RESUMEN
Studies were carried out to determine whether epithelial-mesenchymal transformation (EMT), well described in renal tubular epithelial cells, also occurs in glomerular epithelial cells and whether it is reversible. To this effect, cultured glomerular epithelial cells were incubated with TGF-beta(1) and their transformation into myofibroblasts was studied. At 4 days, the cells altered their phenotype, as shown by a change in shape, an increase in intracellular staining for alpha-smooth muscle actin (alpha-SMA), a decrease in membrane staining for cytokeratin, and an increase in matrix deposition. Changing the medium after 4 days by excluding TGF-beta(1) and adding fetal bovine serum (FBS) [as a source of epidermal growth factor (EGF) and other growth factors] caused the cells to revert to their original epithelial phenotype. By contrast, when the medium was changed in the same manner after 8 days of exposure to TGF-beta(1), the cells did not revert but remained myofibroblastic. Staining the cells for expression of EGF receptor before and after exposure to TGF-beta(1) caused this receptor, originally present on the plasma membrane, to become partly intracellular after 4 days of TGF-beta(1) exposure and completely intracellular after 8 days of TGF-beta(1) exposure. Kidney sections from 2 models of renal mass reduction were stained. Loss of the epithelial marker (podocalyxin) staining and the acquisition of alpha-SMA staining was observed in the glomeruli. It is concluded that EMT takes place in glomerular epithelial cells in vivo and in vitro. In cultured glomerular epithelial cells, the process can be reversed by early, but not late intervention. It seems that TGF-beta(1) exposure progressively downregulates the EGF receptor on the membrane, rendering the cell refractory to EGF signals critical for maintaining the epithelial phenotype.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Podocitos/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Fibroblastos/citología , Infarto/metabolismo , Infarto/patología , Riñón/irrigación sanguínea , Riñón/metabolismo , Riñón/patología , Masculino , Miocitos del Músculo Liso/citología , Nefrectomía , Podocitos/citología , Ratas , Ratas Sprague-Dawley , Sialoglicoproteínas/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/deficiencia , Factor de Crecimiento Transformador beta1RESUMEN
Vascular endothelial growth factor (VEGF) is a potent growth factor that is indispensable for the development of blood vessels in the fetus and for wound healing in adults. VEGF likely plays a role in maintaining the blood vessels once they have been formed. It is not clear, however, whether a low tissue VEGF (caused either by disease or by systemic administration of VEGF antagonists) can cause abnormalities in preexisting blood vessels, especially of wound tissue that requires high local levels of VEGF for healing. The present study investigated the effect of VEGF antagonism on blood vessels of foreign-body granulomas (a model of wound-healing tissue). Granulomas were induced by implanting perforated polyvinyl tubes into the subcutaneous tissue of rats and allowed to develop for 14 days, at which time the implanted tubes were completely encapsulated by the subcutaneous tissue. The encapsulated granulomas consisted of 3 distinct histological layers, of which the middle layer was well perfused by a rich supply of microvessels. Morphologically, the granuloma remained "stable" after developing for 14 days. At 1 week, VEGF levels in the granuloma fluid, which is in equilibrium with the interstitial fluid, were 25 times higher than in the plasma. VEGF levels in the granuloma fluid continued to increase for up to 3 weeks, reflecting the high dependence of the wound tissue on ambient VEGF levels. After injection of the VEGF receptor antagonist in the fully formed granuloma, the preexisting blood vessels in the middle layer regressed and underwent apoptosis, accompanied by expansion of the extracellular matrix (predominately collagen I) into areas normally devoid of matrix. We conclude that wound tissue is sensitive to ambient VEGF levels, and that a low VEGF condition resulting from VEGF receptor antagonism can disrupt the healing of wound tissue.
Asunto(s)
Granuloma de Cuerpo Extraño/fisiopatología , Microcirculación/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/fisiología , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiología , Animales , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Granuloma de Cuerpo Extraño/metabolismo , Granuloma de Cuerpo Extraño/patología , Masculino , Microcirculación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 degrees C, pH 7.4, were Ang 1-9 and Ang II. Quinaprilat did not influence the formation of Ang 1-9, but it inhibited formation of Ang II. The formation of Ang 1-9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1-9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.
Asunto(s)
Carboxipeptidasas A/metabolismo , Riñón/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animales , Riñón/enzimología , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , RatasRESUMEN
BACKGROUND: Relaxin (Rlx), a 6-kD protein hormone, belongs to the insulin growth factor family. We have previously shown that Rlx reduces interstitial fibrosis in a model of chronic papillary necrosis. HYPOTHESIS: The purpose of this study was to extend these observations to a model of renal injury induced by mass reduction. MATERIAL AND METHODS: Renal mass was reduced by either infarction or surgical excision of both poles, with removal of the contralateral kidney. Two weeks later, creatinine clearance was done and animals from both groups implanted with osmotic pumps delivering either Rlx (2 microg/h) or vehicle (Veh). Treatment was continued for 4 weeks. The severity of the glomerular injury was quantified by planimetric measurements. Renal function was assessed by creatinine clearance and plasma creatinine. RESULTS: Rlx significantly decreased systolic blood pressure in animals with infarction. This was accompanied by a decrease in serum creatinine and a slight improvement in creatinine clearance. The severity of the glomerular lesion was reduced by Rlx (sclerosis index, Veh 1.16 +/- 0.13 vs. Rlx 0.74 +/- 0.16, p = 0.037). In the excision group the animals were normotensive. In this group, Rlx treatment was accompanied by a decrease in serum creatinine (Veh 1.01 +/- 0.03 vs. Rlx 0.81 +/- 0.05 mg/dl, p = 0.02) and an increase in GFR (Veh 0.90 +/- 0.14 vs. Rlx 1.33 +/- 0.11 ml/min, p = 0.03). The sclerosis index was also reduced. CONCLUSION: Rlx decreases renal injury by at least two mechanisms, one by lowering blood pressure as seen in the infarction model, the other independent of blood pressure as seen in the normotensive excision model where there was also a significant functional improvement.