RESUMEN
In the renal cortex the peritubular capillary network and the proximal tubular epithelium cooperate in solute and water reabsorption, secretion, and inflammation. However, the mechanisms by which these two cell types coordinate such diverse functions remain to be characterized. Here we investigated the influence of microvascular endothelial cells on proximal tubule cells, using a filter-based, noncontact, close-proximity coculture of the human microvascular endothelial cell line HMEC-1 and the human proximal tubular epithelial cell line HK-2. With the use of DNA microarrays the transcriptomes of HK-2 cells cultured in mono- and coculture were compared. HK-2 cells in coculture exhibited a differential expression of 99 genes involved in pathways such as extracellular matrix (e.g., lysyl oxidase), cell-cell communication (e.g., IL-6 and IL-1 beta), and transport (e.g., GLUT3 and lipocalin 2). HK-2 cells also exhibited an enhanced paracellular gating function in coculture, which was dependent on HMEC-1-derived extracellular matrix. We identified a number of HMEC-1-enriched genes that are potential regulators of epithelial cell function such as extracellular matrix proteins (e.g., collagen I, III, IV, and V, laminin-alpha IV) and cytokines/growth factors (e.g., hepatocyte growth factor, endothelin-1, VEGF-C). This study demonstrates a complex network of communication between microvascular endothelial cells and proximal tubular epithelial cells that ultimately affects proximal tubular cell function. This coculture model and the data described will be important in the further elucidation of microvascular endothelial and proximal tubular epithelial cross talk mechanisms.
Asunto(s)
Comunicación Celular/genética , Endotelio Vascular/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales Proximales/metabolismo , Transcripción Genética/genética , Capilares/metabolismo , Capilares/ultraestructura , Línea Celular , Línea Celular Transformada , Permeabilidad de la Membrana Celular/genética , Técnicas de Cocultivo , Endotelio Vascular/citología , Células Epiteliales/citología , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Riñón/irrigación sanguínea , Riñón/metabolismo , Túbulos Renales Proximales/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
The nephrotoxic potential of the widely used immunosuppressive agent cyclosporine A (CsA) is well recognized. However, the mechanism of renal tubular toxicity is not yet fully elucidated. Chronic CsA nephropathy and renal organ aging share some clinical features, such as renal fibrosis and tubular atrophy, raising the possibility that CsA may exert some of its deleterious effects via induction of a stress-induced senescent phenotype. We investigated this hypothesis in HK-2 cells and primary proximal tubular cells in vitro. CsA induced the production of H2O2, caused cell cycle arrest in the G0/G1 phase, and inhibited DNA synthesis. Furthermore, CsA exposure lead to a reduction of telomere length, increased p53 serine 15 phosphorylation, and caused an upregulation of the cell cycle inhibitor p21(Kip1) (CDKN1A) mRNA levels. CsA caused an increase in p16(INK4a) (CDKN2A) expression after a 13-day exposure in primary proximal tubular cells but not in HK-2 cells. Coincubation of cells with CsA and catalase was able to prevent telomere shortening and partially restored DNA synthesis. In summary, CsA induces cellular senescence in human renal tubular epithelial cells, which can be attenuated by scavenging reactive oxygen species.
Asunto(s)
Ciclosporina/efectos adversos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Túbulos Renales/citología , Envejecimiento/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Humanos , Necrosis , ARN Mensajero/genética , Factores de TiempoRESUMEN
Interstitial inflammation has emerged as a key event in the development of acute renal failure. To gain better insight into the nature of these inflammatory processes, the interplay between tubular epithelial cells, endothelial cells, and neutrophils (PMN) was investigated. A coculture transmigration model was developed, composed of human dermal microvascular endothelial (HDMEC) and human renal proximal tubular cells (HK-2) cultured on opposite sides of Transwell growth supports. Correct formation of an endoepithelial bilayer was verified by light and electron microscopy. The model was used to study the effects of endotoxin (LPS), tumor necrosis factor (TNF)-alpha, and alpha-melanocyte-stimulating hormone (alpha-MSH) by measuring PMN migration and cytokine release. To distinguish between individual roles of microvascular endothelial and epithelial cells in transmigration processes, migration of PMN was investigated separately in HK-2 and HDMEC monolayers. Sequential migration of PMN through endothelium and epithelium could be observed and was significantly increased after proinflammatory stimulation with either TNF-alpha or LPS (3.5 +/- 0.58 and 2.76 +/- 0.64-fold vs. control, respectively). Coincubation with alpha-MSH inhibited the transmigration of PMN through the bilayer after proinflammatory stimulation with LPS but not after TNF-alpha. The bilayers produced significant amounts of IL-8 and IL-6 mostly released from the epithelial cells. Furthermore, alpha-MSH decreased LPS-induced IL-6 secretion by 30% but had no significant effect on IL-8 secretion. We established a transmigration model showing sequential migration of PMN across microvascular endothelial and renal tubular epithelial cells stimulated by TNF-alpha and LPS. Anti-inflammatory effects of alpha-MSH in this bilayer model are demonstrated by inhibition on PMN transmigration and IL-6 secretion.