TNF Determination in Dogs with Acute Renal Failure Induced by Gentamicin Submitted to Hemodialysis
A.M.C. Meneses; J.R.A.C. Brant; A. Melchert; M.E. Saito; J.S.C.T. Caramori; R.C. Gonçalves; M.T.S. Peraçoli; P. Barretti; A.L. Balbi; N.F. Souza; C.C.G. Moraes; R.B.S. Kuroda; D.J.S. Lima; M.J.F.M. Figueiredo; A.C.A. Pereira; M.A.M.K. Alves; R.N. Dias Neto; R.F. Andrade; R.K.G. Bastos; L.H.C. Pereira; A.C.F. Cardoso; L.S. Seixas; E.N.L. Andrade; G.S. Oliveira; K.A. Reis; A.C.C. Lacreta Junior; E.R. Branco; F.C.M. Oliveira; G.R. Galvão; B.M.A. Leandro
Universidade Federal Rural da Amazônia, Instituto da Saúde e Produção Animal, Montese, Belém/Pará/Brazil
Acute renal failure (ARF) is a clinical syndrome characterized by the sudden glomerular filtration decrease, with subsequent toxins accumulation and metabolic regulation of water balance, electrolyte and acid-base loss (Cowgill & Elliot 2004). Prerenal, renal and post-renal disorders can cause ARF in dogs. Hemodynamic instability and decreased cardiac output are the main pre-renal causes that lead to the ARF establishment. When renal perfusion decrease is sustained, the situation may cause ischemic acute tubular necrosis (ATN), which can also occur by nephrotoxic drugs or substances action, including aminoglycoside antibiotics such as gentamicin (Flemenbum 1973). Severe ATN induction may occur in dogs, resulting from gentamicin administration (7mg/kg/day) (Rivers at al. 1996). Melchert (2005) developed a dog ARF model given 45mg/kg/day of gentamicin via sub-dermal. Hemodialysis (HD) is the method that uses an "artificial kidney" to correct fluid composition and body volume imbalances, and eliminates accumulated toxins. It is necessary adequate vascular access, and some equipment, including HD machine, an extracorporeal circuit, dialysis solution designed to reduce osmotic imbalance risk (Stewart 1989) and hemodialysis filters (Cowgill & Langstone 1996; Meneses et al. 2002; Meneses 2003). During HD procedure, there's interaction of the patient's blood components with the extracorporeal circuit, including the dialyzer membrane, with cellular and humoral components of blood activation, causing a number of biological responses (Modi et al. 2001). There are several biological responses produced by dialysis membranes and according to Tizard (2002), immune system cells secrete a large glycoproteins variety, called cytokines, which regulate immune responses through cells signaling. Tumoral necrosis factor (TNF) have two different peptides, with multiple immunological activities, as well as local and systemic inflammatory responses, including effects on lymphoid and non-lymphoid cells (Wang et al. 2003). It is produced by activated macrophages and other cells and has a broad spectrum of biological actions in many target cells, immune or not (Oppenheim et al. 1992). According Basile & Druke (1989), the main changes observed during HD procedure are transient thrombocytopenia, leukopenia and neutropenia, hypoxemia, first use syndrome, amyloidosis and cytokines release such as IL-1 and TNF. This research is justified by the need to obtain answers regarding the dialyzer membranes biocompatibility and TNF use as a biomarker, since the literature is scarce regarding the subject matter, especially in ARF. Moreover, in Brazil, there are no descriptions of such research in dogs, which can bring benefits not only to the veterinary community, but also for application in human medicine.
Materials and Methods
The research was conducted at the Laboratory of the Department of Experimental Medicine, Faculty of Medicine of Botucatu--UNESP, São Paulo, Brazil, with the Ethics Committee in Animal Research approval. Were used 32 dogs, mixed breed, adult, male.16 were clinically healthy and 16 with ARF induced by gentamicin, with body weight ranging from 7 to 14kg, from the University Central Kennel. Animals were divided into four groups: Group1 (8 animals, clinically healthy, received HD treatment using cellulose acetate membrane, every 24 hours, totaling five sessions), group 2 (8 animals, with ARF, received HD treatment using cellulose acetate membrane, every 24 hours, totaling five sessions); grupo3 (8 animals, clinically healthy, received HD treatment using polysulphones membrane, every 24 hours, totaling five sessions) and group 4 (8 animals with ARF, received HD treatment using polysulphones membrane, every 24 hours, totaling five sessions). Nephropathy induction was conducted by gentamicin administration (15mg/kg/TID/IV). ARF establishment was defined when animals had serum creatinine values = 5mg/dL, regardless of urine volume. After anesthesia (propofol--6.6 to 8.8mg/kg/ bolus IV), a double-lumen catheter (8 French) was installed in the right external jugular vein (Meneses et al. 2002). Then, fluoroscopy was performed to verify the correct positioning of the catheter. Before ARF establishment blood samples were collected by jugular venipuncture, and during HD sessions, prescribed according to the needs of each patient, from the extracorporeal circuit (arterial line). TNF detection was performed by biological test and reading through ELISA, at times pre, 5, 30 and post HD. Analysis of variance (parametric or non-parametric) for the design of two factors (group regarding the presence or absence of disease and group on the type of membrane used), together with their tests of multiple comparisons was used (Norman & Trein 1994). All discussions were held on the level of 5% significance.
TNF average at times M0, M5, M30 and M60 in groups 1, 2, 3 and 4 were respectively: 0 in M0, M5, M30, and M60 (Group 1); 434,9 in M0, 572,03 in M5, 719,78 in M30, and 545,59 in M60 (Group 2); 0 in M0, M5, M30, and M60 (Group 3); 523,95 in MO, 369,8 in M5, 389,89 in M30, e 225,28 in M60 (Group 4). Results were expressed in pg/mL.
Discussion and Conclusions
ARF is a clinical syndrome that has many different etiologies and clinical signs. ARF is the leading cause of death in dogs and cats, since the treatment routinely used it is not always efficient, favoring a small and poor prognosis for patients. Recently, peritoneal dialysis (PD) and HD has been a viable alternative for the treatment of dogs and cats with ARF. Concerning TNF determination, there was no detection of this cytokine in normal animals, regardless of the membrane type used, while in the sick animals, the presence of high levels in both HD filters (cellulose acetate and polysulphones). It is noteworthy, however, that levels were higher in the cellulose acetate group. In patients undergoing HD, the detection of high levels of TNF, pro-inflammatory cytokine, can be correlated with a worse prognosis in patients with sepsis and/or the development of multiple organ dysfunctions (Simmons et al. 2004). Generally, as described by Goldstein et al. (2003), the peak production occurs at 30 minutes after the stimuli and it may last for up to 24 hours, a fact similar to that found in our study. The high cytokine levels can also be credited because the animals were uremic, leading to a persistent inflammatory state, this phenomenon called monocytes "pre-activation", leads this cell type to produce large TNF quantities under constant stimulation (Goldstein et al. 2003). This study indicated that occurs TNF levels increase in serum of the ARF animals submitted to HD, reflecting important response to cytokines production in both membranes. HD filters present different biocompatibility degrees, a fact that should be enhanced at the moment of choosing for a certain type of dialyzer membrane. In this study, cellulose acetate membrane proved to be biocompatible so as polysulphones, although the latter have shown slightly higher performance. Relevant factors in choosing membrane type are the individual cost and impact related to its reuse or not. Thus, dialyzer membranes biocompatibility in dogs remains unsolved, opening avenues for the development of new experimental researches, using other markers such as IL-1, IL-6, IL-18 and evaluation of oxidative stress, as well as new types of membranes.
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