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Abstract. 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)×10^-3, n=10 versus (8.7±3.7)×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)×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)×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.

Children have been considered a risk group for lead (Pb) toxicity, mainly because of neurophysiological or neuro-cognitive deficits following Pb exposure. Blood Pb levels (b-Pb) of 100 microg/l currently have been defined as the lowest adverse effect level. The aim of this study was to compare, with the help of urinary markers, the kidney function of children with b-Pb just above this threshold with that of unexposed children, to assess from a nephrological point of view whether the current threshold is justified and whether children really are a particularly vulnerable risk group in terms of Pb-induced kidney damage.

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 histochemi-cal 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.mmHg.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 (μ) was significantly increased in ADR-treated animals, showing no O2-dependence (μ × 10-2: controls (7) 1.3 ± 0.2; ADR 20% O2 (8): 8.1 ± 9.6; ADR 80% O2 (7): 6.9 ± 6.7). ω 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.

The anticancer drug adriamycin (ADR) induces severe nonselective glomerular proteinuria in rats. The proliferation of the glomerular mesangium in chronic ADR nephrosis suggests a disturbed metabolism of extracellular matrix proteins. In the present study the structural protein fibronectin was analyzed by immunological methods in urine and plasma in two rat strains after a single injection of 5 mg of ADR/kg b.wt. After ADR administration the excretion of 220 kd-plasma fibronectin, identified by its electrophoretic mobility and Western blotting, increased. This was confirmed by quantitative measurement (ELISA) of total urinary fibronectin (ADR-treated Munich Wistar Frömter rats: 85.4 +/- 49.5 ng/hr, N = 31; controls: 1.7 +/- 0.7 ng/hr, N = 26; day 7 after ADR injection). Control urines contained distinct fragments of fibronectin with a molecular weight range of 40 to 60 kd. It is suggested that these fragments did not originate from plasma inasmuch as only little correspondence between the fibronectin patterns of urine and plasma was found, and also because in ADR-treated rats the excretion of the fibronectin fragments decreased from day 4 of ADR administration. The data suggest that the urinary fibronectin fragments are released from cellular fibronectin during the normal catabolism of the glomerular extracellular matrices, and that ADR interferes in the metabolism of matrix components. Only as a consequence of an enhanced glomerular permeability is plasma fibronectin excreted in ADR-treated rats.

Vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF) is a selective mitogen for endothelial cells; it increases microvascular permeability and has been shown to relax isolated canine coronary arteries by an endothelium-dependent mechanism. In many tissues VEGF/VPF is expressed after an appropriate stimulus, mostly hypoxia. In the kidney VEGF/VPF is constitutively expressed in glomerular podocytes and epithelia of collecting duct. Glomerular and peritubular capillary endothelia also constitutively express specific VEGF receptors. The in vivo function of renal VEGF/VPF is unknown.

The fine structure of the kidney (glomerulus and archinephric duct) of the hagfish, Myxine glutinosa (L.) was studied in thin sections and by freeze-fracture technique.

The separation of sex dependent urinary proteins of the rat (SDP) by micro-disc electrophoresis results in at least eight well defined protein bands with differing molecular weights. The hepatic origin of a sex dependent urinary protein, named a2u-globulin, has been demonstrated before by other authors applying immunological methods. In the present study, it could been shown that SDP circulate in the plasma at a concentration of 23.8 mg/l. The origin of those protein bands which appear typically upon electrophoresis was still under dispute because they could not been demonstrated in proximal tubular fluid. The present study confirms the extrarenal source of SDP and suggests identity with a2u-globulin. The attempt to track down SDP from plasma to excreted urine demonstrated that, in contrast to proximal fluid, samples from nephron parts distal to the loop of Henle contain large amounts of SDP. An isolated kidney model was used to determine the sieving coefficient and tubular reabsorption of SDP, obtained from male rat urine. We have found a correlation between the sieving coefficient and the molecular weight of SDP. The sieving coefficient ranged from 0.375 to 0.834. The tubular reabsorption which has been determined with an isolated kidney perfused with albumin and erythrocytes also showed variation with regard to molecular weight and was 61.7%, on average.

A variety of sex-dependent urinary proteins of low molecular weight, absent in females and in castrated males, can be identified in male rats by disc electrophoresis. In the urine of male rats of age 5·5 months, albumin comprises only 1-2% of the total protein. Albumin excretion increases greatly with age and associated kidney disease. Total protein excretion, however, stays the same or even decreases slightly as the rat ages, due to a loss of low molecular weight, sex-dependent, proteins. These are virtually absent in senescent rats (38 months of age), although total protein excretion rises tenfold in these animals due to high molecular weight plasma proteins passing into the urine; the glomerular filtration rate decreases to 70% of the value measured at 5·5 months of age.

To elucidate the primary mechanism of high glucose cytotoxicity, the cytoprotective properties of antioxidants against metabolical disorders were assessed in human mesangial cell (HMC) cultures. An 8-day incubation of HMC with high glucose concentration (30 mM) resulted in an extracellular accumulation of the matrixprotein fibronectin (FN), owing to both an expansion of the matrix-associated pericellular FN and a 60% increase of the soluble molecule in the culture medium. The high glucose-induced FN alterations were not due to osmotical effects, as assessed by an iso-osmotic mannitol control. Rather, they are mediated by oxygen-free radicals because the combined treatment of HMC with high glucose and either the antioxidative flavonoid silibinin (given as the water soluble derivative silibinin-C-2,3-dihydrogensuccinate disodium salt) or a radical scavenger cocktail totally prevented the extracellular FN accumulation. This is corroborated further by the determination of malondialdehyde, a product of lipid peroxidation. Incubation of HMC with high glucose resulted in an increase of malondialdehyde in cell homogenates which was completely counteracted by either silibinin or a radical scavenger cocktail. Silibinin alone had no effects on protein synthesis and culture growth. The data presented are compatible with oxidative stress induced by high glucose concentration in HMC cultures. The study further substantiates the proposed role of silibinin in the amelioration of glucose cytotoxicity in renal cells.

Cisplatin is one of the most active cytotoxic agents in the treatment of testicular cancer, but its clinical use is associated with side-effects such as ototoxicity, neurotoxicity and nephrotoxicity. Long-term kidney damage from cisplatin particularly affects the proximal tubular apparatus and can be detected by increased urinary excretion of brush-border enzymes, such as L-alanine-aminopeptidase (AAP), and magnesium. In the current study, the flavonoid silibinin was used as a nephroprotectant for cisplatin-induced nephropathy in a rat animal model. Infusion of silibinin before cisplatin results in a significant decrease in glomerular (indicated by creatinine clearance and serum urea level) and tubular kidney toxicity (excretion of brush-border enzymes and magnesium). Silibinin given alone had no effect on renal function. In order to exclude an inhibition of the anti-tumour activity of cisplatin and 4-hydroperoxy-ifosfamide by co-administration of silibinin, in vitro studies were performed in three established human testicular cancer cell lines. Dose-response curves for cisplatin (3-30 000 nmol) combined with non-toxic silibinin doses (7.25 x 10(-6) or 7.25 x 10(-5) mol l-1) did not deviate significantly from those of cisplatin alone as measured by relative cell survival during a 5 day assay using the sulphorhodamine-B staining technique. Also silibinin did not influence the cytotoxic activity of 4-hydroperoxy-ifosfamide (30-10 000 nmol) in vitro. In summary, these in vitro data rule out a significant inhibition of the anti-tumour activity of the major nephrotoxic components, cisplatin and 4-hydroperoxy-ifosfamide, by co-administration of silibinin in a human germ cell tumour cell line model. Together with these demonstrated cytoprotection effects in the rat animal model, these data form the basis for a randomised clinical trial of silibinin for the protection of cisplatin-associated nephrotoxicity in patients with testicular cancer.

Summary In order to estimate the degree of tubular lesions and to detect a possible glomerular involvement, proteinuria was studied in tubular and/or interstitial nephropathies by determination of total protein and beta-2 microglobulin and by micro-gradientgel electrophoresis. The excretion of albumin, low molecular weight proteins (LMW) and high molecular weight (HMW) proteins was measured in addition. Other proximal tubulus functions such as glucose and phosphate reabsorption were also measured. The clinical entities studied were: chronic postinfectious nephritis, patients with nephropathy following analgesic abuse, tubular glucosuria in pregnancy and cis-platinum treated patients suffering from testicular tumor. The results were interpreted basing on the “selectivity” or urinary protein patterns, which means the relation between LMW and HMW proteins. Micro-gradientgel electrophoresis proved to be a fast and sensitive tool for quantitative as well as qualitative analysis of albumin, LMW and HMW proteins.

Human mesangial cell (HMC) cultures synthesize cellular fibronectin (FN), which is secreted and incorporated into a fibrillar extracellular matrix (ECM). The anticancer drug adriamycin (ADR) induces changes in extracellular FN deposition. As revealed by immunofluorescence staining, a 24 h incubation of the cells with 2 µg ADR/ml resulted in a marked expansion of the pericellular FN fibers, which may be due to either an increased synthesis or a decreased FN degradation. The effects of ADR on FN mRNA were analysed by northern hybridization andin vitro translation. Steady-state FN mRNA levels were significantly increased by 60% following ADR administration. However, yields of radioactivity incorporated into FN by cell-free translation remained constant (2.3±0.7%,n=24, vs controls 2.2±0.8% of total radioactivity,n=23). The quality of translation products was not affected by the drug, whereas translation efficiency of total RNA from ADR-treated HMC was only 75% of controls. The data presented suggest a negative feedback control of FN expression on the level of translation. Extracellular FN accumulation in the experimental model of ADR-induced progressive glomerulopathy therefore cannot be explained by an increased FN synthesis, but is rather regarded a consequence of proteinase inhibition. This assumption is compatible with a diminished number of FN fragments recently demonstrated in the culture medium of ADR-treated HMC, and is further corroborated by the loss of urinary FN degradation products accompanying the onset of proteinuria in ADR-treated rats.

Micropuncture experiments were performed on proximal tubules and collecting ducts of rat kidneys with and without artificial perfusion of surrounding capillaries or vasa recta respectively. Net sodium flux (?Na iso) was estimated under conditions of varying but equal sodium concentrations on both sides of the tubular wall. The transtubular wall equilibrium concentration difference of sodium (?cNa) was also measured in these nephron segments under conditions of zero volume and solute fluxes.

Summary: From earlier microperfusion studies ofD-glucose and water reabsorption in the proximal surface nephron of the rat,D-glucose was found to be removed by a saturable carrier and by an apparent coupling with net fluid reabsorption. Equations appropriate to describe this system were developed. They incorporated carrier-mediatedD-glucose transport, net water transport, and water-coupled solute transport. Water reabsorption was assumed to be constant either per unit surface area, or per unit volume of the nephron, and the rate of carrier-transportedD-glucose was assumed constant per unit length, per unit surface area, or per unit volume of the tubule. The possibility thatD-glucose could be reabsorbed via two carrier systems was also explored analytically. It was observed from this treatment that the fraction ofD-glucose reabsorbed would change if the perfusion rate was changed. With an increase in perfusion rate, a decrease in reabsorbed fraction was seen which indicates that if net fluid reabsorption is proportional to volume, carrier-mediated sugar transport is proportional to surface area or length of the tubule. From these relationshipsJ max, the maximal rate of carrier-transported sugar, was calculated to be 3.3×10−10M/cm2 sec, a value comparable to that reported from other laboratories. The results of this analysis are compatible with the data obtained both by micropuncture experiments during free flow and by glucose clearance studies until theT mG is reached. The possibility that theT mG obtained in clearance studies is due to a decrease in the fraction of fluid reabsorbed in the proximal tubule or to a second saturable carrier is discussed. It is observed that, in either case, if load is increased by increasing the glomerular filtration rate, noT mG would be reached, or stated another way, one would predict from the analysis thatT mG would be proportional to glomerular filtration rate.

Summary: Hexose reabsorption in the kidney was investigated by microperfusion of proximal cortical rat nephrons with Ringer's solution containingD-glucose (2.5, 5.0 and 18.0 mM/l), 3-O-methylD-glucose (4.5 mM/l) orD-fructose (18.0 mM/l) at perfusion rates of 20 or 40×10−6 ml/min. Inulin was used as a reference for fluid reabsorption.

Int the proximal and distal surface tubules isotonic Na reabsorption (F Naout) is impaired by adrenalectomy. In the present experiments the time course of development of transtubular Na concentration differences (Na)P-(Na)TF was studied in the proximal convolution.1.Na concentration was measured in free flow mannitol diuresis. Endproximal (Na)P-(Na)TF averaged 23.0±0.5 meq/l after 8.5±0.6 sec (controls) and 15.2±1.7 meq/l after 13.6±0.5 sec (adrenalectomized rats).2.In modified stopped flow experiments tubular fluid, aspirated at the beginning of the proximal convolution in mannitol diuresis, was injected between two oil columnes and reaspirated after 28–36 sec of contact time. (Na)P-(Na)TF was 30.1±1.4 meq/l and 24.7±2.5 meq/l in the controls and adrenalectomized rats respectively.3.(Na)P-(Na)TF in steady state (i. e. after 60 sec of contact time) were 33.6±0.8 meq/l and 41.8±1.2 meq/l in the 2 groups. From the results we derive at the conclusion that in adrenal insufficiency proximal sodium reabsorption is not only affected by an impairment of the intrinsic transport capacity but also by a proportional decrease of leak permeability for sodium.4.Due to an increased transit time in adrenal insufficiency fractional reabsorption of sodium and of potassium was unchanged in the proximal convolution. Therefore it seems likely that the same compensation mechanism as for sodium holds for potassium and the reabsorption of potassium is impaired in adrenal insufficiency also in the proximal convolution.

In order to analyse the mechanism of inhibition of water diuresis in adrenal insufficiency renal surface tubulus of male albino rats (group I: controls, group II: 8 days or more after bilateral adrenalectomy) have been perfused using the free flow microperfusion technique.

Using the split oil droplet method the effects of adrenal cortical steroids on impaired renal Na transport have been tested in adrenalectomized white rats.

In adrenalectomized rats rate of net transfer of sodium and fluid has been measured using the split oil droplet method. Net reabsorption of sodium was found to be decreased from 8.9 to 4.5×10-5 µEq/mm2 × sec in the proximal convolution and from 1.7 to 1.2×10-5 µEq/mm2 × sec in the distal convolution of surface tubules. This indicates impairment of the local capacity of the epithelium of both tubular segments to transport sodium in adrenal insufficiency. A second pertinent finding was the decreased transit time of Lissamingreen-stained tubular fluid through the proximal convolution in adrenalectomized animals. In contrast to the inhibition of local fluid and sodium reabsorption fractional reabsorption in the proximal convolution in free flow as estimated from transit time and half time of local fluid reabsorption was found to be increased following adrenalectomy. The possible mechanisms of impairment of sodium transport will be discussed.

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