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1.
Endocrinology ; 151(9): 4381-8, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20610569

RESUMO

C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth through a subtype of membranous guanylyl cyclase receptor, GC-B. Although its two cognate natriuretic peptides, ANP and BNP, are cardiac hormones produced from heart, CNP is thought to act as an autocrine/paracrine regulator. To elucidate whether systemic administration of CNP would be a novel medical treatment for chondrodysplasias, for which no drug therapy has yet been developed, we investigated the effect of circulating CNP by using the CNP transgenic mice with an increased circulating CNP under the control of human serum amyloid P component promoter (SAP-Nppc-Tg mice). SAP-Nppc-Tg mice developed prominent overgrowth of bones formed through endochondral ossification. In organ culture experiments, the growth of tibial explants of SAP-Nppc-Tg mice was not changed from that of their wild-type littermates, exhibiting that the stimulatory effect on endochondral bone growth observed in SAP-Nppc-Tg mice is humoral. Then we crossed chondrodysplastic CNP-depleted mice with SAP-Nppc-Tg mice. Impaired endochondral bone growth in CNP knockout mice were considerably and significantly recovered by increased circulating CNP, followed by the improvement in not only their longitudinal growth but also their body weight. In addition, the mortality of CNP knockout mice was greatly decreased by circulating CNP. Systemic administration of CNP might have therapeutic potential against not only impaired skeletal growth but also other aspects of impaired growth including impaired body weight gain in patients suffering from chondrodysplasias and might resultantly protect them from their early death.


Assuntos
Desenvolvimento Ósseo/fisiologia , Condrodisplasia Punctata/sangue , Peptídeo Natriurético Tipo C/sangue , Osteogênese/fisiologia , Animais , Animais Recém-Nascidos , Peso Corporal/genética , Peso Corporal/fisiologia , Desenvolvimento Ósseo/genética , Condrodisplasia Punctata/genética , Condrodisplasia Punctata/mortalidade , Colágeno Tipo II/genética , Colágeno Tipo X/genética , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/farmacologia , Técnicas de Cultura de Órgãos , Osteogênese/genética , Antígeno Nuclear de Célula em Proliferação/análise , Taxa de Sobrevida , Tíbia/efeitos dos fármacos , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismo , Fatores de Tempo
2.
Am J Physiol Endocrinol Metab ; 297(6): E1339-48, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19808910

RESUMO

C-type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. For future clinical application, the efficacy of CNP administration on skeletal growth must be evaluated. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight. These results suggest that CNP administration stimulates skeletal growth without adverse cardiovascular effect, and thus CNP could be a promising remedy targeting achondroplasia.


Assuntos
Desenvolvimento Ósseo/fisiologia , Peptídeo Natriurético Tipo C/sangue , Osteogênese/fisiologia , Absorciometria de Fóton , Animais , Pressão Sanguínea/fisiologia , Densidade Óssea/fisiologia , Feminino , Guanilato Ciclase/metabolismo , Coração/fisiologia , Membro Posterior/anatomia & histologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeo Natriurético Tipo C/genética , Tamanho do Órgão , Proteoglicanas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Kidney Int ; 75(3): 285-94, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19148153

RESUMO

Urinary neutrophil gelatinase-associated lipocalin (Ngal or lipocalin 2) is a very early and sensitive biomarker of kidney injury. Here we determined the origin and time course of Ngal appearance in several experimental and clinically relevant renal diseases. Urinary Ngal levels were found to be markedly increased in lipoatrophic- and streptozotocin-induced mouse models of diabetic nephropathy. In the latter mice, the angiotensin receptor blocker candesartan dramatically decreased urinary Ngal excretion. The reabsorption of Ngal by the proximal tubule was severely reduced in streptozotocin-induced diabetic mice, but upregulation of its mRNA and protein in the kidney was negligible, compared to those of control mice, suggesting that increased urinary Ngal was mainly due to impaired renal reabsorption. In the mouse model of unilateral ureteral obstruction, Ngal protein synthesis was dramatically increased in the dilated thick ascending limb of Henle and N was found in the urine present in the swollen pelvis of the ligated kidney. Five patients with nephrotic syndrome or interstitial nephritis had markedly elevated urinary Ngal levels at presentation, but these decreased in response to treatment. Our study shows that the urinary Ngal level may be useful for monitoring the status and treatment of diverse renal diseases reflecting defects in glomerular filtration barrier, proximal tubule reabsorption, and distal nephrons.


Assuntos
Proteínas de Fase Aguda/metabolismo , Modelos Animais de Doenças , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Lipocalinas/metabolismo , Néfrons/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Injúria Renal Aguda/metabolismo , Proteínas de Fase Aguda/urina , Albuminas/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Benzimidazóis/uso terapêutico , Biomarcadores/urina , Compostos de Bifenilo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/urina , Humanos , Túbulos Renais Proximais/citologia , Lipocalina-2 , Lipocalinas/urina , Alça do Néfron/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nefrite Intersticial/metabolismo , Nefrite Intersticial/urina , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/urina , Proteínas Proto-Oncogênicas/urina , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade , Tetrazóis/uso terapêutico , Fatores de Tempo , Obstrução Ureteral/metabolismo
4.
Am J Physiol Renal Physiol ; 293(4): F1363-72, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17699553

RESUMO

CCN1 (cysteine-rich protein 61; Cyr61) is an extracellular matrix-associated signaling molecule that functions in cell migration, adhesion, and differentiation. We previously reported that CCN1 is induced at podocytes in rat anti-Thy-1 glomerulonephritis, a well-known model of reversible glomerular injury, but its expression and significance in the human kidney remain totally unknown (Sawai K, Mori K, Mukoyama M, Sugawara A, Suganami T, Koshikawa M, Yahata K, Makino H, Nagae T, Fujinaga Y, Yokoi H, Yoshioka T, Yoshimoto A, Tanaka I, Nakao K. J Am Soc Nephrol 14: 1154-1163, 2003). Here we report that, in the human kidney, CCN1 expression was confined to podocytes in normal adult and embryonic glomeruli from the capillary loop stage. Podocyte CCN1 expression was decreased in IgA nephropathy, diabetic nephropathy, and membranous nephropathy, whereas it remained unchanged in minimal change disease and focal segmental glomerulosclerosis. Downregulation of CCN1 was significantly greater in diseased kidneys with severe mesangial expansion. CCN1 protein was also localized in the thick ascending limb of Henle's loop, distal and proximal tubules, and collecting ducts, which was not altered in diseased kidneys. In vitro, recombinant CCN1 protein enhanced endothelial cell adhesion, whereas it prominently inhibited mesangial cell adhesion. CCN1 also completely suppressed mesangial cell migration, suggesting its role as a mesangial-repellent factor. In cultured podocytes, CCN1 markedly induced the expression of cyclin-dependent kinase inhibitor p27(Kip1) as well as synaptopodin in a dose-dependent manner and suppressed podocyte migration. These data indicate that CCN1 is expressed in podocytes, can act on glomerular cells to modulate glomerular remodeling, and is downregulated in diseased kidneys, suggesting that impairment of CCN1 expression in podocytes may contribute to the progression of glomerular disease with mesangial expansion.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Nefropatias/metabolismo , Rim/embriologia , Rim/metabolismo , Adulto , Animais , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Rica em Cisteína 61 , Feminino , Glomerulonefrite por IGA/metabolismo , Glomerulonefrite por IGA/patologia , Humanos , Proteínas Imediatamente Precoces/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , Pessoa de Meia-Idade , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Ratos , Ratos Sprague-Dawley
5.
Nephrol Dial Transplant ; 21(9): 2472-7, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16820374

RESUMO

BACKGROUND: Significance of podocyte injury in the progression of diabetic nephropathy is not well-understood. In this study, we examined whether alteration of gap junction protein connexin43 (Cx43) expression in podocytes is associated with the progression of overt diabetic nephropathy. METHODS: We recruited 29 type 2 diabetic patients with overt nephropathy who underwent renal biopsy. Nephrectomized kidney samples obtained from seven subjects with localized neoplasm and biopsy specimens from five patients diagnosed as minor glomerular abnormalities were used as controls. Cx43 staining on paraffin-embedded kidney sections were studied by immunohistochemistry. RESULTS: In controls, Cx43 was expressed at podocytes in a linear pattern along the glomerular basement membrane. In contrast, downregulation and loss of uniformly linear staining of Cx43 (Cx43 heterogeneity) in podocytes were observed in diabetic nephropathy. Cx43 intensity correlated with current renal function (R = 0.647, P < 0.005), whereas the magnitude of Cx43 heterogeneity correlated well with the degree of future decline in renal function (R = -0.705, P < 0.001). CONCLUSIONS: Alteration of Cx43 expression in podocytes was closely associated with the progression of overt diabetic nephropathy. These results indicate that change in Cx43 expression at podocytes represents a progressive stage in overt diabetic nephropathy and that it may be a convenient way to predict future decline in renal function.


Assuntos
Conexina 43/biossíntese , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Podócitos/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Biópsia , Western Blotting , Células Cultivadas , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/patologia , Progressão da Doença , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Podócitos/patologia , Prognóstico , Estudos Retrospectivos
6.
J Am Soc Nephrol ; 16(9): 2690-701, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15987752

RESUMO

Podocytes play an important role in maintaining normal glomerular function and structure, and podocyte injury leads to proteinuria and glomerulosclerosis. The family of mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase, and p38) may be implicated in the progression of various glomerulopathies, but the role of MAPK in podocyte injury remains elusive. This study examined phosphorylation of p38 MAPK in clinical glomerulopathies with podocyte injury, as well as in rat puromycin aminonucleoside (PAN) nephropathy and mouse adriamycin (ADR) nephropathy. The effect of treatment with FR167653, an inhibitor of p38 MAPK, was also investigated in rodent models. In human podocyte injury diseases, the increased phosphorylation of p38 MAPK was observed at podocytes. In PAN and ADR nephropathy, the phosphorylation of p38 MAPK and ERK was marked but transient, preceding overt proteinuria. Pretreatment with FR167653 (day -2 to day 14, subcutaneously) to PAN or ADR nephropathy completely inhibited p38 MAPK activation and attenuated ERK phosphorylation, with complete suppression of proteinuria. Electron microscopy and immunohistochemistry for nephrin and connexin43 revealed that podocyte injury was markedly ameliorated by FR167653. Furthermore, early treatment with FR167653 effectively prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. In cultured podocytes, PAN or oxidative stress induced the phosphorylation of p38 MAPK along with actin reorganization, and FR167653 inhibited such changes. These findings indicate that the activation of MAPK is necessary for podocyte injury, suggesting that p38 MAPK and, possibly, ERK should become a potential target for therapeutic intervention in proteinuric glomerulopathies.


Assuntos
Síndrome Nefrótica/enzimologia , Síndrome Nefrótica/patologia , Podócitos/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Ativação Enzimática , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Masculino , Camundongos , Síndrome Nefrótica/etiologia , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteinúria/enzimologia , Puromicina Aminonucleosídeo/toxicidade , Ratos , Ratos Endogâmicos WKY , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
7.
J Am Soc Nephrol ; 15(6): 1430-40, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15153554

RESUMO

Connective tissue growth factor (CTGF/CCN2) is one of the candidate factors mediating fibrogenic activity of TGF-beta. It was shown previously that the blockade of CTGF by antisense oligonucleotide (ODN) inhibits TGF-beta-induced production of fibronectin and type I collagen in cultured renal fibroblasts. The in vivo contribution of CTGF in renal interstitial fibrosis, however, remains to be clarified. With the use of a hydrodynamics-based gene transfer technique, the effects of CTGF antisense ODN are investigated in rat kidneys with unilateral ureteral obstruction (UUO). FITC-labeled ODN injection via the renal vein showed that the ODN was specifically introduced into the interstitium. At day 7 after UUO, the gene expression of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen in untreated or control ODN-treated obstructed kidneys was prominently upregulated. CTGF antisense ODN treatment, by contrast, markedly attenuated the induction of CTGF, fibronectin, fibronectin ED-A, and alpha1(I) collagen genes, whereas TGF-beta gene upregulation was not affected. The antisense treatment also reduced interstitial deposition of CTGF, fibronectin ED-A, and type I collagen and the interstitial fibrotic areas. The number of myofibroblasts determined by the expression of alpha-smooth muscle actin was significantly decreased as well. Proliferation of tubular and interstitial cells was not altered with the treatment. These findings indicate that CTGF expression in the interstitium plays a crucial role in the progression of interstitial fibrosis but not in the proliferation of tubular and interstitial cells during UUO. CTGF may become a potential therapeutic target against tubulointerstitial fibrosis.


Assuntos
Fibrose/metabolismo , Proteínas Imediatamente Precoces/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Nefropatias/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Actinas/biossíntese , Animais , Northern Blotting , Western Blotting , Divisão Celular , Fator de Crescimento do Tecido Conjuntivo , Fibronectinas/biossíntese , Técnicas de Transferência de Genes , Imuno-Histoquímica , Rim/metabolismo , Masculino , Microscopia de Fluorescência , Músculo Liso/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/química , Plasmídeos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Transfecção , Regulação para Cima , Obstrução Ureteral
8.
J Am Soc Nephrol ; 14(5): 1154-63, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12707386

RESUMO

Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-beta. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration.


Assuntos
Mesângio Glomerular/citologia , Glomerulonefrite/fisiopatologia , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neovascularização Fisiológica/fisiologia , Indutores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Becaplermina , Divisão Celular/fisiologia , Linhagem Celular Transformada , Movimento Celular/fisiologia , Proteína Rica em Cisteína 61 , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Biblioteca Gênica , Glomerulonefrite/patologia , Camundongos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Antígenos Thy-1/imunologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Regulação para Cima
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