Organic Anion Binding Protein Research Articles

ATP modulates sulfobromophthalein uptake in rat liver plasma membrane vesicles.

The hepatic uptake of the bilirubin-bilirubin-sulfobromophthalein (BSP) group of organic anions is a carrier-mediated process and is accounted for by at least four distinct plasma membrane proteins (bilitranslocase, BSP/bilirubin-binding protein, organic anion-binding protein and the organic anion transport protein). In order to investigate the regulation of basolateral organic anion uptake, BSP transport was measured in rat basolateral liver plasma membrane vesicles in the presence of ATP. ATP significantly stimulated the electroneutral uptake of BSP with an increment in Vmax compared with control (1.57 +/- 0.14 vs 0.73 +/- 0.06 nmol BSP/mg protein per 15 s, respectively; P < 0.001) while the apparent K(m) was not changed significantly (12 +/- 1 vs 12 +/- 2 mumol/L). The stimulatory effect was dose-dependent for ATP (K(m) 1.01 +/- 0.37 mmol/L). ATP had no detectable effect on the electrogenic component of BSP transport. Other nucleotides (ADP, AMP, GTP) and non-hydrolysable ATP did not enhance BSP uptake, suggesting that ATP hydrolysis was necessary for the effect. This was supported by the lack of effect on BSP uptake when ATP was added in the presence of vanadate. The addition of phorbol-12-myristate 13-acetate, an activator of protein kinase C (PKC), increased BSP uptake in a dose-dependent manner in the presence, but not in the absence, of ATP. Incubation of vesicles with staurosporine, an inhibitor of PKC activity, resulted in a dose-dependent inhibition of ATP-sensitive BSP transport. These data indicate that electroneutral BSP hepatic uptake is modulated by ATP. The effect is related to ATP hydrolysis and involves the activity of PKC.

Microsequencing of Organic Anion-Binding Proteins from Basolateral Plasma Membranes in Hepatocytes after Photoaffinity Labelling with Bumetanide and 7, 7-Azotaurocholate

Binding proteins for weak organic acids, in particular bile acids and loop diuretics, were separated by two-dimensional gel electrophoresis after photoaffinity labelling of rat liver basolateral plasma membranes. After staining with Coomassie Blue the protein spots were isolated, electroeluted and concentrated in an SDS gel. The proteins were digested in the gel by endoproteinase Lys-C. Appropriate fragments were separated by HPLC and the sequence was determined. A 57-kD protein with an apparent pi of 5.3, which was marked by photoaffinity labelling with 7,7-azotaurocholate and stained with an antibody against recombinant protein disulfide isomerase (PDI), showed complete identity with PDI in three fragments. [3H]bumetanide, a loop diuretic, marked two 54-kD proteins. One 54-kD protein with an apparent pi of 6.35 showed homology to the tyrosine kinase inhibitor of the insulin receptor. The amino acid sequence of three fragments belonging to the other 54-kD protein with an apparent pi of 6.2 was identical with cytokeratin 8. Two further proteins that were present in large amounts were also sequenced. A 35-kD protein was identified to be arginase. The internal sequences of a 58-kD protein showed 100% homology to endoproteinase ER 60. The results show that the proposed method of isolation of proteins from 2D gels (a) provides sequence data, even if there are only minute amounts of protein obtainable, and (b) allowed identification of yet unknown binding proteins of organic anions in the liver.

Bilitranslocase and sulfobromophthalein/bilirubin-binding protein are both involved in the hepatic uptake of organic anions.

The hepatic uptake of cholephilic organic anions is a carrier-mediated process. Three distinct proteins [bilitranslocase (BTL), sulfobromophthalein (BSP)/bilirubin-binding protein (BBBP), and organic anion-binding protein] have been isolated from the basolateral plasma-membrane domain of the hepatocyte. To investigate the relative role of the first two of them in accounting for the hepatic uptake of organic anions, we measured the initial rates of uptake of 35S-labeled BSP into rat liver plasma-membrane vesicles. Because transport by BTL is electrogenic but transport by BBBP is electroneutral, studies were done either with or without a positive-inside membrane potential produced by adding valinomycin in the presence of an inwardly directed K+ gradient (outside K+ > inside K+). Both electrogenic and electroneutral transport systems followed saturation kinetics. Electroneutral uptake showed an apparent Km of 20 +/- 3 microM (mean +/- SD) and a Vmax of 1.0 +/- 0.13 nmol.(mg of prot)-1.15 sec-1, whereas the electrogenic portion of BSP uptake exhibited a Km of 5.2 +/- 0.8 microM and a Vmax of 1.1 +/- 0.1 nmol.(mg of prot)-1.15 sec-1. In this case, an overshoot was observed 15 sec after valinomycin addition. Electroneutral BSP uptake was inhibited by incubation with anti-BBBP antibody, whereas anti-BTL antibody did not show any inhibitory effect. Conversely, the electrogenic uptake was inhibited by anti-BTL antibody at a BSP concentration of 5 microM; no inhibition was seen either at 20 microM BSP or upon addition of anti-BBBP antibody. From these data we conclude that the hepatic uptake of organic ions occurs via two immunologically distinct carrier proteins (BTL and BBBP) operating in parallel. BTL is a higher affinity electrogenic transporting system of organic ions, whereas BBBP is a lower affinity electroneutral transporter.

Organic anion transport in HepG2 cells: Absence of the high-affinity, chloride-dependent transporter

In previous studies, we identified a 55 kD organic anion–binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity CI−-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin–binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells. Because cell surface organic anion-binding protein and the mitochondrial F1-ATPase β-subunit are immunologically highly cross-reactive, the presence of intracellular but not cell surface organic anion-binding protein immunoreactivity in HepG2 cells suggests that the mitochondrial and cell surface forms of this protein are independently regulated. These studies also indicate that demonstration of sulfobromophthalein transport alone cannot be used as evidence for the presence of the high-affinity transporter. (HEPATOLOGY 1991;14:1217–1223.)

Organic anion transport in HepG2 cells: Absence of the high-affinity, chloride-dependent transporter

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants ( K d = 0.1−0.12 μM) and the binding capacities ( n = 11−15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC 50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5–2.5 and 100 μM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Selective regulation of intrinsic membrane proteins in HepG2.

Expression of three hepatocellular membrane proteins--the asialoglycoprotein receptor (hepatic binding protein) the insulin receptor and organic anion binding protein--have been studied in the HepG2 cell line. HepG2 grown in minimal essential medium supplemented with 10% fetal bovine serum maximally expressed hepatic binding protein and insulin receptor only at confluence while organic anion binding protein appeared independent of the state of cellular proliferation. When cells were grown in medium supplemented with dialyzed fetal bovine serum, adult bovine serum or in chemically defined medium, expression of hepatic binding protein and insulin receptor but not organic anion binding protein was reduced by 60 to 80%. Immunoblotting techniques revealed that cells grown in dialyzed fetal bovine serum contained virtually no mature, glycosylated 45 kD hepatic binding protein, but a small amount of 36 kD protein. Metabolic labeling of cells grown in dialyzed fetal bovine serum with [35S] methionine indicated reduced synthesis of the 45 kD hepatic binding protein and the absence of the 36 kD protein. Restoration of normal expression of hepatic binding protein and insulin receptor was achieved by addition to dialyzed fetal bovine serum of: fetal bovine serum; 2,000 dalton ultrafiltrate of fetal bovine serum, or 300 to 350 dalton fraction of the ultrafiltrate. The normal concentration of organic anion binding protein demonstrable in cells grown in dialyzed fetal bovine serum indicates that the low molecular weight factor(s) is not a generalized modulator of plasma membrane biogenesis. However, its effect on the steady-state level of the hepatic binding protein and insulin receptor, characteristic of mature hepatocytes, suggests a role for this fetal serum factor in hepatocellular differentiation.

Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Comparison of binding of thyroid hormone analogues to hepatic organic anion binding proteins

D v protein and ligandin are two hepatic cytosolic proteins which bind organic anions, including endogenous thyroid hormones. Binding studies were performed using the ANS displacement technique to compare the binding of a variety of thyroid hormone analogues to purified organic anion binder and ligandin. Inhibition of ANS binding by these compounds was competitive. Both proteins bound L- and D-thyroxine with comparable affinity ( K d 30–45 μM), whereas ligandin boudn 3′,3′,5-triiodo- L-thyronine, 3′,3′,5-triiodo- L-thyronine and most analogues with greater affinity. Nevertheless, the order of ligand affinities for both binders was highly correlated, suggesting that the nature of the binding site on both proteins is similar. The binding affinities of these organic anion binders are 2–3 orders of magnitude lower than an hepatic cytosolic thyroid binder reported by others, suggesting that ligandin and organic anion binder may not be important in intracellular thyroid hormone transfer.

Does Z-protein have a role in transport of bilirubin and bromosulfophthalein by isolated perfused rat liver?

Bilirubin and other organic anions are transported in serum avidly bound to albumin from which they are extracted and transferred into the hepatocyte where they bind to cytosolic proteins. Two abundant organic anion binding proteins, ligandin and Z-protein, were previously purified from liver cytosol and characterized. Other studies in isolated perfused rat liver revealed that selectively increased cytosolic ligandin concentration, following phenobarbital treatment or thyroidectomy, directly correlated with net bilirubin uptake which resulted from reduced bilirubin efflux. To clarify the role of Z-protein in hepatic organic anion transport, we have now determined the kinetics of bilirubin and bromosulfophthalein (BSP) uptake in isolated perfused liver of normal rats and compared results to rats in which Z-protein, but not ligandin, was selectively increased following treatment with clofibrate (ethylchlorophenoxy-isobutyrate). These studies revealed that despite a 147% induction of Z-protein in treated animals, there was no effect on influx or efflux of tracer doses of bilirubin or BSP. Addition of albumin to the protein-free 10% fluorocarbon perfusate reduced influx of 3H-bilirubin (p less than 0.03) and tended to reduce influx of BSP. In this situation, there was still no influence of Z-protein concentration on efflux. These studies indicate that Z-protein does not appear to play a role in the hepatic uptake of bilirubin and BSP.

Preparation and metabolism of 125-sulfobromophthalein

Metabolism of 125I-sulfobromophthalein (BSP) prepared by the chloramine-T method was studied in rats. 125I-BSP is removed rapidly from the circulation. However, as compared to BSP, its plasma clearance and biliary excretion are delayed, and its accumulation in the liver is prolonged. Although BSP and 125I-BSP show similar binding to albumin in serum, their binding properties to liver cytosolic proteins and to the liver cell plasma membrane organic anion binding protein (OABP) differ. In contrast to the X-, Y- and Z-protein binding of BSP, 125I-BSP binds predominantly to a high molecular weight protein and only a small proportion of 125I-BSP binds to OABP.

Newly identified organic anion-binding proteins in rat liver cytosol

Previously, all organic anion binding activity in the Y protein (ligandin-containing) fraction of rat liver cytosol has been attributed to the glutathione S-transferases. Gel filtration on Sephadex G-75 superfine has resolved the Y protein fraction into organic anion-binding fractions of two distinct molecular weights: (a) M r 45 000 containing the glutathione S-transferases, and (b) M r 35 000 referred to as Y′. Two proteins (D v and D 1) were purified from the Y′ fraction. D v is a basic protein consisting of four subunits of M r 8000–9000 and selectively binds 1-anilino-8-naphthalene sulfonate and sulfobromophthalein. D 1 is a monomer ( M r 40 000) which exhibits high affinity binding for Rose Bengal. D v protein bound a broad spectrum of organic anions. Antibody raised in rabbits to rat D v showed no cross-reactivity with purified glutathione S-transferases A, B, or C. Thus, we have identified organic anion-binding proteins which are unrelated to the glutathione S-transferases, but which were previously associated with the crude Y-fraction. The relative abundance of these proteins and their binding characteristics suggest their role in hepatic organic anion transport.

Relationship of carbon tetrachloride-induced alterations of ligandin and Z protein to sulfobromophthalein transport in rat liver.

The relationship of the BSP retaining capacity of organic anion binding protein in the liver to the transport of BSP from plasma to bile was examined in CCl4-treated rats. These proteins were separated into ligandin and Z protein by Sephadex G-75 gel chromatography. The BSP retaining capacity of these proteins was significantly decreased by CCl4 administration (2.5 ml/kg, p.o.). At 3 hours after CCl4, this decreased capacity was caused mainly by the decrease in their binding affinity, at 24 hours after CCl4, however, it was accompanied by decreases in both their binding affinity and protein amounts. At 3 hours, the hepatic uptake of BSP evaluated by determination of plasma BSP concentration, the initial disappearance rate (K1), and the hepatic dye content did not differ from those in the controls, and biliary excretion of BSP was unchanged. At 24 hours, hepatic uptake and biliary excretion of BSP as well as bile flow were significantly decreased. From these results, it is suggested that the alteration in the retaining capacity of ligandin and Z protein would not be a determinant factor in the transfer of BSP from plasma to bile.

Binding protein for 1-anilino-8-naphthalenesulfonate in rat liver cytoplasm

The Z-fraction in rat liver cytoplasm, defined first by Levi et al. [J. clin. Invest.48, 2156 (1969)] as an organic anion binding fraction of small molecular weight (10,000–14,000) was further purified by the method of ion-exchange chromatography. The purification was characterized by the use of 1-anilino-8-naphthalenesulfonate (ANS) in sensitively detecting the organic anion binding protein in the Z-fraction. The Z-fraction was found to contain three protein fractions having fairly high binding activities for ANS and sulfobromophthalein (BSP). Among these fractions, the D 2-fraction, which eluted earlier on a DEAE-Sephadex column, had the highest binding affinity for these anions. This D 2-fraction was further purified by CM-Sephadex chromatography. The ANS binding protein, thus purified. was similar in physico-chemical properties to the aminoazodye-binding protein of S 20.w 1.6s (Form III) reported by Ketterer et al. [Biochem. J.155. 511 (1976)]. The binding affinities for both ANS and BSP were little influenced by the purification. By the fluorescence method, about 30 per cent of the protein in the Z-fraction was found to be this binding protein. The binding stoichiometries for the high affinity sites were 0.7 for ANS and 0.4 for BSP.

Identification, purification, and partial characterization of an organic anion binding protein from rat liver cell plasma membrane.

Uptake of bilirubin, sulfobromophthalein (BSP), and other organic anions by the liver is a process with kinetics consistent with carrier mediation. The molecular basis of this transport mechanism is unknown. In the search for the putative organic anion carrier or receptor, the interaction of BSP with rat liver cell plasma membrane (LPM) has been studied. Specific binding of [(35)S]BSP to LPM was determined using a filtration assay. Results revealed high affinity (K(a) = 0.27 muM(-1)), saturable (6.3 nmol/mg protein) binding, which was eliminated after preincubation with trypsin. Although [(35)S]BSP was strongly bound to LPM, the binding was rapidly reversible, preventing direct identification and study of a specific binding site(s). To avoid this problem, a photoaffinity probe was devised, in which [(35)S]BSP is covalently bound to LPM after exposure to ultraviolet light. Subsequent sodium dodecyl sulfate gel electrophoresis and fluorography revealed radioactivity predominantly associated with a single 55,000-mol wt protein. A protein with identical electrophoretic mobility was purified from deoxycholate solubilized LPM after affinity chromatography on glutathione-BSP-agarose gel. This protein migrated as a single band on sodium dodecyl sulfate gel electrophoresis and on urea gel isoelectric focusing. It contained 1-2 residues of sialic acid per 55,000-dalton protein, and was immunologically distinct from rat albumin and ligandin. It bound bilirubin with a K(d) of 20 muM, as determined by tryptophan fluorescence quenching. Although the high affinity of this LPM protein for organic anions suggests that it may function as a hepatocellular organic anion receptor, its role in transport of these compounds is unknown.

Isolation of an organic anion binding protein from rat liver plasma membrane fractions by affinity chromatography

As part of a study of hepatic organic anion transport, solubilized liver plasma membrane proteins were subjected to affinity chromatography on bilirubin- and sulfobromophthalein-labeled agarose columns. Both columns retained a Sudan Black and PAS negative protein of molecular weight 60,000 daltons, which cochromatographed with [ 35S]sulfobromophthalein on Sephadex G-75, and reversibly bound [ 35S]sulfobromophthalein in vitro with high affinity (K a ⋍ 10 7 M −1) and a valence of 2. Erythrocyte ghost membranes did not contain this protein. Sulfobromophthalein-agarose retained two additional smaller proteins which did not cochromatograph with [ 35S]sulfobromophthalein. Their significance is unclear. This study supports the hypothesis that liver cell plasma membranes participate in the hepatic transport of organic anions.

An organic anion binding protein isolated from rat liver cell plasma membrane (LPM)

An organic anion binding protein isolated from rat liver cell plasma membrane (LPM)

Glutathione-S-transferase activity in the rabbit nephron: segmental localization in isolated tubules and formation of thiol adducts of ethacrynic acid.

SummaryLigandin is a major renal organic anion-binding protein and is the major glutathione (GSH)-S-transferase in the rat, monkey, and man. GSH transferase activity was measured in isolated segments of the normal rabbit nephron using 1-chloro-2,4-dinitrobenzene as substrate. Enzyme activity was confined to the proximal convoluted and proximal straight tubules and was not detectable in the loops of Henle and collecting tubules.Incubation of proximal tubules with [14C]ethacrynic acid resulted in formation of glutathione and cysteine adducts. Loops of Henle and collecting tubules formed significantly less glutathione adduct and no other adducts. GSH transferase in the proximal tubule may thus be important in vivo in the formation of thiolated adducts by the kidney.

Ligandin in Perfusates from Transplanted Kidneys: a Test for Tubular Necrosis

Ligandin, an intracellular organic anion-binding protein, having glutathione-S-transferase activity, was detected in concentrated perfusing solutions from 8 of 13 kidneys preserved for homotransplantation. The presence of ligandin in the perfusate correlated well with oliguric acute renal failure following transplantation. Testing the perfusate for ligandin may be useful in predicting tubular damage in renal transplants.