Abmucm
Vol.
JOURNAL
229, No.
up
2, August
Bilirubin activity
PHYSIOLOGY
1975.
Printed
in U.S.A.
UDP-glucuronyltransferase
of Wistar
rat kidney
A. FOLIOT, B. CHRISTOFOROV, E. HOUSSET, AND M. DUBOIS Institut
National
de la Sad
J. P. PETITE,
et de la Recherche Midicale,
FOLIOT, A., B. CHRXSTOFOROV, J. P. PETITE, J. P. ETIENNE, E. HOUSSET, Am M. Ihmxs. Bilirubin UD~-~lucuronyltransferase actiuity of Wdar rat kidney. Am. J. Physiol. 229(Z): 340-343. 1975.Wistar rat kidneys have been shown to possess a bilirubin glucuronyltransferase (BGT) activity capable of conjugating about 35 of the total pool of unconjugated bilirubin within 48 h of being grafted to Gunn rat hosts. Bilirubin conjugated by the kidney is taken up by the liver and excreted in the bile. Except when the bile duct is ligated, no conjugated bilirubin appears in the plasma or urine. Renal BGT activity is about 1,&th of the hepatic activity on a weight basis in Wistar rats. The Gunn rat’s hyperbilirubinemia probably causes an induction of the renal enzyme since its activity doubles in 48 h.
renal
transplantation;
Gunn
rat;
enzyme
induction
THE KIDNEY SEEMS CAP ABLE Of playing an importa .nt role in bilirubin metabolism under certain conditions. Pi mstone et al. (13) have shown the existence of a heme oxygenase in tubular epithelial cells of rat kidn eY capable of transforming hemoglobin to bil .iverdin , which 1s then transformed to unconjugated bilirubin (UCB) by the soluble enzyme biliverdin reductase. According to these authors, renal hemeoxygenase may be as active as the homologous splanchnic or hepatic enzyme during hemoglobinuria. Under similar conditions, dog kidney is also capable of transforming hemoglobin into bilirubin (4). In addition, certain data suggest that the kidney may also be able to conjugate bilirubin. Franc0 et al. (5) have shown that hepatectomized and eviscerated rats retain their capacity to conjugate intravenously injected bilirubin, whereas if they are also bilaterally nephrectomized they totally lose this ability. Barac (1) and Heirwegh and Barac (7), by perfusing either totally eviscerated dogs or isolated dog kidney with blood to which UCB has been added, have provoked a bilirubinuria that greatly surpasses the maximum for UCB in the urine. These authors have identified bilirubin glucuroconjugates in the urine. Finally, Stevenson and Dutton (16) have shown the glucuroconjugation capacity of slices and homogenates of guinea pig kidney in the presence of such substrates as O-aminophenol, O-aminobenzoic acid, and menthol. However, these authors did not use bilirubin as a substrate. In order to study the capacity of rat kidney to conjugate bilirubin in vivo, we have grafted kidneys from normal
J. P. ETIENNE, 75005 Paris, France
Wistar rats to Gunn rat hosts. The homozygous Gunn rat suffers from a nonhemolytic unconjugated bilirubin jaundice, secondary to a total lack of bilirubin glucuronyltransferase (BGT) (8). MATERIALS
AND
METHODS
Graft hosts were 11 male homozygous Gunn rats weighing 450 g and maintained on a constant diet that assured high, stable levels of plasma bilirubin (9). Six male Wistar rats also received grafts and served as controls. Each animal was bilaterally nephrectomized and then received one kidney from a male normal Wistar rat of analogous age and weight. After transplantation, nine of the Gunn rats were housed in metabolic cages permittin? collection of urine and feces and the other two rats had therr bile ducts cannulated. Both Wistar and Gunn rats of age and weight corresponding to the graft hosts served as controls, after being either cannulated or subjected to sham laparotomy. The following parameters were measured in all of the animals just before intervention and again 48 h later: weight, hematocrit, plasma protein, total bilirubin (TB), conjugated bilirubin (CB), and UCB. For the nine animals placed in metabolic cages, the concentration and urinary output of TB, CB, and UCB were determined for the 48 h following the grafts. In this same group, fecal concentration of TB was determined from the 24th to 48th h. Bile was collected in ice-cooled containers in the dark hourly from rats with biliary fistulas, and flow rate, TB, CB, and UCB were determined. YThe BGT activity was measured in the livers and kidneys of six representatives of each of the following categories of rats: 6-wk-old Wistar males, 6-wk-old Wistar females, Wistar males over 2 mo, Wistar females over 2 mo, Gunn homozygous females, and Gunn heterozygous females. Results from another laboratory (12) indicated that there may be a derepression of latent enzymatic activity in tissues of an animal receiving grafts from normal donors. To see if this phenomenon occurred in the present series of experiments, hepatic BGT activity in host Gunn rats was measured 48 h after transplantation. In addition, the BGT activity of the nontransplanted kidney of the Wistar donor was compared with that of the grafted kidney 48 h after transplantation to see if the host’s bilirubin had any effect on the enzyme’s activity. Three other Gunn rats receiving kidney grafts from Wistar rats had their bile ducts ligated at the same time and
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
RENAL
BILIRUBIN
341
UDP-GLUCURONYLTRANSFERASE
were monitored for 72 h for variations in UCB
and CB in
their plasma and urine. Plasma protein was measured by the technique of Gornall et al. (6), TB in plasma, urine, and bile was determined by the method of Jendrassik and Grof (lo), and CB and UCB were assayed by the method of Weber and Schalm (18). Total bilirubin was measured in feces by the technique of Malloy and Evelyn (11). Hepatic and renal BGT was determined by the method of Black et al. (2). The Student f test was used for statistical analysis of results. RESULTS
TABLE 2. Bilirubin excretion in Wistar, and grafted Gunn rats Plasma B ilirubin, mg/lOO mI
Rats
Gunn, BiIe Bilirubin
Concentration, mg/lOO ml
Rate, Erg/h per 100 g body wt
Conjpgatcd, 0
Wistar
1 2
0.5 0*5
12.1 11.2
16.1 16,2
85 80
Gunn
1 2
11.2 14.6
3.0 2.6
4.1 3.5
0 0
Grafted Gunn
I 2
4.0 4.6
23-7 21.1
24.8 21.6
73.8 77.0
Graft tolerance was good as indicated by the small weight loss (mean 30 g in 48 h) and volume of urine (mean 14.6 ml for 24 h). On the other hand, there was a large decrease in hematocrit (mean 46 % before graft, 33 % 48 h later) and a marked diminution of plasma protein levels (72.6 g/liter before graft, 63.9 g/liter 48 h later). Sham-operated Gunn rats, however, showed the same alterations. Autopsies performed 48-240 h after transplantation revealed kidneys that were macroscopically normal l
BILIRUBIN
PLASMA
mgjlOOm1
Gunn
Grafted
G.
Wistar
FIG. 2. Cumulative biliary bilirubin excretion at 6, 24, and 48 h after bile duct cannulation. Shaded portion represents unconjugated bilirubin; unshaded portion represents conjugated bilirubin.
Before
FIO. 1. Plasma retention of conjugated simultaneous renal transplantation and portion represents unconjugated bilk∈ sents conjugated bilirubin.
72h
4Bh
24h
graft
bilirubin in Gunn rats after bile duct ligation. Shaded unshaded portion repre-
TABLE 1 Effect of renal transplantation on plasma, urinary, and fecal bilirubitt in Gunn and Wisbar rats l
Plasma Bilirubin, mg/lOO ml Rats
I Before
Gum Grafted (9) Grafted and bile duct ligated (3) Shamopcrated (6)
graft
48 h after graft
16.1 14.5
zt 1.4 h 0.6
5.5 15.4
=t3 0.7* A 2.1
13.2
& 0.4
12.6
& 0.8
Urinary Bilirubin, 24th-48th h After Graft, mg/24 h
0.90
0 *
Fecal Bilirubin, 24th48th h After Graft, mg/24 h
1.04 0.15
=t 0.17t zt 0.02+
0
0.54
Et 0.04
0 0
0.08 0.07
A 0.05 =f= 0.05
0.12
wutar Grafted (6) Sham operated Values l
P
Vol.
JOURNAL
229, No.
up
2, August
Bilirubin activity
PHYSIOLOGY
1975.
Printed
in U.S.A.
UDP-glucuronyltransferase
of Wistar
rat kidney
A. FOLIOT, B. CHRISTOFOROV, E. HOUSSET, AND M. DUBOIS Institut
National
de la Sad
J. P. PETITE,
et de la Recherche Midicale,
FOLIOT, A., B. CHRXSTOFOROV, J. P. PETITE, J. P. ETIENNE, E. HOUSSET, Am M. Ihmxs. Bilirubin UD~-~lucuronyltransferase actiuity of Wdar rat kidney. Am. J. Physiol. 229(Z): 340-343. 1975.Wistar rat kidneys have been shown to possess a bilirubin glucuronyltransferase (BGT) activity capable of conjugating about 35 of the total pool of unconjugated bilirubin within 48 h of being grafted to Gunn rat hosts. Bilirubin conjugated by the kidney is taken up by the liver and excreted in the bile. Except when the bile duct is ligated, no conjugated bilirubin appears in the plasma or urine. Renal BGT activity is about 1,&th of the hepatic activity on a weight basis in Wistar rats. The Gunn rat’s hyperbilirubinemia probably causes an induction of the renal enzyme since its activity doubles in 48 h.
renal
transplantation;
Gunn
rat;
enzyme
induction
THE KIDNEY SEEMS CAP ABLE Of playing an importa .nt role in bilirubin metabolism under certain conditions. Pi mstone et al. (13) have shown the existence of a heme oxygenase in tubular epithelial cells of rat kidn eY capable of transforming hemoglobin to bil .iverdin , which 1s then transformed to unconjugated bilirubin (UCB) by the soluble enzyme biliverdin reductase. According to these authors, renal hemeoxygenase may be as active as the homologous splanchnic or hepatic enzyme during hemoglobinuria. Under similar conditions, dog kidney is also capable of transforming hemoglobin into bilirubin (4). In addition, certain data suggest that the kidney may also be able to conjugate bilirubin. Franc0 et al. (5) have shown that hepatectomized and eviscerated rats retain their capacity to conjugate intravenously injected bilirubin, whereas if they are also bilaterally nephrectomized they totally lose this ability. Barac (1) and Heirwegh and Barac (7), by perfusing either totally eviscerated dogs or isolated dog kidney with blood to which UCB has been added, have provoked a bilirubinuria that greatly surpasses the maximum for UCB in the urine. These authors have identified bilirubin glucuroconjugates in the urine. Finally, Stevenson and Dutton (16) have shown the glucuroconjugation capacity of slices and homogenates of guinea pig kidney in the presence of such substrates as O-aminophenol, O-aminobenzoic acid, and menthol. However, these authors did not use bilirubin as a substrate. In order to study the capacity of rat kidney to conjugate bilirubin in vivo, we have grafted kidneys from normal
J. P. ETIENNE, 75005 Paris, France
Wistar rats to Gunn rat hosts. The homozygous Gunn rat suffers from a nonhemolytic unconjugated bilirubin jaundice, secondary to a total lack of bilirubin glucuronyltransferase (BGT) (8). MATERIALS
AND
METHODS
Graft hosts were 11 male homozygous Gunn rats weighing 450 g and maintained on a constant diet that assured high, stable levels of plasma bilirubin (9). Six male Wistar rats also received grafts and served as controls. Each animal was bilaterally nephrectomized and then received one kidney from a male normal Wistar rat of analogous age and weight. After transplantation, nine of the Gunn rats were housed in metabolic cages permittin? collection of urine and feces and the other two rats had therr bile ducts cannulated. Both Wistar and Gunn rats of age and weight corresponding to the graft hosts served as controls, after being either cannulated or subjected to sham laparotomy. The following parameters were measured in all of the animals just before intervention and again 48 h later: weight, hematocrit, plasma protein, total bilirubin (TB), conjugated bilirubin (CB), and UCB. For the nine animals placed in metabolic cages, the concentration and urinary output of TB, CB, and UCB were determined for the 48 h following the grafts. In this same group, fecal concentration of TB was determined from the 24th to 48th h. Bile was collected in ice-cooled containers in the dark hourly from rats with biliary fistulas, and flow rate, TB, CB, and UCB were determined. YThe BGT activity was measured in the livers and kidneys of six representatives of each of the following categories of rats: 6-wk-old Wistar males, 6-wk-old Wistar females, Wistar males over 2 mo, Wistar females over 2 mo, Gunn homozygous females, and Gunn heterozygous females. Results from another laboratory (12) indicated that there may be a derepression of latent enzymatic activity in tissues of an animal receiving grafts from normal donors. To see if this phenomenon occurred in the present series of experiments, hepatic BGT activity in host Gunn rats was measured 48 h after transplantation. In addition, the BGT activity of the nontransplanted kidney of the Wistar donor was compared with that of the grafted kidney 48 h after transplantation to see if the host’s bilirubin had any effect on the enzyme’s activity. Three other Gunn rats receiving kidney grafts from Wistar rats had their bile ducts ligated at the same time and
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
RENAL
BILIRUBIN
341
UDP-GLUCURONYLTRANSFERASE
were monitored for 72 h for variations in UCB
and CB in
their plasma and urine. Plasma protein was measured by the technique of Gornall et al. (6), TB in plasma, urine, and bile was determined by the method of Jendrassik and Grof (lo), and CB and UCB were assayed by the method of Weber and Schalm (18). Total bilirubin was measured in feces by the technique of Malloy and Evelyn (11). Hepatic and renal BGT was determined by the method of Black et al. (2). The Student f test was used for statistical analysis of results. RESULTS
TABLE 2. Bilirubin excretion in Wistar, and grafted Gunn rats Plasma B ilirubin, mg/lOO mI
Rats
Gunn, BiIe Bilirubin
Concentration, mg/lOO ml
Rate, Erg/h per 100 g body wt
Conjpgatcd, 0
Wistar
1 2
0.5 0*5
12.1 11.2
16.1 16,2
85 80
Gunn
1 2
11.2 14.6
3.0 2.6
4.1 3.5
0 0
Grafted Gunn
I 2
4.0 4.6
23-7 21.1
24.8 21.6
73.8 77.0
Graft tolerance was good as indicated by the small weight loss (mean 30 g in 48 h) and volume of urine (mean 14.6 ml for 24 h). On the other hand, there was a large decrease in hematocrit (mean 46 % before graft, 33 % 48 h later) and a marked diminution of plasma protein levels (72.6 g/liter before graft, 63.9 g/liter 48 h later). Sham-operated Gunn rats, however, showed the same alterations. Autopsies performed 48-240 h after transplantation revealed kidneys that were macroscopically normal l
BILIRUBIN
PLASMA
mgjlOOm1
Gunn
Grafted
G.
Wistar
FIG. 2. Cumulative biliary bilirubin excretion at 6, 24, and 48 h after bile duct cannulation. Shaded portion represents unconjugated bilirubin; unshaded portion represents conjugated bilirubin.
Before
FIO. 1. Plasma retention of conjugated simultaneous renal transplantation and portion represents unconjugated bilk∈ sents conjugated bilirubin.
72h
4Bh
24h
graft
bilirubin in Gunn rats after bile duct ligation. Shaded unshaded portion repre-
TABLE 1 Effect of renal transplantation on plasma, urinary, and fecal bilirubitt in Gunn and Wisbar rats l
Plasma Bilirubin, mg/lOO ml Rats
I Before
Gum Grafted (9) Grafted and bile duct ligated (3) Shamopcrated (6)
graft
48 h after graft
16.1 14.5
zt 1.4 h 0.6
5.5 15.4
=t3 0.7* A 2.1
13.2
& 0.4
12.6
& 0.8
Urinary Bilirubin, 24th-48th h After Graft, mg/24 h
0.90
0 *
Fecal Bilirubin, 24th48th h After Graft, mg/24 h
1.04 0.15
=t 0.17t zt 0.02+
0
0.54
Et 0.04
0 0
0.08 0.07
A 0.05 =f= 0.05
0.12
wutar Grafted (6) Sham operated Values l
P
