RESUMO
OBJECTIVE: To elucidate the potential function of long non-coding RNA (lncRNA) TUG1 in the progression of diabetic nephropathy (DN) and the underlying mechanism. MATERIALS AND METHODS: Rat diabetes mellitus (DM) model was established by streptozocin (STZ) administration. In vivo levels of TUG1 and relative genes in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway in DM rats and control rats were determined by the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Moreover, levels of kidney weight, 24 h-urine protein, blood urea nitrogen and serum creatinine in DM rats and controls were detected. Mesangial cells were subjected to induction of high-level glucose. Relative levels of TUG1 and relative genes in the PI3K/AKT pathway in mesangial cells were determined as well. Through Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay, the regulatory effect of TUG1 on the proliferative ability of mesangial cells induced with high-level glucose was evaluated. Finally, expression changes in the PI3K/AKT pathway and extracellular matrix (ECM)-related genes in mesangial cells were determined. RESULTS: TUG1 was downregulated in DM rats and mesangial cells induced with high-level glucose. Compared with controls, DM rats presented higher levels of kidney weight, 24 h-urine protein, blood urea nitrogen and serum creatinine, which were markedly reduced after TUG1 overexpression in vivo. Moreover, overexpression of TUG1 downregulated TGF-ß1, FN, and COL-IV, and inhibited the activation of the PI3K/AKT pathway. CONCLUSIONS: TUG1 is downregulated in DN. The overexpression of TUG1 could suppress the proliferation and ECM accumulation of mesangial cells via inhibiting the PI3K/AKT pathway.
Assuntos
Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/genética , Células Mesangiais/citologia , RNA Longo não Codificante/genética , Animais , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Regulação para Baixo , Fibrose , Glucose/farmacologia , Células Mesangiais/química , Células Mesangiais/efeitos dos fármacos , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais , EstreptozocinaRESUMO
Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.
Assuntos
Angiotensina I/química , Sistemas de Liberação de Medicamentos , Vírus da Influenza A/fisiologia , Células Mesangiais/química , Nanopartículas/química , Nanopartículas/uso terapêuticoRESUMO
BACKGROUND: Maternally inherited diabetes and deafness (MIDD), a mitochondrial genetic disorder, typically affects the kidneys and results in end-stage renal disease. Early diagnosis of MIDD is challenging when renal manifestations precede other key clinical features such as diabetes and deafness and/or when the disease is complicated by other renal pathologies. CASE PRESENTATION: Here, we present the case of a 33-year-old Japanese woman who had initially been diagnosed with IgA nephropathy but was found to have MIDD 6 years later. Two renal biopsies were conducted six years apart. While assessment of the first biopsy specimen with the monoclonal antibody (KM55) revealed mesangial IgA deposits (containing the galactose-deficient IgA1 variant [Gd-IgA1]), examination of the second specimen showed no mesangial IgA deposits and newly-developed glomerular global scleroses and tubular damage. Granular swollen epithelial cells (GSECs), characterised by abnormal mitochondria, were observed among the tubules and collecting ducts in both biopsy specimens. Mitochondrial DNA analysis revealed an m.3243A > G mutation. CONCLUSIONS: We rediscovered the usefulness of GSECs as a pathologically distinctive feature of mitochondrial nephropathy and reviewed the literature regarding MIDD complicated by mesangial IgA deposition. Furthermore, we demonstrate that the mesangial IgA deposits in this patient consisted of the galactose-deficient IgA1 variant. The monoclonal antibody (KM55) might be a useful tool to distinguish IgAN from latent IgA deposits.
Assuntos
Surdez/complicações , Surdez/diagnóstico , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Galactose/deficiência , Imunoglobulina A/análise , Células Mesangiais/patologia , Doenças Mitocondriais/complicações , Doenças Mitocondriais/diagnóstico , Adulto , Surdez/genética , Diabetes Mellitus Tipo 2/genética , Feminino , Humanos , Células Mesangiais/química , Células Mesangiais/ultraestrutura , Doenças Mitocondriais/genética , LinhagemRESUMO
BACKGROUND: A link between IgA nephropathy and Crohn's disease has recently been reported. Other researchers hypothesize that intestine-derived IgA complexes deposit in glomerular mesangial cells, eliciting IgA nephropathy. Intestinal mucosal plasma cells mainly secrete IgA2. Nevertheless, IgA1 deposition is strongly implicated as being the primary cause of IgA nephropathy. CASE PRESENTATION: A 46-year-old Japanese man developed IgA nephropathy 29 years ago, following tonsillectomy. As a result, a normal urinalysis was obtained. The patient previously suffered Crohn's disease followed by urinary occult blood and proteinuria six years ago. Exacerbation of IgA nephropathy was highly suspected. Therefore a renal biopsy was performed. A diagnosis of exacerbation of IgA nephropathy with mesangial cell proliferation and fibrotic cellular crescent was based upon the pathological findings. The patient exhibited a positive clinical course and eventually achieved a remission with immunosuppressive therapy including prednisolone treatment. Immunostaining for the detection of IgA subtypes was performed on both of his kidney and excised ileum. The results revealed IgA1 and IgA2 deposition by submucosal cells in intestine. Furthermore, IgA1 deposition of mesangial areas in the patient's kidney, indicated an association of IgA1 with the exacerbation of IgA nephropathy. CONCLUSION: This case represents the possibility that the intestine-derived IgA1 can be the origin of galactose-deficient IgA which is known to cause IgA nephropathy exacerbation.
Assuntos
Doença de Crohn/metabolismo , Glomerulonefrite por IGA/metabolismo , Imunoglobulina A/análise , Mucosa Intestinal/química , Células Mesangiais/química , Doença de Crohn/complicações , Progressão da Doença , Glomerulonefrite por IGA/complicações , Glomerulonefrite por IGA/patologia , Humanos , Íleo , Masculino , Pessoa de Meia-IdadeRESUMO
AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway.
Assuntos
Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Diabetes Mellitus/metabolismo , Glomérulos Renais/química , Obesidade/metabolismo , Esfingomielinas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Glucose/metabolismo , Humanos , Glomérulos Renais/metabolismo , Ácido Láctico/metabolismo , Células Mesangiais/química , Células Mesangiais/citologia , Células Mesangiais/efeitos dos fármacos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esfingomielinas/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The bark of Cinnamomum cassia, called 'Rou-Gui', a traditional spice and medicine in China, is used to treat diseases resulted from kidney yang deficiency, including diabetic nephropathy. The aim of this study is to investigate the anti-diabetic nephropathy activity of Rou-Gui and the active compounds in it. MATERIALS AND METHODS: The air-dried bark of C. cassia was extracted with 90% EtOH, the obtained residue was successively partitioned by petroleum ether, EtOAc, and n-BuOH followed by concentrating to give petroleum ether (RG-1), EtOAc (RG-2), n-BuOH (RG-3), and water fraction (RG-4), respectively. The anti-diabetic nephropathy activity of fraction (RG-1-4) was evaluated in vitro by inhibiting the expression of fibronectin, monocyte chemoattractant protein-1 and interleukin-6 in high-glucose-induced mesangial cells. By bioassay screenings, repeated column chromatography on fractions of RG-1, 2, and 3, led to the isolation of 23 compounds, whose structures were determined by extensive spectroscopic analyses, and the anti-diabetic nephropathy activity of the isolated compounds was also evaluated. RESULTS: Four new sesquiterpenoids, cinnamoids A-D (1-4), a new natural product (5), and 18 known compounds (6-23) were isolated from the EtOH extract of the bark of C. cassia under the bioassay-guided screenings. The anti-diabetic nephropathy activity assay showed that fractions of RG-1, 2, and 3 could significantly inhibit the production of fibronectin, monocyte chemoattractant protein-1 and interleukin-6 in high-glucose-stimulated mesangial cells at the concentration of 50 µg/ml; and sesquiterpenoids 5, 6, 14 and compound 20 could significantly inhibit the expression of fibronectin, monocyte chemoattractant protein-1 and interleukin-6 at the concentration of 50 µM. CONCLUSIONS: The results revealed that sesquiterpenoids may be the active compounds in C. cassia bark on diabetic nephropathy which provided new evidences for the traditional use of this herb to treat diabetic nephropathy and associated kidney diseases.
Assuntos
Cinnamomum aromaticum/química , Nefropatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Casca de Planta/química , Animais , Quimiocina CCL2/análise , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Fibronectinas/análise , Interleucina-6/análise , Células Mesangiais/química , Células Mesangiais/efeitos dos fármacos , Ratos , Sesquiterpenos/isolamento & purificação , Terpenos/isolamento & purificaçãoRESUMO
BACKGROUND: Evidence has accumulated that hypoxia plays a significant role in the pathogenesis and progression of both acute renal injury and chronic renal disease. However, little was known about the effects of hypoxia on lupus nephritis (LN). In the current study, we investigated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in LN. METHODS: Renal biopsies from 22 LN patients and 20 patients with renal carcinoma were obtained. In situ HIF-1α expression was examined by immunohistochemical staining, and the relationship between HIF-1α and clinical/pathological features was analyzed. HIF-1α expression in kidney from both MRL/lpr and C57BL/6 mice was detected by immunohistochemical technology. Dimethyloxaloylglycine (DMOG), an inhibitor of HIF-degrading prolylhydroxylases, was utilized to prevent HIF-1α degradation in mouse mesangial cells (MCs). After DMOG treatment, the proliferation and apoptosis rates of mouse MCs were determined. RESULTS: LN patients showed larger amounts of HIF-1α in both glomerular and tubulointerstitial areas. The levels of intraglomerular HIF-1α were closely associated with renal pathology activity index and clinical manifestations in LN patients. In MRL/lpr mice, intraglomerular HIF-1α-positive cells were also significantly increased. Interestingly, the levels of HIF-1α positively correlated with cell density in glomerulus in both LN patients and MRL/lpr mice. Upon treatment with DMOG, the proliferation of MCs was upregulated, and apoptosis was downregulated. CONCLUSION: HIF-1α is highly expressed in both glomerular and tubulointerstitial tissues in LN, especially in proliferative LN. HIF-1α may promote MCs growth through the induction of proliferation and inhibition of apoptosis, and hence plays an important role in the pathogenesis of LN.
Assuntos
Carcinoma de Células Renais/química , Proliferação de Células , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/química , Nefrite Lúpica/metabolismo , Adulto , Aminoácidos Dicarboxílicos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Imuno-Histoquímica , Túbulos Renais/química , Nefrite Lúpica/complicações , Nefrite Lúpica/patologia , Células Mesangiais/química , Camundongos , Inibidores de Prolil-Hidrolase/farmacologia , Proteinúria/etiologia , Albumina Sérica/metabolismo , Índice de Gravidade de DoençaRESUMO
BACKGROUND AND OBJECTIVES: Several studies have suggested that activation of the complement system is a contributing pathogenic mechanism in IgA nephropathy (IgAN). C4d staining is an inexpensive and easy-to-perform method for the analysis of renal biopsies. This study aimed to assess the clinical and prognostic implications of C4d staining in IgAN. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This retrospective cohort study included 283 patients with IgAN in 11 hospitals in Spain who underwent a renal biopsy between 1979 and 2010. The primary predictor was mesangial C4d staining. Secondary predictors included demographic, clinical, and laboratory characteristics, and Oxford pathologic classification criteria. The primary end point was the cumulative percentage of patients who developed ESRD, defined as onset of chronic dialysis or renal transplantation. C4d was analyzed by immunohistochemical staining using a polyclonal antibody. Kaplan-Meier and Cox proportional hazards analyses were performed to evaluate the effect of C4d staining on renal survival. RESULTS: There were 109 patients (38.5%) and 174 patients (61.5%) who were classified as C4d positive and C4d negative, respectively. Renal survival at 20 years was 28% in C4d-positive patients versus 85% in C4d-negative patients (P<0.001). Independent risk factors associated with ESRD were as follows: proteinuria (hazard ratio [HR] per every 1 g/d increase. 1.16; 95% confidence interval [95% CI], 1.03 to 1.31; P=0.01), eGFR (HR per every 1 ml/min per 1.73 m(2) increase, 0.96; 95% CI, 0.94 to 0.97; P<0.001), T2 Oxford classification (tubular atrophy/interstitial fibrosis, >50%; HR, 4.42; 95% CI, 1.40 to 13.88; P=0.01), and C4d-positive staining (HR, 2.45; 95% CI, 1.30 to 4.64; P=0.01). CONCLUSIONS: C4d-positive staining is an independent risk factor for the development of ESRD in IgAN. This finding is consistent with the possibility that complement activation is involved in the pathogenesis of this disease.
Assuntos
Complemento C4b/análise , Doença Hepática Terminal/fisiopatologia , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/fisiopatologia , Rim/patologia , Células Mesangiais/química , Fragmentos de Peptídeos/análise , Adulto , Biópsia , Progressão da Doença , Doença Hepática Terminal/etiologia , Doença Hepática Terminal/metabolismo , Doença Hepática Terminal/patologia , Feminino , Taxa de Filtração Glomerular , Glomerulonefrite por IGA/complicações , Glomerulonefrite por IGA/metabolismo , Humanos , Hipertelorismo/complicações , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Rim/química , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteinúria/etiologia , Estudos Retrospectivos , Fatores de Risco , Adulto JovemRESUMO
(+)-Lingzhiol and (-)-lingzhiol, a pair of rotary door-shaped meroterpenoidal enantiomers, were isolated from Ganoderma lucidum. Their structures were identified by spectroscopic methods and X-ray diffraction crystallography. Lingzhiol bears an unusual 5/5/6/6 ring system characteristic of sharing a C-3'-C-7' axis. Biological evaluation showed that (+)-lingzhiol or (-)-lingzhiol could selectively inhibit the phosphorylation of Smad3 in TGF-ß1-induced rat renal proximal tubular cells and activate Nrf2/Keap1 in mesangial cells under diabetic conditions.
Assuntos
Células Mesangiais/química , Reishi/química , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/química , Terpenos/química , Fator de Crescimento Transformador beta1/química , Animais , Células Mesangiais/metabolismo , Estrutura Molecular , Fosforilação , Ratos , Reishi/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Difração de Raios XRESUMO
Toxicodenanes A-C (1-3), representing sesquiterpenoids with three new carbon skeletons, were isolated from the dried resin of Toxicodendron vernicifluum. Their structures were identified by spectroscopic data and X-ray diffraction crystallography. Their plausible biosynthetic route was proposed via the isolated intermediate (4). Compounds 2 and 3 could significantly inhibit overproduction of fibronectin, collagen IV, and IL-6 in high-glucose-induced mesangial cells in a dose- and time-dependent manner, showing their potential in diabetic nephropathy.
Assuntos
Colágeno Tipo IV/química , Colágeno Tipo IV/efeitos dos fármacos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/efeitos dos fármacos , Glucose/química , Glucose/metabolismo , Interleucina-6/química , Células Mesangiais/química , Células Mesangiais/efeitos dos fármacos , Sesquiterpenos/química , Toxicodendron/química , Carbono , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/biossíntese , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Difração de Raios XRESUMO
AIM: To investigate the effect of high glucose and mycophenolate (MMF) on the expression of MCP-1 in human mesangial cells (HMCs) and fibronectin (FN). METHODS: The HMCs were divided randomly into five groups: control group (5 mmol/L glucose), high glucose group (30 mmol/L glucose), mannitol group (5 mmol/L glucose and 25 mmol/L mannitol), high glucose+MMF-10 group (30 mmol/L glucose plus 10 µg/mL mycophenolate) and high glucose+MMF-100 group (30 mmol/L glucose plus 100 µg/mL mycophenolate). We detected the levels of MCP-1 and fibronectin in each group at 24 h, 48 h and 72 h, respectively. The expression levels of the MCP-1 mRNA were detected by RT-PCR, and the protein expression of MCP-1 and fibronectin was measured by ELISA. RESULTS: Compared with the control group, the levels of the MCP-1 and FN in high glucose group were significantly increased with the expression peak at 48 h (P<0.01). The MMF with different concentration could inhibit the expression of MCP-1 and FN in time- and dose-dependent manner (P<0.05). CONCLUSION: Mycophenolate could inhibit the expressions of MCP-1 and FN in human mesangial cells and it might be expected to delay the development and progression of glomerular sclerosis and interstitial fibrosis.
Assuntos
Quimiocina CCL2/análise , Fibronectinas/análise , Glucose/farmacologia , Células Mesangiais/efeitos dos fármacos , Ácido Micofenólico/análogos & derivados , Células Cultivadas , Quimiocina CCL2/genética , Humanos , Células Mesangiais/química , Ácido Micofenólico/farmacologia , RNA Mensageiro/análiseRESUMO
The kidney is the major, if not sole, site for the production of 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active form of vitamin D that can stimulate calcium reabsorption in the kidney and may provide renoprotective benefits. The biological effects of 1,25(OH)(2)D(3) are mediated through a nuclear hormone receptor, known as the vitamin D receptor (VDR). It is well accepted that the VDR is present in the distal renal convoluted tubule cells; however, whether VDR is present in other kidney cell types is uncertain. Using a highly specific and sensitive anti-VDR antibody, we determined its distribution in the mouse kidney by immunohistochemistry. Our results show that the VDR is not only present in the distal but is also found in the proximal tubules, but at 24-fold lower levels. The VDR was also found in the macula densa of the juxtaglomerular apparatus, glomerular parietal epithelial cells, and podocytes. In contrast, the VDR is either very low or absent in interstitial fibroblasts, glomerular mesangial cells, and juxtaglomerular cells. Thus, identification of VDR in the proximal tubule, macula densa, and podocytes suggests that 1,25(OH)(2)D(3) plays a direct role in these cells under normal conditions.
Assuntos
Rim/química , Receptores de Calcitriol/análise , Adulto , Animais , Feminino , Fibroblastos/química , Humanos , Imuno-Histoquímica , Sistema Justaglomerular/química , Rim/citologia , Túbulos Renais Distais/química , Túbulos Renais Proximais/química , Masculino , Células Mesangiais/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/química , Receptores de Calcitriol/deficiência , Receptores de Calcitriol/genéticaRESUMO
OBJECTIVE: Myriocin (ISP-1) is a new type of immune inhibitor extracted from cordyceps sinensis. This study was to observe the effects of ISP-1 on the expression of cell cycle regulatory protein D1 (cyclinD1) in high glucose-induced hypertrophy rat glomerular mesangial cells (GMCs). METHODS: Rat GMCs were cultured in vitro and divided into three groups: high glucose (450 mg/dL D-glucose), normal glucose (100 mg/dL D-glucose, control) and ISP-1 (450 mg/dL D-glucose plus 100 µg/mL ISP-1). The protein expression of cyclinD1 was detected by flow cytometry. RESULTS: The expression of cyclinD1 in GMCs in the high glucose group increased significantly in a time-dependent manner compared with that in the control group. ISP-1 treatment significantly inhibited the up-regulated expression of cyclinD1 induced by high concentration glucose, and the expression of cyclinD1 was restored to the level of the control group 48 and 72 hrs after ISP-1 treatment. CONCLUSIONS: High concentration of glucose can up-regulate the expression of cyclinD1 in GMCs. ISP-1 may inhibit the up-regulated expression of cyclinD1, which might contribute to the protective effect of ISP-1 against GMC hypertrophy induced by high glucose.
Assuntos
Ciclina D1/análise , Ácidos Graxos Monoinsaturados/farmacologia , Glucose/toxicidade , Células Mesangiais/patologia , Animais , Fase G1 , Hipertrofia , Células Mesangiais/química , Ratos , Ratos Sprague-DawleyRESUMO
Hyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6-10 mg/dL) for 24-144 h. In further experiments, ihMCs were treated with UA (6-10 mg/dL) for 12 and 24 h simultaneously with losartan (10(-7) mmol/L). Angiotensin II (AII) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased AII protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca(2+)](i), and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. In addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. The latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.
Assuntos
Angiotensina II/fisiologia , Cálcio/análise , Células Mesangiais/efeitos dos fármacos , Ácido Úrico/farmacologia , Angiotensina II/biossíntese , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotelina-1/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Losartan/farmacologia , Células Mesangiais/química , Células Mesangiais/fisiologia , Reação em Cadeia da PolimeraseRESUMO
Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessive disease caused by mutations in a major podocyte protein, nephrin. NPHS1 is associated with heavy proteinuria and the development of glomerular scarring. We studied the cellular and molecular changes affecting the glomerular mesangium in NPHS1 kidneys. Marked hyperplasia of mesangial cells (MC) was mainly responsible for the early mesangial expansion in NPHS1 glomeruli. The levels of the proliferation marker, mindbomb homolog 1 and the major MC mitogen, platelet-derived growth factor, and its receptors, however, were quite normal. Only a small number of cells were positive for CD68 (marker for phagocytic cells) and CD34 (marker for mesenchymal precursor cells) in the NPHS1 mesangium. MCs strongly expressed alpha-smooth muscle actin, indicating myofibloblast transformation. The expression levels of the profibrotic mediators osteopontin and transforming growth factor beta were up-regulated in NPHS1 glomeruli by 3.2 and 1.6-fold, respectively, compared to the controls. The synthesis by MCs of the typical fibroblast products collagen I, fibronectin, and tenascin, however, was low, and the extracellular matrix increase was caused by the accumulation of a normal MC product, collagen IV. The results indicate that severe glomerular sclerosis can develop without major qualitative cellular or molecular changes in the mesangium.
Assuntos
Proliferação de Células , Mesângio Glomerular/patologia , Células Mesangiais/patologia , Síndrome Nefrótica/patologia , Actinas/análise , Adolescente , Antígenos CD/análise , Antígenos CD34/análise , Antígenos de Diferenciação Mielomonocítica/análise , Biópsia , Estudos de Casos e Controles , Criança , Pré-Escolar , Progressão da Doença , Proteínas da Matriz Extracelular/análise , Genótipo , Mesângio Glomerular/química , Mesângio Glomerular/cirurgia , Humanos , Hiperplasia , Imuno-Histoquímica , Lactente , Proteínas de Membrana/genética , Células Mesangiais/química , Pessoa de Meia-Idade , Mutação , Nefrectomia , Síndrome Nefrótica/classificação , Síndrome Nefrótica/congênito , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/cirurgia , Osteopontina/análise , Fenótipo , Fator de Crescimento Derivado de Plaquetas/análise , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Esclerose , Ubiquitina-Proteína Ligases/análiseRESUMO
Cultures of glomerular mesangial cells (MC) of rodent or human origin have been extensively employed in renal research laboratories since the early 1980s. Cultured MC retain extensive analogies with the fairly undifferentiated in vivo phenotype of an intercapillary mesenchymal cell population, i.e., a myofibroblast. MC proliferating in response to mitogens and growth factors can be growth-arrested by withdrawal of serum or 3D culture in collagen gels. They synthesize an extracellular matrix that includes interstitial collagens and has analogies with the glomerular basement membrane; a prominent cytoskeleton acts as a functional contractile apparatus. Cultured MC have been extensively employed as a tool for studying pathophysiological events such as mesangial expansion, scarring, and glomerulosclerosis. Current technology for MC isolation and culture is reviewed, with emphasis on methodological issues relevant to characterization, propagation, and long-term maintenance of homogeneous clones.
Assuntos
Técnicas de Cultura de Células , Células Mesangiais , Animais , Células Cultivadas , Humanos , Células Mesangiais/química , Células Mesangiais/citologia , Ratos , Ratos EndogâmicosRESUMO
OBJECTIVE AND DESIGN: This study investigates the regulatory role of connective tissue growth factor (CTGF) on production of fractalkine, monocyte-chemoattractant protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) in human mesangial cells, and explore the mechanisms of CTGF action. METHODS: Cultured human mesangial cells were treated with CTGF. Expressions of mRNA and proteins of fractalkine, MCP-1 and RANTES were analyzed by real-time polymerase chain reaction (PCR) and by enzyme-linked immunosorbent assay, respectively. Expressions of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB) were assessed by Western blotting. Activities of nuclear factor-KB (NF-KB) were determined by NF-kappaB luciferase reporter assay. RESULTS: CTGF enhanced the mRNA expressions and protein release of fractalkine, MCP-1 and RANTES, and the expressions of phosphorylated ERK1/2, PI3-K and PKB, and activities of NF-KB. Blockade of ERK1/2 inhibited the CTGF-induced expression ofphosphorylated ERK1/2 and NF-kappaB, and partially decreased the expressions of the above chemokines. PI3-K blockade downregulated the CTGF-stimulated expressions of phosphorylated PI3-K, PKB and NF-kappaB but not phosphorylated ERK1/2, partially decreased the expressions of the above chemokines. NF-kappaB blockade abrogated the CTGF-activated NF-kappaB and partially decreased the expressions of the above chemokines. Soluble heparin and K252a, an inhibitor of Trk, blocked CTGF-induced production of the above chemokines and the activation of the above signaling proteins. CONCLUSION: These results demonstrated that CTGF induces production of fractalkine, MCP-1 and RANTES via ERK1/2 and PI3-K/PKB/NF-kappaB-dependent signal pathway mediated by cell surface heparin sulfate proteoglycans and the tyrosine kinase receptor TrkA in human mesangial cells.
Assuntos
Quimiocinas/biossíntese , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Mesângio Glomerular/metabolismo , Células Mesangiais/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocina CCL5/biossíntese , Quimiocina CX3CL1/biossíntese , Mesângio Glomerular/citologia , Humanos , Células Mesangiais/química , RNA Mensageiro/metabolismoRESUMO
The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production.
Assuntos
Colágeno Tipo I/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Células Mesangiais/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Tirosina/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/genética , Genes Reporter , Humanos , Imuno-Histoquímica , Luciferases/metabolismo , Fosforilação , Plasmídeos , Proteínas Recombinantes/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genéticaRESUMO
INTRODUCTION: Abnormal control of the complement alternative pathway (CAP) (factor H, factor I and membrane cofactor protein (MCP) deficiencies) is a well established risk factor for the occurrence of haemolytic uraemic syndrome (HUS). In some instances, HUS may be associated with an unusual glomerulonephritis with isolated C3 deposits (glomerulonephritis C3). We determined whether HUS and glomerulonephritis C3 share common genetic susceptibility factors. METHODS: We identified 19 patients with glomerulonephritis C3. We measured levels of circulating complement components, performed assays for the detection of C3 nephritic factor (C3NeF) and screened factor H, factor I and MCP coding genes for the presence of mutations. RESULTS: Patients were divided in two groups based on renal pathology findings: group I (n = 13) had typical features of type I membranoproliferative glomerulonephritis (glomerulonephritis C3 with membranoproliferative glomerulonephritis (MPGN)) and group II (n = 6) was characterised by mesangial and epimembranous C3 deposits in the absence of mesangial proliferation (glomerulonephritis C3 without MPGN). Mutations in complement regulatory genes were detected in 4/6 patients with glomerulonephritis C3 without MPGN (heterozygous mutations in factor H gene (two patients) with low factor H antigenic level in one case, heterozygous mutations in factor I gene (two patients)) and in only 2/13 patients with glomerulonephritis C3 with MPGN (heterozygous mutations in factor H gene (one patient) and double heterozygous mutation in CD 46 gene (one patient)). In contrast, C3NeF was present in 5/13 patients with glomerulonephritis C3 with MPGN and in 2/6 patients with glomerulonephritis C3 without MPGN, one of whom had a factor H mutation. CONCLUSION: HUS and glomerulonephritis C3 without MPGN share common genetic risk factors. Constitutional or acquired dysregulation of the CAP is probably associated with a wide spectrum of diseases, ranging from HUS to glomerulonephritis C3 with MPGN.
Assuntos
Complemento C3/análise , Via Alternativa do Complemento/genética , Glomerulonefrite/genética , Síndrome Hemolítico-Urêmica/genética , Adolescente , Adulto , Idoso , Criança , Fator Nefrítico do Complemento 3/análise , Fator H do Complemento/análise , Fator H do Complemento/genética , Feminino , Fibrinogênio/análise , Fibrinogênio/genética , Predisposição Genética para Doença , Glomerulonefrite/classificação , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Masculino , Proteína Cofatora de Membrana/genética , Células Mesangiais/química , Células Mesangiais/ultraestrutura , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de RiscoRESUMO
The pathophysiology of the diabetic kidney (e.g., hypertrophy, increase urinary albumin excretion (UAE) is still ill-defined. Parathyroid hormone-related protein (PTHrP) is overexpressed in several nephropathies, but its role remains unclear. We evaluated the effect of high glucose on PTHrP and the PTH1 receptor (PTH1R) protein (by Western blot and immunohistochemistry) in the kidney of mice ith streptozotocin-induced diabetes, and in several mouse renal cells in vitro. Diabetic mice showed a significantly increased renal expression of PTHrP and PTH1R proteins with 2-8 weeks from the onset of diabetes. These animals exhibited an intense immunostaining for both proteins in the renal tubules and glomeruli. Using transgenic mice overexpressing PTHrP targeted to the renal proximal tubule, we found a significant increase in the renal hypertrophy index and in UAE in these diabetic mice relative to their control littermates. Moreover, logistic regression analysis showed a significant association between both PTHrP and PTH1R protein levels and UAE in all diabetic mice throughout the study. High-glucose (25 mm) medium was found to increase PTHrP and PTH1R in tubuloepithelial cells, mesangial cells and podocytes in vitro. Moreover, this increase in PTHrP (but not that of PTH1R) was inhibited by the AT1 receptor antagonist losartan. Collectively, these results indicate that the renal PTHrP/PTH1R system is upregulated in streptozotozin-induced diabetes in mice, and appears to adversely affect the outcome of diabetic renal disease. Our findings also suggest that angiotensin II might have a role in the PTHrP upregulation in this condition.