As células mesangiais (CM) são essenciais para a homeostasia glomerular. Além disso, têm papel crucial no desenvolvimento de glomeroesclerose em nefropatias crônicas. Demonstramos que as isoformas do repressor de transcrição TBX3, TBX3.1 e TBX3 + 2<font face=\"Symbol\">a, foram suprarreguladas precocemente por concentrações baixas de TGF-<font face=\"Symbol\">b1 em CM humanas. A superexpressão das isoformas não alterou a proliferação ou produção de MEC em CM. Entretanto, diminuiu a apoptose induzida pela privação de SBF. Ademais, o silenciamento do gene TBX3 sensibilizou as CM ao estímulo pró-apoptótico causado pela remoção do SBF. Por fim, observamos aumento da expressão protéica de TBX3 nos glomérulos e túbulos em um modelo de nefropatia, guardando correlação temporal com o aumento dos níveis de TGF-<font face=\"Symbol\">b1, colágeno IV e fibronectina. Nossos resultados indicam que o gene TBX3 atua como um fator antiapoptótico nas CM e pode estar envolvido no mecanismo pelo qual TGF-<font face=\"Symbol\">b1 induz glomeruloesclerose e fibrose tubular durante a progressão das nefropatias. / Mesangial cells (MC) are essential for glomerular homeostasis. In addition, MC play a significant role in the development of glomerulosclerosis of chronic nephropathies. We demonstrated that the transcription repressor TBX3 isoforms, TBX3.1 and TBX3 + 2<font face=\"Symbol\">a, were upregulated by low concentrations of TGF-<font face=\"Symbol\">b1. Selective overexpression of the isoforms did not affect MC proliferation or extracellular matrix production. However, TBX3 forced expression decreased apoptosis induced by FBS deprivation. Moreover, TBX3 gene silencing sensitized MC to the proapoptotic stimulus caused by SBF withdrawal. Finally, we observed an increase in TBX3 protein expression in glomerular and tubular regions in a model of chronic nephropathy (5/6 nephrectomy), temporally related to increased expression of TGF-<font face=\"Symbol\">b1, collagen IV and fibronectin. Our results indicate that TBX3 acts as an antiapoptotic factor in MC in vitro and may be involved in the mechanism by which TGF-<font face=\"Symbol\">b1 induces glomerulosclerosis and tubular fibrosis during the progression of nephropathies.

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to breakdown of glomerular peimselectivity and proteinuria. This study addresses mechanisms that limit complement-mediated injury, focusing on Ras. Complement-mediated injury was attenuated in cultured GEC expressing a constitutively active form of Ras (V12Ras), compared with Neo (control) GEC. V12Ras GEC showed constitutive activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways, but inhibition of these pathways did not reverse the protective effect of Ras. V12Ras GEC showed smaller and rounder morphology, decreased F- to G-actin ratio, decreased activity of the Rho GTPase, Rac, and decreased Src activity. In V12Ras GEC, disruption or stabilization of the F-actin cytoskeleton reversed the protective effect of V12Ras on complement-mediated injury. Thus, the protective effect of V12Ras may be dependent on remodeling of the actin cytoskeleton. Furthermore, the reduction of Src activity due to Ras activation may alter the equilibrium in activities of Rho GTPases, a family of proteins known regulate the actin cytoskeleton. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury.

As células mesangiais (CM) são essenciais para a homeostasia glomerular. Além disso, têm papel crucial no desenvolvimento de glomeroesclerose em nefropatias crônicas. Demonstramos que as isoformas do repressor de transcrição TBX3, TBX3.1 e TBX3 + 2<font face=\"Symbol\">a, foram suprarreguladas precocemente por concentrações baixas de TGF-<font face=\"Symbol\">b1 em CM humanas. A superexpressão das isoformas não alterou a proliferação ou produção de MEC em CM. Entretanto, diminuiu a apoptose induzida pela privação de SBF. Ademais, o silenciamento do gene TBX3 sensibilizou as CM ao estímulo pró-apoptótico causado pela remoção do SBF. Por fim, observamos aumento da expressão protéica de TBX3 nos glomérulos e túbulos em um modelo de nefropatia, guardando correlação temporal com o aumento dos níveis de TGF-<font face=\"Symbol\">b1, colágeno IV e fibronectina. Nossos resultados indicam que o gene TBX3 atua como um fator antiapoptótico nas CM e pode estar envolvido no mecanismo pelo qual TGF-<font face=\"Symbol\">b1 induz glomeruloesclerose e fibrose tubular durante a progressão das nefropatias. / Mesangial cells (MC) are essential for glomerular homeostasis. In addition, MC play a significant role in the development of glomerulosclerosis of chronic nephropathies. We demonstrated that the transcription repressor TBX3 isoforms, TBX3.1 and TBX3 + 2<font face=\"Symbol\">a, were upregulated by low concentrations of TGF-<font face=\"Symbol\">b1. Selective overexpression of the isoforms did not affect MC proliferation or extracellular matrix production. However, TBX3 forced expression decreased apoptosis induced by FBS deprivation. Moreover, TBX3 gene silencing sensitized MC to the proapoptotic stimulus caused by SBF withdrawal. Finally, we observed an increase in TBX3 protein expression in glomerular and tubular regions in a model of chronic nephropathy (5/6 nephrectomy), temporally related to increased expression of TGF-<font face=\"Symbol\">b1, collagen IV and fibronectin. Our results indicate that TBX3 acts as an antiapoptotic factor in MC in vitro and may be involved in the mechanism by which TGF-<font face=\"Symbol\">b1 induces glomerulosclerosis and tubular fibrosis during the progression of nephropathies.

Voltage-gated calcium (Cav) channels are a prerequisite for signal transmission at the first olfactory sensory neuron (OSN) synapse within the glomeruli of the main olfactory bulb (MOB). We showed previously that the N-type Cav channel subunit Cav2.2 is present in the vast majority of glomeruli and plays a central role in presynaptic transmitter release. Here, we identify a distinct subset of glomeruli in the MOB of adult mice that is characterized by expression of the P/Q-type channel subunit Cav2.1. Immunolocalization shows that Cav2.1+ glomeruli reside predominantly in the medial and dorsal MOB, and in the vicinity of the necklace glomerular region close to the accessory olfactory bulb. Few glomeruli are detected on the ventral and lateral MOB. Cav2.1 labeling in glomeruli colocalizes with the presynaptic marker vGlut2 in the axon terminals of OSNs. Electron microscopy shows that Cav2.1+ presynaptic boutons establish characteristic asymmetrical synapses with the dendrites of second-order neurons in the glomerular neuropil. Cav2.1+ glomeruli receive axonal input from OSNs that express molecules of canonical OSNs: olfactory marker protein, the ion channel Cnga2, and the phosphodiesterase Pde4a. In the main olfactory epithelium, Cav2.1 labels a distinct subpopulation of OSNs whose distribution mirrors the topography of the MOB glomeruli, that shows the same molecular signature, and is already present at birth. Together, these experiments identify a unique Cav2.1+ multiglomerular domain in the MOB that may form a previously unrecognized olfactory subsystem distinct from other groups of necklace glomeruli that rely on cGMP signaling mechanisms.

No description available.

No description available.

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

<p>Intrauterusna restrikcija rasta (IUGR) se odnosi na stanje u kojem fetus nije u mogućnosti da ostvari svoj genetski potencijal za rast. IUGR je ozbiljan klinički problem i nedavno je povezan sa bolestima odraslog doba kao &scaron;to su hipertenzija, insulin nezavistan diabetes melitus, dislipidemije i ishemijske bolesti srca. Eritropoetin je glavni regulator proliferacije i diferencijacije eritroidnih progenitorskih ćelija zahvaljujući svojoj antiapoptotičkoj aktivnosti. Cilj istraživanja je bio da se ispita uticaj IUGR na bubrege, kao i uticaj eritropotina na bubrege sa IUGR. Eksperimentalna studija je sprovedena u gajili&scaron;tu Pasterovog zavoda u Novom Sadu na 60 mi&scaron;eva rase NMRI. IUGR je izazivana aplikacijom deksametazona gravidnim ženkama. Po rođenju su mladunci bili podeljeni u sedam grupa. Mladuncima je u 1. i 7. danu života davan darbepoetin alfa (DA) u dozama od 1, 4 i 10&mu;g/kg. Dve grupe su predstavljale potomke majki koje su tokom trudnoće dobile DA. Nakon 4 nedelje su uzimani uzorci bubrega i vr&scaron;ena je morfolo&scaron;ka i stereolo&scaron;ka analiza glomerula. Aplikacija deksametazona (100 &mu;g/kg) trudnim mi&scaron;icama dovodi do potomstva sa IUGR. Primena DA kod novorođenih mi&scaron;eva sa IUGR dovodi do bržeg porasta telesne mase u prvih 7 dana života (&bdquo;catch-up― rasta). Mi&scaron;evi rođeni sa IUGR imaju manju povr&scaron;inu glomerula bubrega. Primena DA nakon rođenja i u 7. danu života (4 i 10 &mu;g/kg) kod novorođenih mi&scaron;eva sa IUGR dovodi do hipertrofije glomerula bubrega. IUGR nema uticaja na broj glomerula bubrega mi&scaron;eva. Primena DA nema uticaja na broj glomerula bubrega mi&scaron;eva. Mi&scaron;evi rođeni sa IUGR imaju manju debljinu korteksa bubrega. Primena DA (4 i 10 &mu;g/kg) kod mi&scaron;eva rođenih sa IUGR dovodi do povećanja debljine korteksa bubrega. Davanje DA kod IUGR značajno povećava povr&scaron;inu glomerula i debljinu korteksa bubrega.</p> / <p>Intrauterine growth restriction (IUGR) refers to a condition in which a foetus is not able to achieve its genetic potential for growth. IUGR is a serious clinical problem, and has recently been linked with diseases of adulthood, such as hypertension, insulin-independent diabetes mellitus, dyslipidemia, and ischemic heart disease. Erythropoietin is the major regulator of proliferation and differentiation of erythroid progenitor cells, thanks to its anti-apoptotic activities. The aim of this study was to investigate the effect of IUGR on the kidneys, and the impact of erythropoietin on the kidneys with IUGR. The experimental study was conducted in Pasteur Institute of Novi Sad on 60 mice of NMRI race. IUGR has been imposed with the application of dexamethasone to pregnant females. After birth, the pups were divided into seven groups. DA was administered to the pups on the 1st and 7th day of life (dose 1, 4 and 10 &mu;g/kg). Two groups represented the offspring of the mothers who during pregnancy received DA. After 4 weeks, kidney samples were taken and morphological and stereological analysis of the glomeruli was performed. The application of dexamethasone (100 &mu;g/kg) to pregnant mice leads to their offspring with IUGR. Application of DA to newborn mice with IUGR leads to faster weight gain in the first 7 days of life (&quot;catch-up&quot; growth). Mice born with IUGR have a reduced glomerular surface. Application of DA after birth and on the 7th day of life (4 and 10 &mu;g/kg) in mice with IUGR leads to hypertrophy of the kidney glomeruli. IUGR has no effect on the number of kidney glomeruli. Application of DA has no effect on the number of kidney glomeruli. Mice born with IUGR have a reduced cortical thickness. Application of DA (4 and 10 &mu;g/kg) in mice born with IUGR leads to increased thickness of the kidney cortex. Application of DA to mice with IUGR significantly increases the surface area of the kidney glomeruli and cortical thickness.</p>

Diabetic nephropathy (DN) is one of the leading causes of end stage renal disease (ESRD) in western world and has a trend to spread in developing countries. Pathogenesis of DN is not fully elucidated. Studies of recent years showed that podocyte loss and activation of the rennin-angiotensin system (RAS), especially intra-renal RAS, played important roles in this process. Although renal biopsy is currently the most common way used to determine the expression pattern of podocyte and RAS associated molecules in DN, this invasive procedure has its own risk and is not practical for serial monitoring. We hypothesized that measurement of messenger ribonucleic acid (mRNA) expression of related genes in the urinary sediment might be a useful way to assess the severity of DN. / Firstly, we found that urinary mRNA expressions of podocyte-associated molecules nephrin, podocin, synaptopodin, Wilm's tumor-1 (WT-1) and alpha-actinin-4 were higher in patients with DN than in healthy controls, and urinary nephrin, podocin and synaptopodin expression was related to proteinuria and baseline renal function. In addition, there was a close relationship between urinary mRNA expression of type 2 angiotensin converting enzyme (ACE2), a key element of RAS, and the degrees of proteinuria, renal function and rate of decline of glomerular filtration rate (GFR). Urinary mRNA expression of ACE also inversely correlated with the rate of renal function decline. / In the next step, we studied the change in urinary mRNA expression of nephrin, podocin, synaptopodin, ACE and ACE2 in patients with DN treated with angiotensin converting enzyme inhibitor (ACEI) and addition of angiotensin receptor blocker (ARB). We found that urinary mRNA expression of podocin, synaptopodin and propably nephrin increased with disease progression, and percentage change in urinary podocin expression negatively correlated with rate of decline of GFR. Furthermore, serial measurement of urinary expression of nephrin and possibly synaptopodin may reflect therapeutic response to ARB in these patients. Urinary mRNA expression of ACE and ACE2, however, remained unchanged during the study duration and did not correlate with therapeutic response. / In this series of work, we investigated (i) the relation between the gene expression profile of podocyte-associated molecules and RAS related molecules in the urinary sediment and the severity of DN, including clinically defined parameter of disease severity, histological scarring, and the degree of intra-renal podocyte loss, (ii) the relation between urinary and intra-renal gene expression of patients with DN, (iii) the application of urinary gene expression on the monitoring of disease progression and therapy response of DN. The urinary mRNA expression of related genes was quantified by real-time quantitative polymerase chain reaction (RT Q-PCR). The intra-renal mRNA expression of related genes was studied from the histologic specimens of kidney biopsy by laser catapult microdissection (LCM) and RT Q-PCR. The degree of renal scarring was determined by morphometric analysis. Glomerular podocyte number was determined by stereological study on serial sections of renal biopsy specimen. / Taken together, our results suggest that although urinary mRNA expression of podocyte and RAS associated molecules is not related to intra-renal expression, urinary expression has the potential to be used as a non-invasive tool to assess the severity and progression of DN, and serial measurements of urinary gene expression of podocyte associated molecules may be used to reflect therapy response for patients with DN. Our findings also indicate that the information from urinary gene expression is supplementary to, but not a surrogate of, the data obtained from renal biopsy. / We then examined the relation between urinary gene expression and histological changes in the kidney. We found that urinary WT-1 expression correlated with the degree of kidney fibrosis. Unlike intra-renal expression, urinary mRNA expression of podocyte associated molecules did not correlate with glomerular podocyte number. There was also no association between urinary and intra-renal mRNA expression. / Wang, Gang. / Adviser: Cheuk Chen Szeto. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3423. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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