Angiotensin-converting enzyme inhibitors attenuated advanced glycation end products-induced renal tubular hypertrophy via enhancing nitric oxide signaling.

Advanced glycation end products (AGE) and angiotensin II were closely correlated with the progression of diabetic nephopathy (DN). Nitric oxide (NO) is a protective mediator of renal tubular hypertrophy in DN. Here, we examined the molecular mechanisms of angiotensin-converting enzyme inhibitor (ACEI) and NO signaling responsible for diminishing AGE-induced renal tubular hypertrophy. In human renal proximal tubular cells, AGE decreased NO production, inducible NOS activity, guanosine 3',5'-cyclic monophosphate (cGMP) synthesis, and cGMP-dependent protein kinase (PKG) activation. All theses effects of AGE were reversed by treatment with ACEIs (captopril and enalapril), the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the PKG activator 8-para-chlorophenylthio-cGMPs (8-pCPT-cGMPs). In addition, AGE-enhanced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were clearly reduced by captopril, enalapril, SNAP, and 8-pCPT-cGMPs. The abilities of ACEIs and NO/PKG activation to inhibit AGE-induced hypertrophic growth were verified by the observation that captopril, enalapril, SNAP, and 8-pCPT-cGMPs decreased protein levels of fibronectin, p21 Waf1/Cip1 , and receptor for AGE. The results of the present study suggest that ACEIs significantly reduced AGE-increased ERK/JNK/p38 MAPK activation and renal tubular hypertrophy partly through enhancement of the NO/PKG pathway.

Elucidation of the therapeutic role of mitochondrial biogenesis transducers NRF-1 in the regulation of renal fibrosis.

BACKGROUND: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. MATERIALS AND METHODS: In vitro fibrosis model was established by treatment with transforming growth factor-ß1 (TGF-ß1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). RESULTS: Under renal fibrosis conditions, TGF-ß1 (5ng/ml) increased ROS. Simultaneously, TGF-ß1-induced extracellular fibronectin by ELISA assay. In addition, TGF-ß1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2µg/ml) expression vector dramatically reverse TGF-ß1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2µg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. DISCUSSION: These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.

Treatment with cytokine thymic stromal lymphopoietin short hairpin RNA substantially reduces TGF-ß1-induced interstitial cellular fibrosis.

Thymic stromal lymphopoietin (TSLP) has previously been linked to allergic inflammatory diseases, and tissue fibrosis and organ dysfunction may also arise from such inflammation. It remains unclear, however, whether TSLP plays any role in the occurrence of renal fibrosis, so this study investigated that possibility. An in vitro fibrosis model was established by treating normal rat kidney fibroblast (NRK-49F) cells with transforming growth factor-ß1 (TGF-ß1), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad 7) were investigated. Also, TSLP shRNA was used to silence the effects of TSLP, while an ELISA was conducted to evaluate the fibronectin secretions. The level of fibronectin in the NRK-49F cells was dose- and time-dependently increased by the administration of exogenous TSLP (P<0.05). TSLP also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of Smad7. Interestingly, the application of TSLP shRNA caused a stark reversal of the TGF-ß1-induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. Taken together, these observations provide insights into how extracellular matrices develop and could thus lead to potential therapeutic interventions for the suppression of renal fibrosis.

Hypoxia-induced vascular endothelial growth factor secretion by retinal pigment epithelial cells is inhibited by melatonin via decreased accumulation of hypoxia-inducible factors-1α protein.

BACKGROUND: Hypoxia is the most important stimulus leading to up-regulation of vascular endothelial growth factor (VEGF) in the retina via elevation of hypoxia-inducible factors-1α (HIF-1α) protein. The purpose of this study was to test the effects of melatonin on the expression of VEGF and HIF-1α in the cultured human retinal pigment epithelial (RPE) cells under normoxia and hypoxia. METHOD: An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 ) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without melatonin under normoxia and hypoxia. The protein and RNA levels of VEGF and HIF-1α were measured by ELISA kits and RT-PCR, respectively. RESULT: Hypoxia induced a significant increase of expression and secretion of VEGF and accumulation of HIF-1α protein in RPE cells (P < 0.05). Melatonin at 10-5 to 10-8 M significantly inhibited hypoxia-induced expression, the secretion of VEGF and the accumulation of HIF-1α protein (P < 0.05), but not affected expression of VEGF and HIF-1α under normoxia (P > 0.05). CONCLUSION: This study suggests that melatonin may have potential value in the prevention and treatment of various retinal diseases associated with increase of VEGF, vascular leakage and angiogenesis.

Steap4 attenuates high glucose and S100B-induced effects in mesangial cells.

Six-transmembrane epithelial antigen of prostate 4 (Steap4)-knockout mice develop hyperglycaemia and inflammation whereas Steap4 overexpression attenuates atherosclerosis in diabetic mice. Thus, we studied the roles of Steap4 in high glucose (HG, 27.5 mM) or S100B (1 µM, a ligand for the receptor for advanced glycation end-product or RAGE)-induced effects in mouse mesangial (MES13) cells. We found that HG-induced Steap4 protein expression was dependent on S100B. HG increased cell membrane, but not cytosolic, Steap4 protein expression. HG increased protein-protein interaction between Steap4 and S100B, which was confirmed by mass spectrometry of immunoprecipitated S100B. SP600125, LY294002 and AG490 attenuated S100B-induced Steap4 protein expression or gene transcriptional activity. A mutation in signal transducer and activator of transcription 3 (Stat3) site 2 of the Steap4 promoter constructs resulted in a marked decrease in HG or S100B-induced activation of Steap4 gene transcription. Overexpression of Steap4 attenuates HG or S100B-induced collagen IV, fibronectin and cyclooxygenase 2 protein expression. Overexpression of Steap4 attenuates HG or S100B-induced transforming growth factor-ß (TGF-ß). Moreover, overexpression of Steap4 attenuates S100B-induced signalling. Finally, overexpressing Steap4 attenuated renal expression of fibronectin, S100B, TGF-ß, type IV collagen, p-Akt, p-extracellular signal regulated kinase 1/2 and p-Stat3 in streptozotocin-diabetic mice. Thus, overexpression of Steap4 attenuated HG or S100B-induced effects in MES13 cells and attenuated some of S100B-induced effects in diabetic mouse kidneys.

Cinnamaldehyde and nitric oxide attenuate advanced glycation end products-induced the Jak/STAT signaling in human renal tubular cells.

Cinnamaldehyde is a major and a bioactive compound isolated from the leaves of Cinnamomum osmophloeum kaneh. It possesses anti-diabetic properties in vitro and in vivo and has anti-inflammatory and anti-cancer effects. To explore whether cinnamaldehyde was linked to altered advanced glycation end products (AGE)-mediated diabetic nephropathy, the molecular mechanisms of cinnamaldehyde responsible for inhibition of AGE-reduced nitric oxide (NO) bioactivity in human renal proximal tubular cells were examined. We found that raising the ambient AGE concentration causes a dose-dependent decrease in NO generation. Cinnamaldehyde significantly reverses AGE-inhibited NO generation and induces high levels of cGMP synthesis and PKG activation. Treatments with cinnamaldehyde, the NO donor S-nitroso-N-acetylpenicillamine, and the JAK2 inhibitor AG490 markedly attenuated AGE-inhibited NOS protein levels and NO generation. Moreover, AGE-induced the JAK2-STAT1/STAT3 activation, RAGE/p27(Kip1) /collagen IV protein levels, and cellular hypertrophy were reversed by cinnamaldehyde. The ability of cinnamaldehyde to suppress STAT activation was also verified by the observation that it significantly increased SCOS-3 protein level. These findings indicate for the first time that in the presence of cinnamaldehyde, the suppression of AGE-induced biological responses is probably mediated by inactivating the JAK2-STAT1/STAT3 cascade or activating the NO pathway.

Insight into the modified Ibalizumab-human CD4 receptor interactions: using a computational binding free energy approach.

Antibody drugs are very useful tools for the treatment of many chronic diseases. Recently, however, patients and doctors have encountered the problem of drug resistance. How to improve the affinity of antibody drugs has therefore become a pressing issue. Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1. This study investigates the mutation residues of the complementarity determining regions of Ibalizumab. We propose using the wild and mutations of Ibalizumab-human CD4 receptor complex structures, molecular dynamics techniques, alanine-scanning mutagenesis calculations and solvated interaction energies methods to predict the binding free energy of the Ibalizumab-human CD4 receptor complex structures. This work found that revealed three key positions (31th, 32th and 33th in HCDR-1) of the residues may play an important role in Ibalizumab-human CD4 receptor complex interactions. Therefore, bioengineering substitutions of the three key positions and increasing number of intermolecular interactions (HCDR-1 of Ibalizumab/human CD4 receptor) might improve the binding affinities of this complex structure.

Urinary transforming growth factor α and serum α-fetoprotein as tumor markers of hepatocellular carcinoma.

This case-control study aimed to evaluate the diagnostic application of urinary transforming growth factor (TGF) α and serum α-fetoprotein (AFP) in hepatocellular carcinoma (HCC). TGFα and AFP were determined in 90 pairs of age- and gender-matched patients with cirrhotic HCC and patients with cirrhosis alone and 60 healthy controls. The results indicated that TGFα and AFP levels in patients with HCC were higher than in those with cirrhosis alone or healthy controls (each P = 0.0001). Multivariate analysis indicated that TGFα (odds ratio (OR) 1.03, 95% confidence interval (CI) 1.05-1.16) and AFP (OR 1.03, 95% CI 1.01-1.06) were closely associated, in a dose-related fashion, with the development of HCC. The optimal cutoff values, determined with the receiver operating characteristic (ROC) curves, were 29 µg/g creatinine for TGFα and 100 ng/ml for AFP, respectively. The areas under ROC curve (AUC) were 0.74 for TGFα and 0.78 for AFP, respectively. Both biomarkers showed the same sensitivity (52.2%), high specificity, high positive predictive value, and moderate positive likelihood ratio. Determination of both markers in parallel significantly increased the AUC (0.91) and diagnostic accuracy (92.2%), with a high sensitivity (86.7 %), specificity (97.8%), positive predictive value (PPV; 97.5%), and moderate positive likelihood ratio (PLR; 39.4). Among 31 cirrhotic HCC with AFP ≤ 20 ng/ml, the calculated AUC for TGFα was 0.79, with a sensitivity of 64.5%, specificity of 96.7%, PPV of 87.0%, and PLR of 19.5. In conclusion, urinary TGFα and serum AFP are complementary tumor markers for detection of HCC with low AFP production.

Ubiquitin C-terminal hydrolase-L5 is required for high glucose-induced transforming growth factor-ß receptor I expression and hypertrophy in mesangial cells.

Transforming growth factor-ß (TGF-ß) is pivotal in the pathogenesis of diabetic nephropathy. Type 1 TGF-ß receptor (TGF-ßR1) is degraded by Smad7-dependent ubiquitination-proteasomal pathway, which is deubiquitinated by ubiquitin C-terminal hydrolase-L5 (UCHL5). Therefore, we studied the role of UCHL5 in high glucose (27.8mM)-induced TGF-ßR1 protein expression in mouse mesangial (MES13) cells. UCHL5 short hairpin RNA (shRNA) was used to knock down UCHL5 while LY294002 and the dominant-negative p85 were used to inhibit phosphatidylinositol-3-kinase (PI3K). We found that high glucose increased phospho-Akt, TGF-ßR1 mRNA and protein expression. High glucose also increased UCHL5 protein expression, which was attenuated by LY294002, the dominant-negative p85 and the dominant-negative CREB. High glucose-induced TGF-ßR1 protein expression and TGF-ßR1 protein deubiquitination were attenuated by UCHL5 shRNA. Additionally, high glucose-induced p21(WAF1), fibronectin protein expression and cell hypertrophy were attenuated by UCHL5 shRNA. However, high glucose-induced TGF-ßR1 mRNA, p27(kip1) protein expression and growth inhibition were not affected by UCHL5 shRNA. Finally, glomerular UCHL5 and TGF-ßR1 protein expression were increased in streptozotocin-diabetic rats at 8weeks. We conclude that PI3K-dependent UCHL5 is required for high glucose-induced TGF-ßR1 protein expression in mesangial cells. UCHL5 is also required for high glucose-induced TGF-ßR1 protein deubiquitination, p21(WAF1) and fibronectin protein expression and cell hypertrophy.

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography-tandem mass spectrometry coupled with microscale labeling.

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.

Contributions of active site residues to cofactor binding and catalysis of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase reversely catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. In this study, we investigated the function of active site residues N86, Y155, and K159 in NADH binding and catalysis in the reduction of androstanedione, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching, and anisotropy measurements. The N86A, Y155F, and K159A mutant enzymes decreased the catalytic constant by 37- to 220-fold and increased the dissociation constant by 3- to 75-fold, respectively. Binding of NADH with wild-type and mutant enzymes caused different levels of fluorescence resonance energy transfer, implying a different orientation of nicotinamide ring versus W173. In addition, the enzyme-bound NADH decreased the fluorescence anisotropy value in the order WT>N86A>Y155F>K159A, indicating an increase in the mobility of the bound NADH for the mutants. Data suggest that hydrogen bonding with the hydroxyl group of nicotinamide ribose by K159 and Y155 is important to maintain the orientation of NADH and contributes greatly to the transition-state binding energy to facilitate the catalysis. N86 is important for stabilizing the position of K159. Substitution of alanine for N86 has a minor effect on NADH binding through K159, resulting in a slight increase in the mobility of the bound NADH and decreases in affinity and catalytic constant.

BMP-2 suppresses renal interstitial fibrosis by regulating epithelial-mesenchymal transition.

Dysregulation of epithelial-to-mesenchymal transition (EMT) may contribute to renal fibrogenesis. Our previous study indicated that bone morphogenetic protein-2 (BMP-2) significantly reversed transforming growth factor (TGF)-ß1-induced renal interstitial fibrosis. In this study, we examined the underlying mechanism and elucidate the regulation of EMT process under BMP-2 treatment. Cultured renal interstitial fibroblast (NRK-49F) was treated with TGF-ß1 (10 ng/ml) with or without BMP-2 (10-250 ng/ml) for 24 h. The expression of α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, or Snail transcriptional factors was analyzed by immunofluorescence staining or Western blotting. Cell migration was analyzed by wound-healing assay. NRK-49F treated with TGF-ß1 induced significant EMT including upregulatioin of α-SMA, fibronectin, and snail proteins and down-regulation of E-cadherin. Interestingly, co-treatment with BMP-2 dose-dependently reversed TGF-ß1-induced cellular fibrosis, cell migration, and above EMT change. The above effect was closely correlated with Snail since BMP-2 dose- and time-course dependently induced a significant decrease in the level of Snail. Moreover, Snail siRNA significantly reversed TGF-ß1-induced increases in the level of α-SMA and fibronectin (intracellular and extracellular). We suppose that BMP-2 have the potential to attenuate TGF-ß1-induced renal interstitial fibrosis by attenuating Snail expression and reversing EMT process.

Role of S114 in the NADH-induced conformational change and catalysis of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. In this study, we characterize the role of the conserved residue S114 in cofactor binding and catalysis, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching and anisotropy measurements. The catalytic efficiency of V/K(NADH)Et for wild-type and S114A is 1.5 x10(7) and 3.8 x 10(3) M(-1) s(-1), respectively, suggesting that NADH association to wild-type and S114A mutant enzymes involves two steps, a bimolecular binding step and isomerization. The binding of NADH into a hydrophobic pocket in the active site of wild-type and S114A mutant enzymes restricts its motion and shields the fluorescence quenching from solvent, with an increase in the fluorescence intensity and a blue shift at the maximum wavelength. Furthermore, the binding of NADH leads to the protein fluorescence quenching, mainly due to fluorescence resonance energy transfer to NADH. S114A mutant enzyme decreases 3100-fold in V/Et with no apparent change in K(m) for substrates. Addition of NADH to S114A mutant enzyme induces a secondary structural change. These results suggest that S114 is important to maintain the correct conformation for the nucleotide binding and facilitate the reaction. Substitution of alanine for S114 eliminates the hydrogen bonding interaction with P185, causing a conformational change in a nonproductive binding of NADH and a significant loss of activity.

Advanced glycation end-products activate extracellular signal-regulated kinase via the oxidative stress-EGF receptor pathway in renal fibroblasts.

Advanced glycation end-products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal-regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs-induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)-ROS-EGFR pathway in normal rat kidney interstitial fibroblast (NRK-49F) cells. We found that AGEs (100 microg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short-hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N-acetylcysteine (NAC) attenuated AGEs-induced intracellular ROS. Hydrogen peroxide (5-25 microM) increased RAGE protein level while activating both EGFR and ERK1/2. Low-dose hydrogen peroxide (5 microM) increased whereas high-dose hydrogen peroxide (100 microM) decreased mitogenesis at 3 days. AGEs-activated EGFR and ERK1/2 were attenuated by an anti-oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs-induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs-induced mitogenesis is dependent on the RAGE-ROS-EGFR-ERK1/2 pathway whereas AGEs-activated ERK1/2 is dependent on the RAGE-ROS-EGFR pathway in NRK-49F cells.

Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM.

Effect of osthole on advanced glycation end products-induced renal tubular hypertrophy and role of klotho in its mechanism of action.

BACKGROUND: Osthole has been widely reported to have pharmacological activities such as anti-cancer, anti-inflammation and anti-hyperlipidemic effects. Klotho was identified as an anti-senescence protein in a variety of tissues. Loss of klotho has been associated with chronic kidney disease. However, potential roles and molecular events for osthole and klotho in diabetic nephropathy remain unclear. PURPOSE: In the current study, we undertook to study the effect of osthole on klotho expression in advanced glycation end products (AGE)-cultured human renal proximal tubular cells, and to investigate the molecular mechanisms of osthole and exogenous klotho against AGE-induced renal tubular hypertrophy. METHODS: Cell viability was elucidated by MTT assay. Protein expression was measured by Western blotting. mRNA level was analyzed by real-time PCR. Cellular hypertrophy growth was evaluated by hypertrophy index. Relative cell size was detected by flow cytometry. RESULTS: We found that raising the ambient AGE concentration causes a dose-dependent decrease in klotho synthesis. Osthole significantly increased AGE-inhibited klotho mRNA and protein expression. Osthole and exogenous klotho treatments significantly attenuated AGE-induced Janus kinase 2 (JAK2)-signal transducers and activators of transcription 1 (STAT1) and STAT3 activation. Moreover, protein levels of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 were augmented by osthole and exogenous klotho. The abilities of osthole and exogenous klotho to reverse AGE-induced cellular hypertrophy were verified by the observation that osthole and exogenous klotho inhibited p21Waf1/Cip1/collagen IV/RAGE expression, total protein content, and cell size. CONCLUSION: Consequently, we found that osthole attenuated AGE-induced renal tubular hypertrophy via induction of klotho expression and suppression of the JAK2-STAT1/STAT3 signaling. These results also showed that klotho might be used as a unique molecular target for the treatment of diabetic nephropathy.

Safflower extract: a novel renal fibrosis antagonist that functions by suppressing autocrine TGF-beta.

Progressive renal disease is characterized by the accumulation of extracellular matrix proteins in the renal interstitium. Hence, developing agents that antagonize fibrogenic signals is a critical issue facing researchers. The present study investigated the blood-circulation-promoting Chinese herb, safflower, on fibrosis status in NRK-49F cells, a normal rat kidney interstitial fibroblast, to evaluate the underlying signal transduction mechanism of transforming growth factor-beta (TGF-beta), a potent fibrogenic growth factor. Safflower was characterized and extracted using water. Renal fibrosis model was established both in vitro with fibroblast cells treated with beta-hydroxybutyrate and in vivo using rats undergone unilateral ureteral obstruction (UUO). Western blotting was used to examine protein expression in TGF-beta-related signal proteins such as type I and type II TGF-beta receptor, Smads2/3, pSmad2/3, Smads4, and Smads7. ELISA was used to analyze bioactive TGF-beta1 and fibronectin levels in the culture media. Safflower extract (SE) significantly inhibited beta-HB-induced fibrosis in NRK cells concomitantly with dose-dependent inhibition of the type I TGF-beta1 receptor and its down-stream signals (i.e., Smad). Moreover, SE dose-dependently enhanced inhibitory Smad7. Thus, SE can suppress renal cellular fibrosis by inhibiting the TGF-beta autocrine loop. Moreover, remarkably lower levels of tissue collagen were noted in the nephron and serum TGF-beta1 of UUO rats receiving oral SE (0.15 g/3 ml/0.25 kg/day) compared with the untreated controls. Hence, SE is a potential inhibitor of renal fibrosis. We suggest that safflower is a novel renal fibrosis antagonist that functions by down-regulating TGF-beta signals.

Heat shock protein A1B 1267 polymorphism is highly associated with risk and prognosis of hepatocellular carcinoma: a case-control study.

We conducted a case-control study to elucidate the role of heat shock protein A1B (HSPA1B) 1267 single nucleotide polymorphism (SNP) on the risk and prognosis of hepatocellular carcinoma (HCC). Subjects enrolled included 150 pairs of sex- and age-matched HCC patients and unrelated controls. Genomic DNA was typed for HSPA1B1267 SNP using polymerase chain reaction with restriction fragment length polymorphism. The frequencies of the HSPA1B P2/P2 genotype and the HSPA1B P2 allele in HCC patients were higher than in unrelated controls (each p = 0.0001). Multivariate analysis identified the following independent risk factors for HCC: HSPA1B P1/P2 genotype (odds ratio [OR], 2.34; 95% confidence interval [CI], 1.07-5.11), HSPA1B P2/P2 genotype (OR, 12.06; 95% CI, 4.43-32.79), hepatitis B surface antigen (HBsAg) (OR, 25.95; 95% CI, 11.88-56.68), and antibodies to hepatitis C virus (anti-HCV) (OR, 70.43; 95% CI, 21.89-226.64). There was an additive interaction between HSPA1B P2 allele carriers and the presence of either HBsAg (synergy index = 2.48) or anti-HCV (synergy index = 1.52). However, as HSPA1B1267 SNP is a silent mutation, it is a surrogate genetic marker for increasing risk of HCC. Our findings indicate that patients with chronic hepatitis B/hepatitis C virus infection who harbor this SNP represent a high-risk group for HCC. They should receive more intensive surveillance for early detection of HCC. Moreover, patients with the HSPA1B P2 allele had significantly longer survival (p = 0.002).The limitations of this study include the unknown functional significance of the HSPA1B1267 polymorphism, the relatively small sample size, the fact that this was not a prospective study of cases and controls, and the questionable generalizability of the findings given the specific ethnic composition of the population studied.

Effect of taurine on advanced glycation end products-induced hypertrophy in renal tubular epithelial cells.

Mounting evidence indicates that advanced glycation end products (AGE) play a major role in the development of diabetic nephropathy (DN). Taurine is a well documented antioxidant agent. To explore whether taurine was linked to altered AGE-mediated renal tubulointerstitial fibrosis in DN, we examined the molecular mechanisms of taurine responsible for inhibition of AGE-induced hypertrophy in renal tubular epithelial cells. We found that AGE (but not non-glycated BSA) caused inhibition of cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, bcl-2 protein expression, and mitochondrial cytochrome c release in BSA, AGE, or the antioxidant taurine treatments in these cells. AGE-induced the Raf-1/extracellular signal-regulated kinase (ERK) activation was markedly blocked by taurine. Furthermore, taurine, the Raf-1 kinase inhibitor GW5074, and the ERK kinase inhibitor PD98059 may have the ability to induce cellular proliferation and cell cycle progression from AGE-treated cells. The ability of taurine, GW5074, or PD98059 to inhibit AGE-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of RAGE, p27(Kip1), collagen IV, and fibronectin. The results obtained in this study suggest that taurine may serve as the potential anti-fibrotic activity in DN through mechanism dependent of its Raf-1/ERK inactivation in AGE-induced hypertrophy in renal tubular epithelial cells.

Advanced glycation end-product-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 are dependent on glycogen synthase kinase 3beta in LLC-PK1 cells.

Glycogen synthase kinase 3beta (GSK3beta) is increased by high glucose in mesangial cells. Thus, we studied the role of GSK3beta in advanced glycation end-product (AGE)-induced effects in the proximal tubule-like LLC-PK1 cells. We found that AGE (100 microg/ml) time-dependently (8-48 h) increased phospho-GSK3beta-Tyr216 (active GSK3beta) and time-dependently (4-24 h) decreased phospho-GSK3beta-Ser21/9 (inactive GSK3beta) protein expression. Meanwhile, AGE (100 microg/ml) activated GSK3beta kinase at 8-48 h. AGE (100 microg/ml) dose-dependently (75-100 microg/ml) decreased beta-catenin protein expression but AGE did not decrease beta-catenin protein expression until 48 h. SB216763 (a GSK3beta inhibitor) and GSK3beta shRNA attenuated AGE (100 microg/ml)-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 at 48 h. SB216763 also attenuated AGE-induced type IV collagen. We conclude that AGE activates GSK3beta in LLC-PK1 cells. AGE-inhibited beta-catenin and cyclin D1 protein expression are dependent on GSK3beta. Moreover, AGE-inhibited cell proliferation and AGE-induced type IV collagen protein expression are dependent on GSK3beta.