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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.

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 micrograms 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 and in 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.

Fibronectin (FN) turnover and turnover changes induced by the anticancer drug Adriamycin (ADR) were measured in human mesangial cells (HMC) in vitro. HMC cultures synthesize cellular FN (2.2 +/- 0.3% of total protein synthesis; n = 12) which is secreted and incorporated into a fibrillar extracellular matrix (ECM). A 24 hr incubation of HMC with ADR (0.5-5 micrograms/ml) resulted in an accumulation of FN in the culture medium, with a maximum increase following 5 micrograms/ml (7.3 +/- 2.3 pg/cell vs. controls: 4.4 +/- 1.9 pg/cell; n = 10). Correspondingly, radioactively labeled immunoprecipitable FN was increased in a dosage-dependent manner in the culture medium up to 50% vs. controls. The incorporation of radioactively labeled FN into ECM was significantly increased following 2 micrograms ADR/ml. In accordance, immunofluorescence staining revealed an expansion of pericellular FN fibers in cultures exposed to 2 micrograms ADR/ml. Concomitant with the accumulation of extracellular FN, radioactively labeled FN in the cells was reduced by 22%. Qualitative characterization of FN patterns revealed a diminished number of degradation products in the culture medium of ADR-treated HMC. These data suggest that ADR interferes with the turnover of FN secreted by HMC in vitro in such a way that FN accumulates extracellularly. This in turn leads to a reduced FN synthesis. These findings are compatible with a loss of urinary FN degradation products accompanying the onset of proteinuria in ADR-treated rats.

The concentration of nine endogenous free L-alpha-amino acids (ALA, LEU, ILE, PHE, TYR, LYS, GLU, PRO, GLY) and of taurine were determined simultaneously along the nephron of the rat kidney using free-flow micropuncture techniques without altering plasma amino acid concentration or kidney function. The amount of each amino acid was determined after dansylation (14C-labelled dansyl-chloride) in the micropuncture sample followed by thinlayer chromatography. The main site of reabsorption is the proximal tubule. After 15-20% of the proximal tubule length the bulk of reabsorption has taken place (18.9 plus or minus 3.4% S.E. of the filtered load remaining). Net reabsorption continues to a small but significant extent along the distal nephron (disal tubule and collecting duct). Reabsorption of taurine is less rapid (% remaining of filtered load at the early proximal tubule 37.0 plus or minus 4.6%). The transtubular concentration ratio of all amino acids except taurine follows a homogeneous course. Under the experimental conditions of this study no distction with respect to different systems of reabsorption "neutral", "basic", "acidic", "imino-glycine") could be made.

Glomerular filtration and tubular uptake of plasma proteins have been studied in the rat using micropuncture techniques. 1. Under control conditions the glomerular capillary wall is an effective barrier, only 7.6 microgram/min x 100 g BW albumin have been measured as filtered load. Four to twelve hours after i.v. injection of anti-glomerular-basement membrane serum (anti-GBM-serum sieving coefficient phi and filtered load increased in a dose-dependent manner (phi albumin in controls = 0.27 x 10(-3), after injection of 0.5 ml Antiserum phi = 0.28 x 10(-3) and 1.0 ml Antiserum phi = 2.32 x 10(-3)). 2. The tubular reabsorption capacity is almost reached under control conditions and amounts to 5.6--10.7 microgram/min x 100 g BW for albumin. Only reduced GFR (0.36 +/- 0.07 ml/min x 100 g BW) and reduced tubular flow lead to increased tubular uptake under overload conditions (10.7 vs. 99.0 microgram albumin/min x 100 g BW). 3. Tubular reabsorption of so-called high-molecular-weight proteins seems to be a nonselective mechanism. The ratio Alb/Alb + Glob (89.9--93.1%) did not differ significantly at the individual puncture sites and in the final urine.

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 alpha 2u-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 alpha 2u-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.