Células-tronco (CT) apresentam potencial terapêutico para a doença renal pela possibilidade de regeneração tecidual e recuperação funcional, possivelmente por efeitos parácrinos. Diversos trabalhos mostraram seu efeito renoprotetor em modelo de insuficiência renal aguda. No entanto, existem poucos trabalhos que avaliaram o efeito da CT em doença renal crônica. Neste contexto, a via de inoculação e o número das CT na região da lesão podem desempenhar um papel crucial. Assim, o transplante de CT pela via EV não parece ser o mais apropriado para prover CT em número expressivo no órgão alvo. Uma técnica alternativa consiste em inocular as CT localmente, na região subcapsular renal. O objetivo do presente estudo foi analisar, em modelo experimental de doença renal crônica por nefrectomia 5/6 (Nx), a migração, a distribuição e o possível efeito renoprotetor da inoculação via subcapsular renal de 2 tipos de CT: derivadas da medula óssea (CTdmo) e mesenquimais (CTm). As CT foram coletadas de fêmur e tíbia de ratos doadores através da técnica de flushing. As CTdmo foram isoladas por gradiente de concentração e as CTm pela sua capacidade de aderência ao plástico e ambas marcadas com DAPI para a visualização no tecido. A caracterização das CT foi feita por citometria de fluxo e pela diferenciação celular in vitro. Foram realizados 2 protocolos experimentais. No protocolo I, CTdmo (1x106) foram inoculadas em ratos fêmeas e, no protocolo II, CTm (2x105) foram inoculadas em ratos machos. A região inoculada foi a subcapsula renal e os animais foram acompanhados por 15 e 30 dias. Os animais foram subdivididos nos grupos: Sham, ratos submetidos à cirurgia fictícia; Sham+CT, ratos submetidos à cirurgia fictícia que receberam CT (CTdmo ou CTm); Nx, ratos submetidos a nefrectomia 5/6; Nx+CT, Nx ratos que receberam CT (CTdmo ou CTm). Para avaliar a localização das CTdmo no tecido renal, utilizou-se a coloração de tricrômio de Masson e foi realizada uma análise semiquantitativa para avaliar o grau de infiltração. Foram analisadas a pressão arterial (PA), a albuminúria e a creatinina sérica. Para os animais que receberam CTm foi realizada a análise de parâmetros histológicos e a análise de marcadores inflamatórios, de células em atividade proliferativa, de miofibroblastos e de podócitos. Os resultados do Protocolo I avaliando a análise da infiltração no tecido renal das CTdmo marcadas com DAPI mostrou, em 5 dias, evidente infiltração das células da região subcapsular em sentido ao córtex e medula, inclusive presente em glomérulos. Ratos fêmeas Nx que receberam a inoculação das CTdmo na região da subcapsular renal não apresentaram melhora nos parâmetros que avaliaram a função renal. Protocolo II: as CTm cultivadas mostraram grande capacidade de aderência, crescimento em colônia e de diferenciação em células osteogênicas. A análise por citometria mostrou-se positiva para CD44 e CD90, com uma pequena população de células de CD34, CD45 e CD31, confirmando a presença preponderante de CTm. A inoculação de CTm em ratos Nx proporcionou um bloqueio da progressão da doença renal. Enquanto ratos Nx machos apresentaram elevada PA com 15 e 30 dias (149,6±9,1 e 191,7±2,8 mmHg) a inoculação de CTm promoveu significante redução após 30 dias (145,2±6,8 mmHg; p<0,05 vs Nx). Em ratos Nx foi observado um aumento na creatinina aos 15 e 30 dias (1,13±0,08 e 1,16±0,26 mg/dL) e a inoculação de CTm promoveu uma marcante redução aos 15 dias (0,58±0,03 mg/dL; p<0,05 vs Nx). A albuminúria foi elevada nos ratos Nx aos 15 e 30 dias (41,7±10,8 mg/24h e 138,7±33,6 mg/24h) enquanto os animais do grupo Nx+CTm aos 15 e 30 dias apresentaram diminuição significativa (4,6±1,5 mg/24h e 23,4±7,7 mg/24h; p<0,0001 vs Nx). A glomeruloesclerose do grupo Nx+CTm apresentou aos 30 dias uma redução significativa em relação ao grupo Nx (5,4±2,5% vs 22,0±6,1%, respectivamente; p<0,0001). A análise da fibrose intersticial não revelou diferença após 15 dias e 30 dias no grupo Nx+CTm em relação ao grupo Nx. Com relação ao número de macrófagos, linfócitos e de células em atividade proliferativa, os animais que receberam CTm apresentaram uma discreta diminuição de sua expressão no tecido renal. A expressão de -actina se reduziu significativamente no grupo Nx+CTm. Quanto à expressão de WT-1, específico para podócitos, os animais Nx+CTm tiveram aumento significativo da marcação em relação ao grupo Nx. Em resumo, após a inoculação de CT na região da subcapsula renal, houve marcante migração e distribuição das mesmas em direção à cortical e à medular. A inoculação de CTm proporcionou um efeito renoprotetor no modelo de nefrectomia 5/6. Sendo assim, a inoculação subcapsular renal pode representar uma importante via de inoculação, permitindo assim que um número maior de células atue na proteção da progressão da doença. / Stem cells (SCs) offer therapeutic potential for the treatment of renal diseases, due to the possibility of tissue regeneration and functional recovery. Various studies have shown renoprotection by SCs in experimental models of acute kidney disease. However, only a few studies have studied their effect in chronic kidney disease. The beneficial effect of SCs seems related to their capacity to differentiate or to secrete paracrine/endocrine factors. In this context, the inoculation route or the number of SCs homing in the injured region can play a crucial role. Therefore, transplantation of MSC through the intravenous route does not seem to be best suited for delivery of an important number of cells to the target organ. An alternative technique consists in local delivery of SCs in the subcapsular region of the kidney. The objective of the present study is to analyze the migration, distribution and potential renoprotective effect of the subcapsular inoculation of two types of SC - BSMC and mesenchymal stem cell (MSC) - in an experimental model of chronic kidney disease, the 5/6 nephrectomy (Nx). SCs were collected from the femur and tibia of donor rats by flushing. BSMC were isolated by centrifugation on a concentration gradient and MSCs were isolated by their capacity to adhere to plastic. Both types of SC were stained with DAPI to allow visualization in tissues. SC characterization was carried out by flow cytometry and differentiation in culture. Two experimental procedures were performed. In protocol I, BSMC (106 cells) were injected in female rats and in protocol II, MSCs (2x105 cells) were injected in male rats. Animals were divided into 4 groups: SHAM, sham-operated rats; SHAM+SC, sham-operated rats receiving BSMC or MSCs; Nx, rats undergoing 5/6 nephrectomy; Nx+SC, 5/6 Nx rats receiving BSMC or MSCs. We used Massons Trichrome staining and a semiquantitative analysis according to the degree of infiltration to follow the localization of BSMC in the renal tissue and to quantify their infiltration, respectively. The following parameters were studied: arterial blood pressure (AP), proteinuria (Uprot), albuminuria (Ualb) and serum creatinine (Screat). For the animals receiving SCs, analysis of histology, of inflammatory markers, of proliferating cells and of podocytes was performed. Results from Protocol I assessing DAPI-stained BSMC showed marked infiltration in 5 days from the subcapsular region to the cortex and the medulla, including presence in the glomeruli, over a period of 15 days. Female rats that received subcapsular injection of BSMC did not show improvement of the parameters used to assess kidney function. Protocol II: cultured MSCs demonstrated an ability to adhere to plastic, to grow in colonies and to differentiate in osteogenic cells. Quantitative analysis of cell markers by flow cytometry showed that isolated cells were positive for CD44 and CD90, with a small population of cells positive for CD31, CD34 and CD45, confirming a preponderant presence of MSCs. Inoculation of MSCs in Nx rats blocked the progression of the renal disease. Elevated AP in Nx rats at 15 and 30 days (149.6 ± 9.1 and 191.7 ± 2.8 mm Hg, respectively) was significantly reduced by inoculation of MSCs at 30 days (145.2 ± 6.8 mm Hg, p<0.05 vs Nx). Nx rats showed increased creatinine at 15 and 30 days (1.13 ± 0.08 and 1.16 ± 0.26 mg/dL, respectively) that was significantly reduced by injection of MSCs at 15 days (0.58 ± 0.03 mg/dL, p<0.05 vs Nx). Albuminuria was increased in Nx rats at 15 and 30 days (41.7 ± 10.8 and 138.7 ± 33.6 mg/24h, respectively) and was reduced in the Nx+MSC group at both time points (4.6 ± 1.5, and 23.4 ± 7.7 mg/24h, respectively; p<0.0001 vs Nx). Histologic analysis showed that glomerulosclerosis at 30 days in the Nx+MSC group was significantly reduced as compared to the Nx group (5.4 ± 2.5 % vs 22.0 ± 6.1 %, p<0.0001). Analysis of interstitial fibrosis did not show difference after 15 and 30 days in the Nx+MSC group compared to Nx group. Nx rats receiving MSCs showed slightly decreased inflammation markers, macrophages and lymphocytes, and proliferating cells in the renal tissue when compared to Nx rats. Analysis of myofibroblasts showed a significant decrease in expression of -smooth muscle actin in Nx+MSC rats compared to Nx rats. Podocyte number was analyzed by detection of WT-1, a specific marker. Nx rats receiving MSC had a significantly higher number of podocytes than Nx rats. In conclusion, our results show that after inoculation in the subcapsular region, SCs migrate throughout the cortex in direction of the medulla. Subcapsular inoculation of MSC provides a renoprotective effect in the model of 5/6 nephrectomy. Therefore, subcapsular inoculation could represent an important route of delivery of SCs to the kidney that allows a higher number of cells to act in the protection from progression of the disease.

A doença renal crônica (DRC) gera manifestações orais e alterações salivares nos pacientes, entretanto poucos estudos avaliaram os efeitos desta doença nas glândulas salivares. O objetivo deste trabalho foi avaliar os efeitos da nefropatia crônica experimental na morfologia, metabolismo, estresse oxidativo e concentrações iônicas nas glândulas parótida (PA) e submandibulares (SM) de ratos. A nefropatia crônica experimental foi induzida pelos 5/6 de nefrectomia (grupo DRC) e os resultados foram comparados com os grupos Sham e Controle no tempo experimental de 12 semanas. Foram avaliados os pesos corpóreos iniciais e finais, níveis séricos e urinários de ureia e creatinina, a depuração de creatinina e excreção proteica urinária. A morfologia do córtex renal foi avaliada pela contagem de glomérulos em 30 campos x200 de aumento por grupo, além disso, foram avaliadas a presença de glomeruloesclerose e fibrose intersticial no grupo DRC. Nas glândulas PA e SM, foi realizada contagem de ductos em 30 campos x400 de aumento por grupo e avaliação de fibrose e de glicogênio no parênquima glandular. A análise metabólica avaliou os efeitos da DRC e estímulos simpáticos e parassimpáticos da salivação nos fluxos metabólicos glicolítico, oxidativo e anaplerótico utilizando infusões com [U-13C]Glicose ou [1,6-13C2]Glicose, [2-13C]Glicose e [U-13C]Ácidos graxos, e espectroscopia de ressonância magnética nuclear (RMN) de próton (1H) e carbono 13 (13C) em alta resolução de extratos glandulares como técnica de detecção de incorporação de 13C em metabólitos intermediários. Atividades enzimáticas avaliadas foram: hexoquinase [HK], fosfofrutoquinase-1(PFK-1), piruvato quinase (PK), lactato desidrogenase (LDH), glicose-6-fosfato desidrogenase (G6PD), amilase, peroxidase e catalase. Além disso, foram quantificados os conteúdos de malondialdeído [MDA], glicogênio e concentrações iônicas de cálcio, fósforo e sódio. Foram observadas diminuições significativas dos pesos corpóreos finais, aumentos significativos das concentrações séricas e diminuições das concentrações urinárias de ureia e creatinina, diminuição da depuração de creatinina e aumento da excreção proteica no grupo DRC comparado aos grupos Controle e Sham. Na avaliação do córtex renal encontramos diminuição significativa da contagem glomerular, infiltrado inflamatório, atrofia tubular, glomeruloesclerose e fibrose intersticial. Na avaliação histológica glandular foi observado aumento significativo da contagem de ductos e diminuição de glicogênio na glândula SM. Na análise metabólica observamos a prevalência do fluxo glicolítico nas glândulas salivares. O estímulo salivar promoveu aumentos significativos na razão 12C-Lactato/12C-Alanina nos grupos Controle e Sham, com exceção do grupo DRC PA, onde em repouso se observou um aumento, comparando com os grupos Sham e Controle. No grupo DRC PA foram observadas diminuições significativas dos níveis de 12C-Alanina, 12C-Creatina e 12C-Glicose sem estímulo e aumento de 12C-Acetato com estimulo. No DRC SM detectaram-se aumento dos níveis de 12C-acetato e diminuição significativa da relação 13C3-Lactato/13C3-Acetato pós-estímulo indicativos de inibição do metabolismo oxidativo. Estas alterações nos níveis de metabólitos e suas razões, bem como nos níveis de enriquecimento 13C, denotam alterações profundas a nível metabólico nas glândulas salivares devido a DRC. Nas análises enzimáticas foram encontradas na glândula PA diminuições significativas das atividades da PFK-1 e LDH em relação ao grupo Controle e aumentos significativos da peroxidase e catalase comparado aos grupos Controle e Sham. Na glândula SM foi observada diminuição significativa na atividade enzimática da PFK-1 comparado ao grupo Controle. Além disso, foram observados aumentos significativos do conteúdo de MDA nas glândulas PA e SM e aumento significativo da concentração de sódio na glândula SM comparado aos grupos Controle e Sham. As modificações estruturais, metabólicas, oxidativas e iônica evidenciam a disfunção glandular na DRC e podem estar associadas às alterações salivares que caracterizam a doença. / Chronic kidney disease (CKD) promotes oral manifestations and salivary changes in patients, but few studies have evaluated the effects of this disease in the salivary glands. The aim of this study was to evaluate the effects of experimental chronic nephropathy in morphology, metabolism, oxidative stress and ionic concentrations in parotid (PA) and submandibular glands (SM) of rats. Experimental chronic nephropathy was induced by 5/6 nephrectomy (CKD group) and the results were compared with the Sham and Control groups at 12 weeks experimental period. The initial and final body weights, serum and urinary levels of urea and creatinine, creatinine clearance and protein excretion were evaluated. The morphology of the renal cortex was assessed by counting glomeruli in 30 fields x200 magnification per group, moreover, the presence of glomerulosclerosis and interstitial fibrosis in CKD group were also evaluated. The PA and SM histological evaluation was made by ducts count in 30 fields x400 magnification per group and assessment of fibrosis and glycogen in the glandular parenchyma. Metabolic analysis measured the effects of CKD and sympathetic and parasympathetic stimulation of salivation in glycolytic, oxidative and anaplerotic metabolic fluxes using infusions with [U-13C]Glucose or [1,6-13C2]Glucose, [2-13C]Glucose and [U-13C]Fatty acids, using high resolution proton (1H) and carbon 13 (13C) nuclear magnetic resonance (NMR) spectroscopy of glandular extracts as detection technique of 13C incorporation in intermediary metabolites. Enzyme activities were measured: hexokinase [HK], 1-phosphofructokinase (PFK-1), pyruvate kinase (PK), lactate dehydrogenase (LDH), glucose 6-phosphate dehydrogenase (G6PD), amylase, peroxidase and catalase. Furthermore, the malondialdehyde [MDA], glycogen content and calcium, phosphorus and sodium ionic concentrations were quantified. Significant decreases of final body weights, significant increases in serum concentrations and decreases in urinary concentrations of urea and creatinine, decreased creatinine clearance and increased protein excretion were observed in CKD group comparing with the Control and Sham groups. In the evaluation of the renal cortex, a significant decrease in glomerulus count, inflammatory infiltration, tubular atrophy, glomerulosclerosis and interstitial fibrosis were found. In glandular histological evaluation was observed a significant increase in the ducts count and reduced glycogen content in SM gland. Metabolic analysis revealed the prevalence of glycolytic flux in the salivary glands. The salivary stimulus promoted significant increases of 12C-Lactate/12C-Alanine ratio in the Control and Sham groups, except the DRC PA group, which at rest had an increase of the ratio compared to the sham and control groups. In DRC PA group significant decreases in the levels of 12C-Alanine, 12C-Creatine and 12C-Glucose without stimulation and increase of 12C-Acetate with stimulus were observed. In CKD SM were detected increased levels of 12C-acetate and significant decrease of the 13C-Lactate/13C-Acetate indicative of post-stimulus inhibition of oxidative metabolism. These changes in levels of metabolites and their ratios, as well as the enrichment levels 13C denote major changes at the metabolic level in salivary glands due to DRC. In the enzymatic analyzes, at DRC PA gland were found significant decreases of PFK-1 and LDH activities compared to the Control group and significant increases of peroxidase and catalase compared to control and Sham groups. In DRC SM gland was observed a significant decrease in the enzymatic activity of PFK-1 compared to Control group. In addition, significant increases in MDA content in PA and SM and significant increase in sodium concentration were observed in the SM compared to the control and Sham groups. Structural, metabolic, oxidative stress and ionic changes demonstrate the occurrence of glandular dysfunction in CKD, which might be associated with the salivary changes that characterize the disease.

Células tronco mesenquimais (CTm) apresentam potencial para tratamento da doença renal pela possibilidade de promover regeneração tecidual e recuperação funcional, possivelmente por seus efeitos parácrinos. Na última década, o líquido amniótico foi descrito como uma fonte promissora de extração e isolamento de CTm. Alguns estudos mostraram o efeito renoprotetor das CTm derivadas do líquido amniótico (CTmLA) na doença renal aguda e crônica, quando inoculadas precocemente. Entretanto, ainda não foi estudado o efeito da administração de CTmLA em modelo experimental de doença renal crônica (DRC) com a lesão já estabelecida, situação esta que reproduz melhor a apresentação clínica da doença nos pacientes. Assim, o objetivo do presente estudo foi analisar o efeito da inoculação de CTmLA na região subcapsular renal no modelo de DRC já estabelecido. As CTmLA foram obtidas de pacientes no segundo trimestre de gestação e isoladas através da sua capacidade de aderência ao plástico. A caracterização das CTm foi feita por citometria de fluxo e pela diferenciação celular in vitro. O modelo de DRC utilizado foi o de nefrectomia 5/6 (Nx) que, pela perda de massa renal, evolui com hipertensão arterial, proteinúria, glomeruloesclerose, fibrose intersticial e perda progressiva da função renal. Quinze dias após a indução do modelo, estas alterações já são marcantes e agravam-se com 30 dias. Foram realizados 2 protocolos experimentais: no protocolo I, os animais Nx com DRC estabelecida receberam dose única de CTmLA (5x105) na região subcapsular renal e foram acompanhados por 30 e 60 dias de experimento. No protocolo II, os animais Nx com DRC estabelecida receberam duas doses de CTmLA (5x105) na região subcapsular renal, no 15° e 30° dia após a nefrectomia 5/6, e foram acompanhados por 30 dias, totalizando 60 dias de experimento. Os animais foram subdivididos nos grupos: Sham, ratos submetidos à cirurgia fictícia; Sham+CTmLA, ratos submetidos à Sham que receberam CTmLA; Nx, ratos submetidos à nefrectomia 5/6; Nx+CTmLA, ratos Nx que receberam CTmLA. Para verificar a localização das CTmLA no tecido renal foi realizada a hibridização in situ para cromossomo XY. Foram realizadas análises dos parâmetros clínicos e laboratoriais, além de análise histológica, imunohistoquímica, PCR em tempo real e multiplex. Resultados: as CTmLA cultivadas mostraram grande capacidade de aderência, crescimento em colônia e de diferenciação em células osteogênicas, adipogênicas e condrogênicas. A análise por citometria mostrou-se positiva para CD29, CD44, CD90 e CD105, com uma pequena população de células de CD14, CD34, CD45 e CD117, confirmando a presença preponderante de CTm. Protocolo I: Após 30 dias, a inoculação de CTmLA, dose única, preveniu a elevação da pressão arterial, da proteinúria, da glomeruloesclerose, recuperando a expressão dos marcadores de podócitos, WT-1 e sinaptopodina. Entretanto, não houve efeito benéfico nos níveis de creatinina sérica e na fibrose intersticial, após 30 e 60 dias. O tratamento com CTmLA promoveu uma diminuição marcante do número de macrófagos e uma discreta queda dos leucócitos no infiltrado inflamatório renal, além da diminuição do número de miofibroblastos no interstício renal. Citocinas pró-inflamatórias foram encontradas em menor concentração no tecido renal dos animais que receberam CTmLA (IL-1beta, TNF-alfa, MCP-1 e RANTES). Não houve alteração significativa das citocinas Th1 e Th2, exceto por um aumento da IL-4 nos animais tratados com CTmLA. Os animais que foram acompanhados por 60 dias tiveram uma melhora da proteinúria, da glomeruloesclerose, diminuição do infiltrado de macrófagos e uma melhora da expressão de WT-1. Não foram observadas diferenças estatísticas nos parâmetros de creatinina sérica e fibrose intersticial, aos 30 e 60 dias. Protocolo II: Nos animais que receberam a segunda dose de CTmLA e foram acompanhados por 60 dias observou-se prevenção da elevação da pressão arterial e da proteinúria, além de uma marcante diminuição da fibrose intersticial. Em conclusão, o presente estudo mostrou, pela primeira vez, que a terapia com CTmLA foi capaz de induzir renoproteção nos animais com doença renal crônica estabelecida. O tratamento com CTmLA pode representar uma nova abordagem terapêutica bloqueando a progressão da doença renal crônica / Mesenchymal stem cells (mSC) represent therapeutic potential for the treatment of renal diseases, due to their ability to induce tissue regeneration and functional recovery. Human amniotic fluid stem cells (AFmSC) are a class of fetal, pluripotent stem cells, which present characteristics intermediate between embryonic and adult stem cells. These cells are characterized by the expression of mesenchymal stem cells markers. In addition, they have the ability to differentiate into lineages of all embryonic germ layers. They also show high proliferative rates, but do not induce tumor formation. Therefore, AFmSC are considered to be a very promising cell source and these characteristics have generated a great interest concerning their potential renoprotective effects. The aim of this study was to analyze the effects of AFmSC in an experimental model of chronic kidney disease, the 5/6 nephrectomy model (Nx), after the disease has been established, in order to more closely resemble the clinical settings in humans. AFmSC derived from second-trimester amniocentesis were isolated by plastic adhesion. After 4-7 passages, AFmSC characteristics were confirmed by flow cytometry and by their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages. Two experimental protocols were performed: In protocol I, rats underwent 5/6 nephrectomy (Nx) or sham surgery at day 0, received at day 15 a single dose of hAFmSC (5x105 cells) injected under the renal capsule and were studied at day 30 and 60 days. In protocol II, rats underwent Nx or sham surgery, and received at days 15 and 30, two doses of hAFmSC (5x105 cells) injected under the renal capsule, and were studied at day 60. In both protocols, the animals were subdivided into four groups: Sham, rats submitted to fictitious surgery; Sham+hAFmSC, Sham rats that received hAFmSC; Nx, rats submitted to nephrectomy 5/6; Nx+hAFmSC, Nx rats receiving hAFmSC. The hAFmSC were followed in the renal tissue by in situ hybridization for XY chromosome. In all the groups, clinical and histological parameters were analyzed by immunohistochemistry and real-time PCR. Results: AFmSC cultivated demonstrated an ability to adhere to plastic, to grow in colonies and to differentiate in osteogenic, adipogenic and chondrogenic cells. Quantitative analysis of cell markers by flow cytometry showed that isolated cells were positive for CD29, CD44, CD90 and CD105, with a small population of cells positive for CD14, CD34, CD45 and CD117, confirming a preponderant presence of mSC. Protocol I: After 30 days, the single dose of hAFmSC significantly reduced the blood pressure levels, proteinuria, glomerulosclerosis and improved the expression of podocytes markers, WT-1 and synaptopodin. A marked decrease on the number of macrophages and a discrete decrease of leucocyte infiltration, as well as a reduction of interstitial myofibroblasts was observed. Treatment with hAFmSC significantly reduced some proinflammatory cytokines (IL1beta, TNF-alpha, MCP-1 and RANTES). No significant difference in Th1 or Th2 cytokines was observed, except for IL-4 increase in Nx rats treated with hAFmSC. At 60 days of follow-up, Nx rats treated with hAFmSC presented reduced proteinuria, glomerulosclerosis and macrophages besides increase in WT-1 expression. No improvements were observed on serum creatinine and of interstitial fibrosis, after 30 and 60 days. Protocol II: Inoculation of two doses of hAFmSC in Nx rats improved blood pressure levels, proteinuria and interstitial fibrosis at day 60. In conclusion, the present study demonstrated, for the first time, that hAFmSC induced renoprotection in animals with established chronic kidney disease. Treatment with hAFmSC may represent a novel therapeutic approach for blocking the progression of chronic kidney disease

Introdução: Doador de rim em vida é uma importante fonte de órgão para os pacientes portadores de doença renal crônica (DRC). Os doadores experimentam uma redução abrupta da taxa de filtração glomerular (TFG) e adaptações ao metabolismo mineral demandam estudos nesta população. Nós avaliamos prospectivamente esta adaptação em doadores de rim em vida. Métodos: Entre janeiro de 2010 a agosto de 2011, no hospital das Clínicas de São Paulo e na Universidade de Miami, realizamos a avaliação prospectiva do metabolismo mineral e da função renal por 1 ano em 74 doadores de rim em vida. Medimos a taxa de filtração glomerular (TFG), fósforo (Pi), cálcio (Ca), paratohormônio (PTH), fibroblast Growth Factor 23 (FGF23) e a fração de excreção do fósforo (FePO4) no pré-operatório e nos dias 1, 2, 14, 180 e 360 do pós-operatório. Resultados: Observamos uma redução, aproximadamente, de 40% da TFG nos dois primeiros dias após a cirurgia. No décimo quarto dia após a nefrectomia, observamos o início da recuperação da TFG, chegando ao máximo da recuperação com 1 ano, quando se atingiu 68,6% da função renal se comparado com o dia anterior a doação (75,3 ml/min/1,73m2, p < 0,001). O cálcio sérico apresentou seu nadir no dia 1 (7,99 mg/dL; p < 0,01) e o Pi sérico atingiu seu nadir no dia 2 (2,61 mg/dL; p < 0,01). Já no dia 14, os valores de Ca e Pi retornaram aos valores basais tendo o fósforo evoluído novamente com valores inferiores ao basal no último dia de seguimento (3,36mg/dL; p < 0,001). FGF23 e PTH apresentaram elevação no D1 (111,0144,6 percentil 25-75: 16-63 RU/ml 64,9 30,3pg/mL; p < 0,01). Os valores de FGF23 se mantiveram elevados até o final do estudo enquanto que o PTH retornou aos valores de base no segundo dia e, a partir de então, manteve sem diferença do valores basais até o último dia de estudo. FePO4 elevou de 11,45,2% para 15,28,1% entre o pré-operatório e D365 (p < 0,01). Conclusão: A nefrectomia para doação de rim em 74 pacientes saudáveis elevou os valores de FGF23 durante todo o estudo juntamente com a FePO4. O fósforo, cálcio e PTH séricos apresentaram queda nos seus valores na primeira semana após a nefrectomia, e, com duas semanas após a cirurgia, retornaram aos valores basais mantendo-se estáveis até o final do estudo / Introduction: Living kidney donors (LKDs) experience an abrupt decline in glomerular filtration rate (GFR). Mineral metabolism adaptations in early CKD are still debated and not well studied in LKDs. We prospectively studied acute and long term mineral metabolism adaptation of LKDs. Materials and Methods: We measured renal function and mineral metabolites longitudinally for 1 year (days (D) 1, 2, 14, 180, & 365 post-operatively) in 74 healthy individuals who underwent kidney live donation. Results: eGFR (MDRD) decreased to 59% of its baseline on day 2 and started to increase at day 3, to its maximum at day 360 (75.3±15.6 ml/min/1.73m2, p < 0.01) wile FGF23 increased from 60.6 (25th-75th percentile 19-81 RU/mL) at baseline to 111.0±144.6 (p < 0.01) on day 1 and keep higher than baseline throwout the study. PTH rose maximally on day 1 (64.9 ± 30.3pg/ml) and returned to its base line on D2 and did not change after that. Total serum Calcium levels decreased from 9,40±0,48 mg/dL to a nadir of 7.99±0,51 mg/dL on day 1 (p < 0.001). Serum Phosphate levels reached their nadir on day 2 (2.61±0,52 mg/dL; p < 0.01). At D14 total calcium and phosphate levels had returned to baseline, but phosphate levels returned down on D360 (3.36±0,52 mg/dL; p < 0.001). Phosphate excretion fraction (FePO4) increased from base line (11.4±5.2%) up to 15.2±8.1% until D360 (p < 0.001). Conclusions: Abrupt reduction in eGFR induces physiological increases in FGF23 and PTH, and decreases in serum Ca and Pi in the first week. The changes in FGF23 and Pi urinary fractional excretion of Pi remain modestly yet significantly different from baseline throughout the first year after nephrectomy. Wile Ca, PTH and Pi serum levels are not significantly different from the baseline

Introduction : Les modèles murins sont grandement utilisés dans l’étude des maladies rénales et des pathologies associées. La concentration de la créatinine sérique est un bon indicateur de la filtration glomérulaire et en présence d’insuffisance rénale chronique (IRC), les concentrations de créatinine sérique (et la clairance) reflètent la sévérité de l’IRC. De plus, il a été démontré que l’IRC modifie le métabolisme des médicaments en diminuant l’activité et l’expression des enzymes hépatiques du cytochrome P450 (CYP450). Afin d’étudier la modulation du P450 par l’IRC avec un modèle murin et de confirmer nos résultats chez le rat, nous devons 1) développer un modèle d’IRC chez la souris, 2) mettre au point une technique de dosage des marqueurs de l’IRC et, 3) évaluer l’expression protéique du CYP450 en présence IRC. Matériel et Méthode : Trois modèles chirurgicaux d’IRC chez la souris ont été développés. Une méthode du dosage de la créatinine par chromatographie liquide à haute performance (CLHP) a été mise au point chez la souris et l’expression protéique du P450 a été mesurée par immunobuvardage de type Western. Résultats : Plusieurs paramètres de CLHP comme le pH, la concentration et le débit de la phase mobile modifient le pic d’élution et le temps de rétention de la créatinine. Concernant le modèle expérimental, on observe une perte de poids et une augmentation de la concentration plasmatique de la créatinine chez les souris avec une IRC. De plus, l’expression protéique de plusieurs isoformes du cytochrome P450 est modulée par l’IRC. Nous observons une diminution du CYP 2D de 42% (p < 0,01), du CYP 3A11 de 60% et du CYP 1A de 37% (p <0,01) par rapport aux souris témoins. On ne dénote aucun changement significatif au niveau de l’isoforme 2E1. Conclusion : Il est possible d’induire une insuffisance rénale chronique chez la souris suite à une néphrectomie. La technique de dosage de la créatinine par CLHP est précise et exacte et permet de caractériser la sévérité de l’IRC chez la souris. L’expression protéique du CYP450 est régulée à la baisse dans le foie des souris atteintes d’IRC. / Background: Mice models are widely used in renal studies. Seric creatinine concentration is used to evaluate glomerular filtration rate and is a good marker of chronic renal failure (CRF). It has been shown that CRF diminishes drug metabolism in the rat because of a downregulation of hepatic cytochrome P450 (CYP450) isoforms. To study CYP450 regulation in the mouse model, we needed to 1) develop a model of CRF in the mouse, 2) define a method of dosage of CRF markers, and 3) evaluate CYP450 protein expression in the liver of mice with CRF. Methods: Models of CRF were tested and sub-total nephrectomy was selected because of the efficacy and reproducibility to induce CRF. A high pressure liquid chromatography (HPLC) method for the dosage of creatinine in mice sera was developed. Liver protein expression of CYP1A1, CYP3A11, CYP2D and CYP2E1 was assessed by Western Blot analysis. Results: HPLC parameters such as pH, mobile phase concentration and flow rate modified the elution profile. Weight loss and high seric creatinine concentrations are seen in mice with CRF. Furthermore, protein expression of CYP1A, CYP3A11 and CYP2D was decreased in liver microsomes of mice with CRF by 37%, 60% and 42%, respectively (p<0.01) compared to sham-operated mice. We found no significant difference in the expression of CYP2E1. Conclusions: CRF models are reproducible in the mouse. The HPLC method for creatinine determination is precise and accurate, and can assess the severity of CRF. Hepatic protein expression of CYP450 is modulated in presence of CRF as in the rat.

Introduction: Nous avons déjà montré que l’insuffisance rénale chronique (IRC) entraîne une régulation négative du cytochrome P450 (CYP450) dans le foie et l’intestin de rat. La présente étude cherche à déterminer l’effet de l’IRC sur l’expression des enzymes du CYP450 dans le cerveau de rat. L’expression génique, protéique ainsi que l’activité des isoenzymes du CYP450 ont été analysées dans différentes régions du cerveau (hippocampe, cervelet, cortex et parenchyme cérébral) afin de déterminer l’effet de l’insuffisance rénale chronique sur le métabolisme cérébral des médicaments par le CYP450. Méthodes: Le cerveau entier de rats atteints d’IRC (induite par une néphrectomie sub-totale 5/6) et de rats témoins (laparotomie blanche) a été disséqué en 4 parties (cortex, cervelet, hippocampe et parenchyme cérébral). L’expression protéique et celle de l’ARNm des isoformes 1A, 2C11, 2D, 3A et 4A du cytochrome P450 a été étudiée respectivement par immunobuvardage de type Western et PCR en Temps Réel. L’activité du CYP3A a été mesurée par le métabolisme du DFB en DFH sur des préparations de microsomes de cerveau. Une technique de culture cellulaire d’astrocytes a été mise au point et a permis d’évaluer l’expression des enzymes dans ces cellules suite à l’incubation des astrocytes avec le sérum de rats atteints d’insuffisance rénale chronique. Résultats: Chez les rats atteints d’IRC, les niveaux géniques de CYP1A, 2C et 3A sont diminués d’au moins 40% (p < 0,05) dans presque toutes les parties étudiées. Les niveaux d’ARNm du CYP2D demeurent inchangés. De plus, une diminution significative d’au moins 45% (p < 0,05) de l’expression protéique des CYP1A, 2C et 3A est observée dans presque toutes les structures étudiées. L’activité enzymatique de CYP3A est diminuée significativement dans le cerveau de rats IRC, ainsi que l’expression des enzymes du CYP2C11 dans les astrocytes en culture lorsqu’incubés avec du sérum de rat urémique. Conclusions: Ces études démontrent que le cerveau est également affecté par l’IRC. Ceci se traduit par une diminution de l’expression protéique, génique, ainsi que de l’activité des enzymes du CYP450. Cette diminution pourrait expliquer une augmentation des effets secondaires dans le système nerveux central en IRC. / Background: It has been shown that chronic renal failure (CRF) is associated with a downregulation of liver and intestinal cytochrome P450 (CYP450) in the rat. The present study aimed to investigate the repercussions of CRF on Brain. CYP450 isoenzymes mRNA and protein expression, as well as activity in different brain regions (cortex, cerebellum, hippocampus, and rest of brain parenchyma), have been studied in order to determine the effects of CRF on cerebral drug metabolism by CYP450. Methods: The entire brain of CRF rats (induced by 5/6th nephrectomy) and control rats (sham laparotomy) was dissected into 4 parts (cortex, cerebellum, hippocampus, and rest of brain parenchyma). Protein and mRNA expression of CYP1A, CYP2C, CYP2D, CYP3A, and CYP4A were assessed by Western Blot assay and Real Time PCR, respectively. CYP3A activity was assessed using DFB metabolism into DFH in brain microsomal preparation. Protein and mRNA expression were also assessed in cultured astrocytes incubated with serum from CRF rats. Results: In CRF rats, mRNA levels of CYP1A, CYP2C, and CYP3A were decreased significantly by at least 40% (p < 0.05) in almost all studied brain regions. Protein expression of these isoforms was decreased by at least 45% (p < 0.05) in almost all structures. A significant decrease in CYP3A activity was observed in the brain. The incubation of astrocytes with CRF sera induced a decrease in the protein and mRNA expression of the CYP2C11. Conclusions: CRF is associated with a decrease in some major drug-metabolizing enzymes, which could explain an increase in bioavailability of drugs in the brain.

O fósforo é um dos minerais mais abundantes no corpo além de ser essencial para muitos processos biológicos. A homeostase do fósforo depende da absorção no intestino, da excreção renal, da remodelação óssea e de hormônios como o paratormônio, calcitriol e FGF-23. Nos pacientes com doença renal crônica, a excreção urinária de fósforo está comprometida levando à hiperfosfatemia, que contribui para o aumento da morbidade e mortalidade desses pacientes. A absorção intestinal de fósforo no intestino delgado, particularmente via cotransportadores NaP-IIb e PiT-1, é pouco estudada. O objetivo desse estudo foi avaliar os efeitos de dietas com diferentes concentrações de fósforo na expressão proteica e gênica dos cotransportadores NaP-IIb e PiT-1, bem como na apoptose dos enterócitos dos diferentes segmentos do intestino delgado de animais controles e urêmicos. Estudamos setenta e seis ratos Wistar machos, inicialmente divididos em dois grupos: controles (C) e urêmicos (Nx). Cada grupo foi subdividido em outros três, de acordo com a concentração de fósforo (P) na dieta: dieta Baixa (0,2%), dieta Padrão (0,54%) e dieta Alta (0,9%). Analisamos parâmetros bioquímicos (creatinina, P, Cai, PTH e FGF-23), a expressão proteica dos cotransportadores, através de Western Blotting, ELISA e imunofluorescência, a expressão gênica por PCR em tempo real e a apoptose dos enterócitos pela técnica TUNEL. Os resultados mostraram que os níveis séricos de creatinina, P, PTH e FGF-23 foram significativamente mais elevados nos animais Nx. Nos animais C com dieta baixa em P observamos aumento da expressão proteica do cotransportador NaP-IIb em todos os segmentos do intestino enquanto, no Nx Baixo, a expressão desse cotransportador foi menor somente no jejuno. Quanto ao PiT-1, sua expressão foi menor no íleo do grupo Nx Alto comparado ao seu respectivo controle. A expressão gênica do cotransportador NaP-IIb foi menor no jejuno do Nx Alto e maior no íleo desse mesmo grupo em relação aos seus respectivos controles. A expressão gênica do PiT-1 foi maior em todos os segmentos do grupo Nx Baixo em relação aos seus controles. Detectamos uma correlação direta entre a expressão gênica do NaP-IIb no jejuno com o fósforo sérico. A expressão gênica do PiT-1 no jejuno correlacionou-se com o fósforo sérico e no duodeno e jejuno, com os níveis séricos de FGF-23. No duodeno e no jejuno, a porcentagem de enterócitos apoptóticos foi maior nos animais Nx Alto comparados aos controles, particularmente no duodeno a porcentagem dessas células também foi maior no grupo Nx Baixo e controle padrão. Já no jejuno, tanto o grupo Nx baixo quanto no Nx Padrão observamos menos células apoptóticas que nos seus respectivos controles. Quando analisamos conjuntamente todos os segmentos intestinais o grupo Nx alto apresentou mais células apoptóticas que o controle e o oposto foi observado nos grupos Nx Baixo e Nx Padrão. Em conclusão, dietas com distintas concentrações de fósforo promovem modificações na expressão proteica e gênica dos cotransportadores NaP-IIb e PiT-1 no intestino delgado que não seguem um padrão uniforme. A sobrecarga de fósforo aumenta a porcentagem de enterócitos apoptóticos nos animais urêmicos / Phosphorus is one of the most abundant minerals in the body besides being essential for many biological processes. Phosphorus homeostasis depends on absorption in the small intestine, renal excretion, bone remodeling and hormones such as parathyroid hormone, calcitriol and FGF-23. In patients with chronic kidney disease phosphorus urinary excretion is compromised leading to hyperphosphatemia, which contributes to increased morbidity and mortality of these patients. Intestinal absorption of phosphorus in the small intestine, particularly of NaP-IIb and PiT-1 cotransporters is poorly studied. The aim of this study was to evaluate the effects of diets with different concentrations of phosphorus in protein expression and gene of NaP-IIb and PiT-1 cotransporters as well as in apoptosis of enterocytes of different segments of intestine in control and uremic animals. We studied seventy-six male Wistar rats initially divided into two groups: controls (C) and uremic (Nx). Each group was subdivided into three others, according to the phosphorus concentration in the diet: Low diet (0.2% P), Standard diet (0.54% P) and High diet (0.9% P). We analyzed biochemical parameters (creatinine, P, iCa, PTH and FGF-23), protein expression of cotransporters, using Western Blot, ELISA, immunofluorescence, real-time PCR and apoptosis of enterocytes using the TUNEL technique. Results showed that serum creatinine, P, PTH and FGF-23 were significantly higher in Nx animals. In C animals with a diet low in P we observed increase in protein expression of NaP-IIb cotransporter in all segments of the intestine while in low Nx expression of this cotransporter was lower only in jejunum. As for PiT-1 expression was lower in the ileum of the high Nx group compared to their respective control. Gene expression of NaP-IIb cotransporter was lower in the jejunum of high Nx and higher in the ileum of the same group as compared to their respective controls. PiT-1 gene expression was higher in all segments of the low Nx group compared to their controls. We detected a direct correlation between the gene expression of NaP-IIb in jejunum and serum phosphorus. Gene expression of PiT-1 correlated with serum phosphorus in the jejunum, and correlated with the serum levels of FGF-23 in the jejunum and duodenum. In the duodenum and jejunum the percentage of apoptotic enterocytes was higher in high Nx animals compared to controls; particularly in the duodenum the percentage of these cells was also higher in low Nx group and standard control. In the jejunum in both low Nx group and standard Nx we observed fewer apoptotic cells than in their respective controls. When we analyze all intestinal segments together the high Nx group had more apoptotic cells than the control, and the opposite was observed in the low Nx group and standard Nx group. In conclusion, diets with different phosphorus concentrations promote changes in protein expression and gene of NaP-IIb and PiT-1 cotransporters in the small intestine that do not follow a uniform pattern. Phosphorus overhead increases the percentage of apoptotic enterocytes in uremic animals

Background Renal cell carcinoma (RCC) is the most common type of kidney cancer. Patients diagnosed with metastatic RCC (mRCC) have shorter overall survival compared to those diagnosed at earlier stages. Several targeted therapies, which cost from $7,000 - $16,000 per month have been approved since 2005 to treat mRCC. In addition, there is a growing interest in the use of cytoreductive nephrectomy (CN) with targeted therapies among mRCC patients. However, little is known regarding the economic burden of RCC and role of CN and prescribing patterns of targeted therapies among older mRCC patients. Objectives 1) To assess the economic burden of RCC among older adults in the targeted therapy era 2) To compare the overall survival (OS) and total healthcare cost (THC) among older mRCC patients receiving CN and targeted therapy versus patients receiving targeted therapy alone 3) To describe prescribing patterns of targeted therapies and associated OS and THC among older mRCC patients. Methods This dissertation was conducted using the Surveillance Epidemiology and End Results (SEER) - Medicare linked data. For the first objective, the study included a prevalent cohort of RCC patients from 2013, diagnosed during 2005 - 2013 and continuously enrolled in Medicare. RCC patients were matched to non-cancer beneficiaries using propensity score matching. Generalized linear models estimated the incremental healthcare costs. Incremental total healthcare cost (THC) was multiplied by the estimated number of RCC patients on Medicare to calculate the total economic burden of RCC. For the second objective, we included patients diagnosed with mRCC between 2007-2014 and compared overall survival (OS), and THC between patients who received CN + targeted therapy and targeted therapy alone. A propensity score based inverse probability of treatment weighting (IPTW) method was used to balance the two treatment groups. A Cox proportional hazard model assessed the risk for death and a GLM compared healthcare costs between the groups. For the third objective, patients with mRCC were defined as patients who were diagnosed at stage-IV or at earlier stages but were currently using targeted therapies. Further, we restricted our sample to patients who initiated targeted therapy. We described the frequencies of the most common first and second line targeted therapies. We also described OS and THC per month for clear-cell and non-clear cell mRCC for each therapy and line of therapy. Results The first study included 10,392 each of RCC and control patients. The average THC associated with RCC was $7,419. The average THC was $4,584 for patients diagnosed at stage-I, $4,727 for stage-II, $9,331 for stage-III, and $31,637 for stage-IV. The annual economic burden of RCC on Medicare was estimated to be $1.5 billion. The second study included 471 mRCC patients that received CN + targeted therapy or targeted therapy alone. The median OS from the adjusted survival curves was significantly higher (p Conclusions The economic burden of RCC varied substantially between early stage and metastatic patients. Among mRCC patients, use of CN among targeted therapy users was associated with a higher median OS and similar monthly THC over a lifetime. Sunitinib and everolimus were the most common first and second line targeted therapies among mRCC patients. The descriptive analysis suggested that OS and THC were similar across types of targeted therapy sequences.

Introduction: Nous avons déjà montré que l’insuffisance rénale chronique (IRC) entraîne une régulation négative du cytochrome P450 (CYP450) dans le foie et l’intestin de rat. La présente étude cherche à déterminer l’effet de l’IRC sur l’expression des enzymes du CYP450 dans le cerveau de rat. L’expression génique, protéique ainsi que l’activité des isoenzymes du CYP450 ont été analysées dans différentes régions du cerveau (hippocampe, cervelet, cortex et parenchyme cérébral) afin de déterminer l’effet de l’insuffisance rénale chronique sur le métabolisme cérébral des médicaments par le CYP450. Méthodes: Le cerveau entier de rats atteints d’IRC (induite par une néphrectomie sub-totale 5/6) et de rats témoins (laparotomie blanche) a été disséqué en 4 parties (cortex, cervelet, hippocampe et parenchyme cérébral). L’expression protéique et celle de l’ARNm des isoformes 1A, 2C11, 2D, 3A et 4A du cytochrome P450 a été étudiée respectivement par immunobuvardage de type Western et PCR en Temps Réel. L’activité du CYP3A a été mesurée par le métabolisme du DFB en DFH sur des préparations de microsomes de cerveau. Une technique de culture cellulaire d’astrocytes a été mise au point et a permis d’évaluer l’expression des enzymes dans ces cellules suite à l’incubation des astrocytes avec le sérum de rats atteints d’insuffisance rénale chronique. Résultats: Chez les rats atteints d’IRC, les niveaux géniques de CYP1A, 2C et 3A sont diminués d’au moins 40% (p < 0,05) dans presque toutes les parties étudiées. Les niveaux d’ARNm du CYP2D demeurent inchangés. De plus, une diminution significative d’au moins 45% (p < 0,05) de l’expression protéique des CYP1A, 2C et 3A est observée dans presque toutes les structures étudiées. L’activité enzymatique de CYP3A est diminuée significativement dans le cerveau de rats IRC, ainsi que l’expression des enzymes du CYP2C11 dans les astrocytes en culture lorsqu’incubés avec du sérum de rat urémique. Conclusions: Ces études démontrent que le cerveau est également affecté par l’IRC. Ceci se traduit par une diminution de l’expression protéique, génique, ainsi que de l’activité des enzymes du CYP450. Cette diminution pourrait expliquer une augmentation des effets secondaires dans le système nerveux central en IRC. / Background: It has been shown that chronic renal failure (CRF) is associated with a downregulation of liver and intestinal cytochrome P450 (CYP450) in the rat. The present study aimed to investigate the repercussions of CRF on Brain. CYP450 isoenzymes mRNA and protein expression, as well as activity in different brain regions (cortex, cerebellum, hippocampus, and rest of brain parenchyma), have been studied in order to determine the effects of CRF on cerebral drug metabolism by CYP450. Methods: The entire brain of CRF rats (induced by 5/6th nephrectomy) and control rats (sham laparotomy) was dissected into 4 parts (cortex, cerebellum, hippocampus, and rest of brain parenchyma). Protein and mRNA expression of CYP1A, CYP2C, CYP2D, CYP3A, and CYP4A were assessed by Western Blot assay and Real Time PCR, respectively. CYP3A activity was assessed using DFB metabolism into DFH in brain microsomal preparation. Protein and mRNA expression were also assessed in cultured astrocytes incubated with serum from CRF rats. Results: In CRF rats, mRNA levels of CYP1A, CYP2C, and CYP3A were decreased significantly by at least 40% (p < 0.05) in almost all studied brain regions. Protein expression of these isoforms was decreased by at least 45% (p < 0.05) in almost all structures. A significant decrease in CYP3A activity was observed in the brain. The incubation of astrocytes with CRF sera induced a decrease in the protein and mRNA expression of the CYP2C11. Conclusions: CRF is associated with a decrease in some major drug-metabolizing enzymes, which could explain an increase in bioavailability of drugs in the brain.

Introduction : Les modèles murins sont grandement utilisés dans l’étude des maladies rénales et des pathologies associées. La concentration de la créatinine sérique est un bon indicateur de la filtration glomérulaire et en présence d’insuffisance rénale chronique (IRC), les concentrations de créatinine sérique (et la clairance) reflètent la sévérité de l’IRC. De plus, il a été démontré que l’IRC modifie le métabolisme des médicaments en diminuant l’activité et l’expression des enzymes hépatiques du cytochrome P450 (CYP450). Afin d’étudier la modulation du P450 par l’IRC avec un modèle murin et de confirmer nos résultats chez le rat, nous devons 1) développer un modèle d’IRC chez la souris, 2) mettre au point une technique de dosage des marqueurs de l’IRC et, 3) évaluer l’expression protéique du CYP450 en présence IRC. Matériel et Méthode : Trois modèles chirurgicaux d’IRC chez la souris ont été développés. Une méthode du dosage de la créatinine par chromatographie liquide à haute performance (CLHP) a été mise au point chez la souris et l’expression protéique du P450 a été mesurée par immunobuvardage de type Western. Résultats : Plusieurs paramètres de CLHP comme le pH, la concentration et le débit de la phase mobile modifient le pic d’élution et le temps de rétention de la créatinine. Concernant le modèle expérimental, on observe une perte de poids et une augmentation de la concentration plasmatique de la créatinine chez les souris avec une IRC. De plus, l’expression protéique de plusieurs isoformes du cytochrome P450 est modulée par l’IRC. Nous observons une diminution du CYP 2D de 42% (p < 0,01), du CYP 3A11 de 60% et du CYP 1A de 37% (p <0,01) par rapport aux souris témoins. On ne dénote aucun changement significatif au niveau de l’isoforme 2E1. Conclusion : Il est possible d’induire une insuffisance rénale chronique chez la souris suite à une néphrectomie. La technique de dosage de la créatinine par CLHP est précise et exacte et permet de caractériser la sévérité de l’IRC chez la souris. L’expression protéique du CYP450 est régulée à la baisse dans le foie des souris atteintes d’IRC. / Background: Mice models are widely used in renal studies. Seric creatinine concentration is used to evaluate glomerular filtration rate and is a good marker of chronic renal failure (CRF). It has been shown that CRF diminishes drug metabolism in the rat because of a downregulation of hepatic cytochrome P450 (CYP450) isoforms. To study CYP450 regulation in the mouse model, we needed to 1) develop a model of CRF in the mouse, 2) define a method of dosage of CRF markers, and 3) evaluate CYP450 protein expression in the liver of mice with CRF. Methods: Models of CRF were tested and sub-total nephrectomy was selected because of the efficacy and reproducibility to induce CRF. A high pressure liquid chromatography (HPLC) method for the dosage of creatinine in mice sera was developed. Liver protein expression of CYP1A1, CYP3A11, CYP2D and CYP2E1 was assessed by Western Blot analysis. Results: HPLC parameters such as pH, mobile phase concentration and flow rate modified the elution profile. Weight loss and high seric creatinine concentrations are seen in mice with CRF. Furthermore, protein expression of CYP1A, CYP3A11 and CYP2D was decreased in liver microsomes of mice with CRF by 37%, 60% and 42%, respectively (p<0.01) compared to sham-operated mice. We found no significant difference in the expression of CYP2E1. Conclusions: CRF models are reproducible in the mouse. The HPLC method for creatinine determination is precise and accurate, and can assess the severity of CRF. Hepatic protein expression of CYP450 is modulated in presence of CRF as in the rat.