Yu-Lin Yang

Bone Morphogenetic Protein-2 Antagonizes Renal Interstitial Fibrosis by Promoting Catabolism of Type I Transforming Growth Factor-β Receptors

TGF-beta is a therapeutic target for renal fibrosis. Scientists have long sought ways to antagonize TGF-beta to ameliorate diabetic nephropathy. Bone morphogenetic protein (BMP-2) is a member of the TGF-beta superfamily and is highly regulated in the kidney. Thus, the role of BMP-2 was investigated in NRK-49F cells (rat fibroblasts). We showed that TGF-beta1 induces an increase in fibronectin. Treatment with exogenous BMP-2 or pCMV-BMP-2 significantly reversed the TGF-beta1-induced increase in fibronectin concomitant with a significant decrease in type I TGF-beta receptors (TGF-beta RI). Moreover, BMP-2 significantly shortened the half-life of TGF-beta RI. These results are related to proteosomal activation because MG132, a proteasome inhibitor, abolished BMP-2-mediated degradation of TGF-beta RI. This was confirmed because BMP-2 time course dependently enhanced the ubiquitination level of TGF-beta RI. In addition, Smads would seem to be involved in the interaction of BMP-2 and TGF-beta. We demonstrated that BMP-2 significantly reversed the TGF-beta1-induced increase in pSmad2/3 and reversed the TGF-beta1-induced decrease in inhibitory Smad7. Most importantly, Smad7 small interfering RNA abolished the BMP-2-induced decrease in TGF-beta RI. We evaluated the clinical efficacy of BMP-2 using unilateral ureteral obstruction rats. BMP-2 was administered ip for 7 d. In the unilateral ureteral obstruction kidneys, interstitial fibrosis was prominent. However, treatment with BMP-2 dramatically reduced Massons trichrome staining (collagen) in the interstitial and tubular areas of the kidneys concomitantly with a reduction in TGF-beta RI. These results suggest that BMP-2 acts as a novel fibrosis antagonizing cytokine partly by down-regulating TGF-beta RI and Smads.

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.

Advanced glycation end-product-induced mitogenesis and collagen production are dependent on angiotensin II and connective tissue growth factor in NRK-49F cells.

Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis. Angiotensin II (Ang II) and connective tissue growth factor (CTGF) are involved in the pathogenesis of DN, while Janus kinase 2 (JAK2) is important in advanced glycation end‐product (AGE)‐induced effects in renal interstitial (NRK‐49F) fibroblasts. Thus, we studied the role of Ang II, CTGF, and JAK2 in AGE‐induced effects in NRK‐49F cells. We found that AGE (150 μg/ml) increased mitogenesis and type I collagen production at 7 days while Ang II (10−7M) increased mitogenesis and type I collagen production at 3 days. We also found that AGE (150 μg/ml) increased angiotensinogen protein at 2 days, which was attenuated by AG‐490 (a JAK2 inhibitor). AGE (150 μg/ml) increased CTGF mRNA and protein expression at 3 and 5 days, respectively. Ang II (10−7M) increased CTGF mRNA and protein expression at 1 and 2 days, respectively, which were attenuated by AG‐490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE‐induced CTGF mRNA/protein expression while attenuating AGE‐induced mitogenesis and type I collagen production. AG‐490 and CTGF antisense (but not sense) oligodeoxynucleotide (ODN) attenuated Ang II (10−7M) and AGE‐induced mitogenesis and type I collagen production at 3 and 7 days, respectively. We concluded that AGE (150 μg/ml)‐induced mitogenesis and type I collagen production are dependent on the Ang II‐JAK2‐CTGF pathway in NRK‐49F cells. Moreover, Ang II‐induced mitogenesis and type I collagen production are dependent on the JAK2‐CTGF pathway.

pCMV-BMP-2-Transfected Cell-Mediated Gene Therapy in Anterior Cruciate Ligament Reconstruction in Rabbits

PURPOSE This study investigated the effect of plasmid cytomegalovirus (pCMV)-bone morphogenetic protein 2 (BMP-2) gene therapy on the healing of the tendon-bone interface after anterior cruciate ligament (ACL) reconstruction in rabbits. METHODS The pCMV-BMP-2 was synthesized from full-length human BMP-2 complementary deoxyribonucleic acid, followed by cloning into pCMV Script vector (Clontech Laboratories, Inc., San Jose, CA), and was delivered by a xenogeneic (rat kidney) cell line. The ACL was reconstructed by the transfer of extensor digital tendon in the proximal tibia. In the study group the pCMV-BMP-2 gene-transfected normal rat kidney cells mixed with calcium alginate gel were placed at the tendon-bone interface, whereas no pCMV-BMP-2 was used in the control group. The evaluations included radiography, bone mineral density, magnetic resonance imaging, biomechanical study, histologic examination, and immunohistochemical analysis. RESULTS Bone mineral density showed no significant difference between the groups (P > .05). Magnetic resonance imaging showed significantly better contact between tendon and bone in the study group compared with the control group (P < .0001). In the biomechanical study, significantly higher failure load and maximal graft tension were noted in the study group compared with the control group (P = .034). The modes of graft failure were rupture of the tendon proper in 78% and graft pullout from the bone tunnel in 22% of specimens in the study group versus graft rupture in 22% and graft pullout in 78% in the control group (P = .018). On histologic examination, the study group showed significantly better integration between tendon and bone, as well as more bone tissue around the tendon graft, than the control group (P = .0004). On immunohistochemical analysis, the study group showed significantly higher expressions of von Willebrand factor, vascular endothelial growth factor, proliferation cell nuclear antigen, and BMP-2 than the control group (P < .05). CONCLUSIONS The pCMV-BMP-2 gene therapy significantly improved the healing of tendon to bone and promoted angiogenesis and osteogenesis at the tendon-bone interface after ACL reconstruction in the rabbit model. CLINICAL RELEVANCE Application of pCMV-BMP-2 gene therapy may be an effective adjunct therapy in ACL reconstruction.

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 µg/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 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low‐dose hydrogen peroxide (5 µM) increased whereas high‐dose hydrogen peroxide (100 µM) 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. J. Cell. Biochem. 109: 38–48, 2010.

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.

CD36 is a novel and potential anti‐fibrogenic target in albumin‐induced renal proximal tubule fibrosis

Albumin is not only a risk factor for diabetic nephropathy (DN), but also a therapeutic target. Hence, scientists have long sought ways to elucidate the interactions between albumin and diabetic renal tubule fibrosis. CD36, a surface receptor for thrombospondin‐1, has been reported to interact with latent transforming growth factor‐beta1 (TGF‐β1) and activate its fibrogenic bioactivity. This study elucidates the interactions between CD36 and renal tubule fibrosis. LLC‐PK1 cells were applied to represent renal proximal tubule cells. The expression of CD36 was evaluated by flow cytometry. Fibronectin was assayed by Western blot and enzyme‐linked immunosorbent assay (ELISA). Bioactive TGF‐β1 was assayed by ELISA. We demonstrated that albumin was shown significantly to inhibit cell growth without affecting hypertrophy status since protein content and cell size remained unaffected under albumin treatment. Moreover, albumin dose‐dependently (0, 1, or 10 mg/ml) enhanced the secretion of bioactive TGF‐β1 and fibronectin with the upregulation of CD36. Intriguingly, CD36 siRNA, a potent silencer for CD36 effectively suppressed the albumin‐induced increase in CD36, TGF‐β1, and even fibronectin level. Accordingly, albumin is a pro‐fibrogenic factor for proximal tubule cells since albumin per se markedly upregulated the expression of TGF‐β1 and fibronectin. Most importantly, CD36 may mediate albumin‐induced cellular fibrosis since CD36 siRNA appeared to have anti‐fibrosis effects. This work suggests that CD36 is a novel and potential therapeutic target for diabetic renal tubule fibrosis. J. Cell. Biochem. 101: 735–744, 2007.

Computer-assisted pedicle screw placement for thoracolumbar spine fracture with separate spinal reference clamp placement and registration

BACKGROUND The objective of the study was to improve the accuracy of computer-assisted pedicle screw installation in the spine. This study evaluates the accuracy of computer-assisted pedicle screw placement with separate spinal reference clamp placement and registration on each instrumented vertebra for thoracolumbar spine fractures. METHODS Postoperative radiographs and CT scans assessed the accuracy of pedicle screw placement in 21 adult patients on each instrumented vertebra. Screw placements were graded as good if the screws were placed in the central core of the pedicle and the cancellous portion of the body. Screw placements were graded as fair if the screws were placed slightly eccentrically, causing erosion of the pedicular cortex, and with less than a 2-mm perforation of the pedicular cortex. Screw placements were graded as poor if screws were placed eccentrically with a large portion of the screw extending outside the cortical margin of the pedicle and with more than a 2-mm perforation of the pedicular cortex. RESULTS A total of 140 image-guided pedicle screws were placed in 21 patients: 78 in the thoracic and 62 in the lumbar spine. Of the 140 pedicle screw placements, 96.4% (135/140) were categorized as good; 3.6% (5/140), fair; and 0% were poor. All 5 fair placement screws were placed in the thoracic spine without any mobility. CONCLUSION Separate registration increases accuracy of screw placement in thoracolumbar pedicle instrumentation. Separate spinal reference clamp placement in the instrumented vertebra provides real-time virtual imaging that decreases the possibility of downward displacement during manual installation of the screw.

Leptin and connective tissue growth factor in advanced glycation end-product-induced effects in NRK-49F cells.

Previously, we showed that Janus kinase 2 (JAK2) is important in advanced glycation end‐product (AGE)‐induced effects in renal interstitial (NRK‐49F) fibroblasts. Leptin is a JAK2‐activating cytokine via the long form leptin receptor (Ob‐Rb). Leptin and connective tissue growth factor (CTGF) may be involved in renal fibrosis. However, the relationship between leptin and CTGF in terms of AGE‐induced effects remains unknown. Thus, the effects of AGE (150 μg/ml) and leptin on mitogenesis, CTGF and collagen expression in NRK‐49F cells were determined. We found that leptin and AGE increased mitogenesis and type I collagen protein expression at 3 and 7 days, respectively. AGE increased leptin mRNA and protein expression at 2–3 days. AGE increased CTGF mRNA and protein expression at 3–5 days. AG‐490 (JAK2 inhibitor) abrogated AGE‐induced leptin mRNA and protein expression at 2–3 days. AG‐490 and Ob‐Rb anti‐sense oligodeoxynucleotides (ODN) abrogated AGE‐induced CTGF mRNA and protein expression at 3–5 days. AG‐490 and CTGF anti‐sense ODN abrogated AGE‐induced mitogenesis and collagen protein expression at 7 days. Additionally, leptin dose (0.2–1 μg/ml) and time (1–2 days)‐dependently increased CTGF protein expression. AG‐490 abrogated leptin (1 μg/ml)‐induced CTGF protein expression at 2 days. AG‐490 and CTGF anti‐sense ODN abrogated leptin‐induced mitogenesis and collagen protein expression at 3 days. We concluded that AGE induced JAK2 to increase leptin while leptin induced JAK2 to increase CTGF‐induced mitogenesis and type I collagen protein expression in NRK‐49F cells. Additionally, AGE‐induced mitogenesis and type I collagen protein expression were dependent on leptin‐induced CTGF.