Effects of losartan, in monotherapy or in association with hydrochlorothiazide, in chronic nephropathy resulting from losartan treatment during lactation

Fanelli C, Fernandes BH, Machado FG, Okabe C, Malheiros DM, Fujihara CK, Zatz R. Effects of losartan, in monotherapy or in association with hydrochlorothiazide, in chronic nephropathy resulting from losartan treatment during lactation. Am J Physiol Renal Physiol 301: F580-F587, 2011. First published June 8, 2011; doi:10.1152/ajprenal.00042.2011.-We recently standardized a model (L(Lact)) of severe chronic kidney disease based on impaired nephrogenesis by suppression of angiotensin II activity during lactation (Machado FG, Poppi EP, Fanelli C, Malheiros DM, Zatz R, Fujihara CK. Am J Physiol Renal Physiol 294: F1345-F1353, 2008). In this new study of the L(Lact) model, we sought to gain further insight into renal injury mechanisms associated with this model and to verify whether the renoprotection obtained with the association of the angiotensin II receptor blocker losartan (L) and hydrochlorothiazide (H), which arrested renal injury in the remnant kidney model, would provide similar renoprotection. Twenty Munich-Wistar dams, each nursing six pups, were divided into control, untreated, and L(Lact) groups, given losartan (L; 250 mg.kg(-1).day(-1)) until weaning. The male LLact offspring remained untreated until 7 mo of age, when renal functional and structural parameters were studied in 17 of them, used as pretreatment control (L(Lact)Pre), and followed no further. The remaining rats were then divided among groups L(Lact) + V, untreated; L(Lact) + L, given L (50 mg.kg(-1).day(-1)) now as a therapy; L(Lact) + H, given H (6 mg.kg(-1).day(-1)); and L(Lact) + LH, given L and H. All parameters were reassessed 3 mo later in these groups and in age-matched controls. At this time, L(Lact) rats exhibited hypertension, severe albuminuria, glomerular damage, marked interstitial expansion/inflammation, enhanced cell proliferation, myofibroblast infiltration, and creatinine retention. L monotherapy normalized albuminuria and prevented hypertension and the progression of renal injury, inflammation, and myofibroblast infiltration. In contrast to the remnant model, the LH combination promoted only slight additional renoprotection, perhaps because of a limited tendency to retain sodium in L(Lact) rats.

Influence of Biliary Drainage on the Repair of Hepatic Lesions in Biliary Fibrosis

Background. Bilioduodenal (BD) and biliojejunal (BJ) derivation induce enterobiliary reflux and bile stasis. Decompression of the excluded loop of the Roux-en-Y (BJD) was proposed to minimize these effects. The aim of this study was to compare the influence of these three modalities of biliary bypass on hepatic lesion repair in rats with secondary biliary fibrosis. Materials and Methods. Rats with 15 d of biliary obstruction underwent BD, BJ, and BJD drainage and were compared with a group submitted to simulated operation (SO) and biliary obstruction (CBO). The serum values of total and fractional bilirubin, alkaline phosphatase (ALP), and aminotransferases (AST and ALT), as well as hepatobiliointestinal excretion determined with (99m)Tc-Disida, were used for comparison. In addition, we used morphometric analyses to estimate the mass of the hepatocytes, bile ducts, and liver fibrosis. We also counted hepatic stellate cells (SC). Results. For each of the three modalities of biliary drainage, there were significant reductions in bilirubin, AST, ALP, and the number of SCs. The recovery of the estimated mass of all histologic components occurred only after BJ and BJD; in the BD group, the estimated hepatocyte mass was reduced compared with the SO group. The residual hepatic radioactivity of (99m)Tc-Disida was greater in the BJD group than in the SO group. Conclusions. The interposition of the jejunal loop between the biliary tree and the intestine may slow hepatobiliary clearance of radioactivity, even though it provides the resolution of cholestasis and is effective in recovering from hepatic lesions. (C) 2011 Elsevier Inc. All rights reserved.

HLA polymorphisms as incidence factor in the progression to end-stage renal disease in Brazilian patients awaiting kidney transplant

Chronic renal failure (CRF) leads in the majority of instances to end-stage renal disease (ESRD) requiring renal replacement therapy. Age, gender, genetics, race, hypertension, and smoking among others are factors associated with ESRD. Our interest was to evaluate the possible associations of class I and II HLA antigens with ESRD renal disease independent of other factors, among patients with CRF, having various diagnoses in the Brazilian population of the Sao Paulo state. So 21 HLA-A, 31 HLA-B, and 13 HLA-DR were detected in 105 patients who were compared with 160 healthy controls of both sexes who were not related to the patients evaluated until 2005. We calculated allelic frequencies, haplotypes frequencies, etiological fractions (EF), preventive fractions, and relative risks (RR). We compared demographic data of patients and controls. The antigens positively associated with ESRD were: HLA-A78 (RR = 30.31 and EF = 0.96) and HLA-DR11 (RR = 18.87 and EF = 0.65). The antigens HLAB14 (RR = 29.90 and EF = 0.75) was present at a significantly lower frequency among patients compared with controls. In contrast, no haplotype frequency showed statically significant associations. Further molecular studies may clarify types and subtypes of alleles involved with ESRD progression.

Mast cells, TGF-beta 1 and alpha-SMA expression in IgA nephropathy

IgA nephropathy (IgAN) is a kidney disease with a varying renal prognosis. Recently, many studies have demonstrated that renal alpha-smooth muscle actin (alpha-SMA) and transforming growth factor (TGF-beta 1) expression, as well interstitial mast cell infiltrates could represent a prognostic marker in several renal diseases. The aim of our study was to analyze the prognostic value of mast cell, TGF-beta 1 and alpha-SMA expression in IgAN. A survey of the medical records and renal biopsy reports of 62 patients with a diagnosis of IgAN followed-up from 1987 to 2003 was performed. The mean follow-up time was 74.7 +/- 50.0 months. The immunohistochemical studies were performed using a monoclonal antibody anti-human mast cell tryptase, a polyclonal antibody anti-human TGF-beta 1, and a monoclonal antibody anti-human alpha-SMA. An unfavorable clinical course of IgAN was related to interstitial mast cell infiltrates and alpha-SMA expression in the tubulointerstitial area. Expression of glomerular TGF-beta 1 and alpha-SMA, and interstitial TGF-beta 1 is not correlated with clinical course in IgAN. In conclusion, the increased number of mast cells and higher alpha-SMA expression in the tubulointerstitial area may be predictive factors for the poor prognosis of patients with IgAN.

Slight activation of nuclear factor kappa-B is associated with increased hepatic stellate cell apoptosis in human schistosomal fibrosis

To investigate the relationship between NF-kappa B activation and hepatic stellate cell (HSC) apoptosis in hepatosplenic schistosomiasis, hepatic biopsies from patients with Schistosoma mansoni-induced periportal fibrosis, hepatitis C virus-induced cirrhosis, and normal liver were submitted to alpha-smooth muscle actin (alpha-SMA) and NF-kappa B p65 immunohistochemistry, as well as to NF-kappa B Southwestern histochemistry and TUNEL assay. The numbers of alpha-SMA-positive cells and NF-kappa B- and NF-kappa B p65-positive HSC nuclei were reduced in schistosomal fibrosis relative to liver cirrhosis. In addition, increased HSC NF-kappa B p65 and TUNEL labeling was observed in schistosomiasis when compared to cirrhosis. These results suggest a possible relationship between the slight activation of the NF-kappa B complex and the increase of apoptotic HSC number in schistosome-induced fibrosis, taking place to a reduced HSC number in schistosomiasis in relation to liver cirrhosis. Therefore, the NF-kappa B pathway may constitute an important down-regulatory mechanism in the pathogenesis of human schistosomiasis mansoni, although further studies are needed to refine the understanding of this process. (C) 2009 Elsevier B.V. All rights reserved.

Progression of Lipid Peroxidation Measured as Thiobarbituric Acid Reactive Substances, Damage to DNA and Histopathological Changes in the Liver of Rats Subjected to a Methionine-Choline-Deficient Diet

Methionine-choline-deficient diet represents a model for the study of the pathogenesis of steatohepatitis. Male rats were divided into three groups, the first group receiving a control diet and the other two groups receiving a methionine-choline-deficient diet for 1 month (MCD1) and for 2 months (MCD2), respectively. The livers of the animals were collected for the determination of vitamin E, thiobarbituric acid reactive substances (TBARS), GSH concentration, DNA damages, and for histopathological evaluation. The hepatic TBARS and GSH content was higher (P < 0.05) in the groups receiving the experimental diet (MCD1 and MCD2) compared to control diet, and hepatic vitamin E concentration differed (P < 0.05) between the MCD1 and MCD2 groups, with the MCD2 group presenting a lower concentration. Damage to hepatocyte DNA was greater (P < 0.05) in the MCD2 group (262.80 DNA injuries/100 hepatocytes) compared to MCD1 (136.4 DNA injuries/100 hepatocytes) and control diet (115.83 DNA injuries/100 hepatocytes). Liver histopathological evaluation showed that steatosis, present in experimental groups was micro- and macro-vesicular and concentrated around the centrolobular vein, zone 3, with preservation of the portal space. The inflammatory infiltrate was predominantly periductal and the steatosis and inflammatory infiltrate was similar in the MCD1 and MCD2 groups, although the presence of Mallory bodies was greater in the MCD2 group. The study describes the contribution of a methionine-choline-deficient diet to the progression of steatosis, lipid peroxidation and hepatic DNA damage in rats, serving as a point of reflection about the role of these nutrients in the western diet and the elevated non-alcoholic steatohepatitis rates in humans.

Inhibition of COX 1 and 2 prior to Renal Ischemia/Reperfusion Injury Decreases the Development of Fibrosis

Ischemia and reperfusion injury (IRI) contributes to the development of chronic interstitial fibrosis/tubular atrophy in renal allograft patients, Cyclooxygenase (COX) 1 and 2 actively participate in acute ischemic injury by activating endothelial cells and inducing oxidative stress. Furthermore, blockade of COX I and 2 has been associated with organ improvement after ischemic damage. The aim of this study was to evaluate the role of COX I and 2 in the development of fibrosis by performing a COX I and 2 blockade immediately before IRI We subjected C57BI/6 male mice to 60 min of unilateral renal pedicle occlusion, Prior to surgery mice were either treated with indomethacin (IMT) at days -1 and 0 or were untreated. Blood and kidney samples were collected 6 wks after IRI. Kidney samples were analyzed by real-time reverse transcription-poly me rase chain reaction for expression of transforming growth factor beta (TGF-beta), monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-10, heme oxygenose 1 (HO-1), vimentin, connective-tissue growth factor (CTGF), collagen 1, and bone morphogenic protein 7 (BMP-7), To assess tissue fibrosis we performed morphometric analyses and Sirius red staining. We also performed immunohistochemical analysis of anti-actin smooth muscle, Renal function did not significantly differ between groups. Animals pretreated with IMT showed significantly less interstitial fibrosis than nontreated animals. Gene transcript analyses showed decreased expression of TGF-beta, MCP-1,TNF-alpha, IL-1-beta, vimentin, collagen 1, CTGF and IL-10 mRNA (all P < 0.05), Moreover, HO-I mRNA was increased in animals pretreated with IMT (P < 0.05) Conversely, IMT treatment decreased osteopontin expression and enhanced BMP-7 expression, although these levels did rot reach statistical significance when compared with control expression levels, I he blockade of COX 1 and 2 resulted in less tissue fibrosis, which was associated with a decrease in proinflammatory cytokines and enhancement of the protective cellular response.

Physical therapy assessment tools to evaluate disease progression and phenotype variability in Golden Retriever muscular dystrophy

Dogs suffering from Golden Retriever muscular dystrophy (GRMD) present symptoms that are similar to human patients with Duchenne muscular dystrophy (DMD). Phenotypic variability is common in both cases and correlates with disease progression and response to therapy. Physical therapy assessment tools were used to study disease progression and assess phenotypic variability in dogs with GRMD. At 5 (TO), 9 (T1), 13 (T2) and 17 (T3) months of age, the physical features, joint ranges of motion (ROM), limb and thorax circumferences, weight and creatine kinase (CK) levels were assessed in 11 dogs with GRMD. Alterations of physical features were higher at 13 months, and different disease progression rates were observed. Passive ROM decreased until 1 year old, which was followed by a decline of elbow and tarsal ROM. Limb and thorax circumferences, which were corrected for body weight, decreased significantly between TO and T3. These measurements can be used to evaluate disease progression in dogs with GRMD and to help discover new therapies for DMD patients. (C) 2011 Elsevier Ltd. All rights reserved.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.

In vitro analysis of inhibitory effects of the antibacterial monomer MDPB-containing restorations on the progression of secondary root caries

Objective: This study aimed to analyze in vitro inhibitory effects of restorative materials containing the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) on the formation of artificial secondary root caries lesions. Methods: Class V cavities (2 mm x 2 mm) were prepared in 75 human root fragments. Specimens were randomly divided into five groups (n = 15 fragments per group) and restored as follows: (I) MDPB-free adhesive system + MDPB-free composite (negative control); (II) resin modified glass ionomer (RM-GIC; positive control); (III) MDPB-free adhesive system + MDPB-containing composite (2.83% MDPB); (IV) MDPB-containing adhesive system + MDPB-free composite; M MDPB-containing adhesive system + MDPB-containing composite. Artificial secondary root caries lesions were produced by a biological artificial caries challenge. The restored specimens were immersed into a culture medium containing Streptococcus mutans and sucrose for 15 days. Histological slices (80 +/- 20 mu m) of the specimens were used for measuring the mean depths of the artificial lesions produced in both margins of the restorations using polarized light microscopy. Results were expressed in percentage related to the mean depth of the negative control, considered 100%. Data were compared by ANOVA followed by the Tukey`s test (p <= 0.05). Results: The depths of lesions adjacent to cavities filled with RM-GIC (GII; 85.17 +/- 15.2%) were significantly (p < 0.01) shallower than those adjacent to restorations with MDPB-free composite (GI; 100.00 +/- 10.04%), despite the presence of MDPB in the adhesive system (GIV; 101.95 +/- 21.32%). The depths of lesions adjacent to cavities restored with MDPB-containing composite (GIII; 82.68 +/- 12.81% and GV; 85.65 +/- 15.42%), despite the adhesive system used, were similar to those of RM-GIC (GII). Mean lesions depths in these groups decreased from 13% (GV) to 17% (GIII) in relation to the negative control (GI). Conclusions: MDPB-containing composite inhibits the progression of artificial secondary root caries lesions regardless of adhesive systems. (C) 2009 Elsevier Ltd. All rights reserved.

Altered beta-catenin expression related to cancer progression on actinic cheilitis and squamous cell carcinoma of the lip

beta-Catenin is a bifunctional protein related to cell adhesion and gene transcription when activated by Wnt pathway. Altered expression of beta-catenin was related to loss of differentiation, more aggressive phenotype, increase of tumor invasion, and poor prognosis in a number of different cancers. Actinic cheilitis is caused by excessive exposure to ultraviolet radiation and has a high potential to suffer malignant transformation into squamous cell carcinoma (SCC) of the lip, the most frequent oral malignancy. Studies of oral cancer have shown the correlation of beta-catenin expression and oral SCC prognosis, and loss of membrane expression may be considered as a potential marker for early tumor recurrence. Thirty-five cases of actinic cheilitis and 12 cases of SCC of the lip were select and submitted to immunohistochemical staining using beta-catenin antibody. beta-Catenin was positive on the membrane for all cases. Eighty-five percent of actinic cheilitis cases showed cytoplasmatic staining, and 22% nuclear staining. Eighty-three percent of SCC was positive for beta-catenin, and none of them had nuclear staining. Cytoplasmatic and nuclear staining of beta-catenin on studied cases point to pathway alterations. Results demonstrated that beta-catenin expression is altered on epithelial dysplasia, and it is related to degree of alterations. (C) 2011 Elsevier Inc. All rights reserved.

COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis. Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05). Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam. Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.

Effects of 15-deoxy-Delta(12, 14) prostaglandin J(2) and ciglitazone on human cancer cell cycle progression and death: The role of PPAR gamma

The role of PPAR-gamma in ciglitazone and 15-d PGJ(2)-induced apoptosis and cell cycle arrest of Jurkat (before and after PPAR gamma gene silencing), U937 (express high levels of PPAR gamma) and HeLa (that express very low levels of PPAR gamma) cells was investigated. PPAR gamma gene silencing, per se, induced a G2/M cell arrest, loss of membrane integrity and DNA fragmentation of Jurkat cells, indicating that PPAR gamma is important for this cell survival and proliferation. Ciglitazone-induced apoptosis was abolished after knockdown of PPAR gamma suggesting a PPAR gamma-dependent pro-apoptotic effect. However, ciglitazone treatment was toxic for U937 and HeLa cells regardless of the presence of PPAR gamma. This treatment did not change the cell cycle distribution corroborating with a PPAR gamma-independent mechanism. On the other hand, 15-d PGJ(2) induced apoptosis of the three cancer cell lines regardless of the expression of PPAR gamma. These results suggest that PPAR gamma plays an important role for death of malignant T lymphocytes (Jurkat cells) and PPAR gamma agonists exert their effects through PPAR gamma-dependent and -independent mechanisms depending on the drug and the cell type. (C) 2007 Elsevier B.V. All rights reserved.

Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca(2+) Channels From Secretory Vesicles

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS-C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca(2+)](i)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca(2+)](i). The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca(2+)](i) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes. Diabetes 59:1192-1201, 2010

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.