Delay in Renal Hemodynamic Response to a Meat Meal in Severe Obesity.

BACKGROUND/AIMS: Little information is available about the tubular functions and the renal adjustments that take place in obese subjects after a protein meal. How the excess fat may affect renal response to dietary proteins is currently only partially understood. This paper aims to address (i) whether severe obesity, in the absence of other comorbidities, is responsible of kidney dysfunction at either the glomerular or the tubular level and (ii) whether it compromises renal adaptations to a large protein meal. METHODS: Twenty-eight obese subjects without albuminuria, along with 20 control subjects, age and gender matched, have been studied. The glomerular filtration rate (GFR; inulin clearance), renal plasma flow (p-aminohippurate clearance), the proximal tubular function (lithium clearance), the fractional excretion of sodium (FPRNa) have been measured at the basal level (steady state) and after a protein meal (perturbation). RESULTS: Under steady state conditions, filtration fraction, proximal tubular sodium handling and the FPRNa were not significantly different in non proteinuric obese subjects compared with controls. However, a protein meal led to a delayed glomerular hyperfiltration in obese patients compared with controls. CONCLUSION: This study shows that obese patients, in the absence of significant comorbidities, have a normal proximal tubule Na+ absorption at basal; conversely, these subjects showed a different response to a protein meal compared with normal subjects in terms of changes of GFR. Overall, these results suggest that the modified hemodynamic response to a protein meal might be the earliest hallmark of future kidney dysfunction in obese subjects.

Aptamers: Universal capture units for lateral flow applications.

The present work demonstrates the implementation of aptamers as capture molecules for a wide range of target classes in lateral flow assay applications. The targets were chosen in order to cover a wide range of target classes (small sized - metabolite, medium sized - protein, and large sized - whole cell/spore). For each target class one target molecule was selected as representative and appropriate aptamers were used for lateral flow assay development. The work points out that the implementation of aptamers as capture molecules in a universal lateral flow test platform was successful independent form target size. Furthermore, the limit of detection for p-aminohippuric acid in urine (200 ppm), lysozyme in white wine (20 ppm), and Alicyclobacillus spores in buffered orange juice (>8 CFU/mL) were determined using aptamers as capture molecules. The whole approach is considered as a proof of concept, regarding the ability of aptamers as an alternative to antibodies (in conjunction with directive 2010/63/EU on the protection of animals used for scientific purposes) in lateral flow applications.

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.

Renal effects of carprofen and etodolac in euvolemic and volume-depleted dogs.

OBJECTIVE: To determine the effects of carprofen and etodolac on renal function in euvolemic dogs and dogs with extracellular fluid volume depletion induced via administration of furosemide. ANIMALS: 12 female Beagles. PROCEDURES: Dogs received a placebo, furosemide, carprofen, etodolac, furosemide and carprofen, and furosemide and etodolac. The order in which dogs received treatments was determined via a randomization procedure. Values of urine specific gravity, various plasma biochemical variables, glomerular filtration rate (GFR [urinary clearance of creatinine]), and renal plasma flow (urinary clearance of para-aminohippuric acid) were determined before and after 8 days of drug administration. A washout time of approximately 12 days was allowed between treatment periods. RESULTS: Administration of furosemide, furosemide and carprofen, and furosemide and etodolac caused changes in urine specific gravity and values of plasma biochemical variables. Administration of carprofen or etodolac alone did not have a significant effect on renal plasma flow or GFR. Concurrent administration of furosemide and carprofen or furosemide and etodolac caused a significant decrease in GFR. After 12-day washout periods, mean values of GFR were similar to values before drug administration for all treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated GFR decreased after 8 days of concurrent administration of furosemide and carprofen or furosemide and etodolac to dogs. Administration of preferential cyclooxygenase-2 inhibitors to dogs with extracellular fluid volume depletion or to dogs treated with diuretics may transiently impair renal function.

Metabonomics biomarkers for subacute toxicity screening for benzene exposure in mice.

Benzene is known to produce hematotoxicity in occupational exposure workers. This study examined the utility of metabonomic biomarkers to ascertain subacute toxicity produced by benzene in male C3H/He mice. A 30-d intermittent collection of urine was obtained from mice in this experiment. The relative organ weights, blood parameters, and bone marrow smears were examined to identify specific changes of benzene-induced toxicity. In addition, an integrated analytical approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was developed to map metabolic responses in urine. Five endogenous metabolites, hypoxanthine, spermidine, 4-aminohippuric acid, indolelactic acid, and glutamylphenylalanine, were identified as potential biomarkers of benzene-induced toxicity, indicating that pathways of purine, spermidine, fatty acid, tryptophan, and peptides metabolism might be disturbed in benzene-exposed mice. Our findings showed that the use of urine metabonomics was a more sensitive tool to detect benzene-induced toxicity compared to body weight or blood parameter changes.

Attenuated glomerular arginine transport prevents hyperfiltration and induces HIF-1α in the pregnant uremic rat.

Pregnancy worsens renal function in females with chronic renal failure (CRF) through an unknown mechanism. Reduced nitric oxide (NO) generation induces renal injury. Arginine transport by cationic amino acid transporter-1 (CAT-1), which governs endothelial NO generation, is reduced in both renal failure and pregnancy. We hypothesize that attenuated maternal glomerular arginine transport promotes renal damage in CRF pregnant rats. In uremic rats, pregnancy induced a significant decrease in glomerular arginine transport and cGMP generation (a measure of NO production) compared with CRF or pregnancy alone and these effects were prevented by l-arginine. While CAT-1 abundance was unchanged in all experimental groups, protein kinase C (PKC)-α, phosphorylated PKC-α (CAT-1 inhibitor), and phosphorylated CAT-1 were significantly augmented in CRF, pregnant, and pregnant CRF animals; phenomena that were prevented by coadministrating l-arginine. α-Tocopherol (PKC inhibitor) significantly increased arginine transport in both pregnant and CRF pregnant rats, effects that were attenuated by ex vivo incubation of glomeruli with PMA (a PKC stimulant). Renal histology revealed no differences between all experimental groups. Inulin and p-aminohippurate clearances failed to augment and renal cortical expression of hypoxia inducible factor-1α (HIF-1α) significantly increased in CRF pregnant rat, findings that were prevented by arginine. These studies suggest that in CRF rats, pregnancy induces a profound decrease in glomerular arginine transport, through posttranslational regulation of CAT-1 by PKC-α, resulting in attenuated NO generation. These events provoke renal damage manifested by upregulation of renal HIF-1α and loss of the ability to increase glomerular filtration rate during gestation.

Glomerular hyperfiltration and increased proximal sodium reabsorption in subjects with type 2 diabetes or impaired fasting glucose in a population of the African region.

BACKGROUND. Glomerular hyperfiltration (GHF) is a well-recognized early renal alteration in diabetic patients. As the prevalence of GHF is largely unknown in populations in the African region with respect to normal fasting glucose (NFG), impaired fasting glucose (IFG) and type 2 diabetes [diabetes mellitus (DM)], we conducted a cross-sectional study in the Seychelles islands among families including at least one member with hypertension. METHODS. The glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and proximal tubular sodium reabsorption were measured using inulin, p-aminohippurate (PAH) and endogenous lithium clearance, respectively. Twenty-four-hour urine was collected on the preceding day. RESULTS. Of the 363 participants (mean age 44.7 years), 6.6% had IFG, 9.9% had DM and 63.3% had hypertension. The prevalence of GHF, defined as a GFR >140 ml/min, was 17.2%, 29.2% and 52.8% in NFG, IFG and DM, respectively (P trend <0.001). Compared to NFG, the adjusted odds ratio for GHF was 1.99 [95% confidence interval (CI) 0.73-5.44] for IFG and 5.88 (2.39-14.45) for DM. Lithium clearance and fractional excretion of lithium were lower in DM and IFG than NFG (P < 0.001). CONCLUSION. In this population of African descent, subjects with impaired fasting glucose or type 2 diabetes had a high prevalence of GHF and enhanced proximal sodium reabsorption. These findings provide further insight on the elevated incidence of nephropathy reported among African diabetic individuals.

Renal functional effects of simultaneous bilateral single-tract percutaneous access in pigs.

OBJECTIVE: To present our findings of simultaneous bilateral percutaneous nephrolithotomy (sbPCNL) on bilateral renal haemodynamic and excretory function in an in vivo pig model, as despite sbPCNL being a treatment strategy for patients with bilateral renal stones, the functional response of both kidneys to such a procedure is unknown. MATERIALS AND METHODS: Nine anaesthetized female pigs ( approximately 70 kg) had a single-tract PCNL procedure in the left kidney and then the right kidney in one session (sbPCNL). Percutaneous access was achieved by a 30 F balloon dilator system. Bilateral renal function was measured before, 1.5 and 4.5 h after sbPCNL and included glomerular filtration rate (GFR), effective renal plasma flow (RPF), renal extraction of para-aminohippurate (EPAH, a measure of the efficiency of tubular organic anion transport), urine flow (UV), absolute sodium excretion (UNaV) and fractional sodium excretion (FENa). RESULTS: Both kidneys had similar baseline haemodynamic and excretory function, and showed comparable changes after sbPCNL. Bilateral GFR and RPF decreased by approximately 35% at 1.5 and 4.5 h after sbPCNL; EPAH was reduced to a similar degree in both kidneys at 1.5 h after sbPCNL and remained depressed throughout the observation period; bilateral UV and UNaV progressively decreased by approximately 30% and approximately 60% at 1.5 and 4.5 h after sbPCNL, respectively; bilateral FENa did not significantly change at 1.5 h after sbPCNL but decreased significantly by approximately 50% at 4.5 h. CONCLUSIONS: Both kidneys responded in a similar fashion after sbPCNL, with declines in haemodynamic and excretory function. These bilateral functional responses were comparable to those previously reported after unilateral PCNL, and help to reduce concerns that PCNL of both kidneys in one session could lead to greater functional complications, at least acutely.

Determination of para-aminohippuric acid (PAH) in human plasma and urine by liquid chromatography-tandem mass spectrometry.

In this manuscript, we present a simple and reliable method for the quantitation of para-aminohippuric acid (PAH; 2-(4-aminobenzamido)acetic acid) in human plasma and urine using liquid chromatography coupled to electrospray ionisation low-energy collision-induced dissociation tandem mass spectrometry (HPLC-ESI-CID-MS/MS) analysis (negative ion mode) via multiple reaction monitoring (MRM). Sample preparation involved protein precipitation of plasma samples with acetonitrile and subsequent dilution of urine samples with the mobile phase. The internal standard (IS) adopted was para-aminosalicylic acid (PAS; 4-amino-2-hydroxybenzoic acid). Chromatographic separation was achieved on a Cosmosil HILIC column using an isocratic mobile phase consisting of ammonium acetate buffer (20mM) and acetonitrile (45:55, v/v) at a flow rate of 200microl/min. The transactions monitored were m/z 192.9-->149.1 for PAH and m/z 152.1-->108.1 for IS. Linear calibration curves were generated over the PAH concentration range of 0.2-100mg/L in human plasma and urine. The method was validated for selectivity, accuracy, precision, recovery and stability according to USFDA criteria, and has been successfully applied to a pharmacokinetic study in healthy volunteers administered an intravenous dose of 440mg PAH.

Effect of CP-690,550, an orally active janus kinase inhibitor, on renal function in healthy adult volunteers.

CP-690,550 is a Janus kinase inhibitor being developed to prevent allograft rejection and treat several autoimmune diseases. This study examines the effect of multiple doses of CP-690,550 on renal function in healthy volunteers. Thirty-four volunteers are randomized in a 2:1 ratio in a double-blinded manner to receive CP-690,550 15 mg twice daily or placebo twice daily for 14 days. Volunteers are confined in-house to receive a controlled regimen of water intake and sodium intake of 4 to 5 g/d. The effect of CP-690,550 on glomerular filtration rate (GFR) is measured by iohexol serum clearance, effective renal plasma flow (ERPF) by para-aminohippuric acid (PAH) urinary clearance, and creatinine clearance by 24-hour urine collection on day 1 (predose) and day 15. Steady-state pharmacokinetics and tolerability are assessed. Comparing the day 15 and day 1 (predose) values shows that geometric mean ratios for iohexol serum clearance, PAH urinary clearance, and creatinine clearance are 0.995, 0.925, and 0.948, respectively. When adjusted for the corresponding placebo day ratios, the geometric mean ratios are 1.09, 0.978, and 1.05, respectively. CP-690,550 is well tolerated. These findings indicate that CP-690,550 does not affect GFR, ERPF, or creatinine clearance in healthy volunteers.

On-line automatic SPE-CE coupling for the determination of biological markers in urine.

Automatic SPE has been coupled on-line to CE by a transfer tube and the replenishment system of the CE instrument. The approach allows the target analytes (viz. creatinine, creatine, xanthine, hypoxanthine, uric acid, p-aminohippuric acid and ascorbic acid in urine samples) to be removed from the sample matrix, cleaned up, preconcentrated and injected into the capillary. The detection limits range between 0.14 and 4.50 microg/mL, the quantification limits between 0.45 and 15.0 microg/mL, and linear dynamic ranges - which include the reference healthy human values - from the quantification limits to 1332 microg/mL. The precision, expressed as RSD, ranges between 0.38 and 2.22% for repeatability and between 1.79 and 7.61% for within-laboratory reproducibility. The errors, expressed as RSD for all compounds, range between 0.20 and 6.90%. The time for automatic SPE and that necessary for the individual separation-detection of the target analytes are 13 and 12 min, respectively; the analysis frequency is 5 h(-1). The accuracy of the method and potential matrix effects were studied by using spiked samples and recoveries between 96.00 and 103.07 % were obtained. The proposed method was applied to samples from healthy young students.

Effects of brimonidine ingestion on cardiovascular responses and renal function in conscious dogs.

The effects of brimonidine, an alpha(2)-adrenoceptor agonist, on blood pressure, heart rate, respiratory rate, renal function and some blood parameters were investigated in 10 dogs. Dogs were divided into two groups, low dose (LD; 0.2 mg/kg) and high dose (HD; 0.5 mg/kg) of brimonidine given orally. The alpha(2)-adrenergic antagonist yohimbine hydrochloride was injected to dogs at a dose of 0.1 mg/kg in both groups at the fifth hour after brimonidine administration. The results demonstrated that after administration of brimonidine, mean arterial blood pressure decreased dramatically at 2 h by 23% and 20% in LD and HD groups, respectively. Heart rate was decreased in a similar manner and both remained low at 5 h after brimonidine administration. Respiratory rate was decreased by 50%, while the electrocardiogram showed prolongation of the PR interval. Glomerular filtration rate (GFR) and effective renal blood flow were reduced when measured at 4 h after brimonidine ingestion in both groups, but the effect was more pronounced in the LD group. Brimonidine caused natriuresis and kaliuresis in both LD and HD groups. The packed cell volume was decreased and hyperglycaemia was detected. Most of the effects can be reversed completely after administration of yohimbine. However, yohimbine can restore GFR only partially. These data suggest that brimonidine caused cardiovascular and respiratory depression. The adverse effects of this drug can be antagonized by yohimbine, suggesting that these effects were mediated via the alpha(2)-adrenoceptor.

Simultaneous determination of p-aminobenzoic acid and its metabolites in the urine of volunteers, treated with p-aminobenzoic acid sunscreen formulation.

p-Aminobenzoic acid (PABA) and its metabolites (p-aminohippuric acid, p-acetamidobenzoic acid, and p-acetamidohippuric acid) were detected using high-performance liquid chromatography with an electrochemical (carbon paste) detector (HPLC-ECD). For direct current (dc) mode, with the current at a constant potential, and measurements with suitable experimental parameters, a linear concentration from 0.125 to 1.80 microg/ml was found. The detection limit was approximately 2.0 ng/ml. A carbon paste coulometric detector was used to demonstrate that PABA and its metabolites are electrochemically oxidized in acidic media, and to determine, by analyzing human urine, the percutaneous absorption of PABA and its metabolites. Findings using HPLC-ECD and HPLC with an ultraviolet detector (HPLC-UV) were comparable.

Reducing shock number dramatically decreases lesion size in a juvenile kidney model.

BACKGROUND AND PURPOSE: Adult stone patients are treated with several thousand lithotripter shockwaves (SWs) in order to pulverize a kidney stone. This typical clinical dose assures that the stone will be fractured completely. However, this same dose induces damage to the kidney, especially pediatric-size kidneys. If increasing SW number is known to increase renal injury and functional impairment, will reducing SW number below typical treatment levels significantly decrease kidney damage and hemodynamic changes? MATERIALS AND METHODS: To address this question, one kidney in each of nine juvenile pigs (6-7 weeks old) was treated with 1000 SWs at 24 kV directed at a lower-pole calix with an unmodified HM-3 lithotripter. Parenchymal-lesion size was determined by sectioning the entire kidney and quantitating the amount of hemorrhage in each slice. Renal function was determined before and after SW treatment by inulin clearance, paraaminohippurate (PAH) extraction, and PAH clearance. The resulting morphologic and functional changes were then compared with those of kidneys that had been treated with a typical clinical dose of 2000 SWs (data previously published; J Am Soc Nephrol 2000;11:310). Eleven pigs were utilized as sham-treated controls. RESULTS: Limiting SW number to 1000 significantly reduced the size of the lesion (by 95%) and reduced the degree of functional change (glomerular filtration rate by 38%, PAH extraction by 73%, renal plasma flow by 46%) compared with kidneys receiving 2000 SWs (an adult dose). CONCLUSIONS: These data support the idea that SW number should be reduced to the lowest number that fractures kidney stones in order to minimize renal injury and functional impairment.

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.

The course of diabetic glomerulopathy in patients with type I diabetes: a 6-year follow-up with serial biopsies.

Diabetic nephropathy is a severe complication and few studies have described the early morphological changes over time. Two kidney biopsies were performed, within a 6-year interval, in 29 primarily normoalbuminuric patients, aged 24 years at the second biopsy. These were examined with light and electron microscopy. Glomerular filtration rate, and effective renal plasma flow were determined with inulin and para-aminohippurate clearances. Urinary albumin excretion rate and the 24 ambulatory blood pressure were determined. Ten patients had developed microalbuminuria and/or hypertension; of these, six were treated with antihypertensive medication for 2 years or more. Significant increases were found in night time diastolic blood pressure and decreases in systolic and diastolic dipping. The glomerular volume, mesangial volume, mesangial matrix volume fraction and foot process width increased significantly. The group that was treated later for complications had the worst long-term metabolic control, thicker basement membranes and larger mesangial matrix and volume at the first biopsy, than the persistent normoalbuminuric group. During the follow-up, the untreated group with complications and the persistent normoalbuminuric group showed an increase in morphological parameters, whereas no progression occurred in the treated patients who also improved their metabolic control. In conclusion, the morphological parameters deteriorated in the normoalbuminuric patients and in those with complications, but were unchanged in the small antihypertensive-treated group with improved metabolic control.

Simple HPLC-UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis.

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C(18) column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 microg/ml for iohexol and iothalamate, 5 to 40 microg/ml for PAH and 2.5 to 40 microg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.

Altered renal elimination of organic anions in rats with chronic renal failure.

The progress of chronic renal failure (CRF) is characterized by the development of glomerular and tubular lesions. However, little is known about the expression of organic anions renal transporters. The objective of this work was to study, in rats with experimental CRF (5/6 nephrectomy), the expression of the organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) and their contribution to the pharmacokinetics and renal excretion of p-aminohippurate (PAH). Two groups of animals were used: Sham and CRF. Six months after surgery, systolic blood pressure and plasma creatinine concentrations were significantly higher in CRF groups. CRF rats showed a diminution in: the filtered, secreted and excreted load of PAH; the systemic clearance of PAH; the renal OAT1 expression; and the renal Na-K-ATPase activity. No remarkable modifications were observed in the OAT3 expression from CRF kidneys. The diminution in the systemic depuration and renal excretion of PAH may be explained by the decrease in its filtered and secreted load. The lower OAT1 expression in remnant renal mass of CRF rats or/and the lower activity of Na-K-ATPase might justify, at least in part, the diminished secreted load of this organic anion.

Determination of uric acid and p-aminohippuric acid in human saliva and urine using capillary electrophoresis with electrochemical detection: potential application in fast diagnosis of renal disease.

The monitoring of uric acid (UA) and p-aminohippuric acid (PAH) levels in biological samples is routinely carried out in clinical laboratories as an indication of renal disease. With the aim of investigation of the correlation between the trace amounts of UA and PAH in human saliva or urine and renal diseases, we carried out the determination of UA and PAH in human saliva and urine by using capillary electrophoresis with electrochemical detection (CE-ED) in this work. Under the optimum conditions, UA, PAH and three coexisting analytes could be well separated within 21 min at the separation voltage of 14 kV in 80 mmol/L borax running buffer (pH 9.2). Good linear relationship was established between peak current and concentration of analytes over two orders of magnitude with detection limits (S/N = 3) ranged from 5.01 x 10(-7) to 2.00 x 10(-6) mol/L for all analytes. The result shows that this proposed method could be successfully applied for the study on the correlation between the levels of UA and PAH in human saliva and urine and renal diseases, and provide an alternative and convenient method for the fast diagnosis of renal disease.

Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter.

p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.