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.     

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.     

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.     

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.     

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.     

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.     

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.     

Transcytosis in cultured proximal tubular cells

Summary Studies were designed to examine fluid-phase pinocytosis in proximal tubular cells. Canine proximal tubules were obtained from the band IV of Percoll^® gradient centrifugation of the dispersed renal cortex, and were seeded on collagen-coated polycarbonate membranes. Integrity of monolayers was confirmed by electrophysiologic measurements, and by scanning electron microscopy. At confluence cell monolayers were studied in Ussing chambers. The rate of transfer of a marker of fluidphase pinocytosis, Lucifer Yellow CH, from the luminal to the basolateral bath was three times higher than that occurring in the opposite direction. Fluorescence microscopy demonstrated that Lucifer Yellow was trapped exclusively in the vesicular compartment. Electron microscopy of the monolayers incubated with cationized ferritin added to the luminal or to the basolateral bath revealed that endocytic vesicles were formed only at the luminal surface. Luminal-to-basolateral transfer of Lucifer Yellow was almost completely blocked at 0°C, and was significantly diminished by K⁺ depletion. Transcytosis of Lucifer Yellow was stimulated twofold by 1-oleoyl-2-acetyl-glycerol. Transfer of quin-2 acetoxymethylester across the monolayer was used as a marker of the paracellular pathway, demonstrating the lack of directional selectivity of this transport route. In summary, vectorial fluid-phase pinocytosis in proximal tubular cells represents an additional mechanism contributing to fluid transport in this segment of the nephron.     

Inhibition of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived of soluble survival factors up-regulates Akt and prevents apoptosis

Mouse kidney proximal tubular epithelial (MK-PT) cells die by apoptosis over 7-10 days when deprived of all survival factors. We show here that withdrawal of all survival factors from MK-PT cells is associated with a progressive increase in the activity of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and a progressive decrease in phosphorylated Akt, a kinase critical to cell survival. Pharmacological inhibition of MEK1/2, the immediate upstream kinase for ERK1/2, not only prevented the decrease in phosphorylated Akt, but also prolonged MK-PT cell survival. Inhibition of ERK1/2, by itself, in the absence of any other known survival factors, was as potent as epidermal growth factor in maintaining MK-PT cell viability. ERK1/2 co-immunoprecipitated with Akt in a multimolecular assembly of signaling molecules, containing at a minimum ERK1/2, Akt, Rsk, and 3-phosphoinositide dependent kinase 1 (PDK1). We hypothesize that the kinase Rsk, whose activation requires phosphorylation by both ERK1/2 and PDK1, acts as a bridge bringing ERK1/2 into proximity with PDK1-associated Akt. Although a number of interactions between the Raf-MEK-ERK and PI3K-Akt signaling pathways have been described, our results are the first to show modulation of Akt activity by signaling events originating with ERK1/2. Spontaneous activation of ERK1/2 occurs via MEK1/2 and appears to depend on oxidant stress, accompanying induction of the default pathway of apoptosis. Together, these data suggest that the spontaneous activation of ERK1/2, in the absence of known extracellular stimuli, represents a previously unrecognized major regulatory pathway determining the fate of cells destined to die by the default pathway of apoptosis.     

A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats

Cellular autophagy in convoluted tubules of kidney was studied in 24 rats, killed in pairs at constant time intervals during one diurnal cycle, by (a) morphometric evaluation of tubular cells by the point-counting method in randomly sampled micrographs, and (b) selective search for autophagic vacuoles (AV) directly on the electron microscopy screen. The total area of tubular cells recorded in the electron microscopy sections was 93 X 10(-4) mum2. Since the distal convoluted tubules, covering about 12% of the whole tubulocellular area, contained only 3-4% of all AV, they were omitted from the main calculations. The number of AV per area unit and the total amount of segregated material showed a distinct diurnal rhythm, synchronous for the different types of AV which were distinguished from each other according to their contents. The minimum was found during the night, the maximum during the day. This rhythm appears similar to that described elsewhere in liver cells. The mean segregated fractions were calculated from the relation of segregated to nonsegregated material in proximal convoluted tubular cells. The segregated fraction of the mitochondria was 4.4 X 10(-4). This value could account for the degradation of all mitochondria in a cell within 15 days, i.e., the upper limit of the lifetime of mitochondrial DNA in the cortex of the kidney, if one assumes that a mitochondrion is destroyed within 10 min after being segregated. The degregated fraction of microbodies was 11.7 X 10(-4). This suggests a shorter lifetime of these organelles. It is concluded that cellular autophagy plays a significant role in the turnover of cytoplasmic constituents, including the membranes of the endoplasmic reticulum.     

Active sodium transport in basolateral plasma membrane vesicles from rat kidney proximal tubular cells

Inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can accumulate Na+ actively in two ways. Mode 1, which is K+-independent, is ouabain-insensitive and is inhibited by furosemide and mode 2, which is K+-dependent, is inhibited by ouabain and is insensitive to furosemide. The presence of Mg2+ and ATP in the incubation medium is essential for both modes of Na+ uptake to proceed and in both cases, the nucleotide is hydrolyzed during the process. These results are consistent with the idea of the existence, in these membranes, of two Na+ pumps: one, which can work in the absence of K+ (Na+ pump) and another, which needs K+ to work (Na+ + K+ pump).     

Distinct role of matrix metalloproteinase-3 in kidney injury molecule-1 shedding by kidney proximal tubular epithelial cells

Tubulointerstitial injury is a common pathway in progressive renal impairment and human proximal tubular epithelial cells (PTEC) play a crucial role in this process. Kidney injury molecule-1 (KIM-1) has received increasing attention due to its potential utility as the therapeutic target and biomarker for kidney injury. This study aims to explore the underlying mechanism regulating the release of KIM-1. Cultured primary human PTEC expressed and released KIM-1 from the apical surface through an ectodomain shedding process mediated by matrix metalloproteinase (MMP), independent of gene expression and protein synthesis. The constitutive KIM-1 shedding by PTEC was enhanced in a dose- and time-dependent manner by human serum albumin (HSA) or tumor necrosis factor-α (TNF-α), two important physiological stimuli found during kidney injury. Data from PCR array screening of MMPs gene expression in PTEC following activation by HSA or TNF-α, and from blocking experiments using either synthetic MMP inhibitors or MMP gene knockdown by siRNA, revealed that the constitutive and accelerated shedding of KIM-1 in cultured PTEC was mediated by MMP-3. Furthermore, the up-regulation of MMP-3 and KIM-1 release by PTEC was associated with generation of reactive oxygen species. In a mouse model of acute kidney injury induced by ischemia and reperfusion, increased expression of MMP-3 and KIM-1 as well as their co-localization were observed in kidney from ischemic but not in sham-operated mice. Taken together, these in vitro and in vivo evidences suggest that MMP-3 plays an inductive role in KIM-1 shedding by PTEC.