isoprostanes).Sodium is then returned to systemic circulation by the (Na+ -K+ )- ATPase located at the basolateral membrane.20,21 Glucose and phosphate reabsorption are coupled to sodium transport, via transmembrane co-transporters.22,23 Therefore increased urinary losses of sodium, glucose and phosphate are indicative of severe proximal tubular damage.24 Renal retention of sodium is considered to be responsible for edema and ascites in hepatic cirrhosis.25 The mechanisms of abnormal tubular sodium reabsorption in cirrhosis are multifactorial.All of these mechanisms might be active in RF.4 Hypoglycemia of the RF rats reported in this study is in accordance with the third mechanism related to strong activation of sympathetic nervous system in patients with RF.38 In summary, CCl4 -treatment to cirrhotic rats with RF reduces GFR and filtration fraction and produces proximal tubular damage characterized by increased sodium, glucose and phosphate excretion, decreased renal ATP concentration and reduced free-water clearance.DC animals had severe hypoglycemia.Introduction Alterations in kidney structure and function are frequently found in severe liver disease and once liver function falls below a critical threshold, sodium retention occurs followed by ascites, associated with profound disturbances of splachnic and systemic hemodynamics which in turn may affect renal function.1,2 As disease progresses, constriction of intrarenal vascular system favors marked sodium and water retention, leading to refractory ascites, a progressive rise in plasma creatinine levels and reduction of renal clearances (decompensated cirrhosis).This model is useful to study the pathogenesis of renal failure associated with liver damage when the hepatic function decrease after an acute liver damage.7 In our present work, we used our model to further characterize the systemic and renal hemodynamics, histopathological and functional changes of proximal tubu- (C) 2019, Fundacion Clinica Medica Sur, A.C. Published by Elsevier Espana S.L.U. This is an open access article under the CC BY-NC-ND cells (renal sodium handling, phosphate and glucose excretion rates) as well as the function of cells from the distal nephron (renal water handling), in cirrhotic and in cirrhotic rats with acute liver damage induced by carbon tetrachloride.Decrease in urine volume and in free-water clearance indicated water retention in cirrhotic rats with RF. Pathogenesis of water retention in cirrhosis is complex and probably involves several factors, including increased plasma levels of antidiuretic hormone (ADH), decrease in prostaglandin synthesis and reduced delivery of filtrate to the ascending limb of Henle.31,32 It should be emphasized that increased sodium excretion together with water retention led to dilutional hyponatremia.Sodium, inulin and PAH were measured by flame photometry, anthrone method and spectrophotometry, as previously described.14,15 Renal cortex ATPase activities and tissue ATP concentrations: Renal cortex was separated from medulla and homogenized in solution containing (mM): 240 sucrose, 1 EDTA and 10 Tris-HCl, pH 7.5 (20 % w/v homogenate).Kim SW, Schou UK, Peters CD, de Seigneux S, Kwon TH, Knepper MA, Jonassen TE, et al. Increased apical targeting of renal epithelial sodium channel subunits and decreased expression of type 2 11beta-hydroxysteroid dehydrogenase in rats with CCl4-induced decompensated liver cirrhosis.Moreover, depletion of ATP may increase cytosolic calcium, which activates proteases and phospholipase A2 with increased synthesis of leukotrienes that might induce renal ischemia.3 In accordance, increased production of cysteinyl leukotrienes in RF has been reported.29 Cellular energy depletion results in rapid loss of polarity in proximal tubule cells.Glomerular filtration rate (GFR) and renal plasma flow (RPF) were estimated by inulin and p-aminohippurate clearances, as previously described.14 Arterial blood pressure (BP) was measured with a Statham P-23A transducer and recorded in a RP Beckman Dynograph.Instituto de Enfermedades Cronico-Degenerativas CUCS-Universidad de Guadalajara Guadalajara, Jalisco, Mexico E-mail: [email protected]; [email protected] Fax: 52 (33) 1058 5200 ext 3893 Phone: 52 (33) 1058 5200 ext 3891, 3892 Manuscript received and accepted: 22 July and 20 August 2008 Abstract Relationship between cirrhosis and renal dysfunction is not yet fully understood.Renal hemodynamics Mean arterial (mL min -1 kg-1) Filtration Group Weight (g) pressure (mmHg) C-In C-PAH fraction Control (n = 12) 308 +- 6 110 +- 4 7.35 +- 0.7 13.8 +- 1.4 0.552 +- 0.05 Cirrhotic (n = 9) 340 +- 21 103 +- 8 7.47 +- 1.4 13.9 +- 2.0 0.551 +- 0.09 DC with RF (n = 8) 371 +- 16 75 +- 10 ** 3.54 +- 0.8* 10.7 +- 2.2 0.315 +- 0.03 * DC with RF = decompensated cirrhosis with renal failure; C-In = clearance of inulin; C-PAH = clearance of para-amino-hippurate *p < 0.05 and **p < 0.01, when compared with the control group.4.5 3.0 1.5 0 Albumin (g/100 mL) ** ** Control Cirrhotic D-CR+RF A 80 40 20 0 Total protein (mg/mL) **

Control Cirrhotic D-CR+RF B 60 3200 0 AST (IU/L)

Control Cirrhotic D-CR+RF C 2800 2400 2000 1600 1200 800 400 15 10 5 ALT (IU/L) *

Control Cirrhotic D-CR+RF D 10 20 200 600 1000 1400 1800 2200 0 Table II. The urine/plasma ratio of glucose and phosphates from control, cirrhotic and decompensated cirrhotic rats with renal failure, produced by CCl4 .Group Urine/plasma Urine/plasma glucose ratio phosphate ratio Control (n = 10) 0.026 +- 0.004 3.74 +- 0.34 Cirrhotic (n = 10) 0.196 +- 0.030 * 5.13 +- 0.45 DC with RF (n = 10) 0.561 +- 0.075 ** 21.18 +- [ ] via transmembrane carriers (SGLT 1 and 2) that also reabsorb glucose, coupled to the transport of phosphate or aminoacids, by selective sodium channels or through the paracellular pathway.Ischemia or cellular ATP depletion leads to the rapid redistribution of (Na+ -K+ )-ATPase into the apical membrane domain of the proximal tubule.30 Therefore, following depletion of energy stores, electrolyte (Na, K) gradients collapse and actin-containing microfilaments become disorganized.2.Induction of liver cirrhosis: Liver cirrhosis was achieved in 100 male Wistar rats (40-60 g initial weight; 250 - 350 g final weight) by intraperitoneal injection of 0.2 ml of a mixture of CCl4 (J.T. Baker USA) and mineral oil (Sigma Chemical Company, St. Louis MO) 3 times per week during 8 weeks, progressively increasing CCl4 concentration until the fourth week of treatment.7 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%.In those groups, we studied: (A) glomerular filtration rate (GFR) and plasma renal flow (PRF); (B) renal sodium handling (C) renal cortical Na+ -K+

• ATPase activity and tissue ATP concentrations, (D) renal glucose and phosphate excretion rates and (E) renal water handling.Histological samples examination: Four animals per group (control and CCl4 -treated) were anesthetized with sodium pentobarbital and intravascularly perfused with phosphate buffer solution (PBS), pH 7.4, containing heparine (1,000 UI/L) and procaine (1 g/L) for rinsing of livers and kidneys.Panel A) Serum albumin in control (empty bar), cirrhotic (hatched bar) and rats with acute hepatic and renal damage (fill bar).Panel B) Total plasma protein content, symbols as in panel A: C): Aspartate aminotransferase in the same groups of rats previously described; D): Alanine aminotransferase, ALT.Rincon-Sanchez AR, Covarrubias A, Rivas-Estilla AM, PedrazaChaverri J, Cruz C, Islas-Carbajal MC, Panduro A, et al. PGE2 alleviates kidney and liver damage, decreases plasma renin activity and acute phase response in cirrhotic rats with acute liver damage.This acute treatment change cirrhotic by decompensated cirrosis rats and induce acute renal failure in the animals.7-9 Liver function tests: Liver damage was assessed by measurements of: a) serum albumin,10,11 b) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities12 and c) total proteins.To explain irreversibility of DC damage with RF, it has been also suggested that this illness may be complicated by acute tubular necrosis.3 Histological alterations in the kidney of DC patients with RF, similar to those observed during acute tubular necrosis (ATN), with tubular degeneration and interstitial leukocyte infiltration have been reported.27,28 We observed degenerative changes in cirrhotic and DC rats with RF (Figure 1).Islas-Carbajal MC, Covarrubias A, Grijalva G, Alvarez A, Armendariz-Borunda J, Rincon-Sanchez AR. Nitric oxide synthases inhibition results in renal failure improvement in cirrhotic rats.Camps J, Sola J, Arroyo V, Perez-Ayuso RM, Gaya J, Rivera F, Rodes J. Temporal relationship between the impairment of free water excretion and antidiuretic hormone hypersecretion in rats with experimental cirrhosis.tubules exhibited loss of brush border, cloudy swelling and cellular fragments inside the lumen (arrows) Discussion Hemodynamic and functional renal alterations in DC rats with RF were more severe than in cirrhotic rats.Approximately 10% to 20% cirrhotic patients with ascites spontaneously eliminate relatively large amounts of sodium in the urine.25 Decrease in tubular reabsorption is generally regarded as evidence of intrinsic tubular dysfunction.Kestenbaum B, Sampson JN, Rudser KD, Patterson DJ, Seliger SL, Young B, Sherrard DJ, et al. Serum phosphate levels and mortality risk among people with chronic kidney disease.In spite of diminished filtered sodium load, there was an increment in the fractional excretion of sodium (FeNa), in DC rats with RF. FeNa increased by 135% in DC rats with RF as compared to control rats and, excretion was 68% higher when compared to the cirrhotic group (Figure 3), indicating severe alteration of the tubular handling of sodium.(e) Cirrhotic animals exhibited hyperchromatic podocyte (arrowhead) and large mesangial spaces (small arrow); cloudy swelling (arrows) and hydropic degeneration (double arrow) were observed in the tubules.We observed fall in mean arterial pressure (MAP) probably due to diminished peripheral vascular resistance and significant reduction of GFR and filtration fraction in DC rats with RF, in a similar fashion as reported to cirrhotic rats 17.Halimi C, Bonnard P, Bernard B, Mathurin P, Mofredj A, di Martino V, Demontis R, et al. Effect of terlipressin (Glypressin) on hepatorenal syndrome in cirrhotic patients: results of a multicentre pilot study.Caramelo C, Fernandez-Munoz D, Santos JC, Blanchart A, Rodriguez-Puyol D, Lopez-Novoa JM, Hernando L. Effect of volume expansion on hemodynamics, capillary permeability and renal function in conscious, cirrhotic rats.Moore KP, Taylor GW, Maltby NH, Siegers D, Fuller RW, Dollery CT, Williams R. Increased production of cysteinyl leukotrienes in hepatorenal syndrome.A model of cirrhosis with acute hepatic and renal damage (RF), produced by CCl4 in rats, with hemodynamic and renal functional alterations, similar to those observed in decompensated cirrhosis (DC) in man, was used to study chemical nephrotoxicity in animals.Osmolal (Cosm ) and free-water (CH2O) clearances were calculated as follows: Cosm = [U] osm X Urine flow/[P] osm where [U] osm and [P] osm are urine and plasmatic-osmolalities, respectively, and urine flow is expressed in mL/ min.Rincon AR, Covarrubias A, Pedraza-Chaverri J, Poo JL, Armendariz-Borunda J, Panduro A. Differential effect of CCl4 on renal function in cirrhotic and non-cirrhotic rats.Fernandez-Munoz D, Caramelo C, Santos JC, Blanchart A, Hernando L, Lopez-Novoa JM. Systemic and splanchnic hemodynamic disturbances in conscious rats with experimental liver cirrhosis without ascites.Age and sex matched control animals received the same volume of mineral oil.7 Induction of acute renal damage: At the end of eight weeks of chronic CCl4 treatment and after 6 days of the last chronic CCl4 dose, cirrhotic rats received a single intragastric dose of a 1:1 (v/v) mixture of CCl4 /corn oil (0.5 mL per 100 g body weight).In DC with RF-group, there was severe glomerular damage with increased mesangial space and hyperchromatic nuclei of podocytes; tubules showed cloudy swelling and degeneration (Figure 1f).Excretion of sodium was strikingly increased in DC rats with RF Probably related to decrement in MAP, filtered sodium load decreased in DC rats with RF (756 +- 184 i iEq/min/kg) as compared to control and cirrhotic rats (1032 +- 94 and 1117 +- 214 i iEq/min/kg, respectively).Eur J Gastroenterol Hepatol 1999; 11: 1061-1065.2.3.4.5.6.7.8.9.10.11.12.13.14.15.16.17.18.19.20.21.22.23.24.25.26.27.28.29.30.31.32.33.34.35.36.37.38.

Acute renal failure induced by carbon
tetrachloride in rats with hepatic cirrhosis
Fernando Jaramillo-Juárez;1
María Luisa Rodríguez-Vázquez;1
Ana Rosa Rincón-Sánchez;2# María Consolación Martínez;1
Genaro G. Ortiz;3
Javier Llamas;1
Francisco Anibal Posadas;1
José L. Reyes4
1 Universidad Autónoma de Aguascalientes, Aguascalientes,
México.
2
IECD, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco,
México.
3 Centro de Investigación Biomédica de Occidente-IMSS,
Guadalajara, Jalisco, México.
4 Centro de Investigación y de Estudios Avanzados. Instituto
Politécnico Nacional, México
Address for correspondence:
Ana Rosa Rincón-Sánchez, PhD.
Instituto de Enfermedades Crónico-Degenerativas
CUCS-Universidad de Guadalajara
Guadalajara, Jalisco, México
E-mail: [email protected]; [email protected]
Fax: 52 (33) 1058 5200 ext 3893
Phone: 52 (33) 1058 5200 ext 3891, 3892
Manuscript received and accepted: 22 July and 20 August 2008
Abstract
Relationship between cirrhosis and renal dysfunction is
not yet fully understood. A model of cirrhosis with
acute hepatic and renal damage (RF), produced by
CCl4 in rats, with hemodynamic and renal functional
alterations, similar to those observed in decompensated
cirrhosis (DC) in man, was used to study chemical
nephrotoxicity in animals. We performed in male
Wistar rats hepatic and renal functional and hemodynamic
studies in control, cirrhotic and decompensated
cirrhotic (DC) groups. Cirrhosis was induced with carbon
tetrachloride by chronic administration. Association
between liver and renal functional alterations was
detected in rats with decompensated cirrhosis, showing
fall in mean arterial pressure and reduction of glomerular
filtration rate and filtration fraction. Renal hemodynamics
did not change in cirrhotic rats, similarly to
what occurs in compensated cirrhotic patients. However,
DC rats exhibited increased sodium, glucose and
phosphate urinary excretions and decreased ATP in renal
cortex. DC animals had severe hypoglycemia.
There was an extensive liver fibrosis. Glomeruli had
hypercellularity and tubules showed extensive vacuolization
in cirrhotic and DC rats. The present study
suggests that in this model, damage typical of acute tubular
necrosis ensues in cirrhotic rats. We describe
functional and morphological damage in liver and kidney
in a model of cirrhosis that might predispose to the
development of acute renal failure when an individual
with hepatic damage is exposed in acute way to chemical
toxicants.
Key words: Acute renal failure, kidney, liver, renal
ATP, glomerular filtration rate, hypoglycemia, glycosuria.
Introduction
Alterations in kidney structure and function are frequently
found in severe liver disease and once liver function
falls below a critical threshold, sodium retention occurs
followed by ascites, associated with profound disturbances
of splachnic and systemic hemodynamics which
in turn may affect renal function.1,2 As disease progresses,
constriction of intrarenal vascular system favors marked
sodium and water retention, leading to refractory ascites,
a progressive rise in plasma creatinine levels and reduction
of renal clearances (decompensated cirrhosis). Persistent
renal hypoxia may also induce tubular damage.3
Development of renal failure in patients with liver
failure is frequent, it occurs in approximately the 55% of
patients. This complication may result when the cirrhotic
individual is exposed to xenobiotics, either therapeutic
drugs or environmental pollutants, especially heavy metals.
Renal function is rarely restored in the absence of hepatic
recovery.4 Administration of carbon tetrachloride
(CCl4
) to rats results in a reproducible experimental model
of cirrhosis, that resembles the disease in humans and
provides a tool to study liver-kidney interrelationships.5,6
Previously we have performed an experimental model of
acute liver and renal damage, produced by intragastric
administration of a single dose of CCl4
to cirrhotic rats.
In this experimental model there are hemodynamic and
renal functional alterations similar to those observed in
the human with decompensated cirrhosis. This model is
useful to study the pathogenesis of renal failure associated
with liver damage when the hepatic function decrease
after an acute liver damage.7
In our present work, we used our model to further
characterize the systemic and renal hemodynamics, histopathological
and functional changes of proximal tubu-
© 2019, Fundación Clínica Médica Sur, A.C. Published by Elsevier España S.L.U. This is an open access article under the CC BY-NC-ND
cells (renal sodium handling, phosphate and glucose
excretion rates) as well as the function of cells from the
distal nephron (renal water handling), in cirrhotic and in
cirrhotic rats with acute liver damage induced by carbon
tetrachloride.
Material and methods
Animals:
Male Wistar rats were maintained under 12 h day/
night light cycles fed ad libitum with rodent lab diet
(Ralston Rations-Kansas, KA). Animals were housed in
the animal facility of University Autonomous of Aguascalientes
and all animal studies were conducted in accordance
with the principles and procedures outlined in the
National Institutes of Health’s Guide for the Care and Use
of Laboratory Animals.
Induction of liver cirrhosis: Liver cirrhosis was
achieved in 100 male Wistar rats (40-60 g initial
weight; 250 – 350 g final weight) by intraperitoneal
injection of 0.2 ml of a mixture of CCl4
(J.T. Baker
USA) and mineral oil (Sigma Chemical Company, St.
Louis MO) 3 times per week during 8 weeks, progressively
increasing CCl4 concentration until the fourth
week of treatment.7
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 received the same volume of
mineral oil.7
Induction of acute renal damage: At the end of eight
weeks of chronic CCl4
treatment and after 6 days of the
last chronic CCl4 dose, cirrhotic rats received a single intragastric
dose of a 1:1 (v/v) mixture of CCl4 /corn oil
(0.5 mL per 100 g body weight). This acute treatment
change cirrhotic by decompensated cirrosis rats and induce
acute renal failure in the animals.7-9
Liver function tests: Liver damage was assessed by
measurements of: a) serum albumin,10,11 b) aspartate aminotransferase
(AST) and alanine aminotransferase (ALT)
activities12 and c) total proteins. The method of Lowry et
al. modified by Peterson13 was used for protein determinations
using bovine albumin as standard.
Renal function tests: Animals were divided into three
groups: control, cirrhotic and cirrhotic plus acute renal
damage (decompensated cirrhosis). In those groups, we
studied: (A) glomerular filtration rate (GFR) and plasma
renal flow (PRF); (B) renal sodium handling (C) renal cortical
Na+
-K+

• ATPase activity and tissue ATP concentrations,
  (D) renal glucose and phosphate excretion rates
  and (E) renal water handling.
  Histological samples examination: Four animals per
  group (control and CCl4
  -treated) were anesthetized with
  sodium pentobarbital and intravascularly perfused with
  phosphate buffer solution (PBS), pH 7.4, containing heparine
  (1,000 UI/L) and procaine (1 g/L) for rinsing of livers
  and kidneys. For in situ fixation, tissue was perfused
  with PBS, pH 7.4, containing 10% formaldehyde (v/v).
  Livers and kidneys were removed and fixed for 24 h in
  the same fixing solution. 5-ï ïm sections were stained with
  hematoxylin and eosin. Tissue sections were evaluated
  by optical microscopy. Examiner was unaware of the
  group to which samples belong.
  Renal hemodynamic and sodium handling assays.
  Glomerular filtration rate (GFR) and renal plasma flow
  (RPF) were estimated by inulin and p-aminohippurate
  clearances, as previously described.14 Arterial blood pressure
  (BP) was measured with a Statham P-23A transducer
  and recorded in a RP Beckman Dynograph. Sodium, inulin
  and PAH were measured by flame photometry, anthrone
  method and spectrophotometry, as previously described.14,15
  Renal cortex ATPase activities and tissue ATP concentrations:
  Renal cortex was separated from medulla
  and homogenized in solution containing (mM): 240 sucrose,
  1 EDTA and 10 Tris-HCl, pH 7.5 (20 % w/v homogenate).
  ATPase activities were measured according to
  Quigley and Gotterer 16 ATP concentration was determined
  from renal cortex, according to the method of
  Bucher, modified by Adams.
  Phosphate and glucose excretion: Cirrhotic, DC and
  age-matched control rats were kept in individual metabolic
  cages. Twenty four hour urine samples were collected
  and urinary volume was recorded. Urinary and
  plasma phosphate and glucose were measured by Sumner
  method adapted for microsamples and by the Trinder
  method, respectively. The urinary/plasma phosphate and
  the urinary/plasma glucose ratios were calculated.
  Renal water handling: Water handling was estimated
  by osmolal and free-water clearances. Urine samples were
  collected and total urinary volume of each rat was recorded.
  Plasma and urine osmolalities were measured by
  cryoscopy (OSMETTE, Model 5004). Osmolal (Cosm
  ) and
  free-water (CH2O) clearances were calculated as follows:
  Cosm
  = [U]
  osm X Urine flow/[P]
  osm
  where [U]
  osm
  and [P]
  osm
  are urine and plasmatic-osmolalities,
  respectively, and urine flow is expressed in mL/
  min. Free-water clearance was calculated as follows:
  CH2O = Urine flow - Cosm
  Statistical analyses: Results show means ± SD. Data
  were analyzed using ANOVA followed by Tukey Kramer
  test. Differences were considered significant when p < 0. 05.
  ResultsMorphological studies
  Morphology was normal in livers and kidneys of control
  animals (healthy animals) (Figures 1a and 1d). In
  contrast, cirrhotic and decompensated rats with renal failure
  (RF) showed extensive liver fibrosis confirming that
  hepatic cirrhosis was attained (Figures 1b and 1c). Kidneys
  of cirrhotic and DC with RF-rats showed degenerative
  changes (Figures 1e and 1f). Glomeruli were denser
  in both cirrhotic and DC with RF-rats, than in control
  group. In the cirrhotic group, there was extensive mesangial
  hypercellularity and podocytes showed hyperchromatic
  nuclei. Tubules exhibited fine and extensive vacuolization.
  In DC with RF-group, there was severe glomerular
  damage with increased mesangial space and
  hyperchromatic nuclei of podocytes; tubules showed
  cloudy swelling and degeneration (Figure 1f).
  Blood proteins were reduced and enzymes increased
  in cirrhotic and DC with RF-rats
  As expected, in cirrhotic and DC with RF rats, serum
  albumin concentrations were lower (33.6% and 60%, respectively)
  than in controls. In DC with RF group serum
  albumin further decreased 39.8 %, in relation to cirrhotic
  rats (Figure 2, panel A). Total protein decreased 50 %
  and 33.6 % in the cirrhotic and DC with RF rats, respectively,
  as compared to control values (Figure 2, panel B).
  AST and ALT in DC with RF group and ALT in cirrhotic
  rats increased significantly, compared to controls, and in
  the DC with RF group AST and ALT serum activities further
  increased 255 % and 238 %, as compared with cirrhotic
  rats (Figure 2, panel C and D).
  Arterial blood pressure and glomerular filtration
  decreased in DC with RF rats
  Mean arterial blood pressure (MAP) was similar in control
  and cirrhotic rats. By contrast, we also observed a fall in
  the MAP in the DC rats with RF, suggesting reduction in renal
  perfusion pressure (Table I). GFR also fell by 52 % in
  DC with RF group, as compared to control and cirrhotic animals,
  while no differences were observed between these two
  groups. Moreover, filtration fraction fell by 43% in group of
  DC with RF and did not change in cirrhotic rats (Table I).
  Renal plasma flow (RPF) did not show differences.
  Excretion of sodium was strikingly increased in DC
  rats with RF
  Probably related to decrement in MAP, filtered sodium
  load decreased in DC rats with RF (756 ± 184 ï ïEq/min/kg)
  as compared to control and cirrhotic rats (1032 ± 94 and
  1117 ± 214 ï ïEq/min/kg, respectively). In spite of diminished
  filtered sodium load, there was an increment in the
  fractional excretion of sodium (FeNa), in DC rats with RF.
  FeNa increased by 135% in DC rats with RF as compared
  to control rats and, excretion was 68% higher when compared
  to the cirrhotic group (Figure 3), indicating severe
  alteration of the tubular handling of sodium.
  Severe depletion of ATP renal concentrations in
  cirrhotic and DC with RF rats
  Renal cortical ATP concentrations decreased in both
  CCl4
  -treated groups. We also observed fall of ATP significantly,
  in tissue of cirrhotic DC rats with RF, 62% and
  71%, respectively (Figure 4). By contrast, both groups,
  cirrhotic and DC rats with RF, showed renal cortical ATPases
  (Total, Mg++-dependent and Na+
  -K+
  -dependent)
  that were significantly unaffected. Na+
  -K+
  -ATPase activity
  changed from a control value of 23 ± 3.3 to 32.5 ± 4.6
  and 32.7 ± 10, nmol Pi min-1 mg-1, in cirrhotic and DC
  rats with RF respectively. These findings suggest that
  low tissue concentration of ATP was probably related to
  nucleotide altered synthesis.
  Increased glucose and phosphate urinary excretions
  in DC with RF rats
  However, glucose urine/plasma ratio and phosphate
  urine/plasma ratio were markedly higher in both groups,
  cirrhotic and DC rats with RF, as compared to control values,
  indicating marked urinary losses of glucose and phosphate.
  Indeed, urine/plasma glucose ratio increased 7.5-
  fold in cirrhotic rats and 21.5-fold in DC rats with RF.
  Also, there was a significant difference in the urine/plasma
  glucose ratio values between DC rats and cirrhotic rats
  with RF (p < 0.001) (Table II). Related to phosphate urine/
  plasma ratio, it increased 1.4-fold in cirrhotic rats (not significant)
  and 5.7-fold in DC rats group with RF (Table II).
  On the other hand, the glucose urinary concentration increased
  significantly in both cirrhotic (522 %) and RF rats
  (855 %) (Figure 5B). Because of these changes, serum
  glucose concentration markedly decreased in DC rats
  with RF group (54 %), without significant differences between
  control and cirrhotic rats (Figure 5A).
  Defective renal water handling in cirrhotic and DC
  with RF rats
  Significant decrease in urinary flow was observed in
  both groups cirrhotic and DC rats with RF (3.8 ± 0.35 and
  3.5 ± 0.32 ï ïL/min, respectively) as compared to control
  group (16.3 ± 1.07 ï ïL/min). Urine osmolalities were higher
  in cirrhotic (1597 ± 170) and DC rats with RF (2290 ±
  138), than in control group (595 ± 17 mOsmol/kg). Moreover,
  osmolal clearances were lower in cirrhotic and DC
  rats with RF (20 ± 1.5 and 26 ± 1.6 ï ïL/min, respectively)
  than in control group (33 ± 1.6 ï ïL/min). Free-water clearance
  decreased significantly in DC rats with RF when
  compared to control and cirrhotic group
  Figure 1. Histopathologic evaluation of rat liver and kidney. (a) Control group. Hepatocyte (arrows), kupffer cells (arrowheads), and sinusoidal
  space (asterisk) disclose normal liver organization and structure. (b) Liver exhibited extensive damage with necrosis and fibrosis (arrows),
  hyperplasia of kupffer cells is observed (peak arrow) and most of liver morphology was altered by cirrhosis. (c) Extensive and diffuse damage,
  with vesicular degeneration and necrosis (arrows) was observed in decompensated cirrhosis with renal failure group (dc+rf). (d) No morphological
  abnormalities were observed in the kidney of control animals: glomerular area (asterisk), mesangial space (small arrow), podocyte
  (arrowhead), proximal and distal tubules (arrows). (e) Cirrhotic animals exhibited hyperchromatic podocyte (arrowhead) and large mesangial
  spaces (small arrow); cloudy swelling (arrows) and hydropic degeneration (double arrow) were observed in the tubules. (f) In dc+rf group
  extensive mesangial hypercellularity with hyperchromatic nuclei (small and arrowhead), severe damage with structural modification in all the
  glomerular area were observed. tubules exhibited loss of brush border, cloudy swelling and cellular fragments inside the lumen (arrows)
  Discussion
  Hemodynamic and functional renal alterations in DC
  rats with RF were more severe than in cirrhotic rats. When
  additional acute liver damage was induced in cirrhotic
  rats, intense hemodynamic and renal functional alterations
  ensued. We observed fall in mean arterial pressure
  (MAP) probably due to diminished peripheral vascular
  resistance and significant reduction of GFR and filtration
  fraction in DC rats with RF, in a similar fashion as reported
  to cirrhotic rats 17. In contrast, renal hemodynamics
  did not change in cirrhotic rats, similarly to what occurs
  in compensated cirrhosis in humans.
  Renal response to decreased blood pressure is altered
  in severe liver disease with activation of sympathetic system
  and increased synthesis of several renal vasoconstrictor
  compounds (endothelin, thromboxane A2
  and F2
  -isoprostanes).
  Activated sympathetic nervous system, and
  presumably other renal vasoconstricting agents, cause a
  rightward shift in the autoregulatory curve, making renal
  Table I. Mean arterial blood pressure and renal hemodynamic data from control, cirrhotic and decompensed cirrhotic rats with renal failure,
  produced by CCl4
  .
  Renal hemodynamics
  Mean arterial (mL min -1 kg-1) Filtration
  Group Weight (g) pressure (mmHg) C-In C-PAH fraction
  Control (n = 12) 308 ± 6 110 ± 4 7.35 ± 0.7 13.8 ± 1.4 0.552 ± 0.05
  Cirrhotic (n = 9) 340 ± 21 103 ± 8 7.47 ± 1.4 13.9 ± 2.0 0.551 ± 0.09
  DC with RF (n = 8) 371 ± 16 75 ± 10 ** 3.54 ± 0.8* 10.7 ± 2.2 0.315 ± 0.03 *
  DC with RF = decompensated cirrhosis with renal failure; C-In = clearance of inulin; C-PAH = clearance of para-amino-hippurate
  *p < 0.05 and **p < 0.01, when compared with the control group.
  Figure 2. Hepatic function studies in control, cirrhotic and decompensated cirrhotic rats with renal failure (RF), produced by CCl4
  . Panel
  A) Serum albumin in control (empty bar), cirrhotic (hatched bar) and rats with acute hepatic and renal damage (fill bar).Panel B) Total
  plasma protein content, symbols as in panel A: C): Aspartate aminotransferase in the same groups of rats previously described; D): Alanine
  aminotransferase, ALT. Mean ± SEM are shown, *p < 0.05, **p < 0.01 and ***p < 0.001, when compared with control B) group.
  4.5
  3.0
  1.5
  0
  Albumin (g/100 mL)
  **
  **
  Control Cirrhotic D-CR+RF
  A 80
  40
  20
  0
  Total protein (mg/mL)
  **

Control Cirrhotic D-CR+RF
B
60
3200
0
AST (IU/L)

Control Cirrhotic D-CR+RF
C
2800
2400
2000
1600
1200
800
400
15
10
5
ALT (IU/L)
*

Control Cirrhotic D-CR+RF
D
10
20
200
600
1000
1400
1800
2200
0
Table II. The urine/plasma ratio of glucose and phosphates from
control, cirrhotic and decompensated cirrhotic rats with renal failure,
produced by CCl4
.
Group Urine/plasma Urine/plasma
glucose ratio phosphate ratio
Control (n = 10) 0.026 ± 0.004 3.74 ± 0.34
Cirrhotic (n = 10) 0.196 ± 0.030 * 5.13 ± 0.45
DC with RF (n = 10) 0.561 ± 0.075 ** 21.18 ±
[ ] via transmembrane carriers (SGLT 1 and 2)
that also reabsorb glucose, coupled to the transport of
phosphate or aminoacids, by selective sodium channels
or through the paracellular pathway. Sodium is
then returned to systemic circulation by the (Na+
-K+
)-
ATPase located at the basolateral membrane.20,21 Glucose
and phosphate reabsorption are coupled to sodium
transport, via transmembrane co-transporters.22,23
Therefore increased urinary losses of sodium, glucose
and phosphate are indicative of severe proximal tubular
damage.24
Renal retention of sodium is considered to be responsible
for edema and ascites in hepatic cirrhosis.25 The
mechanisms of abnormal tubular sodium reabsorption in
cirrhosis are multifactorial. Overactivity of the renin-angiotensin-aldosterone
system and the sympathetic nervous
system might play a role in this abnormality.26 Increased
urinary sodium excretion has also been reported
in cirrhosis. Approximately 10% to 20% cirrhotic patients
with ascites spontaneously eliminate relatively
large amounts of sodium in the urine.25 Decrease in tubular
reabsorption is generally regarded as evidence of intrinsic
tubular dysfunction. To explain irreversibility of
DC damage with RF, it has been also suggested that this
illness may be complicated by acute tubular necrosis.3
Histological alterations in the kidney of DC patients with
RF, similar to those observed during acute tubular necrosis
(ATN), with tubular degeneration and interstitial leukocyte
infiltration have been reported.27,28 We observed
degenerative changes in cirrhotic and DC rats with RF
(Figure 1).
Moreover, depletion of ATP may increase cytosolic
calcium, which activates proteases and phospholipase A2
with increased synthesis of leukotrienes that might induce
renal ischemia.3 In accordance, increased production
of cysteinyl leukotrienes in RF has been reported.29
Cellular energy depletion results in rapid loss of polarity
in proximal tubule cells. (Na+
-K+
)-ATPase is located at
the basolateral membrane complex attached to actin cytoskeleton
and is essential for the transtubular vectorial
reabsorption of sodium. Ischemia or cellular ATP depletion
leads to the rapid redistribution of (Na+
-K+
)-ATPase
into the apical membrane domain of the proximal tubule.30
Therefore, following depletion of energy stores,
electrolyte (Na, K) gradients collapse and actin-containing
microfilaments become disorganized.2.
Decrease in urine volume and in free-water clearance
indicated water retention in cirrhotic rats with RF. Pathogenesis
of water retention in cirrhosis is complex and
probably involves several factors, including increased
plasma levels of antidiuretic hormone (ADH), decrease in
prostaglandin synthesis and reduced delivery of filtrate
to the ascending limb of Henle.31,32 It should be emphasized
that increased sodium excretion together with water
retention led to dilutional hyponatremia. Studies in humans
and experimental animals provided strong evidence
that ADH plays a major role in the pathogenesis of
water retention in cirrhosis. Longitudinal studies in rats
with cirrhosis and ascites have shown a chronological relationship
between ADH hypersecretion and impaired
water excretion.33,34 In addition, kidneys from cirrhotic
rats with ascites show increased gene expression of aquaporin-2,
the ADH-regulated water channel.35 In most patients
with ascites free-water clearance is reduced.31 Our
data are in accordance with this report. Water retention
contributes to dilutional hyponatremia observed in patients
with advanced liver disease.36,37
Hypoxic injury to tubular cells represents an early
event in acute renal failure. Our data showed that both,
renal hemodynamics and proximal tubular function
were damaged in DC with RF rats. Persistent renal hypoxia
may favor occurrence of tubular damage associated
to ATP depletion, as shown in this study. In regard
to renal vasoconstriction during RF, sympathetic
axis can be stimulated by three different mechanisms:
a) activation of pressure receptors in response to hypotension,
b) non-volume-dependent hepatic baroreceptors
and c) secondary to metabolic changes like
catecholamines secretion in response to hypoglycemia.
All of these mechanisms might be active in RF.4
Hypoglycemia of the RF rats reported in this study is
in accordance with the third mechanism related to
strong activation of sympathetic nervous system in patients
with RF.38
In summary, CCl4
-treatment to cirrhotic rats with RF
reduces GFR and filtration fraction and produces proximal
tubular damage characterized by increased sodium,
glucose and phosphate excretion, decreased renal
ATP concentration and reduced free-water clearance.
Our study provides a model of liver damage similar to
what occurs in decompensated cirrhotic patients and
brings the possibility to study the overlapping effects
of deleterious agents that might induce renal failure in
them.
Figure 6. Water renal handling in control, cirrhotic and decompensated
cirrhotic rats with renal failure (RF), produced by
CCl4
. Free-water clearances (CH20) were estimated in the three
experimental groups previously described. Mean ± SEM are
shown, *p <
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The relationship between cirrhosis and renal dysfunction is not fully understood. A model of cirrhosis with acute hepatic and renal damage (RF) produced by carbon tetrachloride (CCl 4) in rats was used to study chemical nephrotoxicity in animals. Cirrhosis was induced with carbon tetrachloride by chronic administration. Association between liver and renal functional alterations was detected in rats with decompensated cirrhosis, showing fall in mean arterial pressure and reduction of glomerular filtration rate and filtration fraction. Renal hemodynamics did not change in cirrhotic rats, similar to what occurs in compensated cirrhotic patients. DC rats exhibited increased sodium, glucose, and phosphate urinary excretions and decreased ATP in renal cortex. DC animals had severe hypoglycemia, there was an extensive liver fibrosis, and glomeruli had hypercellularity and tubules showed extensive vacuolization in cirrhotic and DC rats. The present study suggests that in this model, damage typical of acute tubular necrosis ensues in cirrhotic rats. The study describes functional and morphological damage in liver and kidney in a model of cirrhosis that might predispose to the development of acute renal failure when an individual with hepatic damage is exposed in acute way to chemical toxicants.