Renal expression and urinary excretion of aquaporin-2 in postobstructive uropathy in rats.

This work assessed the time course of water renal management together with aquaporin-2 (AQP2) kidney expression and urinary AQP2 levels (AQP2u) in obstructive nephropathy. Adult male Wistar rats were monitored after 1, 2, and 7 days of bilateral ureteral release (bilateral ureteral obstruction (BUO); BUO-1, BUO-2 and BUO-7). Renal water handling was evaluated using conventional clearance techniques. AQP2 levels were assessed by immunoblotting and immunohistochemical techniques. AQP2 expression in apical membranes was downregulated in BUO-1 rats and upregulated both in BUO-2 and BUO-7 animals. AQP2 protein expression in whole cell lysate fraction from kidney cortex and medulla were significantly decreased in all the experimental groups. Concomitantly, mRNA levels of AQP2 decreased in renal medulla of all groups and in renal cortex from BUO-1; however, in renal cortex from BUO-2 and BUO-7 a recovery and an increase in the level of AQP2 mRNA were, respectively, observed. BUO-7 group showed a significant increase in AQP2u. The alterations observed in apical membranes AQP2 expression could explain, at least in part, the evolution time of water kidney management in the postobstructive phase of BUO. Additionally, the AQP2u increase after 7 days of ureteral release may be postulated as a biomarker of improvement in the kidney function.

Hepatic and renal expression of Oatp1 in obstructive uropathy. First detection of Oatp1 in urine, a potential biomarker.

Obstructive renal diseases affect renal function and kidney integrity. Nevertheless, little is known about its systemic or extra-renal effects. The organic anion transporting polypeptide 1 (Oatp1) is a carrier expressed in liver and kidneys. In this study, the hepatic and renal expression of Oatp1 was evaluated in rats with obstructive nephropathy. Moreover, the urinary excretion of Oatp1 (Oatp1u ) was evaluated as a potential biomarker for this pathology. Male Wistar rats with bilateral ureteral obstruction for 5 hours (BUO5), 24 hours (BUO24) or sham operated were used. After 24 hours of ureteral releasing, liver and kidney functional parameters, histopathology, Oatp1 tissular expression and Oatp1u were evaluated. For Oatp1u evaluation two groups were added; BUO1 and BUO2 (1 and 2 hours of ureteral obstruction, respectively). Both liver and kidney functional parameters and histopathological studies showed alterations in BUO5 and BUO24. In hepatic homogenates, Oatp1 significantly decreased in BUO groups and in total liver membranes no modifications were observed. In renal homogenates, Oatp1 significantly decreased in BUO groups, but in apical kidney membranes, its expression was increased. Oatp1u was only detected in BUO groups, even in those (BUO1, BUO2) in which no alterations in the traditional parameters of renal function were observed. Modulations in liver and renal expression of Oatp1 could be an organism strategy to attenuate the effects of the disease and an attempt to maintain the complex organ cross-talk between liver and kidneys. Oatp1u could be a new, early and specific biomarker of obstructive nephropathy.

Erythropoietin alters the pharmacokinetics of organic anions mainly eliminated by the kidney in rats.

Erythropoietin (EPO) is a cytokine originally used for its effects on the hematopoietic system, and is widely prescribed around the world. In the present study, the effects of EPO administration on p-aminohippurate (PAH, a prototype organic anion) pharmacokinetics and on the renal expression of PAH transporters were evaluated. Male Wistar rats were treated with EPO or saline (control group). After 42 h, PAH was administered, and plasma samples were obtained at different time points to determine PAH levels. PAH levels in renal tissue and urine were also assessed. The renal expression of PAH transporters was evaluated by Western blotting. EPO-treated rats showed an increase in PAH systemic clearance, in its elimination rate constant, and in urinary PAH levels, while PAH in renal tissue was decreased. Moreover, EPO administration increased the expression of the transporters of the organic anions evaluated. The EPO-induced increase in PAH clearance is accounted for by the increase in its renal secretion mediated by the organic anion transporters. The goal of this study is to add important information to the wide knowledge gap that exists regarding drug-drug interactions. Owing to the global use of EPO, these results are useful in terms of translation into clinical practice.

Utility of urinary organic anion transporter 5 as an early biomarker of obstructive nephropathy.

Ureteral obstruction is a relevant cause of kidney damage. The traditional parameters used in clinical practice for the detection of renal injury are insensitive and non-specific for the diagnosis of obstructive renal disease. The organic anion transporter 5 (Oat5) is a carrier expressed exclusively in the kidney. In this study, the Oat5 urinary excretion (Oat5u ) was evaluated as a potential biomarker of obstructive nephropathy, comparing it with traditional markers of renal function and with neutrophil gelatinase-associated lipocalin in urine (NGALu ), a more recent biomarker of renal pathology. Bilateral ureteral obstruction (BUO) was induced in male Wistar rats, by complete ligation of ureters for 1 hour (BUO1), 2 hours (BUO2), 5 hours (BUO5), or 24 hours (BUO24). After 24 hours of ureteral releasing, urea and creatinine plasma concentrations, creatinine clearance, urinary total proteins, urinary glucose, and alkaline phosphatase activities in urine were evaluated. Oat5 and NGAL levels were assessed in urine samples by immunoblotting. All parameters of renal function were altered in the BUO24 and some also in BUO5, while the Oat5u increased in all of the experimental groups analyzed. After a long time of ureteral obstruction (BUO24), the urinary excretion of Oat5 markedly increased, in parallel with the alteration in the other parameters evaluated. Nevertheless, in BUO1 and BUO2, Oat5u appeared as the only parameter modified. Therefore, Oat5u could be proposed as a novel early biomarker of ureteral obstruction, with the additional potential to inform about the severity of the obstructive injury suffered by the kidney.

Distribution of the organic anion transporters Oat1 and Oat3 between renal membrane microdomains in obstructive jaundice.

Relation between the renal function and the membrane environment where the organic anion transporters Oat1 and Oat3 are localized is scarce. The aim of this study was to examine the Oat1 and Oat3 distribution in different cellular fractions under physiological conditions as well as the effects of extrahepatic cholestasis on membrane distribution of both proteins. Besides, the potential role of jaundice serum on the Oat1 and Oat3 expression in suspensions of renal tubular cells was evaluated. Cellular and membrane fractions of renal cortex were obtained from control rats to evaluate Oat1 and Oat3 protein expressions. Other rats were subjected to bile duct ligation (BDL) or Sham operation to determine the membrane distribution of Oat1 and Oat3 between lipid raft domains (LRD) and non-LRD. Incubation of renal cortical cells with serum from Sham and BDL were also performed to study Oat1 and Oat3 protein expressions. In physiological conditions, Oat1 and Oat3 were concentrated in LRD. The pathology induced a shift of Oat1 from LRD to non-LRD, while Oat3 showed no changes in its distribution. In cells exposed to BDL serum, Oat1 protein expression in membranes significantly increased. For Oat3, no difference between groups was observed. The Oat1 redistribution to non-LRD in BDL could be favoring the increase in renal transport of organic anions previously observed. This change was specific to Oat1. The in vitro experiment allows to conclude that some component present in BDL serum is responsible for the alterations observed in Oat1 expression in cortical membranes.

Time evolution of methotrexate-induced kidney injury: A comparative study between different biomarkers of renal damage in rats.

Methotrexate (MTX) is commonly used in the treatment of malignant diseases and autoimmune and chronic inflammatory disorders. Along with its effective therapeutic power, MTX has adverse effects on the kidneys. Discovery of new biomarkers is required to improve the early detection of renal damage and optimize the effectiveness of treatments. The aim of this study was to evaluate the time course of MTX-induced nephrotoxicity and to compare the urinary excretion of the organic anion transporter 5 (uOat5) with alterations in other markers of renal function, and to elucidate the possible molecular mechanisms involved in uOat5. Animals were exposed to a unique dose of MTX (80 mg/kg body weight, intraperitoneal). Experiments were carried out at days 2, 4, 8 or 14 after MTX administration. Markers of renal damage, such as creatinine and urea plasma levels, urinary activity of alkaline phosphatase, microalbuminuria, urinary excretion of neutrophil gelatinase-associated lipocalin (uNGAL) and histopathology, were evaluated. Renal organic anion transporter 5 (Oat5) expression and its presence in different urine fraction were assessed by western blotting. uOat5 was significantly increased 2 days after MTX treatment, before than any alteration in other parameters of kidney injury or renal morphology occurred. uNGAL showed an inverted pattern of urinary excretion compared to uOat5. Exosomal pathway is involved in the urinary excretion of Oat5 and depends on the degree of damage induced by MTX. These experimental data allow proposing uOat5 as a potential non-invasive biomarker for early detection of MTX-induced nephrotoxicity.

Novel finding of caveolin-2 in apical membranes of proximal tubule and first detection of caveolin-2 in urine: A promising biomarker of renal disease.

Caveolin-2 (Cav-2) is expressed in a variety of cell tissue, and it has also been found in renal tissue. The expression of Cav-2 in proximal tubules is still unclear. The aim of this study was to carry out a complete evaluation of the expression pattern of Cav-2 in rat renal cortex to clarify and deepen the knowledge about the localization of Cav-2 in the proximal tubules and also to evaluate its presence in urine. Male Wistar rats were used to assess Cav-2 expression by Western blot analysis in homogenates, apical, and basolateral membranes from kidney cortex, in lysates and total plasma membranes from renal cortical cell suspensions, in urine, and in urinary exosomes. Cav-2 was clearly expressed in renal cortex homogenates and in both apical and basolateral membranes isolated from kidney cortex, with a greater expression on the former membranes. It was also observed in lysates and in plasma membranes from cortical cell suspensions. Moreover, Cav-2 was found in urine and in its exosomal fraction. These results confirmed the presence of Cav-2 in proximal tubule cells in the kidney of healthy rats, and showed for the first time its expression at the apical membrane of these cells and in urine. Besides, urinary exosomal pathway could be involved in Cav-2 urinary excretion under normal conditions. We observed an increase in the urinary abundance of Cav-2 in two models of acute kidney injury, and thus we proposed the urinary excretion of Cav-2 as a potential biomarker of kidney injury.

Renal expression and urinary excretion of Na+/dicarboxylate cotransporter 1 (NaDC1) in obstructive nephropathy: a candidate biomarker for this pathology.

Obstructive nephropathy is characterized by alterations in renal function that depends on the degree and type of obstruction. To increase the knowledge about the physiopathological mechanisms involved in the renal damage associated with bilateral ureteral obstruction (BUO), we studied the renal expression and function (as urinary citrate excretion) of sodium-dependent dicarboxylate cotransporter (NaDC1) in rats. In addition, we evaluated the urinary excretion of NaDC1 as a candidate biomarker for this pathology. Male Wistar rats underwent bilateral ureteral obstruction for 1 (BUO1), 2 (BUO2), 5 (BUO5), and 24 (BUO24) h or sham operation. After 24 h of ureteral releasing, traditional parameters of renal function and citrate levels were determined, and NaDC1 levels were evaluated in total renal homogenates, apical plasma membranes, and urine by electrophoresis and Western blotting. Traditional parameters of renal function were only modified in BUO5 and BUO24. The renal expression of NaDC1 was decreased in BUO5 and BUO24, with a concomitant increase in urinary excretion of citrate. Moreover, the urinary excretion of NaDC1 increased after short times of ureteral obstruction (BUO1 and BUO2) and was positively correlated with the time elapsed after obstruction. The results obtained from the renal expression of NaDC1 could explain an adaptive mechanism to prevent the formation of kidney stones by increasing the levels of citrate, a calcium chelator. The urinary excretion of NaDC1 could be postulated as an early biomarker of obstructive nephropathy that also gives information about the duration of the obstruction.

Renal Expression and Urinary Excretion of Na-K-2Cl Cotransporter in Obstructive Nephropathy.

Renal damage due to urinary tract obstruction accounts for up to 30% of acute kidney injury in paediatrics and adults. Bilateral ureteral obstruction (BUO) is associated with polyuria and reduced urinary concentrating capacity. We investigated the renal handling of water and electrolytes together with the renal expression and the urinary excretion of the Na-K-Cl cotransporter (NKCC2) after 1 (BUO-1), 2 (BUO-2), and 7 (BUO-7) days of release of BUO. Immunoblotting and immunohistochemical studies showed that NKCC2 expression was upregulated in apical membranes both from BUO-2 and from BUO-7 rats. The apical membrane expression, where NKCC2 is functional, may be sufficient to normalize water, potassium, sodium, and osmolytes tubular handling. NKCC2 abundance in homogenates and mRNA levels of NKCC2 was significantly decreased in almost all groups suggesting a decrease in the synthesis of the transporter. Urinary excretion of NKCC2 was increased in BUO-7 groups. These data suggest that the upregulation in the expression of NKCC2 in apical membranes during the postobstructive phase of BUO could contribute to improving the excretion of sodium and consequently also the excretion of potassium, osmolytes, and water. Moreover, the increase in urinary excretion of NKCC2 in BUO-7 group could be a potential additional biomarker of renal function recovery.

Pharmacokinetics of the antimicrobial drug Sulfanilamide is altered in a preclinical model of vascular calcification.

In vascular smooth muscle, calcium overload is linked to advancing age. The pharmacokinetics of Sulfanilamide (SA), a compound with antibacterial properties, was evaluated in a preclinical model of vascular calcification. SA was used since it is useful to study possible modifications in the renal and hepatic management of drugs. Vascular calcification was induced by administration of a single high dose of vitamin D3 to rats (treated group) 10 days before the experiments. A parallel control group was processed. The decrease of renal blood flow due to calcification of the renal arteries explains, at least in part, the decrease in the renal clearance of SA observed in treated rats. The liver metabolic function increased in treated rats as demonstrated by increases in plasma appearance rate of acetylated-Sulfanilamide (ASA), hepatic ASA content and hepatic N-acetyltransferase activity. The decrease in renal excretion of SA was not completely compensated by the hepatic metabolism increase, since the elimination rate of SA from the central compartment (K1-0 ) decreased in the treated group. In summary, in this experimental model with sustained arterial calcinosis induced by a single high dose of vitamin D3 10 days before the experiments, the pharmacokinetics of an aminobenzenesulfonamide is modified, at least in part, by the increase in the activity of hepatic N-acetyltransferase and the decrease in renal blood flow. This study emphasizes the importance of considering the presence of vascular calcification when a drug dose scheme is performed, in order to optimize pharmacotherapeutic results.

Impact of the induced organic anion transporter 1 (Oat1) renal expression by furosemide on the pharmacokinetics of organic anions.

AIM: Furosemide is a loop diuretic. Different authors demonstrated that continuous administration of furosemide modulates the expression of organic anion transporters. This study was undertaken to simultaneously evaluate the effects of furosemide pretreatment on organic anion transporter 1 (Oat1) and multidrug resistance protein 2 (Mrp2) renal expressions, on p-aminohippurate (PAH) pharmacokinetics and on renal and urinary PAH levels in rats. METHODS: Male Wistar rats were treated with furosemide (6 mg/100 g body weight per day, subcutaneously, 4 days) (treated group) or saline (control group). On the fifth day, PAH was administered as a bolus infusion in the femoral vein, and plasma samples were obtained from femoral artery at different time points. PAH levels in renal tissue and urine were also assessed. Renal Oat1 and Mrp2 expressions were evaluated by western blotting. RESULTS: Furosemide pretreatment increased both the expression of Oat1 and Mrp2. PAH plasma concentrations decreased following a biexponential function. The furosemide-treated group showed higher PAH plasma levels, a lower systemic clearance and elimination rate constant from the peripheral compartment, indicating that PAH renal elimination was decreased. PAH levels in renal tissue were significantly elevated and in urine appeared to be significantly lower as compared with control animals. CONCLUSIONS: Furosemide pretreatment caused a significant decrease of PAH renal elimination, despite Oat1 and Mrp2 augmented renal expression. The goal of the present study is the addition of important information in the wide gap of knowledge that exists about drug-drug interactions. Because of furosemide worldwide use, the data obtained are interesting and useful in terms of translation to clinical practice.

Expression of renal Oat5 and NaDC1 transporters in rats with acute biliary obstruction.

AIM: To examine renal expression of organic anion transporter 5 (Oat5) and sodium-dicarboxylate cotransporter 1 (NaDC1), and excretion of citrate in rats with acute extrahepatic cholestasis. METHODS: Obstructive jaundice was induced in rats by double ligation and division of the common bile duct (BDL group). Controls underwent sham operation that consisted of exposure, but not ligation, of the common bile duct (Sham group). Studies were performed 21 h after surgery. During this period, animals were maintained in metabolic cages in order to collect urine. The urinary volume was determined by gravimetry. The day of the experiment, blood samples were withdrawn and used to measure total and direct bilirubin as indicative parameters of hepatic function. Serum and urine samples were used for biochemical determinations. Immunoblotting for Oat5 and NaDC1 were performed in renal homogenates and brush border membranes from Sham and BDL rats. Immunohistochemistry studies were performed in kidneys from both experimental groups. Total RNA was extracted from rat renal tissue in order to perform reverse transcription polymerase chain reaction. Another set of experimental animals were used to evaluate medullar renal blood flow (mRBF) using fluorescent microspheres. RESULTS: Total and direct bilirubin levels were significantly higher in BDL animals, attesting to the adequacy of biliary obstruction. An important increase in mRBF was determined in BDL group (Sham: 0.53 ± 0.12 mL/min per 100 g body weight vs BDL: 1.58 ± 0.24 mL/min per 100 g body weight, P < 0.05). An increase in the urinary volume was observed in BDL animals. An important decrease in urinary levels of citrate was seen in BDL group. Besides, a decrease in urinary citrate excretion (Sham: 0.53 ± 0.11 g/g creatinine vs BDL: 0.07 ± 0.02 g/g creatinine, P < 0.05) and an increase in urinary excretion of H(+) (Sham: 0.082 ± 0.03 µmol/g creatinine vs BDL: 0.21 ± 0.04 µmol/g creatinine, P < 0.05) were observed in BDL animals. We found upregulations of both proteins Oat5 and NaDC1 in brush border membranes where they are functional. Immunohistochemistry technique corroborated these results for both proteins. No modifications were observed in Oat5 mRNA and in NaDC1 mRNA levels in kidney from BDL group as compared with Sham ones. CONCLUSION: Citrate excretion is decreased in BDL rats, at least in part, because of the higher NaDC1 expression. Using the outward gradient of citrate generated by NaDC1, Oat5 can reabsorb/eliminate different organic anions of pathophysiological importance.

Altered Renal Expression of Relevant Clinical Drug Transporters in Different Models of Acute Uremia in Rats. Role of Urea Levels.

BACKGROUND/AIMS: Organic anion transporter 1 (Oat1) and 3 (Oat3) are organic anion transporters that play critical roles in the body disposition of numerous clinically important drugs. We investigated the effects of acute uremia on the renal expression of Oat1 and Oat3 in three in vivo experimental models of acute kidney injury (AKI): induced by ischemia, by ureteral obstruction and by the administration of HgCl2. We also evaluated the influence of urea in the expression of these transporters in proximal tubular cells suspensions. METHODS: Membranes were isolated from kidneys of each experimental group and from cell suspensions incubated with different urea concentrations. Oat1 and Oat3 expressions were performed by immunoblotting. RESULTS: A good correlation between uremia and the renal protein expression of Oat1 and Oat3 was observed in vivo. Moreover, the incubation of isolated proximal tubular cells with different concentrations of urea decreases protein expression of Oat1 and Oat3 in plasma membranes in a dose-dependent manner. CONCLUSION: The more severe the renal failure, the more important is the decrease in protein expression of the transporters in renal membranes where they are functional. The in vitro study demonstrates that urea accounts, at least in part, for the decreased expression of Oat1 and Oat3 in proximal tubule plasma membranes.

Protein expression of kidney and liver bilitranslocase in rats exposed to mercuric chloride--a potential tissular biomarker of toxicity.

Bilitranslocase (BTL) is a plasma membrane carrier that transports organic anions of physiological and pharmacological interest. It is expressed in basolateral plasma membrane of kidney and liver. BTL has been recently described as a marker of transition from normal tissue to its neoplastic transformation in human kidney. Inorganic mercury is a major environmental contaminant that produces many toxic effects. Previous reports have described an interaction between BTL and mercuric ions. This study was designed to evaluate the renal and hepatic expression of BTL in rats exposed to a nephrotoxic and hepatotoxic dose of HgCl2. Male rats were treated with a single injection of HgCl2 at a dose of 4mg/kg body wt, i.p. (HgCl2 group). Control rats received the vehicle alone (Control group). Studies were carried out 18h after injection. Afterwards, the kidneys and livers were excised and processed for histopathological studies or immunoblot (homogenates and crude membranes) techniques. In rats treated with HgCl2, immunoblotting showed a significant decrease in the abundance of BTL in homogenates and plasma membranes from kidney and liver. BTL decrease of expression might reflect the grade of damage in renal tubule cells and in hepatocytes. Thus, BTL might be postulated as a new biomarker of tissue toxicity induced by mercury.

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis.

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Expression of kidney and liver bilitranslocase in response to acute biliary obstruction.

BACKGROUND/AIM: It has been recently demonstrated that acute obstructive jaundice is associated with modifications in the renal expression and function of organic anion transporters such as Oat1, Oat3, Oatp1 and Mrp2. This study examined the expression and function of bilitranslocase in liver and kidney from rats with bile duct ligation (BDL). METHODS: Bilitranslocase expression was evaluated in renal homogenates (H), renal basolateral plasma membranes (KBLM) and liver plasma membranes (LPM) by immunoblotting. Bilitranslocase function was studied by measuring the kinetic parameters of electrogenic bromosulfophthalein (BSP) uptake in KBLM and LPM by a spectrophotometric technique. RESULTS: An increased abundance of bilitranslocase in KBLM without modifications in renal H and in LPM from BDL rats was observed compared with Sham rats. BDL rats showed a higher V(max) for BSP uptake in KBLM. No differences between groups were observed for Michaelis-Menten parameters in LPM. CONCLUSION: The higher renal expression and function of bilitranslocase in renal basolateral membranes from rats with obstructive cholestasis might also contribute to the dramatic increase in BSP renal excretion observed in this experimental model. This would be another compensation mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Characterization of the mechanisms involved in the increased renal elimination of bromosulfophthalein during cholestasis: involvement of Oatp1.

The kidneys and liver are the major routes for organic anion elimination. We have recently shown that acute obstructive jaundice is associated with increased systemic and renal elimination of two organic anions, p-aminohippurate and furosemide, principally excreted through urine. This study examined probable adaptive mechanisms involved in renal elimination of bromosulfophthalein (BSP), a prototypical organic anion principally excreted in bile, in rats with acute obstructive jaundice. Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BSP renal clearance was performed by conventional techniques. Renal organic anion-transporting polypeptide 1 (Oatp1) expression was evaluated by immunoblotting and IHC. Excreted, filtered, and secreted loads of BSP were all higher in BDL rats compared with Sham rats. The higher BSP filtered load resulted from the increase in plasma BSP concentration in BDL rats, because glomerular filtration rate showed no difference with the Sham group. The increase in the secreted load might be explained by the higher expression of Oatp1 observed in apical membranes from kidneys of BDL animals. This likely adaptation to hepatic injury, specifically in biliary components elimination, might explain, at least in part, the huge increase in BSP renal excretion observed in this experimental model.

Time course of organic anion excretion in rats with bilateral ureteral obstruction: role of organic anion transporters (Oat1 and Oat3).

BACKGROUND: Urinary tract obstruction is a common cause of renal failure. In this study, we evaluated the time course of P-aminohippurate (PAH) renal excretion and the cortical expression of organic anion transporters (Oat1 and Oat3) at 1 (BUO-1), 2 (BUO-2) and 7 (BUO-7) days after release of 24-hour bilateral ureteral obstruction (BUO) in the rat. METHODS: Conventional clearance technique, differential centrifugation, semiquantitative immunoblotting and immunohistochemical techniques have been employed. RESULTS: These studies showed that Oat1 and Oat3 in basolateral membranes were downregulated both at BUO-1 and BUO-2. Concomitantly, the rats developed a reduction in PAH renal elimination. In contrast, total recovery in PAH renal excretion and in the expression of Oat1 and Oat3 were observed at BUO-7, as compared with the sham group. A direct correlation was observed between the secretory clearance of PAH and Oat1 (r(2) = 0.88) and Oat3 (r(2) = 0.83) expression in basolateral membranes. CONCLUSION: These results indicate that the differential expression of organic anion transporters is one of the main molecular mechanisms contributing to the organic anion excretion modifications observed during the time course of obstructive nephropathy. This study provides evidence regarding the importance of adjusting the dose regimens of negatively charged drugs during the different time phases of this pathology.

Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3.

Pharmacokinetic studies of the drugs administered to subjects with mechanical cholestasis are scarce. The purpose of the present study was to examine the effects of bile duct ligation of 3 days (peak of elevation of serum bile acids and bilirubin) on the systemic and renal PAH clearance and on the expression of cortical renal OAT1 and OAT3 in a rat model. PAH is the prototypical substrate of the renal organic anion transport system. Male Wistar rats underwent a bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BDL rats displayed a significantly lower systemic PAH clearance. Renal studies revealed a reduction in the renal clearance and in the excreted and secreted load of PAH in BDL rats. The OAT1 protein expression in kidney homogenates was not modified, but it decreased in the basolateral membranes from BDL rats. In contrast, OAT3 abundance in both kidney cortex homogenates and in basolateral membranes increased by 3 days after the ligation. Immunocytochemical studies (light microscopic and confocal immunofluorescence microscopic analyses) confirmed the changes in the renal expression of these transport proteins. The present study demonstrates the key role of OAT1 expression in the impaired elimination of PAH after 3 days of obstructive cholestasis.

Expression of rat renal cortical OAT1 and OAT3 in response to acute biliary obstruction.

Renal function in the course of obstructive jaundice has been the subject of great interest; however, little is known about the expression of renal organic anion transporters. The objective of this work was to study, in rats with acute extrahepatic cholestasis, the cortical renal expression of the organic anion transporter 1 (OAT1) and the organic anion transporter 3 (OAT3), in association with the pharmacokinetics and renal excretion of furosemide (FS). Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. All studies were carried out 21 hours after surgery. Rats were anesthetized and the pharmacokinetic parameters of FS and the renal elimination of FS were determined. Afterwards, the kidneys were excised and processed for immunoblot (basolateral membrane and renal homogenates) or immunocytochemical (light microscopic and confocal immunofluorescence microscopic analysis) techniques. The systemic and renal clearance of FS as well as the excreted and secreted load of FS increased in BDL rats. In kidneys from BDL rats, immunoblotting showed a significant increase in the abundance of both OAT1 and OAT3 in homogenates from renal cortex. In basolateral membranes from kidney cortex of BDL rats, OATI abundance was also increased and OAT3 abundance was not modified. Immunocytochemical techniques confirmed these results. In conclusion, acute obstructive jaundice is associated with an upregulation of OAT1 and OAT3, which might explain, at least in part, the increased systemic and renal elimination of FS.