Yung Ho Hsu

Leptin reduces gentamicin-induced apoptosis in rat renal tubular cells via the PI3K-Akt signaling pathway

Leptin, a circulating hormone secreted mainly from adipose tissues, possesses protective effects on many cell types. Serum leptin concentration increases in patients with chronic renal failure and those undergoing maintenance dialysis. Gentamicin, a widely used antibiotic for the treatment of bacterial infection, can cause nephrotoxicity. In the present study, we intended to investigate the influence of leptin on apoptotic pathways and its mechanism in rat renal tubular cells treated with gentamicin. By using Annexin V-FITC/propidium iodide double staining, we found that leptin expressed a dose-dependent protective effect against gentamicin-induced apoptosis in rat renal tubular cells (NRK-52E) within 24h. Pretreatment of the cells with 50 or 100 ng/ml of leptin induced Bcl-2 and Bcl-x(L), increased the phosphorylation of Bad, and decreased the cleaved caspase-3 and caspase-9 in gentamicin-treated NRK-52E cells. Leptin also suppressed the activation of the transcription factor NF-κB and upregulated Akt activation in gentamicin-treated NRK-52E cells. We found that leptin activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway as demonstrated by the suppression of the anti-apoptotic effect of leptin by wortmannin. The treatment of wortmannin suppressed the leptin-induced phospho-Akt, Bcl-2, phospho-Bad as well as Bcl-x(L), and recovered the leptin-reduced cleaved caspase-3 and caspase-9. Based on our results, we suggested that leptin can attenuate gentamicin-induced apoptotic injury in rat renal tubular cells through PI3K/Akt signaling pathway.

Peroxisomal proliferator-activated receptor-α protects renal tubular cells from doxorubicin-induced apoptosis

Peroxisome proliferator-activated receptor-α (PPAR-α) is a transcription factor and has been reported to inhibit cisplatin-mediated proximal tubule cell death. In addition, doxorubicin (Adriamycin)-induced nephrosis in rats is a commonly used experimental model for pharmacological studies of human chronic renal diseases. In this study, we investigated the protective effect of PPAR-α on doxorubicin-induced apoptosis and its detailed mechanism in NRK-52E cells and animal models. The mRNA level of PPAR-α was found to be reduced by doxorubicin treatment in NRK-52E cells. PPAR-α overexpression in NRK-52E cells significantly inhibited doxorubicin-induced apoptosis and the quantity of cleaved caspase-3. Endogenous prostacyclin (PGI2) augmentation, which has been reported to protect NRK-52E cells from doxorubicin-induced apoptosis, induced the translocation and activation of PPAR-α. The transformation of PPAR-α short interfering RNA was applied to silence the PPAR-α gene, which abolished the protective effect of PGI2 augmentation in doxorubicin-treated cells. To confirm the protective role of PPAR-α in vivo, PPAR-α activator docosahexaenoic acid (DHA) was administered to doxorubicin-treated mice, and it has been shown to significantly reduce the doxorubicin-induced apoptotic cells in renal cortex. However, this protective effect of DHA did not exist in PPAR-α-deficient mice. In NRK-52E cells, the overexpression of PPAR-α elevated the activity of catalase and superoxide dismutase and inhibited doxorubicin-induced reactive oxygen species (ROS). PPAR-α overexpression also inhibited the doxorubicin-induced activity of nuclear factor-κB (NF-κB), which was associated with the interaction between PPAR-α and NF-κB p65 subunit as revealed in immunoprecipitation assays. Therefore, PPAR-α is capable of inhibiting doxorubicin-induced ROS and NF-κB activity and protecting NRK-52E cells from doxorubicin-induced apoptosis.

Far-infrared therapy induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in human umbilical vein endothelial cells.

Many studies suggest that far-infrared (FIR) therapy can reduce the frequency of some vascular-related diseases. The non-thermal effect of FIR was recently found to play a role in the long-term protective effect on vascular function, but its molecular mechanism is still unknown. In the present study, we evaluated the biological effect of FIR on vascular endothelial growth factor (VEGF)-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that FIR ranging 3∼10 µm significantly inhibited VEGF-induced proliferation in HUVECs. According to intensity and time course analyses, the inhibitory effect of FIR peaked at an effective intensity of 0.13 mW/cm2 at 30 min. On the other hand, a thermal effect did not inhibit VEGF-induced proliferation in HUVECs. FIR exposure also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinases in HUVECs. FIR exposure further induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO generation in VEGF-treated HUVECs. Both VEGF-induced NO and reactive oxygen species generation was involved in the inhibitory effect of FIR. Nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin abolished the FIR-induced phosphorylation of eNOS and Akt in HUVECs. FIR exposure upregulated the expression of PI3K p85 at the transcriptional level. We further found that FIR exposure induced the nuclear translocation of promyelocytic leukemia zinc finger protein (PLZF) in HUVECs. This induction was independent of a thermal effect. The small interfering RNA transfection of PLZF blocked FIR-increased PI3K levels and the inhibitory effect of FIR. These data suggest that FIR induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in HUVECs.

Peroxisome proliferator-activated receptor alpha plays a crucial role in l-carnitine anti-apoptosis effect in renal tubular cells

BACKGROUND L-carnitine is synthesized mainly in the liver and kidneys from lysine and methionine from dietary sources. Many reports have shown that L-carnitine can protect certain cells against the toxicity of several anticancer and toxic agents, although the detailed mechanism is poorly understood. In this study, we investigated the protective effect of L-carnitine and its molecular mechanism in renal tubular cells undergoing gentamicin-induced apoptosis. METHODS Rat tubular cell line (NRK-52E) and mice were used as the model system. Gentamicin-induced apoptosis in renal tubular cells was examined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling. We introduced short interfering RNA transfection and gene-deficient mice to investigate the protective mechanism of L-carnitine. RESULTS We found that L-carnitine inhibited gentamicin-induced reactive oxygen species generation and correlative apoptotic pathways, resulting in the protection of NRK-52E cells from gentamicin-induced apoptosis. The treatment of L-carnitine also lessened gentamicin-induced renal tubular cell apoptosis in mice. L-carnitine was found to increase the prostacyclin (PGI(2)) generation in NRK-52E cells. The siRNA transfection for PGI(2) synthase significantly reduced L-carnitine-induced PGI(2) and L-carnitines protective effect. We found that the activity of the potential PGI(2) nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARalpha), was elevated by L-carnitine treatment. The siRNA-mediated blockage of PPARalpha considerably reduced the anti-apoptotic effect of L-carnitine. In PPARalpha-deficient mice, L-carnitine treatment also lost the inhibitory effect on gentamicin-induced apoptosis in kidneys. CONCLUSIONS Based on these findings, we suggest that L-carnitine protects renal tubular cells from gentamicin-induced apoptosis through PGI(2)-mediated PPARalpha activation.

MicroRNA-328 inhibits renal tubular cell epithelial-to-mesenchymal transition by targeting the CD44 in pressure-induced renal fibrosis

Epithelial-mesenchymal transition (EMT) occurs in stressed tubular epithelial cells, contributing to renal fibrosis. Initial mechanisms promoting EMT are unknown. Pressure force is an important mechanism contributing to the induction and progression of renal fibrogenesis in ureteric obstruction. In our study of cultured rat renal tubular cells (NRK-52E) under 60 mmHg of pressure, we found that the epithelial marker E-cadherin decreased and mesenchymal markers, e.g., α-smooth muscle actin, fibronectin and Snail, increased. Pressure also induced the expression of connective tissue growth factor and transforming growth factor-β. MicroRNA array assays showed that pressure reduced miR-328 at the initial stage of pressurization. We identified a potential target sequence of miR-328 in rat CD44 3′-untranslated regions. In contrast with the miR-328 expression, CD44 expression was up-regulated at the initial pressurization stage. We also found that miR-328 expression decreased and CD44 increased in ureteric obstruction kidneys in the animal study. CD44 siRNA transfection significantly increased E-cadherin expression and inhibited pressure-induced EMT. Both hyaluronan binding peptide pep-1 and osteopontin neutralizing antibody inhibited pressure-induced EMT. Our results suggest that miR-328-mediated CD44 transient upregulation is an important trigger of the pressure-induced EMT in renal fibrosis.

Rosuvastatin inhibits pressure-induced fibrotic responses via the expression regulation of prostacyclin and prostaglandin E2 in rat renal tubular cells

Statins are reported to alleviate renal fibrosis in animal models with ureteral obstruction. However, the molecular mechanism of this antifibrotic effect is still unclear. Pressure force is an important mechanism contributing to induction and progression of tubulointerstitial fibrogenesis in ureteric obstruction. In this study, we investigated the influence of rosuvastatin on pressure-induced fibrotic responses in rat renal tubular cells (NRK-52E). We established an in vitro pressure culture system to study pressure-induced fibrotic responses in NRK-52E cells. When NRK-52E cells were cultured in the pressure culture system, 60 mm Hg of pressure induced the expression of connective tissue growth factor (CTGF), transforming growth factor (TGF)-β, fibronectin, Smad3, and phospho-Smad3. Rosuvastatin significantly reduced these pressure-induced fibrotic responses at concentrations above 10 μM. Rosuvastatin also reduced the TGF-β-induced expression of fibronectin and CTGF in NRK-52E cells. Pretreatment with rosuvastatin significantly induced prostacyclin (PGI(2)) generation, but reduced pressure-induced prostaglandin E(2) (PGE(2)). PGI(2) synthase small interfering RNA (siRNA) transfection significantly inhibited rosuvastatin-induced peroxisome proliferator-activated receptor α activation. The blockage of peroxisome proliferator-activated receptor α by siRNA transfection reduced the inhibitory effect of rosuvastatin on pressure-induced fibrotic responses. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398), a specific inhibitor of cyclooxygenase-2, diminished pressure-induced PGE(2) generation, and also reduced pressure-induced fibrotic responses. Additionally, PGE(2) decreased the antifibrotic effect of rosuvastatin. In conclusion, rosuvastatin reduces pressure-induced fibrotic responses in renal tubular cells by enhancing the PGI(2)-peroxisome proliferator-activated receptor α pathway and reducing PGE(2) generation.

Increased Long-Term Risk of Dementia in Patients With Carbon Monoxide Poisoning: A Population-Based Study.

Abstract Carbon monoxide (CO) poisoning may cause toxicity of the central nervous system and heart. However, the association between CO poisoning and long-term dementia risk remains unestablished. We investigated the incidence of dementia in patients with CO poisoning in Taiwan and evaluated whether they had a higher risk of dementia than did the general population. A nationwide population-based cohort study was conducted among patients with CO poisoning identified using Taiwans National Health Insurance Research Database (NHIRD) during 2004 to 2013. CO poisoning was defined according to the International Classification of Diseases, Ninth Revision, Clinical Modification codes. The study cohort comprised patients with CO poisoning between 2005 and 2010 (N = 14,590). Each patient was age-, sex-, and index date-matched with 4 randomly selected controls from the comparison cohort (N = 58,360). All patients were followed from the study date until dementia development, death, or the end of 2013. Cox proportional hazards regressions were performed for comparing the hazard ratios for dementia between the 2 cohorts. Incident cases of dementia were identified from the NHIRD. After adjustment for potential confounders, the study cohort was independently associated with a higher dementia risk (adjusted hazard ratio, 2.75; 95% confidence interval, 2.26–3.35). This population-based cohort study indicated that patients with CO poisoning have a higher risk of dementia than do people without CO poisoning.

Protective effects of Zhibai Dihuang Wan on renal tubular cells affected with gentamicin-induced apoptosis

ETHNOPHARMACOLOGICAL RELEVANCE Zhibai Dihuang Wan (ZDW) is an ancient traditional Chinese medicine composed of eight herbal ingredients and has been used to treat chronic kidney inflammation and diabetes for thousands of years. Nonetheless, the influence of ZDW on acute kidney injury is still unknown. We intended to identify the influence of ZDW on cell growth and gentamicin-induced apoptotic injury in renal tubular cells. MATERIALS AND METHODS We extracted ZDW with artificial intestinal fluid and treated rat renal tubular cells (NRK-52E) with various concentrations of the ZDW extraction. Cell proliferation and gentamicin-induced apoptosis of NRK-52E cells were evaluated using real-time proliferation monitoring and annexin V staining, respectively. Western blotting was used to evaluate the levels of Bcl-2 and caspase-3 expression. The effect of ZDW on gentamicin-induced kidney injury was also monitored in mice using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay, and the measurement of serum creatinine and blood urea nitrogen. RESULTS We found that 30 μg/ml of ZDW promoted cell proliferation of the rat renal tubular cells. ZDW also expressed a dose-dependent protective effect against gentamicin-induced apoptosis in the cells. Pretreatment with 3 μg/ml or 30 μg/ml of ZDW maximally increased Bcl-2 and decreased cleaved caspase-3 in the gentamicin-treated NRK-52E cells. Among the herbal ingredients of ZDW, only Phellodendron amurense Rupr., bark (Cortex Phellodendri), and Anemarrhena asphodeloides Bunge, rhizome inhibited both the gentamicin-induced Bcl-2 decrease and cleaved caspase-3 increase. Phellodendron amurense Rupr., bark and Anemarrhena asphodeloides Bunge, rhizome also inhibited gentamicin-induced apoptosis at particular concentrations; however, these two ingredients were less effective than ZDW. In the mouse model of gentamicin-induced nephropathy, the ZDW treatment significantly reduced apoptotic cells in the renal cortex and improved renal function. CONCLUSIONS Our results suggest that ZDW at adequate doses attenuates gentamicin-induced apoptotic injury in renal tubular cells and also protects kidneys from gentamicin-induced injury in mice.

Urotensin II induces transactivation of the epidermal growth factor receptor via transient oxidation of SHP-2 in the rat renal tubular cell line NRK-52E

Urotensin-II (UII) is a potent vasoactive peptide that has been implicated in cardiac fibrosis and renal diseases. However, the role played by UII in renal tissues is largely unknown. In this study, we investigated the effects of human UII (hUII) on rat renal proximal tubular cells of the NRK-52E line and the role of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) in the hUII-induced transactivation of the epidermal growth factor receptor (EGFR). Exposure to hUII at low concentrations significantly induced proliferation in NRK-52E cells; this effect was inhibited by treatment with an ERK1/2 inhibitor (PD98059). UII treatment increased the phosphorylation of EGFR and induced the generation of reactive oxygen species (ROS). Treatment of the ROS scavenger N-acetyl-cysteine (NAC) inhibited EGFR transactivation and ERK phosphorylation induced by hUII. SHP-2 was found to interact with EGFR and be transiently oxidized following the hUII treatment. In SHP-2 knockdown cells, UII-induced phosphorylation of EGFR was less influenced by NAC, and significantly suppressed by heparin binding (HB)-EGF neutralizing antibody. Our data suggest that the ROS-mediated oxidation of SHP-2 is essential for the hUII-induced mitogenic pathway in NRK-52E cells.

Urotensin II exerts antiapoptotic effect on NRK-52E cells through prostacyclin-mediated peroxisome proliferator-activated receptor alpha and Akt activation.

Urotensin II (UII) is a cyclic vasoactive peptide which is mainly expressed in kidneys. Although elevated plasma UII levels are associated with renal impairment, the influence of UII on renal injury is unclear. In this study, we monitored the influence of UII on gentamicin-induced apoptosis in rat tubular cells (NRK-52E). We found that UII significantly reduced gentamicin-induced apoptosis and apoptotic signals. Blocking endogenous UII secretion caused cells to be more susceptible to gentamicin. In gentamicin-treated mice, UII also expressed protective effect on renal tubular cells. UII was also found to induce prostacyclin (PGI2) production, which caused peroxisomal proliferator-activated receptor α (PPARα) activation as revealed by both PGI2 synthase siRNA transfection and piroxicam treatment. Blockage of PPARα by siRNA transfection inhibited UII-induced Akt phosphorylation and the antiapoptotic effect of UII. Our results suggest that UII can protect renal tubular cells from gentamicin-induced apoptosis through PGI2-mediated PPARα and Akt activation.