In vivo experimental groups and protocol
The present experimental protocol was approved by the local Research Ethics Committee (Comissão de Ética para Análise de Projetos de Pesquisa—CAPPesq) and was developed in strict conformity with the international standards for care and manipulation of laboratory animals.
Male Wistar rats aged between 7 and 8 weeks were kept under controlled temperature (23 ± 1 °C), on a 12/12 h’ light/dark cycle with ad libitum access to tap water and HS diet (3.2% Na, Nuvital, Brazil). After 2 weeks of adaptation to HS diet, 60 of these animals were submitted to the NAME experimental model: As previously described, these model of hypertensive nephrosclerosis was induced by the chronic inhibition of endogenous NO, thus stimulating peripheral vasoconstriction4. NO synthesis blockage was obtained by oral daily administration of 70 mg/kg/day of Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME, Sigma-Aldrich #N5751), a L-arginine analogue, diluted on drinking water, associated to the HS diet.
NAME rats were divided among the following 5 groups: NAME: Animals submitted to the NAME model and keep untreated; LOS: NAME animals treated with 50 mg/Kg/day of losartan (LOS, Losartana Potássica 50mg, Medley) diluted in drinking water; MMF: NAME rats treated with 10 mg/Kg/day of Micofenolate Mofetil (MMF, Micofenolato de Mofetila, 500 mg, Accord-Intas Pharmaceuticals), administered daily by oral gavage; TAM: NAME animals receiving 10 mg/Kg/day of Tamoxifen (TAM, Citrato de Tamoxifeno, 20 mg, Sandoz-Novartis) by oral gavage, and LOS + MMF + TAM: NAME rats treated with LOS, MMF and TAM simultaneously. Five additional animals received only HS and were used as Control. An illustrative flow-chart depicting the study design and groups can be seen in Supplemental Material section, on Supplementary Fig. 1A. Additionally, further animals were kept with normal sodium diet (standard rodent chow, Nuvital, Brazil), and used as NS Diet Control for clinical and biochemical analysis.
All groups were followed for 30 days. Body weight was monitored weekly and at the end of this period, systolic blood pressure was evaluated by the tail-cuff pressure method, using a noninvasive system (Visitech Systems, Apex, NC). 24-h urinary albumin excretion rate (24 h-UAE) was analyzed by radial immunodiffusion, as described elsewhere4,20. Animals were anesthetized with an intraperitoneal (IP) injection of 60 mg/kg of sodium pentobarbital and submitted to a ventral laparotomy. Blood samples were collected directly from the abdominal aorta, for renal and hepatic function analyses, followed by euthanasia through overdose of sodium pentobarbital, 80 mg/kg IP. The left kidney was removed and processed for histological and immunohistochemical analysis, while the right kidney was cut into small fragments and rapidly frozen in liquid nitrogen for PCR analysis.
Renal and hepatic function
Blood samples were centrifuged at 2000 rpm for 15 min, at room temperature for serum obtaining. Renal and hepatic function analyses were performed in serum samples of the animals of each experimental group: Blood Urea Nitrogen (SUrea) and serum creatinine concentration (SCreat) were assessed by colorimetric methods (Labtest Diagnóstica do Brasil UREIA CE #37 and CREATININA #35, respectively). Total serum protein (SProt), albumin (SAlb), alanine aminotransferase (ALT) T and alkaline phosphatase (ALK) concentrations were determined using specific colorimetric assay kits (Labtest Diagnóstica do Brasil, #99-1, #19-1, #108-4 and #79-4, respectively).
Histological analysis
As described elsewhere4, kidneys obtained from total nephrectomy were cut in two midcoronal renal slices and pre-fixed with Duboscq-Brazil for 30 min, followed by 24-h post-fixation in buffered 4% formaldehyde. Tissue samples were embedded in paraffin, through conventional techniques, renal tissue sections of 4-μm thickness were obtained and submitted to histological analysis for the assessment of glomerular and tubulointerstitial alterations.
The percentage of glomerulosclerosis and collapsed glomeruli were evaluated in periodic Acid-Schiff (PAS) staining samples, through the analysis of at least 50 randomly sampled glomerular tuft profiles per rat. The criteria used to define sclerotic glomeruli was the presence of segmental hyalinosis lesions, usually with adhesion to Bowman’s capsule. Collapsed glomeruli were defined by their reduced size, wrinkling basement membrane and collapsed capillary loops. Interstitial fibrosis was quantitatively evaluated in Masson-stained sections by a point counting technique4,21.
Immunohistochemical analysis
Immunohistochemistry (IHC) assays were performed to identify interstitial macrophage and T-cell infiltration in renal sections, as well as to evaluate tubulointerstitial cell proliferation and to quantify the percentage of tubulointerstitial area occupied by α-smooth muscle actin (α-SMA), possibly indicating the presence of myofibroblasts in the renal cortex2, collagen I and fibronectin. A mouse monoclonal anti-ED1 antibody (Serotec, Oxford, UK) was used to identify macrophages through the APAAP (alkaline phosphatase anti-alkaline phosphatase) technique. Monoclonal mouse anti-CD3 (Dako, Glostrup, Denmark) and anti-α-SMA (Sigma Chemical CO, St. Louis, USA) antibodies were used, to identify T-cells and myofibroblast, respectively, through a streptavidin–biotin-alkaline phosphatase (Strep-AP) IHC technique. In both APAAP and Strep-AP techniques, the reactions were developed with a fast-red dye solution. Tubulointerstitial cell proliferation was detected by a monoclonal mouse anti-PCNA antibody (Dako, Glostrup, Denmark), while collagen I and fibronectin positivity were detected with polyclonal anti-collagen I (Rockland Immunochemicals, Inc., NY, USA) and anti-fibronectin (Sigma Chemical CO, St. Louis, USA) primary antibodies, using a streptavidin-biotin-horseradish peroxidase (Strep-HRP) IHC technique. Samples were developed with a DAB dye solution.
In vitro experiments
In order to verify the specific antifibrotic activity of TAM alone, and also to investigate if this activity would be somehow inhibited or impaired by the association with other drugs, we performed cell culture experiments using a rat renal fibroblast cell line (NRK-49F; American Type Culture Collection, Manassas, VA). For this purpose, 1 × 105 NRK-49F cells were cultured under 37 °C and 5% CO2 in plastic culture plates with Dulbecco’s Modified Eagle Medium (DMEM-Low glucose, Invitrogen, USA) containing 5% inactivated fetal bovine serum (FBS; Gibco, Carlsbad, MO, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin antibiotic solution (Gibco). Once cells reached 80% of confluence, the culture medium was replaced by DMEM-Low with 100 units/mL penicillin, and 100 mg/mL streptomycin antibiotic solution, plus the specific stimuli, as follows; Control, NRK-49 cells receiving no additional stimuli or treatment diluted in the culture medium, IL-1β + AngII, NRK-49 cells whose culture medium was supplemented with 400 pg/mL of recombinant human IL-1β (PeproTech, Cranbury, NJ, USA) and 1 × 10−7 M human Angiotensin II acetate (Sigma), LOS, IL-1β + AngII cells whose culture medium was further supplemented with 10 µM of Losartan, TAM, IL-1β + AngII cells whose culture medium was further supplemented with 5 µM of Tamoxifen citrate (Sigma) and LOS + TAM, NRK-49 cells receiving all the above mentioned supplements. Cells were kept under the described treatments for 24 h. An illustrative flow-chart of in vitro experiments can be seen in Supplemental Material section, on Supplementary Fig. 1B.
Immunocytochemistry
Immunocytochemistry (ICC) assays were performed to characterize the constitutive expression of vimentin in NRK-49 cells, using a mouse monoclonal anti-vimentin primary antibody (Sigma Chemical CO, St. Louis, USA). The activation of fibroblasts after IL-1β + AngII stimulus, was evaluated through the positivity of these cells for α-SMA, with a mouse monoclonal anti-α-SMA antibody (Sigma Chemical CO, St. Louis, USA). Moreover, ICC was also employed to analyze the expression of collagen I and fibronectin in NRK-49 cells submitted to the different treatments, employing, respectively, the rabbit polyclonal anti-collagen I (Rockland Immunochemicals, Inc., NY, USA) and anti-fibronectin (Sigma Chemical CO, St. Louis, USA) primary antibodies. Vimentin, collagen I and fibronectin ICC were performed through a streptavidin-biotin-alkaline phosphatase (Strep-AP) technique. Reactions were developed with a fast-red dye solution. α-SMA was detected through a streptavidin-biotin-horseradish peroxidase (Strep-HRP) ICC technique, developed with a DAB dye solution.
Real time RT-PCR
Quantitative real-time polymerase chain reaction (PCR) of renal samples of experimental animals, as well as from cultured NRK-49F cells was performed to measure the relative gene expression of TGFβ, SMAD3, SMAD7, Collagen type I, collagen type III and Fibronectin, using Actinβ as a housekeeping control, as previously described. Total RNA extraction was carried out with RNeasy Plus Kit (Qiagen, MD, EUA), following the instructions of the manufacturer. Reverse transcription (RT) was performed with M-MLV enzyme kit (Promega) and qPCR was conducted with the Syber GreenER qPCR Super Mix Universal (Invitrogen), in the StepOne Plus equipment (Applied Biosystetems—Life Technologies). Quantitative comparisons were obtained using the ΔΔCT method (Applied Biosystems, Singapore, Singapore). Primer sequences for amplifying target genes were: Tgfb1 NM_021578.2, left primer: GCTGAACCAAGGAGACGGAA, right primer: CATGAGGAGCAGGAAGGGTC, Smad3 NM_013095.3 left primer: GAGACATTCCACGCTTCACA, right primer: AAAGACCTCCCCTCCAATGT, Smad7 NM_030858.2 left primer: TCTCCCCCTCCTCCTTACTC, right primer: CAGGCTCCAGAAGAAGTTGG, Coll1a1 NM_053304.1 left primer: AGCTGGTGCTAAGGGTGAAG, right primer: GCAATACCAGGAGCACCATT, Coll3a1 NM_053304.1 left primer: AGCTGGTGCTAAGGGTGAAG, right primer: GCAATACCAGGAGCACCATT, Fn1 NM_019143.2 left primer CTCCCGGAACAGATGCAATG, right primer ATCCAGCTGAAGCACTCTGT and Actb NM_031144.3 left primer: AGGGAAATCGTGCGTGACAT, right primer: CCATACCCAGGAAGGAAGGC.
Statistical analysis
Results were presented as mean ± SEM. Differences among all groups were analyzed by one-way ANOVA with Dunnet’s multiple comparison post-test. Means were considered statistically different when p < 0.0522. All statistical analyses were realized using the Graph-Pad Prism™ 5.01 software.
Ethical approval
The present experimental protocol was approved by the local Research Ethics Committee (Comissão de Ética para Análise de Projetos de Pesquisa—CAPPesq) and was developed in strict conformity with the international standards for care and manipulation of laboratory animals.
Statement of adherence to The ARRIVE Guidelines
The present experimental study was reported in accordance with The ARRIVE guidelines 2.0, https://doi.org/10.1371/journal.pbio.3000411, following the ARRIVE Essential 10 requirements.