Hepatocyte Nuclear Factor-1ß Induces Redifferentiation of Dedifferentiated Tubular Epithelial Cells.

Tubular epithelial cells (TECs) can be dedifferentiated by repetitive insults, which activate scar-producing cells generated from interstitial cells such as fibroblasts, leading to the accumulation and deposition of extracellular matrix molecules. The dedifferentiated TECs play a crucial role in the development of renal fibrosis. Therefore, renal fibrosis may be attenuated if dedifferentiated TECs are converted back to their normal state (re-epithelialization). However, the mechanism underlying the re-epithelialization remains to be elucidated. In the present study, TGF-ß1, a profibrotic cytokine, induced dedifferentiation of cultured TECs, and the dedifferentiated TECs were re-epithelialized by the removal of TGF-ß1 stimulation. In the re-epithelialization process, transcription factor hepatocyte nuclear factor 1, beta (HNF-1ß) was identified as a candidate molecule involved in inducing re-epithelialization by means of DNA microarray and biological network analysis. In functional validation studies, the re-epithelialization by TGF-ß1 removal was abolished by HNF-1ß knockdown. Furthermore, the ectopic expression of HNF-1ß in the dedifferentiated TECs induced the re-epithelialization without the inhibition of TGF-ß/Smad signaling, even in the presence of TGF-ß1 stimulation. In mouse renal fibrosis model, unilateral ureteral obstruction model, HNF-1ß expression in the TECs of the kidney was suppressed with fibrosis progression. Furthermore, the HNF-1ß downregulated TECs resulted in dedifferentiation, which was characterized by expression of nestin. In conclusion, HNF-1ß suppression in TECs is a crucial event for the dedifferentiation of TECs, and the upregulation of HNF-1ß in TECs has a potential to restore the dedifferentiated TECs into their normal state, leading to the attenuation of renal fibrosis.

Ghrelin ameliorates catabolic conditions and respiratory dysfunction in a chronic obstructive pulmonary disease model of chronic cigarette smoke-exposed rats.

Cigarette smoking, which is a well-known major risk factor for chronic obstructive pulmonary disease (COPD), causes both pulmonary and extrapulmonary abnormalities. Ghrelin is a gastric peptide that regulates energy homeostasis. In the present study, we investigated the effects of ghrelin on the catabolic changes, respiratory function and emphysema in an animal model of COPD induced by chronic exposure to cigarette smoke. Rats were exposed to cigarette smoke, and they were administered human ghrelin (0.1 or 1 mg/kg, subcutaneous, twice daily) for 12 weeks. Compared with air-exposed rats, body weight gain, food intake, food efficiency, tidal volume, peak expiratory flow rate, and forced expiratory volume at 100 ms were significantly lower, while functional residual capacity, lung capacity, and neutrophils in bronchoalveolar lavage fluid were significantly higher in cigarette smoke-exposed rats. These indicated that the systemic abnormalities associated with COPD developed after the exposure to cigarette smoke. Ghrelin significantly and dose-dependently increased the body weight gain and food efficiency in cigarette smoke-exposed rats. In ghrelin-treated rats, skeletal muscle strength, which tended to be lowered by cigarette smoke exposure, was improved. Ghrelin ameliorated respiratory function and emphysema in a dose-dependent manner, but did not inhibit the increase in neutrophils in the bronchoalveolar lavage fluid. The respiratory functional parameters and lung capacity were significantly correlated with body weight gain. These results suggest that ghrelin inhibited the development of the catabolic changes, respiratory dysfunction, and emphysema that were induced by cigarette smoke exposure in rats, at least in part, through the amelioration of nutritional status.

Chronic exposure to cigarette smoke causes extrapulmonary abnormalities in rats.

Pathophysiological features of chronic obstructive pulmonary disease (COPD) include systemic abnormalities, such as weight loss and skeletal muscle wasting. Although cigarette smoke (CS) is a major risk factor in COPD, the systemic effects of CS exposure remain to be elucidated. In this study, rats were exposed to CS or smoke-free air for 12 weeks. CS-exposed rats developed emphysema and had significantly lower body weight and food intake than control rats. The plasma ghrelin levels significantly increased with an upregulation of gastric ghrelin mRNA expression induced by CS exposure. Further, we observed low plasma insulin-like growth factor-1 levels and high tumor necrosis factor-α levels. A significant reduction of skeletal muscle strength and an increase in the mRNA expression of catabolic factors was observed in CS-exposed rats. These results indicated that chronic CS exposure induced not only pulmonary emphysema but also systemic abnormalities related to muscle catabolism associated with inflammatory responses.

Organ-specific activation of the gastric branch of the efferent vagus nerve by ghrelin in urethane-anesthetized rats.

Ghrelin plays multiple physiological roles such as growth hormone secretion and exerting orexigenic actions; however, its physiological roles in the electrical activity of autonomic nerves remain unclear. Here, we investigated the effects of human ghrelin on several autonomic nerve activities in urethane-anesthetized rats using an electrophysiological method. Intravenous injection of ghrelin at 3 µg/kg significantly and transiently potentiated the efferent activity of the gastric vagus nerve; however, it did not affect the efferent activity of the hepatic vagus nerve. The activated response to ghrelin in the gastric efferent vagus nerve was not affected by the gastric afferent vagotomy, suggesting that this effect was not induced via the gastric afferent vagus nerve. Ghrelin did not affect the efferent activity of the brown adipose tissue, adrenal gland sympathetic nerve, and the renal sympathetic nerve. In addition, rectal temperature and the plasma concentrations of norepinephrine, corticosterone, and renin were also not changed by ghrelin. These findings demonstrate that ghrelin stimulates the gastric efferent vagus nerve in an organ-specific manner without affecting the gastric afferent vagus nerve and that ghrelin does not acutely affect the efferent basal activity of the sympathetic nerve in rats.

Ghrelin improves body weight loss and skeletal muscle catabolism associated with angiotensin II-induced cachexia in mice.

Ghrelin is a gastric peptide that regulates energy homeostasis. Angiotensin II (Ang II) is known to induce body weight loss and skeletal muscle catabolism through the ubiquitin-proteasome pathway. In this study, we investigated the effects of ghrelin on body weight and muscle catabolism in mice treated with Ang II. The continuous subcutaneous administration of Ang II to mice for 6 days resulted in cardiac hypertrophy and significant decreases in body weight gain, food intake, food efficiency, lean mass, and fat mass. In the gastrocnemius muscles of Ang II-treated mice, the levels of insulin-like growth factor 1 (IGF-1) were decreased, and the levels of mRNA expression of catabolic factors were increased. Although the repeated subcutaneous injections of ghrelin (1.0mg/kg, twice daily for 5 days) did not affect cardiac hypertrophy, they resulted in significant body weight gains and improved food efficiencies and tended to increase both lean and fat mass in Ang II-treated mice. Ghrelin also ameliorated the decreased IGF-1 levels and the increased mRNA expression levels of catabolic factors in the skeletal muscle. IGF-1 mRNA levels in the skeletal muscle significantly decreased 24h after Ang II infusion, and this was reversed by two subcutaneous injections of ghrelin. In C2C12-derived myocytes, the dexamethasone-induced mRNA expression of atrogin-1 was decreased by IGF-1 but not by ghrelin. In conclusion, we demonstrated that ghrelin improved body weight loss and skeletal muscle catabolism in mice treated with Ang II, possibly through the early restoration of IGF-1 mRNA in the skeletal muscle and the amelioration of nutritional status.

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin.

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.

The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats.

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor 1a (GHS-R1a), and consists of 28 amino acid residues with octanoyl modification at Ser(3). The previous studies have revealed that N-terminal part of ghrelin including modified Ser(3) is the active core for the activation of GHS-R1a. On the other hand, the role of C-terminal (8-28) region in ghrelin has not been clarified yet. In the present study, we prepared human ghrelin, C-terminal truncated ghrelin derivatives and anamorelin, a small molecular GHS compound which supposedly mimics the N-terminal active core, and examined GHS-R1a agonist activity in vitro, pharmacokinetic (PK) profile and growth hormone (GH) releasing activity in rats. All compounds demonstrated potent GHS-R1a agonist activities in vitro. Although the lack of C-terminal two amino acids did not modify PK profile and GH releasing activity, the deletion of C-terminal 8 and 20 amino acids affected them, and ghrelin(1-7)-Lys-NH(2) exhibited very short plasma half-life and low GH releasing activity in vivo. In rat plasma, ghrelin(1-7)-Lys-NH(2) was degraded more rapidly than ghrelin, suggesting that C-terminal part of ghrelin protected octanoylation of Ser(3) from plasma esterases. Subdiaphragmatic vagotomy significantly attenuated GH response to ghrelin but not to anamorelin. These results suggest that the C-terminal part of ghrelin has an important role in the biological activity in vivo. We also found that ghrelin stimulated GH release mainly via a vagal nerve pathway but anamorelin augmented GH release possibly by directly acting on brain in rats.

Cardiomyocyte proliferation and protection against post-myocardial infarction heart failure by cyclin D1 and Skp2 ubiquitin ligase.

AIMS: Cyclins and other cell-cycle regulators have been used in several studies to regenerate cardiomyocytes in ischaemic heart failure. However, proliferation of cardiomyocytes induced by nuclear-targeted cyclin D1 (D1NLS) stops after one or two rounds of cell cycles due in part to accumulation of p27Kip1, an inhibitor of cyclin-dependent kinase (CDK). Thus, expression of S-phase kinase-associated protein 2 (Skp2), a negative regulator of p27Kip1, significantly enhances the effect of D1NLS and CDK4 on cardiomyocyte proliferation in vitro. Here, we examined whether Skp2 can also improve cardiomyocyte regeneration and post-ischaemic cardiac performance in vivo. METHODS AND RESULTS: Wistar rats underwent ischaemia/reperfusion injury by ligation of the coronary artery followed by injection of adenovirus vectors for D1NLS and CDK4 with or without Skp2. Enhanced proliferation of cardiomyocytes in the presence of Skp2 was demonstrated by increased expression of Ki67, a marker of proliferating cells (1.95% vs. 4.00%), and mitotic phosphorylated histone H3 (0.24% vs. 0.58%). Compared with rats that received only D1NLS and CDK4, expression of Skp2 improved left ventricular function as measured by the maximum and minimum rates of change in left ventricular pressure, the left ventricle end-diastolic pressure, left ventricle end-diastolic volume index, and the lung/body weight ratio. CONCLUSION: Expression of Skp2 enhanced the effect of D1NLS and CDK4 on the proliferation of cardiomyocytes and further contributed to improved post-ischaemic cardiac function. Skp2 might be a versatile tool to improve the effect of cyclins on post-ischaemic regeneration of cardiomyocytes in vivo.

Involvement of MCP-1 in tubulointerstitial fibrosis through massive proteinuria in anti-GBM nephritis induced in WKY rats.

We investigated participation of monocyte chemoattractant protein-1 (MCP-1) in tubulointerstitial fibrosis and correlation between MCP-1 and proteinuria in Wistar-Kyoto (WKY) rats with glomerulonephritis induced by anti-glomerular basement membrane (anti-GBM) antibody. WKY rats showed marked proteinuria and severe glomerular crescent formation at 7 days post antibody injection. At 28 days, tubulointerstitial fibrotic lesions were observed, followed by sustained heavy proteinuria and severe tubulointerstitial fibrosis at 56 days. Histological examination revealed that the overlapped immunoreactivities of MCP-1, rat albumin, and p65NF-kappaB were detected in the same tubular segments of nephritic kidney, and a significant positive correlation was observed between proteinuria and MCP-1 expression in the tubulointerstitial fibrosis. ED-1- and CD8-positive cells were also abundant, and there was a good correlation between monocyte/macrophage recruitment and MCP-1 expression in the tubulointerstitial area. These results suggest that MCP-1 participates in the progression of tubulointerstitial fibrosis, through massive albuminuria, which is accompanied by marked monocyte/macrophage recruitment.

N-acetyl-seryl-aspartyl-lysyl-proline ameliorates the progression of renal dysfunction and fibrosis in WKY rats with established anti-glomerular basement membrane nephritis.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is hydrolyzed by angiotensin-converting enzyme, is a natural regulator of hematopoiesis. Here it is shown that Ac-SDKP inhibits TGF-beta action in mesangial cells. Because TGF-beta is thought to play a pivotal role in the development and progression of glomerulonephritis, the therapeutic effects of Ac-SDKP on an established model of renal dysfunction and histologic alteration in Wistar-Kyoto rats with anti-glomerular basement membrane nephritis was examined. Fourteen days after the induction of anti-glomerular basement membrane nephritis, the rats were treated subcutaneously with Ac-SDKP at a dose of 1 mg/kg per d for 4 wk. Treatment with Ac-SDKP significantly improved proteinuria and renal dysfunction, including increased plasma blood urea nitrogen and creatinine levels and decreased creatinine clearance. Histologic examination showed severe glomerulosclerosis and interstitial fibrosis in the vehicle-treated rats, whereas these histologic injuries were significantly ameliorated in rats that were treated with Ac-SDKP. The histologic improvements were accompanied by the suppression of gene and protein expression of fibronectin, interstitial collagen, and TGF-beta1 in the nephritic kidney. Furthermore, treatment with Ac-SDKP resulted in the inhibition of Smad2 phosphorylation, an increase in Smad7 expression in the kidney, and reduction of macrophage accumulation into the glomeruli and tubulointerstitium in nephritic rats. In conclusion, Ac-SDKP significantly ameliorated the progression of renal dysfunction and fibrosis even after the establishment of nephritis. The inhibitory effect of Ac-SDKP was mediated in part by the inhibition of TGF-beta/Smad signal transduction and the inflammatory response. These findings suggest that Ac-SDKP treatment may be a novel and useful therapeutic strategy for the treatment of progressive renal diseases.

The role of the vagal nerve in peripheral PYY3-36-induced feeding reduction in rats.

Peptide YY (PYY), an anorectic peptide, is secreted postprandially from the distal gastrointestinal tract. PYY(3-36), the major form of circulating PYY, binds to the hypothalamic neuropeptide Y Y2 receptor (Y2-R) with a high-affinity, reducing food intake in rodents and humans. Additional gastrointestinal hormones involved in feeding, including cholecystokinin, glucagon-like peptide 1, and ghrelin, transmit satiety or hunger signals to the brain via the vagal afferent nerve and/or the blood stream. Here we determined the role of the afferent vagus nerve in PYY function. Abdominal vagotomy abolished the anorectic effect of PYY(3-36) in rats. Peripheral administration of PYY(3-36) induced Fos expression in the arcuate nucleus of sham-operated rats but not vagotomized rats. We showed that Y2-R is synthesized in the rat nodose ganglion and transported to the vagal afferent terminals. PYY(3-36) stimulated firing of the gastric vagal afferent nerve when administered iv. Considering that Y2-R is present in the vagal afferent fibers, PYY(3-36) could directly alter the firing rate of the vagal afferent nerve via Y2-R. We also investigated the effect of ascending fibers from the nucleus of the solitary tract on the transmission of PYY(3-36)-mediated satiety signals. In rats, bilateral midbrain transections rostral to the nucleus of the solitary tract also abolished PYY(3-36)-induced reductions in feeding. This study indicates that peripheral PYY(3-36) may transmit satiety signals to the brain in part via the vagal afferent pathway.

Fas signaling induces Akt activation and upregulation of endothelial nitric oxide synthase expression.

A growing body of evidence has shown that Fas, a death receptor, mediates apoptosis-unrelated biological effects. Here, we report that Fas engagement with Fas ligand induced activation of Akt and upregulation of endothelial nitric oxide synthase expression without induction of apoptosis. In the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin, Fas ligand, however, induced apoptosis instead of upregulation of endothelial nitric oxide synthase expression. In vivo, systolic blood pressure was slightly higher in mutant mice with decreased cell surface Fas expression (lpr mice) compared with wild-type mice. In addition, chronic inhibition of nitric oxide synthesis by N(G)-nitro-l-arginine induced a progressive increase in the levels of blood pressure in wild-type mice, whereas no further increase in the levels of blood pressure was observed in lpr mice. Furthermore, acetylcholine caused a lesser endothelium-dependent relaxation of the strips from lpr mice compared with wild-type mice, although the vasoconstrictor potency of phenylephrine was not different between the two groups. These findings indicate that Fas signaling may have a role in the regulation of endothelial function and blood pressure through modulating endothelial nitric oxide synthase expression in the Akt signal-dependent manner.