Shu-Fen Liu

Thrombospondin-1 mediates distal tubule hypertrophy induced by glycated albumin.

Diabetic nephropathy is characterized by early hypertrophy in both glomerular and tubuloepithelial elements. However, no studies to date have established a direct causal link between hyperglycaemia and renal hypertrophy. Our previous studies have found that high glucose does not induce cellular hypertrophy or expression of TGF-beta1 (transforming growth factor-beta1) in distal renal tubule cells [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. In the present study, we used AGEs (advanced glycation end-products) to mimic long-term hyperglycaemia. Similar to glucose, AGEs did not induce TGF-beta1 mRNA in distal renal tubule cells [MDCK (Madin-Darby canine kidney) cells]; however, TGF-beta1 bioactivity was increased significantly. This result indicated post-translational regulation. Since TSP-1 (thrombospondin-1) has been demonstrated to activate latent TGF-beta1 in a variety of systems, the following experiments were performed. We found that AGEs dose-dependently increased both intracellular and extracellular levels of TSP-1. Purified TSP-1, like AGEs, increased the cellular protein content. Furthermore, anti-TSP-1 neutralizing antibodies attenuated the AGE-induced increase in TGF-beta1 bioactivity and hypertrophy. Thus TSP-1 might mediate AGE-induced distal renal tubule hypertrophy. In addition, we observed several putative transcription factor binding sites in the TSP-1 promoter, including those for AP-1 (activator protein-1), CREB (cAMP response element binding protein), NF-kappaB (nuclear factor-kappaB), SRF (serum response factor) and HSF (heat-shock factor), by sequence mapping. We used an enhancer assay to screen possible transcription factors involved. We showed that AP-1 and CREB were specifically induced by AGEs; furthermore, TFD (transcription factor decoy) for AP-1 could attenuate the AGE-induced increases in TSP-1 levels and cellular hypertrophy. Thus regulation of TSP-1 might be critical for hyperglycaemic distal tubule hypertrophy. Furthermore, TSP-1 TFD might be a potential approach to ameliorate diabetic renal hypertrophy.

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)‐β1‐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‐β1 (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‐β1 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‐β1‐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‐β1‐induced increases in the level of α‐SMA and fibronectin (intracellular and extracellular). We suppose that BMP‐2 have the potential to attenuate TGF‐β1‐induced renal interstitial fibrosis by attenuating Snail expression and reversing EMT process. J. Cell. Biochem. 112: 2558–2565, 2011.

Dioscorea alata Attenuates Renal Interstitial Cellular Fibrosis by Regulating Smad- and Epithelial-Mesenchymal Transition Signaling Pathways

Renal interstitial fibrosis is characterized by increased extracellular matrix (ECM) synthesis. Epithelial-mesenchymal transition (EMT) in kidneys is driven by regulated expression of fibrogenic cytokines such as transforming growth factor-beta (TGF-β). Yam, or Dioscorea alata (DA) is an important herb in Chinese medicine widely used for the treatment of clinical diabetes mellitus. However, the fibrosis regulatory effect of DA is unclear. Thus, we examined TGF-β signaling mechanisms against EMT in rat fibroblast cells (NRK-49F). The characterization of DA water-extracts used various methods; after inducing cellular fibrosis in NRK-49F cells by treatment with β-hydroxybutyrate (β-HB) (10 mM), we used Western blotting to examine the protein expression in the TGF-β-related signal protein type I and type II TGF-β receptors, Smads2 and Smad3 (Smad2/3), pSmad2 and Smad3 (pSmad2/3), Smads4, Smads7, and EMT markers. These markers included E-cadherin, alpha-smooth muscle actin (α-SMA), and matrix metalloproteinase-2 (MMP-2). Bioactive TGF-β and fibronectin levels in the culture media were determined using ELISA. Expressions of fibronectin and Snail transcription factor, an EMT-regulatory transcription factor, were assessed by immunofluorescence staining. DA extract dose-dependently (50–200 µg/mL) suppressed β-HB-induced expression of fibronectin in NRK-49F cells concomitantly with the inhibition of Smad2/3, pSmad2/3, and Smad4. By contrast, Smad7 expression was significantly increased. DA extract caused a decrease in α-SMA (α-smooth muscle actin) and MMP-2 levels, and an increase in E-cadherin expression. We propose that DA extract might act as a novel fibrosis antagonist, which acts partly by down regulating the TGF-β/smad signaling pathway and modulating EMT expression.

Albumin induces cellular fibrosis by upregulating transforming growth factor‐beta ligand and its receptors in renal distal tubule cells

Albuminuria is indicative of nephropathy. However, little literature has focused on the role of albumin in renal distal tubule fibrosis. We used a well‐defined distal tubule cell, Madin‐Darby Canine Kidney (MDCK). Proliferation and cytotoxicity were examined. The conditioned supernatant was collected and subjected to ELISA assay for detection of fibronectin and TGF‐β1. Reverse transcription‐PCR and Western blot assay were performed to evaluate the expression of mRNA and protein of two types of TGF‐β receptors (TbetaR). Flow cytometry assay and phosphotyrosine (pY)‐specific antibodies were used to assay the phosphorylation status of TbetaR. We showed that albumin dose dependently (0, 0.1, 1, or 10 mg/ml) inhibited cellular growth in MDCK cells without inducing cellular cytoxicity. In addition, albumin significantly upregulated the secretion of both fibronectin and TGF‐β1 at dose over 1 mg/ml. Moreover, 24 h pretreatment of albumin significantly enhanced exogenous TGF‐β1‐induced secretion of fibronectin. These observations were reminiscent of the implications of TbetaR since TbetaR appears to correlate with the susceptibility of cellular fibrosis. We found that albumin significantly increased protein levels of type I TbetaR (TbetaRI) instead of type II receptors (TbetaRII). In addition, phosphorylation level of TbetaRII of both pY259 and pY424 was significantly enhanced instead of pY336. The novel observation indicates that extreme dose of albumin upregulates TGF‐β autocrine loop by upregulating TGF‐β1, TbetaRI, and the receptor kinase activity of TbetaRII by inducing tyrosine phosphorylation on key amino residue of TbetaRII in renal distal tubule cells. These combinational effects might contribute to the pathogenesis of renal fibrosis. J. Cell. Biochem. 97: 956–968, 2006.

The role of IL-7 in renal proximal tubule epithelial cells fibrosis

BACKGROUND Hyperglycemia is the most important risk factor in the progression of renal fibrosis in diabetic kidney. Based on previous studies, interleukin-7 (IL-7) may exert antifibrotic activities in pulmonary fibrosis model. However, the role of IL-7 in the pathogenesis of renal tubulointerstitial fibrosis remains unclear. Thus, we hereby elucidate the effects of IL-7 in cultured renal proximal tubular epithelial cells (designated as HK-2) treated under hyperglycemic condition. METHODS Cells were cultured in high glucose (27.5mM) for 2 days. Different concentration of IL-7 (10, 50, 100 or 200ng/ml) was added in the last 24h of culture. ELISA was used to evaluate the secreted protein such as fibronectin and TGF-β(1). Western blot was used to examine the EMT marker (including α-smooth muscle actin (α-SMA) and E-cadherin), signal transducer (including Smad Smad2/3 and Smad7) and EMT initiator (e.g. Snail). Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin and Snail). RESULTS We found that IL-7 significantly attenuated high glucose-inhibited cellular growth and high glucose-induced fibrosis. More importantly, high glucose-induced up-regulation of fibronectin, TGF-β, TGF-β RII and pSmad2/3 was markedly inhibited by IL-7. On the contrary, high glucose-induced down-regulation of Smad7 was significantly reversed by IL-7 instead. IL-7 markedly inhibited high glucose-induced increase in α-SMA and Snail and decrease in E-cadherin. CONCLUSION We demonstrate that IL-7 has the potential to inhibit high glucose-induced renal proximal tubular fibrosis partly by modulating Smads and EMT pathway.

Abbott RealTime HBV Assay Is More Sensitive in Detection of Low Viral Load and Little Impacted by Drug Resistant Mutation in Chronic Hepatitis B Patients under Nucleot(s)ide Analogues Therapy

Background Selection of drug-resistant strains may lead to failure of HBV antiviral therapy. There is little information whether there is detection difference in drug resistant mutations between different viral load assays of HBV. Objectives This study is aimed to investigate whether there is drug-resistant strains related detection difference between Abbott RealTime HBV (RealTime) and CobasAmpliPrep/CobasTaqMan HBV assays 2.0 (TaqMan). Study Design One hundred and thirty-four CHB patients who received HBV anti-viral therapy were enrolled. HBV virological markers were tested 3 months apart regularly. Serum HBV DNA levels were determined using the TaqMan and RealTime. YMDD (rt180M and rt204V) mutation was checked in patients who experienced virologic breakthrough (VBT). Results The correlation of HBV DNA observed between the RealTime and TaqMan was good for all 571 samples (R2 = 0.797; P<0.001). However, the correlation in the 434 samples with HBV DNA level <3 log10 IU/ml was not as good as in all samples (R2 = 0.457). Overall, 21.5% of samples had a detection difference of ≥1 log10 IU/ml with 91.9% of these having HBV DNA level <3 log10 IU/ml. Twenty-four patients experiencedVBT. Three of these patients had acquired the YMDD mutation and exhibited discordant viral load results between the two methods tested. In each case, persistent HBV DNA was detected by RealTime and undetectable with TaqMan. Of the patients who experienced a VBT and had acquired YMDD mutation, 4.7% had undetectable HBV DNA by TaqMan while all were detectable with RealTime. Conclusions RealTime assay is more sensitive and is little impacted by the development of drug resistant mutation.

20-Hydroxyecdysone attenuates TGF-β1-induced renal cellular fibrosis in proximal tubule cells

UNLABELLED Renal fibrosis progresses to end stage of diabetes kidney disease, which causes irreversible progressive proximal tubular injury. In a previous study, 20-hydroxyecdysterone (20-HE), a phytoecdysteroid, attenuated renal injury in diabetes models. However, the fibrosis regulatory role remains to be investigated. METHODS The proximal tubular epithelial cells (designated as HK-2) were treated for 48 h with TGF-β1 (5 ng/ml) in different concentrations of 20-HE (0 to 500 nM/ml) in the last 24 h of culture. The extracellular fibronectin was measured by ELISA assay. Western blot and immunofluorescence were used to evaluate the expression of TGF-β1/Smads transducer (including Smad2/3, 4, and 7), epithelial and mesenchymal markers (e.g. E-cadherin and α-smooth muscle actin) and Snail (transcriptional regulators for EMT). RESULTS 20-HE reverses TGF-β1-induced increase in fibronectin (both intracellular and extracellular fibronectin). Simultaneously, 20-HE reverses TGF-β1-induced down-regulation of Smad7. In addition, 20-HE significantly attenuates TGF-β1-induced upregulation of Smad2/3 and pSmad2/3, and downregulation of E-Cadherin. Moreover, 20-HE dramatically suppresses TGF-β1-induced increases in the expression of Snail. CONCLUSION We propose that 20-HE is a potential fibrosis antagonist for renal proximal tubule cells. 20-HE might act through suppressing post-receptor signaling of TGF-β1 and restoring tubule epithelial character by blocking the expression of Snail.

Hyperosmolarity enhanced susceptibility to renal tubular fibrosis by modulating catabolism of type I transforming growth factor‐β receptors

Hyperosmolarity plays an essential role in the pathogenesis of diabetic tubular fibrosis. However, the mechanism of the involvement of hyperosmolarity remains unclear. In this study, mannitol was used to evaluate the effects of hyperosmolarity on a renal distal tubule cell line (MDCK). We investigated transforming growth factor‐β receptors and their downstream fibrogenic signal proteins. We show that hyperosmolarity significantly enhances the susceptibility to exogenous transforming growth factor (TGF)‐β1, as mannitol (27.5 mM) significantly enhanced the TGF‐β1‐induced increase in fibronectin levels compared with control experiments (5.5 mM). Specifically, hyperosmolarity induced tyrosine phosphorylation on TGF‐β RII at 336 residues in a time (0–24 h) and dose (5.5–38.5 mM) dependent manner. In addition, hyperosmolarity increased the level of TGF‐β RI in a dose‐ and time‐course dependent manner. These observations may be closely related to decreased catabolism of TGF‐β RI. Hyperosmolarity significantly downregulated the expression of an inhibitory Smad (Smad7), decreased the level of Smurf 1, and reduced ubiquitination of TGF‐β RI. In addition, through the use of cycloheximide and the proteasome inhibitor MG132, we showed that hyperosmolarity significantly increased the half‐life and inhibited the protein level of TGF‐β RI by polyubiquitination and proteasomal degradation. Taken together, our data suggest that hyperosmolarity enhances cellular susceptibility to renal tubular fibrosis by activating the Smad7 pathway and increasing the stability of type I TGF‐β receptors by retarding proteasomal degradation of TGF‐β RI. This study clarifies the mechanism underlying hyperosmotic‐induced renal fibrosis in renal distal tubule cells. J. Cell. Biochem. 109: 663–671, 2010.

shRNA for Thymic Stromal Lymphopoietin: A Novel Therapeutic Approach for Pulmonary Fibrosis

Thymic stromal lymphopoietin (TSLP) was recently identified as a master switch for Th2 responses. This study discusses the role of TSLP in pulmonary fibrosis. We show that TGF-β1 (a Fibrogenic Growth Factor) up regulates TSLP proteins in human lung fibroblasts (HFL-1) on a dose- and time-course-dependent basis. Additionally, TSLP increases fibronectin expression on a dose- (1 ng/ml to 100 ng/ml) and time-course-dependent basis concomitantly with the upregulation of pSmad2/3 and Smad4, which is the essential downstream signal regulator for TGF-β. Silencing TSLP by TSLP shRNA dramatically reverses TGF-β1-induced cellular fibrosis concomitantly with the suppression of type I TGF-β receptors and pSmad2/3. Parallel results are observed in vivo. Bleomycin-treated C57BL/6 mice show intense staining for TSLP in fibrotic lung tissue by immunohistochemistry. More importantly, Sirius red and H&E staining from bleomycin-treated mice demonstrate that transfection with TSLP shRNA (by intranasal instillation) dramatically decreases both infiltration of inflammatory cells and deposition of collagen compared to the control. Moreover, a whole-body plethysmography test showed that TSLP shRNA transgenic mice significantly attenuate the increase in airway respiratory resistance induced by bleomycin. Thus, it may be possible to use TSLP shRNA as a novel therapeutic approach for treating pulmonary fibrosis by down-regulating TGF-β signal proteins.

Performance characteristics of a real-time RT-PCR assay for quantification of hepatitis C virus RNA in patients with genotype 1 and 2 infections

Abstract Background: Polymerase chain reaction (PCR) methods play an essential role in providing data relating to diagnosis, monitoring and treatment of hepatitis C virus (HCV) infection. The real-time reverse transcription PCR (RT-PCR) assay is an established and promising tool in terms of quantifying HCV RNA for clinical application. This study aimed to evaluate the performance characteristics of a real-time RT-PCR-based test in a clinical setting. Methods: Validation and reproducibility tests were performed using a standard panel. Sera from 197 chronic HCV patients were analyzed by the real-time RT-PCR assay and the results were compared with the Versant bDNA3.0 assay (bDNA3.0). Results: The real-time RT-PCR assay showed an acceptable linear response (r2=0.989–0.995) in the serial dilutions regarding genotypes 1b, 2a, 2b and 1b+2a. HCV viral loads were quantifiable in all 197 patients (100%) by the real-time RT-PCR assay and in 194 (98.5%) by the bDNA3.0. HCV RNA quantification values measured by the real-time RT-PCR and bDNA3.0 assays were positively correlated (Pearsons correlation coefficient r=0.734, p<0.001). The real-time RT-PCR assay values were on average 0.13 logs higher than the bDNA3.0 results. The correlation coefficients with genotypes 1b, 2a, 2b and mixed were 0.737, 0.711, 0.791 and 0.766, respectively (p<0.01). Conclusions: The real-time RT-PCR assay showed comparable performance with bDNA3.0 regarding quantification of HCV viral loads with genotype 1 and 2 HCV infections. Clin Chem Lab Med 2008;46:475–80.