Yuh Mou Sue

The secreted Klotho protein restores phosphate retention and suppresses accelerated aging in Klotho mutant mice

Klotho was identified as the responsible gene in a mutant mouse line whose disruption results in a variety of premature aging-related phenotypes. Nonetheless, the related mechanisms were still unknown. Many studies report that dietary phosphate restriction and genetic ablation of vitamin D pathways indirectly reverse premature aging processes in these mice. Furthermore, transgenic overexpression of klotho in mice extends their life span through inhibition of insulin and IGF1 signaling. We found that intraperitoneal injection of recombinant soluble Klotho protein at dose of 0.02 mg/kg every other day effectively extends the life span of kl/kl mice by 17.4%. Soluble Klotho administration also ameliorated premature aging-related phenotype, such as growth retardation, premature thymus involution and vascular calcification, and effectively enhanced urinary phosphate excretion in kl/kl mice. Klotho treatment attenuated renal fibrosis through down-regulation of transforming growth factor-β signaling as well as reduced cellular senescence through down-regulation of p21-cip1 mRNA levels. In addition, soluble Klotho treatment significantly reduced both renal and aorta calcium deposits. In conclusion, our study shows the therapeutic potential of soluble Klotho protein to treat age-related disorders in mice.

Antioxidation and anti-inflammation by haem oxygenase-1 contribute to protection by tetramethylpyrazine against gentamicin-induced apoptosis in murine renal tubular cells

BACKGROUND Gentamicin, a widely used antibiotic for the treatment of bacterial infection, can cause nephrotoxicity. Tetramethylpyrazine (TMP) is a compound purified from the rhizome of Ligusticum wallichi (called chuanxiong in Chinese). Besides its protection against ischaemia-reperfusion injury and nephritis in mice, we previously reported that TMP reverses gentamicin-induced apoptosis in rat kidneys. Haem oxygenase-1 (HO-1) induction by TMP has also been shown to attenuate myocardial ischaemia/reperfusion injury in rats. METHODS We used rat renal tubular (NRK-52E) cells, transformed cells with HO-1 overexpression or knockdown, and an adenovirus carrying the HO-1 gene (Adv-HO-1) as gene therapy targeting murine kidneys to explore the role of HO-1 in protection by TMP against gentamicin-induced toxicity both in vitro and in vivo. We evaluated the protective effects of HO-1 on several apoptotic parameters induced by gentamicin: cleaved caspases-3 and -9, cycloxygenase-2 (Cox-2) and subcellular localization of nuclear factor kappa B-p65 (NF-kappaB-p65), Bcl-xl and HS-1-associated protein (Hax-1) in NRK-52E cells. RESULTS NRK-52E cells treated with TMP exhibited transcriptional upregulation of the HO-1 protein by approximately twofold. Overexpression of HO-1 in NRK-52E cells significantly increased mitochondrial protein levels of the antiapoptotic molecules, Bcl-xL and Hax-1, and markedly decreased the NADPH oxidase activity and proinflammatory molecules, NF-kappaB-p65 and Cox-2, which might decrease gentamicin-induced activation of caspases-9 and -3. Conversely, NRK-52E cells with HO-1 knockdown significantly exacerbated gentamicin-induced tubular cell apoptosis. Additionally, the concomitant HO-1 induction by TMP was also evident in vivo, and HO-1 therapy markedly attenuated gentamicin-induced renal apoptosis to a similar extent as TMP pretreatment. CONCLUSIONS Collectively, we suggest that HO-1 induced by TMP might, at least in part, protect against gentamicin-induced nephrotoxicity through antiapoptotic and anti-inflammatory mechanisms, and that it may have therapeutic potential for patients with renal disease. This is also the first demonstration that HO-1 increases Hax-1 mitochondrial localization.

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.

PPARδ-mediated p21/p27 induction via increased CREB-binding protein nuclear translocation in beraprost-induced antiproliferation of murine aortic smooth muscle cells

We previously showed that an increase in the peroxisome proliferator-activated receptor-delta (PPARdelta), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPARdelta activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPARdelta activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPARdelta interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPARdelta activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS.

Activation of a nuclear factor of activated T-lymphocyte-3 (NFAT3) by oxidative stress in carboplatin-mediated renal apoptosis

BACKGROUND AND PURPOSE Although carboplatin is currently used as a therapeutic drug for ovarian, breast, and non‐small cell lung cancers, it has serious side effects including renal and cardiac toxicity. Herein, we examined the effect of carboplatin on murine renal tubular cell (RTC) apoptosis both in vivo and in vitro and the underlying molecular mechanisms associated with its activation of the nuclear factor of activated T‐lymphocytes‐3 (NFAT3).

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.

Renin-angiotensin system blockade in heart failure patients on long-term haemodialysis in Taiwan

Heart failure is among the most frequent complications of patients on long‐term haemodialysis. The benefits of renin–angiotensin system (RAS) blockade on the outcomes of these patients have yet to be determined.