Search

Background: The effect of the increasing thickness of the glomerular basement membrane (GBM), which is seen in ageing rats, on the effective hydraulic conductivity (k) of the glomerular capillary wall was studied in Wistar rats aged 2 and 18 months., Methods: With the use of micropuncture techniques, ultrafiltration characteristics of cortical glomeruli were determined in isolated cell-free perfused kidneys. Because the filtration fraction in this preparation is low (3%) as a consequence of high perfusion rates at glomerular filtration rates comparable with in vivo conditions, uniform ultrafiltration conditions are provided over the whole filtering surface. After fixation at a defined perfusion pressure, the surface of glomerular capillaries (S) was obtained morphometrically on light microscopic sections of the glomeruli studied previously., Results: The glomerular ultrafiltration coefficient (K(f)) was 0.025 nl/s.mmHg in young rats and 0.038 nl/s.mmHg in old rats (P<0.0005) and S was 0.140 mm(2) in young and 0.244 mm(2) in old rats (P<0.0005). However, k was not significantly different (18.0 nl/s.mmHg.cm(2) in young and 15.8 nl/s.mmHg.cm(2) in old rats) despite a 2.4-fold increase of GBM thickness as estimated from electron microscopic sections., Conclusions: These findings indicate that the age-dependent increase of GBM thickness in rat kidneys did not substantially increase hydraulic resistance of the glomerular capillary wall.

The determinants of glomerular capillary wall (GCW) permeability to proteins have been subject of controversial discussion. To study this question we have developed a modified isolated perfused rat kidney model in which tubular transport processes are completely blocked by perfusion fixation with glutaraldehyde. This model allows to directly titrate the charge density of the GCW using albumin solutions buffered over a wide pH-range, a manipulation that cannot be performed in the intact kidney. Analyzing the results of these experiments helped to determine a fixed charge density of the GCW of 43 mEq/L. In the present work, we used the isolated perfused fixed rat kidney model to study the influence of this fixed charge on the transglomerular passage of proteins. To do this, the fixed kidney was perfused with albumin solutions containing different isoforms of horseradish peroxidase. The lowest sieving coefficient was obtained with the acidic isoform (0.035+/-0.008, n = 7), while the isoforms at pI 6.85 and 8.45 showed higher sieving coefficients (0.059+/-0.008, n = 7 and 0.090+/-0.008, n = 4, respectively). The highest sieving coefficient (0.59+/-0.031, n = 6) was observed in perfusion experiments of the fixed kidney with cationic HRP (pI > or = 9.30). However, when comparing the sieving coefficients, the highly cationic isoform was excluded because it has a lower molecular weight than the other isoforms. The sieving coefficients of the other isoforms were significantly different (p < 0.05. ANOVA, Scheffé test). In conclusion, the presence of a discrete (even if lower than previously thought) "fixed" charge on the GCW of 43 mEq/L restricts the transglomerular passage of isoforms of horseradish peroxidase by a factor 2-3. These results imply that the influence of charge selectivity has been overstated in the literature.

The charge-related determinants of albumin permeability are the subject of controversial discussion. To study this question we have developed an isolated perfused rat kidney model in which metabolic processes are eliminated by perfusion fixation with glutaraldehyde. The fixed kidneys were perfused with albumin solutions using the following approaches: 1. Modification of the charge of both the glomerular capillary wall (GCW) and albumin using different buffer systems in a pH range spanning the isoelectric points of albumin and the glomerular basement membrane (GBM), the extracellular matrix of the GCW. 2. Modification of the charge of the GCW by perfusing the isolated kidney with cations either before or after fixation. 3. Modification of the charge of albumin by cationization. In the model, the inulin "urine" to perfusate ratio was one. This shows that the tubules have no metabolic activity, that the glomerular filtration rate (GFR) is equal to "urine" flow rate and that the "urine" collected is identical to the ultrafiltrate. Therefore, sieving coefficients in this model can simply be calculated as the ratio between "urine" and perfusate protein concentrations. We could show that: 1. pH has a significant effect on the albumin sieving coefficient: it was maximally increased at pH 4.0 [(70.3 +/- 15.9) x 10(-3), n = 10 versus (8.7 +/- 3.7) x 10(-3), n = 11, at pH 7.4]. Only a pH as low as 4.0 should lead to a pronounced neutralization of the anionic charges of albumin and the GBM; the charge density of the GCW calculated with these data is 43 mEq/l at pH 7.4. 2. Modifying the ionic composition of the GCW with protamine before fixation with glutaraldehyde causes a bigger increase in the glomerular permeability for albumin [(51.2 +/- 22.5) x 10(-3), n = 10, glomerular charge density 21 mEq/l] than modifying the albumin charge by cationization. 3. Modifying the albumin charge by cationization increases the glomerular permeability for albumin [(20.0 +/- 6.7) x 10(-3), n = 8]. These findings support the hypothesis that at the onset of proteinuria changes in the charge and configuration of the GCW could be more important pathogenetic factors than changes in the charge of serum-derived proteins.

Background: The anticancer drug cisplatin is know to have toxic side-effects on different segments of the nephron. The flavonoid silibinin has previously been shown to be protective in models of hepatotoxicity. The aim of the present study was to evaluate, whether silibinin can also ameliorate alterations in renal glomerular and tubular function and tubular morphology induced by cisplatin., Methods: In a rat model renal damage was induced by a single injection of cisplatin (5 mg/kg body weight). The protective effects of silibinin were studied in rats that received the flavonoid (200 mg/kg body weight, i.v.) 1 h prior to the administration of cisplatin. Kidney function was monitored by analysing urinary markers of glomerular and tubular function over a period of 11 days. Animals of a second group, with identical treatment, were sacrificed 4 days after drug application for an evaluation of tubular morphology at the light-microscopical level., Results: Administration of cisplatin caused a decline in kidney function within a day following treatment. Symptoms observed were for example decreases in creatinine clearance and increases in proteinuria, in the urinary activity of the proximal tubular enzymes alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase and in renal magnesium wasting. The effects of cisplatin on creatinine clearance and proteinuria were totally prevented by a pretreatment of the animals with silibinin. Impairment of proximal tubular function was ameliorated, that is enzymuria and magnesium wasting was less pronounced. Silibinin alone had no effect on kidney function. Treatment with silibinin distinctly diminished morphological alterations observed in the S3-segment of the proximal tubule 4 days after cisplatin administration., Conclusion: The effects of cisplatin on glomerular and proximal tubular function as well as proximal tubular morphology could totally or partly be ameliorated by silibinin. It is concluded the silibinin can act as a nephroprotectant and it is suggested that it could have beneficial effects on the kidney in clinical settings.

This study was designed to validate an alternative in vitro system with isolated glomeruli of the Atlantic hagfish Myxine glutinosa as a model to study alterations in glomerular protein metabolisms in pharmaco-toxicology of anticancer drugs. A morphometric characterization of the glomeruli of Myxine glutinosa reveals a calculated glomerular volume of 180 nl/glomerulus. The glomerular extracellular volume, measured as inulin space, is 38.5 nl/glomerulus. Total glomerular protein content of Myxine glutinosa amounts to 3.56 micrograms/glomerulus and total DNA content to 0.44 microgram/glomerulus. Metabolic properties, estimated as glomerular protein synthesis, are comparable with mammalian glomeruli. The glomeruli of Myxine glutinosa are viable in a tissue culture for up to 12 hr. The incorporation rate of radiolabeled amino acids into glomerular, acid-precipitable proteins is almost identical to that of rats (e.g. Myxine glutinosa 1091 +/- 98 DPM/micrograms DNA vs. rat 1340 +/- 84 DPM/micrograms DNA after 4 hr incubation). To evaluate how nephrotoxic substances affect glomerular metabolism in this model, the anticancer drug Adriamycin (ADR) was used to experimentally induce a glomerular lesion. ADR caused an increase in glomerular protein synthesis in isolated glomeruli of Myxine glutinosa, which is in accordance with data found in rats. Cisplatin, in contrast, known to mainly interfere with tubular integrity, had no effect on glomerular protein synthesis, confirming the specificity of the model. The isolated glomeruli of Myxine glutinosa are suggested as a valid alternative multicellular in vitro system for studying alterations in glomerular metabolism under pharmaco-toxicological conditions and for the evaluation of specific target-cell toxicity of selected nephrotoxins.

Using an in vitro microperfusion technique, the filtration characteristics of single isolated glomeruli of the Atlantic hagfish (Myxine glutinosa) were investigated. The suitability of the method as a model to study glomerular function was evaluated. Experiments with a protein-free perfusate led to a filtration coefficient Kf (0.189 +/- 0.181 nl x s-1 x mm Hg-1) that was in the same order of magnitude as determined in vitro for other vertebrates. Morphometric analysis on serial thin sections (1 microns) revealed a glomerular capillary surface (A) of 1.84 +/- 0.72 mm2. A linear relationship between glomerular diameter and A allowed the calculation of the hydraulic conductivity Lp (0.618 +/- 0.384 microliter x s-1 x mmHg-1 x cm-2). The data indicate that the isolated perfused glomerulus of M. glutinosa has filtration characteristics similar to higher vertebrates. The glomeruli of M. glutinosa, which in contrast to glomeruli of other animals are easy to dissect, are a suitable model for the study of glomerular filtration and permeability characteristics of vertebrates. An example of a possible application of the model is a study into the effects of adrenaline on glomerular filtration characteristics. Adrenaline led to increases in ultrafiltration pressure (PUF), single nephron glomerular filtration rate (SNGFR) and filtration fraction (FF); Kf remained unaffected.

The barrier function of glomerular capillaries in vivo, which prevents the leakage of plasma proteins and cellular elements, depends on the basic morphological and electro-chemical fine structure of the glomerular capillary wall, and on a functional barrier maintained by components obtained from blood, which effect the definitive barrier against the leakage of plasma proteins and cellular elements. The functional component of the barrier may explain the variability and some of the phenomena known as functional proteinuria. A certain size and number of morphological "defects" are thought to represent the normal condition, but under pathological conditions they may increase in size and number, resulting in a shift to an increasing permeability for higher molecular mass proteins; also an increase of the size and number of larger defects may enable more red cells to pass the barrier compared with the normal condition. These defects are different from the minimal glomerular lesions which are due to charge defects in the glomerular capillary membrane, primarily the lamina rara interna and the lamina rara externa of the basement membrane.

The glomerular permeability of the isolated perfused kidney is manifold higher than in vivo, although structural changes at the glomerular filtration barrier are not described. It has been proposed that in vivo high molecular weight plasma proteins are concentrated at the glomerular endothelium forming an additional filtration barrier ('concentration polarization'). To test this hypothesis, isolated rat kidneys were perfused with substrate-enriched Krebs-Henseleit solutions containing albumin (50 g/l) or albumin/globulin (40/10 g/l), or with plasma. The pO2 was 675 mm Hg. Furthermore, experiments were carried out with the addition of erythrocytes with and without a reduced O2 supply (pO2 35 mm Hg). Independent of the composition of the perfusate a continuously increasing glomerular permeability was observed immediately after perfusion was started. After about 10 min, a steady state value for the glomerular permeability was reached. The addition of globulin to the perfusate or perfusion with plasma did not prevent the initial increase of the permeability. However, after addition of 5% erythrocytes to the perfusate the increase of permeability was much less pronounced. Neither the reduction of the O2 supply nor the addition of erythrocytes to a higher concentration than 5% had any further effect on the glomerular permeability. These data show that concentration polarization of high molecular weight proteins does not take place at the endothelium of the glomerulus. Independent of their ability to carry O2 erythrocytes play an important role in the glomerular filtration process.

The effect of L-Arg depletion on glomerular hemodynamics and tubular function of isolated rat kidneys perfused with a medium containing 21 amino acids has been studied. A cyclooxygenase inhibitor was added throughout for blockade of prostaglandin synthesis. Arg depletion caused significant (approximately 30%) reductions in renal perfusion flow rate (PFR, 13.9 +/- 1.2 vs. 19.8 +/- 0.6 ml.min-1.g (kidney wt-1), glomerular filtration rate (GFR, 598 +/- 79 vs. 924 +/- 42 microliters.min-1.g kidney wt-1), and urine flow rate (139 +/- 38 vs. 192 +/- 13 microliters.min-1.g kidney wt-1) compared with control kidneys, which were perfused with a physiological concentration of Arg (200 microM). Filtration fraction (FF) increased with Arg depletion (5.1 +/- 0.4 vs. 4.4 +/- 0.4%). Arg-depleted kidneys had a lower absolute sodium (TNa, 75.7 +/- 8.8 vs. 107.9 +/- 6.0 mumol.min-1.g kidney wt-1) and glucose reabsorption (T glucose, 3.7 +/- 0.6 vs. 5.6 +/- 0.5 mumol.min-1.g kidney wt-1), corresponding to a lower sodium and glucose filtration. Potassium handling and reabsorption of free water were not changed. Oxygen consumption (QO2) was lower in Arg-depleted kidneys (4.6 +/- 0.3 vs. 5.5 +/- 0.5 mumol.min-1.g kidney wt-1). The effects of Arg depletion were completely reversed by the addition of Arg (1 mM) at 120 min and partly reversed by the addition of citrulline (1 mM). Ornithine depletion or addition had no effect on PFR, GFR, FF, TNa, T glucose, and QO2. N omega-methyl-L-arginine, a specific inhibitor of nitric oxide endothelium-derived relaxing factor, produced the same effect as Arg depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal adjustments to diets varying in protein and sodium content were studied in young anesthetized Munich-Wistar rats. Diets were as follows: high (50%) protein (HP), normal (24%) protein (NP), reduced (8%) protein without extra salt (RPNS), and RP with extra salt (RPWS, 2.8% extra salt). Rats grew at the same rate on all diets. Glomerular filtration rate (GFR), urea, sodium, and osmolar clearances were measured. In series 1, groups of rats were maintained for 4 wk on the four diets and one group on changed diet (CP2d, RPWS diet for 4 wk changed to NP diet for 2 days). GFR was reduced compared with NP rats by 12% in RPNS and RPWS, and by 16% in CP2d rats, but the differences were not statistically significant. Fractional excretion of urea (FEurea) was significantly changed compared with NP. It was 40% higher in HP rats, and 50 and 56% lower in RPNS and RPWS, respectively. In the CP2d group it had increased to the NP value. In series 2, groups of rats were maintained on the RPNS diet for 1, 2, 3, and 4 wk, respectively. GFR decreased 21.6% after only 1 wk but after 4 wk it was not significantly lower than in the NP group. In contrast, no significant reduction was found in FEurea after 1 wk, whereas it decreased by 40% during the 2nd wk, with no further decrease after 3 and 4 wk. GFR was not directly related to protein or salt intake.(ABSTRACT TRUNCATED AT 250 WORDS)

The glomerular epithelial cells of the Atlantic hagfish (Myxine glutinosa) lack a slit diaphragm which is replaced by filaments. The epithelial cells show numerous occluding junctions (maculae and fasciae occludentes), septate desmosome-like junctions and desmosomes. The present findings are compared with available data on developing and mature glomerular epithelial cells of mammals.

Glomerular albumin filtration was investigated in the isolated perfused rat kidney and compared with in vivo experiments. Applying micropuncture techniques, we obtained samples from the glomerulus or adjacent early proximal convoluted tubules (EPT) of cortical nephrons and analyzed them for albumin using ultramicrodisc electrophoresis. By determining albumin in glomerular filtrate, we could calculate the sieving coefficient (EPTalb/Palb) directly. The control in vivo value was 0.27 +/- 0.05 X 10(-3) (N = 11). In the isolated perfused rat kidney, the sieving coefficient was 2.1 +/- 0.8 X 10(-3), N = 18 (30 g/liter albumin in perfusate) and 2.3 +/- 0.8 X 10(-3), N = 13 (50 g/liter albumin in perfusate), which is approximately eight times the control in vivo value. With elevated renal venous pressure (20 cm H2O), it increased further to 5.4 +/- 1.6 X 10(-3), N = 8. In all experiments, GFR and proximal transit times were similar to the in vivo controls. Although no major morphologic changes could be detected in any instance, the albumin filtration was greatly elevated. These data confirm the role of a functional barrier in the prevention of glomerular albumin filtration.

Changes in the fine structure of the glomerular filtration barrier were studied by perfusion of isolated rat kidneys with varying doses of protamine: 1) at low doses (110-150 micrograms/ml) ultrastructural changes were only detectable after prolonged recirculation (90-120'), 2) at high doses (250-330 micrograms/ml) profound epithelial foot process lesions were observed even after a short perfusion time, 3) at any concentration protamine particles were distributed in the laminae rarae of the GBM, thus visualizing neutralization of fixed negative charges. Reduction of glomerular anionic sites caused pronounced albuminuria even before podocyte morphological alterations were detectable. The effects of protamine on structure and function of the glomerular capillary wall are dose and time dependent. Besides intact morphology of the epithelial cells regular distribution of negative charges is necessary to maintain glomerular barrier function to macromolecules. Foot process alterations are not the primary reason for increased proteinuria but rather a secondary phenomenon.

The early nephrotoxic effect of the antitumor drug adriamycin (ADR) is suggested to be related to the generation of oxygen free radicals. Therefore the O2-dependence and the influence of free radical scavengers were studied in the model of the isolated perfused single glomerulus of Myxine glutinosa and by histochemical demonstration of the glomerular ATP-ase. In Myxine, the glomerular ATP-ase activity was decreased after injection of ADR (5 mg/kg, i.v.). Both ADR-treated Myxine and controls were exposed for 48 h to an artificial atmosphere of 20% O2/80% N2 or 80% O2/20% N2, respectively. After 10 days a significant decrease of the hydraulic conductivity (k) was measured in the experimental group exposed to 80% O2 (k-values expressed as nl/s.mm Hg.mm2: controls (7): 0.059 +/- 0.017; ADR (7): 0.033 +/- 0.026). The reduction of k following the administration of ADR (20 mg/kg) could be prevented by the sulphydryl donor N-acetylcysteine (NAC). The sieving coefficient for albumin (phi) was significantly increased in ADR-treated animals, showing no O2-dependence (phi x 10(-2): controls (7) 1.3 +/- 0.2; ADR 20% O2 (8): 8.1 +/- 9.6; ADR 80% O2 (7): 6.9 +/- 6.7). phi was not affected by NAC. The lipid peroxide levels in liver, kidney and heart of Myxine increased after the administration of ADR, peaking by day 2 to 5. The circulation disorders of ADR-treated Myxine were not due to an accumulation of the drug in the heart, but rather to a lack of the intracellular antioxidant glutathione. It is concluded that the early nephrotoxic effect of ADR, as reflected by a decreased glomerular ATP-ase activity, is mediated by free radical formation. Oxidative stress on membrane compounds seems to reduce the water permeability of the glomerular barrier, while the ADR-induced sieving defect may be due to oxygen independent pathological mechanisms.

Adriamycin (ADR), selectively toxic to glomerular epithelial cells, was administered (5 mg/kg BW, i.v.) to MWF/Ztm rats to study its early effects on glomerular barrier function with respect to albumin and high molecular weight (HMW) proteins. After 7 days of ADR incubation (glomerular filtration rate remains unchanged), protein excretion was significantly increased in treated rats. The proteinuria was due to a nonselective glomerular lesions resulting in an increase in both, but not a changed ratio of HMW proteins to albumin. However, this ADR-induced proteinuria seen in the final urine was not confirmed by free-flow micropuncture studies of superficial glomeruli. The albumin and HMW protein concentrations in samples taken from Bowman's capsular space of ADR-treated rats did not significantly differ from control samples. These data suggest that cortical nephrons are less sensitive to ADR than juxtamedullary nephrons.

Addition of Alcian Blue to the fixative reveals structural elements bearing negatively charged sites within the glomerular capillary wall of the rat kidney. According to previous research (review by Brenner et al., 1977), these charges represent one of the most important functional components of the glomerular filtration barrier. Alcian Blue also demonstrates negatively charged microfibrils (probably glycoproteins) of the mesangial matrix. Preliminary observations of glomeruli of lower vertebrates suggest that there is always a close correlation between the presence of microfibrils and mesangial cells.

The acute heterologous phase of anti-basement membrane glomerulonephritis in rats was studied morphologically and functionally at the single nephron level. In samples drawn from Bowman's space or from the very early segment of the proximal tubule the protein content was measured by ultra micro-disc-electrophoresis. Morphologically, in rats which were treated with anti-glomerular basement-membrane serum (AGBM) no significant changes could be seen in light microscopy whereas in immunohistology the typical, linear, subendothelial fluorescent staining for IgG could be seen. In controls, small amounts of albumin were filtered (7.57 +/- 0.66 mug/min X 100 g body weight, n = 14) but in nephritic animals the filtered load of albumin increased significantly to 58.33 +/- 4.10 mug/min X 100 g body weight, (n = 8). Besides this increased albumin filtration also several serum globulins could be detected, whereas in controls they were missing. Total GFR and proximal passage time did not change significantly. It is concluded that the glomerulus is an effective barrier for protein under control conditions and that it becomes permeable for albumin and higher molecular weight serum proteins following the antigen antibody reaction at the glomerular basement membrane in the AGBM-treated animals.

Alterations in glomerular permeability were studied in female MWF/Ztm rats, newly selected from the Munich Wistar rats with a high number of superficial glomeruli. This strain is characterized by a diminished number of functioning nephrons, an elevated arterial blood pressure and a high proteinuria averaging 24 mg/24 h. Samples of Bowman capsular space (BCS) obtained by free-flow renal micropuncture techniques with pressure control were analyzed for albumin and high-molecular-weight (HMW) proteins by ultramicrodisc electrophoresis. The measured mean albumin and HMW protein concentration in BCS were 1.72 g/l and 0.66 g/l, respectively. The glomerular filtration rate (GFR) averaged 0.74 ml/min/g kidney weight and the single nephron GFR (SNGFR) 48 nl/min. Ultrastructural studies revealed focal bleb-like alterations of the podocytes as has been observed in hyperfiltration states. We suggest that the reduced number of functioning nephrons in the kidneys of these rats could induce glomerular hyperfiltration and permselectivity changes, which cause a high glomerular protein leakage.

In order to decide whether foot process abnormalities in nephrotic states are the cause or the consequence of proteinuria, the polycation protamine has been applied to isolated perfused rat kidneys and functional parameters and morphology have been compared. With 110 micrograms/ml protamine the albumin clearance ratio increased from 2.0 +/- 0.7 x 10(-3) in controls to 14.8 +/- 6.9 x 10(-3) 10-20 min after application and even to 98.0 +/- 30.8 x 10(-3) after 50-80 min. Micropuncture samples collected from early proximal tubules with pressure control were used to calculate the sieving coefficient which correlated with albumin clearance ratios (controls: 1.8 +/- 1.0 x 10(-3); after 10-20 min: 11.8 +/- 6.4 x 10(-3); after 50-80 min: 52.0 +/- 8.5 x 10(-3). Glomerular filtration rate decreased in the early beginning from 1.1 +/- 0.2 ml x min-1 x g-1 kidney to 0.7 +/- 0.2 ml x min-1 x g-1 kidney, but remained constant throughout the whole experiment. Despite the markedly increased albuminuria there were no changes in glomerular foot process morphology with the protamine dose used.

Perfusion fixation of the rat kidneys with aldehydes and alcian blue demonstrates within the glomerular mesangium various components displaying anionic binding sites. These are the surface coat of the mesangial cells, numerous mesangial microfibrils (probably glycoproteins), and polygonal particles (Presumably proteoglycans). These particles lie close to the mesangial cells and form assemblies of various extension within the mesangial matrix as well. In addition, a discontinuous basement membrane can be recognized which surrounds the mesangial cells. In the hagfish glomeruli (fixed without cationic dye), a prominent layer of microfibrils is interposed between mesangial and endothelial cells. Both these cells show incomplete basement membranes.