Amitriptyline aggravates the fibrosis process in a rat model of infravesical obstruction.

Infravesical obstruction (IVO) secondary to benign prostatic hypertrophy can affect up to 50% of men over 50 years old and may cause serious and irreversible alterations throughout the urinary tract, especially in the bladder. Therapeutic approaches are currently limited. Amitriptyline has recently been described as an analgesic, anti-inflammatory and myorelaxant in some experimental models. The objective of this study was to investigate the effects of amitriptyline hydrochloride on the process of fibrosis in a bladder outlet obstruction model in rats. Male Wistar rats were subjected to IVO and studied at intervals of 1 and 14 days postprocedure. The rats were randomly divided into five groups: sham, IVO1-T, IVO1-NT, IVO14-T and IVO14-NT. Bladder tissue was processed for histopathology, immunohistochemistry and RT-PCR. The IVO14 groups presented bladder fibrosis, smooth muscle cell hypertrophy and bladder wall thickening. The IVO14-T group demonstrated a higher intensity of fibrosis, higher macrophage infiltration rate and higher gene expression of Transforming growth factor (TGF) Tgf-ß1. Thus this data shows that in this experimental mode amitriptyline had an amplifying effect on the process of fibrosis as a whole.

Effect of dexamethasone on human osteoblasts in culture: involvement of ß1 integrin and integrin-linked kinase.

Adhesive interactions play a critical role in cell biology, influencing vital processes from proliferation to cell death. Integrins regulate cell-ECM (extracellular matrix) adhesion and must associate with phosphorylating proteins such as ILK (integrin-linked kinase). Dysregulation of ILK expression is associated with anchorage-independent growth, cell survival and inhibition of apoptosis. Glucocorticoids influence differentiation and adhesion of osteoblasts and can affect bone protein synthesis. The objective of this study was to analyse the effect of DEX (dexamethasone) on the biology of osteoblasts, together with its influence on the expression of ILK and ß1 integrin. For this, primary cultures of human osteoblasts were exposed to DEX at 10-9 M (physiological dose) and 10-6 M (pharmacological dose) for 24 and 48 h. Cell viability, apoptosis and cell adhesion were analysed, as well as protein expression of ß1 integrin and ILK. It was observed that cell viability and adhesion were reduced in the cultures evaluated. In comparison with the control cultures, there was slightly less apoptosis in the cultures exposed to the physiological dose and considerably more apoptosis in those exposed to the pharmacological dose. In all treated cultures, protein expression of ILK was slightly higher than in the control cultures, whereas that of ß1 integrin was significantly lower. Both proteins under study were co-localized at the cell periphery in all cultures. Our results suggest that DEX causes osteoblast anoikis, probably due to decreased ß1 integrin expression, which might have had a direct influence upon ILK, reducing its activation and preventing it from playing its characteristic anti-apoptotic role.

Amitriptyline attenuates interstitial inflammation and ameliorates the progression of renal fibrosis.

Amitriptyline is a pleiotropic tricyclic antidepressant, which has anti-oxidant and anti-inflammatory properties. We tested whether amitriptyline might be useful in the treatment of chronic renal disease using the mouse model of unilateral ureteral obstruction. Amitriptyline caused a significant reduction of interstitial fibrosis, determined by Masson's staining, with minimal myofibroblast formation and macrophage infiltration following ureteral obstruction. Using quantitative PCR we found that this treatment significantly reduced the expression of key molecular markers of progressive tubulointerstitial injury such as osteopontin, MCP-1, ICAM-1, and TGF-beta1 compared to their level in a saline-treated control group. Sublethal X-irradiation or mycophenolate mofetil, treatments that reduce inflammation, were comparable to amitriptyline in the reduction of interstitial fibrosis and macrophage infiltration. These studies in animals suggest that amitriptyline is worth testing as a therapeutic agent that might preserve renal function by blocking inflammation and renal fibrosis.

Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Medium Attenuate Fibrosis in an Irreversible Model of Unilateral Ureteral Obstruction.

The therapeutic potential of mesenchymal stem cells (MSCs) and their conditioned medium (MSC-CM) has been extensively studied. MSCs can repair tissue, reduce local inflammation, and modulate the immune response. Persistent renal tubular interstitial inflammation results in fibrosis and leads to chronic kidney disease (CKD). Unilateral ureteral obstruction (UUO) is a very well-accepted renal fibrosis model. In this study, we evaluated factors influenced by the administration of MSCs or MSC-CM in the UUO model. MSCs extracted from rat bone marrow were cultivated in vitro and characterized by flow cytometry and cellular differentiation. Eight groups of female rats were used in experiments (n = 7, each), including Sham, UUO, UUO + MSC (obstruction + MSC), and UUO + CM (obstruction + MSC-CM) for 7 days of obstruction and Sham, UUO, UUO + MSC, and UUO + CM for 14 days of obstruction. The MSCs or MSC-CM was administered via the abdominal vena cava after total ligation of the left ureter. After 7 or 14 days, rats were euthanized, and serum and obstructed kidney samples were collected. MSCs or MSC-CM decreased the expression of molecules, such as Col1a1, α-SMA, and TNF-α. We also observed reductions in the levels of caspase 3, α-SMA, and PCNA in treated animals by immunohistochemistry. Our results suggest that the intravenous administration of MSCs or MSC-CM improves fibrosis progression and factors involved in apoptosis, inflammation, cell proliferation, and epithelial-mesenchymal transition in Wistar rats subjected to UUO, indicating a potential tool for preventing CKD.

Renin-angiotensin system (RAS) blockade attenuates growth and metastatic potential of renal cell carcinoma in mice.

OBJECTIVES: Renal cell carcinoma (RCC) is the most frequent type of cancer among renal neoplasms in adults and responds poorly to radiotherapy and chemotherapy. There is evidence that blockade of the renin-angiotensin system (RAS) might have antineoplastic effects. The aim of this study was to investigate the effects of RAS blockade on RCC in a murine model. METHODS AND MATERIALS: Murine renal cancer cells (Renca) were injected (1 × 10(5)) into the subcapsular space of the left kidney of BALB/c mice (8 wk of age). The animals were divided into 4 groups: a control group (no treatment), angiotensin-receptor blockers group (losartan 100mg/kg/d), angiotensin-converting enzyme inhibitor group (captopril 10mg/kg/d), and angiotensin-receptor blockers +angiotensin-converting enzyme inhibitor group (losartan 100mg/kg/d +captopril 10mg/kg/d). The animals received the drugs by gavage for 21 days after inoculation, beginning 2 days before tumor induction, and were then euthanized. After killing the animals, the kidneys and lungs were removed, weighed, and processed for histopathological and immunohistochemical analyses. Angiogenesis and vascular microvessels were assessed with the antibodies anti-vascular endothelial growth factor and anti-CD34. RESULTS: Angiotensin II-inoculated animals developed renal tumors. Treated animals presented smaller tumors, regardless of the therapeutic regimen, and far fewer lung metastases in both quantity and dimension compared with the controls. The expression of vascular endothelial growth factor and CD34 were significantly decreased in renal tumors of treated animals compared with the controls. CONCLUSIONS: Our findings suggest that blockade of RAS decreases tumor proliferation and metastatic capacity of RCC in this experimental model.

Podocyte Wnt/ß-catenin pathway is activated by integrin-linked kinase in clinical and experimental focal segmental glomerulosclerosis.

BACKGROUND: Changes in podocyte phenotype and function are characteristic of proteinuric glomerular diseases. Integrin-linked kinase (ILK) functions as a common downstream effector in proteinuric diseases. In addition, ILK was shown to interact with the Wnt signaling pathway. Here, we investigated ILK expression as well as its involvement with the Wnt signaling pathway in renal biopsies of patients with primary focal segmental glomerulosclerosis (FSGS), and in a correspondent in vivo model of podocyte lesion. METHODS: Biopsies from 37 patients with primary FSGS were evaluated by immunohistochemistry for ILK, phosphorylated GSK-3ß (pGSK-3ß) and ß-catenin expression. As experimental model, male Wistar rats received 5 injections of puromycin aminonucleoside (PAN) at 2-week intervals, and their kidneys were evaluated for ILK, P-cadherin and pAkt expression as well as ß-catenin and LEF-1 colocalization. RESULTS: Patients presented de novo ILK expression and pGSK-3ß in podocytes. In animals, there was an increase in gene and protein expression of ILK, mainly detected in the podocytes, as well as increased protein expression of pAkt compared with controls. ß-Catenin translocated to the nuclei of podocytes in animals and patients. ß-Catenin colocalized with LEF-1 in the nuclei of podocytes of animals. Gene expression of ß-catenin and P-cadherin in PAN rats was lower compared with controls. CONCLUSIONS: Our findings suggest that activation of ILK activated the Wnt signaling pathway in damaged podocytes. This phenomenon could have an important role in development and/or progression of clinical and experimental FSGS.

Phosphorylation and cytoplasmic localization of MAPK p38 during apoptosis signaling in bone marrow granulocytes of mice irradiated in vivo and the role of amifostine in reducing these effects.

We studied p38 phosphorylation and its intracellular localization during p53 and Puma (a p53 upregulated modulator of apoptosis) apoptotic signaling pathway in bone marrow granulocytes in mice irradiated in vivo and the role of the radioprotector amifostine in ameliorating these responses. Sixty-four C57BL mice were randomly assigned in two non-irradiated (Ami-/rad- and Ami+/rad-) and two irradiated (Ami-/rad+ and Ami+/rad+) groups. Animals received 400mg/kg of amifostine i.p. 30 min prior to a single whole body radiation dose of 7Gy. The experiments were performed using immunohistochemistry for caspase-3, cleaved caspase-3, p53, p-p53 (Ser 15), Puma, p38 and p-p38 (Thr 180/Tyr 182) protein expression. In addition transmission electron microscopy was used for ultrastructural characterization of apoptosis. Data showed that: (i) amifostine significantly reduced the number of apoptotic cells, (ii) p-p53 and Puma proteins were strongly immunostained in granulocytes after irradiation (Ami-/rad+), (iii) amifostine decreased the immunostaining of the proteins (Ami+/rad+), (iv) p38 was immunolocalized in physiological conditions in the nucleus and cytoplasm of granulocytes and neither radiation nor amifostine changed the protein immunostaining or its subcellular distribution, but influenced its activation, (v) radiation-induced p38 phosphorylation and its cytoplasmic accumulation during apoptosis signaling in granulocytes after whole body high radiation dose and amifostine markedly reduced these effects.

Akt pathway activation and increased neuropeptide Y mRNA expression in the rat hippocampus: implications for seizure blockade.

The aim of this study was to analyze the expression of survival-related molecules such Akt and integrin-linked kinase (ILK) to evaluate Akt pathway activation in epileptogenesis process. Furthermore, was also investigated the mRNA expression of neuropeptide Y, a considered antiepileptic neuropeptide, in the pilocarpine-induced epilepsy. Male Wistar rats were submitted to the pilocarpine model of epilepsy. Hippocampi were removed 6h (acute phase), 12h (late acute), 5d (silent) and 60d (chronic) after status epilepticus (SE) onset, and from animals that received pilocarpine but did not develop SE (partial group). Hippocampi collected were used to specify mRNA expression using Real-Time PCR. Immunohistochemistry assay was employed to place ILK distribution in the hippocampus and Western blot technique was used to determine Akt activation level. A decrease in ILK mRNA content was found during acute (0.39+/-0.03) and chronic (0.48+/-0.06) periods when compared to control group (0.87+/-0.10). Protein levels of ILK were also diminished during both periods. Partial group showed increased ILK mRNA expression (0.80+/-0.06) when compared with animals in the acute stage. Silent group had ILK mRNA and immunoreactivity similar to control group. Western blot assay showed an augmentation in Akt activation in silent period (0.52+/-0.03) in comparison with control group (0.44+/-0.01). Neuropeptide Y mRNA expression increased in the partial group (1.67+/-0.22) and in the silent phase (1.45+/-0.29) when compared to control group (0.36+/-0.12). Results suggest that neuropeptide Y (as anticonvulsant) might act in protective mechanisms occurred during epileptic phenomena. Together with ILK expression and Akt activation, these molecules could be involved in hippocampal neuroprotection in epilepsy.