Chronic Kidney Disease (CKD) has high mortality, cardiovascular complications and contributes over 50% of all causes of death in this population. Over 80% of these patients have their treatment subsidized by the Unified Health System (SUS), and 15% of these patients will die on the waiting list for an organ. In Brazil, this incidence and prevalence are increasing, the prognosis remains poor and the disease treatment costs are very high. The CKD induces cellular infiltration into the kidney, mainly macrophages, which produce growth factors and cytokines, that induce apoptosis and proliferation of tubular cells, which promotes activation and proliferation of fibroblasts. Macrophages can adopt various phenotypes that lead to paradigm of development M1 and M2, referred to as classical activation of macrophages (M1 macrophages), or alternative activation of macrophages (M2 macrophages). Depending on the activation state of macrophages, they can play an important role in inflammation and kidney regeneration. M2 Macrophages release inflammatory molecules and growth factors that reduce renal inflammation and promote regeneration. Accordingly, the search for effective therapeutic tools, low cost, no side effects is conceived by many research groups. In this project, we intend to demonstrate that the low level laser (LLLT) fits as a relevant tool to slow progression of CKD. Mice C57/BL6 will be used, CX3CR1 and CCR2-GFP-RFP, male, weighing about 20 grams, provided by the animal facilities of the Federal University of Sao Paulo. The animals will be kept in a animal facility of mice in the Nephrology Department of the Federal University of São Paulo, packed in collective cages containing not more than five animals, with a clear artificial / dark cycle of 12 hours at a constant room temperature of 22 C and food and water supplies are available all the time. On day 0, UUO is performed by complete connection of the ureter with 4-0 surgical silk thread on two points, and an incision between the connection points are made. For each experimental group will be used 10 animals, 5 mice underwent UUO and 5 controls without surgery. The animals are sacrificed at 1, 4 and 7 days after surgery. The LBP (TF Premier, MMOptics, San Carlos-SP-Brazil) will be applied immediately after surgery for the group 1 day, non-invasively in the level density of 150mW, 30 seconds. In four days of group animals will be treated 1x a day every day, and the group of seven days, the animals will be treated from the third day of UUO, with the same dose described above. The renal macrophages would be investigated by flow cytometry (F4 / 80, CD11b, CX3CR1, CCR2, CD86, CD206, MHC class II, p40IL12 and IL10) and confocal imaging. The profile M1 and M2 will be inferred by the gene and protein measures related molecules (iNOS, CD206, arginase 1, CXCL9, CXCL10, among others). Kidney function is measured by creatinine and albuminuria, while renal fibrosis by collagen deposition analysis measures I / IV (PCR, WB and picrosirus). The inflammation will be studied by measurements of gene and protein expression (IL6, TNF-±, IL-1², IL10 and IL13). The specific study of macrophages will be done by adoptive transfer into empty animal, previously depleted of macrophages by use of liposome clodronate. LLLT is also applied in macrophages in vitro prior to transfer (for 30 seconds, 150 mW for 2 days). We believe that LLLT will induce a rapid expression of inflammatory molecules, associated with a profile M2 macrophages and less renal fibrosis.
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