A rapid analytical method for the detection of plasma volume expanders and mannitol based on the urinary saccharides and polyalcohols profile.

A screening procedure specifically developed for the detection of saccharides and polyalcohols in human urine in the framework of doping control analysis is presented. The proposed method, set-up, and validated to detect the abuse of dextran, hydroxyethyl starch and mannitol as a doping practice in sport, involves only one enzymatic hydrolysis step and the direct injection into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The chromatographic conditions were optimized to allow the efficient separation of compounds with the same molecular weight. Good linearity (R(2) 0.990-0.995) and reproducibility of relative retention times (CV% lower than 1) and of relative abundances of characteristic ion transitions (CV% lower than 10) were obtained. The lower limits of detection and quantification were in the range of 30-100 µg/ml. Since the analytes studied are present also in non-doping products (e.g. in fruit as well as in food products and drugs additives), the developed method was also used to establish a range of reference urinary concentrations: 600 doping control samples and 30 samples from volunteers not using any medication were considered. While the hydrolysis products (isomaltose and maltose hydroxyl-ethylated), used as specific markers for the detection of dextran and hydroxyethyl starch abuse, were not detected in urine; mannitol was present in all urines in a concentration range of 30-1200 µg/ml. Since no criteria of positivity for mannitol has been established yet, the results obtained in this study could be considered, in combination with those of previous researches, as a starting point to fix a threshold value for doping control purpose.

Specific screening method for dextran and hydroxyethyl starch in human urine by size exclusion chromatography-in-source collision-induced dissociation-time-of-flight mass spectrometry.

The use of plasma volume expanders (PVE), such as dextran (DEX) and hydroxyethyl starch (HES), is prohibited in sports. DEX is a naturally occurring glucose polymer, whereas HES is synthetically produced from amylopectin starch by substitution with hydroxyethyl groups. In doping control, the commonly applied enzymatic and colorimetric screening methods are lacking adequate specificity for DEX and HES. Also, gas chromatographic-mass spectrometic (GC-MS) screening methods have specificity issues with DEX. In addition, due to the nature of the target compounds, time-consuming derivatisation steps are required in GC-MS. Based on the high molecular weight of carbohydrate polymers excreted in urine after administration of DEX and HES, a screening method was developed involving size exclusion chromatography (SEC) combined with time-of-flight mass spectrometry (TOFMS). By using solely a SEC guard column as an analytical column allowed sufficient chromatographic resolution in a minimal amount of time and with reasonable repeatability (average RSD of 10%). Detector response was linear throughout the measurement range with R(2) > 0.99 for both analytes. Limits of detection were 100 and 250 µg mL(-1) for DEX and HES, respectively. Ion suppression was found to be 52% at maximum. In-source collision-induced dissociation (ISCID) was used to produce characteristic fragments at a mass accuracy better than 2 mDa. The specificity of the SEC-ISCID-TOFMS method was demonstrated with 120 PVE negative doping control samples analyzed in parallel with a routine GC-MS screening method. In addition, seven urine samples from diabetic athletes, causing interpretation problems in routine GC-MS, showed here a definitely negative profile.

Can focused US with a diagnostic US contrast agent favorably affect renal function?

Focused ultrasonography (US) with simultaneous administration of a US microbubble contrast agent was used to transiently increase the glomerular filtration rate while altering the sieving properties of glomeruli in normal rabbits. In its current form, this process has very limited application potential to states of abnormal renal function.

Renal ultrafiltration changes induced by focused US.

PURPOSE: To determine if focused ultrasonography (US) combined with a diagnostic microbubble-based US contrast agent can be used to modulate glomerular ultrafiltration and size selectivity. MATERIALS AND METHODS: The experiments were approved by the animal care committee. The left kidney of 17 healthy rabbits was sonicated by using a 260-kHz focused US transducer in the presence of a microbubble-based US contrast agent. The right kidney served as the control. Three acoustic power levels were applied: 0.4 W (six rabbits), 0.9 W (six rabbits), and 1.7 W (five rabbits). Three rabbits were not treated with focused US and served as control animals. The authors evaluated changes in glomerular size selectivity by measuring the clearance rates of 3000- and 70,000-Da fluorescence-neutral dextrans. The creatinine clearance was calculated for estimation of the glomerular filtration rate. The urinary protein-creatinine ratio was monitored during the experiments. The authors assessed tubular function by evaluating the fractional sodium excretion, tubular reabsorption of phosphate, and gamma-glutamyltransferase-creatinine ratio. Whole-kidney histologic analysis was performed. For each measurement, the values obtained before and after sonication were compared by using the paired t test. RESULTS: Significant (P < .05) increases in the relative (ratio of treated kidney value/nontreated kidney value) clearance of small- and large-molecule agents and the urine flow rates that resulted from the focused US treatments were observed. Overall, 1.23-, 1.23-, 1.61-, and 1.47-fold enhancement of creatinine clearance, 3000-Da dextran clearance, 70 000-Da dextran clearance, and urine flow rate, respectively, were observed. Focal tubular hemorrhage and transient functional tubular alterations were observed at only the highest (1.7-W) acoustic power level tested. CONCLUSION: Glomerular ultrafiltration and size selectivity can be temporarily modified with simultaneous application of US and microbubbles. This method could offer new opportunities for treatment of renal disease.

Disease-dependent mechanisms of albuminuria.

The mechanism of albuminuria is perhaps one of the most complex yet important questions in renal physiology today. Recent studies have directly demonstrated that the normal glomerulus filters substantial amounts of albumin and that charge selectivity plays little or no role in preventing this process. This filtered albumin is then processed by proximal tubular cells by two distinct pathways; dysfunction in either one of these pathways gives rise to discrete forms of albuminuria. Most of the filtered albumin is returned to the peritubular blood supply by a retrieval pathway. Albuminuria in the nephrotic range would arise from retrieval pathway dysfunction. The small quantities of filtered albumin that are not retrieved undergo obligatory lysosomal degradation before urinary excretion as small peptide fragments. This degradation pathway is sensitive to metabolic factors responsible for hypertrophy and fibrosis, particularly molecules such as angiotensin II and transforming growth factor-beta1, whose production is stimulated by hyperglycemic and hypertensive environments. Dysfunction in this degradation pathway leads to albuminuria below the nephrotic range. These new insights into albumin filtration and processing argue for a reassessment of the role of podocytes and the slit diaphragm as major direct determinants governing albuminuria, provide information on how glomerular morphology and "tubular" albuminuria may be interrelated, and offer a new rationale for drug development.

Rapid screening of polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography-tandem mass spectrometry.

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.

Absorption characteristics of model compounds from the small intestinal serosal surface and a comparison with other organ surfaces.

We examined the absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FD-4, MW 4400; FD-10, MW 9500; FD-40, MW 40 500) as model compounds through the small intestinal serosal surface. After application to the rat small intestinal serosal surface using a cylindrical diffusion cell, each compound was absorbed at different rates. The absorption ratios in 6 h after PSP, FD-4, FD-10 and FD-40 application were calculated to be 89.2, 34.6, 14.9 and 2.1% of dose, respectively. Elimination profiles of PSP, FD-4 and FD-10 from the small intestinal serosal surface obeyed first-order kinetics. Moreover, we calculated the apparent permeability coefficient P(app) for comparison to other organ surfaces. The kidney had the highest absorption efficiency, as shown by having more than 1.5 times significantly higher P(app) values of PSP, FD-4 and FD-10. Similar to the other organ surfaces, a correlation was observed between the P(app) of the small intestine and the molecular weight of these hydrophilic compounds. In addition, the small intestine is likely to contribute largely to hydrophilic compound absorption from the peritoneal cavity, judging from absorption clearance, CL(a), calculated using the peritoneal organ surface area.

Identification and quantification of the plasma volume expander dextran in human urine by liquid chromatography-tandem mass spectrometry of enzymatically derived isomaltose.

Plasma volume expanders are used in sports in order to control haematological parameters and/or to mask erythropoietin (EPO) misuse. A reliable method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for doping control purposes, enabling the identification and quantification of the plasma volume expander dextran in human urine. The dextran polymer was enzymatically hydrolysed by alpha-1,6-glucosidase (dextranase) followed by acetylation of the generated isomaltose subunits, allowing the chromatographic separation of different disaccharides, such as lactose, saccharose and isomaltose, as well as the identification and quantification of the analyte in human urine. The method was used to determine the basal concentration of isomaltose resulting from the enzymatic hydrolysis of polymeric 1,6-linked glucose in 238 routine doping control samples. In addition the concentration of dextran measured as isomaltose was estimated in seven urine specimens obtained from patients treated with dextran. Calibration curves for dextran were linear and reproducible. The inter- and intra-assay coefficients of variation for dextran ranged from 4.9 to 7.3% at three concentration levels between 53 and 1186 microg/mL. Recovery ranged from 97 to 112% (mean 106.9%). The assay limit of detection was 3.8 microg/mL and the lower limit of quantification was 12.5 microg/mL. In 96% of the investigated doping control samples, the concentrations of isomaltose were below the LLOQ of 12.5 microg/mL. Even the highest concentrations were approximately 100-300-fold lower than concentrations found in urine samples of patients after intravenous application of dextran. The presented results demonstrate the capability and reliability of the developed LC-MS/MS method for the identification and quantification of dextran in human urine and can be regarded as a method revealing the misuse of dextran in sports.

ACE inhibition improves glomerular size selectivity in patients with idiopathic membranous nephropathy and persistent nephrotic syndrome.

Patients with idiopathic membranous nephropathy (IMN) and persistent nephrotic-range proteinuria are at risk for progression to end-stage renal failure. Whether angiotensin-converting enzyme (ACE) inhibitors are also renoprotective in these patients remains elusive. In 14 patients with IMN (patients) and persistent proteinuria (protein > 3 g/24 h for >6 months), we studied mean arterial pressure (MAP), urinary protein excretion, glomerular filtration rate (GFR), renal plasma flow (RPF), and albumin and neutral dextran fractional clearance after 2 months washout from previous antihypertensive treatment (basal), after 2 months of enalapril (2.5 to 20 mg/d) therapy (posttreatment), and 2 months after enalapril withdrawal (recovery). MAP, proteinuria, and GFR were also measured at the same time points in 6 patients with IMN and persistent overt proteinuria maintained on conventional treatment throughout the study period (controls). Basal MAP, proteinuria, and GFR were similar in the two study groups. However, in patients at the end of the treatment period, MAP (posttreatment, 99.6 +/- 11.2 versus basal, 103.3 +/- 12.1 mm Hg; P < 0.05), proteinuria (posttreatment protein, 5.0 +/- 2.9 versus basal, 7.1 +/- 4.9 g/24 h; P < 0.05), albumin fractional clearance (posttreatment median, 1.7 x 10(-3); range, 0.2 to 22.7 x 10(-3) versus basal median, 4.1 x 10(-3); range, 0.4 to 22. 1 x 10(-3); P < 0.05), and fractional clearance of largest neutral dextrans (radii from 62 to 66 A) were significantly less than basal values. At recovery, MAP significantly increased to 106.6 +/- 11.7 mm Hg (P < 0.001 versus enalapril), but all other parameters remained less than basal values. GFR and RPF were similar at each evaluation. Changes in proteinuria after treatment withdrawal positively correlated (r = 0.72; P < 0.01) with baseline GFR. Theoretical analysis of dextran-sieving data indicated that ACE inhibitor treatment significantly improved glomerular membrane size-selective dysfunction. This effect persisted more than 2 months after treatment withdrawal. No patient had symptomatic hypotension, acute renal function deterioration, or hyperkalemia during enalapril treatment. Thus, in patients with IMN and long-term nephrotic syndrome, ACE inhibitor treatment, but not conventional therapy, improves glomerular barrier size selectivity. The antiproteinuric effect of ACE inhibition is long lasting, especially in patients with more severe renal insufficiency. This is the premise of a long-term renoprotective effect that may limit the need for treatment with more toxic drugs.

A comparison of analytic procedures for measurement of fractional dextran clearances.

Fractional dextran clearances have been extensively used to study glomerular size selectivity. We report on an analysis of different laboratory procedures involved in measuring fractional dextran clearances. The deproteinization of plasma samples by 20% trichloroacetic acid (TCA) revealed a protein contamination of 0.2% +/- 0.3%, whereas both 5% TCA and zinc sulfate deproteinization revealed a significantly higher remaining sample protein content (2.5% +/- 0.4% and 3.4% +/- 0.1%, respectively). Only zinc sulfate revealed incomplete deproteinization of urine samples (0.6% +/- 0.2%). Dextran recovery in plasma and urine supernatants was significantly lower after 5% TCA and zinc sulfate deproteinization when compared with 20% TCA deproteinization. Gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) showed a variance of calibration smaller than 5% over 1 year. The use of 3 different sets of standard dextrans revealed significant differences in calibration. GPC and HPLC followed by anthrone assay showed a comparable variance in dextran concentration in plasma, from 3 to 6 nm (14% to 25%), whereas the variance in urine was lower for the GPC and anthrone assay, especially from 5.4 to 6 nm (23% to 43% versus 50% to 78%). HPLC and online refractometry showed the lowest variance of dextran concentration in plasma, from 3 to 6 nm (<4%), and in urine, from 3 to 5.2 nm (<7%), whereas it showed a higher variance in urine, from 5.4 to 6 nm, in comparison with GPC and HPLC with the anthrone assay. The GPC and anthrone assay revealed higher fractional dextran clearances in comparison with the HPLC and anthrone assay in healthy subjects (3 to 5.4 nm) as well as in patients with nondiabetic proteinuria (4.2 to 5.8 nm), and lower clearances in patients from 3 to 3.4 nm. The HPLC and anthrone assay revealed higher clearances in comparison with HPLC and online refractometry in healthy subjects (3.6 to 5.4 nm) and in patients (3.6 to 5.2 nm). The GPC and anthrone assay revealed characteristic differences in fractional dextran clearances between healthy subjects and patients. The HPLC and anthrone assay showed no significant differences between both groups, whereas HPLC and online refractometry showed only an increased clearance of dextrans from 4.6 to 5.2 nm in patients. Fractional clearances of dextran 5.6 nm as estimated by all 3 dextran assays were not significantly related to the fractional immunoglobulin G clearance or the immunoglobulin-to-albumin clearance index in our patients. Quantitative and qualitative differences in fractional dextran clearances may be induced by differences in laboratory procedures. We recommend sample preparation by 20% TCA deproteinization, frequent calibration with 1 set of dextran standards with low polydispersity, size-exclusion chromatography by GPC, and dextran detection by anthrone assay for optimal measurement of fractional dextran clearances. Even with such an approach, however, the variability in the measurement remains extremely high in the important range of dextrans greater than 5 nm.

Acute renal effects of AT1-receptor blockade after exogenous angiotensin II infusion in healthy subjects.

In 10 healthy normotensive volunteers on a normal sodium diet, we evaluated the renal effects of a single oral dose of 50 mg of irbesartan (SR 47436, BMS 186295), an angiotensin II AT1-receptor antagonist, in baseline conditions and during an exogenous angiotensin II infusion (2.5 ng/kg/min). We used a double-blind, placebo-controlled, crossover design. Hormones, blood pressure, renal hemodynamics, and urinary electrolytes were measured during each phase. To examine further the determinants of glomerular filtration at the microcirculation level, fractional clearance of neutral dextran was performed, and sieving curves were applied on a hydrodynamic model of ultrafiltration. Irbesartan administration was followed by an increase in active renin and plasma angiotensin II concentrations and renal plasma flow without change of systemic blood pressure, glomerular filtration rate, or plasma aldosterone concentration. Irbesartan did not affect either sieving curves or glomerular ultrafiltration determinants. Angiotensin II infusion at 2.5 ng/kg/min elicited a slight pressor response accompanied by a decrease in glomerular filtration rate and renal plasma flow and an enhancement of fractional dextran clearance over the radius range explored (3.4-5.4 nm). The transcapillary glomerular pressure gradient deltaP and the ultrafiltration coefficient kf were computed to increase by 9% and to decrease by 23%, respectively, without change in intrinsic membrane properties. Pretreatment with irbesartan prevented all these effects of angiotensin II.

Molecular weight dependent tissue accumulation of dextrans: in vivo studies in rats.

The effects of molecular weight (M(r)) on the serum and urine pharmacokinetics and tissue distribution of dextrans, potential macromolecular carriers for drug delivery, were studied in rats. A single 5 mg/kg dose of fluorescein-labeled dextrans (FDs) with a M(r) of 4000 (FD-4), 20,000 (FD-20), 70,000 (FD-70), or 150,000 (FD-150) was administered into the tail vein of separate groups of rats. At different times after the administration of each FD, animals were sacrificed, and blood, urine, and various tissues were obtained. The concentrations of FDs in the samples were subsequently determined by using a sensitive and specific high performance size exclusions chromatographic method. Among the tissues studied, high accumulation of dextrans was found only in the liver (liver:serum AUC ratios < or = 29) and spleen (spleen:serum AUC ratios < or = 10), with high concentrations in these tissues persisting even at the last sampling time (96 h). In contrast, the serum concentrations of the studied FDs were not measurable beyond 12 h. The serum and urine kinetics and the liver, spleen, and kidney accumulation of FDs demonstrated a significant degree of M(r) dependency. The total and renal clearance of FDs consistently decreased with an increase in M(r). However, the effects of M(r) on the tissue accumulation of dextrans was tissue dependent. For the liver, the tissue:serum AUC ratios increased from 0.346 for FD-4 to 15.2 for FD-20 and 28.8 for FD-70, while a further increase in M(r) to 150 kDa (FD-150) resulted in lowering the ratio to 8.59 in this tissue. For the spleen, the ratios increased from 0.095 for FD-4 to 9.56 for FD-150.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytical gel filtration of dextran for the study of the glomerular barrier function.

Analytical gel filtration was used for the study of molecular size distribution of clinical dextran in serum and urine for the purpose of evaluations of changes in the human glomerular barrier function. The column was calibrated in terms of solute size using a simple and accurate technique recently described. Only one sample of a dextran possessing a broad molecular mass distribution was necessary for the calibration procedure and the calculations were performed using an ordinary spreadsheet. The accuracy of the calibration, as evaluated by protein samples, is better than 95%. The simplicity makes the method suitable for use in laboratories not normally specializing in analytical gel filtration. Calibration in terms of size is preferably done with respect to viscosity radius to obtain relevant information about the permeability of dextran into porous membranes.

Disposition characteristics of model macromolecules in the perfused rat kidney.

The disposition characteristics of model macromolecules such as dextran (70 kDa), bovine serum albumin (BSA), and their charged derivatives were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery patterns and tissue accumulation of radiolabeled compounds were evaluated under filtering or nonfiltering conditions. In the filtering kidney, cationic macromolecules such as diethylaminoethyl-dextran (DEAE-dex) and cationized BSA (cBSA) accumulated in the kidney to a great extent whereas anionic and neutral macromolecules such as BSA, carboxymethyl-dextran (CM-dex), and dextran showed only small uptake. DEAE-dex and cBSA were distributed to both the medulla and cortex regions of the kidney and their recoveries in the kidney decreased as the injected dose increased. Similar tissue uptake was observed in the nonfiltering kidney perfusion system suggesting that they were mainly taken up by the kidney from the renal capillary side based on electrostatic interaction. In addition, the steady-state distribution volumes of cationic macromolecules calculated from venous outflow patterns were larger than those of the intravascular volume estimated from the distribution volumes of neutral and anionic macromolecules, suggesting their reversible interaction with the vascular wall. On the other hand, dextran derivatives with molecular weight distribution were excreted into urine based on glomerular permselectivity; i.e., cationic DEAE-dex and anionic CM-dex showed enhanced and restricted urinary excretion, respectively, compared with neutral dextran. In contrast, no significant excretion was observed for BSA and cBSA. The utility of the isolated rat kidney perfusion experiment for studying the renal disposition of macromolecular drugs was thus demonstrated.

Extent and course of glomerular injury in human membranous glomerulopathy.

Glomerular permselectivity and dynamics were evaluated serially in 14 nephrotic patients with membranous glomerulopathy (MG). Analysis of transglomerular dextran sieving, before and again after proteinuria remitted, revealed persistent depression by 60-80% of glomerular pore density and the two-kidney ultrafiltration coefficient, Kf. The glomerular filtration rate was lowered by half on each occasion. Morphometric examination of glomeruli in a second group of 16 nephrotic patients with MG revealed a low prevalence of glomerulosclerosis (5 +/- 3%) and a twofold increase in filtration surface due to marked glomerular hypertrophy. Presumably, widening by threefold of the basement membrane and/or epithelial podocytes accounted for the computed reduction in ultrafiltration capacity. There was no correlation between glomerular structure and the subsequent course of MG over the ensuing 24-96 mo. Rather, a twofold expansion of the interstitial compartment predicted those who went on to exhibit progressive renal insufficiency. We conclude that increasing resistance to water flow by walls of patent and perfused glomerular capillaries is the proximate cause of progressive renal insufficiency in MG.

Increasing the plasma half-life of trichosanthin by coupling to dextran.

Trichosanthin (TCS) is a plant protein which has a wide spectrum of pharmacological activities. It was demonstrated recently that this compound suppressed the replication of human immunodeficiency virus (HIV-1) in vitro. The mechanism of action is believed to be inhibition of protein synthesis. Trichosanthin is a low molecular weight protein which is expected to be easily filtered and eliminated through the kidney. To minimize renal loss, the molecular size of trichosanthin can be increased by coupling to dextran. The larger complex will not undergo glomerular filtration and therefore renal loss can be prevented. This study investigates the kidney's role in trichosanthin elimination and the beneficial effect afforded by coupling to dextran in prolonging plasma half-life. For this purpose, a radioimmunoassay has been developed to determine the concentration of TCS in plasma and urine. The sensitivity of this assay is in the nanogram range. Trichosanthin was coupled to dextran T40 by a dialdehyde method and successful coupling was confirmed by gel filtration chromatography. The complex retained specific binding to trichosanthin antibodies with decreased affinity which can be partially reversed after incubation with dextranase; an enzyme that digested dextran. The pharmacokinetics of intravenously administered trichosanthin (0.75 mg/kg) was compared between two groups of rats with normal and impaired renal function (bilateral renal arterial ligation). Rats with ligation showed a decrease in plasma clearance from 4780 +/- 570 to 220 +/- 20 microL/min and an increase in the mean residence time from 9 +/- 1 to 145 +/- 16 min. Despite the several-fold difference in these parameters, recovery of trichosanthin from normal rat urine was only 0.38 +/- 0.05%. This value can be increased by using higher injection doses. The data indicate that the kidney is an important organ for the elimination of trichosanthin. When the dextran-trichosanthin complex was injected into normal rats trichosanthin activity was not detected in the urine. All the pharmacokinetic parameters suggest that the dextran-trichosanthin complex stayed longer in the body and maintained a much higher plasma concentration than trichosanthin.

Effect of dextran sulfate on renal accumulation of gentamicin.

The effect of dextran sulfate of three molecular weights (1000, 5000, and 90,000) on the accumulation of gentamicin in rat kidney was investigated using a continuous infusion technique. During the infusions of both gentamicin and gentamicin-dextran sulfate mixtures, the gentamicin plasma concentration was maintained at 10 microgram/ml. The renal cortical accumulation of gentamicin was significantly lower when dextran sulfate (1000, 5000) was coadministered. The inhibition of cortical gentamicin accumulation increased with increasing dextran sulfate dose, and it was proportional to the amount of dextran sulfate excreted into the urine. Analysis by electrophoresis on cellulose acetate membrane indicated that gentamicin binds to dextran sulfate in rat urine. Therefore, gentamicin-dextran sulfate binding within the lumen of the proximal tubules may reduce the renal reabsorption and possibly the renal toxicity of gentamicin.

Significance of urinary complement components in various glomerular diseases.

The concentrations of two components of the complement system (C1q and C3) were measured in the urine and blood in 10 normal subjects and 134 patients with primary and secondary glomerulonephritis by using a highly sensitive enzyme immunoassay. The values of urinary excretion of C1q and C3 were well correlated to the ratios of fractional clearance of these complement proteins to that of neutral dextran of 55 A, which was used to minimize the influences of glomerular sieving because of their comparable molecular size to these complement components. The rate of renal tubular reabsorption of C1q and C3 were at least 89.2 and 93.4% of filtrated C1q and C3, respectively. Urinary C1q and C3 were excreted significantly in cases of membranoproliferative glomerulonephritis (MPGN), membranous glomerulonephritis, IgA nephropathy with both mesangial and capillary immune complex (IC) deposit and also in case of active lupus nephritis. On the other hand, the concentrations of these complement components were low in case of minimal lesion nephrotic syndrome, mild proliferative glomerulonephritis, inactive lupus nephritis and diabetic nephropathy without any immune staining. There was a significant correlation between the urinary excretion of C1q or C3 and intraglomerular IC deposition, especially IC deposition along the glomerular capillary wall. However, the degree of the excretion of these proteins was not correlated to the degree or permselectivity of proteinuria. The correlations between urinary C1q and C3 were observed in cases of IgA nephropathy with both mesangial and capillary deposit and MPGN, although we couldn't see the correlation in the other glomerular diseases. It is suggested that urinary excretion of such complement components represents the fixation of complement by deposited intraglomerular IC. The measurement of urinary concentration of these complement components provides a new clue to investigation or diagnosis of glomerular diseases.