Chemical synthesis of bilirubin glucuronide conjugates

In dimethylformamide, the two carboxyl groups of bilirubin react with the bifunctional coupling agent, carbonyldiimidazole, to form bilirubin diimidazole, which was isolated and crystallised. The bilirubin diimidazole, termed “activated bilirubin”, was shown to react spontaneously with primary alcohols to form diesters of bilirubin. After addition of the tetrabutyl ammonium salt of glucuronic acid, compounds with chromatographic mobilities similar to those of bilirubin mono- and diglucuronides from bile were formed.Bilirubin diglucuronides were isolated by barium precipitation followed by solvent extraction. The bilirubin diglucuronides were considered to be a mixture of α and β glucuronides esterified at positions 1, 2, 3, or 4 of glucuronic acid because the compound(s) was resistant to hydrolysis with glucuronidase and gave multiple spots by chromatography after diazotization with ethyl anthranilate. The model compounds lauryl glucuronides were synthesized similarly; the most polar product by chromatography had identical chromatographic mobility to synthetic lauryl l-d-glucuronide prepared by reductive debenzylation of lauryl (benzyl (2,3,4-tri-O-benzyl))-d-glucuronide.It is concluded that bilirubin-1-di-β-d-glucuronide can be synthesized when suitable protecting groups for the 2, 3, and 4 hydroxyl groups of glucuronic acid become available.     

Developmental expression of renal organic anion transporters in rat kidney and its effect on renal secretion of phenolsulfonphthalein

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.     

Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy

Fluorofenidone (FD) is a novel pyridone agent with significant antifibrotic effects in vitro. The purpose of this study is to investigate the effects of FD on renal interstitial fibrosis in rats with obstructive nephropathy caused by unilateral ureteral obstruction (UUO). With pirfenidone (PD, 500 mg/kg/day) and enalapril (10 mg/kg/day) as the positive treatment controls, the rats in different experimental groups were administered with FD (500 mg/kg/day) from day 4 to day 14 after UUO. The tubulointerstitial injury, interstitial collagen deposition, and expression of type I and type III collagen, transforming growth factor-β(1) (TGF-β(1)), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), α-smooth muscle actin (α-SMA), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were assessed. FD treatment significantly attenuated the prominently increased scores of tubulointerstitial injury, interstitial collagen deposition, and protein expression of type I and type III collagen in ureter-obstructed kidneys, respectively. As compared with untreated rats, FD also significantly reduced the expression of α-SMA, TGF-β(1), CTGF, PDGF, and inhibitor of TIMP-1 in the obstructed kidneys. Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy through its regulation on fibrogenic growth factors, tubular cell transdifferentiation, and extracellular matrix.     

Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor

1. Conjugated bilirubin is conveniently determined by coupling with the diazonium salt of ethyl anthranilate. 2. This method has been used in the development of assays for UDP-glucuronyltransferase (EC 2.4.1.17), with bilirubin as substrate, in rat liver homogenates, microsomal preparations and partly purified fractions. 3. Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied.     

Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.     

Increased activity and expression of matrix metalloproteinase-9 in a rat model of distal colitis

Matrix metalloproteinases may play a role in tissue remodelling and destruction associated with inflammation. We investigated activity and expression of matrix metalloproteinases in a rat model of colitis and tested the therapeutic potential of a synthetic inhibitor (CGS-27023-A). Colitis was induced by dextran sulphate sodium (at 5% in drinking water for 5 days) in a group of eight rats, whereas a matched control group received plain water. Activity and expression of matrix metalloproteinases were measured in colonic tissue homogenates using zymography and Western blot on days 3 and 5 after induction of colitis. In another set of experiments, two groups of colitic rats (20 per group) were treated with CGS-27023-A (20 mg/kg) or vehicle, respectively. On days 5 and 14, colonic mucosal lesions were blindly scored by microscopic examination. Induction of colitis led to a significant upregulation of matrix metalloproteinase-9 protein and its activity, but no change in matrix metalloproteinase-2 activity was observed. Treatment with CGS-27023-A significantly decreased the extent and severity of epithelial injury but did not influence mucosal repair. We conclude that increased activity of matrix metalloproteinases may contribute to epithelial damage in this model of colitis.     

Effect of obstructive jaundice on the fate of a nephrophilic organic anion in the rat

Renal transtubular transport of phenolsulfophthalein (PSP), a nephrophilic organic anion that circulates bound to albumin, was studied in normal and bile-duct-ligated rats. Intravenously injected PSP disappeared from the circulation more rapidly in bile-duct-ligated jaundiced rats than in intact animals. However, urinary excretion of PSP was significantly lower in the former than in the latter. Kinetic analysis revealed that binding of PSP to plasma protein(s) was significantly lower with jaundiced rats than with intact animals. Addition of albumin to plasma samples from bile-duct-ligated rats markedly increased PSP binding. The decreased PSP binding returned to normal levels after treating the jaundiced plasma with bilirubin oxidase, an enzyme that degrades amphiphilic bilirubin to water soluble metabolites. These results suggest that bilirubin might be the major metabolite that occupied the PSP binding site(s) on albumin in jaundiced rats. When PSP was injected bound to equimolar amount of albumin, the rate of PSP disappearance from the circulation decreased and urinary excretion of the ligand increased markedly; urinary excretion of PSP was significantly larger in bile-duct-ligated rats than in intact animals. These results suggest that the renal transport capacity for amphiphilic organic anions, such as PSP, might be increased compensatively in bile-duct-ligated animals, and that the apparent decrease in renal secretory transport for PSP might result from, at least in part, random distribution of the ligand to extrarenal tissues due to decrease in the binding activity of albumin.     

Renal bilirubin excretion in canine models of jaundice

Renal handling of bilirubin and its relationship to blood bilirubin level were investigated for up to 2 weeks in two models of jaundiced dogs, namely, chronic bile duct ligation (CBDL), which are mildly icteric, and choledococaval anastomosis (CDCA), which develop deep jaundice. The mean (+/- SD) urinary bilirubin excretion in CBDL plateaued at 30.3 +/- 9.3 mg/24 hr whereas in CDCA it continued to increase above the normal bilirubin production rate (56-84 mg/24 hr) up to 130-150 mg/24 hr. The renal clearance of bilirubin in both models was inversely proportional to serum bilirubin concentration. It was approximately twice as high in the CDCA model, which induced also a moderate diuresis. It is suggested that higher serum bilirubin levels in CDCA dogs is due to the increased production of bilirubin which is not compensated by the renal clearance of bilirubin.     

Characterization of L-arginine transporters in rat renal inner medullary collecting duct

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.