Marina Pauwels

HDAC inhibitors in experimental liver and kidney fibrosis

Histone deacetylase (HDAC) inhibitors have been extensively studied in experimental models of cancer, where their inhibition of deacetylation has been proven to regulate cell survival, proliferation, differentiation and apoptosis. This in turn has led to the use of a variety of HDAC inhibitors in clinical trials. In recent years the applicability of HDAC inhibitors in other areas of disease has been explored, including the treatment of fibrotic disorders. Impaired wound healing involves the continuous deposition and cross-linking of extracellular matrix governed by myofibroblasts leading to diseases such as liver and kidney fibrosis; both diseases have high unmet medical needs which are a burden on health budgets worldwide. We provide an overview of the potential use of HDAC inhibitors against liver and kidney fibrosis using the current understanding of these inhibitors in experimental animal models and in vitro models of fibrosis.

Valproic Acid Attenuates Proteinuria and Kidney Injury

Inhibitors of histone deacetylase (HDAC) have anti-inflammatory and antifibrotic effects in several organs and tissues, but their effect on the progression of renal disease is unknown. Here, we studied the effect of valproic acid in adriamycin-induced nephropathy in mice. Administration of valproic acid before kidney injury prevented the development of proteinuria and the onset of glomerulosclerosis. Even after postponing treatment until the peak of adriamycin-induced proteinuria, valproic acid rapidly decreased the quantity of proteinuria and attenuated the progression of renal disease. Valproic acid abrogated the decrease in glomerular acetylation observed during adriamycin-induced nephropathy. Furthermore, valproic acid attenuated the significant upregulation of profibrotic and proinflammatory genes, the deposition of collagen, and the infiltration of macrophages into the kidney. Valproic acid decreased glomerular apoptosis and proliferation induced by adriamycin. Ultrastructural studies further supported the protective effect of valproic acid on podocytes in this model. Taken together, these data suggest that HDACs contribute to the pathogenesis of renal disease and that HDAC inhibitors may have therapeutic potential in CKD.

Different types of peroxisomes in human duodenal epithelium.

Peroxisomes are ubiquitous organelles containing enzyme sequences for beta oxidation of fatty acids, synthesis of bile acids, and ether phospholipids. In the inherited peroxisomal diseases one or more enzymes are deficient in hepatic, renal, and fibroblast peroxisomes. We have examined peroxisomes by light and electron microscopy in 29 duodenal biopsy specimens (21 with normal mucosa) after staining for catalase activity, a marker enzyme. Peroxisomes were most numerous in the apices of the nucleus and at the villus base. Two types were distinguished: rounded to oval forms with a median lesser diameter of 0.23-0.31 microns, and tubular, vermiform organelles 0.1 microns thick and up to 3 microns long. Both types coexist in most patients. Tilting of sections and examination of semithin sections at 120 kV did not show connections between individual organelles. By morphometry, volume density was at least 0.45-0.62% of cellular volume, compared to 1.05% in human liver. In contrast, in four out of five individuals surface density of the peroxisomal membrane was 1.4-2.3 times higher than in control livers; this is expected to favour the exchange of metabolites. We suggest that intestinal peroxisomes contribute substantially to the breakdown of very long chain fatty acids.

Hepatic peroxisomes in adrenoleukodystrophy and related syndromes: cytochemical and morphometric data

Peroxisomes were visualized by cytochemical staining for catalase or/and electron microscopy in liver biopsies of two boys with childhood adrenoleukodystrophy (ALD), and of two girls with autopsy confirmed neonatal adrenoleukodystrophy (NALD). In a third patient previously described as NALD, unusual organelles were seen which may be large abnormal microbodies. Enlarged peroxisomes (determined by morphometry) were also present in the livers of the other two NALD patients. In the ALD patient whose clinical disease was more severe, peroxisomes were larger than in the older ALD case. Catalase staining was diminished and markedly heterogeneous. Additional unusual features such as a separate population of tubular forms, contact with fat droplets, marginal plate and invaginations containing glycogen were seen in the neonatal cases. These data are compared to the enlarged or elongated peroxisomes and heterogeneous staining in the thiolase-deficient “pseudo-Zellweger” patient (Goldfischer et al. 1986) and in 2 siblings with acylCoA oxidase deficiency (Poll-Thé et al. 1986, 1988). Enlarged peroxisomes are a common feature in this group of patients with peroxisomal deficiency disorders, suggesting that increased size and lowered metabolic capacity are associated. Nevertheless a marked morphopathological heterogeneity of peroxisomes thus exists in syndromes described as NALD including previously published cases. Most likely this heterogeneity reflects different enzymatic deficiencies, as confirmed by the biochemical data available. Clinically similar syndromes cover divergent microscopical and enzymatic peroxisomal patterns, and naming of the disease should be adapted to reflect such data. Cytochemical studies are urged in every suspected patient.

Comparison of trichostatin A and valproic acid treatment regimens in a mouse model of kidney fibrosis

Histone deacetylase (HDAC) inhibitors are promising new compounds for the therapy of fibrotic diseases. In this study we compared the effect of two HDAC inhibitors, trichostatin A and valproic acid, in an experimental model of kidney fibrosis. In mice, doxorubicin (adriamycin) can cause nephropathy characterized by chronic proteinuria, glomerular damage and interstitial inflammation and fibrosis, as seen in human focal segmental glomerulosclerosis. Two treatment regimens were applied, treatment was either started prior to the doxorubicin insult or delayed until a significant degree of proteinuria and fibrosis was present. Pre-treatment of trichostatin A significantly hampered glomerulosclerosis and tubulointerstitial fibrosis, as did the pre-treatment with valproic acid. In contrast, the development of proteinuria was only completely inhibited in the pre-treated valproic acid group, and not in the pre-treated trichostatin A animals. In the postponed treatment with valproic acid, a complete resolution of established doxorubicin-induced proteinuria was achieved within three days, whereas trichostatin A could not correct proteinuria in such a treatment regimen. However, both postponed regimens have comparable efficacy in maintaining the kidney fibrosis to the level reached at the start of the treatments. Moreover, not only the process of fibrosis, but also renal inflammation was attenuated by both HDAC inhibitors. Our data confirm a role for HDACs in renal fibrogenesis and point towards a therapeutic potential for HDAC inhibitors. The effect on renal disease progression and manifestation can however be different for individual HDAC inhibitors.

Peroxisomes (microbodies) in human liver: cytochemical and quantitative studies of 85 biopsies.

The number, intracellular distribution, and staining characteristics of human hepatocellular peroxisomes that had been made visible by cytochemical staining for catalase were evaluated in biopsies from 75 patients with hepatic, inflammatory, or malignant disease and ten normal individuals. Intensity of staining was found to be proportional to enzymatic activity by microspectrophotometry. Scanning transmission electron microscopy (STEM) image analysis demonstrated an inverse relationship between peroxisomal size and contrast. Peroxisomes were more abundant, and often concentrated in a perinuclear configuration in cholestatic and cirrhotic livers. Decreased peroxisomal staining was common in cholestasis, cirrhosis, hepatitis, and in almost all patients with malignancies, both with and without hepatic metastases.

Vitamin E Protects Renal Antioxidant Enzymes and Attenuates Glomerulosclerosis in Adriamycin-Treated Rats

Background/Aims: In the rat Adriamycin model of chronic renal failure, the development of glomerulosclerosis and tubulointerstitial lesions is accompanied by decreased activities and mRNA levels of the antioxidant enzymes. In this study, we investigated the effect of oral vitamin E supplementation on antioxidant enzyme activities in both the cortex and isolated glomeruli from Adriamycin-treated rats. Methods: Glomerulosclerosis, tubulointerstitial lesions and ferric iron deposits were evaluated by histochemical staining methods, and antioxidant enzyme activities were measured by spectrophotometry. Results: Vitamin E supplementation of the normal diet attenuates Adriamycin-induced glomerulosclerosis and tubulointerstitial lesions, but not proteinuria and serum total cholesterol, low-density lipoprotein cholesterol, triglycerides and total protein concentrations. In the cortex, vitamin E completely prevented a decrease in enzyme activity for Cu/Zn superoxide dismutase and catalase, and partly for Mn superoxide dismutase and glutathione peroxidase. In the glomeruli, vitamin E completely prevented a decrease in activity for Cu/Zn superoxide dismutase, catalase and glutathione peroxidase, and partly for Mn superoxide dismutase. Conclusion: Dietary supplementation of vitamin E protects the activities of antioxidant enzymes in the kidney cortex and glomeruli, and attenuates the evolution towards terminal renal failure in rats treated with Adriamycin.

Renal antioxidant enzymes and fibrosis-related markers in the rat adriamycin model

Excessive generation of reactive oxygen intermediates can induce changes in the cellular antioxidant defence system. In this study we examine the antioxidant enzyme status and the expression of fibrosis-related marker proteins in the Adriamycin model of chronic renal failure in the rat. Twenty weeks after Adriamycin treatment, rats have overt nephrotic syndrome and renal failure with development of tubulo-interstitial fibrosis and glomerulosclerosis. Lipids accumulate in blood and in both glomeruli and tubulo-interstitial tissue. Desmin and α-smooth muscle actin expression increases in glomeruli and in the tubulo-interstitial area. Renal cortex antioxidant enzyme activities are decreased 20 weeks after Adriamycin injection (to 41% for catalase, to 56% for total superoxide dismutase and to 69% for glutathione peroxidase). The mRNA levels of catalase, Cu/Zn-superoxide dismutase and glutathione peroxidase-1 evaluated by Northern blot are decreased by more than 50% for catalase, Cu/Zn-superoxide dismutase and glutathione peroxidase-1. We conclude that in the rat Adriamycin-induced model of chronic renal failure with fibrosis, the combination of decreased antioxidant enzyme status in renal cortex with high concentrations of lipids in blood and renal tissue facilitates oxidative damage. Development of fibrosis is paralleled by increased expression of desmin and α-smooth muscle actin.

Alterations of hepatocellular peroxisomes in patients with cancer.Catalase cytochemistry and morphometry

Background. Hepatic catalase activity is decreased in patients with malignant diseases, but little is known about the organelles that contain the bulk of catalase: the peroxisomes.

Effect of Mycophenolate Mofetil on Glomerulosclerosis and Renal Oxidative Stress in Rats

Background/Aims: Mycophenolate mofetil (MMF) is known to attenuate glomerulosclerosis in experimental models of renal failure. We investigated whether this is mediated by reduction of oxidative stress. Methods: Effects of MMF on oxidative stress are studied in an experimental rat model (NA model) involving unilateral nephrectomy and two intravenous injections with adriamycin (2 mg/kg). Rats are sacrificed after 2 and 6 weeks. Glomerulosclerosis and tubulointerstitial lesions are demonstrated by histological techniques. Presence of macrophages/monocytes (ED1) and myofibroblasts (α-SMA) is demonstrated by immunohistochemistry. Oxidative stress is evaluated by enzymatic measurements (AOE), spectrofluorometry (TBARS), immunohistochemistry (MDA and HNE) and histology (ferric iron deposition). Results: The NA model shows proteinuria, hypercholesterolemia, beginning glomerulosclerosis, tubulointerstitial sclerosis and tubular dilatation, glomerular, periglomerular and interstitial presence of α-SMA and increased presence of macrophages/monocytes after 6 weeks. Oxidative stress in renal cortex is apparent (increased cortex TBARS concentration, increased glomerular presence of MDA and HNE, decreased activity of antioxidant enzymes, ferric iron deposition in proximal tubules) after 6 weeks. MMF administration results in a decrease of glomerulosclerosis, interstitial sclerosis, glomerular and periglomerular expression of α-SMA and the number of ED1-positive cells in tubulointerstitium and glomeruli. Proteinuria and cholesterolemia are not decreased. TBARS level, and activities of catalase, Mn and Cu/Zn superoxide dismutase as well as the presence of ferric iron in the proximal tubules are not changed by MMF treatment. Cortex activity of glutathione peroxidase returns to normal. Conclusion: MMF has a favorable effect on glomerular and interstitial fibrosis in the NA model of kidney disease, but not on proteinuria and cholesterolemia. Improvement of fibrosis cannot be explained by major changes in oxidative stress or antioxidant defense.