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| Author(s): |
Filipe Miranda de Oliveira Silva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD) |
| Defense date: | 2015-09-02 |
| Examining board members: |
Irene de Lourdes Noronha;
Hugo Abensur;
Eduardo Homsi
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| Advisor: | Irene de Lourdes Noronha |
| Abstract | |
Peritoneal dialysis is an important therapeutic option for patients with stage 5 chronic kidney disease (CKD). However, at medium and long term, morphological and functional changes related to various factors such as bioincompatibility of dialysis solutions and peritoneal infections, among others, establish an inflammatory and fibrotic process in the peritoneal membrane, leading to a loss of dialysis efficiency. The uremic state of these patients aggravates this situation because it intensifies inflammation of the peritoneal membrane. Therapeutic strategies that slow the process of fibrosis of the peritoneal membrane of patients with CKD on dialysis are extremely important. In this context, the present study aimed to establish a model of peritoneal fibrosis associated with CKD with uremia, that mimics the clinical situation, and analyze the effect of two antifibrotic molecules, tamoxifen (TAM) and the BMP7 (bone morphogenic protein-7), in the proposed model. CKD with uremia was induced in male Wistar rats by adenine in the diet during a period of 30 days. After 15 days, with the state of uremia already established, animals received intraperitoneal injections of chlorhexidine gluconate, for the induction of peritoneal fibrosis (PF). Treatment with TAM (10mg/Kg/day by gavage) and BMP7 (30ug/Kg, intraperitoneal injections every 3 days) were initiated along with the induction of peritoneal fibrosis. Six groups were induced: CONTROL, normal animals; CKD, animals with chronic kidney disease; PF, animals with peritoneal fibrosis; CKD / PF, animals with CKD and PF mimicking the clinical situation; CKD / PF + TAM, animals with CKD and PF treated with tamoxifen; and CKD / PF + BMP7, animals with CKD and PF treated with BMP7. During 30 days of the follow-up study, weight, blood pressure, serum urea and creatinine of the animals were verified. At the end of this period, the animals were sacrificed and the peritoneum was removed and subjected to the following analysis: a) histology, to assess the degree of thickening (Masson\'s Trichrome); b) immunohistochemistry, to locate and quantify the presence of inflammatory cells (macrophages, T lymphocytes), myofibroblasts (alfa-smoth muscle actin) and cell proliferation activity (PCNA); c) analysis of pro-inflammatory cytokines (TNF-alfa, IL-1beta and IL-6) both in peritoneal tissue mRNA level through real time PCR as well as on protein level by multiplex; d) real-time PCR to determine the expression of extracellular matrix components (collagen III and fibronectin) and fibrogenic factors (TGF-beta and FSP-1). Furthermore, in order to study the possible signaling pathway associated to peritoneal fibrosis, the expression of SMAD 3 and SMAD 7 in the peritoneum was analyzed via real-time PCR and immunohistochemistry for phosphorylated SMAD 3. Finally, the peritoneal function was assessed by ultrafiltration and mass transferred glucose test (MTG). Data from weight gain showed that while animals from CONTROL and PF groups had significant weight gain (28% and 18% respectively) compared to the first day of protocol, the animals of other groups lost weight significantly, on average, 26% compared to the first day. All animals that received diet rich in adenine developed CKD presented by hypertension, detected on days 15 and 30 of the study (average of 173mmHg and 172mmHg respectively). Confirming the establishment of CKD with uremia, the animals that received diet rich in adenine showed serum urea (average 170 mg/dL on day 15 and 286 mg/dL on day 30) and creatinine (average of 0.97 mg/dL on day 15 and 1.82 mg/dL on day 30) significantly higher in the 15th and 30th days. Treatments with TAM and BMP7 did not influence these parameters. The peritoneal membrane analysis of PF and CKD/PF experimental groups showed a significant thickening of the peritoneal membrane (130 ± 33?m and 132 ± 26?m, respectively; p < 0.001 vs 36 ± 2um and 27 ± 6?m in CONTROL and CKD; p < 0.001) with presence of inflammatory cells. The treatments with TAM and BMP7 were effective in protecting the membrane against the thickening (42 ± 2um and 53±7um, respectively; p < 0.001 vs CKD/PF) and inflammatory infiltrate. Furthermore, with regard to myofibroblasts, effectors cells in the fibrogenesis process detected by alfa-SMA presence, it was found a signicantly expression in the peritoneum of PF and CKD/PF (p < 0.01 vs CONTROLE), and the treatment with TAM and BMP7 significantly protected against the presence of myofibroblasts confirmed by the significantly expression of PCNA. With regard to the detection of pro-inflammatory cytokines, analysis by real-time PCR in the animals of CKD group showed a significant increase in TNF-alfa expression in the peritoneum and IL-1beta compared to the CONTROL group. The expression of these cytokines was also increased in the peritoneum of the CKD/PF group. The treatment with TAM and BMP7 significantly reduced TNF-alfa and IL-1beta expression compared to CKD/PF group (p < 0.01).The repercussion of these results can be observed with the multiplex test in which the presence of these cytokines in its protein form were found in significant quantities in the peritoneum of the animals with uremia associated with PF compared to CONTROL group. The treatment with TAM and BMP7 significantly reduced the presence of these cytokines (p < 0.01 vs CKD/PF). As expected, the mRNA expression of extracellular matrix components was significantly elevated in the PF group and CKD/PF compared to the control group. Indeed, collagen III mRNA expression was higher in PF and CKD/PF group (3.4 ± 1 and 10.3 ± 2.4 UI, respectively; p < 0.01 vs CONTROL) as well as fibronectin (6.5 ± 0.8 and 29.2 ± 1 UI; respectively; p < 0.01 vs CONTROL). The animals treated with TAM and BMP7 significantly blocked the expression of collagen III (1.5 ± 0.9 and 0.2 ± 0.1 UI, respectively; p < 0.01 vs CKD/PF) and fibronectin (6.6±1.2 and 9.7±0.5 UI, respectively; p < 0.01 vs CKD/PF). Further, while in the PF and CKD/PF groups the expression of TGF-beta (13.2 ± 0.9 UI and 30.3 ± 0.1 UI, respectively; p < 0.01) was significantly higher compared to the CONTROL group, TAM and BMP7 decreased the expression of TGF-beta (17.7 ± 0.2 UI and 16.2 ± 0.1UI, respectively; p < 0.01 vs CKD/PF). A similar trend was found with the expression of FSP-1 mRNA with an increase in expression of this gene in PF and CKD/PF groups (p < 0.01 vs CONTROL), with significant blockage in the animals treated with TAM and BMP7 (p < 0.01 vs CKD/PF). Regarding the analysis of signaling pathways possibly involved in the process, SMAD 3 expression was significantly higher in PF and CKD/PF groups (3.7 ± 0.3 UI and 4.6 ± 0.3 UI, respectively) compared to groups CONTROL and CKD (1 ± 0.2 UI and 1.3 ± 0.6 UI, respectively; p < 0,01). Treatments with TAM and BMP7 decreased the expression of SMAD 3 in the peritoneum (1.1 ± 0.6 UI and 1.1 ± 0.7 UI, respectively; p < 0.01 vs CKD/PF). Yet TAM and BMP7 significantly increased the expression of SMAD 7 (2.8 ± 0.5 and 3.7 ± 0.5, respectively; p < 0.01 vs CKD/PF), a regulatory TGF-beta protein capable of blocking the expression of pro-fibrotic and inflammatory factors. Finally, the treated groups had the function of the peritoneum preserved when compared to the PF and CKD / PF groups, checked through the maintenance of the ultrafiltration capacity and reducing MTG. In summary, the animals treated with TAM and BMP7 had the peritoneum protected from thickening, inflammatory infiltrate, presence of myofibroblasts and cellular proliferation. The treatments were also effective in significantly reduce the expression of pro-fibrotic factors and inflammatory cytokines. Regarding SMADs, treatments with TAM and BMP7 were effective in blocking the expression of SMAD 3 and increase SMAD 7 expression. The results of this study suggest that tamoxifen and BMP7 protected the peritoneum in an experimental model of peritoneal fibrosis developed in uremic rats with CKD, possibly due to their anti-inflammatory and anti-fibrotic properties (AU) | |
| FAPESP's process: | 13/16269-9 - Analysis of the effect of tamoxifen and BMP-7 in an experimental model of peritoneal fibrosis in rats with Chronic Kidney Disease |
| Grantee: | Filipe Miranda de Oliveira Silva |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |