Novel Mechanistic PBPK Model to Predict Renal Clearance in Varying Stages of CKD by Incorporating Tubular Adaptation and Dynamic Passive Reabsorption.

Chronic kidney disease (CKD) has significant effects on renal clearance (CLr ) of drugs. Physiologically-based pharmacokinetic (PBPK) models have been used to predict CKD effects on transporter-mediated renal active secretion and CLr for hydrophilic nonpermeable compounds. However, no studies have shown systematic PBPK modeling of renal passive reabsorption or CLr for hydrophobic permeable drugs in CKD. The goal of this study was to expand our previously developed and verified mechanistic kidney model to develop a universal model to predict changes in CLr in CKD for permeable and nonpermeable drugs that accounts for the dramatic nonlinear effect of CKD on renal passive reabsorption of permeable drugs. The developed model incorporates physiologically-based tubular changes of reduced water reabsorption/increased tubular flow rate per remaining functional nephron in CKD. The final adaptive kidney model successfully (absolute fold error (AFE) all < 2) predicted renal passive reabsorption and CLr for 20 permeable and nonpermeable test compounds across the stages of CKD. In contrast, use of proportional glomerular filtration rate reduction approach without addressing tubular adaptation processes in CKD to predict CLr generated unacceptable CLr predictions (AFE = 2.61-7.35) for permeable compounds in severe CKD. Finally, the adaptive kidney model accurately predicted CLr of para-amino-hippuric acid and memantine, two secreted compounds, in CKD, suggesting successful integration of active secretion into the model, along with passive reabsorption. In conclusion, the developed adaptive kidney model enables mechanistic predictions of in vivo CLr through CKD progression without any empirical scaling factors and can be used for CLr predictions prior to assessment of drug disposition in renal impairment.

Proteasome Inhibitors Bortezomib and Carfilzomib Stimulate the Transport Activity of Human Organic Anion Transporter 1.

Organic anion transporter 1 (OAT1), expressed at the basolateral membrane of renal proximal tubule epithelial cells, mediates the renal excretion of many clinically important drugs. Previous study in our laboratory demonstrated that ubiquitin conjugation to OAT1 leads to OAT1 internalization from the cell surface and subsequent degradation. The current study showed that the ubiquitinated OAT1 accumulated in the presence of the proteasomal inhibitors MG132 and ALLN rather than the lysosomal inhibitors leupeptin and pepstatin A, suggesting that ubiquitinated OAT1 degrades through proteasomes. Anticancer drugs bortezomib and carfilzomib target the ubiquitin-proteasome pathway. We therefore investigate the roles of bortezomib and carfilzomib in reversing the ubiquitination-induced downregulation of OAT1 expression and transport activity. We showed that bortezomib and carfilzomib extremely increased the ubiquitinated OAT1, which correlated well with an enhanced OAT1-mediated transport of p-aminohippuric acid and an enhanced OAT1 surface expression. The augmented OAT1 expression and transport activity after the treatment with bortezomib and carfilzomib resulted from a reduced rate of OAT1 degradation. Consistent with this, we found decreased 20S proteasomal activity in cells that were exposed to bortezomib and carfilzomib. In conclusion, this study identified the pathway in which ubiquitinated OAT1 degrades and unveiled a novel role of anticancer drugs bortezomib and carfilzomib in their regulation of OAT1 expression and transport activity. SIGNIFICANCE STATEMENT: Bortezomib and carfilzomib are two Food and Drug Administration-approved anticancer drugs, and proteasome is the drug target. In this study, we unveiled a new role of bortezomib and carfilzomib in enhancing OAT1 expression and transport activity by preventing the degradation of ubiquitinated OAT1 in proteasomes. This finding provides a new strategy in regulating OAT1 function that can be used to accelerate the clearance of drugs, metabolites, or toxins and reverse the decreased expression under disease conditions.

Effects of the nitric oxide synthase inhibitor ronopterin (VAS203) on renal function in healthy volunteers.

AIMS: Reduced nitric oxide (NO) availability may adversely affect renal perfusion and glomerular filtration. The aim of the present study was to characterize in detail the pharmacological effects of VAS203, an inhibitor of NO synthase, on renal haemodynamics in humans. METHODS: This double-blind, randomized, placebo-controlled, cross-over phase-I-study comprised 18 healthy men. Renal haemodynamics were assessed with constant-infusion input-clearance technique with p-aminohippurate and inulin for renal plasma flow (RPF) and glomerular filtration rate (GFR), respectively. After baseline measurement, a constant infusion of the tetrahydrobiopterin analogue ronopterin (VAS203, total 10 mg/kg body weight) or placebo was administered at random order for 6 hours additionally. After a wash-out phase of 28 days, the second course was applied. In parallel, markers of early kidney injury and renal function were assessed repeatedly up to 48 hours after starting VAS203/placebo-infusion. RESULTS: VAS203-infusion resulted in a significant decrease of RPF (P < .0001) and GFR (P < .001) compared to placebo, but magnitude was within the physiological range. RPF and GFR recovered partly 2 hours after end of VAS203-infusion and was normal at beginning of the second infusion period. Compared to placebo, preglomerular resistance (P < .0001), and to lesser extent postglomerular resistance (P < .0001) increased, resulting in a decrease of intraglomerular pressure (P < .01). No treatment related effect on markers of early kidney injury, and on renal function (P for all >.20) have been observed. CONCLUSIONS: Our phase-I-study in healthy humans indicates that VAS203 (10 mg/kg body weight) reduces renal perfusion and glomerular function within the physiological range mainly due to vasoconstriction at the preglomerular site.

Alteration of renal excretion pathways in gentamicin-induced renal injury in rats.

The kidney plays a major part in the elimination of many drugs and their metabolites, and drug-induced kidney injury commonly alters either glomerular filtration or tubular transport, or both. However, the renal excretion pathway of drugs has not been fully elucidated at different stages of renal injury. This study aimed to evaluate the alteration of renal excretion pathways in gentamicin (GEN)-induced renal injury in rats. Results showed that serum cystatin C, creatinine and urea nitrogen levels were greatly increased by the exposure of GEN (100 mg kg-1 ), and creatinine concentration was increased by 39.7% by GEN (50 mg kg-1 ). GEN dose-dependently upregulated the protein expression of rOCT1, downregulated rOCT2 and rOAT1, but not affected rOAT2. Efflux transporters, rMRP2, rMRP4 and rBCRP expressions were significantly increased by GEN(100), and the rMATE1 level was markedly increased by GEN(50) but decreased by GEN(100). GEN(50) did not alter the urinary excretion of inulin, but increased metformin and furosemide excretion. However, GEN(100) resulted in a significant decrease of the urinary excretion of inulin, metformin and p-aminohippurate. In addition, urinary metformin excretions in vivo were significantly decreased by GEN(100), but slightly increased by GEN(50). These results suggested that GEN(50) resulted in the induction of rOCTs-rMATE1 and rOAT3-rMRPs pathway, but not changed the glomerular filtration rate, and GEN(100)-induced acute kidney injury caused the downregulated function of glomerular filtration -rOCTs-rMATE1 and -rOAT1-rMRPs pathway.

OAT1 and OAT3 also mediate the drug-drug interaction between piperacillin and tazobactam.

This study aimed to demonstrate that organic anion transporters (OATs) mediate the drug-drug interaction (DDI) between piperacillin and tazobactam. After co-administration with piperacillin in rats, the AUC of tazobactam in plasma was significantly increased, and t1/2ß was prolonged with significant reduction in plasma clearance, renal clearance and cumulative urinary excretion. In rat and human kidney slices, probenecid, p-aminohippurate and benzylpenicillin inhibited the uptake of piperacillin and tazobactam. Piperacillin significantly inhibited the uptake of tazobactam. Moreover, the uptakes of piperacillin, tazobactam and sulbactam in hOAT1/3-HEK293 cells were significantly higher compared with mock-HEK293 cells, respectively. Piperacillin significantly inhibited the uptake of tazobactam in hOAT1/3-HEK293 cells. The Km values of tazobactam (431 ±â€¯67 µM for hOAT1, 377 ±â€¯63 µM for hOAT3) were significantly higher than those of piperacillin (37 ±â€¯5 µM for hOAT1, 172 ±â€¯28 µM for hOAT3). This suggested that piperacillin has a stronger affinity to hOAT1/3 than tazobactam. Meanwhile, the Km values of tazobactam were increased in the presence of piperacillin with unchanged Vmax. This indicated that piperacillin inhibited the uptake of tazobactam in a competitive manner. In conclusion, piperacillin and tazobactam are the substrates of hOAT1/3, and OAT1/3 mediate the DDI between piperacillin and tazobactam.

U-shaped relationship between serum uric acid levels and intrarenal hemodynamic parameters in healthy subjects.

Hyperuricemia has been reported to affect renal hemodynamics. In a recent study, both low and high levels of serum uric acid (SUA) were found to be associated with loss of kidney function. The goal of this study was to evaluate the relationship between SUA levels and intrarenal hemodynamic parameters in healthy subjects, using plasma clearance of para-aminohippurate (CPAH) and inulin (Cin). Renal and glomerular hemodynamics were evaluated by simultaneous measurements of CPAH and Cin in 48 healthy subjects (54.6 ± 13.4 yr). Intrarenal hemodynamic parameters, including efferent and afferent (Ra) arteriolar resistance, were calculated using Gómez's formulas. Relationships of SUA levels with these intrarenal hemodynamic parameters were examined. In quadratic regression analysis, SUA levels had a significant inverse U-shaped relationship with Cin (P < 0.0001, R2 = 0.350) and CPAH (P = 0.0093, R2 = 0.188) and a U-shaped relationship with Ra (P = 0.0011, R2 = 0.262). In multiple regression analysis with normal (3.5-6.0 mg/dl) and mildly low or high (<3.5 or >6.0 mg/dl) SUA levels entered as dummy variables of zero and one, respectively, mildly low or high SUA levels were significantly and independently associated with Ra (ß = 0.230, P = 0.0403) after adjustment for several factors (R2 = 0.597, P < 0.0001). Both mild hyperuricemia and mild hypouricemia are significantly associated with increased Ra, although weakly. The increase in Ra in subjects with mild hyperuricemia or hypouricemia may be related to renal hemodynamic abnormalities, possibly leading to a decline in renal function.

The Isoquinolone Derived Prolyl Hydroxylase Inhibitor ICA Is a Potent Substrate of the Organic Anion Transporters 1 and 3.

OBJECTIVE: Many cellular responses to hypoxia are mediated by the transcription factor complex hypoxia-inducible factor (HIF). HIF stability is governed by a family of dioxygenases called HIF prolyl hydroxylases (PHDs). Isoquinolone-derived PHD inhibitors, like 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), which stabilize the intracellular HIF-α have been suggested as a potentially beneficial therapeutic strategy for the treatment of disorders associated with ischemia. To stabilize HIF-α, ICA has to be taken up into proximal tubule cells (PCTs) across the basolateral membrane by one of the organic anion transporters 1, 2 or 3 (OAT1, OAT2 or OAT3). The release into the urine across the luminal membrane may be mediated by OAT4. METHOD: To demonstrate interaction of ICA with human OAT1, OAT2, OAT3 and OAT4, ICA was tested on these transporters stably transfected in HEK293 cells by using p-aminohippurate (PAH), cGMP and estrone-3-sulfate (ES) as reference substrates, respectively. RESULTS: Uptakes of PAH and ES in OAT1- and OAT3-transfected HEK293 cells were inhibited by ICA with half-maximal inhibition values of 0.29 ± 0.05 and 2.58 ± 0.16 µM, respectively. OAT2 was less sensitive to ICA. Efflux experiments identified ICA as an OAT1 and OAT3 substrate. Preloading OAT4-transfected HEK293 cells with ICA stimulated ES uptake by 18.3 ± 3.8%. CONCLUSION: The uptake of ICA across the basolateral membrane of PCTs occurs mainly by OAT1 and the efflux into the tubular lumen by OAT4.

Relationship between serum uric acid levels and intrarenal hemodynamic parameters.

BACKGROUND/AIMS: Hyperuricemia has been reported to affect renal hemodynamics in rat models. We evaluate the relationship between serum uric acid and intrarenal hemodynamic parameters in humans, utilizing the plasma clearance of para-aminohippurate (CPAH ) and inulin (Cin). METHODS: Renal and glomerular hemodynamics were assessed by simultaneous measurement of CPAH and Cin in 58 subjects. Of these, 19 subjects were planned to provide a kidney for transplantation; 26 had diabetes without proteinuria; and 13 had mild proteinuria. Renal and glomerular hemodynamics were calculated using Gomez`s formulae. RESULTS: Cin was more than 60 ml/min/1.73m(2) in all subjects. Serum uric acid levels correlated significantly with vascular resistance at the afferent arteriole (Ra) (r = 0.354, p = 0.006) but not with that of the efferent arteriole (Re). Serum uric acid levels (ß = 0.581, p = <0.001) were significantly and independently associated with Ra after adjustment for several confounders (R(2) = 0.518, p = <0.001). CONCLUSIONS: These findings suggest, for the first time in humans, that higher serum uric acid levels are associated significantly with Ra in subjects with Cin > 60 ml/min/1.73m(2). The increase in Ra in subjects with higher uric acid levels may be related to dysfunction of glomerular perfusion.

Renal human organic anion transporter 3 increases the susceptibility of lymphoma cells to bendamustine uptake.

Chronic lymphatic leukemia (CLL) is often associated with nephritic syndrome. Effective treatment of CLL by chlorambucil and bendamustine leads to the restoration of renal function. In this contribution, we sought to elucidate the impact of organic anion transporters (OATs) on the uptake of bendamustine and chlorambucil as a probable reason for the superior efficacy of bendamustine over chlorambucil in the treatment of CLL. We examined the effects of structural analogs of p-aminohippurate (PAH), melphalan, chlorambucil, and bendamustine, on OAT1-mediated [(3)H]PAH uptake and OAT3- and OAT4-mediated [(3)H]estrone sulfate (ES) uptake in stably transfected human embryonic kidney-293 cells. Melphalan had no significant inhibitory effect on any OAT, whereas chlorambucil reduced OAT1-, OAT3-, and OAT4-mediated uptake of PAH or ES down to 14.6%, 16.3%, and 66.0% of control, respectively. Bendamustine inhibited only OAT3-mediated ES uptake, which was reduced down to 14.3% of control cells, suggesting that it interacts exclusively with OAT3. The IC50 value for OAT3 was calculated to be 0.8 µM. Real-time PCR experiments demonstrated a high expression of OAT3 in lymphoma cell lines as well as primary CLL cells. OAT3-mediated accumulation of bendamustine was associated with reduced cell proliferation and an increased rate of apoptosis. We conclude that the high efficacy of bendamustine in treating CLL might be partly contributed to the expression of OAT3 in lymphoma cells and the high affinity of bendamustine for this transporter.

Renal hemodynamic effects of serelaxin in patients with chronic heart failure: a randomized, placebo-controlled study.

BACKGROUND: Serelaxin is a promising therapy for acute heart failure. The renal hemodynamic effects of serelaxin in patients with chronic heart failure are unknown. METHODS AND RESULTS: In this double-blind, randomized, placebo-controlled, multicenter study, patients with New York Heart Association Class II to III chronic heart failure, left ventricular ejection fraction ≤45%, and estimated glomerular filtration rate (GFR) 30 to 89 mL/min per 1.73 m(2) received intravenous serelaxin 30 µg/kg per day or placebo for 24 hours. Primarily, we assessed the difference between serelaxin and placebo on renal plasma flow (para-aminohippuric acid clearance) and GFR (iothalamate clearance) over 8 to 24 hours. All 22 patients from 1 clinical site were excluded from primary analyses before unblinding because of implausible measurements. The primary analysis comprised 65 patients, mean age was 68 (±10) years, 89% were male, mean estimated GFR was 64 (±19) mL/min per 1.73 m(2), and 34% had New York Heart Association Class III symptoms. Renal plasma flow increased by 29% with serelaxin and 14% with placebo (13% relative increase with serelaxin; P=0.0386), whereas GFR changes did not differ significantly during 8 to 24 hours. Filtration fraction increased by 36% with serelaxin and 62% with placebo (16% relative decrease with serelaxin; P=0.0019) during 8 to 24 hours. Changes in systolic blood pressure were largely similar, and creatinine clearance did not differ between groups. Adverse event rates were similar with serelaxin (20.5%) and placebo (25.0%). CONCLUSIONS: In patients with chronic heart failure, serelaxin increased renal plasma flow and reduced the increase in filtration fraction compared with placebo, but did not affect GFR. These results suggest beneficial renal hemodynamic effects in patients with chronic heart failure. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01546532.

Proton pump inhibitors inhibit methotrexate transport by renal basolateral organic anion transporter hOAT3.

The coadministration of methotrexate (MTX) and proton pump inhibitors (PPIs) can result in a pharmacokinetic interaction that delays MTX elimination and subsequently increases the MTX blood concentrations. Human organic anion transporters (hOATs) are responsible for the renal tubular secretion of MTX and are thought to be involved in this drug interaction. The aim of this study was to evaluate the inhibitory potencies of PPIs on hOAT1 and hOAT3, which are the two isoforms of OATs predominantly expressed in kidney proximal tubules. Using stably transfected cell systems that express the uptake transporters human embryonic kidney (HEK)-hOAT1 and HEK-hOAT3, we analyzed the inhibitory potencies of omeprazole, lansoprazole, and pantoprazole on OAT-mediated [(3)H]estrone sulfate (ES), [(3)H]p-aminohippuric acid (PAH), and [(3)H]MTX uptake in vitro. hOAT3 is a high affinity transporter for MTX (Km = 21.17 ± 5.65 µM). Omeprazole, lansoprazole, and pantoprazole inhibited [(3)H]MTX uptake in HEK-hOAT3 cells with an IC50 of 6.8 ± 1.16, 1.14 ± 0.26, and 4.45 ± 1.62 µM, respectively, and inhibited the [(3)H]ES uptake in HEK-hOAT3 cells with an IC50 of 20.59 ± 4.07, 3.96 ± 0.96, and 7.89 ± 2.31 µM, respectively. Furthermore, omeprazole, lansoprazole, and pantoprazole exhibited inhibited PAH uptake on hOAT1 in a concentration-dependent manner (IC50 = 4.32 ± 1.26, 7.58 ± 1.06, and 63.21 ± 4.74 µM, respectively). These in vitro results suggest that PPIs inhibit [(3)H]MTX transport via hOAT3 inhibition, which most likely explains the drug-drug interactions between MTX and PPIs and should be considered for other OATs substrates.

Flavonoids are inhibitors of human organic anion transporter 1 (OAT1)-mediated transport.

Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 µM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of <0.3 and 0.47 µM, respectively. In contrast to the tested flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway.

Differential effect of COX1 and COX2 inhibitors on renal outcomes following ischemic acute kidney injury.

BACKGROUND/AIMS: We have previously shown that 1 mg/kg indomethacin improves expression and functionality of renal organic anion transporters Oat1 and Oat3 after renal ischemia and furthermore improves renal outcome after ischemia. As we detected differential effects of COX1 or COX2 inhibitors on organic anion transport after ischemia and reperfusion in culture, we investigated the effect of the SC560 (COX1 inhibitor) and SC58125 (COX2 inhibitor) on expression of Oat1/3 and renal outcome after ischemic acute kidney injury (iAKI). METHODS: iAKI was induced in rats by bilateral clamping of renal arteries for 45 min. SC560 or SC58125 (1 mg/kg each) were given intraperitoneally as soon as reperfusion started. Sham-treated animals served as controls. Oat1/3 were determined by qPCR and Western blot. Glomerular filtration rate (GFR), p-aminohippurate (PAH) clearance and PAH extraction ratio was determined. All parameters were detected 24 h after ischemia. Renal plasma flow was calculated. RESULTS: In clamped animals SC560 (COX1 inhibitor) restored expression of Oat1/3, as well as renal perfusion. Additionally, SC560 substantially improved kidney function as measured by GFR. Application of the COX2 inhibitor SC58125 did not exert these beneficial effects. CONCLUSION: Our study indicates that COX1 inhibitor SC560 applied after ischemia prevents ischemia-induced downregulation of Oat1/3 during reperfusion and has a substantial protective effect on kidney function. Whether and to what particular extent this apparent improvement of function is mechanistically due to beneficial effects on tubular function, renal perfusion or glomerular filtration will be the scope of future studies.

A plasma concentration of α-ketoglutarate influences the kinetic interaction of ligands with organic anion transporter 1.

The purpose of the present study was to determine whether a physiologic plasma concentration of α-ketoglutarate (αKG) influences the kinetic interaction of ligands with organic anion transporter 1 (OAT1). The effect of extracellular αKG on the kinetics of para-aminohippurate (PAH) and cidofovir transport was examined along with its effect on the potency of 10 drugs in five different classes (uricosuric, nonsteroidal anti-inflammatories, loop diuretics, angiotensin II receptor antagonists, and ß-lactam antibiotics) to inhibit OAT1 expressed in Chinese hamster ovary cells. Extracellular αKG competitively inhibited PAH and cidofovir transport with Ki values (∼5 µM) approximating its unbound plasma concentration (determined by equilibrium dialysis). When PAH was the substrate, extracellular αKG (5 µM) significantly increased IC50 values for some inhibitors (up to 4-fold), such as probenecid, but not for others (an inhibitor-dependent effect). For some inhibitors, a significant increase in IC50 value was observed when cidofovir was the substrate, but not PAH (a substrate-dependent effect). A significant increase in IC50 value was also observed for inhibition of PAH transport by probenecid in renal basolateral membrane vesicles (5.2-fold). The substrate- and inhibitor-dependent effect of extracellular αKG on ligand interactions with OAT1 highlights the complexity of the OAT1 ligand-binding surface. The effect of extracellular αKG on the potency of OAT1 inhibition should be considered when assessing drug-drug interaction potential at the transporter.

Acute kidney injury: arterial spin labeling to monitor renal perfusion impairment in mice-comparison with histopathologic results and renal function.

PURPOSE: To determine if arterial spin-labeling (ASL) magnetic resonance (MR) imaging can show serial changes in renal perfusion in mice with ischemia-induced acute kidney injury (AKI) and to compare imaging results with those of renal histologic examination and inulin and para-aminohippuric acid (PAH) clearance. MATERIALS AND METHODS: In this animal care committee-approved study, AKI was induced in C57Bl/6 mice (n = 26) by clamping the right renal pedicle for 35 minutes for moderate (n = 16) or 45 minutes (n = 11) for severe AKI. Renal perfusion was measured in 10 animals with moderate and seven animals with severe AKI before and at five time points 1-28 days after surgery by using ASL with a 7-T MR imaging unit. Kidney volume loss and histologic evidence of acute tubular injury were assessed. Inulin and PAH clearance was determined in four animals with moderate and six animals with severe AKI to evaluate renal function and plasma flow for statistical analysis. Repeated measures analysis of variance, unpaired t tests, and correlation analysis were used. RESULTS: Renal perfusion values at day 7 were significantly reduced after moderate (56% ± 8; P < .01) and severe (33% ± 6; P < .001) AKI compared with presurgery values. Renal perfusion had returned to baseline levels at day 21 after moderate (96% ± 14) and remained compromised until day 28 after severe (46 % ± 9; P < .05) AKI. At day 28, for moderate versus severe AKI, kidney volume (84% ± 6 vs 60% ± 5; P < .05), degree of tubular injury (5.6% ± 1.8 vs 15.8% ± 2.4; P < .01), and inulin and para-aminohippuric acid clearance (47.5 µL/min ± 5.6 vs 7.3 µL/min ± 2.7; P < .001 and 100.8 µL/min ± 24.3 vs 4.8 µL/min ± 1.0; P < .001, respectively) were significantly different. Relative renal perfusion at days 7-28 significantly correlated with kidney volume loss (P < .01) and tubular injury (P < .05) 4 weeks after AKI. CONCLUSION: ASL allows evaluation of renal perfusion impairment associated with kidney volume loss and histologic changes after AKI in mice and may serve as a noninvasive biomarker for AKI.

Antioxidant and renoprotective effects of Spirogyra neglecta (Hassall) Kützing extract in experimental type 2 diabetic rats.

Spirogyra neglecta extract (SNE) has shown antihyperglycemia and antihyperlipidemia in type 2 diabetic mellitus (T2DM) rats. This study investigated the antioxidant and renoprotective effects of SNE in T2DM rats induced by high-fat diet with low-single dose streptozotocin. T2DM rats were fed daily with SNE (0.25, 0.5, and 1 g/kg BW) for 12 weeks. Renal morphology, malondialdehyde levels, qPCR, and western blotting were analyzed. Renal cortical slices were used to determine renal transport of organic anions, which are estrone sulfate and para-aminohippurate, mediated through organic anion transporter 3-Oat3. Insulin and PKCζ were known to activate Oat3 function while it was inhibited by PKCα. Compared to T2DM, plasma glucose, triglyceride, insulin resistance, renal morphology, and malondialdehyde levels were significantly improved by SNE supplementation. Reduced glutathione peroxidase and nuclear factor κB expressions were related to antioxidant effect of SNE. Oat3 mRNA and protein were not different among groups, but insulin-stimulated rOat3 followed by anion uptakes was abolished in T2DM. This was restored in the slices from SNE treatment. The mechanism of SNE-improved Oat3 was associated with PKCα and PKCζ expressions and activities. These findings indicate that SNE has beneficial effects on renal transport through antioxidant enzymes and PKCs in T2DM rats.

Drugs interacting with organic anion transporter-1 affect uptake of Tc-99m-mercaptoacetyl-triglycine (MAG3) in the human kidney: therapeutic drug interaction in Tc-99m-MAG3 diagnosis of renal function and possible application of Tc-99m-MAG3 for drug development.

INTRODUCTION: Renal uptake of Tc-99m-MG3 involves organic anion transporter (OAT). Treatment with drugs showing OAT affinity might interfere with renal uptake of Tc-99m-MAG3, leading to misinterpretation in Tc-99m-MAG3. This study was conducted to discuss a possible drug interference with Tc-99m-MAG3 diagnosis on OAT sites. METHODS: Renal uptake and plasma clearance of Tc-99m-MAG3 were analyzed in healthy volunteers under control and OAT1 and OAT3 related drug treatment conditions. An in vitro uptake study using OAT1 or OAT3 expressing cells was also conducted. RESULTS: Both PAH and probenecid treatment induced delays in Tc-99m-MAG3 clearance from blood, and reductions in the renal uptake clearance. As a result, the normalized effective renal plasma flow estimated from Tc-99m-MAG3 clearance was significantly underestimated, whereas the glomerular filtration rate estimated from plasma creatinine levels was unchanged. The transport activity of Tc-99m-MAG3 was higher in OAT1-expressing cells than in OAT3-expressing cells. CONCLUSION: Drugs with OAT1 affinity affect the renal uptake of Tc-99m-MAG3 and blood clearance. This might cause misinterpretation of functional diagnosis of the kidney using Tc-99m-MAG3.

Changes in expression of renal Oat1, Oat3 and Mrp2 in cisplatin-induced acute renal failure after treatment of JBP485 in rats.

The purpose of this study is to investigate whether the effect of cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) on acute renal failure (ARF) induced by cisplatin is related to change in expression of renal Oat1, Oat3 and Mrp2 in rats. JBP485 reduced creatinine, blood urea nitrogen (BUN) and indoxyl sulfate (IS) in plasma and malondialdehyde (MDA) in kidney, and recovered the glomerular filtration rate (GFR) and the activity of superoxide dismutase (SOD) in cisplatin-treated rats. The plasma concentration of PAH (para-aminohippurate) determined by LC-MS/MS was increased markedly after intravenous administration of cisplatin, whereas cumulative urinary excretion of PAH and the uptake of PAH in kidney slices were significantly decreased. qRT-PCR and Western-blot showed a decrease in mRNA and protein of Oat1 and Oat3, an increase in mRNA and protein of Mrp2 in cisplatin-treated rats, and an increase in IS (a uremic toxin) after co-treatment with JBP485. It indicated that JBP485 promoted urinary excretion of toxins by upregulating renal Mrp2. This therefore gives in part the explanation about the mechanism by which JBP485 improves ARF induced by cisplatin in rats.

Effect of sulfhydryl modification on rat kidney basolateral plasma membrane transport function.

Transport processes are the hallmark of functioning kidney. Various nephrotoxicants disrupt the transport processes to manifest nephrotoxicity. Of several nephrotoxicants, mercuric chloride (HgCl(2)) depletes the reduced glutathione (GSH) in kidney and has been observed to affect the in vitro p-aminohippurate (PAH) transport by basolateral (BL) membrane vesicles. The role of renal nonprotein sulfhydryls such as, reduced GSH has been demonstrated to affect the PAH transport by BL membrane vesicles. The role of protein sulfhydryls in transport process of PAH by BL membrane is not known. Due to mercury mediated effects on sulfhydryls, the effects of protein-sulfhydryls (-SH) modifying reagents in the current study were investigated on PAH transport by BL membrane. It was observed that modification of -SH by p-chloromercuribenzoate sulphate (pCMBS), and mercuric chloride (HgCl(2)) decreased while recovering the protein -SH with dithiothreitol treatment provided protection against the effects of pCMBS, and HgCl(2) on PAH transport by BL membrane vesicles.

α-Ketoglutarate-related inhibitors of HIF prolyl hydroxylases are substrates of renal organic anion transporters 1 (OAT1) and 4 (OAT4).

2-Oxoglutarate or α-ketoglutarate (αKG) is a substrate of HIF prolyl hydroxylases 1-3 that decrease cellular levels of the hypoxia-inducible factor 1α (HIF-1α) in the presence of oxygen. αKG analogs are applied to stabilize HIF-1α even in the presence of oxygen and thus provide a novel therapeutic option in treating kidney diseases. In the kidneys, the organic anion transporters 1 and 3 (OAT1 and OAT3, respectively) in cooperation with the sodium-dependent dicarboxylate transporter 3 (NaDC3) and the OAT4 might be responsible for the uptake of αKG analogs into and the efflux out of the tubular cells. Using the radiolabelled substrates p-aminohippurate (PAH, OAT1), estrone-3-sulfate (ES; OAT3, OAT4), and succinate (NaDC3), N-oxalylglycine (NOG), dimethyloxalyl glycine (DMOG), 2,4-diethylpyridine dicarboxylate (2,4-DPD), and pyridine-2,4-dicarboxylic acid (PDCA) were tested in cis-inhibition and trans-stimulation experiments. None of these αKG analogs interacted with NaDC3. 2,4-DPD and PDCA inhibited ES uptake by OAT3 moderately. NOG, 2,4-DPD and PDCA, but not DMOG, inhibited PAH uptake by OAT1 significantly. trans-Stimulation experiments and experiments demonstrating stabilization of HIF-1α revealed that NOG and PDCA, but not 2,4-DPD, are translocated by OAT1. All compounds trans-stimulated ES uptake by OAT4, but only PDCA stabilized HIF-1α. The data suggest that OAT1 is involved in the uptake of NOG and PDCA across the basolateral membrane of proximal tubule cells, whereas OAT4 may release these compounds into the primary urine.