RESUMEN
BACKGROUND: An increased number of major histocompatibility complex (MHC) class II-positive cells (OX-6+ cells) were observed in the glomerulus and periglomerular interstitium during the course of anti-glomerular basement membrane (anti-GBM) crescentic glomerulonephritis (GN) in WKY rats. This study aimed to demonstrate that periglomerular OX-6+ cells are dendritic cells (DCs) and to clarify their roles in the pathogenesis of this GN. METHODS: Kidney sections were stained with the OX-6 and the rat DC marker OX-62 by immunohistochemistry, and periglomerular OX-6+ cells were observed by immunoelectron microscopy. Renal mRNA expression for CXCL12 was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization, and that for IL-1beta was examined by in situ hybridization. RESULTS: Immunohistochemistry revealed that most periglomerular OX-6+ cells in this GN were ED-1-negative. OX-62+ cells were observed sparsely in normal kidney interstitium, and considerably more frequently in periglomerular interstitium in this GN. Immunoelectron microscopy confirmed the periglomerular OX-6+ED-1- cells had DC morphology. The increased expression of CXCL12 mRNA in the diseased glomerulus was shown by RT-PCR. By in situ hybridization, CXCL12 mRNA-expressing glomerular cells were the parietal and visceral epithelial cells, which were close to the site of periglomerular OX-6+ cell localization. The intense expression of IL-1beta mRNA by periglomerular cells was demonstrated by in situ hybridization. CONCLUSIONS: The periglomerular distribution of OX-6+ED-1- DCs was demonstrated in anti-GBM crescentic GN in WKY rats. These DCs might be accumulated in periglomerular interstitium by CXCL12, and play a role in the initiation and progression of this GN by producing IL-1beta.
Asunto(s)
Células Dendríticas , Glomerulonefritis/patología , Animales , Glomérulos Renales , Ratas , Ratas Endogámicas WKYRESUMEN
Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.
Asunto(s)
Acuaporinas/deficiencia , Acuaporinas/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Vacuolas/metabolismo , Animales , Acuaporinas/genética , Peso Corporal/genética , Células Cultivadas , Cricetinae , Endosomas/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Túbulos Renales Proximales/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Tamaño de los Órganos/genética , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/ultraestructura , Transporte de ProteínasRESUMEN
BACKGROUND: Fat1 is a member of the cadherin superfamily characterized by its 34 cadherin repeats in the extracellular domain. Fat1 was originally found as a component of the slit diaphragm of podocytes, but its function in podocytes remains obscure. To gain insight into its role in podocytes, we expanded our study of Fat1 expression to puromycin aminonucleoside (PAN) nephrosis, the neonatal kidney, and the primary podocyte culture, where slit diaphragms are absent or disappear. METHODS: Expression of Fat1 was examined in isolated glomeruli of PAN nephrosis by the ribonuclease protection assay and Western blot analysis and in the neonatal kidney by in situ hybridization. Fat1 localization in glomeruli and in the primary culture was confirmed by immunofluorescence or immunoelectron microscopy. RESULTS: In PAN nephrotic rats, glomerular expression of Fat1 increased rather than decreased at both transcript and protein levels in comparison with normal rats. Immunofluorescence microscopy revealed distinct staining for Fat1 along the glomerular capillary wall, where nephrin staining was weakened or disappeared. Immunoelectron microscopy demonstrated significant accumulation of immunogold particles for Fat1 at intercellular junctions newly formed between podocytes in the nephrosis. In the primary culture of podocytes, Fat1 was mainly localized at cell-cell contact sites and in tips of cellular processes. In the neonatal kidney, immature podocytes expressed Fat1 more intensely than mature podocytes as shown by in situ hybridization. Double-labeled immunostaining using anti-pan cadherin antibody revealed that Fat1 in podocytes colocalized with cadherin in immature glomeruli, indicating that junctional complexes of developing podocytes contain Fat1. CONCLUSION: These findings suggest that Fat1 may be a fundamental component of intercellular junctions of podocytes, and may be involved in the initial step of cell contacts of podocytes.
Asunto(s)
Cadherinas/genética , Cadherinas/metabolismo , Comunicación Celular/fisiología , Nefrosis/patología , Nefrosis/fisiopatología , Animales , Animales Recién Nacidos , Antibióticos Antineoplásicos , Células Cultivadas , Femenino , Expresión Génica , Uniones Intercelulares/metabolismo , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Nefrosis/inducido químicamente , Puromicina Aminonucleósido , Ratas , Ratas Endogámicas WKY , Ratas WistarRESUMEN
BACKGROUND: Osteopontin (OPN) is a potent chemoattractant for mononuclear cells that is up-regulated in various inflammatory states of the kidney. The role of OPN and its expression in human renal allograft rejection are unknown. METHODS: We examined by immunohistochemistry and in situ hybridization, renal biopsies from patients with acute rejection (N= 22), protocol biopsies without rejection (N= 9), and perioperative donor biopsies (N= 35) for intrarenal expression of OPN, and its correlation with clinical, laboratory, and histopathologic parameters. In the rejection biopsies, interstitial monocyte/macrophage infiltration, tubulointerstitial cell proliferation/regeneration and apoptosis were investigated. RESULTS: In the majority of rejection biopsies, OPN expression by proximal tubular epithelium was widespread, and tended to be enhanced in the tubules surrounded by numerous inflammatory cells. Conversely, in patients that did not experience episodes of rejection and in donor biopsies, OPN expression by proximal tubules was nil or weak. OPN mRNA was colocalized with its translated protein in the renal tubular epithelium. OPN expression positively correlated with the degree of interstitial inflammation (P < 0.05), CD68+ monocyte infiltration (P < 0.01), Ki-67+ regenerating tubular and interstitial cells (P < 0.05 and P < 0.005, respectively), but not with terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)-positive apoptotic tubular cells. CONCLUSION: These data suggest that inducible expression of OPN in the tubular epithelium may have a pathogenic role in acute renal allograft rejection by mediating interstitial monocyte infiltration and possibly tubular regeneration.
Asunto(s)
Rechazo de Injerto/etiología , Trasplante de Riñón , Sialoglicoproteínas/fisiología , Enfermedad Aguda , Adulto , Anciano , Apoptosis , Femenino , Rechazo de Injerto/metabolismo , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Riñón/metabolismo , Riñón/patología , Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Osteopontina , ARN Mensajero/análisis , Sialoglicoproteínas/análisis , Sialoglicoproteínas/genética , Trasplante HomólogoRESUMEN
Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8(+) T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fcgamma receptors (FcgammaR) in leukocytes recruitment we examined the expression of FcgammaR in glomeruli and the effects of the administration of F(ab')(2) fragment of anti-GBM antibody or FcgammaR blocking on the initiation and progression of this model. A gradual increase of FcgammaR mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')(2) fragment. In vivo blocking of FcgammaR by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of FcgammaR (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry FcgammaR2-expressing cells in glomeruli were identified as macrophages, but not CD8(+) T cells. The expression of FcgammaR1 and 3 was significantly decreased, and that of FcgammaR2 became undetectable in CD8(+) T cell-depleted rats. Thus, CD8(+) T cells may stimulate FcgammaR expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG Fc-FcgammaR interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.
Asunto(s)
Autoanticuerpos/inmunología , Glomerulonefritis Membranosa/inmunología , Macrófagos/inmunología , Receptores de IgG/inmunología , Animales , Membrana Basal/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/ultraestructura , Modelos Animales de Enfermedad , Citometría de Flujo , Expresión Génica , Glomerulonefritis Membranosa/etiología , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Glomérulos Renales/ultraestructura , Depleción Linfocítica , Macrófagos/ultraestructura , Microscopía Fluorescente , Proteinuria/inmunología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas WKY , Receptores de IgG/genéticaRESUMEN
BACKGROUND: Investigated were effects of overexpression of interleukin-10 (IL-10) on the outcome and progression of crescentic glomerulonephritis in Wistar-Kyoto (WKY) rats. METHODS: Rats were singly or simultaneously injected with antiglomerular basement membrane (a-GBM) antibody and adenoviral vector encoding rat IL-10 (Ad-rIL-10) or LacZ (Ad-LacZ) (3 x 1010 pfu/rat) intravenously, and were sacrificed at day 7. Their kidneys and other organs were isolated and examined by histology and immunohistochemistry. The In vivo expression of IL-10 mRNA in the liver of Ad-rIL-10-injected rats was confirmed by both reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay analysis and its translated protein was measured in the serum by enzyme-linked immunosorbent assay (ELISA). RESULTS: The exogenous IL-10 mRNA was strongly expressed in the liver in a dose-dependent manner and was intense at days 4 and 7 but was less intense at day 14. Ad-rIL-10 treatment significantly reduced the incidence of glomerular crescent formation from 67%+/- 1.9% in a-GBM antibody-treated group or 69.8%+/- 1.9% in a-GBM antibody + Ad-LacZ-treated group to 21.6%+/- 1.8% (P < 0.001), the glomerular infiltration of macrophages from 35.7 +/- 6.3 cell s/gcs (a-GBM antibody) or 37.6 +/- 8.6 cells/gcs (both a-GBM antibody + Ad-LacZ) to 17.9 +/- 5.5 cells/gcs (P < 0.001), that of major histocompatibility complex (MHC) class II-positive cells from 14.4 +/- 5.3 cells/gcs (a-GBM antibody) or 15 +/- 4.6 cells/gcs (a-GBM antibody + Ad-LacZ) to 5.7 +/- 2.3 cells/gcs (P < 0.0001) at day 7, the glomerular and immune tissue expression of IL-1beta mRNA, as well as the proteinuria from 159.0 +/- 22.7 mg/24 hours (a-GBM antibody) or 166 +/- 28 mg/24 hours (a-GBM antibody + Ad-LacZ) to 42.2 +/- 35.2 mg/24 hours (P < 0.01) at day 7. The serum creatinine and blood urea nitrogen levels were also reduced from 2.8 +/- 0.1 mg/dL (a-GBM antibody) or 2.8 +/- 0.1 mg/dL (a-GBM antibody + Ad-LacZ) to 1.0 +/- 0.1 mg/dL (P < 0.001) and from 63.2 +/- 8.9 mg/dL (a-GBM antibody) or 61.3 +/- 5.2 mg/dL (a-GBM antibody + Ad-LacZ) to 27.0 +/- 4.5 mg/dL (P < 0.001), respectively. However, the glomerular accumulation of CD8+ T cells was unaffected: 5.4 +/- 1.1 cells/gcs (a-GBM antibody + Ad-rIL-10), 5.9 +/- 1.5 cells/gcs (a-GBM antibody), and 5.8 +/- 1.1 cells/gcs (a-GBM antibody + Ad-LacZ) (P= NS). CONCLUSION: IL-10 gene transfer significantly attenuated the glomerular lesions and injury in the anti-GBM crescentic glomerulonephritis of WKY rats.