O excesso de adenina na dieta (ADE) promove precipitação intrabular de cristais, levando a uma nefrite intersticial progressiva com perda de função renal. Estudo recente demonstrou que esse processo requer ativação do sistema NF-kB. No presente estudo investigamos o possível envolvimento de outros componentes da imunidade inata, além do NF-kB. Verificamos também a hipótese de que a nefropatia associada aos cristais continua a progredir mesmo depois de cessada a sobrecarga de adenina. Foram estudados ratos Munich-Wistar machos e adultos sem tratamento (C) ou recebendo 0.5% de ADE na dieta. Após 1 semana, a ADE foi removida da dieta e os animais foram seguidos por 4 ou 24 semanas. A administração de ADE por 1 semana promoveu uma inflamação intersticial aguda, com perda de função renal, alteração da pressão caudal, sem alterações glomerulares. Os mediadores da imunidade inata, como TLR2, TLR4, inflamassoma NLRP3, IL1beta e IL-6, apresentaram-se ativados sem, no entanto, ativar o sistema NF-kB. Após cessada a sobrecarga de ADE, a inflamação persistiu, com infiltração por macrófagos, expressão elevada de AngII, deposição progressiva de colágeno e, na fase mais tardia, glomeruloesclerose, caracterizando um processo inflamatório crônico, autônomo, que não contou com a participação do eixo NLR/IL1beta. Em contraste, o sistema NF-kB foi ativado, sendo um dos possíveis estímulos a produção intra-renal de AngII. Dois mecanismos patogênicos podem ser identificados neste estudo: 1) agudo, associado à ativação do eixo NLR-IL1beta; 2) crônico, associado à produção de AngII renal e à ativação do sistema NF-kB / Excess adenine in the diet (ADE) promotes intratubular crystal precipitation, leading to progressive interstitial nephritis and loss of renal function. A recent study has shown that this process requires activation of the NF-kB system. In the present study we investigated the possible involvement of other components of innate immunity, in addition to NF-kB, as well as whether nephropathy associated with excess adenine continues to progress even after dietary cessation. Male Munich-Wistar rats without treatment (C) or receiving 0.5% of ADE in the diet were studied. After 1 week, ADE was removed from the diet and the animals were followed for 4 or 24 weeks. Administration of ADE for 1 week promote acute interstitial inflammation, with loss of renal function, alteration of caudal pressure, without glomerular changes. Mediators of innate immunity, such as TLR2, TLR4, NLRP3 inflamassome, IL1beta and IL-6 , were shown to be activated, with no apparent activation of the NF-kB system. In the late phases of the model, the inflammation persisted, with significant infiltration by macrophages, high expression of AngII, progressive collagen deposition and glomerulosclerosis, characterizing a chronic, autonomic inflammatory process that did not involve the participation of the NLR/IL1beta axis. By contrast, the NF-kB system was activated, with intra-renal AngII production as a possible stimulus. Two mechanisms operated this study: 1) an acute one, associated with activation of the NLR-IL1beta axis; 2) a chronic one, associated with intrarenal AngII production and NF-kB activation

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors. Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any preconcentration scheme beyond sample work up by solid phase extraction (SPE). Using this hyphenated approach, submicromolar levels of catecholamines and metanephrines in normal human urine and indolamines in human plasma were detected after the removal of the analytes from their biological matrices and after preconcentration by SPE on mixed mode cation-exchange sorbents. The CE-TOFMS and SPE methods were individualized for each group of compounds. While catecholamines and metanephrines in urine samples were quantitated using 3,4-dihydroxybenzylamine as an internal standard, deuterated isotopes, considered ideal internal standards, were used for the quantitation of indolamines. Because the angiotensin peptides are present in biological fluids at much lower concentrations than the previous two families of analytes, their analysis required the application of additional preconcentration techniques. In this work, the coupling of either of two types of electrophoretic preconcentration methods - field amplified injection (FAI) and isotachophoresis (ITP) - to capillary zone electrophoresis with both UV and MS detection was evaluated. Using FAI-CE-UV, angiotensins were detected at ~1 nM concentrations. Using similar conditions but TOFMS detection, the detection limits were below 10 nM. ITP was evaluated in both single-column and two-column comprehensive arrangements. The detection limits achieved for the ITP-based techniques were approximately one order of magnitude higher than for the FAI-based preconcentration. While the potential usefulness of these techniques was demonstrated using angiotensins standards, substantial additional research would be required to allow these approaches to be applied to plasma as part of clinical assays.