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Previous studies have shown that histochemical modifications of the endoplasmic reticulum in epithelial cells might be related to their transport function. We have examined the effect of sodium maleate, which produces generalized transport derangement reminiscent of Fanconi syndrome, on the organization, morphology and enzyme activities of endoplasmic reticulum in rat kidney cells. The osmium impregnation technique has revealed that apical vacuoles increase in volume and in number in most proximal tubule cells, and contain osmium deposits. Osmium impregnation of the endoplasmic reticulum is much reduced. In vitro studies, performed with isolated microsomes, show NADPH cytochrome c reductase activity in both normal and maleate-treated rats. As revealed by vanadate, Ca+-ATPase activity in isolated microsomes is unnaffected by maleate but the vanadate-insensitive or passive component of calcium uptake increases particularly later in the response. Therefore, the remaining calcium uptake in the presence of vanadate is indeed passive; in vivo maleate administration also appears to increase the passive entry of calcium into the microsomal compartment. The morphological and histochemical alterations of the endoplasmic reticulum cisternae occur rapidly and with a similar time course to the transport defects, suggesting that this organelle plays a role in transcellular transport. Maleate may directly affect the endoplasmic reticulum membranes whereby passive permeability to calcium is increased. The endocytotic apparatus and possibly exocytosis phenomena are modified by maleate as shown by the increased vacuolization and the presence of black osmium deposits in vacuoles.

The effects of two widely used paramagnetic shift reagents for cationic NMR, dysprosium tripolyphosphate [Dy(PPP)2(7-)] and dysprosium triethylenetetramine hexaacetate [Dy(TTHA)3-], on the cell structure of dog and human erythrocytes, dog kidney cortical tubules and rat hepatocytes were investigated. The effect of shift reagents on cell integrity was monitored by measuring the hematocrit values for erythrocytes, by measuring the lactate dehydrogenase (LDH) release and by electron microscopy for cortical tubules and hepatocytes. The quantitation of the dyprosium penetration inside cells was accomplished by atomic absorption, atomic emission and neutron activation. More severe effects were observed with Dy(PPP)2(7-) than with Dy(TTHA)3-, and were dependent on the divalent cation concentration and on the shift reagent concentration. Very serious cell damage was observed after 60 min incubation in the presence of 10 mumol Dy(PPP)2(7-)/mL suspension at low or high divalent cation concentration. The situation was improved at 5 mumol Dy(PPP)2(7-)/mL suspension especially at high divalent cation concentration (2.5 mM). Incubation with Dy3+, PPP5- or Dy(TTHA)3- caused little or no structural effects but dysprosium was found to penetrate slowly inside tubules with Dy(TTHA)3-. Both Dy3+ and Dy(PPP)2(7-) penetrated rapidly inside cells. Dysprosium was found to bind to the isolated cytosol but not to isolated membranes, eliminating the possibility of extracellular membrane binding.

The pattern of transport of penicillin G and carbenicillin was examined directly in the rat kidney by means of micropuncture studies. Samples of plasma, tubular fluid, and urine were assayed for antibiotic content by an agar diffusion technique. Secretion accounted for 67% of the penicillin G but for only 37% of the carbenicillin present in the proximal tubule. No further net secretion of either agent could be detected in the distal nephron. Net secretion of penicillin G decreased from 67% in the distal tubule to 60% in the urine (P less than 0.05%); this reduction correlated with reabsorption of water from the collecting ducts. Both penicillin G and carbenicillin were secreted by the proximal tubule of the rat nephron, but the latter was secreted at a lower rate than the former. A significant fraction of penicillin G was reabsorbed from the collecting ducts under conditions of maximal antidiuresis.

The ultrastructure of collecting duct epithelia was studied with the osmium impregnation technique in renal cortical explants grown in culture in the form of globular bodies. When this technique was applied to 7-day-old globular bodies, the endoplasmic reticulum (ER) of the superficial layer cells was faintly impregnated in the presence or absence of arginine-vasopressin (AVP) in the incubation medium; the ER of the cells located in the core of the globular bodies was densely impregnated with osmium. When these globular bodies were sectioned in 2 fragments and one was incubated in AVP for 30 min while the other was used as a control, a marked increase in osmium impregnation occurred: osmium black deposits were then noted in the lumen of the endoplasmic reticulum of two-thirds of the cells in the superficial layer. Various patterns of impregnation were observed. Cryptlike formations gave rise to mature epithelial cells showing the same pattern of osmium impregnation. When cyclic adenosine monophosphate (cAMP) was substituted for AVP in the incubation medium, the treated globular bodies revealed the same ultrastructural characteristics. Our data suggest that this primary culture of collecting duct epithelia is made up of heterogeneous cells with characteristics of principal and intercalated cells and that the AVP has a stimulatory effect on ER maturation, which is mediated by the adenylate cyclase system.

Multiple factors may modify the pharmacokinetics of aminoglycosides and increase their nephrotoxic potential. In the present study, the influence of Escherichia coli endotoxin on the renal handling of [3H]tobramycin was investigated. The accumulation of [3H]tobramycin in proximal tubules, distal tubules, and collecting ducts was compared in both normal and endotoxin-injected (0.25 mg/kg) rats. Histological observations were also made. Blood pressure and cardiac frequency were recorded, and renal function was evaluated with labeled inulin and p-aminohippuric acid. Following administration of endotoxin, disturbed intrarenal localization of the drug was noted. Grain counts were affected in both proximal and distal tubules. Increased labeling was observed at all time intervals in the proximal tubules. In the distal tubules of endotoxemic animals we could detect higher amounts of drug at 10 and 60 min in the medulla and at 10 min in the cortex. Not all of the tubules were labeled to the same extent. No histological lesion was noted on light microscopy in animals receiving either normal saline or endotoxin. The dose of endotoxin used resulted in very fine physiological disturbance. Both blood pressure and cardiac frequency were minimally affected by endotoxin. Decreases in glomerular filtration rate and renal plasma flow were observed. However, none of these changes was statistically significant. The present study has shown that low doses of endotoxin alter the renal handling of aminoglycosides in the absence of any major physiological disturbance or histological changes. By increasing the total amount of drug within the kidney, endotoxin might increase the nephrotoxic potential of aminoglycosides.

Multiple factors may modify the pharmacokinetics of aminoglycosides and affect their nephrotoxic potential. In the present study, the influence of Escherichia coli pyelonephritis on the renal handling of [3H]tobramycin was investigated. The accumulation of [3H]tobramycin in proximal and distal tubules in both normal and infected rats was compared. Following induction of pyelonephritis, disturbed intrarenal localization of the drug was noted. Grain counts were affected in both proximal and distal tubules. Decreased labeling was observed at all time intervals in the proximal tubules. Electron microscopy showed that radioactivity was associated mostly with lysosomes in both normal and infected rats 1 and 24 h following the injection of the drug. We could detect significantly higher amounts of drug in the distal tubules of the pyelonephritic kidney than the normal levels at 10 min and 24 h postinjection. The drug did not seem to be associated with any particular organelle and was evenly distributed within the distal tubular cells. The present study shows that the transport of tobramycin within the infected nephron is disturbed. These data might shed some light on the influence of infection on the intrarenal pharmacology of aminoglycosides.

Renal tubules of rats were incubated with aminoglycoside (gentamicin or 3H-tobramycin, 10 mg/l) in the presence or absence of Escherichia coli endotoxin (10 mg/l). The kinetics of aminoglycoside uptake by the tubule were only slightly affected by endotoxin. The percentage of serum in the medium affected the tobramycin uptake. This uptake decreased from a mean ratio of concentration in the tubules/concentration in the medium (T/M) of 1.63 in 5% serum to a mean T/M of 0.86 in 10% serum (P less than 0.01).

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3-0.6 micron) of kidney tissue were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80-100 kV. Studies were performed on rat foetuses from 18-21 days of gestation, on newborns, and on 2-20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.

The injection of sodium maleate (200-400 mg/kg) into rats produces aminoaciduria along with glycosuria and phosphaturia, resembling the Fanconi syndrome. This experimental model was studied by means of microinjections into proximal convoluted tubules of the kidney, stop-flow diuresis, and microperfusion of single nephrons. Our results show that, in maleate-treated rats, competition between amino acids or related structures (L-proline, L-OH-proline, and glycine) possesses the same characteristics, and net influx of amino acids appear normal at the proximal nephron. Data obtained by classical stop-flow techniques and single nephron microperfusions also indicate a normal entry of labeled amino acids (L-lysine, glycine, L-valine, L-proline, L-cystine), and 3-0-methyl-D-[3H]glucose and [32P]phosphate from the luminal side of the proximal tubule cell. However, the efflux of molecules from the cell appears enhanced throughout the proximal and distal tubule; molecules that exit at this site are excreted directly into the urine. Our results suggest that the phosphaturia, aminoaciduria, and glycosuria of the experimental Fanconi syndrome can be explained by a modification of the cell membrane permeability (increased efflux) at distal sites of the nephron rather than by a modification of the membrane transport (decreased influx) at the proximal sites, as is currently accepted. Our data also stress the importance of efflux phenomena in membrane transport.

A new histological technique of block staining based on the use of a double lead and copper citrate is described in order to observe thick sections (0.5-1.5 micron) with a transmission electron microscope. With this technique, a network of mitochondrial with many morphological communications was seen in the epithelial cells of the proximal and distal convoluted tubules of the rat nephron. A new schematic view of the proximal and distal tubular cell is presented to illustrate the communications of the chondriome, which could become more or less accentuated following the functional status of the cell.