Normal Rat Kidney Epithelial Cells Research Articles

Real-time label-free monitoring of the cellular response to osmotic stress using conventional and long-range surface plasmons

Cell volume and its regulation are key factors for cellular integrity and also serve as indicators of various cell pathologies. SPR sensors represent an efficient tool for real-time and label-free observations of changes in cell volume and shape. Here, we extend this concept by employing the use of long-range surface plasmons (LRSP). Due to the enhanced penetration depth of LRSP (∼1μm, compared to ∼0.4μm of a conventional surface plasmon), the observation of refractive index changes occurring deeper inside the cells is possible. In this work, the responses of a confluent normal rat kidney (NRK) epithelial cell layer to osmotic stress are studied by both conventional and long-range surface plasmons. Experiments are conducted in parallel using cell layers grown and stimulated under the same conditions to enable direct comparison of the results and discrimination of the osmotic stress-induced effects in different parts of the cell.

Lipopolysaccharide effects on the proliferation of NRK52E cells via alternations in gap-junction function

Gap junctions regulate proper kidney function by facilitating intercellular communication, vascular conduction, and tubular purinergic signaling. However, no clear relationship has been described between gap-junction function and acute kidney injury induced by the endotoxin lipopolysaccharide (LPS). Normal rat kidney epithelial cells (NRK52E cells) were seeded at high and low densities to promote or impede gap-junction formation, respectively, and establish distinctive levels of intercellular communication in culture. Cells were then challenged with LPS at various concentrations (10-1,000 ng/mL). LPS-induced formation and function of gap junctions were assessed by measuring changes in cell proliferation and colony-forming rates, fluorescent dye transmission to adjacent cells, expression levels of connexin43, and repositioning of confluent cells in response to the gap junction inhibitor oleamide or agonist retinoic acid. The cell proliferation rate and colony-forming rate of high- and low-density NRK52E cells were decreased upon LPS challenge, in a dose-dependent manner. The colony-forming rate of confluent high-density cells was significantly lower than that of low-density cells. Oleamide treatment raised the LPS-induced colony-forming rate of high-density cells, whereas retinoic acid decreased the rate. Neither oleamide nor retinoic acid significantly affected the LPS-induced colony-forming rate of low-density cells. Fluorescence transmission of high-density cells was reduced by LPS challenge, in a dose-dependent manner, but inclusion of retinoic acid increased the LPS-induced transmission of fluorescence. LPS challenge of either high- or low-density NRK52E cells resulted in down-regulated connexin43 expression. Gap-junction function plays an important role in concentration-dependent cytotoxic effect of LPS on normal rat kidney cells in vitro.

Role of Reactive Oxygen Species in Transforming Growth Factor-Beta1–Induced Extracellular Matrix Accumulation in Renal Tubular Epithelial Cells

Tubulointerstitial fibrosis, which is characterized by the progressive accumulation of extracellular matrix (ECM), is the main feature of chronic renal allograft dysfunction. Transforming growth factor-β1 (TGF-β1) is the key inducer of tubulointerstitial fibrosis. Plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of ECM degradation, is increasingly recognized to play an important role in renal fibrosis. ECM accumulation is the net result of ECM synthesis and degradation. We previously reported that reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) are required for the TGF-β1–induced epithelial-to-mesenchymal transition in renal proximal tubular epithelial cells. In the present study, we examined the role of the ROS-MAPK pathways in TGF-β1–induced fibronectin and PAI-1 up-regulation in renal tubular epithelial cells. Growth arrested, synchronized normal rat kidney epithelial (NRK-52E) cells were stimulated with TGF-β1 (0.2–20 ng/mL) or H2O2 (1–500 μmol/L) in the presence or absence of inhibitors of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (diphenyleneiodonium [DPI] and apocynin [Apo]) and MAPK (PD98059, an MEK inhibitor, or a p38 MAPK inhibitor) for up to 48 hours. Both TGF-β1 and H2O2 increased fibronectin and PAI-1 secretion in dose-dependent manners. Chemical inhibition of NADPH oxidase, extracellular signal-regulated kinase (ERK), or p38 MAPK all inhibited TGF-β1–induced and H2O2-induced fibronectin and PAI-1 up-regulation. These results suggested that NADPH oxidase-mediated ROS and subsequent ERK and p38 MAPK activation play important roles in ECM accumulation in the renal tubulointerstitium.

Macromolecular Crowding Regulates Assembly of mRNA Stress Granules after Osmotic Stress: NEW ROLE FOR COMPATIBLE OSMOLYTES

The massive uptake of compatible osmolytes such as betaine, taurine, and myo-inositol is a protective response shared by all eukaryotes exposed to hypertonic stress. Their accumulation results mostly from the expression of specific transporters triggered by the transcriptional factor NFAT5/TonEBP. This allows the recovery of the cell volume without increasing intracellular ionic strength. In this study we consider the assembly and dissociation of mRNA stress granules (SGs) in hypertonic-stressed cells and the role of compatible osmolytes. In agreement with in vitro results obtained on isolated mRNAs, both macromolecular crowding and a high ionic strength favor the assembly of SGs in normal rat kidney epithelial cells. However, after hours of constant hypertonicity, the slow accumulation in the cytoplasm of compatible osmolytes via specific transporters both reduces macromolecular crowding and ionic strength, thus leading to the progressive dissociation of SGs. In line with this, when cells are exposed to hypertonicity to accumulate a large amount of compatible osmolytes, the formation of SGs is severely impaired, and cells increase their chances of survival to another hypertonic episode. Altogether, these results indicate that the impact of compatible osmolytes on the mRNA-associated machineries and especially that associated with SGs may play an important role in cell resistance and adaption to hyperosmolarity in many tissues like kidney and liver.

Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian Cells

Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

Gap junctions favor normal rat kidney epithelial cell adaptation to chronic hypertonicity

Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.

Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na(+) affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

Cell growth inhibition and apoptosis by SDS-solubilized single-walled carbon nanotubes in normal rat kidney epithelial cells

Carbon nanotubes (CNTs), promising novel nanomaterials, have been applied to drug delivery and bio-imaging; however, their potential harmful effects on human health and environment have gained much attention recently. In the present study, we investigated cytotoxic effect of solubilized single-walled CNTs (SWCNTs), which were dispersed in water by sodium dodesyl sulfate (SDS), in normal rat kidney epithelial cells (NRK-52E). SDS-SWCNT (0.125-10 μg/mL)-treated NRK-52E cells showed decreased cell viability and enhanced cytotoxicity marker levels following 24-48 h incubation. In addition, SDS-SWCNT treatment evoked the cell growth inhibition: 8 μg/mL SDS-SWCNT induced the growth arrest at G(0)/G(1) phase and levels of cell cycle-related proteins such as CDK2, CDK6 and phosphorylated-retinoblastoma (pRB) were significantly reduced by CNT. Whereas, at higher concentration of SDS-SWCNT, the percentage of cell numbers in apoptotic sub-G(1) phase was substantially increased. Along with these changes, SDS-SWCNT treatment elevated protein levels for p53 and p21 with a concomitant increase in the single strand DNA breakage. Taken together, these results suggest that SDS-solubilized SWCNTs exert genotoxic effect in renal epithelial cells, and p53-dependent signaling can be associated with the growth arrest and apoptosis events upon CNT-induced DNA damage.

Adenovirus-delivered microRNA targeting the vitamin D receptor reduces intracellular Ca2+ concentrations by regulating the expression of Ca2+-transport proteins in renal epithelial cells

What’s known on the subject? and What does the study add? Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone-forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca²⁺ concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed. • To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects. • The adenovirus vector-delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR-dependent epithelial Ca2+ transport proteins were detected using real-time polymerase chain reaction and Western blot assays, respectively. • Fluorescent Ca²⁺ indicator Fluo-4 NW (Fluo-4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500-IX71, Japan) were used to detect the cytosolic free Ca²⁺ concentration at different time points after infection. • The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin-D28k and plasma membrane Ca²⁺-ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. • There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na⁺/Ca²⁺-exchanger (NCX1). • Furthermore, VDR knockdown results in a decrease in intracellular Ca²⁺ concentration ([Ca²⁺]i) in NRK cell lines. • Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin-D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca²⁺]i in NRK cell lines. • The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.

DNA microarray analysis of normal rat kidney epithelial cells treated with cadmium

In order to elucidate the transcriptional response of kidney epithelial cells to cadmium, the gene expression pattern was examined in normal rat kidney epithelial cells (NRK-52E cells) exposed to 50 µM cadmium for 4 hr using DNA microarray. Cadmium was found to increase the expression of 73 genes and decrease the expression of 42 genes in NRK-52E cells before the development of cytotoxicity.

Effect of cadmium chloride on the expression and phosphorylation of mitogen-activated protein kinase in normal rat kidney epithelial cells

To explore the effect of cadmium chloride on the expression and phosphorylation of mitogen-activated protein kinase (MAPK) in normal rat kidney epithelial (NRK) cells. The NRK cells were incubated with cadmium chloride either at respective dose (0, 1, 5, 10, 20, 40 µmol/L) for 24 h or at same dose (10 µmol/L) for respective time (0, 0.5, 1.0, 2.0, 4.0, 8.0 h). Western blotting was applied to test the expression of MAPK in NRK cells (ERK1/2, p38, JNK); and phosphor-specific antibody to detect the phosphorylated MAPK (p-ERK1/2, p-p38, p-JNK). There was no significant difference in the MAPK expression among the groups either treated with different doses or for different time; however, the level of phosphorylated MAPK was comparatively higher than it in control group. There was an obvious expression of p-ERK1/2 at 1.00 ± 0.06 in the group incubated with 10 µmol/L CdCl(2); and the expression in the 20 µmol/L and 40 µmol/L CdCl(2) group was 2.58 ± 0.11, 2.76 ± 0.23 respectively, which was 1.58 and 1.76 times more than it in 10 µmol/L CdCl(2) group. The differences were statistically significant (F = 827.70, P < 0.01). The respective expression of p-p38MAPK in the 20 µmol/L (2.47 ± 0.20)and 40 µmol/L CdCl(2) group (3.73 ± 0.25)was 1.47 and 2.73 times more than it in control group (1.00 ± 0.02), and the differences were also statistically significant (F = 280.06, P < 0.01). There was a dose-effect relationship of the concentration of cadmium in the expression of p-ERK1/2 (r = 0.919, t = 4.69, P = 0.009) and p-p38MAPK (r = 0.945, t = 5.79, P = 0.004). Additionally, phosphorylated MAPK expressed in a time-dependent manner. The expression of p-ERK1/2 was obvious in the group incubated for 1 h (1.26 ± 0.11), and the respective expression in the 4 h group (1.51 ± 0.07) and 8 h group (3.53 ± 0.23) was 1.5 and 3.5 times of it in the control group (1.00 ± 0.02). The differences were statistically significant (F = 427.82, P < 0.001). The expression of p-p38MAPK increased significantly in 1 h group (1.31 ± 0.07); while the respective expression in 4 h group (3.53 ± 0.32) and 8 h group (4.41 ± 0.38) was 3.5 and 4.4 times of it in control group (1.00 ± 0.03). The differences were also statistically significant (F = 280.06, P < 0.001). Cadmium chloride could significantly enhance the phosphorylation of MAPK in NRK cells; and it is probably associated with the activation of MAPK.

Mycophenolic Acid May Delay Allograft Fibrosis by Inhibiting Transforming Growth Factor-β1-Induced Activation of Nox-2 Through the Nuclear Factor-κB Pathway

We evaluated the role of renal tubular Nox-2 in the pathogenesis of epithelial-to-mesenchymal transition (EMT) in kidney allografts. We examined this question in the human kidney allografts with interstitial fibrosis and tubular atrophy not otherwise specified (IFTANOS), in the Fisher to Lewis rat transplant model, and in the in vitro model of transforming growth factor-beta1-induced EMT in normal rat kidney epithelial cells (NRK52E). We first demonstrated that Nox-2 and alpha-smooth muscle actin (SMA) were increased in renal tubules from kidney transplant recipients on calcineurin inhibitors, mycophenolic acid (MPA), and prednisone with IFTANOS, suggestive of EMT (n=6). Next, we examined Nox-2 expression and fibrogenesis in syngeneic transplants, allogeneic transplants treated with MPA 40 mg/kg per 24 hr, and untreated allogeneic transplants for 6 months (n=14 in each group). Immunofluorescent and immunohistochemical studies for Nox-2, alpha-SMA, and E-cadherin showed that similar to patients with IFTANOS, rat allografts had greater tubulointerstitial staining for Nox-2 and alpha-SMA. MPA therapy prevented these changes. Immunoblot analyses examining Nox-2 signaling (phospho-nuclear factor [NF]-kappaB), redox signaling (phospho-smad2), and fibrosis (alpha-SMA and fibronectin) demonstrated that MPA treatment prevented the up-regulation of Nox-2, inhibited p-NF-kappaB and p-smad2, and down-regulated alpha-SMA and fibronectin levels. Finally, we examined Nox-2 signaling in vitro and confirmed that MPA inhibited phospho-NF-kappaB, Nox-2, phospho-smad2, and alpha-SMA during transforming growth factor-beta1-induced EMT of NRK52E cells while reducing Nox-2, vimentin, and fibronectin mRNA levels. MPA may down-regulate Nox-2 activation and EMT through the NF-kappaB pathway in the tubular epithelial cells, suggesting a novel role for this drug independent of its immunosuppressive properties.

Effect of aldosterone and its antagonist spironolactone on epithelial-mesenchymal transition of normal rat kidney epithelial cells in high glucose

To determine the effect of aldosterone and its antagonist, spironolactone on epithelial-mesenchymal transition (EMT) of normal rat kidney epithelial cells (NRK-52E) in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy (DN ) in rats involving aldosterone and spironolacton. NRK-52E cells were simultaneously cultured in the serum-free Dulbecco's modification of Eagle's medium Dulbecco (DMEM) for 12 hours. Then the low glucose (LG) group was cultured in LG (1000 mg/L) DMEM:The high glucose (HG) group was cultured in high glucose (4,500 mg/L) DMEM. The aldosterone (Aldo) groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively. The SP group was cultured in high glucose (4,500 mg/L) DMEM plus 10(-7)mol/L spironolactone. Immunohistochemistry, RT-PCR and Western blot were used to detect E-cadherin and alpha smooth muscle actin(alpha-SMA) mRNA expression. RT-PCR showed that compared with the LG Group, E-cadherin mRNA expression in the HG group was significantly lower, and alpha-SMA mRNA expression was significantly increased(P<0.05). E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group, while the expression of alpha-SMA mRNA was significantly increased in the HG group(P<0.05), with a dose-dependent relationship with aldosterone(r=-0.70,P<0.05;r=0.67, P<0.05). E-cadherin mRNA in the SP group was significantly higher,while alpha-SMA mRNA expression was lower than that in the HG group(P<0.01). Immunohistochemistry and Western blot showed that compared with the LG group, E-cadherin protein expression was significantly reduced, and alpha-SMA expression was significantly increased in the HG group(P<0.01). In the 10 nmol/L Aldo, 50 nmol/L Aldo, and the 100 nmol/L Aldo groups, E-cadherin protein expression was significantly lower, and alpha-SMA protein expression was significantly higher than that in the HG group(P<0.05), with a dose-dependent relationship with aldosterone(r=-0.83,P<0.05;r=0.81, P<0.05). In the SP group, E-cadherin protein expression was higher, and alpha-SMA protein level was lower than that in the HG group(P<0.05). Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu, leading to renal interstitial fibrosis in Diabetic nephropathy. Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT, which may be an important mechanism for the inhibition of renal interstitial fibrosis.

Arylsulfatase B regulates interaction of chondroitin-4-sulfate and kininogen in renal epithelial cells

The enzyme arylsulfatase B ( N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen–bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen–C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen–bradykinin axis and, thereby, may influence blood pressure. Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation.

Temporal ratiometry to assess dynamic concentration distributions of fluorescent molecules in single live cells during continuous diffusional dosing

The introduction of specific molecules into live cells is a widely used approach to probe cellular mechanisms. Recently, we have reported on the sustained dosing of molecules into single cells via a microscopic diffusion port. Here we describe temporal ratiometry, a method to reconstruct intracellular concentration distribution of the delivered molecules as it varies in time during dosing. To characterize this method, we analyzed fluorescence intensity maps obtained during delivery of Lucifer Yellow CH, LY, a polar fluorophore into A7r5 vascular smooth muscle cells, normal rat kidney epithelial cells (NRKE), and MCF-7 human breast cancer cells. Temporal ratiometry indicates a linear increase in concentration of LY in these cells with a nearly uniform distribution during 20 min of delivery. The method cancels the effects of varying cell height and variable accessible volume on the measured intensities at different locations within the cell. Temporal ratiometry will be useful to estimate dynamic changes in intracellular concentration distributions and thus, facilitate the understanding of transport, binding, sequestration, and efflux of molecules introduced into cells.

Kid-1 participates in regulating ERK phosphorylation as a part of the circadian clock output in rat kidney

Circadian clock genes play a role for the regulation of cell cycle, but the factors connecting clock to cell cycle are not fully understood. We found that mRNA of Kid-1— a zinc-finger-type transcriptional repressor was localized to cortical and juxtamedullary segments of tubules but not to glomeruli in the rat kidney. Kid-1 mRNA showed robust circadian oscillation with a peak at ZT16. Under temporal restricted feeding, the phase of the oscillation shifted along with mRNAs of the clock genes—Per1 and Per2. The rhythm of S-phase in cell cycle disappeared in the kidney under the restricted feeding. The level of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was rhythmic with a peak at ZT16 in the kidney. We found that knockdown and overexpression of Kid-1 in NRK52E (normal rat kidney epithelial) cells induced and reduced the phosphorylation of ERK1/2, respectively. The data suggest that clock-controlled Kid-1 regulates the cell cycle of proliferating renal tubular epithelial cells through ERK phosphorylation.

Expression and Purification of Human Keratinocyte Growth Factor 2 by Fusion with SUMO

Small ubiquitin-related modifier (SUMO) fusion system has been shown to be efficient for enhancing expression and preventing degradation of the target protein. We showed herein that SUMO fusion to human keratinocyte growth factor 2 (hKGF-2) gene was feasible and it significantly enhanced protein expression and its efficiency. The fusion DNA fragment composed of SUMO gene, which was fused to hexahistidine tag, and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a-SUMO-hKGF-2. The plasmid was then transformed into Escherichia coli RosettaTM2(DE3), and the recombinant fusion protein SUMO-hKGF-2 was expressed at 30°C for 6 h, with the induction of IPTG at the final concentration of 0.4 mM. The expression level of the fusion protein was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography. After desalting by Sephadex G-25 size exclusion chromatography, the hexahistidine-SUMO-hKGF-2 was digested by SUMO proteases. The recombinant hKGF-2 was purified again with Ni-NTA column and the purity was about 95% with a total yield of 13.9 mg/l culture. The result of mitogenicity assay suggests that the recombinant hKGF-2 can significantly promote the proliferation of normal rat kidney epithelial (NRK-52E) cells.

Protein kinase B/Akt activity is involved in renal TGF-β1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-β (TGF-β1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-β1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-β1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3β phosphorylation. TGF-β1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and α-smooth muscle actin expression, consistent with the fibrotic actions of TGF-β1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-β1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3β, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-β1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

RhoA regulation of NF-κB activation is mediated by COX-2-dependent feedback inhibition of IKK in kidney epithelial cells

Numerous studies have demonstrated a central role of renal tubular epithelial cells in the etiology of kidney injury and disease through the elaboration of inflammatory mediators. However, little is known about the cellular signaling mechanisms involved in this process. In this study we employed normal rat kidney epithelial (NRK52E) cells to identify a novel LPS-induced signaling pathway in which RhoA-mediated AP-1 activity promotes expression of cyclooxygenase-2 (COX-2) with consequent feedback inhibition of NF-kappaB activation through IKKbeta. Inhibition of RhoA signaling using either the RhoA kinase inhibitor Y-27632 or a dominant negative mutant of RhoA (RhoA-DN) dramatically extended the duration of p65-DNA binding, IkappaBalpha phosphorylation, and IKKbeta activity following LPS treatment. Prolongation of events associated with NF-kappaB activation was also observed in cells pretreated and/or cotransfected with the JNK inhibitor SP600125 or deletion mutants of MEKK1 (MEKK1-KD) or Jun (Jun-DN). Conversely, constitutive expression of RhoA prevented NF-kappaB activation by LPS, and this effect was reversed by cotransfection with MEKK1-KD. In addition, we found that the RhoA/AP-1 signaling axis plays a necessary role in COX-2 expression by LPS and that this effect is independent of NF-kappaB activation. Moreover, inhibition of COX-2 activity results in persistent p65-DNA binding, IkappaBalpha phosphorylation, and IKKbeta activity, similar to that observed after prevention of RhoA/AP-1 axis signaling. These findings suggest that COX-2 links the RhoA/AP-1 signaling cascade to NF-kappaB activation, thereby defining a novel integrated model for regulation of the inflammatory response of kidney epithelial cells to LPS and potentially other external stimuli.

Specific distribution of overexpressed aurora B kinase in interphase normal epithelial cells

BackgroundIt is known that aurora B, a chromosomal passenger protein responsible for the proper progression of mitosis and cytokinesis, is overexpressed throughout the cell cycle in cancer cells. Overexpression of aurora B produced multinuclearity and induced aggressive metastasis, suggesting that overexpressed aurora B has multiple functions in cancer development. However, the detailed dynamics and functions of overexpressed aurora B are poorly understood.ResultsWe overexpressed GFP fused aurora B kinase in normal rat kidney epithelial cells. Using spinning disk confocal microscopy, we found that overexpressed aurora B-GFP was predominantly localized in the nucleus and along the cortex as a dot-like or short filamentous structure during interphase. Time-lapse imaging revealed that a cytoplasmic fraction of overexpressed aurora B-GFP was incorporated into the nucleus after cell division. Immunofluorescence studies showed that the nuclear fraction of overexpressed aurora B did not induce ectopic phosphorylation of histone H3 after cell division. The cytoplasmic fraction of overexpressed aurora B-GFP was mainly associated with cortical actin filaments but not stress fibers. Myosin II regulatory light chain, one of the possible targets for aurora B, did not colocalize with cortical aurora B-GFP, suggesting that overexpressed aurora B did not promote phosphorylation of myosin II regulatory light chain in interphase cells.ConclusionWe conclude that overexpressed aurora B has a specific localization pattern in interphase cells. Based on our findings, we propose that overexpressed aurora B targets the nuclear and cortical proteins during interphase, which may contribute to cancer development and tumor metastasis.