Sun-Young Hwang

Ischemia-reperfusion reduces cystathionine-β-synthase-mediated hydrogen sulfide generation in the kidney

Cystathionine-beta-synthase (CBS) catalyzes the rate-limiting step in the transsulfuration pathway for the metabolism of homocysteine (Hcy) in the kidney. Our recent study demonstrates that ischemia-reperfusion reduces the activity of CBS leading to Hcy accumulation in the kidney, which in turn contributes to renal injury. CBS is also capable of catalyzing the reaction of cysteine with Hcy to produce hydrogen sulfide (H(2)S), a gaseous molecule that plays an important role in many physiological and pathological processes. The aim of the present study was to examine the effect of ischemia-reperfusion on CBS-mediated H(2)S production in the kidney and to determine whether changes in the endogenous H(2)S generation had any impact on renal ischemia-reperfusion injury. The left kidney of Sprague-Dawley rat was subjected to 45-min ischemia followed by 6-h reperfusion. The ischemia-reperfusion caused lipid peroxidation and cell death in the kidney. The CBS-mediated H(2)S production was decreased, leading to a significant reduction in the renal H(2)S level. The activity of cystathionine-gamma-lyase, another enzyme responsible for endogenous H(2)S generation, was not significantly altered in the kidney upon ischemia-reperfusion. Partial restoration of CBS activity by intraperitoneal injection of the nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide not only increased renal H(2)S levels but also alleviated ischemia-reperfusion-induced lipid peroxidation and reduced cell damage in the kidney tissue. Furthermore, administration of an exogenous H(2)S donor, NaHS (100 microg/kg), improved renal function. Taken together, these results suggest that maintenance of tissue H(2)S level may offer a renal protective effect against ischemia-reperfusion injury.

Berberine inhibits NADPH oxidase mediated superoxide anion production in macrophages.

Oxidative stress and amplified redox signaling contribute to the pathogenesis of many human diseases including atherosclerosis. The superoxide-generating phagocytic NADPH oxidase is a key source of oxidative stress in the developing atheroma. The aim of the present study was to examine the effect of berberine, a plant-derived alkaloid, on NADPH oxidase-mediated superoxide anion production in macrophages. Lipopolysaccharide (LPS) treatment activated NADPH oxidase in THP-1 monocyte-derived macrophages and increased the intracellular level of superoxide anions. Preincubation of cells with berberine demonstrated a concentration-dependent (10-50 micromol/L) and time-dependent (6-24 h) inhibition of superoxide anion generation in LPS-stimulated macrophages. Cell viability tests confirmed that berberine, at concentrations sufficient for inhibiting NADPH oxidase-mediated superoxide anion generation in macrophages, did not affect cell viability. Real-time PCR analysis revealed that addition of berberine to the culture medium was able to reduce gp91phox mRNA expression in LPS-treated cells. Berberine also restored superoxide dismutase (SOD) activity, which was found to be inhibited by LPS treatment. In conclusion, results from the present study demonstrate that berberine can effectively reduce intracellular superoxide levels in LPS- stimulated macrophages. Such a restoration of cellular redox by berberine is mediated by its selective inhibition of gp91phox expression and enhancement of SOD activity. The therapeutic relevance of berberine in the prevention and management of atherosclerosis remains to be further investigated.

Folic acid supplementation inhibits NADPH oxidase-mediated superoxide anion production in the kidney

Hyperhomocysteinemia, a condition of elevated blood homocysteine (Hcy) levels, is a metabolic disease. It is a common clinical finding in patients with chronic kidney diseases and occurs almost uniformly in patients with end-stage renal disease. Hyperhomocysteinemia is also a risk factor for cardiovascular disease. Our recent studies indicate that hyperhomocysteinemia can lead to renal injury by inducing oxidative stress. Oxidative stress is one of the important mechanisms contributing to Hcy-induced tissue injury. Folic acid supplementation is regarded as a promising approach for prevention and treatment of cardiovascular disease associated with hyperhomocysteinemia due to its Hcy-lowering effect. However, its effect on the kidney is not clear. The aim of this study was to examine the effect of folic acid supplementation on Hcy-induced superoxide anion production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the kidney during hyperhomocysteinemia. Hyperhomocysteinemia was induced in male Sprague-Dawley rats fed a high-methionine diet for 12 wk with or without folic acid supplementation. A group of rats fed a regular diet was used as control. There was a significant increase in levels of superoxide anions and lipid peroxides in kidneys isolated from hyperhomocysteinemic rats. Activation of NADPH oxidase was responsible for hyperhomocysteinemia-induced oxidative stress in the kidney. Folic acid supplementation effectively antagonized hyperhomocysteinemia-induced oxidative stress via its Hcy-lowering and Hcy-independent effect. In vitro study also showed that 5-methyltetrahydrofolate, an active form of folate, effectively reduced Hcy-induced superoxide anion production via NADPH oxidase. Xanthine oxidase activity was increased and superoxide dismutase (SOD) activity was decreased in the kidney of hyperhomocysteinemic rats, which might also contribute to an elevation of superoxide anion level in the kidney. Folic acid supplementation attenuated xanthine oxidase activity and restored SOD activity in the kidney of hyperhomocysteinemic rats. These results suggest that folic acid supplementation may offer renal protective effect against oxidative stress.

Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase during high fat diet feeding

The liver plays a central role in regulating cholesterol homeostasis. High fat diets have been shown to induce obesity and hyperlipidemia. Despite considerable advances in our understanding of cholesterol metabolism, the regulation of liver cholesterol biosynthesis in response to high fat diet feeding has not been fully addressed. The aim of the present study was to investigate mechanisms by which a high fat diet caused activation of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) leading to increased cholesterol biosynthesis. Mice were fed a high fat diet (60% kcal fat) for 5weeks. High fat diet feeding induced weight gain and elevated lipid levels (total cholesterol and triglyceride) in both the liver and serum. Despite cholesterol accumulation in the liver, there was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 and specific protein 1 (Sp1) were also increased in the liver of mice fed a high fat diet. To validate the in vivo findings, HepG2 cells were treated with palmitic acid. Such a treatment activated SREBP-2 as well as increased the mRNA and enzyme activity of HMG-CoA reductase leading to intracellular cholesterol accumulation. Inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. These results suggest that Sp1-mediated SREBP-2 activation contributes to high fat diet induced HMG-CoA reductase activation and increased cholesterol biosynthesis. This may play a role in liver cholesterol accumulation and hypercholesterolemia.

Long-Term High Intake of Whole Proteins Results in Renal Damage in Pigs

Despite evidence of potential antiobesity effects of high-protein (HP) diets, the impact of consuming diets with protein levels at the upper limit of the acceptable macronutrient distribution range (AMDR) on kidney health is unknown. To test whether HP diets affect renal health, whole plant and animal proteins in proportions that mimicked human diets were given to pigs, because their kidneys have a similar anatomy and function to those of humans. Adult female pigs received either normal-protein (NP) or HP (15 or 35% of energy from protein, respectively) isocaloric diets for either 4 or 8 mo. The higher protein in the HP diet was achieved by increasing egg and dairy proteins. Although there were initial differences in body weight and composition, after 8 mo these were similar in pigs consuming the NP and HP diets. The HP compared with NP diet, however, resulted in enlarged kidneys at both 4 and 8 mo. Renal and glomerular volumes were 60-70% higher by the end of the study. These enlarged kidneys had greater evidence of histological damage, with 55% more fibrosis and 30% more glomerulosclerosis. Renal monocyte chemoattractant protein-1 levels also were 22% higher in pigs given the HP diet. Plasma homocysteine levels were higher in the HP pigs at 4 mo and continued to be elevated by 35% at 8 mo of feeding. These findings suggest that long-term intakes of protein at the upper limit of the AMDR from whole protein sources may compromise renal health.

High-fat diet stimulates hepatic cystathionine β-synthase and cystathionine γ-lyase expression.

Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) catalyze homocysteine (Hcy) metabolism via the trans-sulfuration pathway. They are also responsible for hydrogen sulfide (H2S) production via desulfuration reactions. The liver contributes significantly to the regulation of Hcy and H2S homeostasis, which might participate in many physiological and pathological processes. The aim of this study was to investigate the effect of a high-fat diet (HFD) on hepatic CBS and CSE expression and its impact on Hcy and H2S metabolism. Mice (C57BL/6) fed a HFD (60% kcal fat) for 5 weeks developed fatty liver. The mRNA and protein levels of CBS and CSE in the liver were significantly elevated in mice fed a HFD. Subsequently the metabolism of Hcy by CBS and CSE was increased in the liver, and its level decreased in the circulation. Increased CBS and CSE expression also caused a significant elevation in H2S production in the liver. The level of lipid peroxides was elevated, indicating oxidative stress, while the level of total glutathione remained unchanged in the liver of HFD-fed mice. Upregulation of the trans-sulfuration pathway might play an adaptive role against oxidative stress by maintaining total glutathione levels in the liver.