José A. Solís-Herruzo

Effects of lumbar sympathetic block on kidney function in cirrhotic patients with hepatorenal syndrome

We studied the effects of unilateral lumbar sympathetic block on kidney function in eight patients with cirrhosis and hepatorenal syndrome. In five patients with basal glomerular filtration rate (GFR) below 25 ml/min, sympathetic block induced a significant increase in GFR, osmolal clearance, urinary sodium excretion, fractional excretion of filtered sodium (FENa) and effective renal plasma flow (ERPF) and a decrease in plasma renin activity. In the three patients with basal GFR greater than 25 ml/min, sympathetic block produced no significant change in renal function. We conclude that sympathetic block might improve renal function in cirrhotics with hepatorenal syndrome, particularly those with more impaired GFR.

Uric acid and anti-TNF antibody improve mitochondrial dysfunction in ob/ob mice.

The mechanisms responsible for low mitochondrial respiratory chain (MRC) activity in the liver of patients with nonalcoholic steatohepatitis are unknown. In this study, we examined the cause of this dysfunction in ob/ob mice. Forty‐six mice were distributed in six groups: group I: C57BL/6J mice; group II: C57BL/6J Lep(−/−) mice (ob/ob); group III, ob/ob mice treated with manganese [III] tetrakis (5,10,15,20 benzoic acid) porphyrin (MnTBAP); group IV, ob/ob mice treated with IgG1 immunoglobulin; group V, ob/ob mice treated with anti‐TNF antibody; group VI: ob/ob mice treated with uric acid. In liver tissue, we measured MRC activity, fatty acid β‐oxidation, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), 3‐tyrosine‐nitrated proteins, 3‐tyrosine‐nitrated mitochondrial proteins, including cytochrome c and ND4 subunit of complex I. MRC activity was decreased in ob/ob mice. TNF levels, iNOS protein expression, and tyrosine nitrated proteins were markedly increased in the liver of ob/ob mice. In these animals, mitochondrial proteins were markedly tyrosine nitrated, particularly the ND4 subunit of complex I and cytochrome c. Treatment of these animals with uric acid, a peroxynitrite scavenger, anti‐TNF antibody, or MnTBAP decreased tyrosine nitrated proteins, improved the activity of MRC complexes, and led to a marked regression of hepatic steatosis and inflammation. In conclusion, MRC dysfunction and liver lesions found in ob/ob mice are likely to reflect the tyrosine nitration of mitochondrial proteins by peroxynitrite or a peroxynitrite‐derivate radical. Increased hepatic TNF and iNOS expression might enhance peroxynitrite formation and inhibition of MRC complexes. (HEPATOLOGY 2006;44:581–591.)

A pilot trial of fenofibrate for the treatment of non-alcoholic fatty liver disease

BACKGROUND Dyslipidaemia and insulin resistance are two important risk factors for non-alcoholic fatty liver disease. Both factors can improve with fenofibrate. AIMS To evaluate the effect of fenofibrate on the clinical, analytical and histological evolution of patients with non-alcoholic fatty liver disease. SUBJECTS AND METHODS Sixteen consecutive patients with biopsy-confirmed non-alcoholic fatty liver disease were treated with 200mg/day of fenofibrate for 48 weeks. A clinical and biochemical follow-up was done every 3 months. A new liver biopsy was performed in all patients at the end of therapy. RESULTS All patients completed 48 weeks of therapy with fenofibrate, without adverse events. At the end of the study, a significant decrease in triglyceride, glucose, alkaline phosphatase and gamma-glutamyl transpeptidase and an increase of apolipoprotein A1 levels were found. Insulin levels and insulin resistance showed a trend to decrease. Moreover, a reduction in the proportion of patients with abnormal aminotransferase levels (>45IU/L) was observed (alanine aminotransferase: 93.7% vs. 62.5%, p=0.02; aspartate aminotransferase: 50% vs. 18.7%, p=0.02). The body mass index did not show any significant change, but the proportion of patients with metabolic syndrome decreased significantly (43.7% vs. 18.7%, p=0.04). A control biopsy after treatment revealed a decrease in the grade of hepatocellular ballooning degeneration (p=0.03), but the grade of steatosis, lobular inflammation, fibrosis or non-alcoholic fatty liver disease activity score did not change significantly. CONCLUSIONS In patients with non-alcoholic fatty liver disease, treatment with fenofibrate is safe and improves metabolic syndrome, glucose and liver tests. However, its effects on liver histology are minimal.

Clinical and histological efficacy of pegylated interferon and ribavirin therapy of recurrent hepatitis C after liver transplantation

Treatment of recurrent hepatitis C in liver transplant is controversial. The aim of our study was to evaluate the clinical and histological efficacy of pegylated interferon alpha 2b (PEG‐IFN) and ribavirin therapy of recurrent hepatitis C after liver transplantation (LT). We prospectively included 47 liver transplant patients with: 1) a positive test for hepatitis C virus (HCV)‐ribonucleic acid (RNA) in serum; 2) alanine aminotransferase (ALT) >45 UI/mL; and 3) a liver biopsy showing chronic hepatitis without rejection in the previous 2 months. Patients received PEG‐IFN (1.5 μg/kg/week) and ribavirin (800‐1,000 mg/day) for 12 months. Follow‐up was based on biochemical (ALT), virological (RNA‐HCV), and histological (liver biopsy) examinations. Follow‐up lasted a minimum of 6 months after the end of antiviral therapy. Sustained virological response (SVR) was achieved in 23% of the patients. A total of 33 (70%) patients had normalized ALT levels at the end of therapy. Inflammatory portal and lobular score declined significantly in patients with SVR (P < 0.05) but not in nonresponder patients. Fibrosis did not change significantly in either group. SVR was significantly associated with low γ‐glutamyltransferase GGT (P = 0.04) and HCV‐RNA levels (P = 0.03), a virological response at 12 weeks (P = 0.002) and patients compliance (P = 0.04). Ten (21%) patients were withdrawn prematurely due to adverse effects. In conclusion, Therapy with PEG‐IFN and ribavirin achieved SVR and a significant histological improvement in 23% of liver transplant recipients with chronic hepatitis C. Toxicity is an important drawback of this therapy. Liver Transpl 12:1805‐1812, 2006.

Effects of rosiglitazone on the liver histology and mitochondrial function in ob/ob mice.

Insulin resistance is present in almost all patients with nonalcoholic steatohepatitis (NAFLD), and mitochondrial dysfunction likely plays a critical role in the progression of fatty liver into nonalcoholic steatohepatitis. Rosiglitazone, a selective ligand of peroxisome proliferator‐activated receptor gamma (PPARγ), is an insulin sensitizer drug that has been used in a number of insulin‐resistant conditions, including NAFLD. The aim of this study was to analyze the effects of rosiglitazone on the liver histology and mitochondrial function in a model of NAFLD. All studies were carried out in wild‐type and leptin‐deficient (ob/ob) C57BL/6J mice. Ob/ob mice were treated with 1 mg/kg/day, and activity of mitochondrial respiratory chain (MRC), beta‐oxidation, lipid peroxidation, glutathione content in mitochondria, and 3‐tyrosine–nitrated proteins in mitochondria were measured. In addition, histological and ultrastructural changes induced by rosiglitazone were also noted. Rosiglitazone treatment increased liver steatosis, particularly microvesicular steatosis. In these animals, mitochondria were markedly swollen with cristae peripherally placed. In ob/ob mice, this drug increased PPARγ protein expression and lipid peroxide content in liver tissue and decreased glutathione concentration in mitochondria. Rosiglitazone suppressed the activity of complex I of the MRC in ob/ob mice, but did not affect beta‐oxidation. 3‐Tyrosine nitrated mitochondrial proteins, significantly increased in ob/ob mice, were not modified by rosiglitazone treatment. Conclusion: Treatment of ob/ob mice with rosiglitazone did not reverse histological lesions of NAFLD or improve MRC activity. On the contrary, rosiglitazone reduced activity of complex I and increased oxidative stress and liver steatosis. (HEPATOLOGY 2007.)

Decreased bone mineral density after therapy with alpha interferon in combination with ribavirin for chronic hepatitis C

BACKGROUND/AIMS Several thousand patients with chronic hepatitis C have been treated with interferon plus ribavirin. After observing a male patient who developed severe bone loss during this treatment, we studied skeletal status and bone mineral metabolism in patients on therapy with interferon plus ribavirin. METHODS Bone mineral density and biochemical bone markers were studied in 32 male patients (31-58 years old) treated for 12 months with either interferon alone (group 1; n=13) or interferon plus ribavirin (group 2; n= 19). RESULTS Bone mineral density was significantly lower in group 2 (0.877-0.07 g/cm2) than in group 1 (1.108+/-0.08 g/cm2, p<0.001). Likewise, T- and Z-score values were also decreased in group 2 (T: -1.95+/-0.6. Z: -1.76+/-0.51) compared with group 1 (T: 0.19+/-0.6; p<0.001. Z: 0.26+/-0.6; p<0.001). Serum and urine biochemical bone markers were normal in both groups. However, urinary calcium excretion was decreased in patients on combined therapy. CONCLUSION Treatment of chronic hepatitis C with interferon plus ribavirin may induce bone loss. This secondary effect should be investigated during the follow-up of these patients, since they may require therapies aimed at prevention or amelioration of these defects.

Tumor necrosis factor-alpha increases ATP content in metabolically inhibited L929 cells preceding cell death.

The effects of tumor necrosis factor-α (TNF) on ATP levels were studied in metabolically inhibited L929 cells. Treatment of these cells with TNF in the presence of actinomycin D or cycloheximide induces cyclic changes in the intracellular ATP content preceding cell death. After 3 h of incubation, the intracellular ATP content increased by 48 ± 6% (p < 0.001), but at 4 h, it decreased to the control level. Two hours later, it increased again by 23 ± 5% over the control level (p < 0.001). Coinciding with cell death, ATP content decreased progressively until almost complete depletion. These changes in ATP content were associated with parallel alterations in the respiratory coupling and with increased generation of reactive oxygen species. The mechanism by which TNF/actinomycin D or TNF/cycloheximide increased cellular ATP seemed to be dependent on the mitochondrial ATP synthesis and related to the cytotoxic effect of TNF, since blockade of mitochondrial electron transport prevented the increase in cellular ATP, the formation of reactive oxygen species, and the apoptotic cell death caused by TNF. We suggest that the TNF/actinomycin D- or TNF/cycloheximide-induced changes in intracellular ATP levels may be involved in the cytotoxic effect of TNF in metabolically inhibited L929 cells.

Effects of orthotopic liver transplantation on vasoactive systems and renal function in patients with advanced liver cirrhosis.

The effects of Orthotopic liver transplantation (OLT) on renal function and major vasoactive factors was assessed in end-stage cirrhotic patients. Renal function, mean arterial pressure, and plasma vasoactive hormones were measured In 22 cirrhotic patients with refractory ascites before and after OLT. Before OLT, mean arterial pressure, glomerular filtration rate, free water clearance, and fractional sodium excretion serum sodium levels were decreased. In addition, serum creatinine and plasma levels of vasoactive factors were increased. Ten of these patients fulfilled criteria of hepatorenal syndrome (HRS). Nine to 12 months after transplantation, renal function had improved and plasma levels of vasoactive factors had decreased significantly in all patients, including those with HRS. However, glomerular filtration rate remained subnormal and plasma endothelin-1 levels and plasma renin activity remained increased in most of them. In conclusion, OLT improves renal function in patients with end-stage liver cirrhosis, including those with HRS. However, renal function remains subnormal in most of these patients.

High-fat diet decreases activity of the oxidative phosphorylation complexes and causes nonalcoholic steatohepatitis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent histological finding in individuals with abnormal liver-function tests in the Western countries. In previous studies, we have shown that oxidative phosphorylation (OXPHOS) is decreased in individuals with NAFLD, but the cause of this mitochondrial dysfunction remains uncertain. The aims of this study were to determine whether feeding mice a high-fat diet (HFD) induces any change in the activity of OXPHOS, and to investigate the mechanisms involved in the pathogenesis of this defect. To that end, 30 mice were distributed between five groups: control mice fed a standard diet, and mice on a HFD and treated with saline solution, melatonin (an antioxidant), MnTBAP (a superoxide dismutase analog) or uric acid (a scavenger of peroxynitrite) for 28 weeks intraperitoneously. In the liver of these mice, we studied histology, activity and assembly of OXPHOS complexes, levels of subunits of these complexes, gene expression of these subunits, oxidative and nitrosative stress, and oxidative DNA damage. In HFD-fed mice, we found nonalcoholic steatohepatitis, increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, TGFβ1 and collagen α1(I), and increased levels of 3-tyrosine nitrated proteins. The activity and assembly of all OXPHOS complexes was decreased to about 50–60%. The amount of all studied OXPHOS subunits was markedly decreased, particularly the mitochondrial-DNA-encoded subunits. Gene expression of mitochondrial-DNA-encoded subunits was decreased to about 60% of control. There was oxidative damage to mitochondrial DNA but not to genomic DNA. Treatment of HFD-fed mice with melatonin, MnTBAP or uric acid prevented all changes observed in untreated HFD-fed mice. We conclude that a HFD decreased OXPHOS enzymatic activity owing to a decreased amount of fully assembled complexes caused by a reduced synthesis of their subunits. Antioxidants and antiperoxynitrites prevented all of these changes, suggesting that nitro-oxidative stress played a key role in the pathogenesis of these alterations. Treatment with these agents might prevent the development of NAFLD in humans.

Melatonin improves mitochondrial respiratory chain activity and liver morphology in ob/ob mice.

Abstract:  In previous studies, we have shown that mitochondrial respiratory chain (MRC) activity is decreased in patients with nonalcoholic steatohepatitis and in ob/ob mice and that peroxynitrite plays a pathogenic role. The present study examined whether melatonin, a peroxynitrite scavenger, prevents: (i) the in vitro effects of peroxynitrite on normal mitochondrial proteins and (ii) the development of nonalcoholic liver disease, MRC dysfunction and proteomic changes found in the mitochondrial complexes from ob/ob mice. We studied MRC activity, assembly of mitochondrial complexes and its subunits in normal mitochondrial proteins exposed to peroxynitrite in the absence and presence of melatonin. The same studies were done in mitochondrial proteins from ob/ob mice untreated and treated with melatonin. Preincubation of mitochondrial proteins from wild‐type mice with melatonin prevented 3‐tyrosine nitration of these proteins, eliminated the reduction in the MRC activity, the defect in the assembly of mitochondrial complexes and degradation of their subunits induced by peroxynitrite in vitro. Moreover, treatment of ob/ob mice with 10 mg/kg/day melatonin for 12 wk reduced oxidative and nitrosative stress, prevented the loss of MRC activity, protected their complexes and subunits from degradation, and favored assembling of mitochondrial complexes. In addition, this treatment improved fatty liver, decreased hepatic triglyceride concentration and increased apolipoprotein B100 in liver tissue. In conclusion, melatonin prevents the effects of peroxynitrite on mitochondrial proteins in vitro and administration of melatonin to ob/ob mice normalizes liver morphology, mitochondrial dysfunction and assembly of MRC complexes.