Isolated Perfused Rat Kidney Research Articles

Comparison of acute kidney injury following brain death between male and female rats

BackgroundClinical reports associate kidneys from female donors with worse prognostic in male recipients. Brain Death (BD) produces immunological and hemodynamic disorders that affect organ viability. Following BD, female rats are associated with increased renal inflammation interrelated with female sex hormone reduction. Here, the aim was to investigate the effects of sex on BD-induced Acute Kidney Injury (AKI) using an Isolated Perfused rat Kidney (IPK) model. MethodsWistar rats, females, and males (8 weeks old), were maintained for 4h after BD. A left nephrectomy was performed and the kidney was preserved in a cold saline solution (30 min). IPK was performed under normothermic temperature (37°C) for 90 min using WME as perfusion solution. AKI was assessed by morphological analyses, staining of complement system components and inflammatory cell markers, perfusion flow, and creatinine clearance. ResultsBD-male kidneys had decreased perfusion flow on IPK, a phenomenon that was not observed in the kidneys of BD-females (p < 0.0001). BD-male kidneys presented greater proximal (p = 0.0311) and distal tubule (p = 0.0029) necrosis. However, BD-female kidneys presented higher expression of eNOS (p = 0.0060) and greater upregulation of inflammatory mediators, iNOS (p = 0.0051), and Caspase-3 (p = 0.0099). In addition, both sexes had increased complement system formation (C5b-9) (p=0.0005), glomerular edema (p = 0.0003), and nNOS (p = 0.0051). ConclusionThe present data revealed an important sex difference in renal perfusion in the IPK model, evidenced by a pronounced reduction in perfusate flow and low eNOS expression in the BD-male group. Nonetheless, the upregulation of genes related to the proinflammatory cascade suggests a progressive inflammatory process in BD-female kidneys.

Gentamicin Renal Excretion in Rats: Probing Strategies to Mitigate Drug-Induced Nephrotoxicity

The renal excretion of gentamicin, an aminoglycoside antibiotic, was studied in the isolated perfused rat kidney (IPRK) model. Dose-linearity experiments were carried out at four doses (400, 800, 1600, 3200 μg), targeting initial perfusate levels of 5, 10, 20 and 40 μg/ml. Additionally, gentamicin was co-perfused with sodium bicarbonate (0.25 mM) and/or cimetidine (2 mM) to evaluate the effect of urinary alkalization and secretory inhibition on gentamicin excretion and kidney accumulation. Gentamicin displayed net reabsorption in the IPRK, consistent with extensive luminal uptake. Kinetic analysis indicated that luminal transport of gentamicin (kidney ? urine) is the rate-determining step for gentamicin urinary excretion. Clearance and cumulative excretion decreased with increased gentamicin dose. Gentamicin kidney accumulation, estimated by mass balance, ranged from ~20% - 30%. Urinary alkalization significantly increased gentamicin excretion, with no effect on kidney accumulation. Conversely, cimetidine co-administration did not affect gentamicin clearance in the IPRK, but kidney accumulation was significantly reduced. When both sodium bicarbonate and cimetidine were administered together, gentamicin kidney accumulation decreased ~80% with corresponding increases in clearance and excretion ratio (XR) compared to gentamicin alone. A main strategy to reduce the incidence of nephrotoxicity with gentamicin therapy (up to ~25%) involves reducing kidney accumulation of the compound. The results of this research suggest that the combination of urinary alkalization and inhibition of basolateral secretion (blood → kidney) may be a viable approach to mitigate aminoglycoside toxicity, and warrants further investigation.

Renal excretion of apricitabine in rats: ex vivo and in vivo studies

Apricitabine (ATC) is a novel nucleoside reverse transcriptase inhibitor undergoing phase 2/3 clinical development for the treatment of HIV infection. In this investigation, the renal handling of ATC was evaluated in the isolated perfused rat kidney (IPK) model with follow-up in vivo studies. IPK experiments were performed to characterize the renal excretion of ATC, to probe mechanisms of ATC excretion using known inhibitors of organic cation (cimetidine) and organic anion (probenecid) transport systems, and to screen for potential drug-drug interactions between ATC and clinically relevant medications (dapsone, metformin, pentamidine, stavudine, tenofovir and ritonavir). ATC demonstrated net tubular secretion in the IPK with a baseline excretion ratio (XR) of 2.1 ± 0.56. ATC XR decreased 3.6-fold in the presence of cimetidine and 2-fold in the presence of probenecid. Among the clinically relevant medications, metformin produced the greatest inhibitory effect on ATC excretion. In vivo studies were conducted in rats to evaluate ATC disposition upon co-administration with compounds that showed a significant effect on ATC clearance in the IPK model. Co-administration of cimetidine and trimethoprim significantly reduced ATC renal clearance, but resulted in only a moderate increase in plasma exposure. Metformin had no apparent effect on ATC clearance in rats. These findings indicate that the IPK model is more sensitive to secretory inhibition as compared to in vivo. The medications screened showed minimal effects on ATC renal excretion in the IPK, and should thus be excluded as potential in vivo interactants. Overall, this study generated important information on renal handling of ATC to support its development and commercialization.

Renal excretion of clofarabine: Assessment of dose-linearity and role of renal transport systems on drug excretion

The renal excretion of clofarabine was studied in vitro in the isolated perfused rat kidney (IPK) model and in vivo in rats. Clofarabine excretion was studied at four doses (160, 800, 2000 and 4000microg) in the IPK, targeting perfusate levels of 2, 10, 25, 50microg/mL, respectively. Clofarabine (2microg/mL) was also co-perfused with known inhibitors of the, organic cation (cimetidine, ranitidine and tyramine) and organic anion (probenecid, ellagic acid) transport systems. Additionally, the effect of medications including, itraconazole, digoxin, fludarabine and cytarabine (Ara-C) on clofarabine excretion was, evaluated. Based on IPK results, in vivo studies were performed to assess the plasma, pharmacokinetics and urinary recovery of clofarabine (6.5mg/kg, IV) pretreatment, with cimetidine (250mg/kg, IV). Clofarabine clearance was non-linear in the IPK, although at concentrations that were approximately 10- to 100-fold higher than maximum concentrations observed in humans. Excretion ratio data indicated a shift from net, secretion (XR=1.2+/-0.33) to net reabsorption (XR=0.78+/-0.40) at the highest dose, tested. Clofarabine clearance was significantly reduced upon co-administration with, cimetidine (0.83+/-0.22-->0.32+/-0.058mL/min). In vivo data correlated with IPK results as clofarabine clearance decreased 61% (20.2-7.80mL/min/kg), upon co-administration with cimetidine in rats. The results suggest that clofarabine is a substrate for a cimetidine-sensitive organic cation transporter system in the kidney, presumably OCT2. The magnitude of the cimetidine-clofarabine interaction was similar, in IPK and in vivo, demonstrating the utility of the IPK model in characterizing renal, drug excretion and assessing potential drug-drug interactions.

Comparative renal excretion of VX-702, a novel p38 MAPK inhibitor, and methotrexate in the perfused rat kidney model

Context: VX-702 is a novel p38 mitogen-activated protein kinase inhibitor being developed to treat rheumatoid arthritis. Objective: To characterize the renal excretion profile of VX-702 using the isolated perfused rat kidney (IPRK) model. Methods: Studies were performed to assess the dose linearity of VX-702 excretion and to evaluate the effect of inhibitors of organic anion (probenecid) and organic cation (cimetidine) transport systems on VX-702 disposition. VX-702 excretion was studied over a range of doses targeting concentrations between 100 and 600 ng/mL. VX-702 (600 ng/mL) was also co-perfused with probenecid (1 mM) and cimetidine (2 mM). The results were compared to parallel experiments performed with methotrexate (MTX). Results: VX-702 excretion was linear over the range of doses studied, and clearance data were consistent with net reabsorption by the kidney. Transport inhibition studies indicate that VX-702 is not a substrate for renal organic anion and organic cation transport systems. MTX (500 ng/mL) also displayed net reabsorption in the IPRK, but secretory transport was inhibited upon co-administration with probenecid. This finding is consistent with previous IPRK studies that demonstrated inhibitory effects of NSAIDS on MTX excretion. Conclusion: Overall, this study suggests that a renal drug-drug interaction between VX-702 and MTX would be unlikely if these medications were co-administered.

Effect of ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit the renal tubular secretion of methotrexate. However, the relative contribution of the active S- and inactive R-enantiomers is unknown. This study examined the effect of racemic ketoprofen and its enantiomers on the renal disposition of methotrexate in the isolated perfused rat kidney (IPK). Nineteen kidneys were divided between a control and three treatment groups. Controls were perfused with methotrexate alone (25 micrograms mL-1, n = 5) over three 30-min periods. Treatment groups were perfused with methotrexate (25 micrograms m-1) for the first period, followed by a second period of methotrexate (25 micrograms mL-1) plus R- (n = 5), S- (n = 5) or RS-ketoprofen (n = 4) at 25 micrograms mL-1, and a third period of methotrexate (25 micrograms mL-1) plus R-, S- or RS-ketoprofen (50 micrograms mL-1). Perfusate and urine were collected over 10-min intervals. Methotrexate was measured by HPLC and its binding in perfusate by ultrafiltration. The clearance ratio (CR) for methotrexate was obtained by dividing the renal clearance by the product of its fraction unbound and the glomerular filtration rate. During control experiments, there was no significant change in the CR over 90 min. R-, S- and RS-ketoprofen at 50 micrograms mL-1 reduced the CR of methotrexate significantly, but there was no difference between the three groups. While the enantiomers of ketoprofen reduced the renal excretion of methotrexate, the interaction was not enantioselective.

Renal disposition of colistin in the isolated perfused rat kidney.

Nephrotoxicity is an important limitation to the clinical use of colistin against Pseudomonas aeruginosa and other gram-negative pathogens. Previous work reported net tubular reabsorption of colistin by the kidney in vivo, but there is no knowledge of its disposition within the kidney. This study investigated the renal disposition and potential transport mechanisms of colistin in the isolated perfused rat kidney (IPK) model by perfusing with colistin sulfate alone (2 microg/ml) or in the presence of potential inhibitors (tetraethylammonium [TEA], glycine-glycine [Gly-Gly], or hydrochloric acid [HCl]) at three different concentrations. When perfused alone, the renal clearances (CL(R)) for colistin A and B (the major components of colistin) in control kidneys were constant and low (mean values < 0.05 ml/min throughout the perfusion). The mean clearance ratios [CR, defined as CL(R)/(f(u) x GFR), where f(u) is the fraction of drug unbound in perfusate and GFR is the glomerular filtration rate] were significantly less than 1. It was concluded that there is net tubular reabsorption of colistin, and this exceeded the reabsorption of water. Less than 10% eliminated from perfusate was recovered in urine, suggesting considerable renal accumulation of colistin. The CR values for colistin were significantly increased when perfused with TEA (500 microM), Gly-Gly (833 microM), and HCl (2,500, 5,000, and 10,000 microM). It is proposed that renal reabsorption of colistin may involve organic cation transporters (inhibited by TEA) and peptide transporters (inhibited by Gly-Gly) and that the process is sensitive to the pH of urine.

Renal excretion of emtricitabine II. Effect of trimethoprim on emtricitabine excretion: In vitro and in vivo studies

The potential interaction between the nucleoside analog emtricitabine (FTC) and trimethoprim (TMP) was assessed in the isolated perfused rat kidney (IPK) model and in vivo in rats. IPK experiments were performed with FTC alone (2 µg/mL) and in the presence of increasing concentrations of TMP (1–10 µg/mL). TMP inhibited FTC excretion in a concentration dependent manner. The IC50 (TMP concentration associated with a 50% reduction in FTC excretion) was 1.86 ± 0.37 µg/mL. The results were compared to whole animal studies in rats. Animals received an IV dose of FTC (1 mg/kg) with or without pretreatment with TMP (25 mg/kg). TMP coadministration significantly decreased FTC clearance (7.4 ± 1.2 mL/min/kg to 2.7 ± 0.53 mL/min/kg), and elimination half‐life was significantly increased (58 ± 12 min to 215 ± 44 min). A good correlation was obtained between IPK findings and in vivo data, as FTC renal clearance was reduced ∼60% in the presence of TMP in both studies. Based on this investigation, TMP would be expected to inhibit the renal excretion of FTC when the two compounds are coadministered, resulting in increased plasma exposure of FTC. However, the clinical significance of this finding remains to be elucidated. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5411–5420, 2008

Renal excretion of emtricitabine I: Effects of organic anion, organic cation, and nucleoside transport inhibitors on emtricitabine excretion

The excretion of emtricitabine (FTC) was characterized using isolated perfused rat kidney (IPK) model. Studies were performed to assess the dose‐linearity of FTC excretion, to evaluate the effect of inhibitors of organic anion (probenecid, PBC), organic cation (tetraethylammonium, TEA; cimetidine, CMD) and nucleoside (uridine, URD) transport systems on FTC excretion, and to determine the potential interaction between FTC and trimethoprim (TMP). FTC excretion was studied over a range of doses (80–1600 µg), targeting concentrations encompassing the therapeutic range of FTC (1–20 µg/mL). FTC (2 µg/mL) was also coperfused with PBC (500 µM), TEA (500 µM), CMD (2 mM), URD (500 µM), and TMP (13.7 µM). FTC dose‐linearity studies revealed that excretion parameters were not significantly different among dosing groups. Of the transport inhibitors tested, FTC XR decreased more than twofold in the presence of CMD (0.32 ± 0.099). PBC, TEA, and URD had no observed effect on FTC excretion. TMP coadministration significantly inhibited FTC excretion (XR = 0.43 ± 0.052). The results suggest that FTC renal transport is likely mediated by a CMD‐sensitive organic cation transporter (OCT) in the kidney. TMP may inhibit the renal excretion of FTC when the two compounds are coadministered in vivo. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5401–5410, 2008

Determination of colistin in human plasma, urine and other biological samples using LC–MS/MS

A liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed to quantify colistin in human plasma and urine, and perfusate and urine from the isolated perfused rat kidney (IPK). Solid phase extraction (SPE) preceded chromatography on a Synergi Fusion-RP column with a mobile phase of acetonitrile, water and acetic acid (80/19/1) at 0.2 mL/min. Ions were generated using electrospray ionization and detected in the positive-ion mode. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 0.028 μg/mL (human plasma, IPK perfusate and urine)/0.056 μg/mL (human urine) to 1.78 μg/mL (all four media) for colistin A sulfate; corresponding values for colistin B sulfate were 0.016/0.032 to 1.01 μg/mL. Accuracy and precision were within 10%. The LLOQ for colistin A sulfate was 0.028 μg/mL in human plasma, IPK perfusate and urine and 0.056 μg/mL in human urine; corresponding values for colistin B sulfate were 0.016 and 0.032 μg/mL. The low sample volume, short analysis time and low LLOQ are ideal for pre-clinical and human pharmacokinetic studies of colistin.

Effects of Trimethoprim on the Clearance of Apricitabine, a Deoxycytidine Analog Reverse Transcriptase Inhibitor, and Lamivudine in the Isolated Perfused Rat Kidney

Apricitabine (ATC) is a novel deoxycytidine analog reverse transcriptase inhibitor in development for the treatment of human immunodeficiency virus infection. Studies were performed to characterize the excretion of ATC and its metabolite, BCH-335 (-1-(2-hydroxymethyl-[1,3]oxathiolan-4-yl)-1H-pyrimidine-2,4-dione), in the isolated perfused rat kidney (IPK). A second objective was to investigate the effect of trimethoprim on ATC excretion because trimethoprim inhibits the excretion of lamivudine, structurally similar to ATC, in the IPK. ATC excretion was nonlinear at doses of 80 to 1600 microg. The excretion ratio (ratio of clearance to glomerular filtration rate, assuming negligible protein binding) was greater than 1.0, indicating net tubular secretion. In contrast, the excretion of BCH-335 was independent of the dose of BCH-335. Concomitant administration of ATC and BCH-335 did not affect the excretion of either compound. Trimethoprim significantly inhibited the excretion of both ATC and BCH-335, with IC(50) values of 0.45 and 0.54 microg/ml, respectively. In the presence of trimethoprim, the excretion ratios for both compounds were less than 1.0, indicating tubular reabsorption. Trimethoprim inhibited the excretion of ATC and lamivudine to similar extents. Following concomitant administration of ATC, lamivudine, and trimethoprim, there was no evidence of an interaction between ATC and lamivudine. These results suggest that ATC undergoes active tubular secretion in the kidney. Because the renal excretion of both ATC and lamivudine is inhibited by trimethoprim to similar extents, in clinical practice exposure to ATC, it would be expected to be increased in the presence of therapeutic concentrations of trimethoprim to a similar extent as has been shown previously for lamivudine.

Hypoxia-inducible factors and tubular cell survival in isolated perfused kidneys

Adaptation to hypoxic environment is conferred through hypoxia-inducible transcription factors (HIFs). We have previously shown that the HIF system is transiently activated in vivo in radiocontrast-induced acute renal failure, associated with profound hypoxia in the renal medulla. Medullary thick ascending limbs (mTALs), the most affected nephron segments in this model, were virtually unable to mount an adaptive HIF response. Here, we study correlations between oxygenation, HIF activation, and cell viability in a related ex vivo model, the isolated perfused rat kidney (IPK). In IPKs perfused with cell-free oxygenated medium, severe medullary hypoxic damage developed, affecting 42+/-9% of mTALs in the mid-inner stripe. HIF-1alpha tubular immunostaining was noted with a zonal and tubular pattern largely similar to our findings in vivo: in 34+/-3% of collecting ducts (CDs) within the mid-inner stripe and extensively in the papillary tip, whereas mTALs were all HIF-negative. In IPKs supplemented with RBCs (improved oxygen supply), mTAL damage was totally prevented and CDs' HIF expression was attenuated (22+/-4%). By contrast, although measures designed to reduce medullary hypoxia by decreasing tubular reabsorptive activity (furosemide, ouabain, or high-albumin-non-filtering system) reduced mTAL damage, all paradoxically resulted in increased HIF expression in CDs (51+/-4%), and 17+/-3% of mTALs became immunostained as well. Our data confirm that CDs and mTALs have markedly different HIF responses, which correlate with their viability under hypoxic stress. mTALs transcriptional adaptation occurs within a narrow hypoxic range, and it appears that workload reduction can shift mTALs into this window of opportunity for HIF activation and survival.

Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion

This study evaluated whether cytoskeletal alterations during the ischemic conditions associated with kidney preservation could determine apoptosis. Cytoskeletal alterations are among the main effects of ischemia and may induce apoptosis. Rat kidneys were preserved in University of Wisconsin (UW) solution for 24 h. Some groups of animals underwent 45 min of warm ischemia (WI) to evaluate its effect on both the actin cytoskeleton and apoptosis (assessed by caspase-3 activity and TUNEL staining). Swinholide A (SwinA) and Latrunculin B (LB), two actin cytoskeleton-targeted agents, were administered to assess the effect of direct actin disruption on apoptosis. Jasplakinolide (JP), a compound that stabilizes actin filaments, was administered to evaluate the effect of actin stabilization. Apoptosis was evaluated at 3 h of ex vivo reperfusion using the isolated perfused rat kidney (IPK) model. Apoptosis increased during reperfusion with WI or administration of actin disruptor agents. Administration of stabilizing agents reversed apoptosis in kidneys that had previously undergone WI or had received an actin disruptor agent. The disruption of the actin cytoskeleton during ischemic conditions associated with kidney preservation induces apoptosis upon reperfusion through caspase-3 activation.

Characterization of the urinary albumin degradation pathway in the isolated perfused rat kidney

This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.

Application of the Isolated Perfused Rat Kidney Model to Assess Gender Effects on Drug Excretion

ABSTRACTPurpose. To study the effect of gender on the renal disposition of two organic anions, p-aminohippuric acid (PAH) and furosemide (FSM) in the isolated perfused rat kidney (IPK). Methods. IPK experiments (3–4 per treatment group) were conducted using kidneys from male and female Sprague Dawley rats. PAH was administered as a continuous infusion (with loading dose, targeted steady-state concentration 10 ug/mL). FSM was added as a bolus dose (2.65 mg, targeted concentration 33 ug/mL). Urine was collected in 10-min. intervals and perfusate was sampled at the midpoint of each collection period. Control (drug naïve) perfusions were performed for both genders. PAH and FSM were measured by HPLC. Kidney viability (GFR [estimated using inulin clearance], sodium reabsorption, glucose reabsorption) was monitored continuously during each perfusion experiment (2-h duration). Results. Good kidney function was maintained across all study groups, and lower GFR estimates in female kidneys were due to differences in kidney weight. For PAH, kidney weight corrected renal clearance (0.88 ± 0.37 mL/min/g vs. 0.59 ± 0.19 mL/min/g) and excretion ratio (3.8 ± 1.7 vs. 2.2 ± 0.72) were significantly higher in male kidneys. For FSM, renal clearance was significantly lower in female (0.10 ± 0.05 mL/min/g) compared to male kidneys (0.15 ± 0.07 mL/min/g). Mass balance analysis showed that FSM cumulative urinary excretion was significantly higher and kidney accumulation was significantly lower in experiments with male kidneys. Conclusions. The study demonstrates that the IPK is a useful model to assess gender effects on renal drug disposition. The renal excretion of organic anions is reduced in female rats, possibly due to gender differences in expression and/or activity of membrane transporters (both basolateral and luminal) in the kidney.

Iron chelators do not reduce cold-induced cell injury in the isolated perfused rat kidney model

In vitro, cold-induced injury is an important contributor to renal tubular cell damage. It is mediated by iron-dependent formation of reactive oxygen species and can be prevented by iron chelation. We studied whether iron chelators can prevent cold-induced damage in the isolated perfused rat kidney (IPK) model both after cold perfusion (CP) and after cold storage (CS). We hypothesized that in the CP model iron-dependent cold-induced injury is more pronounced, since oxygen is constantly provided. The IPK was either flushed with University of Wisconsin (UW) solution and stored for 4, 18 or 24 h at 4 degrees C or perfused during 4 h at 4 degrees C with UW for machine perfusion. The iron chelators 2,2'-dipyridyl or desferal, or the negative control 4,4'-dipyridyl were added during the cold perfusion. Kidney function was measured during 2 h reperfusion at 37.5 degrees C and compared to a control group (without cold preservation). Compared to control perfusion, kidney function was decreased in all experimental protocols. glomerular filtration rate and FR(H2O) were significantly decreased, while FE(gluc) and FE(Na) were higher after 4 h CS and CP. After 4 h CP, also renal vascular resistance was increased. Addition of 2,2'-dipyridyl did not improve kidney function after either CS or CP. Prolonged periods of CS worsened kidney function. The addition of 2,2'-dipyridyl or desferal did not improve kidney function after longer periods of CS. Addition of an iron chelator to the preservation solution UW did not improve kidney function after both CS and CP. Iron chelation is not able to prevent cold-induced damage in the isolated perfused rat kidney.

MR microscopy and microspectroscopy of the intact kidney

AbstractMagnetic resonance (MR) techniques have become essential in clinical practice and a broad range of research areas. We begin with a review of the potential and limitations for resolution improvements by MR techniques. The kidney has distinct regional structural, functional and biochemical variability. The isolated perfused rat kidney (IPRK) retains renal function while eliminating movement and susceptibility boundaries which severely limit the potential of MR techniques. The IPRK, with a length of less than 20 mm in the longest axis, will be used to illustrate the potential resolution of different MR techniques and the different biological information that can be obtained. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson 22A: 50–59, 2004.

The Isolated Perfused Rat Kidney Model: A Useful Tool for Drug Discovery and Development

Over the past three decades, the Isolated Perfused Rat Kidney (IPK) has been used to study numerous aspects of renal drug disposition. Among the available ex-vivo methods to study renal transport, the IPK allows for elucidation of the overall contributions of renal transport mechanisms on drug excretion. Therefore, IPK studies can provide a bridge between in vitro findings and in vivo disposition. This review paper begins with a detailed overview of IPK methodology (system components, surgical procedure, study design). Various applications of the IPK are then presented. These applications include characterizing renal excretion mechanisms, screening for clinically significant drug interactions, studying renal drug metabolism, and correlating renal drug disposition with drug-induced changes in kidney function. Lastly, the role of IPK studies in drug development is discussed. Demonstrated correlations between IPK data and clinical outcomes make the IPK model a potentially useful tool for drug discovery and evaluation.

Characterization of pentamidine excretion in the isolated perfused rat kidney.

To study the renal excretion and kidney accumulation of pentamidine, a potentially nephrotoxic compound, in the isolated perfused rat kidney (IPK). IPK experiments (3-4 per treatment group) were conducted using male Sprague-Dawley rats (250-350 g). Dose proportionality studies were carried out over a pentamidine dosing range of 80-4000 microg, designed to target initial perfusate concentrations from 1 to 50 microg/mL. Separate interaction experiments were conducted between pentamidine (800 microg) and tetraethylammonium (dose 8000 microg) or dideoxyinosine (dose 80 microg). Inulin was used as a glomerular filtration rate (GFR) marker. Control (drug-naive) perfusions were also carried out. Pentamidine was analysed in perfusate, kidney and urine samples by HPLC. Inulin was measured by a colorimetric method. Pentamidine CLR (1.1 +/- 0.6 to 0.05 +/- 0.03 mL/min) and excretion ratio (3.6 +/- 1.5 to 0.56 +/- 0.15) significantly decreased over the range of doses studied. Significant reductions in viability parameters (GFR, Na reabsorption) were noted in kidneys perfused with high dose pentamidine (4000 microg). Tetraethylammonium co-administration reduced pentamidine renal excretion, resulting in significantly greater kidney accumulation of pentamidine and reduced kidney function. Dideoxyinosine administration had minimal effects on pentamidine disposition. Pentamidine renal transport involves a combination of mechanisms (filtration, secretion and passive reabsorption). Dose proportionality studies demonstrated non-linear excretion of pentamidine. Inhibition of pentamidine renal clearance by tetraethylammonium was consistent with decreased luminal transport. The detrimental effects of pentamidine on kidney function were the result of significant kidney accumulation of drug. The potential exists for drug-drug interactions between pentamidine and organic cations, increasing the risk of drug-induced nephrotoxicity.

ATP-Dependent K+ Channels in Renal Ischemia Reperfusion Injury

ATP-dependent K+ channels (KATP) account for most of the recycling of K+ which enters the proximal tubules cell via Na, K-ATPase. In the mitochondrial membrane, opening of these channels preserves mitochondrial viability and matrix volume during ischemia. We examined KATP channel modulation in renal ischemia-reperfusion injury (IRI), using an isolated perfused rat kidney (IPRK) model, in control, IRI, IRI + 200 µM diazoxide (a KATP opener), IRI + 10 µM glibenclamide (a KATP blocker) and IRI + 200 µM diazoxide + 10 µM glibenclamide groups. IRI was induced by 2 periods of warm ischemia, followed by 45 min of reperfusion. IRI significantly decreased glomerular filtration rate (GFR) and increased fractional excretion of sodium (FENa) (p<0.01). Neither diazoxide nor glibenclamide had an effect on control kidney function other than an increase in renal vascular resistance produced by glibenclamide. Pretreatment with 200 µM diazoxide reduced the postischemic increase in FENa (p<0.05). Adding 10 µM glibenclamide inhibited the diazoxide effect on postischemic FENa (p<0.01). Histology showed that kidneys pretreated with glibenclamide demonstrated an increase in injury in the thick ascending limb of outer medulla (p<0.05). Glibenclamide significantly decreased post ischemic renal vascular resistance (p<0.05), but had no significant effect on other renal function parameters. Our results suggest that sodium reabsorption is improved by KATP activation and blockade of KATP channels during IRI has an injury enhancing effect on renal epithelial function and histology. This may be mediated through KATP modulation in cell and/or mitochondrial inner membrane.