RESUMO
S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.
Assuntos
Adenina , Insuficiência Renal Crônica , Adenina/efeitos adversos , Animais , Fibrose , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência Renal Crônica/patologia , Esfingosina/farmacologia , Receptores de Esfingosina-1-FosfatoRESUMO
OBJECTIVE: End-stage renal disease associates with catabolism and sarcopenia. Hypothetically, peroral supplemental nutrition over 6 months prevents catabolism in hemodialysis patients. DESIGN: Prospective randomized pilot study (ClinicalTrials.gov Identifier: NCT00687050). SUBJECTS: Twenty-three hemodialysis patients (15 males and 7 females) with or without human immunodeficiency virus (HIV) infection of 2 ambulatory hemodialysis centers. INTERVENTION: HIV-positive hemodialysis patients (n = 7, Group 1) were started on supplemental nutrition drinks (250 kcal/day), HIV-negative hemodialysis patients (n = 16, Group 2) were randomized to supplemental nutrition drinks (250 kcal/day) or received none. MAIN OUTCOME MEASURES: Body impedance analysis, anthropometric measures, magnetic resonance imaging results for mid-iliopsoas muscle cross-sectional area and laboratory parameters including albumin, cytokines at baseline, and at 6 months follow-up. RESULTS: Seven patients in Group 1 (mean age: 50.6 ± 9.6 years) and 16 patients in Group 2 (mean age: 54.0 ± 13.3 years) were recruited. Serum creatinine (Group 1: 6.4 ± 3.0 mg/dL; Group 2: 10.7 ± 2.5 mg/dL; P < .01), Body impedance analysis-derived phase angle alpha (Group 1: 5.1 ± 1.2; Group 2: 6.9 ± 1.6; P < .01), mid-arm circumference (Group 1: 26.1 ± 1.3 cm; Group 2: 29.6 ± 2.4 cm; P < .01) were less in Group 1 versus Group 2 patients at baseline suggesting that HIV-positive hemodialysis patients had a poorer nutritional status at baseline. At 6-month follow-up, mortality was higher in Group 1 patients (29%) than in Group 2 patients (6%). There was no significant treatment effect on nutritional status in survivors of Group 1 or in the supplemental nutrition arm of Group 2 when compared with baseline or to untreated controls. CONCLUSIONS: A new oral supplemental nutrition over 6 months had no treatment effect in surviving HIV-positive hemodialysis patients or in maintenance hemodialysis patients without HIV infection. The limitations of this study were small study size and unexpected high mortality among HIV-positive hemodialysis patients.
Assuntos
Caquexia/prevenção & controle , Infecções por HIV/terapia , Falência Renal Crônica/terapia , Apoio Nutricional , Diálise Renal/efeitos adversos , Adolescente , Adulto , Idoso , Composição Corporal , Proteína C-Reativa/metabolismo , Caquexia/complicações , Impedância Elétrica , Feminino , Seguimentos , Infecções por HIV/complicações , Humanos , Interleucina-1beta/sangue , Interleucina-6/sangue , Falência Renal Crônica/complicações , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Avaliação Nutricional , Estado Nutricional , Projetos Piloto , Estudos Prospectivos , Sarcopenia/complicações , Sarcopenia/prevenção & controle , Albumina Sérica/metabolismo , Fator de Necrose Tumoral alfa/sangue , Adulto JovemRESUMO
The development of new strategies to preserve renal function after acute kidney injury (AKI) is necessary due to limited clinical intervention options. The organ-protective effects of mesenchymal stromal/stem cells (MSCs) and their conditioned medium (CM) have been investigated demonstrating that both separately promoted tubular recovery and ameliorated the outcome of AKI. Nevertheless, strategies to optimise the regenerative potential of both are highly needed. Here we investigated the effects of CM from adipose-derived MSCs (ASCs) preincubated in a hypoxic environment (Hyp). Protective factors were investigated by PCR analysis and a protein array in vitro. The expression of 64 of the 308 proteins assayed was found to be more than two-fold increased after Hyp. CM of Hyp-pretreated ASCs (pCM) was used to enhance regeneration in a mouse model of cisplatin-induced AKI (cisAKI). Renal function was assessed by measurements of markers for AKI and serum cytokine levels. The pCM significantly ameliorated serum creatinine and neutrophil gelatinase-associated lipocalin values, and also the levels of inflammatory cytokines IL-1ß and IL-6 in the serum of mice with AKI. Our work clearly showed that a Hyp preconditioning significantly increases the release of protective factors in ASCs and enhances the therapeutic effects of CM in cisAKI in mice.
Assuntos
Injúria Renal Aguda/prevenção & controle , Células-Tronco Adultas/transplante , Antineoplásicos/toxicidade , Cisplatino/toxicidade , Injúria Renal Aguda/induzido quimicamente , Tecido Adiposo Branco/patologia , Células-Tronco Adultas/fisiologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultivo Condicionados , Interleucinas/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Clinical relevance of ELISA- and single-antigen bead assay (SAB)-detected pretransplant HLA antibodies (SAB-HLA-Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death-censored graft loss was found in patients with pretransplant SAB-HLA-Ab [HR: 4.46; 95% confidence interval (CI): 1.47-13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB-HLA-Ab but without SAB-detected donor-specific Ab (SAB-DSA) (HR: 4.91; 95% CI of 1.43-16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA-DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long-term graft loss in patients with pretransplant SAB-HLA, even in the absence of DSA. SAB-HLA-Ab-positive patients, being negative in ELISA or CDC assay, might profit from a well-HLA-DR-matched graft and intensified immunosuppression.
Assuntos
Anticorpos/sangue , Sobrevivência de Enxerto , Antígenos HLA/imunologia , Transplante de Rim , Insuficiência Renal/cirurgia , Adulto , Idoso , Biópsia , Função Retardada do Enxerto/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunização , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Proteinúria/sangue , Insuficiência Renal/imunologia , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Doadores de TecidosRESUMO
Expression of kidney injury molecule-1 (Kim-1) is rapidly upregulated following tubular injury, constituting a biomarker for acute kidney damage. We examined the renal localization of Kim-1 expression in PKD/Mhm (polycystic kidney disease, Mannheim) (cy/+) rats (cy: mutated allel, +: wild type allel), an established model for autosomal dominant polycystic kidney disease, with chronic, mainly proximal tubulointerstitial alterations. For immunohistochemistry or Western blot analysis, kidneys of male adult heterozygously-affected (cy/+) and unaffected (+/+) littermates were perfusion-fixed or directly removed. Kim-1 expression was determined using peroxidase- or fluorescence-linked immunohistochemistry (alone or in combination with markers for tubule segments or differentiation). Compared to (+/+), only in (cy/+) kidneys, a chronic expression of Kim-1 could be detected by Western blot analysis, which was histologically confined to an apical cellular localization in areas of cystically-transformed proximal tubules with varying size and morphology, but not in distal tubular segments. Kim-1 was expressed by cystic epithelia exhibiting varying extents of dedifferentiation, as shown by double labeling with aquaporin-1, vimentin or osteopontin, yielding partial cellular coexpression. In this model, in contrast to other known molecules indicating renal injury and/or repair mechanisms, the chronic renal expression of Kim-1 is strictly confined to proximal cysts. Its exact role in interfering with tubulo-interstitial alterations in polycystic kidney disease warrants future investigations.
Assuntos
Moléculas de Adesão Celular/metabolismo , Túbulos Renais Proximais/metabolismo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Animais , Biomarcadores/metabolismo , Desdiferenciação Celular , Modelos Animais de Doenças , Túbulos Renais Proximais/patologia , Masculino , Especificidade de Órgãos , Ratos , Regulação para CimaRESUMO
BACKGROUND: We examined the value of the novel acute kidney injury (AKI) markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in acute postrenal impairment. These biomarkers have been evaluated in prerenal and intrarenal AKI so far, but not in human acute postrenal kidney injury. With regard to multimorbid and critically ill patients the discrimination of different AKI origins often remains a challenge. As the trend goes towards a diagnostic panel of AKI markers, we hereby aim to contribute to evaluate further options of discrimination in an observational case-control study. MATERIALS AND METHODS: Blood and urine samples were obtained from 53 patients with acute obstructive nephropathy secondary to ureteral calculi and 52 age-matched healthy controls. Serum NGAL (sNGAL), urinary NGAL (uNGAL) and urinary KIM-1 (uKIM-1) levels were determined using a commercially available ELISA kit, creatinine applying the Jaffé's method. RESULTS: While urinary levels of KIM-1 were not significantly different between patients with obstructive nephropathy and controls, a striking increase in sNGAL (P < 0·001) and uNGAL (P < 0·01) levels was detected in the obstructive nephropathy group. Within the obstructive nephropathy group, sNGAL (P = 0·01) and uNGAL (P = 0·049) but not uKIM-1 correlated positively with the white blood cell count and uNGAL correlated positively (P = 0·002) with the extent of leucocyturia. CONCLUSIONS: High levels of sNGAL and uNGAL observed in stone-induced acute obstructive nephropathy may represent a valuable marker of postrenal AKI. Low uKIM-1 levels may help to discriminate postrenal AKI events using a panel of markers in this setting.
Assuntos
Injúria Renal Aguda/diagnóstico , Proteínas de Fase Aguda/metabolismo , Lipocalinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Virais/metabolismo , Injúria Renal Aguda/etiologia , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Lipocalina-2 , Masculino , Cólica Renal/etiologia , Cálculos Ureterais/complicações , Obstrução Ureteral/etiologiaRESUMO
Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.
Assuntos
Carcinoma , Neoplasias do Colo , Humanos , RNA Mensageiro/genética , Caspase 7 , Ubiquitina-Proteína Ligases/metabolismo , RNA , Neoplasias do Colo/genética , Linhagem Celular Tumoral , Ubiquitina , Apoptose/genética , Proteínas com Motivo Tripartido/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Several studies revealed alterations of single sphingolipid species, such as chain length-specific ceramides, in plasma and serum of patients with kidney diseases. Here, we investigated whether such alterations occur in kidney tissue from patients and mice suffering from renal fibrosis, the common endpoint of chronic kidney diseases. METHODS: Human fibrotic kidney samples were collected from nephrectomy specimens with hydronephrosis and/or pyelonephritis. Healthy parts from tumor nephrectomies served as nonfibrotic controls. Mouse fibrotic kidney samples were collected from male C57BL/6J mice treated with an adenine-rich diet for 14 days or were subjected to 7 days of unilateral ureteral obstruction (UUO). Kidneys of untreated mice and contralateral kidneys (UUO) served as respective controls. Sphingolipid levels were detected by LC-MS/MS. Fibrotic markers were analyzed by TaqMan® analysis and immunohistology. RESULTS: Very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 were significantly downregulated in both fibrotic human kidney cortex and fibrotic murine kidney compared to respective control samples. These effects correlate with upregulation of COL1α1, COL3α1 and αSMA expression in fibrotic human kidney cortex and fibrotic mouse kidney. CONCLUSION: We have shown that very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 are consistently downregulated in fibrotic kidney samples from human and mouse. Our findings support the use of in vivo murine models as appropriate translational means to understand the involvement of ceramides in human kidney diseases. In addition, our study raises interesting questions about the possible manipulation of ceramide metabolism to prevent progression of fibrosis and the use of ceramides as potential biomarkers of chronic kidney disease.
Assuntos
Ceramidas/metabolismo , Hidronefrose/metabolismo , Pielonefrite/metabolismo , Esfingolipídeos/metabolismo , Obstrução Ureteral/metabolismo , Actinas/genética , Actinas/metabolismo , Adenina/administração & dosagem , Idoso , Animais , Biomarcadores/metabolismo , Ceramidas/classificação , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Regulação da Expressão Gênica , Humanos , Hidronefrose/induzido quimicamente , Hidronefrose/genética , Hidronefrose/patologia , Rim/metabolismo , Rim/patologia , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pielonefrite/induzido quimicamente , Pielonefrite/genética , Pielonefrite/patologia , Esfingolipídeos/classificação , Obstrução Ureteral/genética , Obstrução Ureteral/patologiaRESUMO
Angiotensin (Ang) II-induced fibrosis of the kidney is characterized by the enhanced expression of profibrotic and proinflammatory genes, including the serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2). In addition to transcriptional regulation, both genes are subject to post-transcriptional control by AU-rich destabilizing elements that reside within the 3' untranslated region of the mRNA. We demonstrated that the continuous infusion of AngII in rats induced fibrosis concomitant with a significant increase in glomerular PAI-1 and COX-2 expression levels. Using RNA pull-down assays and electromobility shift assays, we demonstrated the increased binding of the ubiquitous RNA-binding protein human-antigen R (HuR) to the mRNAs of both PAI-1 and COX-2 in the cytoplasmic fractions of renal homogenates from AngII-treated rats. Actinomycin D experiments in rat mesangial cells revealed that AngII stabilizes both mRNAs via HuR as proven by small interfering RNA. Mechanistically, AngII promotes an increase in nucleo-cytoplasmic HuR shuttling, which was blocked by the PKC inhibitor rottlerin and the type-I AngII (AT(1)) receptor antagonist valsartan but was unaffected by both AT(2) receptor antagonists PD123319 and CGP42112. Co-immunoprecipitation revealed that AngII treatment caused an increase in nuclear PKC-delta concomitant with binding to nuclear HuR both in vitro and in vivo. The post-transcriptional regulation of PAI-1 and COX-2 by PKC-delta-dependent HuR shuttling may contribute to the pathogenesis of hypertensive nephrosclerosis triggered by AngII.
Assuntos
Angiotensina II/metabolismo , Ciclo-Oxigenase 2/metabolismo , Nefropatias/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Angiotensina II/farmacologia , Animais , Antígenos de Superfície/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ensaio de Desvio de Mobilidade Eletroforética , Fibrose , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Hipertensão/complicações , Imuno-Histoquímica , Imunoprecipitação , Nefropatias/etiologia , Masculino , RNA Mensageiro , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The importance of the Notch signaling in the development of glomerular diseases has been recently described. Therefore we analyzed in podocytes the expression and activity of ADAM10, one important component of the Notch signaling complex. METHODS: By Western blot, immunofluorescence and immunohistochemistry analysis we characterized the expression of ADAM10 in human podocytes, human urine and human renal tissue. RESULTS: We present evidence, that differentiated human podocytes possessed increased amounts of mature ADAM10 and released elevated levels of L1 adhesion molecule, one well known substrate of ADAM10. By using specific siRNA and metalloproteinase inhibitors we demonstrate that ADAM10 is involved in the cleavage of L1 in human podocytes. Injury of podocytes enhanced the ADAM10 mediated cleavage of L1. In addition, we detected ADAM10 in urinary podocytes from patients with kidney diseases and in tissue sections of normal human kidney. Finally, we found elevated levels of ADAM10 in urinary vesicles of patients with glomerular kidney diseases. CONCLUSIONS: The activity of ADAM10 in human podocytes may play an important role in the development of glomerular kidney diseases.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Nefropatias/enzimologia , Glomérulos Renais/enzimologia , Proteínas de Membrana/metabolismo , Podócitos/enzimologia , Proteínas ADAM/genética , Proteínas ADAM/urina , Proteína ADAM10 , Adulto , Idoso , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/urina , Linhagem Celular , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Nefropatias/genética , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/urina , Pessoa de Meia-Idade , Podócitos/metabolismo , RNA Interferente Pequeno/metabolismo , Urina/citologiaRESUMO
Hypertensive nephrosclerosis is characterized by activation of the renin-angiotensin-aldosterone system in combination with an inflammatory response characterized by an infiltration of T-cells and mononuclear cells, which release proinflammatory cytokines like IL-1beta/TNFalpha. In various models of experimental hypertensive disease the chemokine osteopontin (OPN) enhances further leukocyte infiltration. Therefore, we investigated the induction of OPN expression in renal mesangial cells (MCs) by aldosterone and the inflammatory cytokines IL-1beta/TNFalpha. Incubation with aldosterone resulted in a time- and concentration-dependent increase in OPN mRNA and protein. OPN mRNA expression followed a biphasic time course with an early increase between 4 and 8 h and the second phase starting at 14 h. The early phase was independent of protein synthesis, indicating a direct effect of aldosterone. Aldosterone-mediated induction of OPN was prevented by spironolactone, indicative of a receptor-mediated aldosterone effect. The mineralocorticoid receptor (MR) was identified in MCs by RT-PCR and immunoprecipitation, and shown to interact with a putative aldosterone-response element of the OPN promoter. The proinflammatory cytokines IL-1beta and TNFalpha only marginally affected OPN expression in MCs. However, coincubation of aldosterone and the cytokines synergistically increased OPN mRNA and protein levels. Since the synergistic effect on OPN mRNA was inhibited by diphenyleneiodonium, we assume an involvement of reactive oxygen species (ROS). We conclude that the chemokine OPN is a target gene of aldosterone in renal MCs, which is activated via the MR, and that proinflammatory cytokines enhance aldosterone-dependent OPN expression. In vivo, this may result in further leukocyte infiltration aggravating hypertensive nephrosclerosis.
Assuntos
Aldosterona/farmacologia , Interleucina-1beta/farmacologia , Células Mesangiais/efeitos dos fármacos , Osteopontina/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Aldosterona/fisiologia , Animais , Quimiotaxia de Leucócito , Sinergismo Farmacológico , Hipertensão/complicações , Células Mesangiais/metabolismo , Nefroesclerose/etiologia , Nefroesclerose/fisiopatologia , Oniocompostos/farmacologia , Osteopontina/genética , Osteopontina/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Mineralocorticoides/efeitos dos fármacos , Receptores de Mineralocorticoides/fisiologia , Sistema Renina-Angiotensina/fisiologiaRESUMO
The chemokine CXCL16 plays an important role in the recruitment of leukocytes to sites of inflammation influencing the course of experimental glomerulonephritis. Here we show that human kidneys highly express CXCL16 in the distal tubule, connecting tubule and principal cells of the collecting duct with weak expression in the thick ascending limb of Henle. Beside the membrane localization, a soluble form of CXCL16 can be proteolytically released which acts as a chemotactic factor. In human renal tissue the expression pattern of the disintegrin-like metalloproteinase ADAM10 is similar to that of CXCL16, suggesting ADAM10 can potentially cleave CXCL16 in vivo. When we tested this in primary tubular cells we found that blockade of ADAM10 activity inhibited the IFN-gamma induced release of soluble CXCL16. Acute tubular damage in renal allografts was associated with elevated urinary CXCL16 and this correlated with focally increased apical CXCL16 expression in the distal tubules and collecting ducts. Renal allograft biopsies, with a histopathological diagnosis of acute interstitial rejection, showed increased basolateral ADAM10 expression together with high numbers of infiltrating T cells. Our results suggest that CXCL16 and ADAM10 are involved in the recruitment of T cells to the kidney and play an important role in inflammatory kidney diseases.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Quimiocinas CXC/metabolismo , Transplante de Rim/imunologia , Rim/metabolismo , Proteínas de Membrana/metabolismo , Receptores Depuradores/metabolismo , Proteínas ADAM/imunologia , Proteína ADAM10 , Adulto , Idoso , Secretases da Proteína Precursora do Amiloide/imunologia , Quimiocina CXCL16 , Quimiocinas CXC/imunologia , Quimiocinas CXC/urina , Quimiotaxia de Leucócito , Feminino , Expressão Gênica , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Humanos , Túbulos Renais/patologia , Masculino , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Receptores Depuradores/imunologia , Solubilidade , Linfócitos T/fisiologiaRESUMO
Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação da Expressão Gênica , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Animais , Modelos Animais de Doenças , Fibrose , Rim/metabolismo , Rim/patologia , Masculino , Rim Policístico Autossômico Dominante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
BACKGROUND: The inducible cyclooxygenase (COX)-2 is a target of immunosuppressive drugs routinely administered to patients after transplantation. This study investigates a potential involvement of COX-2 in transplant rejection. Therefore, we examined the expression of COX-2 in biopsies obtained for diagnostic purposes. METHODS: COX-2 was detected by immunohistochemistry and in situ hybridization. Congruent staining was obtained by both methods: in specimens of a kidney explanted as the result of vascular rejection, tubular epithelial cells and endothelial cells stained positively for COX-2. Furthermore, in appendiceal specimens obtained at surgery, epithelial cells of the crypts, interstitial cells, and mesothelial cells were positive by both methods, affirming the specificity of the antibody. RESULTS: Compared with healthy control subjects, intensive staining of COX-2 was observed in most of the 28 biopsies obtained from patients diagnosed with vascular rejection combined with cellular interstitial rejection and tubulitis. Glomeruli and the macula densa area were essentially negative compared with prominent staining in cortical and medullary epithelial cells of the tubuli. Staining was distinct with individual positive cells in the tubular cross sections. Few arteries expressed COX-2 in intimal cells. Less prominent expression of COX-2 was detected in the biopsies of six kidneys obtained from patients diagnosed with acute tubular necrosis. CONCLUSION: This is the first report to show the up-regulation of COX-2 in human transplanted kidneys, despite ongoing immunosuppressive treatment. It remains to be established whether the up-regulation of COX-2 is part of the rejection process or has to be considered implicated in renal preservative mechanisms.
Assuntos
Rejeição de Enxerto/patologia , Isoenzimas/metabolismo , Transplante de Rim/patologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Apendicite/enzimologia , Apendicite/patologia , Biomarcadores/análise , Biópsia , Ciclo-Oxigenase 2 , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/imunologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/genética , Rim/enzimologia , Córtex Renal/patologia , Glomérulos Renais/patologia , Transplante de Rim/imunologia , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/genética , Valores de Referência , Urotélio/enzimologia , Urotélio/patologiaRESUMO
A local renin-angiotensin system (RAS), including specific angiotensin II receptor subtypes, is present in the rat ovary. Immunohistochemistry using a polyclonal antibody and mRNA in situ hybridization were performed on perfusion-fixed, paraffin-embedded ovaries obtained from untreated sexually mature, normally cycling rats. Immunofluorescent staining revealed an exclusive and distinct labeling of follicular granulosa cells showing a plaque-like expression pattern at the cell borders, being detectable in different stages of atretic degeneration. On adjacent sections the expression of the respective mRNA could be shown in granulosa cells of the same follicle. The AT2 receptor may be implicated in the ovarian atretic process by influencing follicular cell-cell communication.
Assuntos
Células da Granulosa/metabolismo , RNA Mensageiro/biossíntese , Receptor Tipo 2 de Angiotensina/biossíntese , Animais , Feminino , Imuno-Histoquímica , Hibridização In Situ , Ratos , Ratos Sprague-Dawley , Receptor Tipo 2 de Angiotensina/genéticaRESUMO
BACKGROUND: Osteopontin (Opn) is highly upregulated in many different animal models of renal disease, where it is suspected to participate in progression of the disease. In some models, angiotensin II (Ang II) seems to induce the elevated Opn production. Therefore, we examined the regulation of Opn in two-kidney, one-clip (2K1C) hypertensive rats, in which Ang II mediates the elevated blood pressure. METHODS: At days 7, 14, and 28, the clipped and nonclipped kidneys of hypertensive or sham-operated rats were analyzed for osteopontin protein, mRNA expression and mononuclear cell infiltration by imumunohistochemistry, in situ hybridization, and Northern blot analysis. Rats were treated with the Ang II type 1 receptor antagonist Valsartan starting 14 days after clipping. RESULTS: In sham-operated rats, Opn was mainly localized to cells of the thin ascending limbs of the outer medulla. No significant Opn staining was observed in cortical tubules. Focally defined tubular cortical Opn staining was observed in clipped and contralateral kidneys of hypertensive animals at days 14 and 28. Osteopontin protein expression correlated with the mRNA expression detected by in situ hybridization and Northern blot. Treatment with Valsartan reduced osteopontin staining by 51%, mRNA by 47%, and mononuclear cell number by 97% in nonclipped kidneys compared to untreated two-kidney, one-clip animals. In clipped kidneys, however, Opn protein and mRNA expression was not reduced, but a 240% increase in interstitial mononuclear cell number was observed. CONCLUSIONS: Osteopontin is involved in the induction of nephrosclerosis in renovascular hypertension, probably by a mechanism augmenting monocyte infiltration. Angiotensin II appears to be an important inducer of Opn in the nonclipped kidney. Ischemic conditions may regulate Opn expression in the clipped kidney.
Assuntos
Hipertensão Renal/fisiopatologia , Rim/fisiologia , Sialoglicoproteínas/genética , Valina/análogos & derivados , Antagonistas de Receptores de Angiotensina , Animais , Anti-Hipertensivos/farmacologia , Expressão Gênica , Hipertensão Renal/tratamento farmacológico , Rim/citologia , Masculino , Monócitos , Osteopontina , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina , Instrumentos Cirúrgicos , Tetrazóis/farmacologia , Valina/farmacologia , ValsartanaRESUMO
PURPOSE: We investigated whether the recently established biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), may help to diagnose acute urinary tract infections (UTI) in adults and are able to distinguish between upper or lower localization. METHODS: NGAL levels were measured in blood and urine, and KIM-1 concentrations in urine of 97 subjects. We recruited age- and gender-matched groups of 30 patients with acute upper UTI and 29 patients with acute lower UTI as well as 38 healthy controls. NGAL and KIM-1 were determined by ELISA, serum and urine creatinine applying the Jaffé's method. RESULTS: Urinary NGAL (uNGAL) was significantly increased in patients with upper as well as with lower UTI compared with the healthy controls (P < 0.01; P < 0.05). Accordingly, uNGAL normalized on urinary creatinine (uNGAL/uCrea) was markedly higher in patients with lower UTI compared with the control group (P < 0.05), and uNGAL/uCrea levels were still raised in patients with upper UTI, though not reaching statistical significance (P = 0.07). However, neither uNGAL nor uNGAL/uCrea levels displayed significant differences between patients with upper or lower UTI (P = 0.75; P = 0.97). uKIM-1 levels were close to the detection limit and too low to reliably reveal differences between the three groups. CONCLUSIONS: While uKIM-1 seems not to serve as a helpful biomarker in this setting, increased levels of uNGAL indicate inflammatory processes in the urinary tract in adults. However, the determination of uNGAL levels does not allow to differentiate between upper and lower UTI.
Assuntos
Proteínas de Fase Aguda/urina , Lipocalinas/sangue , Lipocalinas/urina , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/urina , Infecções Urinárias/sangue , Infecções Urinárias/urina , Doença Aguda , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Creatinina/sangue , Creatinina/urina , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Feminino , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Lipocalina-2 , Masculino , Glicoproteínas de Membrana/urina , Receptores ViraisRESUMO
We previously demonstrated that the widely used immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506), independent of immunophilin binding, can activate profibrogenic transforming growth factor ß (TGFß)/Smad signaling cascades in rat renal mesangial cells (MC). Here we report that both peptidyl-prolyl cis/trans isomerase (PPIase) inhibitors activate the extracellular-signaling regulated kinase (ERK) a member of the mitogen activated protein kinase (MAPK) and induce a rapid and transient increase in ERK phosphorylation. The MEK inhibitor U0126, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), a cell-permeant superoxide dismutase (SOD) and stigmatellin, an inhibitor of mitochondrial cytochrome bc1 complex strongly attenuated the increase in ERK1/2 phosphorylation triggered by PPIase inhibitors. Moreover, neutralizing antibodies against heparin binding-epidermal growth factor (HB-EGF), and inhibition of the EGF receptor by either small interfering (si)RNA or AG1478, demonstrate that ERK activation by both PPIase inhibitors is mediated via HB-EGF-induced EGF receptor (EGFR) tyrosine kinase activation. The strong inhibitory effects achieved by GM6001 and TAPI-2 furthermore implicate the involvement of a desintegrin and metalloproteinase 17 (ADAM17). Concomitantly, the PPIase inhibitor-induced ADAM17 secretase activity was significantly reduced by SOD and stigmatellin thus suggesting that mitochondrial ROS play a primary role in PPIase inhibitor-induced and ADAM17-mediated HB-EGF shedding. Functionally, both immunosuppressants caused a strong increase in MC proliferation which was similarly impeded when cells were treated in the presence of NAC, TAPI-2 or AG1478, respectively. Our data suggest that CsA and FK506, via ROS-dependent and ADAM17-catalyzed HB-EGF shedding induce the mitogenic ERK1/2 signaling cascade in renal MC.
Assuntos
Ciclosporina/farmacologia , Receptores ErbB/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tacrolimo/farmacologia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/enzimologia , Células Mesangiais/metabolismo , RatosRESUMO
OBJECTIVES: We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression. Furthermore, we investigated whether a particular kidney donor ABCC2 genotype is associated with delayed graft function in patients. METHODS: A new MRP2-deficient rat strain was established. Renal cross-transplantations were performed between congenic MRP2-deficient and wild-type rats. Renal disposition of MRP2 substrates was investigated in native and transplanted rats. Proteomic analyses and transcriptional profiling were performed in rat kidney graft cortices. Ninety-eight human kidney donor-recipient pairs were genotyped for five ABCC2 polymorphisms. The relationship between delayed graft function and ABCC2 genetic variants in donors and recipients was analyzed by backward stepwise logistic regression. RESULTS: In rats, the absence of renal MRP2 reduced renal bilirubin glucuronide excretion at pathologic plasma concentrations, modified renal p-aminohippurate excretion and did not affect renal morphine-6-glucuronide excretion. Renal MRP2 deficiency led to renal cortical protein or mRNA upregulation of glutathione transferase isoenzymes, glutaredoxin 2, and heme oxygenase-1. In patients, a particular donor ABCC2 genotype was associated with an increased incidence of delayed graft function. CONCLUSION: Kidney graft-specific MRP2 deficiency has mild effects on the renal excretion of some organic solutes under experimental conditions and induces a protein and gene expression pattern indicative of activated antioxidant defense mechanisms. This suggests that MRP2 is a determinant of the redox status in tubular epithelial cells and thus of the susceptibility to renal damage under conditions of treatment with multiple drugs and increased oxygen radical formation.
Assuntos
Genótipo , Transplante de Rim , Rim/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Doadores de Tecidos , Animais , Animais Congênicos , Perfilação da Expressão Gênica , Variação Genética , Glutarredoxinas/metabolismo , Glutationa Transferase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Logísticos , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Polimorfismo Genético , Proteoma/análise , Proteômica , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Transcrição Gênica , Regulação para CimaRESUMO
BACKGROUND: Aldosterone contributes substantially to cardiac and renal injury by acting on target cells not involved in the regulation of salt and water balance. The profibrotic protein connective tissue growth factor (CTGF) has been identified as one of the target proteins of aldosterone. However, the molecular mechanisms of aldosterone-mediated CTGF induction have not been characterized. METHODS: Mesangial cells were treated with aldosterone or dexamethasone. CTGF expression was characterized at the mRNA and protein level. Translocation of the glucocorticoid receptor (GR) was detected by immunocytochemistry and by Western blotting. RESULTS: Aldosterone and dexamethasone induced CTGF at the mRNA and protein level in a time- and concentration-dependent manner. Specific antagonists of the mineralocorticoid receptor, spironolactone, canrenoate or eplerenone, did not inhibit CTGF induction. However, inhibition of the GR by RU486 prevented dexamethasone-as well as aldosterone-induced CTGF expression, indicating the importance of the GR in aldosterone-mediated regulation of CTGF. This notion was confirmed by translocation of the GR to the nucleus upon stimulation with aldosterone. CONCLUSIONS: CTGF is a functional target of aldosterone in mesangial cells, but aldosterone-induced CTGF gene expression is not directly mediated by the mineralocorticoid receptor.