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1.
Aging (Albany NY) ; 12(17): 17601-17624, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32858527

RESUMO

Healthy aging is typified by a progressive and absolute loss of podocytes over the lifespan of animals and humans. To test the hypothesis that a subset of glomerular parietal epithelial cell (PEC) progenitors transition to a podocyte fate with aging, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice were generated. PECs were inducibly labeled with a tdTomato reporter, and podocytes were constitutively labeled with an EGFP reporter. With advancing age (14 and 24 months) glomeruli in the juxta-medullary cortex (JMC) were more severely injured than those in the outer cortex (OC). In aged mice (24m), injured glomeruli with lower podocyte number (41% decrease), showed more PEC migration and differentiation to a podocyte fate than mildly injured or healthy glomeruli. PECs differentiated to a podocyte fate had ultrastructural features of podocytes and co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of mesangial (Perlecan) or endothelial (ERG) cells. PECs differentiated to a podocyte fate did not express CD44, a marker of PEC activation. Taken together, we demonstrate that a subpopulation of PECs differentiate to a podocyte fate predominantly in injured glomeruli in mice of advanced age.

2.
Kidney Int ; 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32592814

RESUMO

Glomerular podocytes undergo structural and functional changes with advanced age, that; increase susceptibility of aging kidneys to worse outcomes following superimposed glomerular; diseases. To delineate transcriptional changes in podocytes in aged mice, RNA-seq was performed on isolated populations of reporter-labeled (tdTomato) podocytes from multiple young (two to three months) and advanced aged mice (22 to 24 months, equivalent to 70 plus year old humans). Of the 2,494 differentially expressed genes, 1,219 were higher and 1,275 were lower in aged podocytes. Pathway enrichment showed that major biological processes increased in aged podocytes included immune responses, non-coding RNA metabolism, gene silencing and MAP kinase signaling. Conversely, aged podocytes showed downregulation of developmental, morphogenesis and metabolic processes. Canonical podocyte marker gene expression decreased in aged podocytes, with increases in apoptotic and senescence genes providing a mechanism for the progressive loss of podocytes seen with aging. In addition, we revealed aberrations in the podocyte autocrine signaling network, identified the top transcription factors perturbed in aged podocytes, and uncovered candidate gene modulations that might promote healthy aging in podocytes. The transcriptional signature of aging is distinct from other kidney diseases. Thus, our study provides insights into biomarker discovery and molecular targeting of the aging process itself within podocytes.

3.
Nat Immunol ; 20(10): 1393-1403, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31477919

RESUMO

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.


Assuntos
Linfócitos B/imunologia , Cadeias kappa de Imunoglobulina/genética , Células Precursoras de Linfócitos B/fisiologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores CXCR4/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Células Cultivadas , Quimiocina CXCL12/metabolismo , Feminino , Fatores Reguladores de Interferon/genética , Linfopoese , Masculino , Camundongos , Receptores de Antígenos de Linfócitos B/genética , Receptores CXCR4/genética , Recombinação Genética
4.
Kidney Int ; 96(3): 597-611, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200942

RESUMO

Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA|LC1|tdTomato|Nphs1-FLPo|FRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman's capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.

5.
Oncogene ; 37(46): 6069-6082, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29991800

RESUMO

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells.


Assuntos
Adenoviridae/genética , Proteínas Reguladoras de Apoptose/genética , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Glioma/tratamento farmacológico , Proteínas Proto-Oncogênicas/genética , Tamoxifeno/farmacologia , Regulação para Cima/genética , Proteína X Associada a bcl-2/genética , Células A549 , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Glioma/genética , Células HEK293 , Humanos , Camundongos , Terapia Viral Oncolítica/métodos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
6.
Kidney Int ; 93(5): 1240-1246, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29580637

RESUMO

Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow-colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.


Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Transdiferenciação Celular , Glomerulosclerose Segmentar e Focal/metabolismo , Podócitos/metabolismo , Renina/metabolismo , Células-Tronco/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Genes Reporter , Glomerulosclerose Segmentar e Focal/patologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Masculino , Camundongos Transgênicos , Microscopia de Fluorescência , Fenótipo , Podócitos/patologia , Renina/genética , Células-Tronco/patologia
7.
Am J Physiol Renal Physiol ; 315(1): F97-F109, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29412700

RESUMO

Blocking the renin-angiotensin-aldosterone system (RAAS) remains a mainstay of therapy in hypertension and glomerular diseases. With the population aging, our understanding of renin-producing cells in kidneys with advanced age is more critical than ever. Accordingly, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R mice to permanently fate map cells of renin lineage (CoRL). The number of Td-tomato-labeled CoRL decreased significantly in aged mice (24 mo of age) compared with young mice (3.5 mo of age), as did renin mRNA levels. To determine whether aged CoRL responded less to RAAS blockade, enalapril and losartan were administered over 25 days following uninephrectomy in young and aged mice. The number of CoRL increased in young mice in response to enalapril and losartan. However, this was significantly lower in aged mice compared with young mice due to limited proliferation, but not recruitment. Gene expression analysis of laser-captured CoRL showed a substantial increase in mRNA levels for proapoptotic and prosenescence genes, and an increase in a major prosenescence protein on immunostaining. These results show that CoRL are lower in aged mice and do not respond to RAAS inhibition to the same extent as young mice.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Linhagem da Célula , Enalapril/farmacologia , Rim/efeitos dos fármacos , Losartan/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Renina/metabolismo , Fatores Etários , Envelhecimento , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Feminino , Genes Reporter , Rim/metabolismo , Rim/patologia , Rim/cirurgia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos Transgênicos , Nefrectomia
8.
Cancer Lett ; 417: 75-88, 2018 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-29269086

RESUMO

KISS1 tumor suppressor protein regulates cancer cell invasion via MMP9 metalloproteinase. Downregulation of KISS1 gene expression promotes progression of breast cancer and melanoma, resulting in the development of distant metastases. In the current study, we investigated whether restoration of KISS1 expression in KISS1-deficient human metastatic breast cancer cells holds potential as an advanced anticancer strategy. To this end we engineered an infectivity-enhanced conditionally-replicative human adenovirus type 5 encoding KISS1 as an "arming" transgene in the Ad5 E3 region for an ectopic KISS1 expression in transduced cancer cells. The oncolytic potential of the vector was examined using brain-invading metastatic clones of CN34 and MDA-MB-231 breast cancer cells, which supported high levels of AdKISS1 replication, correlating with a robust CRAd-mediated cytotoxicity. Secretion of cellular factors responsible for tumor angiogenesis, cell-to-cell communication and anti-tumoral immune responses upon KISS1 expression in breast cancer cells was analyzed by a RayBiotech Kiloplex Quantibody array. Overall, our results indicate that KISS1 transgene expression provides an important benefit for CRAd-mediated cytotoxicity in breast cancer cells and holds potential as an anticancer treatment in conjunction with oncolytic virotherapy of breast and other metastatic cancers.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Kisspeptinas/genética , Neovascularização Patológica/genética , Células A549 , Adenoviridae/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Células Cultivadas , Genes Supressores de Tumor , Vetores Genéticos/genética , Humanos , Kisspeptinas/metabolismo , Neovascularização Patológica/metabolismo , Terapia Viral Oncolítica/métodos
9.
Stem Cell Reports ; 9(4): 1152-1166, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28966119

RESUMO

Wilms' tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1fl/fl reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1fl/fl mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.


Assuntos
Linhagem da Célula , Podócitos/metabolismo , Renina/genética , Proteínas WT1/genética , Animais , Biomarcadores , Movimento Celular/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Deleção de Genes , Testes de Função Renal , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Knockout , Podócitos/citologia , Renina/metabolismo , Proteínas WT1/metabolismo
10.
Autophagy ; 13(11): 1905-1923, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28981380

RESUMO

Formation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis.


Assuntos
Astrócitos/patologia , Autofagia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/secundário , Quimiocina CXCL12/metabolismo , Kisspeptinas/metabolismo , MicroRNAs/metabolismo , Adulto , Idoso , Animais , Astrócitos/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Microglia/metabolismo , Microglia/patologia , Pessoa de Meia-Idade , Ensaios Antitumorais Modelo de Xenoenxerto
11.
PLoS One ; 12(3): e0173891, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28329012

RESUMO

Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.


Assuntos
Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Renina/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Linhagem da Célula , Movimento Celular , Claudina-1/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia Intravital , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Fator de Transcrição PAX8/metabolismo , Proteínas Repressoras/metabolismo , Processos Estocásticos
12.
Oncotarget ; 8(16): 25989-25999, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-27517625

RESUMO

Glioblastoma multiforme (GBM) is a rapidly progressive brain tumor with a median survival of 15-19 months. Therapeutic resistance and recurrence of the disease is attributed to cancer stem cells (CSC). Here, we report that CMV70-3P miRNA encoded by CMV increases GBM CSC stemness. Inhibition of CMV70-3P expression using oligo inhibitors significantly attenuated the ability of primary glioma cells to proliferate and form neurospheres. At the molecular level, we show that CM70-3P increases expression of cellular SOX2. Collectively, these findings indicate that CMV70-3P is a potential regulator of CMV- mediated glioma progression and cancer stemness.


Assuntos
Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Glioma/etiologia , Glioma/metabolismo , MicroRNAs , Células-Tronco Neoplásicas/metabolismo , RNA Viral , Antígeno AC133/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Expressão Gênica , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Fenótipo , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética
13.
Kidney Int ; 91(4): 896-913, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27998643

RESUMO

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wild-type mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain), and collagen IV staining were lower in CD44 knockout compared with wild-type mice with FSGS. Parietal epithelial cells had lower migration from Bowman's capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cell migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44-overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression, and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cell phenotype.


Assuntos
Matriz Extracelular/enzimologia , Glomerulosclerose Segmentar e Focal/enzimologia , Receptores de Hialuronatos/metabolismo , Glomérulos Renais/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Podócitos/enzimologia , Albuminúria/enzimologia , Albuminúria/genética , Albuminúria/prevenção & controle , Animais , Movimento Celular , Células Cultivadas , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Predisposição Genética para Doença , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Receptores de Hialuronatos/genética , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Masculino , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fenótipo , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fatores de Tempo , Transfecção
14.
Arthritis Rheumatol ; 68(11): 2740-2751, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27159593

RESUMO

OBJECTIVE: In lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ adaptive immune cell networks that amplify local tissue damage. Since conventional therapy appears ineffective for severe TII, and these patients often progress to renal failure, understanding in situ mechanisms might reveal new therapeutic targets. This study was undertaken to assess whether dysregulated apoptotic regulators maintain local adaptive immunity and drive inflammation in TII. METHODS: This study utilized novel computational approaches that, when applied to multicolor confocal images, quantified apoptotic regulator protein expression in selected lymphocyte subsets. This approach was validated using laser-capture microdissection (LCM) coupled to quantitative polymerase chain reaction (qPCR). Furthermore, the consequences of dysregulated apoptotic mediator expression were explored in a murine model of lupus nephritis. RESULTS: Analyses of renal biopsy tissue from patients with lupus nephritis and those with mixed cellular renal allograft rejection revealed that the B cell lymphoma 2 protein (Bcl-2) was frequently expressed in infiltrating lymphocytes, whereas expression of myeloid cell leukemia 1 was low. In contrast, the reciprocal pattern of expression was observed in tonsil germinal centers. These results were consistent with RNA expression data obtained using LCM and qPCR. Bcl-2 was also highly expressed in tubulointerstitial infiltrates in (NZB × NZW)F1 (NZB/NZW) mice. Furthermore, treatment of NZB/NZW mice with ABT-199, a selective oral inhibitor of Bcl-2, prolonged survival and prevented proteinuria and development of TII in a lupus prevention model. Interestingly, glomerular immune complexes were partially ameliorated by ABT-199 treatment, and serum anti-double-stranded DNA antibody titers were unaffected. CONCLUSION: These data demonstrate that Bcl-2 is an attractive therapeutic target in patients with lupus nephritis who manifest TII.


Assuntos
Apoptose , Nefrite Lúpica/metabolismo , Linfócitos/metabolismo , Nefrite Intersticial/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Imunidade Adaptativa/imunologia , Animais , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Feminino , Centro Germinativo/metabolismo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Inflamação , Rim/efeitos dos fármacos , Rim/imunologia , Rim/metabolismo , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/imunologia , Glomérulos Renais/metabolismo , Transplante de Rim , Microdissecção e Captura a Laser , Nefrite Lúpica/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos NZB , Microscopia Confocal , Microscopia de Fluorescência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Nefrite Intersticial/imunologia , Tonsila Palatina , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia
15.
J Am Soc Nephrol ; 27(12): 3611-3627, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27080979

RESUMO

Because adult podocytes cannot proliferate and are therefore unable to self-renew, replacement of these cells depends on stem/progenitor cells. Although podocyte number is higher after renin-angiotensin-aldosterone system (RAAS) inhibition in glomerular diseases, the events explaining this increase are unclear. Cells of renin lineage (CoRL) have marked plasticity, including the ability to acquire a podocyte phenotype. To test the hypothesis that RAAS inhibition partially replenishes adult podocytes by increasing CoRL number, migration, and/or transdifferentiation, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R CoRL reporter mice to induce permanent labeling of CoRL with red fluorescent protein variant tdTomato. We then induced experimental FSGS, typified by abrupt podocyte depletion, with a cytopathic antipodocyte antibody. RAAS inhibition by enalapril (angiotensin-converting enzyme inhibitor) or losartan (angiotensin-receptor blocker) in FSGS mice stimulated the proliferation of CoRL, increasing the reservoir of these cells in the juxtaglomerular compartment (JGC). Compared with water or hydralazine, RAAS inhibition significantly increased the migration of CoRL from the JGC to the intraglomerular compartment (IGC), with more glomeruli containing RFP+CoRL and, within these glomeruli, more RFP+CoRL. Moreover, RAAS inhibition in FSGS mice increased RFP+CoRL transdifferentiation in the IGC to phenotypes, consistent with those of podocytes (coexpression of synaptopodin and Wilms tumor protein), parietal epithelial cells (PAX 8), and mesangial cells (α8 integrin). These results show that in the context of podocyte depletion in FSGS, RAAS inhibition augments CoRL proliferation and plasticity toward three different glomerular cell lineages.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Linhagem da Célula , Enalapril/farmacologia , Losartan/farmacologia , Podócitos/citologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Renina/fisiologia , Animais , Linhagem da Célula/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Masculino , Camundongos , Podócitos/efeitos dos fármacos
16.
Am J Physiol Renal Physiol ; 310(11): F1397-413, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27076646

RESUMO

The current studies used genetic fate mapping to prove that adult podocytes can be partially replenished following depletion. Inducible NPHS2-rtTA/tetO-Cre/RS-ZsGreen-R reporter mice were generated to permanently label podocytes with the ZsGreen reporter. Experimental focal segmental glomerulosclerosis (FSGS) was induced with a cytotoxic podocyte antibody. On FSGS day 7, immunostaining for the podocyte markers p57, synaptopodin, and podocin were markedly decreased by 44%, and this was accompanied by a decrease in ZsGreen fluorescence. The nuclear stain DAPI was absent in segments of reduced ZsGreen and podocyte marker staining, which is consistent with podocyte depletion. Staining for p57, synaptopodin, podocin, and DAPI increased at FSGS day 28 and was augmented by the ACE inhibitor enalapril, which is consistent with a partial replenishment of podocytes. In contrast, ZsGreen fluorescence did not return and remained significantly low at day 28, indicating replenishment was from a nonpodocyte origin. Despite administration of bromodeoxyuridine (BrdU) thrice weekly throughout the course of disease, BrdU staining was not detected in podocytes, which is consistent with an absence of proliferation. Although ZsGreen reporting was reduced in the tuft at FSGS day 28, labeled podocytes were detected along the Bowman's capsule in a subset of glomeruli, which is consistent with migration from the tuft. Moreover, more than half of the migrated podocytes coexpressed the parietal epithelial cell (PEC) proteins claudin-1, SSeCKS, and PAX8. These results show that although podocytes can be partially replenished following abrupt depletion, a process augmented by ACE inhibition, the source or sources are nonpodocyte in origin and are independent of proliferation. Furthermore, a subset of podocytes migrate to the Bowman's capsule and begin to coexpress PEC markers.


Assuntos
Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Animais , Cápsula Glomerular/metabolismo , Cápsula Glomerular/patologia , Modelos Animais de Doenças , Glomerulosclerose Segmentar e Focal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glomérulos Renais/patologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Podócitos/patologia
17.
BMC Nephrol ; 17: 5, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26746687

RESUMO

BACKGROUND: Recent studies indicate that mural cells of the preglomerular vessels, known as cells of renin lineage (CoRL), contribute to repair and regeneration of injured kidney glomeruli. However, their potential roles in tubulointerstitial disease are less understood. The aim of this study was to better understand CoRL number and distribution following UUO so that future mechanistic studies could be undertaken. METHODS: We mapped the fate of CoRL in adult Ren1cCreER x Rs-tdTomato-R reporter mice that underwent UUO. Kidney biopsies from sham and UUO-subjected mice on days 3, 7, and 14 were evaluated by immunohistochemistry. RESULTS: In sham animals, CoRL were restricted to juxtaglomerular location. At day 7 following UUO, CoRL increased two-fold, were perivascular in location, and co-expressed pericyte markers (PDGFßR, NG2), but did not express renin. At day 14 post UUO, labeled CoRL detached from vessels and were present in the interstitium, in areas of fibrosis, where they now expressed the myofibroblast marker alpha-smooth muscle actin. The increase in CoRL was likely due to proliferation as marked by BrdU labeling, and migration from the cortex. Following UUO starting from day 3, active hypoxia inducible factor-2α was detected in nuclei in labeled CoRL, in the cortex, but not those cells found in medulla. CONCLUSIONS: We have demonstrated that arteriolar CoRL are potential kidney progenitors that may contribute to the initial vascular regeneration. However, in chronic kidney injury (≥14 days post UUO), perivascular CoRL transition to myofibroblast-like cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Rim/citologia , Rim/metabolismo , Pericitos/metabolismo , Insuficiência Renal Crônica/metabolismo , Actinas/metabolismo , Animais , Antígenos/metabolismo , Diferenciação Celular , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Feminino , Fibrose , Camundongos , Pericitos/citologia , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Insuficiência Renal Crônica/etiologia , Renina/metabolismo , Obstrução Ureteral/complicações
18.
Cancer Lett ; 365(2): 240-50, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26052095

RESUMO

MMP14 (MT1-MMP) is a cell membrane-associated proteinase of the extracellular matrix, whose biological roles vary from angiogenesis to cell proliferation and survival. We recently found a direct correlation between MMP14 expression levels in brain tumors of glioma patients and the disease progression. By using gene silencing as an experimental approach we found that MMP14 knockdown decreases production of pro-angiogenic factors such as VEGF and IL8 and thereby suppresses angiogenesis in glioma tumors. Although the clinical relevance of MMP14 down-regulation and its possible implications for glioma therapy in humans remain unclear, we observed a significant improvement in animal survival upon down-regulation of MMP14 in murine intracranial glioma xenografts infected with MMP14 shRNA-expressing CRAd. We further found that down-regulation of MMP14 in gliomas by combinational treatment with CRAd-S-5/3 and Marimastat, a chemical inhibitor of metalloproteinases, augments suppression of pro-angiogenic factors, caused by the replication-competent adenovirus. We also demonstrated that delivery of MMP14-targeting shRNA by a fiber-modified adenoviral vector to the glioma cells effectively suppresses their proliferation in vitro and in vivo. Thus our data indicate that inhibition of MMP14 expression in tumors in combination with glioma virotherapy could be effectively utilized to suppress angiogenesis and neovascularization of glioma tumors by decreasing production of pro-angiogenic factors.


Assuntos
Adenoviridae/genética , Neoplasias Encefálicas/genética , Glioma/genética , Metaloproteinase 14 da Matriz/genética , Terapia Viral Oncolítica , Idoso , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Vetores Genéticos/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Interleucina-8/biossíntese , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Neovascularização Patológica/genética , Interferência de RNA , RNA Interferente Pequeno , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/biossíntese
19.
Aging (Albany NY) ; 7(6): 370-82, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26081073

RESUMO

With increasing age, the kidney undergoes characteristic changes in the glomerular and tubulo-interstitial compartments, which are ultimately accompanied by reduced kidney function. Studies have shown age-related loss of peritubular vessels. Normal peritubular vessel tone, function and survival depend on neighboring pericytes. Pericyte detachment leads to vascular damage, which can be accompanied by their differentiation to fibroblasts and myofibroblasts, a state that favors matrix production. To better understand the fate of pericytes in the aged kidney, 27 month-old mice were studied. Compared to 3 month-old young adult mice, aged kidneys showed a substantial decrease in capillaries, identified by CD31 staining, in both cortex and medulla. This was accompanied by a marked decrease in surrounding NG2+ / PDGFRß+ pericytes. This decrease was more pronounced in the medulla. Capillaries devoid of pericytes were typically dilated in aged mice. Aged kidneys were also characterized by interstitial fibrosis due to increased collagen-I and -III staining. This was accompanied by an increase in the number of pericytes that acquired a pro-fibrotic phenotype, identified by increased PDGFRß+ / αSMA+ staining. These findings are consistent with the decline in kidney interstitial pericytes as a critical step in the development of changes to the peritubular vasculature with aging, and accompanying fibrosis.


Assuntos
Envelhecimento/fisiologia , Rim/irrigação sanguínea , Rim/citologia , Pericitos/citologia , Animais , Antígenos/genética , Antígenos/metabolismo , Capilares/anatomia & histologia , Capilares/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Insuficiência Renal
20.
Am J Physiol Renal Physiol ; 309(4): F341-58, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26062877

RESUMO

Modified vascular smooth muscle cells of the kidney afferent arterioles have recently been shown to serve as progenitors for glomerular epithelial cells in response to glomerular injury. To determine whether such cells of renin lineage (CoRL) serve as progenitors for other cells in kidney disease characterized by both glomerular and tubulointerstitial injury, permanent genetic cell fate mapping of adult CoRL using Ren1cCreER × Rs-tdTomato-R reporter mice was performed. TdTomato-labeled CoRL were almost completely restricted to the juxtaglomerular compartment in healthy kidneys. Following 2 wk of antibody-mediated focal segmental glomerulosclerosis (FSGS) or 16 wk of ⅚ nephrectomy-induced chronic kidney diseases, tdTomato-mapped CoRL were identified in both interstitial and glomerular compartments. In the interstitium, PDGFß receptor (R)-expressing cells significantly increased, and a portion of these expressed tdTomato. This was accompanied by a decrease in native pericyte number, but an increase in the number of tdTomato cells that coexpressed the pericyte markers PDGFß-R and NG2. These cells surrounded vessels and coexpressed the pericyte markers CD73 and CD146, but not the endothelial marker ERG. Within glomeruli of reporter mice with the ⅚ nephrectomy model, a subset of labeled CoRL migrated to the glomerular tuft and coexpressed podocin and synaptopodin. By contrast, labeled CoRL were not detected in glomerular or interstitial compartments following uninephrectomy. These observations indicate that in addition to supplying new adult podocytes to glomeruli, CoRL have the capacity to become new adult pericytes in the setting of interstitial disease. We conclude that CoRL have the potential to function as progenitors for multiple adult cell types in kidney disease.


Assuntos
Células-Tronco Adultas/metabolismo , Linhagem da Célula , Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/metabolismo , Nefrite Intersticial/metabolismo , Células-Tronco Pluripotentes/metabolismo , Podócitos/metabolismo , Insuficiência Renal Crônica/metabolismo , Renina/metabolismo , Células-Tronco Adultas/ultraestrutura , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Movimento Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genes Reporter , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Glomérulos Renais/ultraestrutura , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nefrite Intersticial/genética , Nefrite Intersticial/patologia , Pericitos/metabolismo , Pericitos/ultraestrutura , Fenótipo , Células-Tronco Pluripotentes/ultraestrutura , Podócitos/ultraestrutura , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Renina/genética
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