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Studies during the past decade have led to the recognition of a fundamental, widely expressed mechanism of structural damage in energy-deprived cells, which is suppressed by physiologic levels of glycine and is independent of Ca2+ availability or alterations of cytosolic free Ca2+. To gain insight into this process, Madin-Darby canine kidney (MDCK) cells were depleted of adenosine triphosphate (ATP) by a mitochondrial uncoupler in glucose-free medium, and intracellular free Ca2+ was clamped at 100 nM to avoid calcium cytotoxicity. Although the ATP-depleted cells swelled and blebbed and their plasma membranes appeared to be under tension, they nevertheless became permeable to macromolecules. The plasma membranes of these cells retained structural continuity, as determined by morphologic observations, and confocal microscopy of a plasma membrane protein label (Biotin: Ultra Avidin-Texas Red) and a lipid label (NBD-sphingomyelin). Using fluoresceinated dextrans of graded molecular size, membrane permselectivity was examined noninvasively by confocal microscopy. Measured as inside/outside ratios of fluorescence intensity, the permeability indices showed progressively greater restriction to diffusion of increasingly larger dextran molecules across plasma membranes, with sharp break-points between 70,000 and 145,000 daltons (d). The results indicated that the membranes behaved as if they were perforated by water-filled channels or "pores," with size-exclusion limits of molecular dimensions. The membrane defects evolved from small pores permeable only to propidium iodide (668 d) and the smallest dextran (4,000 d), before enlarging with time to become permeable to larger dextrans. Inclusion of glycine during ATP depletion did not affect cell swelling or blebbing but completely prevented the development of permeability defects. Treatment of cells before ATP depletion with a membrane-impermeant homobifunctional "nearest neighbor" cross-linking agent, 3,3' dithiobis(sulfosuccinimidylpropionate), suppressed the development of permeability defects, even in the absence of glycine. These observations suggest that the cellular abnormality that is suppressed by glycine involves rearrangement of plasma membrane proteins to form water-filled pores large enough to leak macromolecules.

Treatment of two cultured renal tubule epithelial cell lines, MDCK and LLC-PK1, with ionomycin produced rapidly evolving models of lethal cell injury characterized by increases of cytosolic free calcium to the microM level within 15 minutes followed by lactate dehydrogenase release and failure to exclude vital dyes that began between 30 and 60 minutes and became extensive after 60 minutes. The pattern of injury was similar when the mitochondrial uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, was added to ionomycin. Carbonyl cyanide-m-chlorophenylhydrazone alone produced severe ATP depletion but not lactate dehydrogenase release. Inclusion of glycine in the experimental medium at concentrations ranging from 0.25 mM to 5 mM did not affect the increases of cytosolic free calcium or ATP depletion but was protective against enzyme release and failure to exclude vital dyes for 180 minutes. Maximal protection was achieved at glycine concentrations between 1 and 5 mM. Several other small neutral amino acids including alanine, beta-alanine, L-serine, 1-aminocyclopropane-1-carboxylic acid, and alpha-aminoisobutyric acid also had protective effects but, glucose, pyruvate, glutamate, glutamine, leucine, valine, and taurine did not. These data indicate that potent protective effects of glycine and other small neutral amino acids previously shown in fresh tubule preparations are fully expressed in cultured tubule cells of diverse origin when appropriate acute injury models are used and the protective effects are sustained for long durations. The suitability of cultured cell lines for prolonged exposure studies will provide a powerful way of further exploring mechanisms of these effects.

Synthetic polycations bind to glomerular polyanions (GPA) and increase permeability to macromolecules and immune complexes. Platelet factor 4 and other platelet cationic proteins also bind to GPA and may play a role in immune complex deposition. Here we examine the potential of locally released cationic proteins to bind to GPA after immune complex-induced platelet activation within the renal microvasculature. Rabbits were immunized against bovine serum albumin (BSA), and BSA (2 or 4 mg/ml in buffered saline) was infused into the left renal artery to deliver 8 or 16 mg of BSA over 20 minutes. Thirty minutes later, kidneys were removed and tissue processed for light, immunofluorescence, and electron microscopy for the assessment of glomerular alterations and the localization of immune complexes (IgG and BSA), platelet factor 4, and platelet cationic proteins. GPA was measured by quantitative ultrastructural assessment of polyethyleneimine binding sites. Glomerular capillaries contained large intraluminal immune complexes and platelet aggregates. Also, numerous deposits were observed within subendothelial and subepithelial aspects of the glomerular basement membrane (GBM). Immunofluorescence and immunocytochemistry revealed prominent localization of platelet factor 4, platelet cationic proteins, IgG and BSA within peripheral capillary walls of glomeruli concomitant with a reduction in GPA. Glomeruli of controls or contralateral kidneys did not show GBM localization of immune complexes or platelet proteins. Thus, nascent formation of immune complexes in capillaries was associated with platelet activation and deposition of endogenous cationic proteins in the GBM. This mechanism may be involved in neutralization of GPA and mediation of increased permeability, which leads to GBM deposition of immune complexes.

The aminonucleoside of puromycin induces proteinuria and renal damage when given to rats. Aminonucleoside of puromycin was administered to male Wistar-Furth rats as a single intravenous injection in a dose of 15 mg. per 100 gm. of body weight. The animals were studied 9 days later when the mean urinary protein was 175 mg. per 24 hours. Evidence of glomerular epithelial cell injury included massive obliteration of foot processes, appearance of microvilli, protein reabsorption droplets, extreme attenuation of cytoplasm with formation of blebs, and focal detachment of epithelial cells from glomerular basement membrane. An increase in both the amount of mesangial matrix and the number of mesangial cells was also observed. The fractional clearance (C/GFR) of anionic horseradish peroxidase had increased 18.5 times as compared to control values and was nearly equal to the C/GFR of neutral horseradish peroxidase in the experimental rats. The C/GFR of cationic horseradish peroxidase was decreased by one-third so that it approximated the C/GFRs of both anionic and neutral horseradish peroxidase. These findings indicate a nearly complete loss of the charge-selective barrier to filtration. In addition, C/GFRs of tritiated uncharged dextrans with a range of molecular radii from 18 to 58 Angstrom (A) were determined. The C/GFRs of dextrans (alpha e less than 30 A) were decreased in the experimental rats as compared to C/GFRs of dextrans of corresponding molecular size in control rats. However, the C/GFRs of dextrans (alpha e greater than 38A) were increased in experimental as compared to control rats. Further, both anionic and cationic ferritin (alpha e = 61 A) were observed in the urinary space near denuded areas of glomerular basement membrane. These results indicate that the size-selective properties of the glomerular barrier to filtration have been modified with decreased C/GFR of small molecules and increased C/GFR of large molecules. Thus, the proteinuria of aminonucleoside nephrosis in rats occurs secondary to alterations in both the charge- and size-selective barriers to glomerular filtration.

The objectives of this study were to evaluate the effects of food restriction (without protein or phosphorus restriction) and protein restriction (without the restriction of other nutrients or calories) on the outcome of the remnant kidney model of chronic renal failure in rats. After 5/6 nephrectomy, rats were assigned to one of the following dietary groups: group I (control-ad libitum) consumed a 21% casein diet ad libitum; group II (food restriction with protein restriction) consumed 36% less calories, protein and minerals than group I; group III (food restriction without protein restriction) consumed 36% less calories and minerals than group I, but equivalent amounts of protein; group IV (protein restriction) consumed 38% less protein than group I, but equivalent amounts of calories and minerals; group V (NaCl restriction) consumed 40% less sodium chloride than group I, but equivalent amounts of all other nutrients. All groups consumed equivalent amounts of calcium, phosphorus and vitamins. Groups II and III experienced retardation of growth in comparison to groups I, IV and V. The food-restricted groups II and III, but not groups IV and V, had less proteinuria than group I 20 weeks postablation. By 21 weeks postablation, the kidneys from group I showed severe parenchymal damage, characteristic of end-stage renal pathology. These changes were prevented in the food-restricted groups II and III, but not in groups IV and V. The percentage of glomeruli with severe structural damage was less in groups II (27.3 +/- 8.8) and III (26.9 +/- 7.5) compared with group I (72.4 +/- 7.8). In contrast, the corresponding values in groups IV and V were not significantly different from group I. Interstitial volume (the percentage of tubulointerstitium which is interstitium) which was proportional to the severity of tubular damage was significantly lower in groups II (25.1 +/- 4.5) and III (20.4 +/- 2.8) when compared with groups I (48.1 +/- 3.0), IV (44.4 +/- 6.6), or V (41.9 +/- 4.2). An interstitial volume less than 30 correlated with well preserved renal histology, whereas a value greater than 40 was indicative of end-stage renal pathology. These results indicate that the restriction of carbohydrate, fat, and minerals (except for calcium and phosphorus) retarded growth and prevented the development of end-stage renal pathology in the remnant kidney model of chronic renal failure in rats, regardless of whether protein was restricted or not.(ABSTRACT TRUNCATED AT 400 WORDS)

Left kidneys of rats were made ischemic for 25 minutes and proximal tubule brush border alterations studied in the S1 and S2 segments. Scanning electron microscopy revealed that brush border microvilli became unstable, fused with one another, and were interiorized into proximal tubule cytoplasm soon after reflow of blood following ischemia. Rapid regeneration followed; scanning electron microscopy showed that regeneration occurred in a fashion whereby clusters of microvilli in flower-like configurations were extruded from the cell interior toward the surface. Such unique patterns of microvillus formation have not been reported before. Activity of the brush border enzymes, alkaline phosphatase and maltase, were not significantly depressed throughout the cycle of brush border loss and regeneration. Likewise, there were no alterations in the activity of beta-glucuronidase, a lysosomal enzyme. Alkaline phosphatase cytochemistry showed that microvillus membranes that were interiorized into the cell cytoplasm retained enzyme activity on their surfaces during the early period of brush border loss as well as during regeneration. These results strongly suggest that in reversibly injured proximal tubule cells regeneration of the brush border occurs primarily by a process of recycling of damaged, previously incorporated membrane. The nature of the initial membrane damage and the mechanism of recycling remain unknown.

Perfusion of rat kidneys with polycations (protamine sulfate, poly-L-lysine), resulted in glomerular epithelial alterations very similar to those observed in proteinuric states, particularly rat aminonucleoside nephrosis. Such changes did not occur after exposure to neutral or anionic macromolecules (poly-DL-alanine, myoglobin, heparin, poly-L-glutamic acid and ovalbumin). Morphigenetic factors in the polycation-induced lesion included retraction and flattening of foot processes, narrowing of filtration slits, formation of occluding junctions between foot processes and cell swelling. The associated suppression of histochemically demonstrable glomerular polyanion suggested that neutralization of cell surface anionic sites was an important factor in the causation of the lesion, which was reversible by reperfusion with heparin. Observations by freeze-fracture confirmed the similarity of the polycation-induced lesion to the epithelial changes in rat aminonucleoside nephrosis. Following exposure to polycations there was also "staining" of anionic sites on epithelial and endothelial cell membranes and glomerular basement membrane. Reperfusion of protamine-treated kidneys with heparin resulted in restoration of previously suppressed colloidal iron staining and the formation of spherical, electron-dense (heparin-protamine) complexes within the glomerular filter.