RESUMEN
Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF). Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-ß1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses. Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-ß1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein. Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/genética , Riñón/patología , Proteínas Klotho/deficiencia , Nefritis Intersticial/enzimología , Obstrucción Ureteral/enzimología , Animales , Modelos Animales de Enfermedad , Fibrosis , Humanos , Hidrazonas/administración & dosificación , Hidroxiurea/administración & dosificación , Hidroxiurea/análogos & derivados , Inyecciones Intraperitoneales , Túbulos Renales/citología , Proteínas Klotho/efectos de los fármacos , RatonesRESUMEN
Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.
Asunto(s)
Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Riñón/enzimología , Nefritis Intersticial/enzimología , Peroxidasa/metabolismo , Peroxidasas/metabolismo , Obstrucción Ureteral/enzimología , Animales , Movimiento Celular , Modelos Animales de Enfermedad , Peroxidasa del Eosinófilo/deficiencia , Peroxidasa del Eosinófilo/genética , Eosinófilos/patología , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Femenino , Fibrosis , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis Intersticial/etiología , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasas/deficiencia , Peroxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/patología , PeroxidasinaRESUMEN
Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the in vivo significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of Sphk2-/- mice showed reduced renal damage and diminished levels of the renal injury markers TGFß1 and αSMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed Sphk2-/- kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNFα, CXCL1 and ILß1, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in Sphk2-/- bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.
Asunto(s)
Macrófagos/fisiología , Nefritis Intersticial/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Actinas/biosíntesis , Actinas/genética , Animales , Microambiente Celular , Citocinas/biosíntesis , Citocinas/genética , Fibrosis , Regulación de la Expresión Génica/inmunología , Glucólisis , Riñón/enzimología , Riñón/patología , Lisofosfolípidos/sangre , Lisofosfolípidos/fisiología , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Nefritis Intersticial/etiología , Nefritis Intersticial/inmunología , Nefritis Intersticial/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Isoformas de Proteínas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Esfingosina/análogos & derivados , Esfingosina/sangre , Esfingosina/fisiología , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Crecimiento Transformador beta1/genética , Obstrucción Ureteral/complicacionesRESUMEN
SUV39H1, the histone methyltransferase (HMTase) of histone H3 lysine 9 trimethylation (H3K9me3), is a known transcriptional repressor of inflammatory genes. The effect of SUV39H1 on inflammatory gene promoters under high-glucose stimulation in vascular smooth muscle cells (VSMCs), macrophages, and cardiomyocytes has been studied, but how SUV39H1 functions in renal tubules under diabetic conditions is unclear. Renal biopsy specimens of ten diabetic nephropathy (DN) subjects and seven non-DN minimal change diseases (MCD) subjects were collected. SUV39H1, IL-6, and MCP-1 expression in renal tissues were measured using immunohistochemical, while SUV39H1, H3K9me3, IL-6, and MCP-1 in human proximal tubular epithelial cells (HK-2) under varying glucose conditions were assayed by Western blot and ELISA. SUV39H1 was overexpressed in HK-2 cells; the regulation of SUV39H1 and H3K9me3 on NF-κB, IL-6, MCP-1, caspase 3, and apoptosis was measured. SUV39H1 was expressed more in diabetic human renal tubules. HK-2 cells with high glucose up-regulated IL-6 and MCP-1 in a dose- and time-dependent manner, and SUV39H1 expression was reduced with greater glucose and prolonged stimulation. Expression of H3K9me3 was synchronized with SUV39H1. Moreover, overexpression of SUV39H1 in high glucose environment was accompanied with increased H3K9me3 and decreased inflammation and apoptosis. SUV39H1 dysregulation may be involved in DN progression. Overexpression of SUV39H1 may reduce renal inflammation and apoptosis via epigenetic modulation, thus plays a protective role in DN.
Asunto(s)
Glucemia/metabolismo , Nefropatías Diabéticas/enzimología , Células Epiteliales/enzimología , Túbulos Renales/enzimología , Metiltransferasas/metabolismo , Nefritis Intersticial/enzimología , Proteínas Represoras/metabolismo , Adulto , Apoptosis , Estudios de Casos y Controles , Caspasa 3/metabolismo , Línea Celular , Microambiente Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Epigénesis Genética , Células Epiteliales/patología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Túbulos Renales/patología , Masculino , Metiltransferasas/genética , Persona de Mediana Edad , FN-kappa B/metabolismo , Nefritis Intersticial/sangre , Nefritis Intersticial/genética , Nefritis Intersticial/patología , Proteínas Represoras/genética , Factores de TiempoRESUMEN
The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.
Asunto(s)
Daño del ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Nefritis Intersticial/enzimología , Nefritis Intersticial/genética , Poliploidía , Animales , Células Cultivadas , Reparación del ADN/genética , Desoxirribonucleasas/metabolismo , Exodesoxirribonucleasas , Técnicas de Sustitución del Gen , Riñón/patología , Ratones , Enzimas Multifuncionales , Nefritis Intersticial/fisiopatologíaRESUMEN
Karyomegalic interstitial nephritis (KIN) is a chronic interstitial nephropathy characterized by tubulointerstitial nephritis and formation of enlarged nuclei in the kidneys and other tissues. We recently reported that recessive mutations in the gene encoding FANCD2/FANCI-associated nuclease 1 (FAN1) cause KIN in humans. FAN1 is a major component of the Fanconi anemia-related pathway of DNA damage response (DDR) signaling. To study the pathogenesis of KIN, we generated a Fan1 knockout mouse model, with abrogation of Fan1 expression confirmed by quantitative RT-PCR. Challenging Fan1-/- and wild-type mice with 20 mg/kg cisplatin caused AKI in both genotypes. In contrast, chronic injection of cisplatin at 2 mg/kg induced KIN that led to renal failure within 5 weeks in Fan1-/- mice but not in wild-type mice. Cell culture studies showed decreased survival and reduced colony formation of Fan1-/- mouse embryonic fibroblasts and bone marrow mesenchymal stem cells compared with wild-type counterparts in response to treatment with genotoxic agents, suggesting that FAN1 mutations cause chemosensitivity and bone marrow failure. Our data show that Fan1 is involved in the physiologic response of kidney tubular cells to DNA damage, which contributes to the pathogenesis of CKD. Moreover, Fan1-/- mice provide a new model with which to study the pathomechanisms of CKD.
Asunto(s)
Endodesoxirribonucleasas/genética , Mutación , Nefritis Intersticial/enzimología , Nefritis Intersticial/genética , Animales , Modelos Animales de Enfermedad , Exodesoxirribonucleasas , Ratones , Ratones Noqueados , Enzimas Multifuncionales , Insuficiencia Renal Crónica/etiologíaRESUMEN
AIM: In addition to lowering blood glucose in patients with type 2 diabetes mellitus, dipeptidyl peptidase 4 (DPP4) inhibitors have been shown to be antifibrotic and anti-inflammatory. We have previously shown that DPP4 inhibition in human kidney proximal tubular cells exposed to high glucose reduced fibrotic and inflammatory markers. Hence, we wanted to demonstrate renoprotection in an in vivo model. METHODS: We used a type 1 diabetic animal model to explore the renoprotective potential of saxagliptin independent of glucose lowering. We induced diabetes in enos -/- mice using streptozotocin and matched glucose levels using insulin. Diabetic mice were treated with saxagliptin and outcomes compared with untreated diabetic mice. RESULTS: We provide novel data that saxagliptin limits renal hypertrophy, transforming growth factor beta-related fibrosis and NF-κBp65-mediated macrophage infiltration. Overall, there was a reduction in histological markers of tubulointerstitial fibrosis. There was no reduction in albuminuria or glomerulosclerosis. CONCLUSION: Our findings highlight the potential of DPP4 inhibition as additional therapy in addressing the multiple pathways to achieve renoprotection in diabetic nephropathy.
Asunto(s)
Adamantano/análogos & derivados , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Nefropatías Diabéticas/prevención & control , Dipéptidos/farmacología , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Riñón/efectos de los fármacos , Nefritis Intersticial/prevención & control , Adamantano/farmacología , Albuminuria/enzimología , Albuminuria/prevención & control , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/genética , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Fibronectinas/metabolismo , Fibrosis , Glomerulonefritis/enzimología , Glomerulonefritis/prevención & control , Hipertrofia , Insulina/sangre , Riñón/enzimología , Riñón/patología , Masculino , Ratones Noqueados , Nefritis Intersticial/enzimología , Nefritis Intersticial/genética , Nefritis Intersticial/patología , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo III/genética , Fosforilación , Transducción de Señal/efectos de los fármacos , Proteína Smad2 , Proteína smad3/metabolismo , Estreptozocina , Factor de Transcripción ReIA/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
TGFß1/Smad, Wnt/ß-catenin and snail1 are preferentially activated in renal tubular epithelia after injury, leading to epithelial-mesenchymal transition (EMT). The stress response is coupled to EMT and kidney injury; however, the underlying mechanism of the stress response in EMT remains elusive. AMP-activated protein kinase (AMPK) signalling is responsive to stress and regulates cell energy balance and differentiation. We found that knockdown of AMPKα, especially AMPKα2, enhanced EMT by up-regulating ß-catenin and Smad3 in vitro. AMPKα2 deficiency enhanced EMT and fibrosis in a murine unilateral ureteral obstruction (UUO) model. AMPKα2 deficiency also increased the expression of chemokines KC and MCP-1, along with enhanced infiltration of inflammatory cells into the kidney after UUO. CK2ß interacted physically with AMPKα and enhanced AMPKα Thr172 phosphorylation and its catalytic activity. Thus, activated AMPKα signalling suppresses EMT and secretion of chemokines in renal tubular epithelia through interaction with CK2ß to attenuate renal injury.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Quinasa de la Caseína II/metabolismo , Transdiferenciación Celular , Células Epiteliales/enzimología , Transición Epitelial-Mesenquimal , Túbulos Renales Proximales/enzimología , Nefritis Intersticial/prevención & control , Proteínas Quinasas Activadas por AMP/deficiencia , Proteínas Quinasas Activadas por AMP/genética , Animales , Quinasa de la Caseína II/genética , Línea Celular , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Células Epiteliales/inmunología , Células Epiteliales/patología , Fibrosis , Humanos , Mediadores de Inflamación/metabolismo , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis Intersticial/enzimología , Nefritis Intersticial/genética , Nefritis Intersticial/inmunología , Nefritis Intersticial/patología , Unión Proteica , Interferencia de ARN , Proteína smad3/metabolismo , Transfección , Obstrucción Ureteral/complicaciones , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
AIM: FTY720, a new immunomodulatory drug with low cytotoxicity, is currently used to treat multiple sclerosis. In this study, we investigated the effects of FTY720 on inflammatory cell infiltration in albumin overload-induced nephropathy of rats. METHODS: Male Wistar rats were subjected to right-side nephrectomy and divided into 3 groups. One week after the surgery, albumin overload (AO) group was treated with BSA (5 g·kg(-1)·d(-1), ip) for 9 weeks; AO+FTY720 group was given BSA (5 g·kg(-1)·d(-1), ip) plus FTY720 (0.5 g·kg(-1)·d(-1), ip) for 9 weeks; and control group received daily ip injection of equivalent volume of saline. All rats were killed 9 weeks after nephrectomy. RESULTS: AO rats exhibited gradually increased urinary protein excretion accompanied by elevated urinary N-acetyl-ß-O-glucosaminidase activity, and both reached their peak values at week 7. Furthermore, AO significantly increased lymphocytes and monocytes in circulation and the inflammatory cells recruited to tubulointerstitium, and the expression of inflammatory cytokines MCP-1, TNF-α and IL-6, as well as sphingosine 1-phosphate (S1P) receptors S1pr1 and S1pr3, and S1P-synthesizing enzyme sphingosine kinase 1 (Sphk1) in the kidney. Concomitant administration of FTY720 significantly attenuated all the AO-induced pathological changes. CONCLUSION: FTY720 alleviates tubulointerstitium inflammation in an AO rat model of nephropathy via down-regulation of the Sphk1 pathway.
Asunto(s)
Albuminuria/tratamiento farmacológico , Antiinflamatorios/farmacología , Inmunosupresores/farmacología , Túbulos Renales/efectos de los fármacos , Nefritis Intersticial/prevención & control , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Acetilglucosaminidasa/orina , Albuminuria/enzimología , Albuminuria/patología , Albuminuria/orina , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Clorhidrato de Fingolimod , Mediadores de Inflamación/metabolismo , Túbulos Renales/enzimología , Túbulos Renales/patología , Linfocitos/efectos de los fármacos , Linfocitos/enzimología , Lisofosfolípidos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Nefritis Intersticial/enzimología , Nefritis Intersticial/patología , Nefritis Intersticial/orina , Ratas Wistar , Receptores de Lisoesfingolípidos/efectos de los fármacos , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/metabolismo , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato , Factores de TiempoRESUMEN
An autoantibody against carbonic anhydrase II was identified in a case of acute tubulointerstitial nephritis induced by famotidine, which inhibits carbonic anhydrase II in addition to the gastric proton pump. The patient's serum reacted with distal nephron homogenates at the same molecular weight as purified carbonic anhydrase II, and immunohistochemistry using the patient's serum showed staining at the distal nephron. Carbonic anhydrase II may be a causative antigen in the famotidine-induced acute tubulointerstitial nephritis.
Asunto(s)
Autoanticuerpos/biosíntesis , Anhidrasa Carbónica II/inmunología , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/enzimología , Autoanticuerpos/sangre , Femenino , Humanos , Persona de Mediana Edad , Nefritis Intersticial/inmunologíaRESUMEN
OBJECTIVES: Excess mineralocorticoids such as deoxycorticosterone acetate (DOCA) together with salt are known to cause tubulointerstitial fibrosis, but the mechanisms underlying fibrosis progression are unclear. Therefore, we investigated the role of matrix metalloproteinase 2 (MMP2) in the epithelial-mesenchymal transition and fibrosis progression. METHODS: Uninephrectomized rats drank 0.9% NaCl and 0.3% KCl solution and were treated with DOCA alone, DOCA + spironolactone, or vehicle for 1, 4, or 8 weeks. SBP, kidney function and morphology, and kidney and urine MMP2 activity were compared among the groups. RESULTS: At week 4, the DOCA-treated group exhibited hypertension, tubulointerstitial fibrosis, increased MMP2 activity in the kidney and urine, and overexpression of MMP2 in proximal tubule cells and MMP14 in apical membranes; these results were more pronounced at week 8. At week 8, the proximal tubule cell apicolateral surface proteins villin, claudin 2, and E-cadherin were downregulated, and the mesenchymal marker α-smooth muscle actin was upregulated in the tubulointerstitium of DOCA-treated rats. These DOCA/salt-induced changes (except for hypertension) and fibrosis progression observed at week 8 were reversed by TISAM (a selective MMP2 inhibitor), which was administered from week 4 to week 8. All of the effects of DOCA/salt at week 8 were attenuated by spironolactone. CONCLUSION: Eight weeks of treatment with DOCA/salt activated MMP2, primarily on the apical surface of proximal tubule cells, which induced epithelial-mesenchymal transition from the luminal side and promoted tubulointerstitial fibrosis progression. These MMP2-induced changes occurred via downstream processes regulated by mineralocorticoid receptors.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Fibrosis/patología , Hipertensión/patología , Túbulos Renales Proximales/patología , Metaloproteinasa 2 de la Matriz/metabolismo , Cloruro de Sodio/farmacología , Animales , Colágeno/metabolismo , Desoxicorticosterona/farmacología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Quimioterapia Combinada , Fibrosis/inducido químicamente , Fibrosis/enzimología , Hipertensión/inducido químicamente , Hipertensión/enzimología , Riñón/metabolismo , Riñón/patología , Túbulos Renales Proximales/enzimología , Masculino , Nefrectomía , Nefritis Intersticial/inducido químicamente , Nefritis Intersticial/enzimología , Nefritis Intersticial/patología , Ratas , Ratas Sprague-Dawley , Espironolactona/farmacologíaRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) contributes to necrotic cell death and inflammation in several disease models; however, the role of PARP1 in fibrogenesis remains to be defined. Here, we tested whether PARP1 was involved in the pathogenesis of renal fibrosis using the unilateral ureteral obstruction (UUO) mouse model. UUO was performed by ligation of the left ureter near the renal pelvis in Parp1-knockout (KO) and wild-type (WT) male mice. After 10 days of UUO, renal PARP1 expression and activation were strongly increased by 6- and 13-fold, respectively. Interstitial fibrosis induced by UUO was significantly attenuated in Parp1-KO kidneys compared with that in WT kidneys at 10 days, but not at 3 days, based on collagen deposition, α-smooth muscle actin (α-SMA), and fibronectin expression. Intriguingly, the UUO kidneys in Parp1-KO mice showed a dramatic decrease in infiltration of neutrophil and reduction in expression of proinflammatory proteins including intercellular adhesion molecule-1, tumor necrosis factor-α, inducible nitric oxide synthase, and toll-like receptor 4 as well as phosphorylation of nuclear factor-κB p65, but not transforming growth factor-ß1 (TGF-ß1) at both 3 and 10 days. Pharmacological inhibition of PARP1 in rat renal interstitial fibroblast (NRK-49F) cell line or genetic ablation in primary mouse embryonic fibroblast cells did not affect TGF-ß1-induced de novo α-SMA expression. Parp1 deficiency significantly attenuated UUO-induced histological damage in the kidney tubular cells, but not apoptosis. These data suggest that PARP1 induces necrotic cell death and contributes to inflammatory signaling pathways that trigger fibrogenesis in obstructive nephropathy.
Asunto(s)
Riñón/enzimología , Riñón/patología , Nefritis Intersticial/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Obstrucción Ureteral/enzimología , Actinas/metabolismo , Animales , Apoptosis , Fibrosis , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Necrosis , Nefritis Intersticial/patología , Poli(ADP-Ribosa) Polimerasa-1 , Factor de Crecimiento Transformador beta1/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Histone deacetylase (HDAC) regulates gene expression by modifying chromatin structure. Although changes in the expression and activities of HDAC may affect the course of kidney disease, the role of HDAC in tubulointerstitial injury has not been explored. We therefore investigated the alterations in HDAC expression and determined the effects of HDAC inhibition on the tubulointerstitial injury induced by unilateral ureteral obstruction. The induction of HDAC1 and HDAC2, accompanied by a decrease in histone acetylation was observed in kidneys injured by ureteral obstruction. Immunohistochemical analysis revealed that HDAC1 and HDAC2 were induced in renal tubular cells. Treatment with an HDAC inhibitor, trichostatin A (TSA), attenuated macrophage infiltration and fibrotic changes in tubulointerstitial injury induced by ureteral obstruction. The induction of colony-stimulating factor-1 (CSF-1), a chemokine known to be involved in macrophage infiltration in tubulointerstitial injury, was reduced in injured kidneys from mice treated with TSA. TSA, valproate, and the knockdown of HDAC1 or HDAC2 significantly reduced CSF-1 induced by TNF-alpha in renal tubular cells. These results suggest that tubular HDAC1 and HDAC2, induced in response to injury, may contribute to the induction of CSF-1 and the initiation of macrophage infiltration and profibrotic responses. These findings suggest a potential of HDAC inhibition therapy aimed at reducing inflammation and fibrosis in tubulointerstitial injury.
Asunto(s)
Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Túbulos Renales/enzimología , Túbulos Renales/patología , Nefritis Intersticial/enzimología , Nefritis Intersticial/patología , Acetilación , Animales , Modelos Animales de Enfermedad , Fibrosis , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Ácidos Hidroxámicos/farmacología , Túbulos Renales/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nefritis Intersticial/etiología , Obstrucción Ureteral/complicacionesRESUMEN
The aim of the study was to evaluate tubulointerstitial immunoexpression of MMPs and TIMPs in chronic renal allograft injury, and to assess any relationships between immunoexpression of MMPs and interstitial monocytes/ macrophages and mast cells. Immunohistochemistry with antibodies against MMP-2, MMP-2, MMP-12, TIMP-1, TIMP-2, CD68, and mast cells tryptase was carried out on 17 renal biopsy specimens from patients with chronic allograft injury, and on 11 control kidney tissues. In renal specimens in chronic allograft injury increased expression of MMP-2, MMP-12 and TIMP-1 was noted. The immunoexpression of MMP-9 and TIMP-2 was low, and did not differ in comparison with controls. Increased immunoexpression of MMP-12 was positively correlated with the number of interstitial CD68 + cells. The correlation between immunostaining of MMP-12 and mast cells tended to be positive, however it did not reach statistical significance. There were significant positive correlations between immunostaining of MMP-9 and CD68+ cells, as well as between MMP-9 and the number of mast cells. In conclusion our study revealed that the remodelling of kidney structure in patients with chronic allograft injury is associated with dysregulation of MMPs and TIMPs, and may suggest that interstitial monocytes/macrophages and mast cells may cooperate with MMPs in pathogenesis of renal fibrosis.
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Rechazo de Injerto/enzimología , Trasplante de Riñón , Metaloproteinasas de la Matriz/metabolismo , Adulto , Biomarcadores/metabolismo , Biopsia , Enfermedad Crónica , Femenino , Rechazo de Injerto/patología , Humanos , Técnicas para Inmunoenzimas , Riñón , Túbulos Renales/enzimología , Túbulos Renales/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Mastocitos/metabolismo , Mastocitos/patología , Monocitos/metabolismo , Monocitos/patología , Nefritis Intersticial/enzimología , Nefritis Intersticial/patología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
Immunoglobulin A (IgA) nephropathy is an important cause of end-stage kidney disease (ESKD). Tubulointerstitial inflammation and subsequent fibrosis appear to be a major contributor of the disease progression to ESKD; however, the underlying mechanism is poorly understood. Herein, we report that a unique feature of CYLD expression in kidneys of patients with IgA nephropathy and a CYLD-mediated negative regulation of inflammatory responses in human tubular epithelial cells. Immunochemical staining revealed that CYLD was predominantly expressed in renal tubular epithelial cells in 81% of the patients (37 cases) with proteinuric IgA nephropathy. Patients with positive CYLD had significantly less tubulointerstitial lesions and higher estimated glomerular filtration rate (eGFR) levels when compared with those negative. Logistic regression analysis indicated that eGFR was a predictor for the CYLD expression. In cultured human tubular epithelial HK-2 cells, tumor necrosis factor-alpha (TNFalpha) up-regulated CYLD expression. Adenoviral knockdown of CYLD did not affect albumin-, hydrogen peroxide (H(2)O(2))-, tunicamycin- or thapsigargin-induced cell death; however, it enhanced TNFalpha-induced expression of intracellular adhesion molecule (ICAM)-1 as well as activation of c-Jun N-terminal kinase (JNK). Moreover, monocyte adhesion to the TNFalpha-inflamed HK-2 cells was significantly increased by the CYLD shRNA approach. Taken together, our results suggest that CYLD negatively regulates tubulointertitial inflammatory responses via suppressing activation of JNK in tubular epithelial cells, putatively attenuating the progressive tubulointerstitial lesions in IgA nephropathy.
Asunto(s)
Glomerulonefritis por IGA/enzimología , Túbulos Renales/enzimología , Nefritis Intersticial/enzimología , Proteínas Supresoras de Tumor/fisiología , Adhesión Celular , Enzima Desubiquitinante CYLD , Técnicas de Silenciamiento del Gen , Glomerulonefritis por IGA/genética , Glomerulonefritis por IGA/patología , Humanos , Túbulos Renales/patología , MAP Quinasa Quinasa 4/metabolismo , Monocitos/enzimología , Monocitos/fisiología , Nefritis Intersticial/genética , Nefritis Intersticial/patología , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Chronic inflammation is a major outcome determinant in several renal disorders. Induction of monocyte chemoattractant protein (MCP)-1 expression in tubular epithelial cells contributes importantly to the recruitment of inflammatory cells from the circulation toward the damaged tubulo-interstitium. Because the MCP-1 gene contains several c-Jun binding sites, we hypothesized that the c-Jun NH(2)-terminal kinase (JNK) pathway regulates MCP-1 expression and subsequently tubulo-interstitial inflammation. This was investigated in cultured rat tubular epithelial cells (NRK-52E) and in the rat unilateral ischemia/reperfusion (I/R) model. In NRK-52E cells, the JNK inhibitor anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125) reduced interleukin-1beta-, transforming growth factor-beta-, or bovine serum albumin-induced MCP-1 expression in a potent manner (up to 150-fold). In the rat I/R model, JNK activation was low in controls but induced in tubular cells from 30 min after I/R. The extent of JNK activation correlated with interstitial macrophage accumulation. Treatment with SP600125 (30 mg/kg/day i.p. for 4 days) reduced renal c-Jun activation; MCP-1, osteopontin, and vimentin expression; and interstitial macrophage and T-cell accumulation (all p < 0.05). In human renal disease, we also found induction of JNK activation, which correlated strongly with interstitial macrophage accumulation, tubulointerstitial fibrosis, and renal function loss. In conclusion, these data indicate that the JNK pathway plays an important role in renal inflammation, at least in part through induction of MCP-1 gene expression in tubular epithelial cells.
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Inflamación/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Túbulos Renales/enzimología , Nefritis Intersticial/enzimología , Adolescente , Adulto , Animales , Antracenos/farmacología , Western Blotting , Línea Celular , Quimiocina CCL2/genética , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Activación Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/inmunología , Células Epiteliales/patología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/patología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Enfermedades Renales/enzimología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/inmunología , Túbulos Renales/patología , Macrófagos/citología , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Nefritis Intersticial/inmunología , Nefritis Intersticial/patología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Daño por Reperfusión/enzimología , Daño por Reperfusión/inmunología , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Linfocitos T/inmunología , Adulto JovenRESUMEN
Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.
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Riñón/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nefritis Intersticial/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Obstrucción Ureteral/enzimología , Animales , Apoptosis , Activación Enzimática , Fibrosis , Riñón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Nefritis Intersticial/etiología , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/patologíaRESUMEN
OBJECTIVE: The present study investigated the relationships between reactive oxygen species (ROS), interstitial fibrosis, and renal proximal tubular cell (RPTC) apoptosis in type 2 diabetic db/db mice and in db/db transgenic (Tg) mice overexpressing rat catalase (rCAT) in their RPTCs (db/db rCAT-Tg). RESEARCH DESIGN AND METHODS: Blood pressure, blood glucose, and albuminuria were monitored for up to 5 months. Kidneys were processed for histology and apoptosis studies (terminal transferase-mediated dUTP nick-end labeling or immunostaining for active caspase-3 and Bax). Real-time quantitative PCR assays were used to quantify angiotensinogen (ANG), p53, and Bax mRNA levels. RESULTS: db/db mice developed obesity, hyperglycemia, hypertension, and albuminuria. In contrast, db/db rCAT-Tg mice became obese and hyperglycemic but had normal blood pressure and attenuated albuminuria compared with db/db mice. Kidneys from db/db mice displayed progressive glomerular hypertrophy, glomerulosclerosis, interstitial fibrosis, and tubular apoptosis and increased expression of collagen type IV, Bax, and active caspase-3, as well as increased ROS production. These changes, except glomerular hypertrophy, were markedly attenuated in kidneys of db/db rCAT-Tg mice. Furthermore, ANG, p53, and Bax mRNA expression was increased in renal proximal tubules of db/db mice but not of db/db rCAT-Tg mice. CONCLUSIONS: Our results indicate a crucial role for intra-renal ROS in the progression of hypertension, albuminuria, interstitial fibrosis, and tubular apoptosis in type 2 diabetes and demonstrate the beneficial effects of suppressing ROS formation.
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Catalasa/genética , Diabetes Mellitus Tipo 2/fisiopatología , Túbulos Renales Proximales/enzimología , Nefritis Intersticial/genética , Animales , Apoptosis , Catalasa/metabolismo , Bovinos , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Nefropatías Diabéticas , Etiquetado Corte-Fin in Situ , Túbulos Renales Proximales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nefritis Intersticial/enzimología , Obesidad/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Accumulating evidence suggests a pathogenic role of chronic hypoxia in various kidney diseases. Chronic hypoxia in the kidney was induced by unilateral renal artery stenosis, followed 7 days later by observation of tubulointerstitial injury. Proteomic analysis of the hypoxic kidney found various altered proteins. Increased proteins included lipocortin-5, calgizzarin, ezrin, and transferrin, whereas the decreased proteins were alpha(2u)-globulin PGCL1, eukaryotic translation elongation factor 1alpha(2), and Cu/Zn superoxide dismutase (SOD1). Among these proteins, we focused on Cu/Zn-SOD, a crucial antioxidant. Western blot analysis and real-time quantitative PCR analysis confirmed the downregulation of Cu/Zn-SOD in the chronic hypoxic kidney. Furthermore, our laser capture microdissection system showed that the expression of Cu/Zn-SOD was predominant in the tubulointerstitium and was decreased by chronic hypoxia. The tubulointerstitial injury estimated by histology and immunohistochemical markers was ameliorated by tempol, a SOD mimetic. This amelioration was associated with a decrease in levels of the oxidative stress markers 4-hydroxyl-2-nonenal and nitrotyrosine. Our in vitro studies utilizing cultured tubular cells revealed a role of TNF-alpha in downregulation of Cu/Zn-SOD. Since the administration of anti-TNF-alpha antibody ameliorated Cu/Zn-SOD suppression, TNF-alpha seems to be one of the suppressants of Cu/Zn-SOD. In conclusion, our proteomic analysis revealed a decrease in Cu/Zn-SOD, at least partly by TNF-alpha, in the chronic hypoxic kidney. This study, for the first time, uncovered maladaptive suppression of Cu/Zn-SOD as a mediator of a vicious cycle of oxidative stress and subsequent renal injury induced by chronic hypoxia.