Rat Renal Proximal Tubule Cells Research Articles

Critical subcellular targets of cisplatin and related platinum analogs in rat renal proximal tubule cells

The clinical usefulness of chemotherapeutic agents containing the platinum moiety is often limited by their nephrotoxicity. To investigate the mechanism of nephrotoxicity, and to assess the effects of platinum analogs on specific organelles and basal protein synthesis, biochemical and ultrastructural analyses were performed in rat renal proximal tubule cells (RPTCs). Neutral red (NR) uptake was used to measure lysosomal function, and conversion of MTT to formazan used to assess mitochondrial function. Despite their differential toxicity, cisplatin, carboplatin and CI-973 caused similar progressive inhibition of specific functions, suggesting they may share a common mechanism of nephrotoxicity. Protein synthesis was the earliest indicator of toxicity, followed by NR uptake and MTT conversion. Fluorescent probes for lysosomes (acridine orange) and mitochondria (rhodamine 123) confirmed that cisplatin's toxicity to RPTCs was delayed and cumulative. Condensation of nucleolar components and fragmentation of RER were observed in RPTCs treated for as little as two hours. Since the nucleolus is the site of ribosome biogenesis, the early inhibition of protein synthesis by cisplatin may arise from disruption of this region. In contrast, mitochondrial dysfunction and swelling were late-stage events, and are therefore unlikely to be the primary targets of nephrotoxic platinum compounds.

Transplacental effects of gentamicin on endocytosis in rat renal proximal tubule cells.

Changes in kidney maturation in utero have been reported after gentamicin administration to pregnant rats. While the proteinuria commonly observed could be related to modifications of the glomerular basement membrane, perturbed renal protein handling could be accounted for by changes in the proximal tubular cells. Therefore, we studied the effect of gentamicin on the renal handling and transport of proteins in proximal tubular cells using the horseradish peroxidase, a fluid-phase marker, as a probe. Gentamicin was administered intraperitoneally to pregnant Wistar rats (75 mg/kg body weight per day) and neonatal kidneys were studied 1 day after birth. In proximal tubular cells of the deep cortical area, containing the fully matured nephrons of neonates, the transport and digestion of reabsorbed peroxidase was considerably reduced compared with controls where peroxidase reached lysosomes after endocytosis. Urinary protein excretion increased in treated animals. We conclude that gentamicin, entering the proximal tubular cells via the endocytic pathway, decreases the tubular reabsorption of proteins, thus increasing urinary protein excretion.

Protein kinase C activity in rat renal proximal tubule cells.

The presence of protein kinase C (PKC) in proximal tubule cells of the rat kidney is established by means of immunodetection and by the demonstration of calcium- and phospholipid-dependent, staurosporine-inhibitable histone phosphorylation. The calcium-dependence of renal PKC is described. Maximal activation of the enzyme (178.2 and 258.8 pmol P1 mg-1 min-1 for cytosol and membrane respectively) was achieved with 5 microM of Ca2+. Phorbol 12, 13 dibutyrate (PDBu) translocated PKC from cytosol to membrane in a dose- and time-dependent fashion, while 4 alpha-phorbol 12,13-didecanoate produced no significant effect on translocation. Cytosolic PKC activity was compared in immature and mature tissues (10- and 40-day-old kidneys). Basal activity was found to be significantly higher (P less than 0.05) in immature cells (272.8 vs. 157.5 pmol Pi mg-1 min-1). PDBu at 10(-6) M for 15 min reduced immunoreactivity in the soluble fraction of both groups, which was accompanied by a significant decrease in kinase activity. We speculate that the high PKC activity in the infant kidney plays a role in cell growth.

Glutathione depletion, lipid peroxidation, DNA double-strand breaks and the cytotoxicity of 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone in rat renal cortical cells

The mechanisms involved in the cytotoxicity of 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone, a model compound for hydroquinone derived mercapturic acids, were investigated in rat renal proximal tubule cells. 2-Bromo-3-( N-acetylcystein- S-yl)hydroquinone induced a time- and concentration-dependent decrease in cell viability and in the levels of cellular glutathione. Antioxidants such as N,N′-diphenyl- p-phenylene diamine and ascorbic acid and the iron chelator desferrioxamine very efficiently protected the cells from 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone without influencing glutathione depletion. The acetoxymethyl ester of the Ca 2+ chelator Quin-2, the inhibitor of the Ca 2+- and Mg 2+- dependent endonucleases, aurintricarboxylic acid and the poly(ADP-ribose)-polymerase inhibitor 3-aminobenzamide also ameliorated 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone cytotoxicity. Moreover, 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone depleted Ca 2+ from isolated kidney mitochondria, increased the amount of malondialdehyde in rat kidney cells and induced DNA double-strand breaks in renal cells in culture. These results suggest that renal cells oxidize 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone to the corresponding quinone; this soft electrophile reacts rapidly with glutathione, thus depleting cellular glutathione concentrations as indicated by the tentative identification of a 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone thioether in the incubation medium of renal cells treated with the mercapturate. As a result of the massive glutathione depletion, peroxidative mechanisms then cause an elevation of the cytosolic concentrations of ionized calcium; impairment of the ability of the mitochondria to sequester Ca 2+ plays an important role in the elevation of the Ca 2+ concentration. Finally, activation of Ca 2+- and Mg 2+-dependent endonucleases results in DNA damage and cell death.

Membrane receptors involved in endocytosis in rat renal proximal tubule

In conclusion the present study has demonstrated the localization of 3 receptors for endocytosis in rat renal proximal tubule cells. The three receptors are located in the membranes of the vacuolar compartment involved in endocytosis in these cells and in addition in dense apical tubules responsible for membrane recycling in the proximal tubule.

An Immunocytochemical Study on Distinct Intracellular Localization of Cathepsin E and Cathepsin D in Human Gastric Cells and Various Rat Cells

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.

Cytotoxicity of cysteine S-conjugates: Structure-activity relationships

The cytotoxicity of cysteine S-conjugates was investigated in freshly isolated rat renal proximal tubule cells. The study was designed to determine the contribution of the thiols and of the acylating intermediates formed by cysteine conjugate β-lyase to the initiation of cytotoxicity. Cell viability was determined by trypan blue exclusion and by lactate dehydrogenase leakage. The S-conjugates S-(1,2,2-trichlorovinyl)- l-cysteine, S-(1,2,3,3,3-pentachloroprop-1-enyl)- l-cysteine and S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)- l-cysteine, at a concentration of 0.2 mM, reduced cell viability compared to controls from 85% to <50% after 3 h. The α-chlorinated enethiols formed from these S-conjugates are transformed to acylating intermediates. The S-conjugate S-(2-chlorovinyl)- l-cysteine forms an enethiol, which cannot transform to an acylating intermediate and did not reduce cell viability at 0.2 mM; at 1 mM, it resulted in a very slight reduction of cell viability after 3 h. S-(pentachlorophenyl)- l-cysteine and S-benzyl- l-cysteine, which form stable thiols after metabolism by β-lyase, were not cytotoxic at a concentration of 1 mM. The direct acting S-(2-chloroethyl)- l-cysteine (0.2 mM) reduced cell viability after 3 h from 85% to 90% (control) to 40%. The results obtained suggest that reactions of the initial thiol-metabolites with biological macromolecules do not contribute to the induction of cytotoxicity by cysteine S-conjugates and indicate that acylating intermediates formed by cysteine conjugate R-lyase induce cytotoxic effects by non-selective acylation of cellular macromolecules.

Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells.

Epidermal growth factor (EGF) is a potent mitogen that rapidly activates plasma membrane Na+-H+ exchangers, thereby causing intracellular alkalinization. The rise in intracellular pH (pHi) may be an important signal for cell growth. However, recent studies have dissociated Na+-H+ exchange activity and/or alkalinization from cellular proliferation. We have studied the role of EGF in the growth of rat renal proximal tubule (PT) cells in primary culture and monitored the early effects of EGF on pHi in these cells using microfluorimetry and the pHi probe, 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). EGF increased DNA synthesis in growing PT cells and produced transient alkalinization (2-3 min) due to activation of Na+-H+ exchange. In contrast, in the absence of extracellular Na+, EGF administration caused pHi to decrease. This acidification was prevented by 2-deoxy-D-glucose and 6-aminonicotinamide, inhibitors of glucose utilization and the hexose monophosphate shunt (HMP), respectively. EGF was also found to stimulate HMP shunt activity in PT cells using an isotopic method for distinguishing between glucose utilization through the HMP shunt vs. glycolysis. Because EGF caused both cytoplasmic acidifying (HMP activation) and alkalinizing (Na+-H+ exchange activation) processes, we propose that the primary role for the activation of Na+-H+ exchange during growth may be to extrude acid from the cell in order to maintain pHi at levels permissive for cell growth.

Luminal uptake and intracellular transport of insulin in renal proximal tubules

It is generally accepted that proteins taken up from the renal tubular fluid are transported into lysosomes in proximal tubule cells. Recently, however, it has been postulated that insulin in isolated perfused rat kidneys did not accumulate in lysosomes but to a certain degree in the Golgi region. The present study was undertaken to investigate the intracellular handling of biologically unaltered insulin in rat renal proximal tubule cells. Rats were prepared for in vivo micropuncture and either a colloidal gold insulin complex or insulin monoiodinated in the A-14 position (125I-insulin) was microinfused into proximal tubules. After 5, 10, 25 or 60 min the tubules were fixed by microinfusion of glutaraldehyde and processed for electron microscopy or electron microscope autoradiography. A qualitative analysis of tubules infused with colloidal gold insulin or 125I-insulin showed that insulin was taken up by endocytosis and transported to lysosomes, and a quantitative autoradiographic analysis of the 125I-insulin microinfused tubules showed that the grain density after five min was significantly increased for endocytic vacuoles and for lysosomes. After 60 min the grain density was still significant over lysosomes. The accumulation of grains was non-significant over all other areas analyzed at any time. This study shows that insulin is taken up from the luminal side of the proximal tubule by endocytosis and transported to the lysosomes. There was no significant transport to the Golgi region.

Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells

A renal brush border fraction was isolated from newborn Sprague-Dawley rats, and its morphological and enzymatic characteristics were studied in comparison to that from the adult. Definite microvillar structures are seen by electron microscopy, and brush border preparations from the newborn are enriched in known marker enzymes. Though morphological development is more advanced and enzyme specific activities are greater in the adult, polyacrylamide gel electrophoresis of membrane proteins reveal no significant change in pattern with increasing age. These studies suggest that the brush border of the proximal tubule cell is present at birth as a significantly developed structure.

The Fine Structure of Rat Renal Microbodies and Cytoplasmic Bodies Following Perfusion Fixation

Microbodies have received extensive morphological and cytochemical investigation since they were first described by Rhodin in 1954. To our knowledge, however, all investigations of microbodies and cytoplasmic bodies of rat renal proximal tubule cells have employed immersion fixation. Tisher, et al. have shown convincing evidence of fine structural alteration of microbodies in rhesus monkey kidney following immersion fixation; these alterations were not encountered when in vivo intravascular perfusion was employed. In view of these studies, and the fact that techniques for perfusion fixation have been established specifically for the rat kidney by Maunsbach, it seemed desirable to employ perfusion fixation to study the fine structure and distribution of microbodies and cytoplasmic bodies within the rat renal proximal tubule.