Chih-Sung Hsieh

Effects of maternal l-citrulline supplementation on renal function and blood pressure in offspring exposed to maternal caloric restriction: The impact of nitric oxide pathway

Maternal undernutrition can cause reduced nephron number and glomerular hypertrophy, consequently leading to adult kidney disease. We intended to elucidate whether NO deficiency evolves to kidney disease vulnerability in offspring from mothers with caloric restriction diets and whether maternal L-citrulline (L-Cit) supplementation can prevent this. Using a rat model with 50% caloric restriction, four groups of 3-month-old male offspring were sacrificed to determine their renal outcome: control, caloric restriction (CR), control treated with 0.25% L-citrulline solution during the whole period of pregnancy and lactation (Cit), and CR treated in the same way (CR+Cit group). The CR group had low nephron numbers, increased glomerular diameter, and an increased plasma creatinine level compared with the control group. Maternal L-Cit supplementation prevented these effects. The CR+Cit and Cit groups developed hypertension beginning at 4 and 8weeks of age, respectively. Plasma asymmetric and symmetric dimethylarginine (ADMA and SDMA) levels were increased, but L-arginine/ADMA ratios (AAR) were decreased in the CR group vs the control group. This was prevented by maternal L-Cit supplementation. Renal cortical neuronal NOS-alpha (nNOSalpha) protein abundance was significantly decreased in the Cit and CR+Cit groups. Collectively, reduced nephron number, reduced renal nNOSalpha expression, increased ADMA, and decreased AAR contribute to the developmental programming of adult kidney disease and hypertension. Although maternal L-Cit supplementation prevents caloric restriction-induced low nephron number and renal dysfunction, it also induces hypertension.

Melatonin blocks oxidative stress-induced increased asymmetric dimethylarginine

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, and its increase is associated with many systemic diseases. We recently found that increases in plasma and hepatic ADMA levels were associated with oxidative stress in young bile-duct-ligation (BDL) rats; these increases were prevented by melatonin therapy. Therefore, we used an in vivo BDL model and in vitro cultured hepatocytes to elucidate the protective mechanisms of melatonin against oxidative stress-induced increase in ADMA. We found that the presence of reactive oxygen species (ROS) in young rats with BDL leads to downregulation of dimethylarginine dimethyaminohydrolase (DDAH)-1 and -2 as well as DDAH activity. Melatonin prevented ADMA increases in the liver mainly by regulating DDAH-1 and -2. The expression and activity of DDAH were suppressed in vitro by superoxide and hydrogen peroxide (H(2)O(2)) in a time-dependent manner, whereas melatonin could block H(2)O(2)-induced downregulation of DDAH-2 as well as decreased DDAH activity, thereby preventing increases in hepatic ADMA. Our findings reveal a mechanistic basis of DDAH downregulation by ROS and suggest that melatonin might be a potential therapy for various diseases with elevated cellular ADMA.

Roles of nitric oxide and asymmetric dimethylarginine in pregnancy and fetal programming.

Nitric oxide (NO) regulates placental blood flow and actively participates in trophoblast invasion and placental development. Asymmetric dimethylarginine (ADMA) can inhibit NO synthase, which generates NO. ADMA has been associated with uterine artery flow disturbances such as preeclampsia. Substantial experimental evidence has reliably supported the hypothesis that an adverse in utero environment plays a role in postnatal physiological and pathophysiological programming. Growing evidence suggests that the placental nitrergic system is involved in epigenetic fetal programming. In this review, we discuss the roles of NO and ADMA in normal and compromised pregnancies as well as the link between placental insufficiency and epigenetic fetal programming.

Melatonin prevents increased asymmetric dimethylarginine in young rats with bile duct ligation

Abstract:  Identifying and treating kidney injury in cirrhosis is important. Bile duct ligation (BDL) is a commonly used cholestatic liver disease model. We hypothesized that asymmetric dimethylarginine (ADMA) is involved in BDL‐induced oxidative stress and kidney injury, which can be prevented by melatonin. We also intended to elucidate whether increased ADMA is due to increased protein arginine methyltransferase‐1 (PRMT1, ADMA‐synthesizing enzyme) and/or decreased dimethylarginine dimethylaminohydrolase (DDAH, ADMA‐metabolizing enzyme). Three groups of young rats were studied, sham (N = 7), untreated BDL rats (N = 9), and melatonin‐treated BDL rats (N = 6, BDL + M). Melatonin‐treated BDL rats received daily melatonin 1 mg/kg/day via intraperitoneal injection. One‐third of the young BDL rats died compared with none in the BDL + M group. All surviving rats were killed 14 days after surgery. BDL rats had higher plasma aspartate aminotransferase, alanine aminotransferase, direct and total bilirubin, and ammonia levels than shams. They also had kidney injury characterized by increased tubulointerstitial injury scores and plasma creatinine and symmetric dimethylarginine levels, which melatonin prevented. Plasma ADMA levels were elevated in BDL rats, combined with increased hepatic PRMT1 and decreased renal DDAH activity. In addition, melatonin increased hepatic DDAH2 expression, increased DDAH activity and concomitantly decreased ADMA contents in both the liver and kidney. In conclusion, melatonin therapy decreased mortality and prevented kidney injury induced by BDL via reduction of ADMA (by increasing DDAH activity) and oxidative stress.

The interaction between high ammonia diet and bile duct ligation in developing rats: assessment by spatial memory and asymmetric dimethylarginine

Bile duct ligation (BDL) in developing rats causes cholestasis, impaired liver function and cognition. Because both nitric oxide (NO) and ammonia are implicated in hepatic encephalopathy (HE), we hypothesized that asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor, and ammonia affect cognition in young rats with BDL. Four groups of young male Sprague–Dawley rats ages 17 days were used: rat underwent laparotomy (SC group), rat underwent laparotomy plus a 30% ammonium acetate diet (SC + HA group), rat underwent BDL (BDL group), rats underwent BDL plus high ammonia diet (BDL + HA group). Spatial memory was assessed by Morris water maze task. Plasma was collected for biochemical and ADMA analyses. Liver and brain cortex were collected for determination of protein arginine methyltransferase‐1 (PRMT1, ADMA‐synthesizing enzyme) and dimethylarginine dimethylaminohydrolase (DDAH, ADMA‐metabolizing enzyme). We found BDL group had significantly higher plasma direct/total bilirubin, aspartate aminotransferase, alanine aminotransferase, ADMA, liver p22phox, and worse spatial performance as compared with SC group. High ammonia diet increased plasma ammonia and ADMA concentration, and aggravated spatial deficit in the presence of BDL‐induced cholestasis. We conclude plasma ADMA plays a role in BDL‐induced spatial deficit. High ammonia aggravated the spatial deficits encountered in cholestatic young rats.

Alterations in NADPH oxidase expression and blood–brain barrier in bile duct ligation-treated young rats: Effects of melatonin

Bile duct ligation (BDL)-treated rats exhibit cholestasis and increased systemic and brain oxidative stress. Activation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and disruption of the blood-brain barrier (BBB) are implicated as the pathogenetic mechanisms of brain dysfunction in BDL-treated adult rats. Young rats underwent sham ligation or BDL at day 17 for 2 or 4weeks. Treatment group rats were administered melatonin between day 17 and 45. We found a progressive increase in prefrontal cortex NADPH-dependent superoxide anion (O(2)(-)) production and increased expression of NADPH oxidase subunits in either the prefrontal cortex or the hippocampus in BDL-treated young rats. In addition, expression of intercellular adhesion molecule-1 (ICAM) and tissue plasminogen activator (t-PA) genes were increased in either the prefrontal cortex or the hippocampus. Prefrontal cortex capillary junctional complex proteins expressions including occludin, claudin-5, platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin were not altered. Melatonin lowered the prefrontal cortex NADPH-dependent O(2)(-) production and t-PA gene expression. We conclude that alterations in NADPH oxidase expression and BBB are involved in brain dysfunction in BDL-treated young rats. In addition, melatonin regulates NADPH oxidase activity and t-PA gene expression.

Sex Differences of Oxidative Stress to Cholestatic Liver and Kidney Injury in Young Rats

BACKGROUND Sexual dimorphism plays a role in the liver and in renal injuries. However, whether sex is a risk factor in bile duct ligation (BDL) in young rats has never been examined. METHODS Six male and six female rats treated with BDL were sacrificed 2 weeks after surgery and were designated as BDL-M and BDL-F groups. The other six male and six female rats that received sham ligation were designated as sham-M and sham-F groups. Plasma biochemistry and liver and kidney asymmetric dimethylarginine (ADMA)-related molecules were examined. RESULTS Both BDL-M and BDL-F groups had elevated plasma aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, and transforming growth factor-β1 levels. The BDL-F group had lower plasma AST and ALT levels than the BDL-M group. The BDL-M and BDL-F groups had elevated plasma ADMA levels. The cationic amino acid transporter 1 (CAT1) level was increased in the BDL-F group as compared to the sham-F group, whereas the CAT2 level was reduced in the both BDL-M and BDL-F groups. CONCLUSION We found that young male rats were prone to higher degrees of biochemical liver and kidney injury to cholestasis. Sex differences in modulation of oxidative stress markers, such as ADMA, may play a role. Our results support careful monitoring and optimal treatment of cholestatic disease, especially in young male patients.

Programming Effects of Prenatal Glucocorticoid Exposure with a Postnatal High-Fat Diet in Diabetes Mellitus

Increasing evidence has shown that many chronic diseases originate from early life, even before birth, through what are termed as fetal programming effects. Glucocorticoids are frequently used prenatally to accelerate the maturation of the lungs of premature infants. High-fat diets are associated with insulin resistance, but the effects of prenatal glucocorticoid exposure plus a postnatal high-fat diet in diabetes mellitus remain unclear. We administered pregnant Sprague-Dawley rats’ intraperitoneal dexamethasone (0.1 mg/kg body weight) or vehicle at gestational days 14–20. Male offspring were administered a normal or high-fat diet starting from weaning. We assessed the effects of prenatal steroid exposure plus postnatal high-fat diet on the liver, pancreas, muscle and fat at postnatal day 120. At 15 and 30 min, sugar levels were higher in the dexamethasone plus high-fat diet (DHF) group than the vehicle plus high-fat diet (VHF) group in the intraperitoneal glucose tolerance test (IPGTT). Serum insulin levels at 15, 30 and 60 min were significantly higher in the VHF group than in the vehicle and normal diet group. Liver insulin receptor and adenosine monophosphate-activated protein kinase mRNA expressions and protein levels were lower in the DHF group. Insulin receptor and insulin receptor substrate-1 mRNA expressions were lower in the epididymal adipose tissue in the VHF and DHF groups. “Programming” of liver or epididymal adipose tissue resulted from prenatal events. Prenatal steroid exposure worsened insulin resistance in animals fed a high-fat diet.

Endotoxemia exacerbates kidney injury and increases asymmetric dimethylarginine in young bile duct-ligated rats.

ABSTRACT Cirrhosis increases the risk of kidney injury and sepsis, leading to increased mortality. Elevated levels of plasma asymmetric dimethylarginine (ADMA) occur in patients critically ill with cirrhosis, renal failure, and sepsis. We used a rat model of cirrhosis with superimposed sepsis to assess the relationship of plasma and tissue ADMA profiles with acute kidney injury and survival. Seventeen-day-old male Sprague-Dawley rats (n = 37) were randomly assigned to four groups: (1) sham operation plus diet control (n = 6); (2) bile duct ligation (BDL, n = 8); (3) sham operation plus lipopolysaccharide (LPS, n = 9); and (4) BDL plus LPS (n = 14). Lipopolysaccharide was given by intraperitoneal injection (1 mg/kg in saline) 3 h before sacrifice. All rats were sacrificed 14 days after surgery. Lipopolysaccharide increased the rate of BDL-associated death and dysfunction of the liver and kidneys. These results were supported by increased levels of plasma ADMA and a decreased L-arginine/ADMA ratio (AAR). Plasma and tissue levels of ADMA and AAR were not correlated. Lipopolysaccharide restored BDL-induced ADMA level elevation in the liver but increased ADMA level in the kidneys. Lipopolysaccharide increased hepatic AAR, decreased renal AAR, and paradoxically mediated the expression of neuronal nitric oxide synthase-&bgr; in the liver and kidneys. A novel mechanism underlies the LPS-mediated L-arginine–ADMA–nitric oxide pathway activation and exacerbation of kidney injury and mortality in our BDL model. In the presence of cirrhosis with superimposed sepsis, simultaneous lowering of ADMA levels and enhancement of L-arginine levels to restore plasma and renal AARs may be an optimal strategy for the treatment of kidney injury.

Effects of Hepatocyte CD14 Upregulation during Cholestasis on Endotoxin Sensitivity

Cholestasis is frequently related to endotoxemia and inflammatory response. Our previous investigation revealed a significant increase in plasma endotoxin and CD14 levels during biliary atresia. We therefore propose that lipopolysacharides (LPS) may stimulate CD14 production in liver cells and promote the removal of endotoxins. The aims of this study are to test the hypothesis that CD14 is upregulated by LPS and investigate the pathophysiological role of CD14 production during cholestasis. Using Western blotting, qRT-PCR, and promoter activity assay, we demonstrated that LPS was associated with a significant increase in CD14 and MD2 protein and mRNA expression and CD14 promoter activity in C9 rat hepatocytes but not in the HSC-T6 hepatic stellate cell line in vitro. To correlate CD14 expression and endotoxin sensitivity, in vivo biliary LPS administration was performed on rats two weeks after they were subjected to bile duct ligation (BDL) or a sham operation. CD14 expression and endotoxin levels were found to significantly increase after LPS administration in BDL rats. These returned to basal levels after 24 h. In contrast, although endotoxin levels were increased in sham-operated rats given LPS, no increase in CD14 expression was observed. However, mortality within 24 h was more frequent in the BDL animals than in the sham-operated group. In conclusion, cholestasis and LPS stimulation were here found to upregulate hepatic CD14 expression, which may have led to increased endotoxin sensitivity and host proinflammatory reactions, causing organ failure and death in BDL rats.