RESUMEN
Parathyroid hormone (PTH) is essential for calcium homeostasis and its action is mediated by the PTH type 1 receptor (PTHR1), a class B G-protein-coupled receptor. Hypoparathyroidism and osteoporosis can be treated with PTH injections; however, no orally effective PTH analogue is available. Here we show that PCO371 is a novel, orally active small molecule that acts as a full agonist of PTHR1. PCO371 does not affect the PTH type 2 receptor (PTHR2), and analysis using PTHR1-PTHR2 chimeric receptors indicated that Proline 415 of PTHR1 is critical for PCO371-mediated PTHR1 activation. Oral administration of PCO371 to osteopenic rats provokes a significant increase in bone turnover with limited increase in bone mass. In hypocalcemic rats, PCO371 restores serum calcium levels without increasing urinary calcium, and with stronger and longer-lasting effects than PTH injections. These results strongly suggest that PCO371 can provide a new treatment option for PTH-related disorders, including hypoparathyroidism.
Asunto(s)
Hipoparatiroidismo/tratamiento farmacológico , Imidazolidinas/síntesis química , Receptor de Hormona Paratiroídea Tipo 1/agonistas , Compuestos de Espiro/síntesis química , Animales , Perros , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Imidazolidinas/farmacología , Masculino , Estructura Molecular , Mutación , Glándulas Paratiroides/efectos de los fármacos , Glándulas Paratiroides/cirugía , Ratas , Compuestos de Espiro/farmacologíaRESUMEN
Diabetic nephropathy is the leading cause of end-stage renal disease. It is pathologically characterized by the accumulation of extracellular matrix in the mesangium, of which the main component is α1/α2 type IV collagen (Col4a1/a2). Recently, we identified Smad1 as a direct regulator of Col4a1/a2 under diabetic conditions in vitro. Here, we demonstrate that Smad1 plays a key role in diabetic nephropathy through bone morphogenetic protein 4 (BMP4) in vivo. Smad1-overexpressing mice (Smad1-Tg) were established, and diabetes was induced by streptozotocin. Nondiabetic Smad1-Tg did not exhibit histological changes in the kidney; however, the induction of diabetes resulted in an â¼1.5-fold greater mesangial expansion, consistent with an increase in glomerular phosphorylated Smad1. To address regulatory factors of Smad1, we determined that BMP4 and its receptor are increased in diabetic glomeruli and that diabetic Smad1-Tg and wild-type mice treated with a BMP4-neutralizing antibody exhibit decreased Smad1 phosphorylation and â¼40% less mesangial expansion than those treated with control IgG. Furthermore, heterozygous Smad1 knockout mice exhibit attenuated mesangial expansion in the diabetic condition. The data indicate that BMP4/Smad1 signaling is a critical cascade for the progression of mesangial expansion and that blocking this signal could be a novel therapeutic strategy for diabetic nephropathy.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Matriz Extracelular/metabolismo , Riñón/metabolismo , Proteína Smad1/metabolismo , Animales , Proteína Morfogenética Ósea 4/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Matriz Extracelular/patología , Riñón/patología , Ratones , Ratones Noqueados , Fosforilación , Proteína Smad1/genéticaRESUMEN
The human fibroblast growth factor 23 (hFGF23) and its autosomal dominant hypophosphatemic rickets (ADHR) mutant genes were incorporated into animals by naked DNA injection to investigate the action on phosphate homeostasis in vivo. The hFGF23 mutants (R176Q, R179Q, and R179W) markedly reduced serum phosphorus (6.2-6.9 mg/dl) compared with the plasmid MOCK (8.5 mg/dl). However, native hFGF23 did not affect serum phosphorus (8.6 mg/dl). Both hFGF23 and hFGF23R179Q mRNAs were expressed more than 100-fold in the liver 4 days after injection, however, the C-terminal portion of hFGF23 was detected only in the serum from hFGF23R179Q-injected animals (1109 pg/ml). hFGF23R179Q mutant was secreted as a 32-kDa protein, whereas, native hFGF23 was detected as a 20-kDa protein in the cell-conditioned media. These results suggest the hFGF23R179Q protein is resistant to intracellular proteolytic processing. The hFGF23R179Q suppressed Na/P(i) co-transport activities both in kidney and in small intestine by 45 and 30%, respectively, as well as serum 1alpha,25-dihydroxyvitamin D(3) to less than 15 pg/ml. However, it had little effect on serum parathyroid hormone (PTH). Infusion of hFGF23R179Q protein normalized serum phosphorus in thyroparathyroidectomized rats without affecting serum calcium. Taken together, the FGF23 mutants reduce both phosphate uptake in intestine and phosphate reabsorption in kidney, independent of PTH action.
Asunto(s)
Colecalciferol/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Mutación , Sodio/metabolismo , Adenina/farmacología , Animales , Transporte Biológico , Western Blotting , Células COS , Clonación Molecular , Creatinina/metabolismo , ADN Complementario/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Técnicas de Transferencia de Gen , Humanos , Hígado/metabolismo , Masculino , Ratones , Nefritis/sangre , Fosfatos/metabolismo , Plásmidos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Factores de TiempoRESUMEN
Osteoclast differentiation factor, ODF, also called RANKL, TRANCE, or OPGL, is a key molecule for osteoclast differentiation and activation, and is thought to act as a membrane-associated molecule in bone remodeling. Recent study suggested that soluble ODF (sODF) released from T cells also has some roles in bone resorption. To investigate the physiological and pathological function of sODF, we generated two types of transgenic mice overexpressing sODF. Mice overexpressing sODF ubiquitously from the early developmental stage died at the late fetal stage. The other type of mice, expressing sODF only in the liver after birth, grew to maturity with normal body size and weight. However, they exhibited a marked decrease in bone mineral density with aging compared with their non-transgenic littermates, and in addition, the strength of their femurs was extremely reduced. Histological analysis showed that the trabecular bone mass was decreased at 6 weeks of age and was sparse at age 3-4 months. The number of osteoclasts was significantly increased, while the number of osteoblasts was not altered on the surface of young trabecular bone. These results indicate that excessive production of sODF causes osteoporosis by accelerated osteoclastogenesis. The transgenic mouse overexpressing sODF in the liver could serve as a useful animal model for studying bone remodeling and evaluating therapeutic agents for osteoporosis.