Inhibition of cellular autophagy in proximal tubular cells of the kidney in streptozotocin-diabetic and uninephrectomized rats.

To examine the significance of anti-catabolism in renal hypertrophy, cellular autophagy was investigated by electron microscopic morphometry in proximal tubular cells (PTCs) of the outer cortex of the rat kidney after the induction of diabetes mellitus by streptozotocin (STZ) and after unilateral nephrectomy. Adult male Sprague-Dawley rats were divided into three groups and killed by retrograde perfusion fixation, 1, 2 and 3 days after the induction of diabetes (group D; n = 24), after unilateral nephrectomy (group N; n = 24) and after combined treatment (group DN; n = 24). Untreated, age-matched litter mates served as controls (group C; n = 24). By comparison with these controls, the left kidney to initial body weight ratio was increased by 8, 23, and 15% in group D animals, by 8, 23, and 24% in group N animals, and by 10, 21, and 25% in group DN animals at the first, second and third day, respectively. Quantitative evaluation of large test areas showed that the volume and numerical densities of autophagic vacuoles (AVs) in PTCs were significantly lower in these hypertrophed kidneys than in the controls. The average reduction in AV volume density was about 65% in group D animals, about 50% in group N animals and about 75% in group DN animals. These data show that autophagic degradation of cytoplasmic components in PTCs is inhibited in renal hypertrophy independently of the growth stimulus, i.e. uninephrectomy or diabetes. Since insulin per se inhibits cellular autophagy in PTCs, the expected effect of insulin dificiency seems to be counteracted by as yet undefined stimuli that may be related to metabolic work load.     

Inhibition of cellular autophagy in kidney tubular cells stimulated to grow by unilateral nephrectomy

Cytoplasmic growth (hypertrophy) presupposes a positive metabolic balance brought about by increased anabolic and/or decreased catabolic processes. Degradation of cytoplasmic components takes place in autophagic vacuoles (AVs) whose volume fraction may be taken as a measure of the relative rate of degradation of cytoplasmic components. Male adult Sprague-Dawley rats (n = 80) were unilaterally nephrectomized (n = 40) or sham-operated (n = 40) and were killed 3.5-57.5 h p.o. The volume density of AVs in parenchymal cells of renal cortical convoluted tubules was determined morphometrically by systematic evaluation of large test fields in the electron microscope. During compensatory renal growth, the volume densities of autophagic vacuoles were reduced at day 0 (3.5-8 h p.o.), day 1 (20.5-33.5 h p.o.) and day 2 (44.5-57.5 h p.o.) by 49% (p less than 0.01), 43% (p less than 0.05), and 19% (n.s.), respectively, when compared with sham-operated controls. No decrease, and even an increase, in the AV-volume fraction was found in liver parenchymal cells of the unilaterally nephrectomized animals. This indicates that inhibition of autophagy is not a general response after unilateral nephrectomy, but is confined to the growing kidney, where it may represent a significant factor in the increase of cytoplasmic mass.     

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats (SHR-Okamoto strain adult rats) was measured with conventional 3 mol KCl microelectrodes, in vivo. Peritubular cell membrane potential was not different in SHR (-66.5 ± 0.7 mV) as compared with normotensive control Wistar rats (-67.5 ± 1.2 mV). To test the effects of possible altered sodium membrane transport in SHR on proximal tubule peritubular membrane potential, we allowed SHR and control rats to drink 1% NaCl for two weeks. Again, proximal tubule peritubular membrane potential was not different in SHR on 1% NaCl (-67.0 ± 1.0 mV) as compared with control rats on 1% NaCl (-64.7 ± 1.3 mV). From these results we concluded that peritubular membrane potential in kidney proximal tubular cells of SHR was not different from normotensive Wistar control rats, and if some alteration of sodium transport in kidney proximal tubular cells of SHR could exist, that was not possible to evaluate from the measurements of peritubular membrane potential in kidney proximal tubular cells.     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.     

Effects of cumene hydroperoxide on cellular cation composition in frog kidney proximal tubular cells

Effects of cumene hydroperoxide were studied on the peritubular membrane potential and cellular cation composition in frog kidney proximal tubular cells. After perfusion of isolated frog kidneys for 30 min with 1.3x10(-4) mol l(-1) cumene hydroperoxide Ringer solution, the peritubular membrane potential gradually declined. The ouabain-like effects were demonstrated on cell Na and K activities after 1 h of perfusion with cumene hydroperoxide. The peritubular apparent transference number for potassium was decreased. Intracellular pH was not altered in the presence of cumene hydroperoxide. Intracellular free Ca(2+) concentration increased slowly and moderately. The concentration of the malondialdehyde in the kidney homogenates, measured as an index of lipid peroxidation, was increased. A previously observable effect of cumene hydroperoxide on the peritubular membrane potential was prevented by oxygen radical scavengers.     

Diffusive water permeability in isolated kidney proximal tubular cells: Nature of the cellular water pathways

Summary The diffusive water permeability (P _d ) of the plasma membrane of proximal kidney tubule cells was measured using a¹H-NMR technique. The values obtained for the exchange time (T _ex) across the membrane were independent of the cytocrit and of the Mn²⁺ concentration (in the range 2.5 to 5mm). At 25°C the calculatedP _d value was (per cm² of outer surface area without taking into account membrane invaginations) 197±17 m/sec. This value equals 22.3±1.9 m/sec when the invaginations are taken into account. Cell exposure to 2.5mm parachloromercuribenzenesulfonic acid,pCMBS, (for 20 to 35 min) reducedP _d to 45% of its control value. Fivemm dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2±1.0 kcal/mol under control conditions. It increased to 9.1±2.2 kcal/mol in the presence of 2.5mm pCMBS. Using our previous values for the osmotic water permeability (P _os) in proximal straight tubular cells theP _os/P _d ratio equals 18±1, under control conditions, and 3.2±0.3 in the presence ofpCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed bypCMBS. Assuming laminar flow (within the pore), fromP _os/P _d of 13 to 18 an unreasonably large pore radius of 12 to 15 Å is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore. The water permeability remaining in the presence ofpCMBS indicates water permeation through the lipid bilayer. There are similarities between these results and those obtained in human red blood cells and in the apical cell membrane of the toad urinary bladder.     

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a ₁/₂-adrenergic receptor (AR) agonist) and iodoclonidine (an ₂-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (₁-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (-AR blocker), atenolol (₁-AR blocker), yohimbine (₂-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (₂-AR blocker) and prazosin (₁-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP 254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5-flanking region of the ANG gene and subsequently stimulates the gene expression.     

Cytotoxicity of alkylating agents in isolated rat kidney proximal tubular and distal tubular cells

Patterns of chemical-induced cytotoxicity in different regions of the nephron were studied with freshly isolated proximal tubular and distal tubular cells from rat kidney. Three model alkylating agents, methyl vinyl ketone, allyl alcohol, and N-dimethylnitrosamine, were used as test chemicals. Methyl vinyl ketone and a metabolite of allyl alcohol, acrolein, are Michael acceptors that bind to cellular protein sulfhydryl groups and GSH. N-Dimethylnitrosamine binds to cellular protein and DNA. Lactate dehydrogenase leakage was used to assess irreversible cellular injury. Distal tubular cells were more susceptible than proximal tubular cells to injury produced by methyl vinyl ketone or allyl alcohol while the two cell populations were equally susceptible to injury produced by N-dimethylnitrosamine. Preincubation of both proximal tubular and distal tubular cells with GSH protected them from methyl vinyl ketone- and allyl alcohol-induced cytotoxicity but had no effect on N-dimethylnitrosamine-induced cytotoxicity. Similarly, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, altered cellular GSH status. As with GSH status, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, caused pronounced inhibitory effects on mitochondrial function, as evidenced by ATP depletion and inhibition of cellular oxygen consumption. These results demonstrate that alkylating agents are cytotoxic to both proximal tubular and distal tubular cells, and that interaction with cellular GSH is a factor determining nephron cell type specificity of injury.     

Morphological effects of cadmium on proximal tubular cells in rats

Twenty-four male rats of the Wistar strain divided into four groups were injected sc with a dose of 0.8, 1.5, and 3.0 mg Cd/kg body wt as CdCl₂ in saline, and saline alone to the control rats, three times a week for 3 wk. Cadmium levels of whole kidney homogenate, supernatant (cytosol), precipitate, and metallothionein (MT) fraction were measured. Histological changes of the renal proximal tubules were investigated by optical and electron microscopy. In the kidneys, Cd levels were increased with the increment of Cd dosage; 80–90% of Cd was contained in cytosol, and 55–75% was in MT fraction. Non-MT-Cd reached a maximum in the 1.5 mg Cd group, whereas that of the 3.0 mg Cd group showed some decline. With increasing Cd doses, the size of nuclei and nucleoli in the cells of proximal tubule showed significant enlargement and also an increase in the number of nucleoli on light microscopy. At higher doses, chromatin condensation of the tubular nuclei and vacuolar degeneration of the tubular cells were evident. On electron microscopy, perichromatin granules of the proximal tubular nuclei were increased in number, especially in the rats of Cd 0.8 mg and 1.5 mg/kg groups. As the Cd doses increased, ring-shaped nucleoli were increased in number and nucleolar segregation was observed more clearly. Moreover, in the 3.0 mg/kg Cd group, nuclear indentation and nucleoli containing compact dense granules were observed. In the cytoplasm, there was an increase of lysosomes, myelin bodies, ring-shaped mitochondria, and vesiculation; ultimate changes were degeneration and cell necrosis. The injured cells were heterogenously distributed in each nephron and this heterogeneity was attributed in the difference in Cd content and cell cycle in each cell of the nephron.     

Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.     

Digalactosylceramide is the receptor for staphylococcal enterotoxin-B in human kidney proximal tubular cells

We have characterized a glycosphingolipid (GSL) receptor forStaphylococcus enterotoxin-B (SEB) in cultured human kidneyproximal tubular (PT) cells. Solid-phase binding of [¹²⁵I]SEBto the GSL receptor was concentration dependent and was notdisplaceable by two structurally related toxins, such as staphylococcalenterotoxin-A and toxic shock syndrome toxin-1. Rat kidney cellsdid not bind [¹²⁵I]SEB. However, when the rat kidney cells werepre-incubated with digalactosylceramide, there was a concentration-dependentbinding of [¹²⁵I]SEB. Trimethylsilyl derivatization of methylglycosides, followed by gas-liquid chromatography-mass spectrometry(GC-MS), revealed that galactose was the major sugar componentof this putative receptor GSL. The sphingosines present in thisGSL were d18:2, d22:2 and d23:0; the fatty acids present werepalmitate, oleate and stearate. Permethvlation of alditol acetatesand GC-MS revealed two predominant sugars, namely 2, 3, 4 and6 tetramethylgalactital and 2, 3 and 6 trimethylgalactital.The GSL receptor for SEB was sensitive to a-galactosidase, andresistant to (3-galactosidase and fi-glucosidase. Taken together,our studies reveal that the tentative structure of the receptorfor SEB in human kidney PT cells is CerGal     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor β₂ whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

Metformin (Met), an AMP-activated protein kinase (AMPK) inducer, is primarily transported by organic cation transporters expressed at the surface of renal proximal tubular epithelial cells. However, the implication of Met in renal function remains poorly understood. Interestingly, AICAR, another AMPK inducer, has been shown to inhibit the Unfolded Protein Response (UPR) generated by tunicamycin in cardiomyocytes in an AMPK-kinase dependent fashion suggesting metformin may also block the UPR. In this work, we have examined the effect of metformin on the expression of UPR-related markers (GRP94 and CHOP) induced by glucosamine (GlcN), 2-deoxyglucose (2-DOG) and tunicamycin (TUNI) in renal proximal tubular epithelial cells and in murine mesangial cells. Met attenuated GRP94 and CHOP expression induced by GlcN and 2-DOG, but not TUNI only in renal epithelial cells, even though the AMPK activation was observed in both renal epithelial and mesangial cells. Met did not require the contribution of its AMPK kinase inducing activity to block UPR markers expression. This report has identified a novel inhibitory function of metformin on UPR, which may have a beneficial impact on kidney homeostatic function.     

Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulateproduction of guanosine 3',5'-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate(J_va). Comparedwith control solutions, SNP(10⁴ M) decreasedJ_va by 23% whenadministered luminally and by 35% when added to the peritubularperfusate. Stimulation of fluid uptake by luminal angiotensin II (ANGII; 10⁹ M) was abolished bySNP (10⁴ and10⁶ M). In proximal tubulesuspensions, ANF (10⁶ M)increased cGMP concentration to 143%, whereas SNP(10⁶,10⁵,10⁴,10³ M) raised cGMP to 231, 594, 687, and 880%, respectively.S-nitroso-N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANFonly affected ANG II-stimulated transport may be because of productionof higher concentrations of cGMP by SNP.

     

Peritubular Na-K exchange ion pump in maleate-treated frog kidney proximal tubular cells

• 1.1. After perfusion of isolated frog kidneys for 1 hr with 10⁻³ or 10⁻² M maleate Ringer, the peritubular membrane potential gradually declined in a dose-dependent manner.
• 2.2. The ouabain-like effects of maleate on cell Na and K activities were dose-dependent and smaller than the effects of zero K or 10⁻⁴M ouabain. Intracellular pH was not altered in the presence of 10⁻²M maleate.
• 3.3. The driving force for Na entry into the cell was reduced, respectively, to 81.4 and 58.4% (of control) in the presence of 10⁻³ and 10⁻² M maleate.
• 4.4. There was no histochemically detectable inhibition of proximal tubule Na-K ATPase activity during 3 hr of perfusion with 10⁻² M maleate.

     

Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells

A method for preparation of highly purified basolateral plasma membranes from rat kidney proximal tubular cells is reported. These membranes were assayed for the presence of vesicles as well as for their orientation. (Na+ + K+)-ATPase activity and [3H]ouabain binding studies with membranes treated with or without SDS revealed that the preparation consisted of almost 100% vesicles. The percentage of inside-out vesicles was found to be approx. 70%. This percentage was determined measuring the (Na+ + K+)-ATPase activity in K+-loaded vesicles and in membranes treated with or without trypsin and SDS. These membranes represent a very efficient tool to assay the correlation between active transport and ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells.     

Sugar uptake into a primary culture of dog kidney proximal tubular cells

We describe the functional characteristics of a new primary culture system derived from a suspension of dog proximal tubular cells. The culture system is maintained on alpha minimal essential medium with 15% fetal calf serum supplementation. At confluency the cultured cells demonstrate the following: (i) typical epithelial morphology using light microscopy, with multiple dome formation inhibited by ouabain; (ii) strong binding with a polyclonal antibody directed against dog proximal tubular brush border membrane antigens; (iii) high concentration of alkaline phosphatase activity by histochemical staining; and (iv) 25-hydroxycholecalciferol (25(OH)D3)-24-hydroxylase activity. Sugar transport was assessed using alpha-methyl-D-glucopyranoside (alpha MG), a nonmetabolizable analog of D-glucose, as well as L-glucose, and 3-O-methyl-D-glucose (3OMG). The transport of alpha MG was stereospecific; temperature sensitive; inhibited strongly by phlorizin but not by cytochalasin B, phloretin, or 3OMG; and Na dependent. The transport of 3OMG is stereospecific, temperature sensitive, and inhibited strongly by phloretin and cytochalasin B, but not by phlorizin. Despite the apparent heterogeneity of cell type, this primary culture system exhibits many features of normal dog proximal tubule function.