Jau-Shyang Huang

Effect of Lactobacillus reuteri GMNL-263 treatment on renal fibrosis in diabetic rats.

Hyperglycemia is the most important factor in the progression of renal fibrosis in diabetic kidney. Prevention and treatment of renal fibrosis may improve diabetic nephropathy. To explore whether probiotic Lactobacillus reuteri GMNL-263 treatment was linked to altered hyperglycemia-mediated renal fibrosis in diabetic kidney, the mechanisms of L. reuteri GMNL-263 treatment responsible for the inhibition of renal fibrosis in streptozotocin (STZ)-induced diabetic rats were examined. Diabetic rats were induced by intraperitoneal injection of STZ (50 mg/kg). Induction of diabetes was confirmed by measurement of the blood glucose using the glucose oxidase method, and hyperglycemic rats with levels >16 mmol/L were used. We found that L. reuteri GMNL-263 treatment caused reduction of glycated hemoglobin and blood glucose levels in STZ-induced diabetic rats for 28 days (all p<0.05). Treatment with L. reuteri GMNL-263 increased body weight but decreased kidney weight in diabetic rats as compared to diabetic control (p<0.05). In diabetic renal cortex, the Janus kinase 2/signal transducers and activators of transcription 1 (but not extracellular signal-regulated kinase/c-Jun N-terminal kinase/p38 mitogen-activated protein kinase) activation was markedly blocked by L. reuteri GMNL-263 treatment. The ability of L. reuteri GMNL-263 treatment to inhibit renal fibrosis was verified by the observation that it significantly decreased protein levels of plasminogen activator inhibitor-1, p21(Waf1/Cip1), α-smooth muscle actin, and fibronectin in diabetic renal cortex. The results obtained in this study indicate that L. reuteri GMNL-263 treatment may protect STZ-induced diabetic rats from hyperglycemia-enhanced renal fibrosis.

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.

Effects of Nitric Oxide and Antioxidants on Advanced Glycation End Products-Induced Hypertrophic Growth in Human Renal Tubular Cells

The accumulation of advanced glycation end products (AGE) is a key mediator of renal tubular hypertrophy in diabetic nephropathy (DN). Reactive oxygen species and nitric oxide (NO) were involved in the progression of DN. In this study, the molecular mechanisms of NO and antioxidants responsible for inhibition of AGE-induced renal tubular hypertrophy were examined. We found that AGE (but not nonglycated bovine serum albumin) significantly suppressed the NO/cGMP/PKG signaling in human renal proximal tubular cells. NO donors S-nitroso-N-acetylpenicillamine (SNAP)/sodium nitroprusside (SNP) and antioxidants N-acetylcysteine (NAC)/taurine treatments significantly attenuated AGE-inhibited NO production, cGMP synthesis, and inducible NO synthase/cGMP-dependent protein kinase (PKG) activation. Moreover, AGE-induced extracellular signal-regulated kinase/c-Jun N-terminal kinase/p38 mitogen-activated protein kinase activation was markedly blocked by antireceptor for AGE (RAGE), SNAP, SNP, NAC, and taurine. The abilities of NO and antioxidants to inhibit AGE/RAGE-induced hypertrophic growth were verified by the observation that SNAP, SNP, NAC, and taurine inhibited fibronectin, p21(Waf1/Cip1), and RAGE expression. Therefore, antioxidants significantly attenuated AGE/RAGE-enhanced cellular hypertrophy partly through induction of the NO/cGMP/PKG signaling.

Klotho attenuates high glucose-induced fibronectin and cell hypertrophy via the ERK1/2-p38 kinase signaling pathway in renal interstitial fibroblasts.

Although exogenous klotho attenuates renal fibrosis, it is not known if exogenous klotho attenuates diabetic nephropathy (DN). Thus, we studied the anti-fibrotic mechanisms of klotho in terms of transforming growth factor-β (TGF-β) and signaling pathways in high glucose (HG, 30 mM)-cultured renal interstitial fibroblast (NRK-49F) cells. We found that HG increased klotho mRNA and protein expression. HG also activated TGF-β Smad2/3 signaling and activated extracellular signal-regulated kinase (ERK1/2) and p38 kinase signaling. Exogenous klotho (400 pM) attenuated HG-induced TGF-β bioactivity, type II TGF-β receptor (TGF-βRII) protein expression and TGF-β Smad2/3 signaling. Klotho also attenuated HG-activated ERK1/2 and p38 kinase. Additionally, klotho and inhibitors of ERK1/2 or p38 kinase attenuated HG-induced fibronectin and cell hypertrophy. Finally, renal tubular expression of klotho decreased in the streptozotin-diabetic rats at 8 weeks. Thus, exogenous klotho attenuates HG-induced profibrotic genes, TGF-β signaling and cell hypertrophy in NRK-49F cells. Moreover, klotho attenuates HG-induced fibronectin expression and cell hypertrophy via the ERK1/2 and p38 kinase-dependent pathways.

Cinnamaldehyde impairs high glucose-induced hypertrophy in renal interstitial fibroblasts

Cinnamaldehyde is a major and a bioactive compound isolated from the leaves of Cinnamomum osmophloeum kaneh. To explore whether cinnamaldehyde was linked to altered high glucose (HG)-mediated renal tubulointerstitial fibrosis in diabetic nephropathy (DN), the molecular mechanisms of cinnamaldehyde responsible for inhibition of HG-induced hypertrophy in renal interstitial fibroblasts were examined. We found that cinnamaldehyde caused inhibition of HG-induced cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, cleaved poly(ADP-ribose) polymerase (PARP) protein expression, and mitochondrial cytochrome c release in HG or cinnamaldehyde treatments in these cells. HG-induced extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) (but not the Janus kinase 2/signal transducers and activators of transcription) activation was markedly blocked by cinnamaldehyde. The ability of cinnamaldehyde to inhibit HG-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of collagen IV, fibronectin, and alpha-smooth muscle actin (alpha-SMA). The results obtained in this study suggest that cinnamaldehyde treatment of renal interstitial fibroblasts that have been stimulated by HG reduces their ability to proliferate and hypertrophy through mechanisms that may be dependent on inactivation of the ERK/JNK/p38 MAPK pathway.

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‐β), 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 β‐hydroxybutyrate and in vivo using rats undergone unilateral ureteral obstruction (UUO). Western blotting was used to examine protein expression in TGF‐β‐related signal proteins such as type I and type II TGF‐β receptor, Smads2/3, pSmad2/3, Smads4, and Smads7. ELISA was used to analyze bioactive TGF‐β1 and fibronectin levels in the culture media. Safflower extract (SE) significantly inhibited β‐HB‐induced fibrosis in NRK cells concomitantly with dose‐dependent inhibition of the type I TGF‐β1 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‐β autocrine loop. Moreover, remarkably lower levels of tissue collagen were noted in the nephron and serum TGF‐β1 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‐β signals. J. Cell. Biochem. 104: 908–919, 2008.

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.

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/p27Kip1/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. J. Cell. Biochem. 116: 1028–1038, 2015.

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

Transforming growth factor-β (TGF-β) is pivotal in the pathogenesis of diabetic nephropathy. Type 1 TGF-β receptor (TGF-βR1) 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-βR1 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-βR1 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-βR1 protein expression and TGF-βR1 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-βR1 mRNA, p27(kip1) protein expression and growth inhibition were not affected by UCHL5 shRNA. Finally, glomerular UCHL5 and TGF-βR1 protein expression were increased in streptozotocin-diabetic rats at 8weeks. We conclude that PI3K-dependent UCHL5 is required for high glucose-induced TGF-βR1 protein expression in mesangial cells. UCHL5 is also required for high glucose-induced TGF-βR1 protein deubiquitination, p21(WAF1) and fibronectin protein expression and cell hypertrophy.