V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells

The ubiquitous vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

Is the acetylcholine releasing protein mediatophore present in rat brain?

Mediatophore is a protein purified from the nerve terminal membranes of Torpedo electric organ. It confers to artificial membranes a calcium-dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for the Torpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The activity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.     

Regulation of glucocorticoid receptors and Na−K ATPase activity by hydrocortisone in proximal tubular epithelial cells

Summary The effect of hydrocortisone (HC) in modulating glucocorticoid receptors (GR) and sodium-potassium adenosine triphosphatase (Na−K ATPase) activity was studied in primary cultures of immunoisolated murine proximal tubular epithelial cells (PTEC). Utilizing monoclonal antibody against stage-specific embryonic antigen-1, a homogeneous population of PTEC was obtained in high yield. The cells were cultured to confluence and further treated for 48 h in serum-free growth medium containing no HC (control); 50 nM HC; or 50 nM HC plus 20 nM of the antiglucocorticoid, RU 38486. PTEC treated with 50 nM HC had 56% of GR binding and 160% Na−K ATPase activity as compared to controls (P<0.01). GR binding was abolished by incubation in RU 38486 whereas Na−K ATPase fell below control values (P<0.05). Brief incubations of HC-treated PTEC with 0.5 mM ouabain resulted in a fall in GR binding without a change in Na−K ATPase activity. These data indicate that in PTEC, HC regulates GR binding and they suggest that stimulation of Na−K ATPase activity is a direct biological response to this receptor-hormone interaction. Thus, primary cultures of immunoaffinity-isolated PTEC offer a good model system for investigating the molecular basis underlying the regulation of GR binding and postreceptor events influenced by glucocorticoids.     

Ectopic expression of Ligand-of-Numb protein X promoted TGF-β induced epithelial to mesenchymal transition of proximal tubular epithelial cells

Ligand-of-Numb protein X (LNX) was initially characterized as a RING finger type E3 ubiquitin ligase that targeted the intrinsic cell fate determinant Numb for ubiquitination dependent degradation. However, the physiological function of LNX remains largely unknown. In the present study, we demonstrate that ectopic expression of LNX in human proximal tubular epithelial cells (HK-2 cells) significantly enhanced TGF-β1 induced epithelial to mesenchymal transition (EMT). The EMT-promoting effect of LNX manifested as strong inhibition of E-cadherin expression, enhanced expression of vimentin, fibronectin or PAI-1, and increased cell migration. This function of LNX was shown to be independent of its ligase activity because ectopic expression of a mutant form of LNX (C48ALNX) that lacks E3 ligase activity had the similar effect as the wild-type LNX. Overexpression of E-cadherin could inhibit LNX augmented EMT. This study suggests a potential role for LNX in promoting EMT in human proximal tubular epithelial cells.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.     

Effects of testosterone and 17, β– estradiol on the polyamine metabolism in cultivated normal rat kidney epithelial cells

Summary. Ornithine decarboxylase (ODC) and diamine oxidase (DAO) are important enzymes involved in the metabolism of polyamines (putrescine, spermidine and spermine). The influence of testosterone (T) and 17, β– estradiol (E₂) on the activity of ODC and DAO was examined in cultivated normal rat kidney (NRK) epithelial cells. The results showed an increase in enzyme activities 4 hours or 12 hours after hormonal treatment. Both T and E₂ led to a significant increase (1.6-fold) in ODC protein level as compared to the controls. Cellular concentration of spermidine and spermine increased (2.2- and 2.6-fold respectively) 4 hours after T addition. A higher levels in concentrations of putrescine (1.4-fold) and spermine (1.5-fold) 12 hours after E₂ treatment were observed. These results suggest that the biosynthesis and terminal oxidation of the polyamines in NRK epithelial cells are androgen- and estrogen-mediated and depend on the hormonal sensitivity of the cells. Received April 5, 1999, Accepted December 20, 1999     

Is rat skeletal muscle hexokinase an allosteric enzyme?

A recent report by M. Gregoriou, I. P. Trayer, and A. Cornish-Bowden (1986, Eur. J. Biochem. 161, 171-176) showed that the mechanism for rat skeletal muscle hexokinase contains two allosteric sites: one for ATP and one for glucose 6-phosphate. In this report, we show that the allosteric mechanism is at variance with a large amount of kinetic data for the skeletal muscle hexokinase reaction in the literature. In addition, the allosteric mechanism conflicts with isotope exchange at chemical equilibrium data reported by M. Gregoriou, I. P. Trayer, and A. Cornish-Bowden (1983, Eur. J. Biochem. 134, 283-288).     

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233–1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.     

Caveolins redistribute in uterine epithelial cells during early pregnancy in the rat: An epithelial polarisation strategy?

At the time of implantation, uterine luminal epithelial cells undergo a dramatic change in all plasma membrane domains. Changes in the basolateral plasma membrane at the time of implantation include progression from smooth to highly tortuous, as well as a loss of integrin-based focal adhesions. Another aspect of the basolateral plasma membrane that has not been studied in uterine epithelial cells are caveolae, which are omega-shaped invaginations of the plasma membrane known to be involved in endocytosis and contribute to membrane curvature. The current study investigated caveolin, a major protein of caveolae, to explore the possible roles that they play in the remodelling of the basolateral plasma membrane of uterine epithelial cells during early pregnancy in the rat. Morphological caveolae were found at the time of implantation and were significantly increased compared to day 1 of pregnancy. Caveolins 1 and 2 were found to shift to the basolateral plasma membrane of uterine epithelial cells at the time of implantation as well as when treated with progesterone alone, and in combination with oestrogen. A statistically significant increase in the amount of caveolin-1 and a decrease in caveolin-2 protein in uterine epithelial cells was observed at the time of implantation. Caveolin-1 also co-immunoprecipitated with integrin β1 on day 1 of pregnancy, which is a protein that has been reported to be found in integrin-based focal adhesions at the basolateral membrane on day 1 of pregnancy. The localisation and expression of caveolin-1 at the time of implantation is consistent with the presence and increase of morphological caveolae seen at this time. The localisation and expression of caveolins 1 and 2 in luminal uterine epithelium at the time of implantation suggest a role in trafficking proteins and the maintenance of a polarised epithelium.     

Oxidative stress plays a permissive role in α2-adrenoceptor-mediated events in immortalized SHR proximal tubular epithelial cells

Objective To test the hypothesis that the identification of mutation in the carboxypeptidase E (CPE) gene which leads to marked hyperproinsulinaemia is consistent with a possible role for mutations in CPE in the development of coronary heart disease. Methods The study subjects consisted of 51 consecutive patients (34 males and 17 females) who will undergo coronary angiography for suspected or known coronary atherosclerosis. Coronary heart disease (CHD) was defined as having a luminal diameter stenosis ≥50% in at least one of three major coronary arteries by coronary angiography or based on the Rose Questionnaire. The insulin and proinsulin level were measured using highly sensitive two-site sandwich ELISA methods. Screening for mutations of the eight exons of the CPE gene was performed by polymerase chain reaction followed by bidirectional sequencing. Results We scanned eight exons and exon–intron junctional region. Overall, we found 12 distinct variants in the intron region and three variants in the exon region. Among the 15 variants, 10 mutations were rare. The further explored study reveal that the above five non-rare variants would not affect the level of glucose, insulin, and proinsulin. However, the results suggest that the prevalence of the coronary heart disease was significant difference between the wild type group and mutant type group according to the A4545G (P = 0.020). The results from the logistic regression reveal that the subjects with the CPE mutation of A4545G, the odds ratio for the coronary heart disease was 0.196 (95% CI: 0.046 to 0.830, P = 0.027). Conclusions In the present study, the mutation of CPE gene would not affect the level of glucose, insulin, and proinsulin. The hypothesis of a possible role for mutations in CPE in the development of coronary heart disease needs further study.     

Kidney proximal tubule cells: epithelial cells without EGTA-extractable annexins?

The expression and the subcellular localizations of annexins I, II, IV, VI, and XIII in renal epithelial cells were investigated, using immunological techniques with specific monoclonal antibodies. Upon performing Western blotting experiments, no annexins VI and XIII were detected in kidney, whereas annexins I, II, and IV were. Immunofluorescence labelling procedure performed on thin frozen renal sections showed the presence of these three annexins along the plasma membrane of the collecting duct cells with a restricted expression of annexin I at principal cells. Annexin I was also found present in some glomerular cells. None of these annexins, however, were detected in the proximal tubular cells upon performing immunofluorescence labelling and electrophoretic analysis on an EGTA (ethylenebis(oxyethylenenitrilo)tetraacetic acid)-extractable annexin fraction prepared from freshly isolated cells. This is the first time a mammalian epithelial cell has been found to express non-typical annexin (at least partly solubilized with EGTA). However, when these cells were grown in primary culture, they were found to express annexins I, II, IV, and V. As well as being located along the basolateral membrane, annexins I and II are also present on vesicles, which suggests that these annexins may be involved in vesicular traffic under cell culture conditions.     

Is sodium current present in human sinoatrial node cells?

Pacemaker activity of the sinoatrial node has been studied extensively in various animal species, but is virtually unexplored in man. As such, it is unknown whether the fast sodium current (I_Na) plays a role in the pacemaker activity of the human sinoatrial node. Recently, we had the unique opportunity to perform patch-clamp experiments on single pacemaker cells isolated from a human sinoatrial node. In 2 out of the 3 cells measured, we observed large inward currents with characteristics of I_Na. Although we were unable to analyze the current in detail, our findings provide strong evidence that I_Na is present in human sinoatrial node pacemaker cells, and that this I_Na is functionally available at potentials negative to -60 mV.     

Is the mechanical activity of epithelial cells controlled by deformations or forces?

The traction forces developed by cells depend strongly on the substrate rigidity. In this letter, we characterize quantitatively this effect on MDCK epithelial cells by using a microfabricated force sensor consisting in a high-density array of soft pillars whose stiffness can be tailored by changing their height and radius to obtain a rigidity range from 2 nN/microm up to 130 nN/microm. We find that the forces exerted by the cells are proportional to the spring constant of the pillars meaning that, on average, the cells deform the pillars by the same amount whatever their rigidity. The relevant parameter may thus be a deformation rather than a force. These dynamic observations are correlated with the reinforcement of focal adhesions that increases with the substrate rigidity.     

Downregulation of integrin β4 decreases the ability of airway epithelial cells to present antigens

Airway epithelial cells have been demonstrated to be accessory antigen presentation cells (APC) capable of activating T cells and may play an important role in the development of allergic airway inflammation of asthma. In asthmatic airways, loss of expression of the adhesion molecule integrin β4 (ITGB4) and an increase in Th2 inflammation bias has been observed in our previous study. Given that ITGB4 is engaged in multiple signaling pathways, we studied whether disruption of ITGB4-mediated cell adhesion may contribute to the adaptive immune response of epithelial cells, including their ability to present antigens, induce the activate and differentiate of T cells. We silenced ITGB4 expression in bronchial epithelial cells with an effective siRNA vector and studied the effects of ITGB4 silencing on the antigen presentation ability of airway epithelial cells. T cell proliferation and cytokine production was investigated after co-culturing with ITGB4-silenced epithelial cells. Surface expression of B7 homologs and the major histocompatibility complex (MHC) class II was also detected after ITGB4 was silenced. Our results demonstrated that silencing of ITGB4 resulted in impaired antigen presentation processes and suppressed T cell proliferation. Meanwhile, decrease in Th1 cytokine production and increase in Th17 cytokine production was induced after co-culturing with ITGB4-silenced epithelial cells. Moreover, HLA-DR was decreased and the B7 homologs expression was different after ITGB4 silencing. Overall, this study suggested that downregulation of ITGB4 expression in airway epithelial cells could impair the antigen presentation ability of these cells, which further regulate airway inflammation reaction in allergic asthma.