ABSTRACT MOLECULAR MECHANISMS AND GENE SIGNATURES INVOVLED IN CALCINEURIN INHIBITOR NEPHROTOXICITY IN KIDNEY ALLOGRAFT By Huong Le Diem Nguyen, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physiology at Virginia Commonwealth University. Virginia Commonwealth University, 2011. Major Director: Valeria Mas, Ph.D. Associate Professor, Department of Surgery and Pathology Director of Molecular Transplant Research Laboratory, Division of Transplant Calcineurin inhibitors (CNI), cyclosporin A and tacrolimus, are potent immunosuppressive agents but induce toxicities causing damages and graft dysfunction, and have been suggested to contribute to late-term loss of graft in kidney transplant recipients. Even though insights on mechanism of CNI nephrotoxicity have been uncovered, prevention and treatment of these toxicities remain a major challenge in the clinical administration of CNI due to low dose-toxicity correlation, difficulty in establishing a differential patho-histological diagnosis, and varying individual susceptibility. We hypothesize that CNI nephrotoxicity follows distinct disease pathways and is characterized by significant gene signatures that differentiate it from other conditions such as acute rejection and chronic allograft dysfunction. Moreover, we postulate that CNI-induced toxicity profiles contribute to the IF/TA signatures. Microarray analysis and gene annotation were done on the study database included of tissues diagnosed with CNI nephrotoxicity (n = 9), interstitial fibrosis/tubular atrophy (IF/TA, n=10), and normal allografts (NA, n = 8). All samples were histologically classified based on the revised Banff ‘07 criteria for renal allograft pathology. Top-scored biological networks in CNI tissues were related to metabolic disease, cellular development, renal necrosis, apoptosis cell-death, immunological disease, inflammatory disease, and many others. Canonical pathway analysis emphasized oxidative stress response mediated by NRF2 and various cell-death signaling pathways including 14-3-3 signaling pathway, p53 signaling pathway, and TGF-β signaling pathway. Profiling of differentially expressed genes was done based on their statistical significance and biological relevance to the unique pathology of CNI nephrotoxicity. Among these, three genes RGS1, CXCR4, and TGIF1 were further quantitatively evaluated using real time-PCR. Between CNI group and normal allograft, t-test results showed only RGS1 gene expression level was statistically significant. Between IF/TA group in normal allograft, both RGS1 and CXCR4 showed statistical significance. The calculated relative fold changes revealed an up-regulated pattern of RGS1 and CXCR4 expression in association with pathological groups (CNI and IF/TA). We did not, however, find any association between the expression of TGIF1 in either CNI group or IF/TA group.

O timerosal é um agente antisséptico utilizado em vacinas como conservante. Devido a presença de etilmercúrio (EtHg) em sua composição (com aproximadamente 49% de mercúrio (Hg) em peso), uma preocupação existe em relação aos possíveis efeitos tóxicos em humanos. No entanto, pouco se sabe sobre o perfil cinético do EtHg em mamíferos. Neste sentido, este trabalho teve como objetivo avaliar a distribuição tecidual e meias-vidas do Hg, seu metabolismo no sangue (conversão a mercúrio inorgânico) e nefrotoxicidade após exposição ao timerosal, utilizando modelos in vivo e in vitro. Para isto, o trabalho foi dividido em 3 estudos: (I) camundongos machos Swiss foram expostos a 20 ?g de Hg sob a forma de timerosal via intramuscular. Sangue, cérebro, coração, rim e fígado foram coletados após 0,5; 1; 8; 16; 144; 720 e 1980 horas (h) da exposição (n=4) e analisados quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (II) alíquotas (n=4) de sangue total, plasma e eritrócitos humanos foram expostas a timerosal ou EtHg (3 mg/l) durante 24 h e analisadas quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (III) células HK2 foram expostas durante 24 h a timerosal (0 ?M a 2 ?M) e avaliadas quanto à viabilidade e proliferação celular, apoptose, expressão de proteínas Bax e TGF-?1, saúde mitocondrial e concentrações de fibronectina no meio. Verificou-se que o transporte de EtHg do músculo para os tecidos e a sua conversão em Hg inorgânico (Hgi) ocorrem rapidamente. Após 0,5 h da exposição ao timerosal, as concentrações mais altas de ambos EtHg e Hgi foram encontradas no rins (> 70% do Hg total no corpo do animal). O cérebro apresentou uma menor contribuição para a carga corporal de Hg (<1,0% do Hg total no corpo do animal). Após trinta dias da exposição ao timerosal, houve excreção considerável de Hg e o fígado apresentou a maior parte do Hg ainda restante no corpo dos animais. As meias-vidas foram estimadas (em dias) em 8,8; 10,7; 7,8; 7,7 e 45,2; para o sangue, cérebro, coração, fígado e rim, ii respectivamente. Sugere-se que a extensão da conversão de EtHg a Hgi é modulada em parte pela partição do EtHg no plasma e no sangue, uma vez que o EtHg é rapidamente convertido a Hgi nas células vermelhas, mas não no plasma. O mecanismo de dealquilação em células vermelhas parece ser mediado pela reação de Fenton (formação de radicais hidroxil). Ainda, EtHg/timerosal diminuiu a viabilidade celular e mitose, promoveu a apoptose, prejudicou o estado de transição de permeabilidade mitocondrial, aumentou a expressão de Bax e TGF-?1 e secreção de fibronectina. Coletivamente, os resultados demonstram que a cinética do timerosal (EtHg) está mais próxima a do Hgi - e não do MeHg - e o rim deve ser considerado um alvo potencial de toxicidade do EtHg, já que é o órgão exposto às maiores concentrações de Hg , e onde o metal apresenta sua maior meia-vida biológica. Adicionalmente, o EtHg/timerosal demonstrou ser um agente antiproliferativo, apoptótico e pró-fibrótico em células humanas renais. / Thimerosal is an antiseptic agent used in vaccines as a preservative. Due to the presence of ethylmercury (EtHg) in its composition (approximately 49% mercury (Hg) by weight), there is a concern regarding possible toxic effects in humans. However, little is known about the kinetic profile of EtHg in mammals. Thus, this study aimed to evaluate the tissue distribution and half-life of Hg, its metabolism in blood (conversion to inorganic mercury) and nephrotoxicity after exposure to thimerosal, using in vivo and in vitro models. The work was divided into three studies: (I) Swiss male mice were exposed to 20 ?g of Hg in the form of thimerosal intramuscularly. Blood, brain, heart, kidney and liver were collected after 0.5, 1, 8, 16, 144, 720 and 1980 hours (h) of exposure (n=4) and analyzed for concentrations of Hg species by HPLC-ICP-MS; (II) aliquots (n=4) of whole blood, plasma and erythrocytes were exposed to thimerosal or EtHg (3 mg/l) for 24 h and analyzed for Hg species concentrations by HPLC-ICP-MS; (III) HK2 cells were exposed for 24 h to thimerosal (0 ?M to 2 ?M) and evaluated for cell viability and proliferation, apoptosis, Bax and TGF-?1 expression, mitochondrial health and concentrations of fibronectin in the medium. It has been found that the transport of EtHg from muscle to tissues and its conversion into inorganic Hg (Hgi) occur quickly. After 0.5 h of exposure to thimerosal, higher concentrations of both EtHg and Hgi were found in the kidneys (> 70% of total Hg in the animal body). The brain showed a minor contribution to the body burden of Hg (<1.0% of total Hg in the animal body). Thirty days after exposure to thimerosal, there was considerable excretion of Hg and liver had the most Hg still remaining in the animal body. Half-lives were estimated (in days) at 8.8; 10.7; 7.8; 7.7 and 45.2; for blood, brain, heart, liver and kidney, respectively. It is suggested that the extent of conversion of the EtHg into Hgi is modulated in part by the partition of EtHg in plasma and blood, since EtHg is rapidly converted into Hgi in red cells but not in plasma. The mechanism of dealkylation in red cells seems to be mediated by the Fenton reaction iv (formation of hydroxyl radicals). Additionally, EtHg/thimerosal decreased cell viability and mitosis, promoted apoptosis, impaired mitochondrial permeability transition state, increased expression of Bax and TGF-?1 and fibronectin secretion in the media. Collectively, the results demonstrate that the kinetics of thimerosal (EtHg) is closer to the Hgi - and not the MeHg - and kidney should be considered a potential target for EtHg toxicity, since it is exposed to the highest concentrations of Hg and it is the tissue where the metal has a greater biological half-life. Additionally, EtHg/thimerosal was shown to be an antiproliferative, apoptotic and pro-fibrotic agent in human kidney cells.

Quinones represent an important class of endogenous compounds such as neurotransmitters and coenzyme Q10, electrophilic xenobiotics and environmental toxicants that have known reactivity based on their ability to redox cycle and generate oxidative stress, as well as to alkylate target proteins. 1,4-Benzoquinone (BQ) is a reactive quinone that we have used to help predict target residue covalent binding by such compounds. Hydroquinone glutathione conjugates (HQ-GSH) cause renal cell necrosis by producing reactive oxygen species (ROS) and by adducting proteins preferentially localized in the S3 segment of the renal proximal tubules. In vitro experimentation using model peptides and proteins have identified cysteine, lysine, arginine, and glutamic acid as amino acids targeted for quinone-thioether adduction. By mimicking a standard protein digestion protocol (100 mM ammonium bicarbonate pH 7.5, or 50 mM Tris-HCl pH 7.5), we demonstrated that cysteine-BQ adducts are unstable. Taken together, these results indicate that BQ-adduct formation on cysteine residues may be a transient interaction, where physiological conditions may play a role in adduct stability. In vivo experimentation following administration of 2-(glutathion-S-yl)HQ (MGHQ, 400 μmol/kg, iv, 2 hr) to Long Evans rats identified the specific site of quinone-thioether protein adduction on a number of proteins. Urinary proteins were isolated, and either trypsin digested en masse and analyzed by multi-dimensional protein identification technology (MuDPIT) or, following SDS-PAGE, single immunopositive bands were excised, trypsin digested and analysed by LC-MSMS. Following site-specific identification of adducts, 3-dimensional protein modeling of adducts on the protein was performed as a way to reveal the potential structural consequence of the modification on 3D structure. The outer stripe of the outer medulla (OSOM) is the target site of protein adduction caused by quinone-thioethers. Using a 2DGE-Western blot approach, in combination with an extensive knowledge of quinol-thioether chemistry, LC-MSMS, and the latest MSMS analysis software, we identified the specific amino acid site of adduction on 17 unique peptides from 34 target proteins within the OSOM. Of the 22 bands analyzed, adducted peptides were identified in 11 of them. Many of the target proteins identified have previously been identified as a target of other electrophiles, producing additional evidence that such protein adduction is selective rather than random. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response.

Made available in DSpace on 2015-07-22T20:50:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-26. Added 1 bitstream(s) on 2015-08-11T03:26:28Z : No. of bitstreams: 1 Publico-322.pdf: 393779 bytes, checksum: e3edd8a910aaf2a4ada36110dfd5e404 (MD5) / Introdução: As polimixinas são antimicrobianos antigos, e que caíram em desuso por muitos anos pelos relatos de toxicidade, principalmente nefrotoxicidade e neurotoxicidade. O surgimento de bactérias gram negativas multirresistentes em especial P aeruginosa e A baumanii em todo o mundo é uma realidade e temos observado seu crescimento em inquéritos epidemiológicos internacionais a partir dos anos 90. Não há estudos que demonstrem a taxa de disfunção renal nos pacientes transplantados pelo uso da polimixina, porém sabe-se que a função renal contribui de forma estatisticamente significante para a sobrevida dos receptores a longo prazo. Métodos: Foram levantados retrospectivamente os prontuários de todos os pacientes transplantados de órgãos sólidos, e que utilizaram polimixinas, durante o período janeiro 2001 a dezembro 2007 em 2 hospitais de ensino na cidade de São Paulo, Brasil. O objetivo principal do estudo foi definir o percentual de nefrotoxicidade apresentado pelos pacientes. Com o objetivo de avaliar essa variável escolhemos 2 definições de toxicidade renal (critério 1 e critério 2) e as aplicamos em todos os pacientes estudados com o objetivo de compará-las entre si e com a literatura. Critério 1 foi definido como creatinina sérica > 2 mg/dl após introdução da polimixina em pacientes com disfunção renal aguda, ou aumento de 50% da creatinina sérica em relação aos valores pré-polimixina naqueles com disfunção renal prévia. Associa-se à definição em qualquer das 2 situações acima a diminuição do clearance de creatinina estimado pela metodologia de Cockcroft & Gault em 50%, ou evolução para terapia dialítica. Critério 2 foi definido como qualquer aumento de creatinina sérica. Resultados: Foram identificados 92 pacientes transplantados de órgãos sólidos que utilizaram polimixinas. Em sua maioria eram transplantados de rim, ou rim/pâncreas (90,2%), cujo enxerto foi recebido de doador falecido em 70,7% dos casos. O principal diagnóstico foi infecção do trato urinário (ITU) (41,3%), seguido de infecção do sítio cirúrgico (17,4%), e pneumonia (16,3%). P aeruginosa foi o agente etiológico mais freqüente, presente em 76,1% dos isolados. Houve cura microbiológica em 25 pacientes (100%), cura clínica de 71 pacientes (77,2%), e mortalidade hospitalar em 21 pacientes (22,8%). Quarenta e quatro pacientes (47,8%) apresentaram nefrotoxicidade por qualquer dos 2 critérios adotados, 30 pacientes (32,6%) pelo critério 1, e 44 pacientes (47,8%) pelo critério 2. A análise multivariada demonstrou associação estatisticamente significante entre ITU e proteção para nefrotoxicidade [p 0,02; OR 0,24; IC 95% (0,07 – 0,86)] e média de tempo de utilização da polimixina maior como fator de risco para disfunção renal [p 0,03; OR 1,06; IC 95% (1,0 – 1,13)] pelo critério 1. Conclusão: A taxa de disfunção renal de 32,6% ainda é alta, porém menor que as inicialmente descritas na década de 60 e 70. A utilização da polimixina é eficaz principalmente no tratamento de ITU em pacientes transplantados de órgãos sólidos. A utilização de polimixina deve ser realizada por menor tempo possível e, quando não existir outro antimicrobiano possível. / Introduction: Polymyxins are old antimicrobials which had their use discontinued for many years because of nephrotoxicity and neurotoxicity description. The development of multirresistant gram negative bacteria in special P aeruginosa and A baumanii all over the world is a matter of fact and we have observed its growth in international epidemiologic surveys since the 90’s. Until now we don´t have studies that demonstrate renal dysfunction tax in transplanted patients because of polymyxin use, nevertheless we know that renal function contribute in a statistical significant manner for receptors survive in long term. Methods: We have retrospectively searched for all solid organ transplanted patients and who have used polymyxins from January 2001 to December 2007 in 2 teaching hospitals in São Paulo city, Brazil. The main study objective was to define the nephrotoxicity percentage. For evaluating this variable we choosed 2 renal function definitions (first criteria and second criteria) and applied them in all studied patients with the objective of comparing them each other and with the literature. First criteria was defined as serum creatinine > 2 mg/dl after polymyxin introduction in those patients with acute renal dysfunction, or 50% serum creatinine increase in relation to value before polymyxin was given in those patients with previous nephotoxicity. In both situations described above we also considered renal dysfunction if 50% decrease in estimated creatinine clearance by Cockcroft & Gault methodology occurred, or progression to dialysis therapy. Second criteria was defined as any serum creatinine increase. Results: We identified 92 solid organ transplanted patients who used polymyxins. The majority of them received renal or renal/pancreas grafts (90.2%), and the organs transplanted were from deceased donors in 70,7%. The main site of infection were urinary tract infection (UTI) (41.3%), followed by surgical site infection (SSI) (17.4%) and pneumonia (16.3%). P aeruginosa were the main etiologic agent present in 76.1% of isolates. Microbiologic cure occurred in 25 patients (100%), clinical cure in 71 patients (77.2%), and in hospital mortality occurred in 21 patients (22.8%). Fourty four patients (47.8%) presented nephrotoxicity according to any of the 2 adopted criteria, 30 patients (32.6%) according to the first criteria, and 44 patients (47.8%) according to the second criteria. Multivariate analysis show statistical significant association among UTI and protection for renal dysfunction [p 0.02; OR 0.24; IC 95% (0.07 – 0.86)], and greater mean polymyxin utilization time (p 0.03) as a risk factor for renal dysfunction by the first criteria. Conclusions: The 32.6% percentage of renal dysfunction is still high, but lower than that reported in the 60’s and 70’s. Polymyxin utilization is effective principally for the treatment of UTI in solid organ transplanted patients, its use should be judicious and for shorter time as possible. / TEDE / BV UNIFESP: Teses e dissertações

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Gentamicina (GM) à um aminoglicosÃdeo amplamente utilizado contra infecÃÃes por microrganismos gram-negativos. LesÃo renal aguda (LRA) à a principal limitaÃÃo para a sua eficÃcia terapÃutica, ocorrendo em 10-20% dos pacientes. O evento capital à produÃÃo de espÃcies reativa de oxigÃnio (EROs) e alteraÃÃo na dinÃmica intra-renal e glomerular. O gengibre à fonte de substÃncias bioativas que podem ter efeito satisfatÃrio na renoproteÃÃo por aÃÃo antioxidante. A fraÃÃo ZOEB4 à enriquecida com os compostos com [6]-, [8]- e [10]- gingerol, isolada a partir do extrato do Zingiber officinale Roscoe, parece ser promissora diante desta nefrotoxidade, podendo auxiliar o rim na resposta antioxidante endÃgena, diminuindo o dano oxidativo e atenuando a resposta prÃ-inflamatÃria. O presente estudo visou investigar os possÃveis efeitos protetores da fraÃÃo ZOEB4 em um quadro de LRA pela administraÃÃo de GM 100 mg/kg i.p. Foram utilizados ratos Wistar, adultos machos, divididos em 6 grupos. Os grupos controles foram induzidos com NaCl 0,9% por 7dias e tratados oralmente com tween-80 2%, ou com a fraÃÃo ZOEB4 25 mg/kg; grupos induzidos a nefrotoxicidade com GM 100mg/kg por 7dias acrescido do tratamento oral com a fraÃÃo ZOEB4(6,25, 12,5 ou 25mg/kg) ou com tween 80 2%. O tratamento oral ocorreu durante 5 dias a partir do 5 dia de induÃÃo. Ao termino de cada tratamento foram coletados plasma, urina e rins para as anÃlises. Foram avaliados os parÃmetros bioquÃmicos indicativos de funÃÃo renal, funÃÃo tubular, perfil oxidativo, atividade das enzimas antioxidantes, a transcriÃÃo gÃnica de mediadores prÃ-inflamatÃrios atravÃs da reaÃÃo da polimerase em cadeia em tempo real (qPCR), alÃm da anÃlise histopatolÃgica. A GM alterou consideravelmente quase que todos os parÃmetros investigados: Clcr (0,7Â0,1 mL/min), ureia (73,1Â7,5 mg/dL) proteÃna urinÃria (93,5Â9,2 mg/dL), FENa (3,3Â0,5 %) e FEK (78,76 11,7 %), MDA renal(1,7Â0,3 nM/mg de prot.), GSH (101,6 23,4Âg/mg prot.), SOD (52,47Â7,7 U/mg prot.) e TNF-&#945; (3,2 Â0,7) estabelecendo assim, dano renal nos animais. O tratamento com a fraÃÃo ZOEB4 na dose de 25mg/kg desencadeou proteÃÃo diante desta nefrotoxicidade, revertendo a diminuiÃÃo do Clcr (1,3 0,2 mL/min), diminuindo os nÃveis sÃrico de ureia (41,7Â0,03 mg/dL), de proteÃna urinÃria (51,36Â3,2 mg/dL), da FENa (1,82  0,21 %) e FEK (48,8Â4,6 %), MDA (0,67Â0,2 nM/mg de prot.), alÃm de aumentar os nÃveis de GSH (250,2Â27,7 ug/mg prot.) e a atividade da SOD (112,2Â6,4 U/mg prot.) e, inibiÃÃo da transcriÃÃo de TNF-&#945; (1,5Â0,2). Desta forma, proporcionando proteÃÃo a funÃÃo renal. Estes resultados, acrescentado aos relatos de estudos prÃvios realizados com os compostos desta planta, indicam um futuro promissor para a utilizaÃÃo dos gingerois como um coadjuvante no tratamento diante de LRAs induzidas por aminoglicosÃdeos.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Kidneys are vulnerable to chemical agent-induced injuries. Children (neonates and infants) are a particular at risk age group because they have renal functions less developed than that of adults. The exposure of children to medicines considered to be nephrotoxic agents - namely the aminoglycosides, nonsteroidal anti-inflammatory drugs (NSAID) and angiotensin converting enzyme (ACE) inhibitory drugs - should be avoided whenever possible. Hospitalized children are pointed out to be the group at greatest risk of nephrotoxicity due to their high level of exposure to these medicines as well as the frequent and improper use of non standardized medicines in this age range. To determine the prevalence of use of potentially-nephrotoxic drugs (PND) and the frequency of concomitant risk factors in hospitalized children younger than 2 years old in a medical ward in a pediatric hospital in Fortaleza, Brazil. A prospective, observational, follow-up study was developed. All children younger than 2 years old admitted to the general ward were included and followed-up. Sociodemographic data, pathological and pharmacotherapeutic antecedents were recorded, as well as information about the use of drugs and the attendance of risk factors for nephrotoxicity associated with aminoglycosides, NSAID and ACE inhibitors. Also, the occurrence of adverse events was identified. Data was taken from medical records and interviews with the mothers of the children. Statistical analysis involved cumulative frequency, cumulative percentage, central tendency measures, Student âtâ test and ANOVA. During the study period (September/2005 to March/2006), 120 admissions were recorded. Three patients were excluded because of incomplete data. The results represented 117 admissions that affected 103 different children. The prevalence of the use of PND was 96,6%. A total of 1065 drugs were used, 69% with potential intrinsic nephrotoxicity based on available literature. The mean number of PND used was 6,3  4,0 per patient. The PND most frequently used were: metamizole (10,1%), ranitidine (6,2%) and prednisone (5,1%). Around 18% of children used aminoglycosides, 65,8% and 4,3% had taken NSAID and ACE inhibitory drugs respectively. A total of 368 risk factors for nephrotoxicity were detected (3,5Â1,8 risk factors/patient). The most frequent factors were: the use of at least one PND (30,7%), the use of 2 or more PND (28,3%) and the use of NSAID concomitantly with that of potassium-rich salt substitutes(10%).The PND use was considered high when compared with published data from this studied age group. The frequency of risk factors for nephrotoxicity also reached considerable levels. It would be important to know if there exist safer therapeutic alternatives and what preventative measures could be adopted in each case. The contribution of a clinical pharmacist to a safe pharmacotherapy for hospitalized children would be a strategy for reducing PND-associated risk. / Os rins sÃo bastante vulnerÃveis a danos produzidos por agentes quÃmicos. Dentre as substÃncias nefrotÃxicas estÃo os medicamentos, os quais merecem destaque devido à ampla exposiÃÃo aos mesmos. Alguns grupos, como aminoglicosÃdeos, antiinflamatÃrios nÃo-esteroidais (AINE) e inibidores da enzima conversora de angiotensina (IECA) sÃo muito conhecidos pelo seu potencial nefrotÃxico intrÃnseco. As crianÃas menores (neonatos e lactentes) sÃo dignas de atenÃÃo especial no que se refere a essa questÃo, porque freqÃentemente usam medicamentos e, alÃm disso, a capacidade funcional de seus rins à menor do que a dos adultos. A melhor maneira de tratar a questÃo da nefrotoxicidade à prevenindo-a. Determinar a prevalÃncia de uso de medicamentos potencialmente nefrotÃxicos (MPN) e observar a presenÃa de condiÃÃes que favorecem ao desenvolvimento da nefrotoxicidade (fatores de risco) em crianÃas menores de dois anos de idade internadas em enfermaria geral de um hospital pediÃtrico em Fortaleza â Brasil. Estudo observacional, prospectivo, de seguimento de pacientes. Todas as crianÃas menores de dois anos admitidas na enfermaria âEâ foram incluÃdas e monitorizadas. InformaÃÃes sociodemogrÃficas, antecedentes patolÃgicos e farmacolÃgicos foram registrados, bem como informaÃÃes sobre o uso de medicamentos, presenÃa de fatores de risco para nefrotoxicidade associada a aminoglicosÃdeo, AINE e IECA, e ocorrÃncia de eventos adversos. Os dados foram coletados dos prontuÃrios mÃdicos e atravÃs de entrevista com os responsÃveis pelas crianÃas, sendo analisados estatisticamente usando medidas de freqÃÃncia, tendÃncia central e os testes âtâ de Students e Anova. Durante o perÃodo de estudo (setembro/2005 a marÃo/2006), ocorreu um total de 120 admissÃes na enfermaria; trÃs dos pacientes foram excluÃdos do estudo porque tinham dados incompletos. Os resultados se referem a 117 admissÃes correspondentes a 103 crianÃas. A prevalÃncia de uso de MPN foi de 96,6%. Do total de 1065 itens de prescriÃÃo consumidos, 68,6% tinham potencial nefrotÃxico intrÃnseco. O nÃmero mÃdio de MPN utilizados foi 6,3  4,0 por paciente. Dentre os MPN mais usados estavam: dipirona (10,1%), ranitidina (6,2%) e prednisona (5,1%). Dois por cento das crianÃas usaram aminoglicosÃdeos, 7,3% usaram AINE e 0,8% utilizaram IECA. Foram detectados 368 fatores de risco para nefrotoxicidade, com uma mÃdia de 3,15  1,8 fatores de risco/paciente. Os fatores de risco mais freqÃentes foram: uso de, no mÃnimo, um MPN (30,7% do total de fatores); uso de 2 ou mais MPN concomitantemente (28,3%) e o uso de AINE concomitante ao uso de suplementos de potÃssio (10%). O uso de MPN na faixa etÃria estudada foi considerado elevado. A freqÃÃncia de fatores de risco para nefrotoxicidade tambÃm ocorreu em nÃveis preocupantes. Seria importante conhecer se existiam alternativas mais seguras em cada caso e que medidas preventivas poderiam ser adotadas. A inclusÃo do farmacÃutico clÃnico na atenÃÃo a crianÃas hospitalizadas seria uma estratÃgia com grande potencial de impacto na reduÃÃo de riscos associados aos MPN.

O timerosal é um agente antisséptico utilizado em vacinas como conservante. Devido a presença de etilmercúrio (EtHg) em sua composição (com aproximadamente 49% de mercúrio (Hg) em peso), uma preocupação existe em relação aos possíveis efeitos tóxicos em humanos. No entanto, pouco se sabe sobre o perfil cinético do EtHg em mamíferos. Neste sentido, este trabalho teve como objetivo avaliar a distribuição tecidual e meias-vidas do Hg, seu metabolismo no sangue (conversão a mercúrio inorgânico) e nefrotoxicidade após exposição ao timerosal, utilizando modelos in vivo e in vitro. Para isto, o trabalho foi dividido em 3 estudos: (I) camundongos machos Swiss foram expostos a 20 ?g de Hg sob a forma de timerosal via intramuscular. Sangue, cérebro, coração, rim e fígado foram coletados após 0,5; 1; 8; 16; 144; 720 e 1980 horas (h) da exposição (n=4) e analisados quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (II) alíquotas (n=4) de sangue total, plasma e eritrócitos humanos foram expostas a timerosal ou EtHg (3 mg/l) durante 24 h e analisadas quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (III) células HK2 foram expostas durante 24 h a timerosal (0 ?M a 2 ?M) e avaliadas quanto à viabilidade e proliferação celular, apoptose, expressão de proteínas Bax e TGF-?1, saúde mitocondrial e concentrações de fibronectina no meio. Verificou-se que o transporte de EtHg do músculo para os tecidos e a sua conversão em Hg inorgânico (Hgi) ocorrem rapidamente. Após 0,5 h da exposição ao timerosal, as concentrações mais altas de ambos EtHg e Hgi foram encontradas no rins (> 70% do Hg total no corpo do animal). O cérebro apresentou uma menor contribuição para a carga corporal de Hg (<1,0% do Hg total no corpo do animal). Após trinta dias da exposição ao timerosal, houve excreção considerável de Hg e o fígado apresentou a maior parte do Hg ainda restante no corpo dos animais. As meias-vidas foram estimadas (em dias) em 8,8; 10,7; 7,8; 7,7 e 45,2; para o sangue, cérebro, coração, fígado e rim, ii respectivamente. Sugere-se que a extensão da conversão de EtHg a Hgi é modulada em parte pela partição do EtHg no plasma e no sangue, uma vez que o EtHg é rapidamente convertido a Hgi nas células vermelhas, mas não no plasma. O mecanismo de dealquilação em células vermelhas parece ser mediado pela reação de Fenton (formação de radicais hidroxil). Ainda, EtHg/timerosal diminuiu a viabilidade celular e mitose, promoveu a apoptose, prejudicou o estado de transição de permeabilidade mitocondrial, aumentou a expressão de Bax e TGF-?1 e secreção de fibronectina. Coletivamente, os resultados demonstram que a cinética do timerosal (EtHg) está mais próxima a do Hgi - e não do MeHg - e o rim deve ser considerado um alvo potencial de toxicidade do EtHg, já que é o órgão exposto às maiores concentrações de Hg , e onde o metal apresenta sua maior meia-vida biológica. Adicionalmente, o EtHg/timerosal demonstrou ser um agente antiproliferativo, apoptótico e pró-fibrótico em células humanas renais. / Thimerosal is an antiseptic agent used in vaccines as a preservative. Due to the presence of ethylmercury (EtHg) in its composition (approximately 49% mercury (Hg) by weight), there is a concern regarding possible toxic effects in humans. However, little is known about the kinetic profile of EtHg in mammals. Thus, this study aimed to evaluate the tissue distribution and half-life of Hg, its metabolism in blood (conversion to inorganic mercury) and nephrotoxicity after exposure to thimerosal, using in vivo and in vitro models. The work was divided into three studies: (I) Swiss male mice were exposed to 20 ?g of Hg in the form of thimerosal intramuscularly. Blood, brain, heart, kidney and liver were collected after 0.5, 1, 8, 16, 144, 720 and 1980 hours (h) of exposure (n=4) and analyzed for concentrations of Hg species by HPLC-ICP-MS; (II) aliquots (n=4) of whole blood, plasma and erythrocytes were exposed to thimerosal or EtHg (3 mg/l) for 24 h and analyzed for Hg species concentrations by HPLC-ICP-MS; (III) HK2 cells were exposed for 24 h to thimerosal (0 ?M to 2 ?M) and evaluated for cell viability and proliferation, apoptosis, Bax and TGF-?1 expression, mitochondrial health and concentrations of fibronectin in the medium. It has been found that the transport of EtHg from muscle to tissues and its conversion into inorganic Hg (Hgi) occur quickly. After 0.5 h of exposure to thimerosal, higher concentrations of both EtHg and Hgi were found in the kidneys (> 70% of total Hg in the animal body). The brain showed a minor contribution to the body burden of Hg (<1.0% of total Hg in the animal body). Thirty days after exposure to thimerosal, there was considerable excretion of Hg and liver had the most Hg still remaining in the animal body. Half-lives were estimated (in days) at 8.8; 10.7; 7.8; 7.7 and 45.2; for blood, brain, heart, liver and kidney, respectively. It is suggested that the extent of conversion of the EtHg into Hgi is modulated in part by the partition of EtHg in plasma and blood, since EtHg is rapidly converted into Hgi in red cells but not in plasma. The mechanism of dealkylation in red cells seems to be mediated by the Fenton reaction iv (formation of hydroxyl radicals). Additionally, EtHg/thimerosal decreased cell viability and mitosis, promoted apoptosis, impaired mitochondrial permeability transition state, increased expression of Bax and TGF-?1 and fibronectin secretion in the media. Collectively, the results demonstrate that the kinetics of thimerosal (EtHg) is closer to the Hgi - and not the MeHg - and kidney should be considered a potential target for EtHg toxicity, since it is exposed to the highest concentrations of Hg and it is the tissue where the metal has a greater biological half-life. Additionally, EtHg/thimerosal was shown to be an antiproliferative, apoptotic and pro-fibrotic agent in human kidney cells.

Introduction: Over the past few decades, nanoparticles (NPs) have gained immeasurable interest in the field of drug delivery. Various NP formulations have been disseminated in drug development in an attempt to increase efficacy, safety and tolerability of incorporated drugs. In this context, NP formulations that increase solubility, control release, and/or affect the in vivo disposition of drugs, were developed to improve the pharmacokinetic and pharmacodynamic properties of encapsulated drugs.Areas covered: In this article, important properties related to NP function such as particle size, surface charge and shape are disseminated. Also, the current understanding of how NP characteristics affect particle uptake and targeted delivery is elucidated. Selected NP systems currently used in delivery of drugs in biological systems and their production methods are discussed as well. Emphasis is placed on current NP formulations that are shown to reduce drug-induced adverse renal complications.Expert opinion: Formulation designs utilizing NP-encapsulated drugs offer alternative pharmacotherapy options with improved safety profiles for current and emerging drugs. NPs have been shown to increase the therapeutic index of several entrapped drugs mostly by decreasing drug localization and side effects on organs. Recent studies on NP-encapsulated chemotherapeutic and antibiotic medications show enhanced therapeutic outcomes by altering drug degradation, increasing systemic circulation and/or enhancing cell specific targeting. They may also reduce the distribution of encapsulated drugs into the kidneys and attenuate drug-associated adverse renal complications. The usefulness of NP formulation in reducing the nephrotoxicity of nonsteroidal anti-inflammatory drugs is an underexplored territory that deserves more attention.

Many drugs and environmental chemicals, such as antibiotics and chemotherapeutic agents, are nephrotoxic (toxic to the kidney) and are a common cause of acute kidney injury and chronic kidney disease. Conventional tissue models for assessment of drug-induced nephrotoxicity rely on animals or simple cell culture models, which lack tissue characteristics of the human kidney required to accurately predict a drug’s effect in clinical trials. Microfluidic kidney-on-chips can generate tissue with improved human relevance compared to traditional models, however, generally lack high-throughput and multiparametric data collection capabilities for evaluation of nephrotoxic drug exposures. Standard data collection techniques remain limited to fluorescent imaging or colorimetric assays that often focus on single endpoints, are invasive due to the addition of labels, and fail to capture dynamic changes in tissue function. Additionally, conventional toxicological readouts rely on bulk measures of injury, such as cell death, which are less sensitive than sub-lethal changes in cell function and morphology that occur prior to cell death. Due to the challenges above, there is a need for new measurement approaches that enable collection of kinetic, multi-parametric, and sub-lethal readouts of injury in kidney-on-chip systems. In this work, we developed and characterized several measurement approaches for evaluation of tissue function in kidney-on-chip systems and assessment of drug-induced nephrotoxicity. In chapter 2, we developed a novel optical-based oxygen sensing technique for measurement of sub-lethal mitochondrial dysfunction in an array of kidney-on-chips. In chapter 3, we investigated an approach for simultaneous transepithelial electrical resistance (TEER) sensing and flow control to enable near-continuous monitoring of tissue barrier function under different flow conditions. In chapter 4, we demonstrated the use of different data collection modalities, including multiple sensors, fluorescent imaging, and colorimetric-based assays, to generate multi-parametric readouts for evaluation of drug-induced nephrotoxicity in kidney-on-chips. / 2024-05-24T00:00:00Z

Background: Tenofovir disoproxil fumarate (TDF) has high antiretrovirus (ARV) activity and available in fixed dose combination (FDC). However, it has been found to cause renal dysfunction. Objectives: To document the prevalence, incidence, pattern of occurence and associated factors of nephrotoxicity in patients initiated on TDF based ART regimen in Mitchell's Plain CHC ARV Clinic and make recommendations. Methodology: The study was conducted by reviewing retrospective records of all ARV naïve HIV positive adults initiated on TDF based ARV regimen from January 2016 to June 2016. The creatinine clearance (CrCl) was calculated from follow up parameters till June 2018. Results: 87 patients were included in the study and 56% were female. The mean age was 34 years. Majority, 83%, had normal renal function at ART initiation. Older age [OR = 1.11; 95% CI (1.03–1.19), p =0.005], was associated with an increased probability of non-normal renal function at baseline. The incidence of CrCl < 90ml/min were 1.5% at 1 month post ARV initiation, 3.3% at 4 months, 6.1% at 12 months and 2.8% at 24 months while the prevalence were 10.5%,11.5%, 20.4% and 16.7% respectively. Older age and male gender were independently associated with prevalence of renal impairment. Conclusion: Renal dysfunction in patients initiated on TDF based regimen in this study varied and were relatively small when compared to the prevalence of renal dysfunction at initiation. Majority of the decline in CrCl were transient and patients were found to have recovered after further follow up. It is recommended that the frequency of renal function monitoring in patients on TDF regimen be done within programmatic guidelines based on patients' risk factors and potential poor outcomes.